HPV8-E6 Interferes with Syntenin-2 Expression through Deregulation of Differentiation, Methylation and Phosphatidylinositide-Kinase Dependent Mechanisms.

PubMed

Marx, Benjamin; Miller-Lazic, Daliborka; Doorbar, John; Majewski, Slawomir; Hofmann, Kay; Hufbauer, Martin; Akgül, Baki

2017-01-01

The E6 oncoproteins of high-risk human papillomaviruses (HPV) of genus alpha contain a short peptide sequence at the carboxy-terminus, the PDZ binding domain, with which they interact with the corresponding PDZ domain of cellular proteins. Interestingly, E6 proteins from papillomaviruses of genus beta (betaPV) do not encode a comparable PDZ binding domain. Irrespective of this fact, we previously showed that the E6 protein of HPV8 (betaPV type) could circumvent this deficit by targeting the PDZ protein Syntenin-2 through transcriptional repression (Lazic et al., 2012). Despite its high binding affinity to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ), very little is known about Syntenin-2. This study aimed to extend the knowledge on Syntenin-2 and how its expression is controlled. We now identified that Syntenin-2 is expressed at high levels in differentiating and in lower amounts in keratinocytes cultured in serum-free media containing low calcium concentration. HPV8-E6 led to a further reduction of Syntenin-2 expression only in cells cultured in low calcium. In the skin of patients suffering from Epidermodysplasia verruciformis, who are predisposed to betaPV infection, Syntenin-2 was expressed in differentiating keratinocytes of non-lesional skin, but was absent in virus positive squamous tumors. Using 5-Aza-2'-deoxycytidine, which causes DNA demethylation, Syntenin-2 transcription was profoundly activated and fully restored in the absence and presence of HPV8-E6, implicating that E6 mediated repression of Syntenin-2 transcription is due to promoter hypermethylation. Since Syntenin-2 binds to PI(4,5)P 2 , we further tested whether the PI(4,5)P 2 metabolic pathway might govern Syntenin-2 expression. PI(4,5)P 2 is generated by the activity of phosphatidylinositol-4-phosphate-5-kinase type I (PIP5KI) or phosphatidylinositol-5-phosphate-4-kinase type II (PIP4KII) isoforms α, β and γ. Phosphatidylinositide kinases have recently been identified as regulators of gene transcription. Surprisingly, transfection of siRNAs directed against PIP5KI and PIP4KII resulted in higher Syntenin-2 expression with the highest effect mediated by siPIP5KIα. HPV8-E6 was able to counteract siPIP4KIIα, siPIP4KIIβ and siPIP5KIγ mediated Syntenin-2 re-expression but not siPIP5KIα. Finally, we identified Syntenin-2 as a key factor regulating PIP5KIα expression. Collectively, our data demonstrates that Syntenin-2 is regulated through multiple mechanisms and that downregulation of Syntenin-2 expression may contribute to E6 mediated dedifferentiation of infected skin cells.

Identification of the Interactome of a Palmitoylated Membrane Protein, Phosphatidylinositol 4-Kinase Type II Alpha.

PubMed

Gokhale, Avanti; Ryder, Pearl V; Zlatic, Stephanie A; Faundez, Victor

2016-01-01

Phosphatidylinositol 4-kinases (PI4K) are enzymes responsible for the production of phosphatidylinositol 4-phosphates, important intermediates in several cell signaling pathways. PI4KIIα is the most abundant membrane-associated kinase in mammalian cells and is involved in a variety of essential cellular functions. However, the precise role(s) of PI4KIIα in the cell is not yet completely deciphered. Here we present an experimental protocol that uses a chemical cross-linker, DSP, combined with immunoprecipitation and immunoaffinity purification to identify novel PI4KIIα interactors. As predicted, PI4KIIα participates in transient, low-affinity interactions that are stabilized by the use of DSP. Using this optimized protocol we have successfully identified actin cytoskeleton regulators-the WASH complex and RhoGEF1, as major novel interactors of PI4KIIα. While this chapter focuses on the PI4KIIα interactome, this protocol can and has been used to generate other membrane interactome networks.

Dual control of cardiac Na+–Ca2+ exchange by PIP2: electrophysiological analysis of direct and indirect mechanisms

PubMed Central

Yaradanakul, Alp; Feng, Siyi; Shen, Chengcheng; Lariccia, Vincenzo; Lin, Mei-Jung; Yang, Jinsong; Kang, T M; Dong, Ping; Yin, Helen L; Albanesi, Joseph P; Hilgemann, Donald W

2007-01-01

Cardiac Na+–Ca2+ exchange (NCX1) inactivates in excised membrane patches when cytoplasmic Ca2+ is removed or cytoplasmic Na+ is increased. Exogenous phosphatidylinositol-4,5-bis-phosphate (PIP2) can ablate both inactivation mechanisms, while it has no effect on inward exchange current in the absence of cytoplasmic Na+. To probe PIP2 effects in intact cells, we manipulated PIP2 metabolism by several means. First, we used cell lines with M1 (muscarinic) receptors that couple to phospholipase C's (PLCs). As expected, outward NCX1 current (i.e. Ca2+ influx) can be strongly inhibited when M1 agonists induce PIP2 depletion. However, inward currents (i.e. Ca2+ extrusion) without cytoplasmic Na+ can be increased markedly in parallel with an increase of cell capacitance (i.e. membrane area). Similar effects are incurred by cytoplasmic perfusion of GTPγS or the actin cytoskeleton disruptor latrunculin, even in the presence of non-hydrolysable ATP (AMP-PNP). Thus, G-protein signalling may increase NCX1 currents by destabilizing membrane cytoskeleton–PIP2 interactions. Second, to increase PIP2 we directly perfused PIP2 into cells. Outward NCX1 currents increase as expected. But over minutes currents decline substantially, and cell capacitance usually decreases in parallel. Third, using BHK cells with stable NCX1 expression, we increased PIP2 by transient expression of a phosphatidylinositol-4-phosphate-5-kinase (hPIP5KIβ) and a PI4-kinase (PI4KIIα). NCX1 current densities were decreased by > 80 and 40%, respectively. Fourth, we generated transgenic mice with 10-fold cardiac-specific overexpression of PI4KIIα. This wortmannin-insensitive PI4KIIα was chosen because basal cardiac phosphoinositides are nearly insensitive to wortmannin, and surface membrane PI4-kinase activity, defined functionally in excised patches, is not blocked by wortmannin. Both phosphatidylinositol-4-phosphate (PIP) and PIP2 were increased significantly, while NCX1 current densities were decreased by 78% with no loss of NCX1 expression. Most mice developed cardiac hypertrophy, and immunohistochemical analysis suggests that NCX1 is redistributed away from the outer sarcolemma. Cholera toxin uptake was increased 3-fold, suggesting that clathrin-independent endocytosis is enhanced. We conclude that direct effects of PIP2 to activate NCX1 can be strongly modulated by opposing mechanisms in intact cells that probably involve membrane cytoskeleton remodelling and membrane trafficking. PMID:17540705

The Btk-dependent PIP5K1γ lipid kinase activation by Fas counteracts FasL-induced cell death.

PubMed

Rossin, Aurélie; Lounnas, Nadia; Durivault, Jérôme; Miloro, Giorgia; Gagnoux-Palacios, Laurent; Hueber, Anne-Odile

2017-11-01

The Fas/FasL system plays a critical role in death by apoptosis and immune escape of cancer cells. The Fas receptor being ubiquitously expressed in tissues, its apoptotic-inducing function, initiated upon FasL binding, is tightly regulated by several negative regulatory mechanisms to prevent inappropriate cell death. One of them, involving the non-receptor tyrosine kinase Btk, was reported mainly in B cells and only poorly described. We report here that Btk negatively regulates, through its tyrosine kinase activity, the FasL-mediated cell death in epithelial cell lines from colon cancer origin. More importantly, we show that Btk interacts not only with Fas but also with the phosphatidylinositol-4-phosphate 5-kinase, PIP5K1γ, which, upon stimulation by Fas ligand, is responsible of a rapid and transient synthesis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ). This production requires both the presence and the tyrosine kinase activity of Btk, and participates in the negative regulation of FasL-mediated cell death since knocking down PIP5K1γ expression significantly strengthens the apoptotic signal upon FasL engagement. Altogether, our data demonstrate the cooperative role of Btk and PIP5K1γ in a FasL-induced PI(4,5)P 2 production, both proteins participating to the threshold setting of FasL-induced apoptotic commitment in colorectal cell lines.

Phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)phosphate in plant tissues. [Pisum sativum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Irvine, R.F.; Letcher, A.J.; Lander, D.J.

1989-03-01

Pea (Pisum sativum) leaf discs or swimming suspensions of Chlamydomonas eugametos were radiolabeled with ({sup 3}H)myo-inositol or ({sup 32}P)Pi and the lipids were extracted, deacylated, and their glycerol moieties removed. The resulting inositol trisphosphate and bisphosphate fractions were examined by periodate degradation, reduction and dephosphorylation, or by incubation with human red cell membranes. Their likely structures were identified as D-myo-inositol(1,4,5)trisphosphate and D-myo-inositol(1,4,)-bisphosphate. It is concluded that plants contain phosphatidylinositol(4)phosphate and phosphatidylinositol(4,5)bisphosphate; no other polyphosphoinositides were detected.

Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development

PubMed Central

Stith, Bradley J.

2015-01-01

This review emphasizes how lipids regulate membrane fusion and the proteins involved in three developmental stages: oocyte maturation to the fertilizable egg, fertilization and during first cleavage. Decades of work show that phosphatidic acid (PA) releases intracellular calcium, and recent work shows that the lipid can activate Src tyrosine kinase or phospholipase C during Xenopus fertilization. Numerous reports are summarized to show three levels of increase in lipid second messengers inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol (DAG) during the three different developmental stages. In addition, possible roles for PA, ceramide, lysophosphatidylcholine, plasmalogens, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylinositol 4,5-bisphosphate, membrane microdomains (rafts) and phosphatidylinositol 3,4,5-trisphosphate in regulation of membrane fusion (acrosome reaction, sperm-egg fusion, cortical granule exocytosis), inositol 1,4,5-trisphosphate receptors, and calcium release are discussed. The role of six lipases involved in generating putative lipid second messengers during fertilization is also discussed: phospholipase D, autotaxin, lipin1, sphingomyelinase, phospholipase C, and phospholipase A2. More specifically, proteins involved in developmental events and their regulation through lipid binding to SH3, SH4, PH, PX, or C2 protein domains is emphasized. New models are presented for PA activation of Src (through SH3, SH4 and a unique domain), that this may be why the SH2 domain of PLCγ is not required for Xenopus fertilization, PA activation of phospholipase C, a role for PA during the calcium wave after fertilization, and that calcium/calmodulin may be responsible for the loss of Src from rafts after fertilization. Also discussed is that the large DAG increase during fertilization derives from phospholipase D production of PA and lipin dephosphorylation to DAG. PMID:25748412

Mapping of Functional Domains of the Lipid Kinase Phosphatidylinositol 4-Kinase Type III Alpha Involved in Enzymatic Activity and Hepatitis C Virus Replication

PubMed Central

Harak, Christian; Radujkovic, Danijela; Taveneau, Cyntia; Reiss, Simon; Klein, Rahel; Bressanelli, Stéphane

2014-01-01

ABSTRACT The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an endoplasmic reticulum (ER)-resident enzyme that synthesizes phosphatidylinositol 4-phosphate (PI4P). PI4KIIIα is an essential host factor for hepatitis C virus (HCV) replication. Interaction with HCV nonstructural protein 5A (NS5A) leads to kinase activation and accumulation of PI4P at intracellular membranes. In this study, we investigated the structural requirements of PI4KIIIα in HCV replication and enzymatic activity. Therefore, we analyzed PI4KIIIα mutants for subcellular localization, reconstitution of HCV replication in PI4KIIIα knockdown cell lines, PI4P induction in HCV-positive cells, and lipid kinase activity in vitro. All mutants still interacted with NS5A and localized in a manner similar to that of the full-length enzyme, suggesting multiple regions of PI4KIIIα are involved in NS5A interaction and subcellular localization. Interestingly, the N-terminal 1,152 amino acids were dispensable for HCV replication, PI4P induction, and enzymatic function, whereas further N-terminal or C-terminal deletions were deleterious, thereby defining the minimal PI4KIIIα core enzyme at a size of ca. 108 kDa. Additional deletion of predicted functional motifs within the C-terminal half of PI4KIIIα also were detrimental for enzymatic activity and for the ability of PI4KIIIα to rescue HCV replication, with the exception of a proposed nuclear localization signal, suggesting that the entire C-terminal half of PI4KIIIα is involved in the formation of a minimal enzymatic core. This view was supported by structural modeling of the PI4KIIIα C terminus, suggesting a catalytic center formed by an N- and C-terminal lobe and an armadillo-fold motif, which is preceded by three distinct alpha-helical domains probably involved in regulation of enzymatic activity. IMPORTANCE The lipid kinase PI4KIIIα is of central importance for cellular phosphatidylinositol metabolism and is a key host cell factor of hepatitis C virus replication. However, little is known so far about the structure of this 240-kDa protein and the functional importance of specific subdomains regarding lipid kinase activity and viral replication. This work focuses on the phenotypic analysis of distinct PI4KIIIα mutants in different biochemical and cell-based assays and develops a structural model of the C-terminal enzymatic core. The results shed light on the structural and functional requirements of enzymatic activity and the determinants required for HCV replication. PMID:24920820

Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets

PubMed Central

Mohan, Nimmy; AP, Sudheesh; Francis, Nimmy; Anderson, Richard; Laishram, Rakesh S.

2015-01-01

Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P2) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P2 as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3′-end processing. PMID:26138484

PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway.

PubMed

Sun, H; Lesche, R; Li, D M; Liliental, J; Zhang, H; Gao, J; Gavrilova, N; Mueller, B; Liu, X; Wu, H

1999-05-25

To investigate the molecular basis of PTEN-mediated tumor suppression, we introduced a null mutation into the mouse Pten gene by homologous recombination in embryonic stem (ES) cells. Pten-/- ES cells exhibited an increased growth rate and proliferated even in the absence of serum. ES cells lacking PTEN function also displayed advanced entry into S phase. This accelerated G1/S transition was accompanied by down-regulation of p27(KIP1), a major inhibitor for G1 cyclin-dependent kinases. Inactivation of PTEN in ES cells and in embryonic fibroblasts resulted in elevated levels of phosphatidylinositol 3,4,5,-trisphosphate, a product of phosphatidylinositol 3 kinase. Consequently, PTEN deficiency led to dosage-dependent increases in phosphorylation and activation of Akt/protein kinase B, a well-characterized target of the phosphatidylinositol 3 kinase signaling pathway. Akt activation increased Bad phosphorylation and promoted Pten-/- cell survival. Our studies suggest that PTEN regulates the phosphatidylinositol 3,4, 5,-trisphosphate and Akt signaling pathway and consequently modulates two critical cellular processes: cell cycle progression and cell survival.

Sac1--Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cai, Yiying; Deng, Yongqiang; Horenkamp, Florian

2014-08-25

Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot–Marie–Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1–Vps74 interface results in a broader distribution of phosphatidylinositolmore » 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.« less

Selectins and chemokines use shared and distinct signals to activate β2 integrins in neutrophils

PubMed Central

Yago, Tadayuki; Zhang, Nan; Zhao, Liang; Abrams, Charles S.

2018-01-01

Rolling neutrophils receive signals while engaging P- and E-selectin and chemokines on inflamed endothelium. Selectin signaling activates β2 integrins to slow rolling velocities. Chemokine signaling activates β2 integrins to cause arrest. Despite extensive study, key aspects of these signaling cascades remain unresolved. Using complementary in vitro and in vivo assays, we found that selectin and chemokine signals in neutrophils triggered Rap1a-dependent and phosphatidylinositol-4-phosphate 5-kinase γ (PIP5Kγ90)–dependent pathways that induce integrin-dependent slow rolling and arrest. Interruption of both pathways, but not either pathway alone, blocked talin-1 recruitment to and activation of integrins. An isoform of PIP5Kγ90 lacking the talin-binding domain (PIP5Kγ87) could not activate integrins. Chemokines, but not selectins, used phosphatidylinositol-4,5-bisphosphate 3-kinase γ (PI3Kγ) in cooperation with Rap1a to mediate integrin-dependent slow rolling (at low chemokine concentrations), as well as arrest (at high chemokine concentrations). High levels of chemokines activated β2 integrins without selectin signals. When chemokines were limiting, they synergized with selectins to activate β2 integrins. PMID:29592875

Selectins and chemokines use shared and distinct signals to activate β2 integrins in neutrophils.

PubMed

Yago, Tadayuki; Zhang, Nan; Zhao, Liang; Abrams, Charles S; McEver, Rodger P

2018-04-10

Rolling neutrophils receive signals while engaging P- and E-selectin and chemokines on inflamed endothelium. Selectin signaling activates β2 integrins to slow rolling velocities. Chemokine signaling activates β2 integrins to cause arrest. Despite extensive study, key aspects of these signaling cascades remain unresolved. Using complementary in vitro and in vivo assays, we found that selectin and chemokine signals in neutrophils triggered Rap1a-dependent and phosphatidylinositol-4-phosphate 5-kinase γ (PIP5Kγ90)-dependent pathways that induce integrin-dependent slow rolling and arrest. Interruption of both pathways, but not either pathway alone, blocked talin-1 recruitment to and activation of integrins. An isoform of PIP5Kγ90 lacking the talin-binding domain (PIP5Kγ87) could not activate integrins. Chemokines, but not selectins, used phosphatidylinositol-4,5-bisphosphate 3-kinase γ (PI3Kγ) in cooperation with Rap1a to mediate integrin-dependent slow rolling (at low chemokine concentrations), as well as arrest (at high chemokine concentrations). High levels of chemokines activated β2 integrins without selectin signals. When chemokines were limiting, they synergized with selectins to activate β2 integrins. © 2018 by The American Society of Hematology.

Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets.

PubMed

Mohan, Nimmy; Sudheesh, A P; Francis, Nimmy; Anderson, Richard; Laishram, Rakesh S

2015-08-18

Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P2) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P2 as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3'-end processing. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes

PubMed Central

Cestari, Igor; Stuart, Ken

2015-01-01

African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing. PMID:25964327

GABARAP-mediated targeting of PI4K2A/PI4KIIα to autophagosomes regulates PtdIns4P-dependent autophagosome-lysosome fusion.

PubMed

Albanesi, Joseph; Wang, Hanzhi; Sun, Hui-Qiao; Levine, Beth; Yin, Helen

2015-11-02

For decades, phosphatidylinositol 4-phosphate (PtdIns4P) was considered primarily as a precursor in the synthesis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P 2 ). More recently, specific functions for PtdIns4P itself have been identified, particularly in the regulation of intracellular membrane trafficking. PI4K2A/PI4KIIα (phosphatidylinositol 4-kinase type 2 α), one of the 4 enzymes that catalyze PtdIns4P production in mammalian cells, promotes vesicle formation from the trans-Golgi network (TGN) and endosomes. We recently identified a novel function for PI4K2A-derived PtdIns4P, as a facilitator of autophagosome-lysosome (A-L) fusion. We further showed that that this function requires the presence of the autophagic adaptor protein GABARAP (GABA[A] receptor-associated protein), which binds to PI4K2A and recruits it to autophagosomes. The mechanism whereby GABARAP-PI4K2A-PtdIns4P promotes A-L fusion remains to be defined. Based on other examples of phosphoinositide involvement in membrane trafficking, we speculate that it acts by recruiting elements of the membrane docking and fusion machinery.

Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow.

PubMed

Berk, B C; Corson, M A; Peterson, T E; Tseng, H

1995-12-01

Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.

Golgi-Associated Protein Kinase C-ε Is Delivered to Phagocytic Cups: Role of Phosphatidylinositol 4-Phosphate.

PubMed

Hanes, Cheryl M; D'Amico, Anna E; Ueyama, Takehiko; Wong, Alexander C; Zhang, Xuexin; Hynes, W Frederick; Barroso, Margarida M; Cady, Nathaniel C; Trebak, Mohamed; Saito, Naoaki; Lennartz, Michelle R

2017-07-01

Protein kinase C-ε (PKC-ε) at phagocytic cups mediates the membrane fusion necessary for efficient IgG-mediated phagocytosis. The C1B and pseudosubstrate (εPS) domains are necessary and sufficient for this concentration. C1B binds diacylglycerol; the docking partner for εPS is unknown. Liposome assays revealed that the εPS binds phosphatidylinositol 4-phosphate (PI4P) and PI(3,5)P 2 Wortmannin, but not LY294002, inhibits PKC-ε concentration at cups and significantly reduces the rate of phagocytosis. As Wortmannin inhibits PI4 kinase, we hypothesized that PI4P mediates the PKC-ε concentration at cups and the rate of phagocytosis. PKC-ε colocalizes with the trans -Golgi network (TGN) PI4P reporter, P4M, suggesting it is tethered at the TGN. Real-time imaging of GFP-PKC-ε-expressing macrophages revealed a loss of Golgi-associated PKC-ε during phagocytosis, consistent with a Golgi-to-phagosome translocation. Treatment with PIK93, a PI4 kinase inhibitor, reduces PKC-ε at both the TGN and the cup, decreases phagocytosis, and prevents the increase in capacitance that accompanies membrane fusion. Finally, expression of the Golgi-directed PI4P phosphatase, hSac1-K2A, recapitulates the PIK93 phenotype, confirming that Golgi-associated PI4P is critical for efficient phagocytosis. Together these data are consistent with a model in which PKC-ε is tethered to the TGN via an εPS-PI4P interaction. The TGN-associated pool of PKC-ε concentrates at the phagocytic cup where it mediates the membrane fusion necessary for phagocytosis. The novelty of these data lies in the demonstration that εPS binds PI4P and PI(3,5)P 2 and that PI4P is necessary for PKC-ε localization at the TGN, its translocation to the phagocytic cup, and the membrane fusion required for efficient Fc [γ] receptor-mediated phagocytosis. Copyright © 2017 by The American Association of Immunologists, Inc.

Ste12/Fab1 phosphatidylinositol-3-phosphate 5-kinase is required for nitrogen-regulated mitotic commitment and cell size control

PubMed Central

Schauries, Marie; Kaczmarek, Adrian; Franz-Wachtel, Mirita; Du, Wei; Krug, Karsten; Maček, Boris; Petersen, Janni

2017-01-01

Tight coupling of cell growth and cell cycle progression enable cells to adjust their rate of division, and therefore size, to the demands of proliferation in varying nutritional environments. Nutrient stress promotes inhibition of Target Of Rapamycin Complex 1 (TORC1) activity. In fission yeast, reduced TORC1 activity advances mitotic onset and switches growth to a sustained proliferation at reduced cell size. A screen for mutants, that failed to advance mitosis upon nitrogen stress, identified a mutant in the PIKFYVE 1-phosphatidylinositol-3-phosphate 5-kinase fission yeast homolog Ste12. Ste12PIKFYVE deficient mutants were unable to advance the cell cycle to reduce cell size after a nitrogen downshift to poor nitrogen (proline) growth conditions. While it is well established that PI(3,5)P2 signalling is required for autophagy and that Ste12PIKFYVE mutants have enlarged vacuoles (yeast lysosomes), neither a block to autophagy or mutants that independently have enlarged vacuoles had any impact upon nitrogen control of mitotic commitment. The addition of rapamycin to Ste12PIKFYVE deficient mutants reduced cell size at division to suggest that Ste12PIKFYVE possibly functions upstream of TORC1. ste12 mutants display increased Torin1 (TOR inhibitor) sensitivity. However, no major impact on TORC1 or TORC2 activity was observed in the ste12 deficient mutants. In summary, Ste12PIKFYVE is required for nitrogen-stress mediated advancement of mitosis to reduce cell size at division. PMID:28273166

Phosphatidylinositol 3-kinase activity in murine motoneuron disease: the progressive motor neuropathy mouse.

PubMed

Wagey, R; Lurot, S; Perrelet, D; Pelech, S L; Sagot, Y; Krieger, C

2001-01-01

A murine model of motoneuron disease, the pmn/pmn mouse, shows a reduction in the retrograde transport of fluorescent probes applied directly onto the cut end of sciatic nerve. Brain-derived neurotrophic factor (BDNF), when co-applied with fluorescent tracers, increases the number of retrograde labelled motoneurons. We demonstrate here that spinal cord tissue from pmn/pmn mice had significantly reduced phosphatidylinositol 3-kinase activity and expression in the particulate fraction compared to controls, without changes in the activities or expression of the downstream kinases, protein kinase B/Akt or Erk1. Systemic administration of BDNF augmented phosphatidylinositol 3-kinase specific activity in spinal cord tissue from pmn/pmn and control mice, with a greater elevation in the particulate fractions of pmn/pmn mice than in controls. We examined the effect of inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase on the retrograde labelling of motoneurons, 24h following the direct application of inhibitors and Fluorogold to the cut end of sciatic nerve in control and pmn/pmn mice (labelling index). The mitogen-activated protein kinase kinase inhibitor PD 98059 had no effect on the labelling index in control or pmn/pmn mice. In the absence of exogenous BDNF, phosphatidylinositol 3-kinase inhibitors reduced the number of labelled motoneurons in control mice, without changing the labelling index in pmn/pmn. Co-application of phosphatidylinositol 3-kinase inhibitors with BDNF to the cut end of sciatic nerve blocked the action of BDNF on retrograde labelling in pmn/pmn mice. These results indicate that the retrograde labelling of motoneurons is mediated by phosphatidylinositol 3-kinase-dependent and -independent pathways. In pmn/pmn mice, phosphatidylinositol 3-kinase activity in spinal neurons is below the level required for optimal retrograde labelling of motoneurons and labelling can be augmented by the administration of growth factors stimulating phosphatidylinositol 3-kinase activity. The data indicate that phosphatidylinositol 3-kinase activity is important in the uptake and/or retrograde transport of substances by motoneurons and is altered in this model of motoneuron diseases.

GABARAPs regulate PI4P-dependent autophagosome:lysosome fusion.

PubMed

Wang, Hanzhi; Sun, Hui-Qiao; Zhu, Xiaohui; Zhang, Li; Albanesi, Joseph; Levine, Beth; Yin, Helen

2015-06-02

The Atg8 autophagy proteins are essential for autophagosome biogenesis and maturation. The γ-aminobutyric acid receptor-associated protein (GABARAP) Atg8 family is much less understood than the LC3 Atg8 family, and the relationship between the GABARAPs' previously identified roles as modulators of transmembrane protein trafficking and autophagy is not known. Here we report that GABARAPs recruit palmitoylated PI4KIIα, a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) and binds GABARAPs, from the perinuclear Golgi region to autophagosomes to generate PI4P in situ. Depletion of either GABARAP or PI4KIIα, or overexpression of a dominant-negative kinase-dead PI4KIIα mutant, decreases autophagy flux by blocking autophagsome:lysosome fusion, resulting in the accumulation of abnormally large autophagosomes. The autophagosome defects are rescued by overexpressing PI4KIIα or by restoring intracellular PI4P through "PI4P shuttling." Importantly, PI4KIIα's role in autophagy is distinct from that of PI4KIIIβ and is independent of subsequent phosphatidylinositol 4,5 biphosphate (PIP2) generation. Thus, GABARAPs recruit PI4KIIα to autophagosomes, and PI4P generation on autophagosomes is critically important for fusion with lysosomes. Our results establish that PI4KIIα and PI4P are essential effectors of the GABARAP interactome's fusion machinery.

Enterovirus 3A Facilitates Viral Replication by Promoting Phosphatidylinositol 4-Kinase IIIβ–ACBD3 Interaction

PubMed Central

Xiao, Xia; Lei, Xiaobo; Zhang, Zhenzhen; Ma, Yijie; Qi, Jianli; Wu, Chao; Xiao, Yan; Li, Li

2017-01-01

ABSTRACT Like other enteroviruses, enterovirus 71 (EV71) relies on phosphatidylinositol 4-kinase IIIβ (PI4KB) for genome RNA replication. However, how PI4KB is recruited to the genome replication sites of EV71 remains elusive. Recently, we reported that a host factor, ACBD3, is needed for EV71 replication by interacting with viral 3A protein. Here, we show that ACBD3 is required for the recruitment of PI4KB to RNA replication sites. Overexpression of viral 3A or EV71 infection stimulates the interaction of PI4KB and ACBD3. Consistently, EV71 infection induces the production of phosphatidylinositol-4-phosphate (PI4P). Furthermore, PI4KB, ACBD3, and 3A are all localized to the viral-RNA replication sites. Accordingly, PI4KB or ACBD3 depletion by small interfering RNA (siRNA) leads to a reduction in PI4P production after EV71 infection. I44A or H54Y substitution in 3A interrupts the stimulation of PI4KB and ACBD3. Further analysis suggests that stimulation of ACBD3-PI4KB interaction is also important for the replication of enterovirus 68 but disadvantageous to human rhinovirus 16. These results reveal a mechanism of enterovirus replication that involves a selective strategy for recruitment of PI4KB to the RNA replication sites. IMPORTANCE Enterovirus 71, like other human enteroviruses, replicates its genome within host cells, where viral proteins efficiently utilize cellular machineries. While multiple factors are involved, it is largely unclear how viral replication is controlled. We show that the 3A protein of enterovirus 71 recruits an enzyme, phosphatidylinositol 4-kinase IIIβ, by interacting with ACBD3, which alters cellular membranes through the production of a lipid, PI4P. Consequently, the viral and host proteins form a large complex that is necessary for RNA synthesis at replication sites. Notably, PI4KB-ACBD3 interaction also differentially mediates the replication of enterovirus 68 and rhinovirus 16. These results provide new insight into the molecular network of enterovirus replication. PMID:28701404

Identification of human Phosphatidyl Inositol 5-Phosphate 4-Kinase as an RNA binding protein that is imported into Plasmodium falciparum.

PubMed

Vindu, Arya; Dandewad, Vishal; Seshadri, Vasudevan

2018-04-06

Plasmodium falciparum is a causative agent for malaria and has a complex life cycle in human and mosquito hosts. Translation repression of specific set of mRNA has been reported in gametocyte stages of this parasite. A conserved element present in the 3'UTR of some of these transcripts was identified. Biochemical studies have identified components of the RNA storage and/or translation inhibitor complex but it is not yet clear how the complex is specifically recruited on the RNA targeted for translation regulation. We used the 3'UTR region of translationally regulated transcripts to identify Phosphatidyl-inositol 5-phosphate 4-kinase (PIP4K2A) as the protein that associates with these RNAs. We further show that recombinant PIP4K2A has the RNA binding activity and can associate specifically with Plasmodium 3'UTR RNAs. Immunostainings show that hPIP4K2A is imported into the Plasmodium parasite from RBC. These results identify a novel RNA binding role for PIP4K2A that may play a role in Plasmodium propagation. Copyright © 2018 Elsevier Inc. All rights reserved.

The Novel Poly(A) Polymerase Star-PAP is a Signal-Regulated Switch at the 3′-end of mRNAs

PubMed Central

Li, Weimin; Laishram, Rakesh S.; Anderson, Richard A.

2013-01-01

The mRNA 3′-untranslated region (3′-UTR) modulates message stability, transport, intracellular location and translation. We have discovered a novel nuclear poly(A) polymerase termed Star-PAP (nuclear speckle targeted PIPKIα regulated-poly(A) polymerase) that couples with the transcriptional machinery and is regulated by the phosphoinositide lipid messenger phosphatidylinositol-4,5-bisphosphate (PI4,5P2), the central lipid in phosphoinositide signaling. PI4,5P2 is generated primarily by type I phosphatidylinositol phosphate kinases (PIPKI). Phosphoinositides are present in the nucleus including at nuclear speckles compartments separate from known membrane structures. PIPKs regulate cellular functions by interacting with PI4,5P2 effectors where PIPKs generate PI4,5P2 that then modulates the activity of the associated effectors. Nuclear PIPKIα interacts with and regulates Star-PAP, and PI4,5P2 specifically activates Star-PAP in a gene- and signaling-dependent manner. Importantly, other select signaling molecules integrated into the Star-PAP complex seem to regulate Star-PAP activities and processivities toward RNA substrates, and unique sequence elements around the Star-PAP binding sites within the 3′-UTR of target genes contribute to Star-PAP specificity for processing. Therefore, Star-PAP and its regulatory molecules form a signaling nexus at the 3′-end of target mRNAs to control the expression of select group of genes including the ones involved in stress responses. PMID:23306079

A novel IRS-1-associated protein, DGKζ regulates GLUT4 translocation in 3T3-L1 adipocytes

PubMed Central

Liu, TingYu; Yu, BuChin; Kakino, Mamoru; Fujimoto, Hitoshi; Ando, Yasutoshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

2016-01-01

Insulin receptor substrates (IRSs) are major targets of insulin receptor tyrosine kinases. Here we identified diacylglycerol kinase zeta (DGKζ) as an IRS-1-associated protein, and examined roles of DGKζ in glucose transporter 4 (GLUT4) translocation to the plasma membrane. When DGKζ was knocked-down in 3T3-L1 adipocytes, insulin-induced GLUT4 translocation was inhibited without affecting other mediators of insulin-dependent signaling. Similarly, knockdown of phosphatidylinositol 4-phosphate 5-kinase 1α (PIP5K1α), which had been reported to interact with DGKζ, also inhibited insulin-induced GLUT4 translocation. Moreover, DGKζ interacted with IRS-1 without insulin stimulation, but insulin stimulation decreased this interaction. Over-expression of sDGKζ (short-form DGKζ), which competed out DGKζ from IRS-1, enhanced GLUT4 translocation without insulin stimulation. Taking these results together with the data showing that cellular PIP5K activity was correlated with GLUT4 translocation ability, we concluded that IRS-1-associated DGKζ prevents GLUT4 translocation in the absence of insulin and that the DGKζ dissociated from IRS-1 by insulin stimulation enhances GLUT4 translocation through PIP5K1α activity. PMID:27739494

A PI4P-driven electrostatic field controls cell membrane identity and signaling in plants

PubMed Central

Simon, Mathilde Laetitia Audrey; Platre, Matthieu Pierre; Marquès-Bueno, Maria Mar; Armengot, Laia; Stanislas, Thomas; Bayle, Vincent; Caillaud, Marie-Cécile; Jaillais, Yvon

2016-01-01

Many signaling proteins permanently or transiently localize to specific organelles for function. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. Here, we found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PI4P). Our results further reveal that, contrarily to other eukaryotes, PI4P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID, as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATORs (MAKRs) family, which are involved in brassinosteroid and receptor-like kinase signaling. We anticipate that this PI4P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition. PMID:27322096

A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants.

PubMed

Simon, Mathilde Laetitia Audrey; Platre, Matthieu Pierre; Marquès-Bueno, Maria Mar; Armengot, Laia; Stanislas, Thomas; Bayle, Vincent; Caillaud, Marie-Cécile; Jaillais, Yvon

2016-06-20

Many signalling proteins permanently or transiently localize to specific organelles. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. Here, we found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PtdIns(4)P). Our results further reveal that, contrarily to other eukaryotes, PtdIns(4)P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATOR (MAKR) family, which are involved in brassinosteroid and receptor-like kinase signalling. We anticipate that this PtdIns(4)P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition.

Targeting Plasmodium PI(4)K to eliminate malaria.

PubMed

McNamara, Case W; Lee, Marcus Cs; Lim, Chek Shik; Lim, Siau Hoi; Roland, Jason; Simon, Oliver; Yeung, Bryan Ks; Chatterjee, Arnab K; McCormack, Susan L; Manary, Micah J; Zeeman, Anne-Marie; Dechering, Koen J; Kumar, Tr Santha; Henrich, Philipp P; Gagaring, Kerstin; Ibanez, Maureen; Kato, Nobutaka; Kuhen, Kelli L; Fischli, Christoph; Nagle, Advait; Rottmann, Matthias; Plouffe, David M; Bursulaya, Badry; Meister, Stephan; Rameh, Lucia; Trappe, Joerg; Haasen, Dorothea; Timmerman, Martijn; Sauerwein, Robert W; Suwanarusk, Rossarin; Russell, Bruce; Renia, Laurent; Nosten, Francois; Tully, David C; Kocken, Clemens Hm; Glynne, Richard J; Bodenreider, Christophe; Fidock, David A; Diagana, Thierry T; Winzeler, Elizabeth A

2013-12-12

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

Targeting Plasmodium PI(4)K to eliminate malaria

NASA Astrophysics Data System (ADS)

McNamara, Case W.; Lee, Marcus C. S.; Lim, Chek Shik; Lim, Siau Hoi; Roland, Jason; Nagle, Advait; Simon, Oliver; Yeung, Bryan K. S.; Chatterjee, Arnab K.; McCormack, Susan L.; Manary, Micah J.; Zeeman, Anne-Marie; Dechering, Koen J.; Kumar, T. R. Santha; Henrich, Philipp P.; Gagaring, Kerstin; Ibanez, Maureen; Kato, Nobutaka; Kuhen, Kelli L.; Fischli, Christoph; Rottmann, Matthias; Plouffe, David M.; Bursulaya, Badry; Meister, Stephan; Rameh, Lucia; Trappe, Joerg; Haasen, Dorothea; Timmerman, Martijn; Sauerwein, Robert W.; Suwanarusk, Rossarin; Russell, Bruce; Renia, Laurent; Nosten, Francois; Tully, David C.; Kocken, Clemens H. M.; Glynne, Richard J.; Bodenreider, Christophe; Fidock, David A.; Diagana, Thierry T.; Winzeler, Elizabeth A.

2013-12-01

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

Suppression of Chemotaxis by SSeCKS via Scaffolding of Phosphoinositol Phosphates and the Recruitment of the Cdc42 GEF, Frabin, to the Leading Edge

PubMed Central

Ko, Hyun-Kyung; Guo, Li-wu; Su, Bing; Gao, Lingqiu; Gelman, Irwin H.

2014-01-01

Chemotaxis is controlled by interactions between receptors, Rho-family GTPases, phosphatidylinositol 3-kinases, and cytoskeleton remodeling proteins. We investigated how the metastasis suppressor, SSeCKS, attenuates chemotaxis. Chemotaxis activity inversely correlated with SSeCKS levels in mouse embryo fibroblasts (MEF), DU145 and MDA-MB-231 cancer cells. SSeCKS loss induced chemotactic velocity and linear directionality, correlating with replacement of leading edge lamellipodia with fascin-enriched filopodia-like extensions, the formation of thickened longitudinal F-actin stress fibers reaching to filopodial tips, relative enrichments at the leading edge of phosphatidylinositol (3,4,5)P3 (PIP3), Akt, PKC-ζ, Cdc42-GTP and active Src (SrcpoY416), and a loss of Rac1. Leading edge lamellipodia and chemotaxis inhibition in SSeCKS-null MEF could be restored by full-length SSeCKS or SSeCKS deleted of its Src-binding domain (ΔSrc), but not by SSeCKS deleted of its three MARCKS (myristylated alanine-rich C kinase substrate) polybasic domains (ΔPBD), which bind PIP2 and PIP3. The enrichment of activated Cdc42 in SSeCKS-null leading edge filopodia correlated with recruitment of the Cdc42-specific guanine nucleotide exchange factor, Frabin, likely recruited via multiple PIP2/3-binding domains. Frabin knockdown in SSeCKS-null MEF restores leading edge lamellipodia and chemotaxis inhibition. However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin. Consistent with the notion that chemotaxis is controlled by SSeCKS-PIP (vs. -Src) scaffolding activity, constitutively-active phosphatidylinositol 3-kinase could override the ability of the Src inhibitor, SKI-606, to suppress chemotaxis and filopodial enrichment of Frabin in SSeCKS-null MEF. Our data suggest a role for SSeCKS in controlling Rac1 vs. Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement. PMID:25356636

Mutation in fission yeast phosphatidylinositol 4-kinase Pik1 is synthetically lethal with defect in telomere protection protein Pot1.

PubMed

Sugihara, Asami; Nguyen, Luan Cao; Shamim, Hossain Mohammad; Iida, Tetsushi; Nakase, Mai; Takegawa, Kaoru; Senda, Mitsuhisa; Jida, Shohei; Ueno, Masaru

2018-02-19

Fission yeast Pik1p is one of three phosphatidylinositol 4-kinases associated with the Golgi complex, but its function is not fully understood. Deletion of pot1 + causes telomere degradation and chromosome circularization. We searched for the gene which becomes synthetically lethal with pot1Δ. We obtained a novel pik1 mutant, pik1-1, which is synthetically lethal with pot1Δ. We found phosphoinositol 4-phosphate in the Golgi was reduced in pik1-1. To investigate the mechanism of the lethality of the pot1Δ pik1-1 double mutant, we constructed the nmt-pot1-aid pik1-1 strain, where Pot1 function becomes low by drugs, which leads to telomere loss and chromosome circularization, and found pik1-1 mutation does not affect telomere resection and chromosome circularization. Thus, our results suggest that pik1 + is required for the maintenance of circular chromosomes. Copyright © 2018 Elsevier Inc. All rights reserved.

WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy

PubMed Central

Bakula, Daniela; Müller, Amelie J.; Zuleger, Theresia; Takacs, Zsuzsanna; Franz-Wachtel, Mirita; Thost, Ann-Katrin; Brigger, Daniel; Tschan, Mario P.; Frickey, Tancred; Robenek, Horst; Macek, Boris; Proikas-Cezanne, Tassula

2017-01-01

Autophagy is controlled by AMPK and mTOR, both of which associate with ULK1 and control the production of phosphatidylinositol 3-phosphate (PtdIns3P), a prerequisite for autophagosome formation. Here we report that WIPI3 and WIPI4 scaffold the signal control of autophagy upstream of PtdIns3P production and have a role in the PtdIns3P effector function of WIPI1-WIPI2 at nascent autophagosomes. In response to LKB1-mediated AMPK stimulation, WIPI4-ATG2 is released from a WIPI4-ATG2/AMPK-ULK1 complex and translocates to nascent autophagosomes, controlling their size, to which WIPI3, in complex with FIP200, also contributes. Upstream, WIPI3 associates with AMPK-activated TSC complex at lysosomes, regulating mTOR. Our WIPI interactome analysis reveals the scaffold functions of WIPI proteins interconnecting autophagy signal control and autophagosome formation. Our functional kinase screen uncovers a novel regulatory link between LKB1-mediated AMPK stimulation that produces a direct signal via WIPI4, and we show that the AMPK-related kinases NUAK2 and BRSK2 regulate autophagy through WIPI4. PMID:28561066

Phosphatidylinositol 3,4,5-trisphosphate modulation in SHIP2-deficient mouse embryonic fibroblasts.

PubMed

Blero, Daniel; Zhang, Jing; Pesesse, Xavier; Payrastre, Bernard; Dumont, Jacques E; Schurmans, Stéphane; Erneux, Christophe

2005-05-01

SHIP2, the ubiquitous SH2 domain containing inositol 5-phosphatase, includes a series of protein interacting domains and has the ability to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]in vitro. The present study, which was undertaken to evaluate the impact of SHIP2 on PtdIns(3,4,5)P(3) levels, was performed in a mouse embryonic fibroblast (MEF) model using SHIP2 deficient (-/-) MEF cells derived from knockout mice. PtdIns(3,4,5)P(3) was upregulated in serum stimulated -/- MEF cells as compared to +/+ MEF cells. Although the absence of SHIP2 had no effect on basal PtdIns(3,4,5)P(3) levels, we show here that this lipid was significantly upregulated in SHIP2 -/- cells but only after short-term (i.e. 5-10 min) incubation with serum. The difference in PtdIns(3,4,5)P(3) levels in heterozygous fibroblast cells was intermediate between the +/+ and the -/- cells. In our model, insulin-like growth factor-1 stimulation did not show this upregulation. Serum stimulated phosphoinositide 3-kinase (PI 3-kinase) activity appeared to be comparable between +/+ and -/- cells. Moreover, protein kinase B, but not mitogen activated protein kinase activity, was also potentiated in SHIP2 deficient cells stimulated by serum. The upregulation of protein kinase B activity in serum stimulated cells was totally reversed in the presence of the PI 3-kinase inhibitor LY-294002, in both +/+ and -/- cells. Altogether, these data establish a link between SHIP2 and the acute control of PtdIns(3,4,5)P(3) levels in intact cells.

Amplification of Chromosome 1q Genes Encoding the Phosphoinositide Signalling Enzymes PI4KB, AKT3, PIP5K1A and PI3KC2B in Breast Cancer

PubMed Central

Waugh, Mark G.

2014-01-01

Little is known about the possible oncogenic roles of genes encoding for the phosphatidylinositol 4-kinases, a family of enzymes that regulate an early step in phosphoinositide signalling. To address this issue, the mutational status of all four human phosphatidylinositol 4-kinases genes was analyzed across 852 breast cancer samples using the COSMIC data resource. Point mutations in the phosphatidylinositol 4-kinase genes were uncommon and appeared in less than 1% of the patient samples however, 62% of the tumours had increases in gene copy number for PI4KB which encodes the phosphatidylinositol 4-kinase IIIbeta isozyme. Extending this analysis to subsequent enzymes in the phosphoinositide signalling cascades revealed that the only PIP5K1A, PI3KC2B and AKT3 genes exhibited similar patterns of gene copy number variation. By comparison, gene copy number increases for established oncogenes such as EGFR and HER2/Neu were only evident in 20% of the samples. The PI4KB, PIP5K1A, PI3KC2B and AKT3 genes are related in that they all localize to chromosome 1q which is often structurally and numerically abnormal in breast cancer. These results demonstrate that a gene quartet encoding a potential phosphoinositide signalling pathway is amplified in a subset of breast cancers. PMID:25368680

Nuclear Phosphatidylinositol-Phosphate Type I Kinase α-Coupled Star-PAP Polyadenylation Regulates Cell Invasion

PubMed Central

A.P., Sudheesh

2017-01-01

ABSTRACT Star-PAP, a nuclear phosphatidylinositol (PI) signal-regulated poly(A) polymerase (PAP), couples with type I PI phosphate kinase α (PIPKIα) and controls gene expression. We show that Star-PAP and PIPKIα together regulate 3′-end processing and expression of pre-mRNAs encoding key anti-invasive factors (KISS1R, CDH1, NME1, CDH13, FEZ1, and WIF1) in breast cancer. Consistently, the endogenous Star-PAP level is negatively correlated with the cellular invasiveness of breast cancer cells. While silencing Star-PAP or PIPKIα increases cellular invasiveness in low-invasiveness MCF7 cells, Star-PAP overexpression decreases invasiveness in highly invasive MDA-MB-231 cells in a cellular Star-PAP level-dependent manner. However, expression of the PIPKIα-noninteracting Star-PAP mutant or the phosphodeficient Star-PAP (S6A mutant) has no effect on cellular invasiveness. These results strongly indicate that PIPKIα interaction and Star-PAP S6 phosphorylation are required for Star-PAP-mediated regulation of cancer cell invasion and give specificity to target anti-invasive gene expression. Our study establishes Star-PAP–PIPKIα-mediated 3′-end processing as a key anti-invasive mechanism in breast cancer. PMID:29203642

Nuclear Phosphatidylinositol-Phosphate Type I Kinase α-Coupled Star-PAP Polyadenylation Regulates Cell Invasion.

PubMed

A P, Sudheesh; Laishram, Rakesh S

2018-03-01

Star-PAP, a nuclear phosphatidylinositol (PI) signal-regulated poly(A) polymerase (PAP), couples with type I PI phosphate kinase α (PIPKIα) and controls gene expression. We show that Star-PAP and PIPKIα together regulate 3'-end processing and expression of pre-mRNAs encoding key anti-invasive factors ( KISS1R , CDH1 , NME1 , CDH13 , FEZ1 , and WIF1 ) in breast cancer. Consistently, the endogenous Star-PAP level is negatively correlated with the cellular invasiveness of breast cancer cells. While silencing Star-PAP or PIPKIα increases cellular invasiveness in low-invasiveness MCF7 cells, Star-PAP overexpression decreases invasiveness in highly invasive MDA-MB-231 cells in a cellular Star-PAP level-dependent manner. However, expression of the PIPKIα-noninteracting Star-PAP mutant or the phosphodeficient Star-PAP (S6A mutant) has no effect on cellular invasiveness. These results strongly indicate that PIPKIα interaction and Star-PAP S6 phosphorylation are required for Star-PAP-mediated regulation of cancer cell invasion and give specificity to target anti-invasive gene expression. Our study establishes Star-PAP-PIPKIα-mediated 3'-end processing as a key anti-invasive mechanism in breast cancer. Copyright © 2018 A.P. and Laishram.

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

PubMed Central

Yu, Xinlei; Long, Yun Chau; Shen, Han-Ming

2015-01-01

Autophagy is an evolutionarily conserved and exquisitely regulated self-eating cellular process with important biological functions. Phosphatidylinositol 3-kinases (PtdIns3Ks) and phosphoinositide 3-kinases (PI3Ks) are involved in the autophagic process. Here we aim to recapitulate how 3 classes of these lipid kinases differentially regulate autophagy. Generally, activation of the class I PI3K suppresses autophagy, via the well-established PI3K-AKT-MTOR (mechanistic target of rapamycin) complex 1 (MTORC1) pathway. In contrast, the class III PtdIns3K catalytic subunit PIK3C3/Vps34 forms a protein complex with BECN1 and PIK3R4 and produces phosphatidylinositol 3-phosphate (PtdIns3P), which is required for the initiation and progression of autophagy. The class II enzyme emerged only recently as an alternative source of PtdIns3P and autophagic initiator. However, the orthodox paradigm is challenged by findings that the PIK3CB catalytic subunit of class I PI3K acts as a positive regulator of autophagy, and PIK3C3 was thought to be an amino acid sensor for MTOR, which curbs autophagy. At present, a number of PtdIns3K and PI3K inhibitors, including specific PIK3C3 inhibitors, have been developed for suppression of autophagy and for clinical applications in autophagy-related human diseases. PMID:26018563

Phosphoinositides and membrane curvature switch the mode of actin polymerization via selective recruitment of toca-1 and Snx9

PubMed Central

Gallop, Jennifer L.; Walrant, Astrid; Cantley, Lewis C.; Kirschner, Marc W.

2013-01-01

The membrane–cytosol interface is the major locus of control of actin polymerization. At this interface, phosphoinositides act as second messengers to recruit membrane-binding proteins. We show that curved membranes, but not flat ones, can use phosphatidylinositol 3-phosphate [PI(3)P] along with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to stimulate actin polymerization. In this case, actin polymerization requires the small GTPase cell cycle division 42 (Cdc42), the nucleation-promoting factor neural Wiskott–Aldrich syndrome protein (N-WASP) and the actin nucleator the actin-related protein (Arp) 2/3 complex. In liposomes containing PI(4,5)P2 as the sole phosphoinositide, actin polymerization requires transducer of Cdc42 activation-1 (toca-1). In the presence of phosphatidylinositol 3-phosphate, polymerization is both more efficient and independent of toca-1. Under these conditions, sorting nexin 9 (Snx9) can be implicated as a specific adaptor that replaces toca-1 to mobilize neural Wiskott–Aldrich syndrome protein and the Arp2/3 complex. This switch in phosphoinositide and adaptor specificity for actin polymerization from membranes has implications for how different types of actin structures are generated at precise times and locations in the cell. PMID:23589871

Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells

PubMed Central

Venkatareddy, Madhusudan; Verma, Rakesh; Kalinowski, Anne; Patel, Sanjeevkumar R.; Shisheva, Assia

2016-01-01

The mechanisms by which the glomerular filtration barrier prevents the loss of large macromolecules and simultaneously, maintains the filter remain poorly understood. Recent studies proposed that podocytes have an active role in both the endocytosis of filtered macromolecules and the maintenance of the filtration barrier. Deletion of a key endosomal trafficking regulator, the class 3 phosphatidylinositol (PtdIns) 3-kinase vacuolar protein sorting 34 (Vps34), in podocytes results in aberrant endosomal membrane morphology and podocyte dysfunction. We recently showed that the vacuolation phenotype in cultured Vps34–deficient podocytes is caused by the absence of a substrate for the Vps34 downstream effector PtdIns 3-phosphate 5-kinase (PIKfyve), which phosphorylates Vps34-generated PtdIns(3)P to produce PtdIns (3,5)P2. PIKfyve perturbation and PtdIns(3,5)P2 reduction result in massive membrane vacuolation along the endosomal system, but the cell-specific functions of PIKfyve in vivo remain unclear. We show here that the genetic deletion of PIKfyve in endocytically active proximal tubular cells resulted in the development of large cytoplasmic vacuoles caused by arrested endocytic traffic progression at a late-endosome stage. In contrast, deletion of PIKfyve in glomerular podocytes did not significantly alter the endosomal morphology, even in age 18-month-old mice. However, on culturing, the PIKfyve-deleted podocytes developed massive cytoplasmic vacuoles. In summary, these data suggest that glomerular podocytes and proximal tubules have different requirements for PIKfyve function, likely related to distinct in vivo needs for endocytic flux. PMID:26825532

Membrane-trafficking sorting hubs: cooperation between PI4P and small GTPases at the trans-Golgi Network

PubMed Central

Santiago-Tirado, Felipe H.; Bretscher, Anthony

2011-01-01

Cell polarity in eukaryotes requires constant sorting, packaging, and transport of membrane-bound cargo within the cell. These processes occur in two sorting hubs: the recycling endosome for incoming material, and the trans-Golgi Network for outgoing. Phosphatidylinositol 3-phosphate and 4–5 phosphate are enriched at the endocytic and exocytic sorting hubs, respectively, where they act together with small GTPases to recruit factors to segregate cargo and regulate carrier formation and transport. In this review, we summarize the current understanding of how these lipids and GTPases directly regulate membrane trafficking, emphasizing the recent discoveries of phosphatidylinositol 4-phosphate functions at the trans-Golgi Network. PMID:21764313

Effects of antiglaucoma drugs on [32P]orthophosphate incorporation into phospholipids of cat iris and ciliary process.

PubMed

Yorio, T; DeLoach, G; Satumtira, N

1985-01-01

The effects of antiglaucoma drugs on [32P]-orthophosphate incorporation into phospholipids of iris and ciliary process were investigated. Both iris and ciliary process rapidly incorporated 32Pi into the major phospholipids, with the acidic phosphoinositides demonstrating a greater labelling than phosphatidylcholine, indicating a greater turnover. The muscarinic agonists, carbachol and pilocarpine, stimulated 32Pi-labelling of phosphatidylinositol (PI) and phosphatidic acid (PA) in both iris and ciliary process. These effects were blocked by atropine, suggesting that the response was mediated through muscarinic receptors. The beta blocking ocular hypotensive drugs, propranolol, timolol and atenolol, produced varying effects on 32P incorporation into phospholipids of iris and ciliary process. Propranolol stimulated 32Pi-labelling into phosphatidylinositol 4', 5' bisphosphate (PIP2), phosphatidylinositol 4' phosphate (PIP), PI and PA. Timolol decreased 32Pi-incorporation into PIP2 and PI, whereas atenolol, a selective beta 1 antagonist, had no significant effect on 32Pi-labelling of phospholipids. The above findings on propranolol agree with previous observations which demonstrated that propranolol redirects glycerolipid metabolism through multiple effects on the enzymes in phospholipid biosynthesis, particularly in stimulating phosphatidylinositol kinases. The results with timolol suggest that this drug may decrease phosphoinositide hydrolysis. The effects of these ocular hypotensive, non-selective beta blocking drugs on phospholipid turnover may ultimately limit the accumulation of breakdown products which could serve as cellular messengers.

Analysis of Phosphatidic Acid Binding and Regulation of PIPKI In Vitro and in Intact Cells.

PubMed

Tay, L W R; Wang, Z; Du, G

2017-01-01

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a lipid second messenger that regulates a wide array of essential cellular events, such as signal transduction, vesicle trafficking, actin cytoskeleton dynamics, adhesion, and motility. To control the spatiotemporal production of PI(4,5)P2, the activity of type 1 phosphotidylinositol-4-phosphate-5-kinases (PIPKIs) is tightly regulated by small GTPases and another signaling lipid, phosphatidic acid (PA). It is of interest that PI(4,5)P2 is also a critical cofactor for the activation of the PA-generating enzyme, phospholipase D (PLD). It has been proposed that the reciprocal stimulation of PLD and PIPKI enzymes enables a rapid feedforward stimulation loop for the localized and acute generation of signaling lipids that are critical for the regulation of actin cytoskeletal reorganization and membrane trafficking. Here, we outline the methods for the expression and purification of PIPKIγ from bacteria, determination of direct PA binding, and activation of PIPKIγ using in vitro liposomes assays, and examination of actin cytoskeletal reorganization promoted by the PA-PIPKIγ signaling in intact cells using fluorescent microscopy. © 2017 Elsevier Inc. All rights reserved.

c-Fos-activated synthesis of nuclear phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] promotes global transcriptional changes.

PubMed

Ferrero, Gabriel O; Renner, Marianne L; Gil, Germán A; Rodríguez-Berdini, Lucia; Caputto, Beatriz L

2014-08-01

c-Fos is a well-recognized member of the AP-1 (activator protein-1) family of transcription factors. In addition to this canonical activity, we previously showed that cytoplasmic c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. c-Fos associates with particular enzymes of the lipid synthesis pathway at the endoplasmic reticulum and increases the Vmax of the reactions without modifying the Km values. This lipid synthesis activation is associated with events of differentiation and proliferation that require high rates of membrane biogenesis. Since lipid synthesis also occurs in the nucleus, and different phospholipids have been assigned transcription regulatory functions, in the present study we examine if c-Fos also acts as a regulator of phospholipid synthesis in the nucleus. Furthermore, we examine if c-Fos modulates transcription through its phospholipid synthesis activator capacity. We show that nuclear-localized c-Fos associates with and activates PI4P5K (phosphatidylinositol-4-monophosphate 5-kinase), but not with PI4KIIIβ (type IIIβ phosphatidylinositol 4-kinase) thus promoting PtdIns(4,5)P₂ (phosphatidylinositol 4,5-bisphosphate) formation, which, in turn, promotes transcriptional changes. We propose c-Fos as a key regulator of nuclear PtdIns(4,5)P₂ synthesis in response to growth signals that results in c-Fos-dependent transcriptional changes promoted by the newly synthesized lipids.

Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling.

PubMed

Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi; Asbury, Charles L; Hille, Bertil; Koh, Duk-Su

2016-03-01

Activated Gq protein-coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein-tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein-coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to secondary depletion of PIP2. Indeed, artificial recruitment of PIP5K removed the secondary loss of PIP2 completely. Altogether, our experimental and theoretical approaches demonstrate roles and dynamics of the protein kinases, β-arrestin, and PIP5K in the desensitization of PAR2. © 2016 Jung et al.

Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling

PubMed Central

Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi; Asbury, Charles L.; Hille, Bertil

2016-01-01

Activated Gq protein–coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein–tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein–coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to secondary depletion of PIP2. Indeed, artificial recruitment of PIP5K removed the secondary loss of PIP2 completely. Altogether, our experimental and theoretical approaches demonstrate roles and dynamics of the protein kinases, β-arrestin, and PIP5K in the desensitization of PAR2. PMID:26927499

Directional control of WAVE2 membrane targeting by EB1 and phosphatidylinositol 3,4,5-triphosphate.

PubMed

Takahashi, Kazuhide; Tanaka, Tacu; Suzuki, Katsuo

2010-03-01

Membrane targeting of WAVE2 along microtubules is mediated by a motor protein kinesin and requires Pak1, a downstream effector of Rac1. However, the mechanism by which WAVE2 targeting to the leading edge is directionally controlled remains largely unknown. Here we demonstrate that EB1, a microtubule plus-end-binding protein, constitutively associates with stathmin, a microtubule-destabilizing protein, in human breast cancer cells. Stimulation of the cells with insulin-like growth factor I (IGF-I) induced Pak1-dependent binding of the EB1-stathmin complex to microtubules that bear WAVE2 and colocalization of the complex with WAVE2 at the leading edge. Depletion of EB1 by small interfering RNA (siRNA) abrogated the IGF-I-induced WAVE2 targeting and stathmin binding to microtubules. On the other hand, chemotaxis chamber assays indicated that the IGF-I receptor (IGF-IR) was locally activated in the region facing toward IGF-I. In addition, IGF-I caused phosphatidylinositol 3-kinase (PI 3-kinase)-dependent production of phosphatidylinositol 3,4,5-triphosphate (PIP3) near activated IGF-IR and WAVE2 colocalization with it. Collectively, WAVE2-membrane targeting is directionally controlled by binding of the EB1-stathmin complex to WAVE2-bearing microtubules and by the interaction between WAVE2 and PIP3 produced near IGF-IR that is locally activated by IGF-I.

Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO.

PubMed

Reidick, Christina; El Magraoui, Fouzi; Meyer, Helmut E; Stenmark, Harald; Platta, Harald W

2014-12-23

The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes-autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept.

Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

PubMed Central

Reidick, Christina; El Magraoui, Fouzi; Meyer, Helmut E.; Stenmark, Harald; Platta, Harald W.

2014-01-01

The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept. PMID:25545884

A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria

PubMed Central

Mbengue, Alassane; Bhattacharjee, Souvik; Pandharkar, Trupti; Liu, Haining; Estiu, Guillermina; Stahelin, Robert V.; Rizk, Shahir; Njimoh, Dieudonne L.; Ryan, Yana; Chotivanich, Kesinee; Nguon, Chea; Ghorbal, Mehdi; Lopez-Rubio, Jose-Juan; Pfrender, Michael; Emrich, Scott; Mohandas, Narla; Dondorp, Arjen M.; Wiest, Olaf; Haldar, Kasturi

2015-01-01

Artemisinins are the corner stone of anti-malarial drugs1. Emergence and spread of resistance to them2–4 raises risk of wiping out recent gains achieved in reducing world-wide malaria burden and threatens future malaria control and elimination on a global level. Genome wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance5–10. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase as well as its lipid product phosphatidylinositol 3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signaling, where transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination. PMID:25874676

Association of Factor V Secretion with Protein Kinase B Signaling in Platelets from Horses with Atypical Equine Thrombasthenia.

PubMed

Norris, J W; Pombo, M; Shirley, E; Blevins, G; Tablin, F

2015-01-01

Two congenital bleeding diatheses have been identified in Thoroughbred horses: Glanzmann thrombasthenia (GT) and a second, novel diathesis associated with abnormal platelet function in response to collagen and thrombin stimulation. Platelet dysfunction in horses with this second thrombasthenia results from a secretory defect. Two affected and 6 clinically normal horses. Ex vivo study. Washed platelets were examined for (1) expression of the αIIb-β3 integrin; (2) fibrinogen binding capacity in response to ADP and thrombin; (3) secretion of dense and α-granules; (4) activation of the mammalian target of rapamycin (mTOR)-protein kinase B (AKT) signaling pathway; and (5) cellular distribution of phosphatidylinositol-4-phosphate-3-kinase, class 2B (PIK3C2B) and SH2 containing inositol-5'-phosphatase 1 (SHIP1). Platelets from affected horses expressed normal amounts of αIIb-β3 integrin and bound fibrinogen normally in response to ADP, but bound 80% less fibrinogen in response to thrombin. α-granules only released 50% as much Factor V as control platelets, but dense granules released their contents normally. Protein kinase B (AKT) phosphorylation was reduced after thrombin activation, but mTOR Complex 2 (mTORC2) and phosphoinositide-dependent kinase 1 (PDK1) signaling were normal. SH2-containing inositol-5'-phosphatase 1 (SHIP1) did not localize to the cytoskeleton of affected platelets and was decreased overall consistent with reduced AKT phosphorylation. Defects in fibrinogen binding, granule secretion, and signal transduction are unique to this thrombasthenia, which we designate as atypical equine thrombasthenia. Copyright © The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of American College of Veterinary Internal Medicine.

Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses.

PubMed

Barbosa, Inês C R; Shikata, Hiromasa; Zourelidou, Melina; Heilmann, Mareike; Heilmann, Ingo; Schwechheimer, Claus

2016-12-15

Polar transport of the phytohormone auxin through PIN-FORMED (PIN) auxin efflux carriers is essential for the spatiotemporal control of plant development. The Arabidopsis thaliana serine/threonine kinase D6 PROTEIN KINASE (D6PK) is polarly localized at the plasma membrane of many cells where it colocalizes with PINs and activates PIN-mediated auxin efflux. Here, we show that the association of D6PK with the basal plasma membrane and PINs is dependent on the phospholipid composition of the plasma membrane as well as on the phosphatidylinositol phosphate 5-kinases PIP5K1 and PIP5K2 in epidermis cells of the primary root. We further show that D6PK directly binds polyacidic phospholipids through a polybasic lysine-rich motif in the middle domain of the kinase. The lysine-rich motif is required for proper PIN3 phosphorylation and for auxin transport-dependent tropic growth. Polybasic motifs are also present at a conserved position in other D6PK-related kinases and required for membrane and phospholipid binding. Thus, phospholipid-dependent recruitment to membranes through polybasic motifs might not only be required for D6PK-mediated auxin transport but also other processes regulated by these, as yet, functionally uncharacterized kinases. © 2016. Published by The Company of Biologists Ltd.

Differential Association of Phosphatidylinositol 3-Kinase, SHIP-1, and PTEN with Forming Phagosomes

PubMed Central

Kamen, Lynn A.; Levinsohn, Jonathan

2007-01-01

In macrophages, enzymes that synthesize or hydrolyze phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] regulate Fcγ receptor-mediated phagocytosis. Inhibition of phosphatidylinositol 3-kinase (PI3K) or overexpression of the lipid phosphatases phosphatase and tensin homologue (PTEN) and Src homology 2 domain-containing inositol phosphatase (SHIP-1), which hydrolyze PI(3,4,5)P3 to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], respectively, inhibit phagocytosis in macrophages. To examine how these enzymes regulate phagosome formation, the distributions of yellow fluorescent protein (YFP) chimeras of enzymes and pleckstrin homology (PH) domains specific for their substrates and products were analyzed quantitatively. PTEN-YFP did not localize to phagosomes, suggesting that PTEN regulates phagocytosis globally within the macrophage. SHIP1-YFP and p85-YFP were recruited to forming phagosomes. SHIP1-YFP sequestered to the leading edge and dissociated from phagocytic cups earlier than did p85-cyan fluorescent protein, indicating that SHIP-1 inhibitory activities are restricted to the early stages of phagocytosis. PH domain chimeras indicated that early during phagocytosis, PI(3,4,5)P3 was slightly more abundant than PI(3,4)P2 at the leading edge of the forming cup. These results support a model in which phagosomal PI3K generates PI(3,4,5)P3 necessary for later stages of phagocytosis, PTEN determines whether those late stages can occur, and SHIP-1 regulates when and where they occur by transiently suppressing PI(3,4,5)P3-dependent activities necessary for completion of phagocytosis. PMID:17442886

Tomato pistil factor STIG1 promotes in vivo pollen tube growth by binding to phosphatidylinositol 3-phosphate and the extracellular domain of the pollen receptor kinase LePRK2

USDA-ARS?s Scientific Manuscript database

The speed of pollen tube growth is a major determinant of reproductive success in flowering plants. Tomato (Solanum lycopersicum) STIGMA-SPECIFIC PROTEIN1 (STIG1), a small Cys-rich protein from the pistil, was previously identified as a binding partner of the pollen receptor kinase LePRK2 and shown ...

Development of highly sensitive cell-based AKT kinase ELISA for monitoring PI3K beta activity and compound efficacy.

PubMed

Yanamandra, Mahesh; Kole, Labanyamoy; Giri, Archana; Mitra, Sayan

2017-01-01

Phosphatidylinositol-3 kinase (PI3K) pathway regulates multiple cellular functions involving cell survival, growth, motility proliferation, apoptosis, and adhesion. These are deregulated in various diseases such as cancer, atherosclerosis, and inflammation. PI3Ks phosphorylate phosphatidylinositol 4,5-biphosphate (PIP2) yielding phosphatidylinositol 3, 4, 5 triphosphate (PIP3) which in turn activate AKT kinase (serine/threonine kinase), the central enzyme in regulation of metabolic functions. Due to their implications in disease pathophysiology, PI3K/AKT inhibitors became attractive targets for pharmaceutical industries. In order to assess the functional response generated by PI3K inhibitors, an appropriate cell-based screening system is essential in any screening cascade. Here we report the development of highly sensitive in-vitro cell-based kinase ELISA which quantifies the phosphorylated AKT kinase (serine 473) and total AKT kinase directly within the cells upon compound treatment. PI3Kβ overexpressing NIH3T3 cells stimulated by lysophosphatidic acid was used for PI3K/Akt pathway activation. Assay performance reliability and robustness were determined by percentage coefficient of variation (%CV) and Z factor which demonstrated an excellent agreement with assay guidelines. This 96-well plate medium throughput assay methodology was used to screen novel molecules and proved a commendable tool to study the mechanism of action property and target engagement of novel PI3K inhibitors in drug discovery.

Molecular Basis for Association of PIPKIγ-p90 with Clathrin Adaptor AP-2*

PubMed Central

Kahlfeldt, Nina; Vahedi-Faridi, Ardeschir; Koo, Seong Joo; Schäfer, Johannes G.; Krainer, Georg; Keller, Sandro; Saenger, Wolfram; Krauss, Michael; Haucke, Volker

2010-01-01

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the Iγ-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P2 metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKIγ-p90 associates with both the μ and β2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKIγ-p90 tail binds to a cognate recognition site on the sandwich subdomain of the β2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2μ, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKIγ-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKIγ tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2β and AP-2μ. Our data also suggest that interactions between AP-2 and the tail domain of PIPKIγ-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKIγ-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P2 synthesis during clathrin-mediated SV endocytosis. PMID:19903820

Pleiotropic Alterations in Lipid Metabolism in Yeast sac1 Mutants: Relationship to “Bypass Sec14p” and Inositol Auxotrophy

PubMed Central

Rivas, Marcos P.; Kearns, Brian G.; Xie, Zhigang; Guo, Shuling; Sekar, M. Chandra; Hosaka, Kohei; Kagiwada, Satoshi; York, John D.; Bankaitis, Vytas A.

1999-01-01

SacIp dysfunction results in bypass of the requirement for phosphatidylinositol transfer protein (Sec14p) function in yeast Golgi processes. This effect is accompanied by alterations in inositol phospholipid metabolism and inositol auxotrophy. Elucidation of how sac1 mutants effect “bypass Sec14p” will provide insights into Sec14p function in vivo. We now report that, in addition to a dramatic accumulation of phosphatidylinositol-4-phosphate, sac1 mutants also exhibit a specific acceleration of phosphatidylcholine biosynthesis via the CDP-choline pathway. This phosphatidylcholine metabolic phenotype is sensitive to the two physiological challenges that abolish bypass Sec14p in sac1 strains; i.e. phospholipase D inactivation and expression of bacterial diacylglycerol (DAG) kinase. Moreover, we demonstrate that accumulation of phosphatidylinositol-4-phosphate in sac1 mutants is insufficient to effect bypass Sec14p. These data support a model in which phospholipase D activity contributes to generation of DAG that, in turn, effects bypass Sec14p. A significant fate for this DAG is consumption by the CDP-choline pathway. Finally, we determine that CDP-choline pathway activity contributes to the inositol auxotrophy of sac1 strains in a novel manner that does not involve obvious defects in transcriptional expression of the INO1 gene. PMID:10397762

Structural basis for phosphatidylinositol-phosphate biosynthesis

NASA Astrophysics Data System (ADS)

Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

2015-10-01

Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.

HIP1 and HIP1r stabilize receptor tyrosine kinases and bind 3-phosphoinositides via epsin N-terminal homology domains.

PubMed

Hyun, Teresa S; Rao, Dinesh S; Saint-Dic, Djenann; Michael, L Evan; Kumar, Priti D; Bradley, Sarah V; Mizukami, Ikuko F; Oravecz-Wilson, Katherine I; Ross, Theodora S

2004-04-02

Huntingtin-interacting protein 1-related (HIP1r) is the only known mammalian relative of huntingtin-interacting protein 1 (HIP1), a protein that transforms fibroblasts via undefined mechanisms. Here we demonstrate that both HIP1r and HIP1 bind inositol lipids via their epsin N-terminal homology (ENTH) domains. In contrast to other ENTH domain-containing proteins, lipid binding is preferential to the 3-phosphate-containing inositol lipids, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,5-bisphosphate. Furthermore, the HIP1r ENTH domain, like that of HIP1, is necessary for lipid binding, and expression of an ENTH domain-deletion mutant, HIP1r/deltaE, induces apoptosis. Consistent with the ability of HIP1r and HIP1 to affect cell survival, full-length HIP1 and HIP1r stabilize pools of growth factor receptors by prolonging their half-life following ligand-induced endocytosis. Although HIP1r and HIP1 display only a partially overlapping pattern of protein interactions, these data suggest that both proteins share a functional homology by binding 3-phosphorylated inositol lipids and stabilizing receptor tyrosine kinases in a fashion that may contribute to their ability to alter cell growth and survival.

Two-ligand priming mechanism for potentiated phosphoinositide synthesis is an evolutionarily conserved feature of Sec14-like phosphatidylinositol and phosphatidylcholine exchange proteins.

PubMed

Huang, Jin; Ghosh, Ratna; Tripathi, Ashutosh; Lönnfors, Max; Somerharju, Pentti; Bankaitis, Vytas A

2016-07-15

Lipid signaling, particularly phosphoinositide signaling, plays a key role in regulating the extreme polarized membrane growth that drives root hair development in plants. The Arabidopsis AtSFH1 gene encodes a two-domain protein with an amino-terminal Sec14-like phosphatidylinositol transfer protein (PITP) domain linked to a carboxy-terminal nodulin domain. AtSfh1 is critical for promoting the spatially highly organized phosphatidylinositol-4,5-bisphosphate signaling program required for establishment and maintenance of polarized root hair growth. Here we demonstrate that, like the yeast Sec14, the AtSfh1 PITP domain requires both its phosphatidylinositol (PtdIns)- and phosphatidylcholine (PtdCho)-binding properties to stimulate PtdIns-4-phosphate [PtdIns(4)P] synthesis. Moreover, we show that both phospholipid-binding activities are essential for AtSfh1 activity in supporting polarized root hair growth. Finally, we report genetic and biochemical evidence that the two-ligand mechanism for potentiation of PtdIns 4-OH kinase activity is a broadly conserved feature of plant Sec14-nodulin proteins, and that this strategy appeared only late in plant evolution. Taken together, the data indicate that the PtdIns/PtdCho-exchange mechanism for stimulated PtdIns(4)P synthesis either arose independently during evolution in yeast and in higher plants, or a suitable genetic module was introduced to higher plants from a fungal source and subsequently exploited by them. © 2016 Huang, Ghosh, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Acetylcholine but not adenosine triggers preconditioning through PI3-kinase and a tyrosine kinase.

PubMed

Qin, Qining; Downey, James M; Cohen, Michael V

2003-02-01

Adenosine and acetylcholine (ACh) trigger preconditioning by different signaling pathways. The involvement of phosphatidylinositol 3-kinase (PI3-kinase), a protein tyrosine kinase, and Src family tyrosine kinase in preconditioning was evaluated in isolated rabbit hearts. Either wortmannin (PI3-kinase blocker), genistein (tyrosine kinase blocker), lavendustin A (tyrosine kinase blocker), or 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2; Src family tyrosine kinase blocker) was given for 15 min to bracket a 5-min infusion of either adenosine or ACh (trigger phase). The hearts then underwent 30 min of regional ischemia. Infarct size for ACh alone was 9.3 +/- 3.5% of the risk zone versus 34.3 +/- 4.1% in controls. All four inhibitors blocked ACh-induced protection. When wortmannin or PP2 was infused only during the 30-min ischemic period (mediator phase), ACh-induced protection was not affected (7.4 +/- 2.1% and 9.7 +/- 1.7% infarction, respectively). Adenosine-triggered protection was not blocked by any of the inhibitors. Therefore, PI3-kinase and at least one protein tyrosine kinase, probably Src kinase, are involved in the trigger phase of ACh-induced, but not adenosine-induced, preconditioning. Neither PI3-kinase nor Src kinase is a mediator of the protection of ACh.

GATA4-mediated cardiac hypertrophy induced by D-myo-inositol 1,4,5-tris-phosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu Zhiming; Zhu Shanjun; Liu Daoyan

2005-12-16

We evaluated the effects of D-myo-inositol 1,4,5-tris-phosphate on cardiac hypertrophy. D-myo-inositol 1,4,5-tris-phosphate augmented cardiac hypertrophy as evidenced by its effects on DNA synthesis, protein synthesis, and expression of immediate-early genes c-myc and c-fos, {beta}-myosin heavy chain, and {alpha}-actin. The administration of D-myo-inositol 1,4,5-tris-phosphate increased the expression of nuclear factor of activated T-cells and cardiac-restricted zinc finger transcription factor (GATA4). Real-time quantitative RT-PCR showed that D-myo-inositol 1,4,5-tris-phosphate-induced GATA4 mRNA was significantly enhanced even in the presence of the calcineurin inhibitor, cyclosporine A. The effect of D-myo-inositol 1,4,5-tris-phosphate was blocked after inhibition of inositol-trisphosphate receptors but not after inhibition of c-Raf/mitogen-activated proteinmore » kinase kinase (MEK)/mitogen-activated protein kinase (ERK) or p38 mitogen-activated protein kinase pathways. The study shows that D-myo-inositol 1,4,5-tris-phosphate-induced cardiac hypertrophy is mediated by GATA4 but independent from the calcineurin pathway.« less

Mechanism of Poliovirus Resistance to Host Phosphatidylinositol-4 Kinase III β Inhibitor.

PubMed

Arita, Minetaro

2016-02-12

Phosphatidylinositol-4 kinase III β (PI4KB) and oxysterol-binding protein (OSBP) family I have been identified as the major targets of anti-enterovirus drug candidates. Resistance mutations in poliovirus (PV) to these inhibitors have been identified in viral 3A protein, represented by a G5318A (3A-Ala70Thr) mutation, but the mechanism of viral resistance to host PI4KB/OSBP inhibitors remained unknown. In this study, we found that a G5318A mutation enhances the basal levels of phosphatidylinositol 4-phosphate (PI4P) and of the 3A protein and decreases the levels of the 3AB protein during PV replication. The 3A protein acted as a major effector responsible for the resistance to PI4KB inhibitor, but did not enhance the PI4KB activity in vitro in contrast to the 2C, 2BC, 3AB, and 3D proteins. The 3AB protein acted as the primary target of a G5318A mutation and also as an effector. We identified novel resistance mutations to a PI4KB inhibitor [C5151U (3A-T14M) and C5366U (3A-H86Y) mutations] and found that there is a positive correlation between the extent of the resistance phenotype and the levels of the 3A proteins. These results suggested that the 3A protein overproduced by enhanced processing of the 3AB protein with the resistance mutations overcomes the inhibitory effect of PI4KB inhibitor on PV replication independently of the hyperactivation of the PI4KB/OSBP pathway.

Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase.

PubMed

Bago, Ruzica; Malik, Nazma; Munson, Michael J; Prescott, Alan R; Davies, Paul; Sommer, Eeva; Shpiro, Natalia; Ward, Richard; Cross, Darren; Ganley, Ian G; Alessi, Dario R

2014-11-01

The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates PtdIns (phosphatidylinositol) at endosomal membranes to generate PtdIns(3)P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing PtdIns(3)P-binding PX (phox homology) and FYVE domains. In the present study, we describe a highly selective and potent inhibitor of Vps34, termed VPS34-IN1, that inhibits Vps34 with 25 nM IC50 in vitro, but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific PtdIns(3)P-binding probe from endosome membranes, within 1 min, without affecting the ability of class I PI3K to regulate Akt. Moreover, we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3), the only protein kinase known to interact specifically with PtdIns(3)P via its N-terminal PX domain, might be controlled by Vps34. Mutations disrupting PtdIns(3)P binding ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop [PDK1 (phosphoinositide-dependent kinase 1) site] and hydrophobic motif (mammalian target of rapamycin site) residues. VPS34-IN1 induced a rapid ~50-60% loss of SGK3 phosphorylation within 1 min. VPS34-IN1 did not inhibit activity of the SGK2 isoform that does not possess a PtdIns(3)P-binding PX domain. Furthermore, class I PI3K inhibitors (GDC-0941 and BKM120) that do not inhibit Vps34 suppressed SGK3 activity by ~40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ~80-90%. These data suggest SGK3 phosphorylation and hence activity is controlled by two pools of PtdIns(3)P. The first is produced through phosphorylation of PtdIns by Vps34 at the endosome. The second is due to the conversion of class I PI3K product, PtdIns(3,4,5)P3 into PtdIns(3)P, via the sequential actions of the PtdIns 5-phosphatases [SHIP1/2 (Src homology 2-domain-containing inositol phosphatase 1/2)] and PtdIns 4-phosphatase [INPP4B (inositol polyphosphate 4-phosphatase type II)]. VPS34-IN1 will be a useful probe to delineate physiological roles of the Vps34. Monitoring SGK3 phosphorylation and activity could be employed as a biomarker of Vps34 activity, in an analogous manner by which Akt is used to probe cellular class I PI3K activity. Combining class I (GDC-0941) and class III (VPS34-IN1) PI3K inhibitors could be used as a strategy to better analyse the roles and regulation of the elusive class II PI3K.

Involvement of Sac1 phosphoinositide phosphatase in the metabolism of phosphatidylserine in the yeast Saccharomyces cerevisiae.

PubMed

Tani, Motohiro; Kuge, Osamu

2014-04-01

Sac1 is a phosphoinositide phosphatase that preferentially dephosphorylates phosphatidylinositol 4-phosphate. Mutation of SAC1 causes not only the accumulation of phosphoinositides but also reduction of the phosphatidylserine (PS) level in the yeast Saccharomyces cerevisiae. In this study, we characterized the mechanism underlying the PS reduction in SAC1-deleted cells. Incorporation of (32) P into PS was significantly delayed in sac1∆ cells. Such a delay was also observed in SAC1- and PS decarboxylase gene-deleted cells, suggesting that the reduction in the PS level is caused by a reduction in the rate of biosynthesis of PS. A reduction in the PS level was also observed with repression of STT4 encoding phosphatidylinositol 4-kinase or deletion of VPS34 encoding phophatidylinositol 3-kinase. However, the combination of mutations of SAC1 and STT4 or VPS34 did not restore the reduced PS level, suggesting that both the synthesis and degradation of phosphoinositides are important for maintenance of the PS level. Finally, we observed an abnormal PS distribution in sac1∆ cells when a specific probe for PS was expressed. Collectively, these results suggested that Sac1 is involved in the maintenance of a normal rate of biosynthesis and distribution of PS. Copyright © 2014 John Wiley & Sons, Ltd.

SIRT1 Regulates Thyroid-Stimulating Hormone Release by Enhancing PIP5Kγ Activity through Deacetylation of Specific Lysine Residues in Mammals

PubMed Central

Akieda-Asai, Sayaka; Zaima, Nobuhiro; Ikegami, Koji; Kahyo, Tomoaki; Yao, Ikuko; Hatanaka, Takahiro; Iemura, Shun-ichiro; Sugiyama, Rika; Yokozeki, Takeaki; Eishi, Yoshinobu; Koike, Morio; Ikeda, Kyoji; Chiba, Takuya; Yamaza, Haruyoshi; Shimokawa, Isao; Song, Si-Young; Matsuno, Akira; Mizutani, Akiko; Sawabe, Motoji; Chao, Moses V.; Tanaka, Masashi; Kanaho, Yasunori; Natsume, Tohru; Sugimura, Haruhiko; Date, Yukari; McBurney, Michael W.; Guarente, Leonard; Setou, Mitsutoshi

2010-01-01

Background SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there. Methodology/Principal Findings Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K)γ was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kγ and enhanced PIP5Kγ enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kγ knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kγ, PI(4,5)P2, and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice. Conclusions/Significance Our findings indicated that the control of TSH release by the SIRT1-PIP5Kγ pathway is important for regulating the metabolism of the whole body. PMID:20668706

Platelets lacking PIP5KIγ have normal integrin activation but impaired cytoskeletal-membrane integrity and adhesion

PubMed Central

Wang, Yanfeng; Zhao, Liang; Suzuki, Aae; Lian, Lurong; Min, Sang H.; Wang, Ziqian; Litvinov, Rustem I.; Stalker, Timothy J.; Yago, Tadayuki; Klopocki, Arkadiusz G.; Schmidtke, David W.; Yin, Helen; Choi, John K.; McEver, Rodger P.; Weisel, John W.; Hartwig, John H.; Abrams, Charles S.

2013-01-01

Three isoforms of phosphatidylinositol-4-phosphate 5-kinase (PIP5KIα, PIP5KIβ, and PIP5KIγ) can each catalyze the final step in the synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn can be either converted to second messengers or bind directly to and thereby regulate proteins such as talin. A widely quoted model speculates that only p90, a longer splice form of platelet-specific PIP5KIγ, but not the shorter p87 PIP5KIγ, regulates the ligand-binding activity of integrins via talin. However, when we used mice genetically engineered to lack only p90 PIP5KIγ, we found that p90 PIP5KIγ is not critical for integrin activation or platelet adhesion on collagen. However, p90 PIP5KIγ-null platelets do have impaired anchoring of their integrins to the underlying cytoskeleton. Platelets lacking both the p90 and p87 PIP5KIγ isoforms had normal integrin activation and actin dynamics, but impaired anchoring of their integrins to the cytoskeleton. Most importantly, they formed weak shear-resistant adhesions ex vivo and unstable vascular occlusions in vivo. Together, our studies demonstrate that, although PIP5KIγ is essential for normal platelet function, individual isoforms of PIP5KIγ fulfill unique roles for the integrin-dependent integrity of the membrane cytoskeleton and for the stabilization of platelet adhesion. PMID:23372168

Phosphatidylserine Stimulation of Drs2p·Cdc50p Lipid Translocase Dephosphorylation Is Controlled by Phosphatidylinositol-4-phosphate*

PubMed Central

Jacquot, Aurore; Montigny, Cédric; Hennrich, Hanka; Barry, Raphaëlle; le Maire, Marc; Jaxel, Christine; Holthuis, Joost; Champeil, Philippe; Lenoir, Guillaume

2012-01-01

Here, Drs2p, a yeast lipid translocase that belongs to the family of P4-type ATPases, was overexpressed in the yeast Saccharomyces cerevisiae together with Cdc50p, its glycosylated partner, as a result of the design of a novel co-expression vector. The resulting high yield allowed us, using crude membranes or detergent-solubilized membranes, to measure the formation from [γ-32P]ATP of a 32P-labeled transient phosphoenzyme at the catalytic site of Drs2p. Formation of this phosphoenzyme could be detected only if Cdc50p was co-expressed with Drs2p but was not dependent on full glycosylation of Cdc50p. It was inhibited by orthovanadate and fluoride compounds. In crude membranes, the phosphoenzyme formed at steady state at 4 °C displayed ADP-insensitive but temperature-sensitive decay. Solubilizing concentrations of dodecyl maltoside left this decay rate almost unaltered, whereas several other detergents accelerated it. Unexpectedly, the dephosphorylation rate for the solubilized Drs2p·Cdc50p complex was inhibited by the addition of phosphatidylserine. Phosphatidylserine exerted its anticipated accelerating effect on the dephosphorylation of Drs2p·Cdc50p complex only in the additional presence of phosphatidylinositol-4-phosphate. These results explain why phosphatidylinositol-4-phosphate tightly controls Drs2p-catalyzed lipid transport and establish the functional relevance of the Drs2p·Cdc50p complex overexpressed here. PMID:22351780

Cdk5 Phosphorylation of ErbB4 is Required for Tangential Migration of Cortical Interneurons

PubMed Central

Rakić, Sonja; Kanatani, Shigeaki; Hunt, David; Faux, Clare; Cariboni, Anna; Chiara, Francesca; Khan, Shabana; Wansbury, Olivia; Howard, Beatrice; Nakajima, Kazunori; Nikolić, Margareta; Parnavelas, John G.

2015-01-01

Interneuron dysfunction in humans is often associated with neurological and psychiatric disorders, such as epilepsy, schizophrenia, and autism. Some of these disorders are believed to emerge during brain formation, at the time of interneuron specification, migration, and synapse formation. Here, using a mouse model and a host of histological and molecular biological techniques, we report that the signaling molecule cyclin-dependent kinase 5 (Cdk5), and its activator p35, control the tangential migration of interneurons toward and within the cerebral cortex by modulating the critical neurodevelopmental signaling pathway, ErbB4/phosphatidylinositol 3-kinase, that has been repeatedly linked to schizophrenia. This finding identifies Cdk5 as a crucial signaling factor in cortical interneuron development in mammals. PMID:24142862

Identification of a conserved 8 aa insert in the PIP5K protein in the Saccharomycetaceae family of fungi and the molecular dynamics simulations and structural analysis to investigate its potential functional role.

PubMed

Khadka, Bijendra; Gupta, Radhey S

2017-08-01

Homologs of the phosphatidylinositol-4-phosphate-5-kinase (PIP5K), which controls a multitude of essential cellular functions, contain a 8 aa insert in a conserved region that is specific for the Saccharomycetaceae family of fungi. Using structures of human PIP4K proteins as templates, structural models were generated of the Saccharomyces cerevisiae and human PIP5K proteins. In the modeled S. cerevisiae PIP5K, the 8 aa insert forms a surface exposed loop, present on the same face of the protein as the activation loop of the kinase domain. Electrostatic potential analysis indicates that the residues from 8 aa conserved loop form a highly positively charged surface patch, which through electrostatic interaction with the anionic portions of phospholipid head groups, is expected to play a role in the membrane interaction of the yeast PIP5K. To unravel this prediction, molecular dynamics (MD) simulations were carried out to examine the binding interaction of PIP5K, either containing or lacking the conserved signature insert, with two different membrane lipid bilayers. The results from MD studies provide insights concerning the mechanistic of interaction of PIP5K with lipid bilayer, and support the contention that the identified 8 aa conserved insert in fungal PIP5K plays an important role in the binding of this protein with membrane surface. Proteins 2017; 85:1454-1467. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins

USDA-ARS?s Scientific Manuscript database

The internalization of oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors’ cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants ...

PI(5)P Regulates Autophagosome Biogenesis

PubMed Central

Vicinanza, Mariella; Korolchuk, Viktor I.; Ashkenazi, Avraham; Puri, Claudia; Menzies, Fiona M.; Clarke, Jonathan H.; Rubinsztein, David C.

2015-01-01

Summary Phosphatidylinositol 3-phosphate (PI(3)P), the product of class III PI3K VPS34, recruits specific autophagic effectors, like WIPI2, during the initial steps of autophagosome biogenesis and thereby regulates canonical autophagy. However, mammalian cells can produce autophagosomes through enigmatic noncanonical VPS34-independent pathways. Here we show that PI(5)P can regulate autophagy via PI(3)P effectors and thereby identify a mechanistic explanation for forms of noncanonical autophagy. PI(5)P synthesis by the phosphatidylinositol 5-kinase PIKfyve was required for autophagosome biogenesis, and it increased levels of PI(5)P, stimulated autophagy, and reduced the levels of autophagic substrates. Inactivation of VPS34 impaired recruitment of WIPI2 and DFCP1 to autophagic precursors, reduced ATG5-ATG12 conjugation, and compromised autophagosome formation. However, these phenotypes were rescued by PI(5)P in VPS34-inactivated cells. These findings provide a mechanistic framework for alternative VPS34-independent autophagy-initiating pathways, like glucose starvation, and unravel a cytoplasmic function for PI(5)P, which previously has been linked predominantly to nuclear roles. PMID:25578879

Fluorescent Inhibitors as Tools To Characterize Enzymes: Case Study of the Lipid Kinase Phosphatidylinositol 4-Kinase IIIβ (PI4KB).

PubMed

Humpolickova, Jana; Mejdrová, Ivana; Matousova, Marika; Nencka, Radim; Boura, Evzen

2017-01-12

The lipid kinase phosphatidylinositol 4-kinase IIIβ (PI4KB) is an essential host factor for many positive-sense single-stranded RNA (+RNA) viruses including human pathogens hepatitis C virus (HCV), Severe acute respiratory syndrome (SARS), coxsackie viruses, and rhinoviruses. Inhibitors of PI4KB are considered to be potential broad-spectrum virostatics, and it is therefore critical to develop a biochemical understanding of the kinase. Here, we present highly potent and selective fluorescent inhibitors that we show to be useful chemical biology tools especially in determination of dissociation constants. Moreover, we show that the coumarin-labeled inhibitor can be used to image PI4KB in cells using fluorescence-lifetime imaging microscopy (FLIM) microscopy.

A Novel Positive Feedback Loop Mediated by the Docking Protein Gab1 and Phosphatidylinositol 3-Kinase in Epidermal Growth Factor Receptor Signaling

PubMed Central

Rodrigues, Gerard A.; Falasca, Marco; Zhang, Zhongtao; Ong, Siew Hwa; Schlessinger, Joseph

2000-01-01

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4,5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR. PMID:10648629

In vivo, Pikfyve generates PI(3,5)P2, which serves as both a signaling lipid and the major precursor for PI5P

PubMed Central

Zolov, Sergey N.; Bridges, Dave; Zhang, Yanling; Lee, Wei-Wei; Riehle, Ellen; Verma, Rakesh; Lenk, Guy M.; Converso-Baran, Kimber; Weide, Thomas; Albin, Roger L.; Saltiel, Alan R.; Meisler, Miriam H.; Russell, Mark W.; Weisman, Lois S.

2012-01-01

Mutations that cause defects in levels of the signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] lead to profound neurodegeneration in mice. Moreover, mutations in human FIG4 predicted to lower PI(3,5)P2 levels underlie Charcot–Marie–Tooth type 4J neuropathy and are present in selected cases of amyotrophic lateral sclerosis. In yeast and mammals, PI(3,5)P2 is generated by a protein complex that includes the lipid kinase Fab1/Pikfyve, the scaffolding protein Vac14, and the lipid phosphatase Fig4. Fibroblasts cultured from Vac14−/− and Fig4−/− mouse mutants have a 50% reduction in the levels of PI(3,5)P2, suggesting that there may be PIKfyve-independent pathways that generate this lipid. Here, we characterize a Pikfyve gene-trap mouse (Pikfyveβ-geo/β-geo), a hypomorph with ∼10% of the normal level of Pikfyve protein. shRNA silencing of the residual Pikfyve transcript in fibroblasts demonstrated that Pikfyve is required to generate all of the PI(3,5)P2 pool. Surprisingly, Pikfyve also is responsible for nearly all of the phosphatidylinositol-5-phosphate (PI5P) pool. We show that PI5P is generated directly from PI(3,5)P2, likely via 3′-phosphatase activity. Analysis of tissues from the Pikfyveβ-geo/β-geo mouse mutants reveals that Pikfyve is critical in neural tissues, heart, lung, kidney, thymus, and spleen. Thus, PI(3,5)P2 and PI5P have major roles in multiple organs. Understanding the regulation of these lipids may provide insights into therapies for multiple diseases. PMID:23047693

Modulation of phosphorylation of tocopherol and phosphatidylinositol by hTAP1/SEC14L2-mediated lipid exchange

USDA-ARS?s Scientific Manuscript database

The vitamin E derivative, alpha-tocopheryl phosphate (aTP), is detectable in cultured cells, plasma and tissues in small amounts, suggesting the existence of enzyme(s) with a-tocopherol (aT) kinase activity. Here, we characterize the production of aTP from aT and [g-32P]-ATP in primary human coronar...

Muscarinic modulation of TREK currents in mouse sympathetic superior cervical ganglion neurons.

PubMed

Rivas-Ramírez, P; Cadaveira-Mosquera, A; Lamas, J A; Reboreda, A

2015-07-01

Muscarinic receptors play a key role in the control of neurotransmission in the autonomic ganglia, which has mainly been ascribed to the regulation of potassium M-currents and voltage-dependent calcium currents. Muscarinic agonists provoke depolarization of the membrane potential and a reduction in spike frequency adaptation in postganglionic neurons, effects that may be explained by M-current inhibition. Here, we report the presence of a riluzole-activated current (IRIL ) that flows through the TREK-2 channels, and that is also inhibited by muscarinic agonists in neurons of the mouse superior cervical ganglion (mSCG). The muscarinic agonist oxotremorine-M (Oxo-M) inhibited the IRIL by 50%, an effect that was abolished by pretreatment with atropine or pirenzepine, but was unaffected in the presence of himbacine. Moreover, these antagonists had similar effects on single-channel TREK-2 currents. IRIL inhibition was unaffected by pretreatment with pertussis toxin. The protein kinase C blocker bisindolylmaleimide did not have an effect, and neither did the inositol triphosphate antagonist 2-aminoethoxydiphenylborane. Nevertheless, the IRIL was markedly attenuated by the phospholipase C (PLC) inhibitor ET-18-OCH3. Finally, the phosphatidylinositol-3-kinase/phosphatidylinositol-4-kinase inhibitor wortmannin strongly attenuated the IRIL , whereas blocking phosphatidylinositol 4,5-bisphosphate (PIP2 ) depletion consistently prevented IRIL inhibition by Oxo-M. These results demonstrate that TREK-2 currents in mSCG neurons are inhibited by muscarinic agonists that activate M1 muscarinic receptors, reducing PIP2 levels via a PLC-dependent pathway. The similarities between the signaling pathways regulating the IRIL and the M-current in the same neurons reflect an important role of this new pathway in the control of autonomic ganglia excitability. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Neurotrophic Effect of Citrus 5-Hydroxy-3,6,7,8,3′,4′-Hexamethoxyflavone: Promotion of Neurite Outgrowth via cAMP/PKA/CREB Pathway in PC12 Cells

PubMed Central

Lai, Hui-Chi; Wu, Ming-Jiuan; Chen, Pei-Yi; Sheu, Ting-Ting; Chiu, Szu-Ping; Lin, Meng-Han; Ho, Chi-Tang; Yen, Jui-Hung

2011-01-01

5-Hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5-OH-HxMF), a hydroxylated polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. In this research, we report the first investigation of the neurotrophic effects and mechanism of 5-OH-HxMF in PC12 pheochromocytoma cells. We found that 5-OH-HxMF can effectively induce PC12 neurite outgrowth accompanied with the expression of neuronal differentiation marker protein growth-associated protein-43(GAP-43). 5-OH-HxMF caused the enhancement of cyclic AMP response element binding protein (CREB) phosphorylation, c-fos gene expression and CRE-mediated transcription, which was inhibited by 2-naphthol AS-E phosphate (KG-501), a specific antagonist for the CREB-CBP complex formation. Moreover, 5-OH-HxMF-induced both CRE transcription activity and neurite outgrowth were inhibited by adenylate cyclase and protein kinase A (PKA) inhibitor, but not MEK1/2, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) or calcium/calmodulin-dependent protein kinase (CaMK) inhibitor. Consistently, 5-OH-HxMF treatment increased the intracellular cAMP level and downstream component, PKA activity. We also found that addition of K252a, a TrKA antagonist, significantly inhibited NGF- but not 5-OH-HxMF-induced neurite outgrowth. These results reveal for the first time that 5-OH-HxMF is an effective neurotrophic agent and its effect is mainly through a cAMP/PKA-dependent, but TrKA-independent, signaling pathway coupling with CRE-mediated gene transcription. A PKC-dependent and CREB-independent pathway was also involved in its neurotrophic action. PMID:22140566

Phosphatidylinositol 4-Kinase IIIβ Is Required for Severe Acute Respiratory Syndrome Coronavirus Spike-mediated Cell Entry*

PubMed Central

Yang, Ning; Ma, Ping; Lang, Jianshe; Zhang, Yanli; Deng, Jiejie; Ju, Xiangwu; Zhang, Gongyi; Jiang, Chengyu

2012-01-01

Phosphatidylinositol kinases (PI kinases) play an important role in the life cycle of several viruses after infection. Using gene knockdown technology, we demonstrate that phosphatidylinositol 4-kinase IIIβ (PI4KB) is required for cellular entry by pseudoviruses bearing the severe acute respiratory syndrome-coronavirus (SARS-CoV) spike protein and that the cell entry mediated by SARS-CoV spike protein is strongly inhibited by knockdown of PI4KB. Consistent with this observation, pharmacological inhibitors of PI4KB blocked entry of SARS pseudovirions. Further research suggested that PI4P plays an essential role in SARS-CoV spike-mediated entry, which is regulated by the PI4P lipid microenvironment. We further demonstrate that PI4KB does not affect virus entry at the SARS-CoV S-ACE2 binding interface or at the stage of virus internalization but rather at or before virus fusion. Taken together, these results indicate a new function for PI4KB and suggest a new drug target for preventing SARS-CoV infection. PMID:22253445

Chronic alteration in phosphatidylinositol 4,5-bisphosphate levels regulates capsaicin and mustard oil responses

PubMed Central

Patil, Mayur J.; Belugin, Sergei; Akopian, Armen N.

2011-01-01

There is an agreement that acute (in minutes) hydrolysis and accumulation of phosphatidylinositol 4,5-bisphosphate (PIP2) modulate TRPV1 and TRPA1 activities. Since inflammation results in PIP2 depletion, persisting for long periods (hours-to-days) in pain models and in clinic, we examined whether chronic depletion and accumulation of PIP2 affects capsaicin and mustard oil responses. In addition we also wanted to evaluate whether the effects of PIP2 depend on TRPV1 and TRPA1 co-expression, and whether the PIP2 actions vary in expression cells versus sensory neurons. Chronic PIP2 production was stimulated by over-expression of phosphatidylinositol-4-phosphate-5-kinase, while PIP2-specific phospholipid 5′-phosphatase was selected to reduce plasma membrane levels of PIP2. Our results demonstrate that capsaicin (100 nM; CAP) responses and receptor tachyphylaxis are not significantly influenced by chronic changes in PIP2 levels in wild-type (WT) or TRPA1 null-mutant sensory neurons, as well as CHO cells expressing TRPV1 alone or with TRPA1. However, low concentrations of CAP (20 nM) produced a higher response after PIP2 depletion in cells containing TRPV1 alone, but not TRPV1 together with TRPA1. Mustard oil (25 μM; MO) responses were also not affected by PIP2 in WT sensory neurons and cells co-expressing TRPA1 and TRPV1. In contrast, PIP2 reduction leads to pronounced tachyphylaxis to MO in cells with both channels. Chronic effect of PIP2 on TRPA1 activity depends on presence of the TRPV1 channel and cell type (CHO vs. sensory neurons). In summary, chronic alterations in PIP2 levels regulate magnitude of CAP and MO responses, as well as MO-tachyphylaxis. This regulation depends on co-expression profile of TRPA1 and TRPV1 and cell type. PMID:21337373

Chronic alteration in phosphatidylinositol 4,5-biphosphate levels regulates capsaicin and mustard oil responses.

PubMed

Patil, Mayur J; Belugin, Sergei; Akopian, Armen N

2011-06-01

There is an agreement that acute (in minutes) hydrolysis and accumulation of phosphatidylinositol 4,5-bisphosphate (PIP(2) ) modulate TRPV1 and TRPA1 activities. Because inflammation results in PIP(2) depletion, persisting for long periods (hours to days) in pain models and in the clinic, we examined whether chronic depletion and accumulation of PIP(2) affect capsaicin (CAP) and mustard oil (MO) responses. In addition, we wanted to evaluate whether the effects of PIP(2) depend on TRPV1 and TRPA1 coexpression and whether the PIP(2) actions vary in expression cells vs. sensory neurons. Chronic PIP(2) production was stimulated by overexpression of phosphatidylinositol-4-phosphate-5-kinase, and PIP(2) -specific phospholipid 5'-phosphatase was selected to reduce plasma membrane levels of PIP(2) . Our results demonstrate that CAP (100 nM) responses and receptor tachyphylaxis are not significantly influenced by chronic changes in PIP(2) levels in wild-type (WT) or TRPA1 null-mutant sensory neurons as well as CHO cells expressing TRPV1 alone or with TRPA1. However, low concentrations of CAP (20 nM) produced a higher response after PIP(2) depletion in cells containing TRPV1 alone but not TRPV1 together with TRPA1. MO (25 μM) responses were also not affected by PIP(2) in WT sensory neurons and cells coexpressing TRPA1 and TRPV1. In contrast, PIP(2) reduction leads to pronounced tachyphylaxis to MO in cells with both channels. Chronic effect of PIP(2) on TRPA1 activity depends on presence of the TRPV1 channel and cell type (CHO vs. sensory neurons). In summary, chronic alterations in PIP(2) levels regulate magnitude of CAP and MO responses as well as MO tachyphylaxis. This regulation depends on coexpression profile of TRPA1 and TRPV1 and cell type. Copyright © 2011 Wiley-Liss, Inc.

A-kinase anchoring protein 150 mediates transient receptor potential family V type 1 sensitivity to phosphatidylinositol-4,5-bisphosphate.

PubMed

Jeske, Nathaniel A; Por, Elaine D; Belugin, Sergei; Chaudhury, Sraboni; Berg, Kelly A; Akopian, Armen N; Henry, Michael A; Gomez, Ruben

2011-06-08

A-kinase anchoring protein 150 (AKAP150) is a scaffolding protein that controls protein kinase A- and C-mediated phosphorylation of the transient receptor potential family V type 1 (TRPV1), dictating receptor response to nociceptive stimuli. The phospholipid phosphatidylinositol-4,5-bisphosphate (PIP(2)) anchors AKAP150 to the plasma membrane in naive conditions and also affects TRPV1 activity. In the present study, we sought to determine whether the effects of PIP(2) on TRPV1 are mediated through AKAP150. In trigeminal neurons and CHO cells, the manipulation of cellular PIP(2) led to significant changes in the association of AKAP150 and TRPV1. Following PIP(2) degradation, increased TRPV1:AKAP150 coimmunoprecipitation was observed, resulting in increased receptor response to capsaicin treatment. Phospholipase C activation in neurons isolated from AKAP150(-/-) animals indicated that PIP(2)-mediated inhibition of TRPV1 in the whole-cell environment requires expression of the scaffolding protein. Furthermore, the addition of PIP(2) to neurons isolated from AKAP150 wild-type mice reduced PKA sensitization of TRPV1 compared with isolated neurons from AKAP150(-/-) mice. These findings suggest that PIP(2) degradation increases AKAP150 association with TRPV1 in the whole-cell environment, leading to sensitization of the receptor to nociceptive stimuli.

Regulation and physiological functions of mammalian phospholipase C.

PubMed

Nakamura, Yoshikazu; Fukami, Kiyoko

2017-04-01

Phospholipase C (PLC) is a key enzyme in phosphoinositide metabolism. PLC hydrolyses phosphatidylinositol 4,5-bis-phosphate to generate two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol, that generate diverse cellular responses. PLC is activated by various signalling molecules, including Ca2+, heterometric G proteins, small G proteins, and receptor/non-receptor tyrosine kinases. In addition to their enzymatic activity, some PLC subtypes also function as a guanine nucleotide exchange factor, GTPase-activating protein, and adaptor protein, independent of their lipase activity. There are 13 PLC isozymes in mammals, and they are categorized into six classes based on structure. Generation and analysis of genetically modified mice has revealed the unexpectedly diverse physiological functions of PLC isozymes. Although all PLC isozymes catalyze the same reaction, each PLC isozyme has unique physiological functions. This review focuses on the regulation and physiological functions of PLCs. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

The machinery at endoplasmic reticulum-plasma membrane contact sites contributes to spatial regulation of multiple Legionella effector proteins.

PubMed

Hubber, Andree; Arasaki, Kohei; Nakatsu, Fubito; Hardiman, Camille; Lambright, David; De Camilli, Pietro; Nagai, Hiroki; Roy, Craig R

2014-07-01

The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.

Do phosphoinositides regulate membrane water permeability of tobacco protoplasts by enhancing the aquaporin pathway?

PubMed

Ma, Xiaohong; Shatil-Cohen, Arava; Ben-Dor, Shifra; Wigoda, Noa; Perera, Imara Y; Im, Yang Ju; Diminshtein, Sofia; Yu, Ling; Boss, Wendy F; Moshelion, Menachem; Moran, Nava

2015-03-01

Enhancing the membrane content of PtdInsP 2 , the already-recognized protein-regulating lipid, increased the osmotic water permeability of tobacco protoplasts, apparently by increasing the abundance of active aquaporins in their membranes. While phosphoinositides are implicated in cell volume changes and are known to regulate some ion channels, their modulation of aquaporins activity has not yet been reported for any organism. To examine this, we compared the osmotic water permeability (P f) of protoplasts isolated from tobacco (Nicotiana tabacum) cultured cells (NT1) with different (genetically lowered or elevated relative to controls) levels of inositol trisphosphate (InsP3) and phosphatidyl inositol [4,5] bisphosphate (PtdInsP2). To achieve this, the cells were transformed with, respectively, the human InsP3 5-phosphatase ('Ptase cells') or human phosphatidylinositol (4) phosphate 5-kinase ('PIPK cells'). The mean P f of the PIPK cells was several-fold higher relative to that of controls and Ptase cells. Three results favor aquaporins over the membrane matrix as underlying this excessive P f: (1) transient expression of the maize aquaporin ZmPIP2;4 in the PIPK cells increased P f by 12-30 μm s(-1), while in the controls only by 3-4 μm s(-1). (2) Cytosol acidification-known to inhibit aquaporins-lowered the P f in the PIPK cells down to control levels. (3) The transcript of at least one aquaporin was elevated in the PIPK cells. Together, the three results demonstrate the differences between the PIPK cells and their controls, and suggest a hitherto unobserved regulation of aquaporins by phosphoinositides, which could occur through direct interaction or indirect phosphoinositides-dependent cellular effects.

Phosphatidylinositol 3-Kinase and Protein Kinase C as Molecular Determinants of Chemoresistance in Breast Cancer

DTIC Science & Technology

2004-07-01

medium (Cambrex, San Diego, CA) supplemented with bovine pituitary extract . cancer contained approximately one-half the level of cer- 3-[4,5...rill et al., 1988) as modified by Yoon et al. (1999) was used. Lipid Cytochrome c Release. Cells (4 X 106) were harvested with 0.5% extract aliquots... extracted in chloroform, and the organic phase was dried under a nitrogen z 80- stream. The lipids contained in the organic phase extract were re- 70

Involvement of the PI3K/Akt/GSK3β pathway in photodynamic injury of neurons and glial cells

NASA Astrophysics Data System (ADS)

Komandirov, M. A.; Knyazeva, E. A.; Fedorenko, Y. P.; Rudkovskii, M. V.; Stetsurin, D. A.; Uzdensky, A. B.

2010-10-01

Photodynamic treatment causes intense oxidative stress and kills cells. It is currently used in neurooncology. However, along with tumor it damages surrounding healthy neuronal and glial cells. In order to study the possible role of the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β signaling pathway in photodynamic damage to normal neurons and glia, we used isolated crayfish stretch receptor that consists only of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens (100 nM). The laser diode (670nm, 0.4W/cm2) was used as a light source. Application of specific inhibitors of the enzymes involved in this pathway showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Protein kinase Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3β participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β pathway was not involved as a whole in photodynamic injury of crayfish neurons and glial cells but its components, protein kinase Akt and glycogen synthase kinase-3β, independently and cell-specifically regulated photoinduced death of neurons and glial cells. These data showed that in this system necrosis was not non-regulated and catastrophic mode of cell death. It was controlled by some signaling proteins. The obtained results may be used for search of pharmacological agents that selectively modulate injury of normal neurons and glial cells during photodynamic therapy of brain tumors.

Involvement of the PI3K/Akt/GSK3β pathway in photodynamic injury of neurons and glial cells

NASA Astrophysics Data System (ADS)

Komandirov, M. A.; Knyazeva, E. A.; Fedorenko, Y. P.; Rudkovskii, M. V.; Stetsurin, D. A.; Uzdensky, A. B.

2011-03-01

Photodynamic treatment causes intense oxidative stress and kills cells. It is currently used in neurooncology. However, along with tumor it damages surrounding healthy neuronal and glial cells. In order to study the possible role of the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β signaling pathway in photodynamic damage to normal neurons and glia, we used isolated crayfish stretch receptor that consists only of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens (100 nM). The laser diode (670nm, 0.4W/cm2) was used as a light source. Application of specific inhibitors of the enzymes involved in this pathway showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Protein kinase Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3β participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, the phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β pathway was not involved as a whole in photodynamic injury of crayfish neurons and glial cells but its components, protein kinase Akt and glycogen synthase kinase-3β, independently and cell-specifically regulated photoinduced death of neurons and glial cells. These data showed that in this system necrosis was not non-regulated and catastrophic mode of cell death. It was controlled by some signaling proteins. The obtained results may be used for search of pharmacological agents that selectively modulate injury of normal neurons and glial cells during photodynamic therapy of brain tumors.

Simultaneous inhibition of pan-phosphatidylinositol-3-kinases and MEK as a potential therapeutic strategy in peripheral T-cell lymphomas.

PubMed

Martín-Sánchez, Esperanza; Rodríguez-Pinilla, Socorro M; Sánchez-Beato, Margarita; Lombardía, Luis; Domínguez-González, Beatriz; Romero, Diana; Odqvist, Lina; García-Sanz, Pablo; Wozniak, Magdalena B; Kurz, Guido; Blanco-Aparicio, Carmen; Mollejo, Manuela; Alves, F Javier; Menárguez, Javier; González-Palacios, Fernando; Rodríguez-Peralto, José Luis; Ortiz-Romero, Pablo L; García, Juan F; Bischoff, James R; Piris, Miguel A

2013-01-01

Peripheral T-cell lymphomas are very aggressive hematologic malignancies for which there is no targeted therapy. New, rational approaches are necessary to improve the very poor outcome in these patients. Phosphatidylinositol-3-kinase is one of the most important pathways in cell survival and proliferation. We hypothesized that phosphatidylinositol-3-kinase inhibitors could be rationally selected drugs for treating peripheral T-cell lymphomas. Several phosphatidylinositol-3-kinase isoforms were inhibited genetically (using small interfering RNA) and pharmacologically (with CAL-101 and GDC-0941 compounds) in a panel of six peripheral and cutaneous T-cell lymphoma cell lines. Cell viability was measured by intracellular ATP content; apoptosis and cell cycle changes were checked by flow cytometry. Pharmacodynamic biomarkers were assessed by western blot. The PIK3CD gene, which encodes the δ isoform of phosphatidylinositol-3-kinase, was overexpressed in cell lines and primary samples, and correlated with survival pathways. However, neither genetic nor specific pharmacological inhibition of phosphatidylinositol-3-kinase δ affected cell survival. In contrast, the pan-phosphatidylinositol-3-kinase inhibitor GDC-0941 arrested all T-cell lymphoma cell lines in the G1 phase and induced apoptosis in a subset of them. We identified phospho-GSK3β and phospho-p70S6K as potential biomarkers of phosphatidylinositol-3-kinase inhibitors. Interestingly, an increase in ERK phosphorylation was observed in some GDC -0941-treated T-cell lymphoma cell lines, suggesting the presence of a combination of phosphatidylinositol-3-kinase and MEK inhibitors. A highly synergistic effect was found between the two inhibitors, with the combination enhancing cell cycle arrest at G0/G1 in all T-cell lymphoma cell lines, and reducing cell viability in primary tumor T cells ex vivo. These results suggest that the combined treatment of pan-phosphatidylinositol-3-kinase + MEK inhibitors could be more effective than single phosphatidylinositol-3-kinase inhibitor treatment, and therefore, that this combination could be of therapeutic value for treating peripheral and cutaneous T-cell lymphomas.

Carcinogenesis of PIK3CA

PubMed Central

2013-01-01

PIK3CA is the most frequently mutated oncogene in human cancers. PIK3CA is phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha. It controls cell growth, proliferation, motility, survival, differentiation and intracellular trafficking. In most of human cancer alteration occurred frequently in the alpha isoform of phosphatidylinositol 3 kinase. PIK3CA mutations were most frequent in endometrial, ovarian, colorectal, breast, cervical, squamous cell cancer of the head and neck, chondroma, thyroid carcinoma and in cancer family syndrome. Inhibition of PI3K signaling can diminish cell proliferation, and in some circumstances, promote cell death. Consequently, components of this pathway present attractive targets for cancer therapeutics. A number of PI3K pathway inhibitors have been developed and used. PI3K inhibitors (both pan-PI3K and isoform-specific PI3K inhibitors), dual PI3K-mTOR inhibitors that are catalytic site inhibitors of the p110 isoforms and mTOR (the kinase component of both mTORC1 and mTORC2), mTOR catalytic site inhibitors, and AKT inhibitors are the most advanced in the clinic. They are approved for the treatment of several carcinomas. PMID:23768168

Erk1/2 and Akt kinases are involved in the protective effect of aniracetam in astrocytes subjected to simulated ischemia in vitro.

PubMed

Gabryel, Bozena; Pudelko, Anna; Malecki, Andrzej

2004-06-28

The present study focused on the mechanism of cytoprotective effect of aniracetam on the primary rat astrocyte cultures exposed to simulated ischemia conditions in vitro. To study these mechanisms, the aniracetam-mediated modulation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/Akt kinase pathways was determined. Simulated in vitro ischemia caused death of approximately 35% of astrocytes via apoptosis and decreased cell viability about 50% at 8 h. Exposure to aniracetam at concentrations of 0.1-10 microM in these conditions significantly decreased the number of apoptotic cells. Moreover, the intensification of 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide (MTT) conversion and the decrease of lactate dehydrogenase (LDH) release after 1 and 10 microM aniracetam treatment were observed indicating a significant increase in cell viability. When cultured astrocytes were incubated during 8 h simulated ischemia with [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] (U0126), an extracellular regulated kinase 1 and 2 (Erk1/2) inhibitor or wortmannin, a phosphatidylinositol 3-kinase (PI3 kinase)/Akt inhibitor, the cell apoptosis was accelerated. These effects of used kinase inhibitors (both U0126 and wortmannin) were antagonized by adding 1 and 10 microM aniracetam to the culture medium. In addition, aniracetam significantly stimulated of phospho-Erk1/2 kinase and phospho-Akt expression. Maximum levels of Erk1/2 and Akt activation were observed as a result of treatment with 10 microM aniracetam. U0126 and wortmannin markedly attenuated the effects of aniracetam on expression of activated kinases. Results of the present study indicate that both Erk1/2 and PI 3-K/Akt kinase pathways are vital for cytoprotective effect of aniracetam.

mTORC1 activity repression by late endosomal phosphatidylinositol 3,4-bisphosphate.

PubMed

Marat, Andrea L; Wallroth, Alexander; Lo, Wen-Ting; Müller, Rainer; Norata, Giuseppe Danilo; Falasca, Marco; Schultz, Carsten; Haucke, Volker

2017-06-02

Nutrient sensing by mechanistic target of rapamycin complex 1 (mTORC1) on lysosomes and late endosomes (LyLEs) regulates cell growth. Many factors stimulate mTORC1 activity, including the production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] by class I phosphatidylinositol 3-kinases (PI3Ks) at the plasma membrane. We investigated mechanisms that repress mTORC1 under conditions of growth factor deprivation. We identified phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ], synthesized by class II PI3K β (PI3KC2β) at LyLEs, as a negative regulator of mTORC1, whereas loss of PI3KC2β hyperactivated mTORC1. Growth factor deprivation induced the association of PI3KC2β with the Raptor subunit of mTORC1. Local PI(3,4)P 2 synthesis triggered repression of mTORC1 activity through association of Raptor with inhibitory 14-3-3 proteins. These results unravel an unexpected function for local PI(3,4)P 2 production in shutting off mTORC1. Copyright © 2017, American Association for the Advancement of Science.

Involvement of protein kinase B and mitogen-activated protein kinases in experimental normothermic liver ischaemia-reperfusion injury.

PubMed

Cursio, R; Filippa, N; Miele, C; Van Obberghen, E; Gugenheim, J

2006-06-01

This study evaluated the role of protein kinase B (PKB), phosphatidylinositol 3-kinase (PI3-K), Bcl-2-associated death protein (BAD) and mitogen-activated protein kinases (MAPKs) in normothermic ischaemia-reperfusion (IR)-induced apoptosis in rat liver. Rats were divided into two groups that received either phosphate-buffered saline (control) or the caspase inhibitor Z-Asp-2,6-dichorobenzoyloxymethylketone (Z-Asp-cmk), injected intravenously 2 min before the induction of 120 min of normothermic liver ischaemia. Liver apoptosis was assessed by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method. PI3-K, PKB, BAD and MAPK activities were measured in ischaemic and non-ischaemic lobes at various times after reperfusion. The number of TUNEL-positive cells was significantly decreased after pretreatment with Z-Asp-cmk. In controls, PI3-K and PKB activities and BAD phosphorylation were inhibited in ischaemic liver lobes. The MAPKs (extracellular signal-regulated kinases, c-Jun N-terminal kinase and p38) showed different patterns of activation during IR. PKB activity was not modified by pretreatment with Z-Asp-cmk. Induction of apoptosis during IR liver injury might be triggered by inactivation of the antiapoptotic PI3-K-PKB pathway and activation of the proapoptotic MAPKs. Copyright (c) 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

Anti-hepatitis C virus activity and toxicity of type III phosphatidylinositol-4-kinase beta inhibitors.

PubMed

Lamarche, M J; Borawski, J; Bose, A; Capacci-Daniel, C; Colvin, R; Dennehy, M; Ding, J; Dobler, M; Drumm, J; Gaither, L A; Gao, J; Jiang, X; Lin, K; McKeever, U; Puyang, X; Raman, P; Thohan, S; Tommasi, R; Wagner, K; Xiong, X; Zabawa, T; Zhu, S; Wiedmann, B

2012-10-01

Type III phosphatidylinositol-4-kinase beta (PI4KIIIβ) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIβ inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIβ in lymphocyte proliferation. Previously proposed functions of PI4KIIIβ in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIβ inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C.

Anti-Hepatitis C Virus Activity and Toxicity of Type III Phosphatidylinositol-4-Kinase Beta Inhibitors

PubMed Central

LaMarche, M. J.; Borawski, J.; Bose, A.; Capacci-Daniel, C.; Colvin, R.; Dennehy, M.; Ding, J.; Dobler, M.; Drumm, J.; Gaither, L. A.; Gao, J.; Jiang, X.; Lin, K.; McKeever, U.; Puyang, X.; Raman, P.; Thohan, S.; Tommasi, R.; Wagner, K.; Xiong, X.; Zabawa, T.; Zhu, S.

2012-01-01

Type III phosphatidylinositol-4-kinase beta (PI4KIIIβ) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIβ inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIβ in lymphocyte proliferation. Previously proposed functions of PI4KIIIβ in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIβ inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C. PMID:22825118

3′ Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP)

PubMed Central

Kurokawa, Tatsuki; Takasuga, Shunsuke; Sakata, Souhei; Yamaguchi, Shinji; Horie, Shigeo; Homma, Koichi J.; Sasaki, Takehiko; Okamura, Yasushi

2012-01-01

Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5′ position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] upon voltage depolarization. However, it is unclear whether VSPs also have 3′ phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P3. TLC assay showed that the 3′ phosphate of PI(3,4,5)P3 was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] was removed by VSPs. Monitoring of PI(3,4)P2 levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PHTAPP1-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5′ phosphatase activity of VSP toward PI(3,4,5)P3. However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P3 is dephosphorylated at the 5′ position, PI(3,4)P2 is then dephosphorylated at the 3′ position. These results suggest that substrate specificity of the VSP changes with membrane potential. PMID:22645351

3' Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP).

PubMed

Kurokawa, Tatsuki; Takasuga, Shunsuke; Sakata, Souhei; Yamaguchi, Shinji; Horie, Shigeo; Homma, Koichi J; Sasaki, Takehiko; Okamura, Yasushi

2012-06-19

Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5' position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] upon voltage depolarization. However, it is unclear whether VSPs also have 3' phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P(3). TLC assay showed that the 3' phosphate of PI(3,4,5)P(3) was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] was removed by VSPs. Monitoring of PI(3,4)P(2) levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PH(TAPP1)-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5' phosphatase activity of VSP toward PI(3,4,5)P(3). However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P(3) is dephosphorylated at the 5' position, PI(3,4)P(2) is then dephosphorylated at the 3' position. These results suggest that substrate specificity of the VSP changes with membrane potential.

Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: A sup 31 P NMR study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shashidhar, M.S.; Kuppe, A.; Volwerk, J.J.

1990-09-04

The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by {sup 31}P NMR. {sup 31}P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are {minus}0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. The authors also report the new and unexpected observation that themore » phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by {sup 31}P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B.cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. They propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.« less

Study of ATM Phosphorylation by Cdk5 in Neuronal Cells.

PubMed

She, Hua; Mao, Zixu

2017-01-01

The phosphatidylinositol-3-kinase-like kinase ATM (ataxia-telangiectasia mutated) plays a central role in coordinating the DNA damage responses including cell cycle checkpoint control, DNA repair, and apoptosis. Mutations of ATM cause a spectrum of defects ranging from neurodegeneration to cancer predisposition. We previously showed that Cdk5 (cyclin-dependent kinase 5) is activated by DNA damage and directly phosphorylates ATM at serine 794 in postmitotic neurons. Phosphorylation at serine 794 precedes and is required for ATM autophosphorylation at serine 1981, and activates ATM kinase activity. Cdk5-ATM pathway plays a crucial role in DNA damage-induced neuronal injury. This chapter describes protocols used in analyzing ATM phosphorylation by Cdk5 in CGNs (cerebellar granule neurons) and its effects on neuronal survival.

Insulin-like growth factor-mediated muscle differentiation: collaboration between phosphatidylinositol 3-kinase-Akt-signaling pathways and myogenin.

PubMed

Tureckova, J; Wilson, E M; Cappalonga, J L; Rotwein, P

2001-10-19

The differentiation and maturation of skeletal muscle require interactions between signaling pathways activated by hormones and growth factors and an intrinsic regulatory network controlled by myogenic transcription factors. Insulin-like growth factors (IGFs) play key roles in muscle development in the embryo and in regeneration in the adult. To study mechanisms of IGF action in muscle, we developed a myogenic cell line that overexpresses IGF-binding protein-5. C2BP5 cells remain quiescent in low serum differentiation medium until the addition of IGF-I. Here we use this cell line to identify signaling pathways controlling IGF-mediated differentiation. Induction of myogenin by IGF-I and myotube formation were prevented by the phosphatidylinositol (PI) 3-kinase inhibitor, LY294002, even when included 2 days after growth factor addition, whereas expression of active PI 3-kinase could promote differentiation in the absence of IGF-I. Differentiation also was induced by myogenin but was blocked by LY294002. The differentiation-promoting effects of IGF-I were mimicked by a modified membrane-targeted inducible Akt-1 (iAkt), and iAkt was able to stimulate differentiation of C2 myoblasts and primary mouse myoblasts incubated with otherwise inhibitory concentrations of LY294002. These results show that an IGF-regulated PI 3-kinase-Akt pathway controls muscle differentiation by mechanisms acting both upstream and downstream of myogenin.

Dual regulation of TRPV1 by phosphoinositides.

PubMed

Lukacs, Viktor; Thyagarajan, Baskaran; Varnai, Peter; Balla, Andras; Balla, Tamas; Rohacs, Tibor

2007-06-27

The membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PIP2] regulates many ion channels. There are conflicting reports on the effect of PtdIns(4,5)P2 on transient receptor potential vanilloid 1 (TRPV1) channels. We show that in excised patches PtdIns(4,5)P2 and other phosphoinositides activate and the PIP2 scavenger poly-Lys inhibits TRPV1. TRPV1 currents undergo desensitization on exposure to high concentrations of capsaicin in the presence of extracellular Ca2+. We show that in the presence of extracellular Ca2+, capsaicin activates phospholipase C (PLC) in TRPV1-expressing cells, inducing depletion of both PtdIns(4,5)P2 and its precursor PtdIns(4)P (PIP). The PLC inhibitor U73122 and dialysis of PtdIns(4,5)P2 or PtdIns(4)P through the patch pipette inhibited desensitization of TRPV1, indicating that Ca2+-induced activation of PLC contributes to desensitization of TRPV1 by depletion of PtdIns(4,5)P2 and PtdIns(4)P. Selective conversion of PtdIns(4,5)P2 to PtdIns(4)P by a rapamycin-inducible PIP2 5-phosphatase did not inhibit TRPV1 at high capsaicin concentrations, suggesting a significant role for PtdIns(4)P in maintaining channel activity. Currents induced by low concentrations of capsaicin and moderate heat, however, were potentiated by conversion of PtdIns(4,5)P2 to PtdIns(4)P. Increasing PtdIns(4,5)P2 levels by coexpressing phosphatidylinositol-4-phosphate 5-kinase inhibited TRPV1 at low but not at saturating capsaicin concentrations. These data show that at low capsaicin concentrations and other moderate stimuli, PtdIns(4,5)P2 partially inhibits TRPV1 in a cellular context, but this effect is likely to be indirect, because it is not detectable in excised patches. We conclude that phosphoinositides have both inhibitory and activating effects on TRPV1, resulting in complex and distinct regulation at various stimulation levels.

Fish Oil Supplementation in Humans: Effects on Platelet Responses, Phospholipid Composition and Metabolism.

NASA Astrophysics Data System (ADS)

Skeaff, Clark Murray

Platelets are believed to play a significant role in the development of occlusive vascular diseases. Epidemiological reports have correlated the high intake of marine foods, rich in omega3 fatty acids, with diminished platelet responses and a low incidence of arterial thrombosis and myocardial infarction. The activation of platelet responses is mediated by the accelerated metabolism of membrane phospholipid; therefore, it was of interest to examine, in human volunteers, the effect of a dietary fish oil concentrate (MaxEPA), enriched in omega 3 polyunsaturated fatty acids, on platelet aggregation and phospholipid composition/metabolism. For the complete separation of cellular phospholipids, a one-dimensional thin-layer chromatography system using silica-gel pre-coated glass plates was developed. The solvent system consisted of CHCl_3/CH_3OH/CH _3COOH/H_2O (50/37.5/3.5/2.0, by vol), required approximately 90-120 minutes for full phospholipid separation, and was highly reproducible even under conditions of variable humidity and temperature. The consumption of a fish oil concentrate (MaxEPA) for 6 weeks (3.6 g of 20:5omega 3 and 2.4 g of 22:6omega3 per day) diminished both the collagen- and platelet activating factor-induced maximum aggregation responses in washed human platelet suspensions by 50.1% and 27.2%, respectively, as compared to initial unsupplemented baseline responses. Thrombin -induced aggregation remained unchanged. Thrombin stimulation of intact human platelets produced a significant decrease in the mass of phosphatidylinositol in plasma membrane. In platelets pre-labelled with (2-^3H) glycerol and stimulated with either thrombin or low-dose collagen, the loss of (^3H) phosphatidylinositol did not differ between those subjects consuming olive oil or fish oil. Likewise, the thrombin-stimulated accumulation of diacylglycerol, an activator of protein kinase C, was unaffected by fish oil consumption. The ratio of collagen -induced increase in radioactivity associated with ( ^3H) PIP_2/( ^3H) PIP was 0.41 in fish oil consumers and 1.14 in olive oil consumers. These results are consistent with a dampened collagen-induced phosphatidylinositol 4 -phosphate kinase activity in platelets of healthy individuals consuming dietary fish oil. This effect may be eicosanoid -related based on work with BW 755C, a dual inhibitor of the cyclooxygenase and lipoxygenase enzymes. The relevance of these findings to the altered production of inositol 1,4,5 trisphosphate remains to be determined.

Dysregulation of the IGF-I/PI3K/AKT/mTOR signaling pathway in autism spectrum disorders.

PubMed

Chen, Jianling; Alberts, Ian; Li, Xiaohong

2014-06-01

The IGF-I/PI3K/AKT/mTOR signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation, motility, survival, metabolism and protein synthesis. Insulin-like growth factor-I (IGF-I) is synthesized in the liver and fibroblasts, and its biological actions are mediated by the IGF-I receptor (IGF-IR). The binding of IGF-I to IGF-IR leads to the activation of phosphatidylinositol 3-kinase (PI3K). Activated PI3K stimulates the production of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] and phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3]. The PH domain of AKT (protein kinase B, PKB) (v-AKT murine thymoma viral oncogene homolog) binds to PI(4,5)P2 and PI(3,4,5)P3, followed by phosphorylation of the Thr308 and Ser473 regulatory sites. Tuberous sclerosis complex 1 (TSC1) and TSC2 are upstream regulators of mammalian target of rapamycin (mTOR) and downstream effectors of the PI3K/AKT signaling pathway. The activation of AKT suppresses the TSC1/TSC2 heterodimer, which is an upstream regulator of mTOR. Dysregulated IGF-I/PI3K/AKT/mTOR signaling has been shown to be associated with autism spectrum disorders (ASDs). In this review, we discuss the emerging evidence for a functional relationship between the IGF-I/PI3K/AKT/mTOR pathway and ASDs, as well as a possible role of this signaling pathway in the diagnosis and treatment of ASDs. Copyright © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

Partial characterization of the cross-reacting determinant, a carbohydrate epitope shared by decay accelerating factor and the variant surface glycoprotein of the African Trypanosoma brucei.

PubMed

Shak, S; Davitz, M A; Wolinsky, M L; Nussenzweig, V; Turner, M J; Gurnett, A

1988-03-15

The variant surface glycoprotein (VSG) of the African trypanosome is anchored in the cell membrane by a complex glycan attached to phosphatidylinositol. The carboxyl terminal portion of VSG contains a cryptic carbohydrate epitope, the cross-reacting determinant (CRD), that is revealed only after removal of the diacylglycerol by phosphatidylinositol-specific phospholipase C (PIPLC) or VSG lipase. Recently, we have shown that after hydrolysis by PIPLC, decay-accelerating factor (DAF)--a mammalian phosphatidylinositol-anchored protein--also contains the CRD epitope. Using a two site immunoradiometric assay in which the capturing antibody is a monoclonal antibody to DAF and the revealing antibody is anti-CRD, we now show that sugar phosphates significantly inhibited the binding of anti-CRD antibody to DAF released by PIPLC. DL-myo-inositol 1,2-cyclic phosphate was the most potent inhibitor of binding (IC50 less than 10(-8) M). Other sugar phosphates, such as alpha-D-glucose-1-phosphate, which also possess adjacent hydroxyl and phosphate moieties in cis also inhibited binding at low concentrations (IC50 = 10(-5) to 10(-4) M). In contrast, sugar phosphates which do not possess adjacent hydroxyl and phosphate moieties in cis and simple sugars weakly inhibited binding (IC50 greater than 10(-3) M). These results suggest that myo-inositol 1,2-cyclic phosphate contributes significantly to the epitope recognized by the anti-CRD antibody and is consistent with analysis of the carboxyl terminus of VSG, which also suggested the presence of the cyclic inositol phosphate. In light of the recent findings that human serum contains a glycan-phosphatidyl-inositol-specific phospholipase D, which converts DAF from a hydrophobic to a hydrophilic form lacking the CRD, the observation that the phosphate is crucial for expression of the epitope may be relevant in understanding the origin of CRD-negative DAF in urine and plasma.

Nanoscale Landscape of Phosphoinositides Revealed by Specific Pleckstrin Homology (PH) Domains Using Single-molecule Superresolution Imaging in the Plasma Membrane*

PubMed Central

Ji, Chen; Zhang, Yongdeng; Xu, Pingyong; Xu, Tao; Lou, Xuelin

2015-01-01

Both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are independent plasma membrane (PM) determinant lipids that are essential for multiple cellular functions. However, their nanoscale spatial organization in the PM remains elusive. Using single-molecule superresolution microscopy and new photoactivatable fluorescence probes on the basis of pleckstrin homology domains that specifically recognize phosphatidylinositides in insulin-secreting INS-1 cells, we report that the PI(4,5)P2 probes exhibited a remarkably uniform distribution in the major regions of the PM, with some sparse PI(4,5)P2-enriched membrane patches/domains of diverse sizes (383 ± 14 nm on average). Quantitative analysis revealed a modest concentration gradient that was much less steep than previously thought, and no densely packed PI(4,5)P2 nanodomains were observed. Live-cell superresolution imaging further demonstrated the dynamic structural changes of those domains in the flat PM and membrane protrusions. PI4P and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) showed similar spatial distributions as PI(4,5)P2. These data reveal the nanoscale landscape of key inositol phospholipids in the native PM and imply a framework for local cellular signaling and lipid-protein interactions at a nanometer scale. PMID:26396197

Phosphatidylinositol kinase. A component of the chromaffin-granule membrane

PubMed Central

Phillips, John H.

1973-01-01

Phosphorylation of bovine chromaffin granules by ATP leads to the formation of diphosphoinositide in the granule membrane. Both phosphatidylinositol kinase and its substrate are components of this membrane, and triphosphoinositide is not formed under the conditions of the assay. The reaction is Mg2+-dependent and is stimulated by Mn2+ and F− ions. The initial reaction is rapid, with a broad pH profile and a `transition' temperature for its activation energy at 27°C. The apparent Km for ATP is 5μm. ATP, N-ethylmaleimide, Cu2+ ions and NaIO4 are inhibitory. The phospholipids of chromaffin-granule membranes have been analysed: 6.8% of the lipid P is found in phosphatidylinositol, and only 2–3% in phosphatidylserine. Comparison of the rate of phosphorylation of intact and lysed granules suggests that the sites for phosphorylation are on the outer (cytoplasmic) surface of the granules, and diphosphoinositide may therefore make an important contribution to the charge of the chromaffin granule in vivo. PMID:4360713

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

PubMed Central

Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

2014-01-01

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

Exercise activates the phosphatidylinositol 3-kinase pathway.

PubMed

Chen, Michael J; Russo-Neustadt, Amelia A

2005-04-27

Physical exercise is known to enhance psychological well-being and coping capacity. Voluntary physical exercise in rats also robustly and rapidly up-regulates hippocampal brain-derived neurotrophic factor (BDNF) mRNA levels, which are potentiated following a regimen of chronic antidepressant treatment. Increased BDNF levels are associated with enhanced activity of cyclic AMP response element binding protein (CREB). So far, relatively little is known about the intracellular signaling mechanisms mediating this effect of exercise. We wished to explore the possibility that exercise and/or antidepressant treatment activate the hippocampal phosphatidylinositol-3 (PI-3) kinase pathway, which mediates cellular survival. In young male Sprague-Dawley rats, we examined the effects of 2 weeks of daily voluntary wheel-running activity and/or tranylcypromine (n = 7 per group) on the levels of the active forms of protein-dependent kinase-1 (PDK-1), PI-3 kinase, phospho-thr308-Akt, phospho-ser473-Akt, and phospho-glycogen synthase kinase-3beta (GSK3beta; inactive form), as well as BDNF, activated CREB, and the phospho-Trk receptor, in the rat hippocampus, and compared these with sedentary saline-treated controls. Immunoblotting analyses revealed that in exercising rats, there was a significant increase in PI-3 kinase expression (4.61 times that of controls, P = 0.0161) and phosphorylation of PDK-1 (2.73 times that of controls, P = 0.0454), thr308-Akt (2.857 times that of controls, P = 0.0082), CREB (60.27 times that of controls, P = 0.05), and Trk (35.3 times that of controls, P < 0.0001) in the hippocampi of exercising animals; BDNF was also increased (3.2 times that of controls), but this was not statistically significant. In rats receiving both exercise and tranylcypromine, BDNF (4.51 times that of controls, P = 0.0068) and PI-3 kinase (4.88 times that of controls, P = 0.0103), and the phospho- forms of Trk (13.67 times that of controls, P = 0.0278), thr308-Akt (3.644 times that of controls, P = 0.0004), GSK-3beta (2.93 times that of controls, P = 0.026), and CREB (88.97 times that of controls, P = 0.0053) were significantly increased. These results suggest that the exercise-induced expression of BDNF is associated with the increased expression of several key intermediates of the PI-3 kinase/Akt pathway, which is known for its role in enhancing neuronal survival.

Role of protein kinase A and class II phosphatidylinositol 3-kinase C2β in the downregulation of KCa3.1 channel synthesis and membrane surface expression by lyso-globotriaosylceramide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Ju Yeon; Park, Seonghee, E-mail: sp@ewha.ac.kr

The intermediate conductance calcium-activated potassium channel (KCa3.1) mediates proliferation of many cell types including fibroblasts, and is a molecular target for intervention in various cell proliferative diseases. Our previous study showed that reduction of KCa3.1 channel expression by lyso-globotriaosylceramide (lyso-Gb3) inhibits differentiation into myofibroblasts and collagen synthesis, which might lead to development of ascending thoracic aortic aneurysm secondary to Fabry disease. However, how lyso-Gb3 downregulates KCa3.1 channel expression is unknown. Therefore, we aimed to investigate the underlying mechanisms of lyso-Gb3-mediated KCa3.1 channel downregulation, focusing on the cAMP signaling pathway. We found that lyso-Gb3 increased the intracellular cAMP concentration by upregulationmore » of adenylyl cyclase 6 and inhibited ERK 1/2 phosphorylation through the protein kinase A (PKA) pathway, leading to the inhibition of KCa3.1 channel synthesis, not the exchange protein directly activated by cAMP (Epac) pathway. Moreover, lyso-Gb3 suppressed expression of class II phosphatidylinositol 3-kinase C2β (PI3KC2β) by PKA activation, which reduces the production of phosphatidylinositol 3-phosphate [PI(3)P], and the reduced membrane surface expression of KCa3.1 channel was recovered by increasing the intracellular levels of PI(3)P. Consequently, our findings that lyso-Gb3 inhibited both KCa3.1 channel synthesis and surface expression by increasing intracellular cAMP, and controlled surface expression through changes in PI3KC2β-mediated PI(3)P production, suggest that modulation of PKA and PI3KC2β activity to control of KCa3.1 channel expression can be an alternative important target to attenuate ascending thoracic aortic aneurysms in Fabry disease. - Highlights: • Lyso-Gb3 causes elevation of intracellular cAMP. • Lyso-Gb3 inhibits the ERK 1/2 phosphorylation through PKA, thereby reducing KCa3.1 channel synthesis. • Lyso-Gb3 reduces PI3KC2β-mediated intracellular PI(3)P production. • Lyso-Gb3 reduces both surface and total expression of the KCa3.1 channel. • Increasing intracellular levels of PI(3)P only recovers the reduced surface expression.« less

Phosphatidylinositol-4-kinase type II alpha contains an AP-3-sorting motif and a kinase domain that are both required for endosome traffic.

PubMed

Craige, Branch; Salazar, Gloria; Faundez, Victor

2008-04-01

The adaptor complex 3 (AP-3) targets membrane proteins from endosomes to lysosomes, lysosome-related organelles and synaptic vesicles. Phosphatidylinositol-4-kinase type II alpha (PI4KIIalpha) is one of several proteins possessing catalytic domains that regulate AP-3-dependent sorting. Here we present evidence that PI4KIIalpha uniquely behaves both as a membrane protein cargo as well as an enzymatic regulator of adaptor function. In fact, AP-3 and PI4KIIalpha form a complex that requires a dileucine-sorting motif present in PI4KIIalpha. Mutagenesis of either the PI4KIIalpha-sorting motif or its kinase-active site indicates that both are necessary to interact with AP-3 and properly localize PI4KIIalpha to LAMP-1-positive endosomes. Similarly, both the kinase activity and the sorting signal present in PI4KIIalpha are necessary to rescue endosomal PI4KIIalpha siRNA-induced mutant phenotypes. We propose a mechanism whereby adaptors use canonical sorting motifs to selectively recruit a regulatory enzymatic activity to restricted membrane domains.

Integration of Golgi trafficking and growth factor signaling by the lipid phosphatase SAC1

PubMed Central

Blagoveshchenskaya, Anastasia; Cheong, Fei Ying; Rohde, Holger M.; Glover, Greta; Knödler, Andreas; Nicolson, Teresa; Boehmelt, Guido; Mayinger, Peter

2008-01-01

When a growing cell expands, lipids and proteins must be delivered to its periphery. Although this phenomenon has been observed for decades, it remains unknown how the secretory pathway responds to growth signaling. We demonstrate that control of Golgi phosphatidylinositol-4-phosphate (PI(4)P) is required for growth-dependent secretion. The phosphoinositide phosphatase SAC1 accumulates at the Golgi in quiescent cells and down-regulates anterograde trafficking by depleting Golgi PI(4)P. Golgi localization requires oligomerization of SAC1 and recruitment of the coat protein (COP) II complex. When quiescent cells are stimulated by mitogens, SAC1 rapidly shuttles back to the endoplasmic reticulum (ER), thus releasing the brake on Golgi secretion. The p38 mitogen-activated kinase (MAPK) pathway induces dissociation of SAC1 oligomers after mitogen stimulation, which triggers COP-I–mediated retrieval of SAC1 to the ER. Inhibition of p38 MAPK abolishes growth factor–induced Golgi-to-ER shuttling of SAC1 and slows secretion. These results suggest direct roles for p38 MAPK and SAC1 in transmitting growth signals to the secretory machinery. PMID:18299350

Upregulation of Phosphatidylinositol 3-Kinase (PI3K) Enhances Ethylene Biosynthesis and Accelerates Flower Senescence in Transgenic Nicotiana tabacum L.

PubMed

Dek, Mohd Sabri Pak; Padmanabhan, Priya; Sherif, Sherif; Subramanian, Jayasankar; Paliyath, And Gopinadhan

2017-07-15

Phosphatidylinositol 3-kinase (PI3K) is a key enzyme that phosphorylates phosphatidylinositol at 3'-hydroxyl position of the inositol head group initiating the generation of several phosphorylated phosphatidylinositols, collectively referred to as phosphoinositides. The function of PI3K in plant senescence and ethylene signal transduction process was studied by expression of Solanum lycopersicum PI3K in transgenic Nicotiana tabacum , and delineating its effect on flower senescence. Detached flowers of transgenic tobacco plants with overexpressed Sl - PI3K (OX) displayed accelerated senescence and reduced longevity, when compared to the flowers of wild type plants. Flowers from PI3K-overexpressing plants showed enhanced ethylene production and upregulated expression of 1-aminocyclopropane-1-carboxylic acid oxidase 1 ( ACO1 ). Real time polymerase chain reaction (PCR) analysis showed that PI3K was expressed at a higher level in OX flowers than in the control. Seedlings of OX-lines also demonstrated a triple response phenotype with characteristic exaggerated apical hook, shorter hypocotyls and increased sensitivity to 1-aminocyclopropane-1-carboxylate than the control wild type seedlings. In floral tissue from OX-lines, Solanum lycopersicum phosphatidylinositol 3-kinase green fluorescent protein (PI3K-GFP) chimera protein was localized primarily in stomata, potentially in cytoplasm and membrane adjacent to stomatal pores in the guard cells. Immunoblot analysis of PI3K expression in OX lines demonstrated increased protein level compared to the control. Results of the present study suggest that PI3K plays a crucial role in senescence by enhancing ethylene biosynthesis and signaling.

A genome-wide detection of copy number variation using SNP genotyping arrays in Beijing-You chickens.

PubMed

Zhou, Wei; Liu, Ranran; Zhang, Jingjing; Zheng, Maiqing; Li, Peng; Chang, Guobin; Wen, Jie; Zhao, Guiping

2014-10-01

Copy number variation (CNV) has been recently examined in many species and is recognized as being a source of genetic variability, especially for disease-related phenotypes. In this study, the PennCNV software, a genome-wide CNV detection system based on the 60 K SNP BeadChip was used on a total sample size of 1,310 Beijing-You chickens (a Chinese local breed). After quality control, 137 high confidence CNVRs covering 27.31 Mb of the chicken genome and corresponding to 2.61 % of the whole chicken genome. Within these regions, 131 known genes or coding sequences were involved. Q-PCR was applied to verify some of the genes related to disease development. Results showed that copy number of genes such as, phosphatidylinositol-5-phosphate 4-kinase II alpha, PHD finger protein 14, RHACD8 (a CD8α- like messenger RNA), MHC B-G, zinc finger protein, sarcosine dehydrogenase and ficolin 2 varied between individual chickens, which also supports the reliability of chip-detection of the CNVs. As one source of genomic variation, CNVs may provide new insight into the relationship between the genome and phenotypic characteristics.

Downregulation of RBO-PI4KIIIα Facilitates Aβ42 Secretion and Ameliorates Neural Deficits in Aβ42-Expressing Drosophila.

PubMed

Zhang, Xiao; Wang, Wen-An; Jiang, Li-Xiang; Liu, Hai-Yan; Zhang, Bao-Zhu; Lim, Nastasia; Li, Qing-Yi; Huang, Fu-De

2017-05-10

Phosphoinositides and their metabolizing enzymes are involved in Aβ 42 metabolism and Alzheimer's disease pathogenesis. In yeast and mammals, Eighty-five requiring 3 (EFR3), whose Drosophila homolog is Rolling Blackout (RBO), forms a plasma membrane-localized protein complex with phosphatidylinositol-4-kinase Type IIIα (PI4KIIIα) and a scaffold protein to tightly control the level of plasmalemmal phosphatidylinositol-4-phosphate (PI 4 P). Here, we report that RBO binds to Drosophila PI4KIIIα, and that in an Aβ 42 -expressing Drosophila model, separate genetic reduction of PI4KIIIα and RBO, or pharmacological inhibition of PI4KIIIα ameliorated synaptic transmission deficit, climbing ability decline, premature death, and reduced neuronal accumulation of Aβ 42 Moreover, we found that RBO-PI4KIIIa downregulation increased neuronal Aβ 42 release and that PI4P facilitated the assembly or oligomerization of Aβ 42 in/on liposomes. These results indicate that RBO-PI4KIIIa downregulation facilitates neuronal Aβ 42 release and consequently reduces neuronal Aβ 42 accumulation likely via decreasing Aβ 42 assembly in/on plasma membrane. This study suggests the RBO-PI4KIIIα complex as a potential therapeutic target and PI4KIIIα inhibitors as drug candidates for Alzheimer's disease treatment. SIGNIFICANCE STATEMENT Phosphoinositides and their metabolizing enzymes are involved in Aβ 42 metabolism and Alzheimer's disease pathogenesis. Here, in an Aβ 42 -expressing Drosophila model, we discovered and studied the beneficial role of downregulating RBO or its interacting protein PI4KIIIα-a protein that tightly controls the plasmalemmal level of PI 4 P-against the defects caused by Aβ 42 expression. Mechanistically, RBO-PI4KIIIα downregulation reduced neuronal Aβ 42 accumulation, and interestingly increased neuronal Aβ 42 release. This study suggests the RBO-PI4KIIIα complex as a novel therapeutic target, and PI4KIIIα inhibitors as new drug candidates. Copyright © 2017 the authors 0270-6474/17/374928-14$15.00/0.

Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

PubMed Central

Wold, Mitchell S.; Gong, Shiaoching; Phillips, Greg R.; Dou, Zhixun; Zhao, Yanxiang; Heintz, Nathaniel; Zong, Wei-Xing; Yue, Zhenyu

2014-01-01

Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis. PMID:25275521

Insulin-induced activation of glycerol-3-phosphate acyltransferase by a chiro-inositol-containing insulin mediator is defective in adipocytes of insulin-resistant, type II diabetic, Goto-Kakizaki rats.

PubMed

Farese, R V; Standaert, M L; Yamada, K; Huang, L C; Zhang, C; Cooper, D R; Wang, Z; Yang, Y; Suzuki, S; Toyota, T

1994-11-08

Type II diabetic Goto-Kakizaki (GK) rats were insulin-resistant in euglycemic-hyperinsulinemic clamp studies. We therefore examined insulin signaling systems in control Wistar and diabetic GK rats. Glycerol-3-phosphate acyltransferase (G3PAT), which is activated by headgroup mediators released from glycosyl-phosphatidylinositol (GPI), was activated by insulin in intact and cell-free adipocyte preparations of control, but not diabetic, rats. A specific chiro-inositol-containing inositol phosphoglycan (IPG) mediator, prepared from beef liver, bypassed this defect and comparably activated G3PAT in cell-free adipocyte preparations of both diabetic GK and control rats. A myo-inositol-containing IPG mediator did not activate G3PAT. Relative to control adipocytes, labeling of GPI by [3H]glucosamine was diminished by 50% and insulin failed to stimulate GPI hydrolysis in GK adipocytes. In contrast to GPI-dependent G3PAT activation, insulin-stimulated hexose transport was intact in adipocytes and soleus and gastrocnemius muscles of the GK rat, as was insulin-induced activation of mitogen-activated protein kinase and protein kinase C. We conclude that (i) chiro-inositol-containing IPG mediator activates G3PAT during insulin action, (ii) diabetic GK rats have a defect in synthesizing or releasing functional chiro-inositol-containing IPG, and (iii) defective IPG-regulated intracellular glucose metabolism contributes importantly to insulin resistance in diabetic GK rats.

Insulin-induced activation of glycerol-3-phosphate acyltransferase by a chiro-inositol-containing insulin mediator is defective in adipocytes of insulin-resistant, type II diabetic, Goto-Kakizaki rats.

PubMed Central

Farese, R V; Standaert, M L; Yamada, K; Huang, L C; Zhang, C; Cooper, D R; Wang, Z; Yang, Y; Suzuki, S; Toyota, T

1994-01-01

Type II diabetic Goto-Kakizaki (GK) rats were insulin-resistant in euglycemic-hyperinsulinemic clamp studies. We therefore examined insulin signaling systems in control Wistar and diabetic GK rats. Glycerol-3-phosphate acyltransferase (G3PAT), which is activated by headgroup mediators released from glycosyl-phosphatidylinositol (GPI), was activated by insulin in intact and cell-free adipocyte preparations of control, but not diabetic, rats. A specific chiro-inositol-containing inositol phosphoglycan (IPG) mediator, prepared from beef liver, bypassed this defect and comparably activated G3PAT in cell-free adipocyte preparations of both diabetic GK and control rats. A myo-inositol-containing IPG mediator did not activate G3PAT. Relative to control adipocytes, labeling of GPI by [3H]glucosamine was diminished by 50% and insulin failed to stimulate GPI hydrolysis in GK adipocytes. In contrast to GPI-dependent G3PAT activation, insulin-stimulated hexose transport was intact in adipocytes and soleus and gastrocnemius muscles of the GK rat, as was insulin-induced activation of mitogen-activated protein kinase and protein kinase C. We conclude that (i) chiro-inositol-containing IPG mediator activates G3PAT during insulin action, (ii) diabetic GK rats have a defect in synthesizing or releasing functional chiro-inositol-containing IPG, and (iii) defective IPG-regulated intracellular glucose metabolism contributes importantly to insulin resistance in diabetic GK rats. PMID:7972005

Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eyre, Nicholas S., E-mail: nicholas.eyre@adelaide.edu.au; Centre for Cancer Biology, SA Pathology, Adelaide; Hampton-Smith, Rachel J.

Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using ‘APEX2’-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using amore » customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα. - Highlights: • NS5A residues Ser-222, Ser-235 and Thr-348 are phosphorylated during HCV infection. • Phosphorylation of Ser-235 is essential to HCV RNA replication. • Mutation of Ser-235 alters replication compartment localization and morphology. • Phosphatidylinositol-4 kinase III alpha is important for Ser-235 phosphorylation.« less

The identification of raft-derived tau-associated vesicles that are incorporated into immature tangles and paired helical filaments.

PubMed

Nishikawa, T; Takahashi, T; Nakamori, M; Hosomi, N; Maruyama, H; Miyazaki, Y; Izumi, Y; Matsumoto, M

2016-12-01

Neurofibrillary tangles (NFTs), a cardinal pathological feature of neurodegenerative disorders, such as Alzheimer's disease (AD) are primarily composed of hyper-phosphorylated tau protein. Recently, several other molecules, including flotillin-1, phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] and cyclin-dependent kinase 5 (CDK5), have also been revealed as constituents of NFTs. Flotillin-1 and PtdIns(4,5)P2 are considered markers of raft microdomains, whereas CDK5 is a tau kinase. Therefore, we hypothesized that NFTs have a relationship with raft domains and the tau phosphorylation that occurs within NFTs. We investigated six cases of AD, six cases of other neurodegenerative diseases with NFTs and three control cases. We analysed the PtdIns(4,5)P2-immunopositive material in detail, using super-resolution microscopy and electron microscopy to elucidate its pattern of expression. We also investigated the spatial relationship between the PtdIns(4,5)P2-immunopositive material and tau kinases through double immunofluorescence analysis. Pretangles contained either paired helical filaments (PHFs) or PtdIns(4,5)P2-immunopositive small vesicles (approximately 1 μm in diameter) with nearly identical topology to granulovacuolar degeneration (GVD) bodies. Various combinations of these vesicles and GVD bodies, the latter of which are pathological hallmarks observed within the neurons of AD patients, were found concurrently in neurons. These vesicles and GVD bodies were both immunopositive not only for PtdIns(4,5)P2, but also for several tau kinases such as glycogen synthase kinase-3β and spleen tyrosine kinase. These observations suggest that clusters of raft-derived vesicles that resemble GVD bodies are substructures of pretangles other than PHFs. These tau kinase-bearing vesicles are likely involved in the modification of tau protein and in NFT formation. © 2015 The Authors Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British.

AKAP150 mediates TRPV1 sensitivity to phosphatidylinositol-4, 5-bisphosphate

PubMed Central

Jeske, Nathaniel A.; Por, Elaine D.; Belugin, Sergei; Chaudhury, Sraboni; Berg, Kelly A.; Akopian, Armen N.; Henry, Michael A.; Gomez, Ruben

2011-01-01

A-kinase anchoring protein 150 (AKAP150) is a scaffolding protein that controls protein kinase A- and C-mediated phosphorylation of the transient receptor potential family V type 1 (TRPV1), dictating receptor response to nociceptive stimuli. The phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) anchors AKAP150 to the plasma membrane in naïve conditions, and also affects TRPV1 activity. In the present study, we sought to determine whether the effects of PIP2 on TRPV1 are mediated through AKAP150. In trigeminal neurons and CHO cells, the manipulation of cellular PIP2 led to significant changes in the association of AKAP150 and TRPV1. Following PIP2 degradation, increased TRPV1:AKAP150 co-immunoprecipitation was observed, resulting in increased receptor response to capsaicin treatment. Phospholipase C activation in neurons isolated from AKAP150−/− animals indicated that PIP2 -mediated inhibition of TRPV1 in the whole cell environment requires expression of the scaffolding protein. Furthermore, the addition of PIP2 to neurons isolated from AKAP150 wild-type mice reduced PKA-sensitization of TRPV1 compared to isolated neurons from AKAP150−/− mice. These findings suggest that PIP2 degradation increases AKAP150 association with TRPV1 in the whole cell environment, leading to sensitization of the receptor to nociceptive stimuli. PMID:21653872

On the role of phosphatidylinositol 3-kinase, protein kinase b/Akt, and glycogen synthase kinase-3β in photodynamic injury of crayfish neurons and glial cells.

PubMed

Komandirov, Maxim A; Knyazeva, Evgeniya A; Fedorenko, Yulia P; Rudkovskii, Mikhail V; Stetsurin, Denis A; Uzdensky, Anatoly B

2011-10-01

Photodynamic treatment that causes intense oxidative stress and cell death is currently used in neurooncology. However, along with tumor cells, it may damage healthy neurons and glia. To study the involvement of signaling processes in photodynamic injury or protection of neurons and glia, we used crayfish mechanoreceptor consisting of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens. Application of specific inhibitors showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3β participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β pathway was not involved as a whole in photodynamic injury of crayfish neurons and glia but its components, Akt and glycogen synthase kinase-3β, independently and cell specifically regulated death of neurons and glial cells. According to these data, necrosis in this system was a controlled but not a non-regulated cell death mode. The obtained results may be used for the search of pharmacological agents selectively modulating death and survival of normal neurons and glial cells during photodynamic therapy of brain tumors.

Regulation of the plasma membrane type III phosphatidylinositol 4-kinase by positively charged compounds.

PubMed

Yang, W; Boss, W F

1994-08-15

The effects of positively charged compounds on a plasma membrane, type III phosphatidylinositol 4-kinase were studied. To determine whether the enzyme would respond differently to the compounds in a membrane-associated versus a soluble state, both the plasma membrane and solubilized (released by 0.01% (v/v) Triton X-100) PI 4-kinase were used. Spermidine, spermine, polylysine, cardiotoxin, melittin, and histone stimulated the solubilized PI 4-kinase but had little effect on or weakly stimulated the membrane-associated PI 4-kinase. Polyarginine inhibited membrane-associated PI 4-kinase 75% and inhibited the solubilized PI 4-kinase 30%, indicating that charge alone was not sufficient for activation. Polyarginine also eliminated the activation of the solubilized PI 4-kinase by a PI 4-kinase activator protein, PIK-A49. Calmodulin, a common calcium-binding protein, at micromolar levels strongly inhibited solubilized PI 4-kinase activity but did not inhibit membrane-associated PI 4-kinase activity. The inhibition of the solubilized PI 4-kinase by calmodulin was calcium independent. Calcium alone (1 microM-0.1 mM) inhibited PI 4-kinase activity only slightly (< 30%). The differential effects of the positively charged compounds on the solubilized and membrane-associated PI 4-kinase were not due to substrate availability because both enzymes were assayed in the presence of excess PI (0.6 mM) and 0.3% (v/v) Triton X-100. The data suggest that positively charged compounds affected the enzyme activity not only by interacting with the substrates or products of the reaction but also by interacting with the PI 4-kinase or regulatory components in the plasma membrane.

Nitroglycerin drives endothelial nitric oxide synthase activation via the phosphatidylinositol 3-kinase/protein kinase B pathway

PubMed Central

Mao, Mao; Sudhahar, Varadarajan; Ansenberger-Fricano, Kristine; Fernandes, Denise C.; Tanaka, Leonardo Y.; Fukai, Tohru; Laurindo, Francisco R.M.; Mason, Ronald P.; Vasquez-Vivar, Jeannette; Minshall, Richard D.; Stadler, Krisztian; Bonini, Marcelo G.

2012-01-01

Nitroglycerin (GTN) has been clinically used to treat angina pectoris and acute heart episodes for over 100 years. The effects of GTN have long been recognized and active research has contributed to the unraveling of numerous metabolic routes capable of converting GTN to the potent vasoactive messenger nitric oxide. Recently, the mechanism by which minute doses of GTN elicit robust pharmacological responses was revisited and eNOS activation was implicated as an important route mediating vasodilation induced by low GTN doses (1–50 nM). Here, we demonstrate that at such concentrations the pharmacologic effects of nitroglycerin are largely dependent on the phosphatidylinositol 3-kinase, Akt/PKB, and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signal transduction axis. Furthermore, we demonstrate that nitroglycerin-dependent accumulation of 3,4,5-InsP3, probably because of inhibition of PTEN, is important for eNOS activation, conferring a mechanistic basis for GTN pharmacological action at pharmacologically relevant doses. PMID:22037515

Low PIP4K2B expression in human breast tumors correlates with reduced patient survival: A role for PIP4K2B in the regulation of E-cadherin expression.

PubMed

Keune, Willem-Jan; Sims, Andrew H; Jones, David R; Bultsma, Yvette; Lynch, James T; Jirström, Karin; Landberg, Goran; Divecha, Nullin

2013-12-01

Phosphatidylinositol-5-phosphate (PtdIns5P) 4-kinase β (PIP4K2B) directly regulates the levels of two important phosphoinositide second messengers, PtdIns5P and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P2]. PIP4K2B has been linked to the regulation of gene transcription, to TP53 and AKT activation, and to the regulation of cellular reactive oxygen accumulation. However, its role in human tumor development and on patient survival is not known. Here, we have interrogated the expression of PIP4K2B in a cohort (489) of patients with breast tumor using immunohistochemical staining and by a meta-analysis of gene expression profiles from 2,999 breast tumors, both with associated clinical outcome data. Low PIP4K2B expression was associated with increased tumor size, high Nottingham histological grade, Ki67 expression, and distant metastasis, whereas high PIP4K2B expression strongly associated with ERBB2 expression. Kaplan-Meier curves showed that both high and low PIP4K2B expression correlated with poorer patient survival compared with intermediate expression. In normal (MCF10A) and tumor (MCF7) breast epithelial cell lines, mimicking low PIP4K2B expression, using short hairpin RNA interference-mediated knockdown, led to a decrease in the transcription and expression of the tumor suppressor protein E-cadherin (CDH1). In MCF10A cells, knockdown of PIP4K2B enhanced TGF-β-induced epithelial to mesenchymal transition (EMT), a process required during the development of metastasis. Analysis of gene expression datasets confirmed the association between low PIP4K2B and low CDH1expression. Decreased CDH1 expression and enhancement of TGF-β-induced EMT by reduced PIP4K2B expression might, in part, explain the association between low PIP4K2B expression and poor patient survival.

Complete inhibition of creatine kinase in isolated perfused rat hearts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fossel, E.T.; Hoefeler, H.

1987-01-01

Transient exposure of an isolated isovolumic perfused rat heart to low concentrations (0.5 mM) of perfusate-born iodoacetamide resulted in complete inhibition of creatine kinase and partial inhibition of glyceraldehyde-3-phosphate dehydrogenase in the heart. At low levels of developed pressure, hearts maintained mechanical function, ATP, and creatine phosphate levels at control values. However, iodoacetamide-inhibited hearts were unable to maintain control values of end diastolic pressure or peak systolic pressure as work load increased. Global ischemia resulted in loss of all ATP without loss of creatine phosphate, indicating lack of active creatine kinase. These results indicate that isovolumic perfused rat hearts aremore » able to maintain normal function and normal levels of high-energy phosphates without active creatine kinase at low levels of developed pressure. /sup 31/P-NMR of the heart was carried out.« less

Binding of Diphtheria Toxin to Phospholipids in Liposomes

NASA Astrophysics Data System (ADS)

Alving, Carl R.; Iglewski, Barbara H.; Urban, Katharine A.; Moss, Joel; Richards, Roberta L.; Sadoff, Jerald C.

1980-04-01

Diphtheria toxin bound to the phosphate portion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyl phosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol% (compared to dimyristoyl phosphatidylcholine) of dipalmitoyl phosphatidic acid, dicetyl phosphate, phosphatidylinositol phosphate, cardiolipin, or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristol phosphatidylcholine / cholesterol liposomes did not result in toxin binding. The calcium salt of dipalmitoyl phosphatidic acid was more effective than the sodium salt, and the highest level of binding occurred with liposomes consisting only of dipalmitoyl phosphatidic acid (calcium salt) and cholesterol. Binding of toxin to liposomes was dependent on pH, and the pattern of pH dependence varied with liposomes having different compositions. Incubation of diphtheria toxin with liposomes containing dicetyl phosphate resulted in maximal binding at pH 3.6, whereas binding to liposomes containing phosphatidylinositol phosphate was maximal above pH 7. Toxin did not bind to liposomes containing 20 mol% of a free fatty acid (palmitic acid) or a sulfated lipid (3-sulfogalactosylceramide). Toxin binding to dicetyl phosphate or phosphatidylinositol phosphate was inhibited by UTP, ATP, phosphocholine, or p-nitrophenyl phosphate, but not by uracil. We conclude that (a) diphtheria toxin binds specifically to the phosphate portion of certain phospholipids, (b) binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction, and (c) binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. We propose that a minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid), or that some other phosphorylated membrane molecule (such as a phosphoprotein) may be important in the initial binding of diphtheria toxin to cells.

Contrasting effects of phosphatidylinositol 4,5‐bisphosphate on cloned TMEM16A and TMEM16B channels

PubMed Central

Ta, Chau M; Acheson, Kathryn E; Rorsman, Nils J G; Jongkind, Remco C

2017-01-01

Background and Purpose Ca2+‐activated Cl− channels (CaCCs) are gated open by a rise in intracellular Ca2+ concentration ([Ca2+]i), typically provoked by activation of Gq‐protein coupled receptors (GqPCR). GqPCR activation initiates depletion of plasmalemmal phosphatidylinositol 4,5‐bisphosphate (PIP2). Here, we determined whether PIP2 acts as a signalling lipid for CaCCs coded by the TMEM16A and TMEM16B genes. Experimental Approach Patch‐clamp electrophysiology, in conjunction with genetically encoded systems to control cellular PIP2 content, was used to define the mechanism of action of PIP2 on TMEM16A and TMEM16B channels. Key Results A water‐soluble PIP2 analogue (diC8‐PIP2) activated TMEM16A channels by up to fivefold and inhibited TMEM16B by ~0.2‐fold. The effects of diC8‐PIP2 on TMEM16A currents were especially pronounced at low [Ca2+]i. In contrast, diC8‐PIP2 modulation of TMEM16B channels did not vary over a broad [Ca2+]i range but was only detectable at highly depolarized membrane potentials. Modulation of TMEM16A and TMEM16B currents was due to changes in channel gating, while single channel conductance was unaltered. Co‐expression of TMEM16A or TMEM16B with a Danio rerio voltage‐sensitive phosphatase (DrVSP), which degrades PIP2, led to reduction and enhancement of TMEM16A and TMEM16B currents respectively. These effects were abolished by an inactivating mutation in DrVSP and antagonized by simultaneous co‐expression of a phosphatidylinositol‐4‐phosphate 5‐kinase that catalyses PIP2 formation. Conclusions and Implications PIP2 acts as a modifier of TMEM16A and TMEM16B channel gating. Drugs interacting with PIP2 signalling may affect TMEM16A and TMEM16B channel gating and have potential uses in basic science and implications for therapy. PMID:28616863

Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate.

PubMed

Franke, T F; Kaplan, D R; Cantley, L C; Toker, A

1997-01-31

The regulation of the serine-threonine kinase Akt by lipid products of phosphoinositide 3-kinase (PI 3-kinase) was investigated. Akt activity was found to correlate with the amount of phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) in vivo, and synthetic PtdIns-3,4-P2 activated Akt both in vitro and in vivo. Binding of PtdIns-3,4-P2 occurred within the Akt pleckstrin homology (PH) domain and facilitated dimerization of Akt. Akt mutated in the PH domain was not activated by PI 3-kinase in vivo or by PtdIns-3, 4-P2 in vitro, and it was impaired in binding to PtdIns-3,4-P2. Examination of the binding to other phosphoinositides revealed that they bound to the Akt PH domain with much lower affinity than did PtdIns-3,4-P2 and failed to increase Akt activity. Thus, Akt is apparently regulated by the direct interaction of PtdIns-3,4-P2 with the Akt PH domain.

Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools*

PubMed Central

Goto, Asako; Charman, Mark; Ridgway, Neale D.

2016-01-01

Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50–70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT. PMID:26601944

Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools.

PubMed

Goto, Asako; Charman, Mark; Ridgway, Neale D

2016-01-15

Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50-70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4-kinase.

PubMed

Paquet, Tanya; Le Manach, Claire; Cabrera, Diego González; Younis, Yassir; Henrich, Philipp P; Abraham, Tara S; Lee, Marcus C S; Basak, Rajshekhar; Ghidelli-Disse, Sonja; Lafuente-Monasterio, María José; Bantscheff, Marcus; Ruecker, Andrea; Blagborough, Andrew M; Zakutansky, Sara E; Zeeman, Anne-Marie; White, Karen L; Shackleford, David M; Mannila, Janne; Morizzi, Julia; Scheurer, Christian; Angulo-Barturen, Iñigo; Martínez, María Santos; Ferrer, Santiago; Sanz, Laura María; Gamo, Francisco Javier; Reader, Janette; Botha, Mariette; Dechering, Koen J; Sauerwein, Robert W; Tungtaeng, Anchalee; Vanachayangkul, Pattaraporn; Lim, Chek Shik; Burrows, Jeremy; Witty, Michael J; Marsh, Kennan C; Bodenreider, Christophe; Rochford, Rosemary; Solapure, Suresh M; Jiménez-Díaz, María Belén; Wittlin, Sergio; Charman, Susan A; Donini, Cristina; Campo, Brice; Birkholtz, Lyn-Marie; Hanson, Kirsten K; Drewes, Gerard; Kocken, Clemens H M; Delves, Michael J; Leroy, Didier; Fidock, David A; Waterson, David; Street, Leslie J; Chibale, Kelly

2017-04-26

As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment. Copyright © 2017, American Association for the Advancement of Science.

Inhibitory Effect of Vascular Endothelial Growth Factor on the Slowly Activating Delayed Rectifier Potassium Current in Guinea Pig Ventricular Myocytes.

PubMed

Lin, Zhenhao; Xing, Wenlu; Gao, Chuanyu; Wang, Xianpei; Qi, Datun; Dai, Guoyou; Zhao, Wen; Yan, Ganxin

2018-01-26

Vascular endothelial growth factor (VEGF) exerts a number of beneficial effects on ischemic myocardium via its angiogenic properties. However, little is known about whether VEGF has a direct effect on the electrical properties of cardiomyocytes. In the present study, we investigated the effects of different concentrations of VEGF on delayed rectifier potassium currents (I K ) in guinea pig ventricular myocytes and their effects on action potential (AP) parameters. I K and AP were recorded by the whole-cell patch clamp method in ventricular myocytes. Cells were superfused with control solution or solution containing VEGF at different concentrations for 10 minutes before recording. Some ventricular myocytes were pretreated with a phosphatidylinositol 3-kinase inhibitor for 1 hour before the addition of VEGF. We found that VEGF inhibited the slowly activating delayed rectifier potassium current (I K s ) in a concentration-dependent manner (18.13±1.04 versus 12.73±0.34, n=5, P =0.001; 12.73±0.34 versus 9.05±1.20, n=5, P =0.036) and prolonged AP duration (894.5±36.92 versus 746.3±33.71, n=5, P =0.021). Wortmannin, a phosphatidylinositol 3-kinase inhibitor, eliminated these VEGF-induced effects. VEGF had no significant effect on the rapidly activating delayed rectifier potassium current (I K r ), resting membrane potential, AP amplitude, or maximal velocity of depolarization. VEGF inhibited I K s in a concentration-dependent manner through a phosphatidylinositol 3-kinase-mediated signaling pathway, leading to AP prolongation. The results indicate a promising therapeutic potential of VEGF in prevention of ventricular tachyarrhythmias under conditions of high sympathetic activity and ischemia. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria.

PubMed

Mbengue, Alassane; Bhattacharjee, Souvik; Pandharkar, Trupti; Liu, Haining; Estiu, Guillermina; Stahelin, Robert V; Rizk, Shahir S; Njimoh, Dieudonne L; Ryan, Yana; Chotivanich, Kesinee; Nguon, Chea; Ghorbal, Mehdi; Lopez-Rubio, Jose-Juan; Pfrender, Michael; Emrich, Scott; Mohandas, Narla; Dondorp, Arjen M; Wiest, Olaf; Haldar, Kasturi

2015-04-30

Artemisinins are the cornerstone of anti-malarial drugs. Emergence and spread of resistance to them raises risk of wiping out recent gains achieved in reducing worldwide malaria burden and threatens future malaria control and elimination on a global level. Genome-wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by the PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase, as well as its lipid product phosphatidylinositol-3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signalling in which transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination.

Phosphorylation regulates the sensitivity of voltage-gated Kv7.2 channels towards phosphatidylinositol-4,5-bisphosphate.

PubMed

Salzer, Isabella; Erdem, Fatma Asli; Chen, Wei-Qiang; Heo, Seok; Koenig, Xaver; Schicker, Klaus W; Kubista, Helmut; Lubec, Gert; Boehm, Stefan; Yang, Jae-Won

2017-02-01

Phosphatidylinositol-4,5-bisphosphate (PIP 2 ) is a key regulator of many membrane proteins, including voltage-gated Kv7.2 channels. In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP 2 -binding domains in Kv7.2. Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP 2 . Dephosphorylation of Kv7.2 affected channel inhibition via M 1 muscarinic receptors, but not via bradykinin receptors. Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP 2 , thereby ensuring the tight regulation of the channel via G protein-coupled receptors. The function of numerous ion channels is tightly controlled by G protein-coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol-4,5-bisphosphate (PIP 2 ). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP 2 and through phosphorylation. Using liquid chromatography-coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2-binding domains. To evaluate the effect of phosphorylation on PIP 2 -mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A 5 mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP 2 depletion via the voltage-sensitive phosphatase Dr-VSP than were wild-type channels. In vitro phosphorylation assays with the purified C-terminus of Kv7.2 revealed that CDK5, p38 MAPK, CaMKIIα and PKA were able to phosphorylate the five serines. Inhibition of these protein kinases reduced the sensitivity of wild-type but not mutant Kv7.2 channels towards PIP 2 depletion via Dr-VSP. In superior cervical ganglion neurons, the protein kinase inhibitors attenuated Kv7 current regulation via M 1 receptors, but left unaltered the control by B2 receptors. Our results revealed that the phosphorylation status of serines located within a putative PIP 2 -binding domain determined the phospholipid sensitivity of Kv7.2 channels and supported GPCR-mediated channel regulation. © 2016 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Protein kinase C (PKC) phosphorylates human platelet inositol trisphosphate 5/sup +/-/-phosphomonoesterase (IP/sub 3/ 5'-p'tase) increasing phosphatase activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Connolly, T.M.; Majerus, P.W.

1986-05-01

Phosphoinositide breakdown in response to thrombin stimulation of human platelets generates messenger molecules that activate PKC (diglyceride) and mobilize Ca/sup + +/ (inositol tris-phosphates). The water soluble products of phospholipase C-mediated metabolism of phosphatidylinositol 4,5-diphosphate are inositol 1,4,5 P/sub 3/ (IP/sub 3/) and inositol 1:2-cyclic 4,5 P/sub 3/ (cIP/sub 3/). A specific phosphatase, IP/sub 3/ 5'-p'tase, cleaves the 5 phosphate from IP/sub 3/ or cIP/sub 3/ to form IP/sub 2/ or cIP/sub 2/ and P/sub i/, none of which mobilizes Ca/sup + +/. Thus, the IP/sub 3/ 5'-p'tase may regulate cellular responses to IP/sub 3/ or cIP/sub 3/. The authorsmore » find that IP/sub 3/ 5'-p'tase isolated from human platelets is phosphorylated by rat brain PKC, resulting in a 4-fold increase in IP/sub 3/ 5'-p'tase activity. The authors phosphorylated IP/sub 3/ 5'-p'tase using ..gamma.. /sup 32/P-ATP and found that the labeled enzyme comigrated on SDS-PAGE with the previously described 40K protein phosphorylated in response to thrombin stimulation of platelets. The similarity of the PKC-phosphorylated IP/sub 3/ 5'-p'tase observed in vitro and the thrombin-stimulated phosphorylated 40K protein known to be phosphorylated by PKC in vivo, suggests that these proteins may be the same. These results suggest that platelet Ca/sup + +/ mobilization maybe regulated by PKC phosphorylation of the IP/sub 3/ 5'-p'tase and can explain the observation that phorbol ester treatment of intact human platelets results in decreased production of IP/sub 3/ and decreased Ca/sup + +/ mobilization upon subsequent thrombin addition.« less

Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A2 and activation by a 70 kDa protein.

PubMed

Gross, W; Yang, W; Boss, W F

1992-02-19

Plasma membranes were isolated from carrot (Daucus carota L.) cells grown in suspension culture and treated with phospholipase A2 from snake or bee venom for 10 min. As a result of this treatment, phosphatidylinositol kinase activity was recovered in the soluble fraction. There was no detectable diacylglycerol kinase or phosphatidylinositol monophosphate kinase activity released from the membranes after the phospholipase A2 treatment. Treating the plasma membranes with phospholipase C or D did not release PI kinase activity. The phospholipase A2-released PI kinase was activated over 2-fold by a heat stable, soluble 70 kDa protein. The partially purified 70 kDa activator increases the Vmax but does not affect the Km of the phospholipase A2-released PI kinase.

Cysteinyl leukotrienes promote human airway smooth muscle migration.

PubMed

Parameswaran, Krishnan; Cox, Gerard; Radford, Katherine; Janssen, Luke J; Sehmi, Roma; O'Byrne, Paul M

2002-09-01

Cysteinyl leukotrienes promote airway smooth muscle (ASM) contraction and proliferation. Little is known about their role in ASM migration. We investigated this using cultured human ASMs (between the second and fifth passages) obtained from the large airways of resected nonasthmatic lung. Platelet-derived growth factor-BB (1 ng/ml) promoted significant (3.5-fold) ASM migration of myocytes across collagen-coated 8- micro m polycarbonate membranes in Transwell culture plates. Leukotriene E(4) (10(-7), 10(-8), 10(-9) M) did not demonstrate a chemotactic effect; it did promote chemokinesis. Priming by leukotriene E(4) (10(-7) M) significantly augmented the directional migratory response to platelet-derived growth factor (1.5-fold, p < 0.05). This was blocked by montelukast (10(-6) M), demonstrating the effect to be mediated by the cysteinyl leukotriene receptor. The "priming effect" was also partially attenuated by prostaglandin E(2) (10(-7) M). Whereas both the chemokinetic and the chemotactic "primed" responses were equally attenuated by a p38 mitogen-activated protein kinase inhibitor (SB203580, 25 micro M) and by a Rho-kinase inhibitor (Y27632, 10 micro M), the chemotactic response showed greater inhibition than chemokinesis by a phosphatidylinositol-3 kinase inhibitor (LY294002, 50 micro M). These experiments suggest that cysteinyl leukotrienes play an augmentary role in human ASM migration. The phosphatidylinositol-3 kinase pathway is a key signaling mechanism in the chemotactic migration of ASM cells in response to cysteinyl leukotrienes.

Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

PubMed

Varley, C L; Royds, J A; Brown, B L; Dobson, P R

2001-01-01

We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway. Copyright 2001 S. Karger AG, Basel

Acetylcholine-dependent upregulation of TASK-1 channels in thalamic interneurons by a smooth muscle-like signalling pathway.

PubMed

Leist, Michael; Rinné, Susanne; Datunashvili, Maia; Aissaoui, Ania; Pape, Hans-Christian; Decher, Niels; Meuth, Sven G; Budde, Thomas

2017-09-01

The ascending brainstem transmitter acetylcholine depolarizes thalamocortical relay neurons while it induces hyperpolarization in local circuit inhibitory interneurons. Sustained K + currents are modulated in thalamic neurons to control their activity modes; for the interneurons the molecular nature of the underlying ion channels is as yet unknown. Activation of TASK-1 K + channels results in hyperpolarization of interneurons and suppression of their action potential firing. The modulation cascade involves a non-receptor tyrosine kinase, c-Src. The present study identifies a novel pathway for the activation of TASK-1 channels in CNS neurons that resembles cholinergic signalling and TASK-1 current modulation during hypoxia in smooth muscle cells. The dorsal part of the lateral geniculate nucleus (dLGN) is the main thalamic site for state-dependent transmission of visual information. Non-retinal inputs from the ascending arousal system and inhibition provided by γ-aminobutyric acid (GABA)ergic local circuit interneurons (INs) control neuronal activity within the dLGN. In particular, acetylcholine (ACh) depolarizes thalamocortical relay neurons by inhibiting two-pore domain potassium (K 2P ) channels. Conversely, ACh also hyperpolarizes INs via an as-yet-unknown mechanism. By using whole cell patch-clamp recordings in brain slices and appropriate pharmacological tools we here report that stimulation of type 2 muscarinic ACh receptors induces IN hyperpolarization by recruiting the G-protein βγ subunit (Gβγ), class-1A phosphatidylinositol-4,5-bisphosphate 3-kinase, and cellular and sarcoma (c-Src) tyrosine kinase, leading to activation of two-pore domain weakly inwardly rectifying K + channel (TWIK)-related acid-sensitive K + (TASK)-1 channels. The latter was confirmed by the use of TASK-1-deficient mice. Furthermore inhibition of phospholipase Cβ as well as an increase in the intracellular level of phosphatidylinositol-3,4,5-trisphosphate facilitated the muscarinic effect. Our results have uncovered a previously unknown role of c-Src tyrosine kinase in regulating IN function in the brain and identified a novel mechanism by which TASK-1 channels are activated in neurons. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Drosophila Mtm and class II PI3K coregulate a PI(3)P pool with cortical and endolysosomal functions.

PubMed

Velichkova, Michaella; Juan, Joe; Kadandale, Pavan; Jean, Steve; Ribeiro, Inês; Raman, Vignesh; Stefan, Chris; Kiger, Amy A

2010-08-09

Reversible phosphoinositide phosphorylation provides a dynamic membrane code that balances opposing cell functions. However, in vivo regulatory relationships between specific kinases, phosphatases, and phosphoinositide subpools are not clear. We identified myotubularin (mtm), a Drosophila melanogaster MTM1/MTMR2 phosphoinositide phosphatase, as necessary and sufficient for immune cell protrusion formation and recruitment to wounds. Mtm-mediated turnover of endosomal phosphatidylinositol 3-phosphate (PI(3)P) pools generated by both class II and III phosphatidylinositol 3-kinases (Pi3K68D and Vps34, respectively) is needed to down-regulate membrane influx, promote efflux, and maintain endolysosomal homeostasis. Endocytosis, but not endolysosomal size, contributes to cortical remodeling by mtm function. We propose that Mtm-dependent regulation of an endosomal PI(3)P pool has separable consequences for endolysosomal homeostasis and cortical remodeling. Pi3K68D depletion (but not Vps34) rescues protrusion and distribution defects in mtm-deficient immune cells and restores functions in other tissues essential for viability. The broad interactions between mtm and class II Pi3K68D suggest a novel strategy for rebalancing PI(3)P-mediated cell functions in MTM-related human disease.

Nrbf2 protein suppresses autophagy by modulating Atg14L protein-containing Beclin 1-Vps34 complex architecture and reducing intracellular phosphatidylinositol-3 phosphate levels.

PubMed

Zhong, Yu; Morris, Deanna H; Jin, Lin; Patel, Mittul S; Karunakaran, Senthil K; Fu, You-Jun; Matuszak, Emily A; Weiss, Heidi L; Chait, Brian T; Wang, Qing Jun

2014-09-19

Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1(-/-);Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Interference of P-REX2a may inhibit proliferation and reverse the resistance of SGC7901 cells to doxorubicin.

PubMed

Ai, Yaowei; Zhou, Qiaohui; Li, Ling; Pan, Zhihong; Guo, Mingwen; Han, Jingbo

2018-03-01

Drug resistance inhibits the efficacy of doxorubicin in gastric cancer. Phosphatidylinositol 3,4,5-trisphosphate RAC exchanger 2a (P-REX2a) activates the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway by binding to and inactivating phosphatase and tensin homolog (PTEN), which functions as a tumor promoter in a number of types of cancer. However, there is no research concerning the association between P-REX2a expression and drug resistance in gastric cancer. In the present study, the expression of P-REX2a in clinical gastric cancer tissues was detected, and the mechanism of doxorubicin resistance in the gastric cancer cell line SGC7901 was investigated. Using reverse transcription-quantitative polymerase chain reaction and western blotting, it was demonstrated that the mRNA and protein expression of P-REX2a was increased in gastric cancer tissues. MTT assays were also used to determine proliferation, and proliferation was revealed to be reduced following transfection of P-REX2a small interfering (si)RNA. When the cells were treated with 0.3 µM doxorubicin for 24 h, the rate of apoptosis in the siRNA-transfected groups significantly increased and no marked changes in of PTEN and Akt expression were observed. By contrast, the activity of PTEN increased, and the expression of p-Akt (S473) decreased in the P-REX2a siRNA-transfected group compared with the control. The detection of PTEN enzymatic activity in the present study was based on phosphatidylinositol-3,4,5-trisphosphate. Therefore, it was concluded that P-REX2a may participate in the generation of resistance to doxorubicin in gastric cancer, and this may be associated with the upregulation of the PI3K/Akt signaling pathway via inactivation of PTEN.

A molecular signaling model of platelet phosphoinositide and calcium regulation during homeostasis and P2Y1 activation.

PubMed

Purvis, Jeremy E; Chatterjee, Manash S; Brass, Lawrence F; Diamond, Scott L

2008-11-15

To quantify how various molecular mechanisms are integrated to maintain platelet homeostasis and allow responsiveness to adenosine diphosphate (ADP), we developed a computational model of the human platelet. Existing kinetic information for 77 reactions, 132 fixed kinetic rate constants, and 70 species was combined with electrochemical calculations, measurements of platelet ultrastructure, novel experimental results, and published single-cell data. The model accurately predicted: (1) steady-state resting concentrations for intracellular calcium, inositol 1,4,5-trisphosphate, diacylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylinositol phosphate, and phosphatidylinositol 4,5-bisphosphate; (2) transient increases in intracellular calcium, inositol 1,4,5-trisphosphate, and G(q)-GTP in response to ADP; and (3) the volume of the platelet dense tubular system. A more stringent test of the model involved stochastic simulation of individual platelets, which display an asynchronous calcium spiking behavior in response to ADP. Simulations accurately reproduced the broad frequency distribution of measured spiking events and demonstrated that asynchronous spiking was a consequence of stochastic fluctuations resulting from the small volume of the platelet. The model also provided insights into possible mechanisms of negative-feedback signaling, the relative potency of platelet agonists, and cell-to-cell variation across platelet populations. This integrative approach to platelet biology offers a novel and complementary strategy to traditional reductionist methods.

Protein kinase activity of the glycolytic enzyme PGK1 regulates autophagy to promote tumorigenesis.

PubMed

Qian, Xu; Li, Xinjian; Lu, Zhimin

2017-07-03

Macroautophagy/autophagy is a cellular defense response to stress conditions and is crucial for cell homeostasis maintenance. However, the precise mechanism underlying autophagy initiation, especially in response to glutamine deprivation and hypoxia, is yet to be explored. We recently discovered that PGK1 (phosphoglycerate kinase 1), a glycolytic enzyme, functions as a protein kinase, phosphorylating BECN1/Beclin 1 to initiate autophagy. Under glutamine deprivation or hypoxia stimulation, PGK1 is acetylated at K388 by NAA10/ARD1 in an MTOR-inhibition-dependent manner, leading to the interaction between PGK1 and BECN1 and the subsequent phosphorylation of BECN1 at S30 by PGK1. This phosphorylation enhances ATG14-associated PIK3C3/VPS34-BECN1-PIK3R4/VPS15 complex activity, thereby increasing phosphatidylinositol-3-phosphate (PtdIns3P) generation in the initiation stage of autophagy. Furthermore, NAA10-dependent PGK1 acetylation and PGK1-dependent BECN1 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumor formation. Our work reveals the important dual roles of PGK1 as a glycolytic enzyme and a protein kinase in the mutual regulation of cell metabolism and autophagy in maintaining cell homeostasis.

The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake.

PubMed

Isakoff, S J; Taha, C; Rose, E; Marcusohn, J; Klip, A; Skolnik, E Y

1995-10-24

Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells.

Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP•Mg2+ and 5'-OH oligonucleotide and a product complex with GDP•Mg2+ and 5'-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoacceptor recognition.

PubMed

Das, Ushati; Wang, Li Kai; Smith, Paul; Jacewicz, Agata; Shuman, Stewart

2014-01-01

Clostridium thermocellum polynucleotide kinase (CthPnk), the 5' end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from an NTP donor to a 5'-OH polynucleotide acceptor. Here we report the crystal structures of CthPnk-D38N in a Michaelis complex with GTP•Mg(2+) and a 5'-OH oligonucleotide and a product complex with GDP•Mg(2+) and a 5'-PO4 oligonucleotide. The O5' nucleophile is situated 3.0 Å from the GTP γ phosphorus in the Michaelis complex, where it is coordinated by Asn38 and is apical to the bridging β phosphate oxygen of the GDP leaving group. In the product complex, the transferred phosphate has undergone stereochemical inversion and Asn38 coordinates the 5'-bridging phosphate oxygen of the oligonucleotide. The D38N enzyme is poised for catalysis, but cannot execute because it lacks Asp38-hereby implicated as the essential general base catalyst that abstracts a proton from the 5'-OH during the kinase reaction. Asp38 serves as a general acid catalyst during the 'reverse kinase' reaction by donating a proton to the O5' leaving group of the 5'-PO4 strand. The acceptor strand binding mode of CthPnk is distinct from that of bacteriophage T4 Pnk.

Binding of peptides with basic and aromatic residues to bilayer membranes: phenylalanine in the myristoylated alanine-rich C kinase substrate effector domain penetrates into the hydrophobic core of the bilayer.

PubMed

Zhang, Wenyi; Crocker, Evan; McLaughlin, Stuart; Smith, Steven O

2003-06-13

Electrostatic interactions with positively charged regions of membrane-associated proteins such as myristoylated alanine-rich C kinase substrate (MARCKS) may have a role in regulating the level of free phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in plasma membranes. Both the MARCKS protein and a peptide corresponding to the effector domain (an unstructured region that contains 13 basic residues and 5 phenylalanines), MARCKS-(151-175), laterally sequester the polyvalent lipid PI(4,5)P2 in the plane of a bilayer membrane with high affinity. We used high resolution magic angle spinning NMR to establish the location of MARCKS-(151-175) in membrane bilayers, which is necessary to understand the sequestration mechanism. Measurements of cross-relaxation rates in two-dimensional nuclear Overhauser enhancement spectroscopy NMR experiments show that the five Phe rings of MARCKS-(151-175) penetrate into the acyl chain region of phosphatidylcholine bilayers containing phosphatidylglycerol or PI(4,5)P2. Specifically, we observed strong cross-peaks between the aromatic protons of the Phe rings and the acyl chain protons of the lipids, even for very short (50 ms) mixing times. The position of the Phe rings implies that the adjacent positively charged amino acids in the peptide are close to the level of the negatively charged lipid phosphates. The deep location of the MARCKS peptide in the polar head group region should enhance its electrostatic sequestration of PI(4,5)P2 by an "image charge" mechanism. Moreover, this location has interesting implications for membrane curvature and local surface pressure effects and may be relevant to a wide variety of other proteins with basic-aromatic clusters, such as phospholipase D, GAP43, SCAMP2, and the N-methyl-d-aspartate receptor.

Role of the Phosphoinositide 3-Kinase-Akt-Mammalian Target of the Rapamycin Signaling Pathway in Long-Term Potentiation and Trace Fear Conditioning Memory in Rat Medial Prefrontal Cortex

ERIC Educational Resources Information Center

Sui, Li; Wang, Jing; Li, Bao-Ming

2008-01-01

Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt (also known as protein kinase B, PKB), mammalian target of rapamycin (mTOR), the 70-kDa ribosomal S6 kinase (p70S6k), and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), may play important roles in long-term synaptic plasticity and memory in many…

PDK1-mediated activation of MRCKα regulates directional cell migration and lamellipodia retraction

PubMed Central

Gagliardi, Paolo Armando; di Blasio, Laura; Puliafito, Alberto; Seano, Giorgio; Sessa, Roberto; Chianale, Federica; Leung, Thomas; Bussolino, Federico

2014-01-01

Directional cell migration is of paramount importance in both physiological and pathological processes, such as development, wound healing, immune response, and cancer invasion. Here, we report that 3-phosphoinositide-dependent kinase 1 (PDK1) regulates epithelial directional migration and invasion by binding and activating myotonic dystrophy kinase–related CDC42-binding kinase α (MRCKα). We show that the effect of PDK1 on cell migration does not involve its kinase activity but instead relies on its ability to bind membrane phosphatidylinositol (3,4,5)-trisphosphate. Upon epidermal growth factor (EGF) stimulation, PDK1 and MRCKα colocalize at the cell membrane in lamellipodia. We demonstrate that PDK1 positively modulates MRCKα activity and drives its localization within lamellipodia. Likewise, the retraction phase of lamellipodia is controlled by PDK1 through an MRCKα-dependent mechanism. In summary, we discovered a functional pathway involving PDK1-mediated activation of MRCKα, which links EGF signaling to myosin contraction and directional migration. PMID:25092657

Direct and indirect effects of RNA interference against pyridoxal kinase and pyridoxine 5'-phosphate oxidase genes in Bombyx mori.

PubMed

Huang, ShuoHao; Yao, LiLi; Zhang, JianYun; Huang, LongQuan

2016-08-01

Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize pyridoxal 5'-phosphate by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. Currently, little is known on how pyridoxal 5'-phosphate biosynthesis is regulated, and pyridoxal 5'-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of pyridoxal 5'-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase has a greater impact on the gene transcription of enzymes using pyridoxal 5'-phosphate as a cofactor in Bombyx mori. Our study suggests that pyridoxal 5'-phosphate biosynthesis and dynamic balance may be regulated by genetic networks. Copyright © 2016 Elsevier B.V. All rights reserved.

The dual PH domain protein Opy1 functions as a sensor and modulator of PtdIns(4,5)P₂ synthesis.

PubMed

Ling, Yading; Stefan, Christopher J; Macgurn, Jason A; Audhya, Anjon; Emr, Scott D

2012-06-29

Phosphatidylinositol-4,5-bisphosphate, PtdIns(4,5)P(2), is an essential signalling lipid that regulates key processes such as endocytosis, exocytosis, actin cytoskeletal organization and calcium signalling. Maintaining proper levels of PtdIns(4,5)P(2) at the plasma membrane (PM) is crucial for cell survival and growth. We show that the conserved PtdIns(4)P 5-kinase, Mss4, forms dynamic, oligomeric structures at the PM that we term PIK patches. The dynamic assembly and disassembly of Mss4 PIK patches may provide a mechanism to precisely modulate Mss4 kinase activity, as needed, for localized regulation of PtdIns(4,5)P(2) synthesis. Furthermore, we identify a tandem PH domain-containing protein, Opy1, as a novel Mss4-interacting protein that partially colocalizes with PIK patches. Based upon genetic, cell biological, and biochemical data, we propose that Opy1 functions as a coincidence detector of the Mss4 PtdIns(4)P 5-kinase and PtdIns(4,5)P(2) and serves as a negative regulator of PtdIns(4,5)P(2) synthesis at the PM. Our results also suggest that additional conserved tandem PH domain-containing proteins may play important roles in regulating phosphoinositide signalling.

Targeting the phosphatidylinositol 3-kinase/Akt/mechanistic target of rapamycin signaling pathway in B-lineage acute lymphoblastic leukemia: An update.

PubMed

Simioni, Carolina; Martelli, Alberto M; Zauli, Giorgio; Vitale, Marco; McCubrey, James A; Capitani, Silvano; Neri, Luca M

2018-04-18

Despite considerable progress in treatment protocols, B-lineage acute lymphoblastic leukemia (B-ALL) displays a poor prognosis in about 15-20% of pediatric cases and about 60% of adult patients. In addition, life-long irreversible late effects from chemo- and radiation therapy, including secondary malignancies, are a growing problem for leukemia survivors. Targeted therapy holds promising perspectives for cancer treatment as it may be more effective and have fewer side effects than conventional therapies. The phosphatidylinositol 3-phosphate kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway is a key regulatory cascade which controls proliferation, survival and drug-resistance of cancer cells, and it is frequently upregulated in the different subtypes of B-ALL, where it plays important roles in the pathophysiology, maintenance and progression of the disease. Moreover, activation of this signaling cascade portends a poorer prognosis in both pediatric and adult B-ALL patients. Promising preclinical data on PI3K/Akt/mTOR inhibitors have documented their anticancer activity in B-ALL and some of these novel drugs have entered clinical trials as they could lead to a longer event-free survival and reduce therapy-associated toxicity for patients with B-ALL. This review highlights the current status of PI3K/Akt/mTOR inhibitors in B-ALL, with an emphasis on emerging evidence of the superior efficacy of synergistic combinations involving the use of traditional chemotherapeutics or other novel, targeted agents. © 2018 Wiley Periodicals, Inc.

Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636

PubMed Central

Sbrissa, Diego; Ikonomov, Ognian C.; Filios, Catherine; Delvecchio, Khortnal

2012-01-01

PIKfyve is an essential mammalian lipid kinase with pleiotropic cellular functions whose genetic knockout in mice leads to preimplantation lethality. Despite several reports for PIKfyve-catalyzed synthesis of phosphatidylinositol 5-phosphate (PtdIns5P) along with phosphatidylinositol-3,5-biphosphate [PtdIns(3,5)P2] in vitro and in vivo, the role of the PIKfyve pathway in intracellular PtdIns5P production remains underappreciated and the function of the PIKfyve-synthesized PtdIns5P pool poorly characterized. Hence, the recently discovered potent PIKfyve-selective inhibitor, the YM201636 compound, has been solely tested for inhibiting PtdIns(3,5)P2 synthesis. Here, we have compared the in vitro and in vivo inhibitory potency of YM201636 toward PtdIns5P and PtdIns(3,5)P2. Unexpectedly, we observed that at low doses (10–25 nM), YM201636 inhibited preferentially PtdIns5P rather than PtdIns(3,5)P2 production in vitro, whereas at higher doses, the two products were similarly inhibited. In cellular contexts, YM201636 at 160 nM inhibited PtdIns5P synthesis twice more effectively compared with PtdIns(3,5)P2 synthesis. In 3T3L1 adipocytes, human embryonic kidney 293 and Chinese hamster ovary (CHO-T) cells, levels of PtdIns5P dropped by 62–71% of the corresponding untreated controls, whereas those of PtdIns(3,5)P2 fell by only 28–46%. The preferential inhibition of PtdIns5P versus PtdIns(3,5)P2 at low doses of YM201636 was explored to probe contributions of the PIKfyve-catalyzed PtdIns5P pool to insulin-induced actin stress fiber disassembly in CHO-T cells, GLUT4 translocation in 3T3L1 adipocytes, and induction of aberrant cellular vacuolation in these or other cell types. The results provide the first experimental evidence that the principal pathway for PtdIns5P intracellular production is through PIKfyve and that insulin effect on actin stress fiber disassembly is mediated entirely by the PIKfyve-produced PtdIns5P pool. PMID:22621786

An update on gain-of-function mutations in primary immunodeficiency diseases.

PubMed

Jhamnani, Rekha D; Rosenzweig, Sergio D

2017-12-01

Most primary immunodeficiencies described since 1952 were associated with loss-of-function defects. With the advent and popularization of unbiased next-generation sequencing diagnostic approaches followed by functional validation techniques, many gain-of-function mutations leading to immunodeficiency have also been identified. This review highlights the updates on pathophysiology mechanisms and new therapeutic approaches involving primary immunodeficiencies because of gain-of-function mutations. The more recent developments related to gain-of-function primary immunodeficiencies mostly involving increased infection susceptibility but also immune dysregulation and autoimmunity, were reviewed. Updates regarding pathophysiology mechanisms, different mutation types, clinical features, laboratory markers, current and potential new treatments on patients with caspase recruitment domain family member 11, signal transducer and activator of transcription 1, signal transducer and activator of transcription 3, phosphatidylinositol-4,5-biphosphate 3-kinase catalytic 110, phosphatidylinositol-4,5-biphosphate 3-kinase regulatory subunit 1, chemokine C-X-C motif receptor 4, sterile α motif domain containing 9-like, and nuclear factor κ-B subunit 2 gain-of-function mutations are reviewed for each disease. With the identification of gain-of-function mutations as a cause of immunodeficiency, new genetic pathophysiology mechanisms unveiled and new-targeted therapeutic approaches can be explored as potential rescue treatments for these diseases.

Brain-Derived Neurotrophic Factor (BDNF) and Traumatic Brain Injury (Head and Spinal)

DTIC Science & Technology

2000-01-01

phosphatidylinositol 3-kinase are involved in brain-derived neurotrophic factor- mediated survival and neuritogenesis of the neuroblastoma cell line ... SH - SY5Y , J. Neurochem. 73 (1999) 1409-1421. 15. Gottshalk, W.A., Jiang, H., Tartaglia, N., Feng, L., Figurov, A., Lu, B., Signaling mechanisms...NT-6), and neurotrophin-7 (NT-7) (4, 5, 24, 80). Neurotrophins are believed to promote their cell survival, growth, and differentiation effects

The mechanisms behind decreased internalization of angiotensin II type 1 receptor.

PubMed

Bian, Jingwei; Zhang, Suli; Yi, Ming; Yue, Mingming; Liu, Huirong

2018-04-01

The internalization of angiotensin II type 1 receptor (AT 1 R) plays an important role in maintaining cardiovascular homeostasis. Decreased receptor internalization is closely related to cardiovascular diseases induced by the abnormal activation of AT 1 R, such as hypertension. However, the mechanism behind reduced AT 1 R internalization is not fully understood. This review focuses on four parts of the receptor internalization process (the combination of agonists and receptors, receptor phosphorylation, endocytosis, and recycling) and summarizes the possible mechanisms by which AT 1 R internalization is reduced based on these four parts of the process. (1) The agonist has a large molecular weight or a stronger ability to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5) P 2 ), which can increase the consumption of PtdIns (4,5) P 2 . (2) AT 1 R phosphorylation is weakened because of an abnormal function of phosphorylated kinase or changes in phospho-barcoding and GPCR-β-arrestin complex conformation. (3) The abnormal formation of vesicles or AT 1 R heterodimers with fewer endocytic receptors results in less AT 1 R endocytosis. (4) The enhanced activity and upregulated expression of small GTP-binding protein 4 (Rab4) and 11 (Rab11), which regulate receptor recycling, and phosphatidylinositol 3-kinase increase AT 1 R recycling. In addition, lower expression of AT 1 R-associated protein (ATRAP) or higher expression of AT 1 R-associated protein 1 (ARAP1) can reduce receptor internalization. Copyright © 2018 Elsevier Inc. All rights reserved.

Targeted suppression of AR-V7 using PIP5K1α inhibitor overcomes enzalutamide resistance in prostate cancer cells.

PubMed

Sarwar, Martuza; Semenas, Julius; Miftakhova, Regina; Simoulis, Athanasios; Robinson, Brian; Gjörloff Wingren, Anette; Mongan, Nigel P; Heery, David M; Johnsson, Heather; Abrahamsson, Per-Anders; Dizeyi, Nishtman; Luo, Jun; Persson, Jenny L

2016-09-27

One mechanism of resistance of prostate cancer (PCa) to enzalutamide (MDV3100) treatment is the increased expression of AR variants lacking the ligand binding-domain, the best characterized of which is AR-V7. We have previously reported that Phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), is a lipid kinase that links to CDK1 and AR pathways. The discovery of PIP5Kα inhibitor highlight the potential of PIP5K1α as a drug target in PCa. In this study, we show that AR-V7 expression positively correlates with PIP5K1α in tumor specimens from PCa patients. Overexpression of AR-V7 increases PIP5K1α, promotes rapid growth of PCa in xenograft mice, whereas inhibition of PIP5K1α by its inhibitor ISA-2011B suppresses the growth and invasiveness of xenograft tumors overexpressing AR-V7. PIP5K1α is a key co-factor for both AR-V7 and AR, which are present as protein-protein complexes predominantly in the nucleus of PCa cells. In addition, PIP5K1α and CDK1 influence AR-V7 expression also through AKT-associated mechanism dependent on PTEN-status. ISA-2011B disrupts protein stabilization of AR-V7 which is dependent on PIP5K1α, leading to suppression of invasive growth of AR-V7-high tumors in xenograft mice. Our study suggests that combination of enzalutamide and PIP5K1α may have a significant impact on refining therapeutic strategies to circumvent resistance to antiandrogen therapies.

Targeted suppression of AR-V7 using PIP5K1α inhibitor overcomes enzalutamide resistance in prostate cancer cells

PubMed Central

Sarwar, Martuza; Semenas, Julius; Miftakhova, Regina; Simoulis, Athanasios; Robinson, Brian; Wingren, Anette Gjörloff; Mongan, Nigel P.; Heery, David M.; Johnsson, Heather; Abrahamsson, Per-Anders; Dizeyi, Nishtman; Luo, Jun; Persson, Jenny L.

2016-01-01

One mechanism of resistance of prostate cancer (PCa) to enzalutamide (MDV3100) treatment is the increased expression of AR variants lacking the ligand binding-domain, the best characterized of which is AR-V7. We have previously reported that Phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), is a lipid kinase that links to CDK1 and AR pathways. The discovery of PIP5Kα inhibitor highlight the potential of PIP5K1α as a drug target in PCa. In this study, we show that AR-V7 expression positively correlates with PIP5K1α in tumor specimens from PCa patients. Overexpression of AR-V7 increases PIP5K1α, promotes rapid growth of PCa in xenograft mice, whereas inhibition of PIP5K1α by its inhibitor ISA-2011B suppresses the growth and invasiveness of xenograft tumors overexpressing AR-V7. PIP5K1α is a key co-factor for both AR-V7 and AR, which are present as protein-protein complexes predominantly in the nucleus of PCa cells. In addition, PIP5K1α and CDK1 influence AR-V7 expression also through AKT-associated mechanism dependent on PTEN-status. ISA-2011B disrupts protein stabilization of AR-V7 which is dependent on PIP5K1α, leading to suppression of invasive growth of AR-V7-high tumors in xenograft mice. Our study suggests that combination of enzalutamide and PIP5K1α may have a significant impact on refining therapeutic strategies to circumvent resistance to antiandrogen therapies. PMID:27588408

Cholesteryl ester transfer protein inhibition enhances endothelial repair and improves endothelial function in the rabbit.

PubMed

Wu, Ben J; Shrestha, Sudichhya; Ong, Kwok L; Johns, Douglas; Hou, Liming; Barter, Philip J; Rye, Kerry-Anne

2015-03-01

High-density lipoproteins (HDLs) can potentially protect against atherosclerosis by multiple mechanisms, including enhancement of endothelial repair and improvement of endothelial function. This study asks if increasing HDL levels by inhibiting cholesteryl ester transfer protein activity with the anacetrapib analog, des-fluoro-anacetrapib, enhances endothelial repair and improves endothelial function in New Zealand White rabbits with balloon injury of the abdominal aorta. New Zealand White rabbits received chow or chow supplemented with 0.07% or 0.14% (wt/wt) des-fluoro-anacetrapib for 8 weeks. Endothelial denudation of the abdominal aorta was carried out after 2 weeks. The animals were euthanized 6 weeks postinjury. Treatment with 0.07% and 0.14% des-fluoro-anacetrapib reduced cholesteryl ester transfer protein activity by 81±4.9% and 92±12%, increased plasma apolipoprotein A-I levels by 1.4±0.1-fold and 1.5±0.1-fold, increased plasma HDL-cholesterol levels by 1.8±0.2-fold and 1.9±0.1-fold, reduced intimal hyperplasia by 37±11% and 51±10%, and inhibited vascular cell proliferation by 25±6.1% and 35±6.7%, respectively. Re-endothelialization of the injured aorta increased from 43±6.7% (control) to 69±6.6% and 76±7.7% in the 0.07% and 0.14% des-fluoro-anacetrapib-treated animals, respectively. Aortic ring relaxation and guanosine 3',5'-cyclic monophosphate production in response to acetylcholine were also improved. Incubation of HDLs from the des-fluoro-anacetrapib-treated animals with human coronary artery endothelial cells increased cell proliferation and migration relative to control. These effects were abolished by knockdown of scavenger receptor-B1 and PDZ domain-containing protein 1 and by pharmacological inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt. Increasing HDL levels by inhibiting cholesteryl ester transfer protein reduces intimal thickening and regenerates functional endothelium in damaged New Zealand White rabbit aortas in an scavenger receptor-B1-dependent and phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt-dependent manner. © 2015 American Heart Association, Inc.

Dynamics of Phosphoinositide-Dependent Signaling in Sympathetic Neurons

PubMed Central

Kruse, Martin; Vivas, Oscar; Traynor-Kaplan, Alexis

2016-01-01

In neurons, loss of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] leads to a decrease in exocytosis and changes in electrical excitability. Restoration of PI(4,5)P2 levels after phospholipase C activation is therefore essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We measured dynamic changes of PI(4,5)P2, phosphatidylinositol 4-phosphate, diacylglycerol, inositol 1,4,5-trisphosphate, and Ca2+ upon muscarinic stimulation in sympathetic neurons from adult male Sprague-Dawley rats with electrophysiological and optical approaches. We used this kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show and explain faster synthesis of PI(4,5)P2 in sympathetic neurons than in electrically nonexcitable tsA201 cells. They can be used to understand dynamic effects of receptor-mediated phospholipase C activation on excitability and other PI(4,5)P2-dependent processes in neurons. SIGNIFICANCE STATEMENT Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a minor phospholipid in the cytoplasmic leaflet of the plasma membrane. Depletion of PI(4,5)P2 via phospholipase C-mediated hydrolysis leads to a decrease in exocytosis and alters electrical excitability in neurons. Restoration of PI(4,5)P2 is essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We studied the dynamics of phosphoinositide metabolism in sympathetic neurons upon muscarinic stimulation and used the kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show a several-fold faster synthesis of PI(4,5)P2 in sympathetic neurons than in an electrically nonexcitable cell line, and provide a framework for future studies of PI(4,5)P2-dependent processes in neurons. PMID:26818524

Sequence analysis of the PIP5K locus in Eimeria maxima provides further evidence for eimerian genome plasticity and segmental organization.

PubMed

Song, B K; Pan, M Z; Lau, Y L; Wan, K L

2014-07-29

Commercial flocks infected by Eimeria species parasites, including Eimeria maxima, have an increased risk of developing clinical or subclinical coccidiosis; an intestinal enteritis associated with increased mortality rates in poultry. Currently, infection control is largely based on chemotherapy or live vaccines; however, drug resistance is common and vaccines are relatively expensive. The development of new cost-effective intervention measures will benefit from unraveling the complex genetic mechanisms that underlie host-parasite interactions, including the identification and characterization of genes encoding proteins such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K). We previously identified a PIP5K coding sequence within the E. maxima genome. In this study, we analyzed two bacterial artificial chromosome clones presenting a ~145-kb E. maxima (Weybridge strain) genomic region spanning the PIP5K gene locus. Sequence analysis revealed that ~95% of the simple sequence repeats detected were located within regions comparable to the previously described feature-rich segments of the Eimeria tenella genome. Comparative sequence analysis with the orthologous E. maxima (Houghton strain) region revealed a moderate level of conserved synteny. Unique segmental organizations and telomere-like repeats were also observed in both genomes. A number of incomplete transposable elements were detected and further scrutiny of these elements in both orthologous segments revealed interesting nesting events, which may play a role in facilitating genome plasticity in E. maxima. The current analysis provides more detailed information about the genome organization of E. maxima and may help to reveal genotypic differences that are important for expression of traits related to pathogenicity and virulence.

Phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in hyperinsulinemic db/db mice.

PubMed

Nishida, Hidenori; Sohara, Eisei; Nomura, Naohiro; Chiga, Motoko; Alessi, Dario R; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

2012-10-01

Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently identified with-no-lysine kinase (WNK)-oxidative stress-responsive kinase 1 (OSR1)/STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NaCl cotransporter (NCC) phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both SpakT243A/+ and Osr1T185A/+ knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the phosphatidylinositol 3-kinase/Akt signaling cascade in the kidney in response to hyperinsulinemia. A phosphatidylinositol 3-kinase inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific phosphatidylinositol 3-kinase inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions, such as the metabolic syndrome.

Arabidopsis Type II Phosphatidylinositol 4-Kinase PI4Kγ5 Regulates Auxin Biosynthesis and Leaf Margin Development through Interacting with Membrane-Bound Transcription Factor ANAC078

PubMed Central

Tan, Shu-Tang; Xue, Hong-Wei

2016-01-01

Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5–1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5–1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5–1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5–1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors. PMID:27529511

A Tumor-Associated Mutation of FYVE-CENT Prevents Its Interaction with Beclin 1 and Interferes with Cytokinesis

PubMed Central

Sagona, Antonia P.; Nezis, Ioannis P.; Bache, Kristi G.; Haglund, Kaisa; Bakken, Anne Cathrine; Skotheim, Rolf I.; Stenmark, Harald

2011-01-01

The tumor suppressor activity of Beclin 1 (BECN1), a subunit of class III phosphatidylinositol 3-kinase complex, has been attributed to its regulation of apoptosis and autophagy. Here, we identify FYVE-CENT (ZFYVE26), a phosphatidylinositol 3-phosphate binding protein important for cytokinesis, as a novel interacting protein of Beclin 1. A mutation in FYVE-CENT (R1945Q) associated with breast cancer abolished the interaction between FYVE-CENT and Beclin 1, and reduced the localization of these proteins at the intercellular bridge during cytokinesis. Breast cancer cells containing the FYVE-CENT R1945Q mutation displayed a significant increase in cytokinetic profiles and bi - multinuclear phenotype. Both Beclin 1 and FYVE-CENT were found to be downregulated in advanced breast cancers. These findings suggest a positive feedback loop for recruitment of FYVE-CENT and Beclin 1 to the intercellular bridge during cytokinesis, and reveal a novel potential tumor suppressor mechanism for Beclin 1. PMID:21455500

A tumor-associated mutation of FYVE-CENT prevents its interaction with Beclin 1 and interferes with cytokinesis.

PubMed

Sagona, Antonia P; Nezis, Ioannis P; Bache, Kristi G; Haglund, Kaisa; Bakken, Anne Cathrine; Skotheim, Rolf I; Stenmark, Harald

2011-03-24

The tumor suppressor activity of Beclin 1 (BECN1), a subunit of class III phosphatidylinositol 3-kinase complex, has been attributed to its regulation of apoptosis and autophagy. Here, we identify FYVE-CENT (ZFYVE26), a phosphatidylinositol 3-phosphate binding protein important for cytokinesis, as a novel interacting protein of Beclin 1. A mutation in FYVE-CENT (R1945Q) associated with breast cancer abolished the interaction between FYVE-CENT and Beclin 1, and reduced the localization of these proteins at the intercellular bridge during cytokinesis. Breast cancer cells containing the FYVE-CENT R1945Q mutation displayed a significant increase in cytokinetic profiles and bi-multinuclear phenotype. Both Beclin 1 and FYVE-CENT were found to be downregulated in advanced breast cancers. These findings suggest a positive feedback loop for recruitment of FYVE-CENT and Beclin 1 to the intercellular bridge during cytokinesis, and reveal a novel potential tumor suppressor mechanism for Beclin 1.

Regulated Assembly of Vacuolar ATPase Is Increased during Cluster Disruption-induced Maturation of Dendritic Cells through a Phosphatidylinositol 3-Kinase/mTOR-dependent Pathway*

PubMed Central

Liberman, Rachel; Bond, Sarah; Shainheit, Mara G.; Stadecker, Miguel J.; Forgac, Michael

2014-01-01

The vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation. PMID:24273170

Insulin and leptin induce Glut4 plasma membrane translocation and glucose uptake in a human neuronal cell line by a phosphatidylinositol 3-kinase- dependent mechanism.

PubMed

Benomar, Yacir; Naour, Nadia; Aubourg, Alain; Bailleux, Virginie; Gertler, Arieh; Djiane, Jean; Guerre-Millo, Michèle; Taouis, Mohammed

2006-05-01

The insulin-sensitive glucose transporter Glut4 is expressed in brain areas that regulate energy homeostasis and body adiposity. In contrast with peripheral tissues, however, the impact of insulin on Glut4 plasma membrane (PM) translocation in neurons is not known. In this study, we examined the role of two anorexic hormones (leptin and insulin) on Glut4 translocation in a human neuronal cell line that express endogenous insulin and leptin receptors. We show that insulin and leptin both induce Glut4 translocation to the PM of neuronal cells and activate glucose uptake. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, totally abolished insulin- and leptin-dependent Glut4 translocation and stimulation of glucose uptake. Thus, Glut4 translocation is a phosphatidylinositol 3-kinase-dependent mechanism in neuronal cells. Next, we investigated the impact of chronic insulin and leptin treatments on Glut4 expression and translocation. Chronic exposure of neuronal cells to insulin or leptin down-regulates Glut4 proteins and mRNA levels and abolishes the acute stimulation of glucose uptake in response to acute insulin or leptin. In addition, chronic treatment with either insulin or leptin impaired Glut4 translocation. A cross-desensitization between insulin and leptin was apparent, where exposure to insulin affects leptin-dependent Glut4 translocation and vice versa. This cross-desensitization could be attributed to the increase in suppressor of cytokine signaling-3 expression, which was demonstrated in response to each hormone. These results provide evidence to suggest that Glut4 translocation to neuronal PM is regulated by both insulin and leptin signaling pathways. These pathways might contribute to an in vivo glucoregulatory reflex involving a neuronal network and to the anorectic effect of insulin and leptin.

PI-273, a Substrate-Competitive, Specific Small-Molecule Inhibitor of PI4KIIα, Inhibits the Growth of Breast Cancer Cells.

PubMed

Li, Jiangmei; Gao, Zhen; Zhao, Dan; Zhang, Lunfeng; Qiao, Xinhua; Zhao, Yingying; Ding, Hong; Zhang, Panpan; Lu, Junyan; Liu, Jia; Jiang, Hualiang; Luo, Cheng; Chen, Chang

2017-11-15

While phosphatidylinositol 4-kinase (PI4KIIα) has been identified as a potential target for antitumor therapy, the clinical applications of PI4KIIα are limited by a lack of specific inhibitors. Here we report the first small-molecule inhibitor (SMI) of human PI4KIIα. Docking-based and ligand-based virtual screening strategies were first employed to identify promising hits, followed by two rounds of kinase activity inhibition validation. 2-(3-(4-Chlorobenzoyl)thioureido)-4-ethyl-5-methylthiophene-3-carboxamide (PI-273) exhibited the greatest inhibitory effect on PI4KIIα kinase activity (IC 50 = 0.47 μmol/L) and suppressed cell proliferation. Surface plasmon resonance and thermal shift assays indicated that PI-273 interacted directly with PI4KIIα. Kinetic analysis identified PI-273 as a reversible competitive inhibitor with respect to the substrate phosphatidylinositol (PI), which contrasted with most other PI kinase inhibitors that bind the ATP binding site. PI-273 reduced PI4P content, cell viability, and AKT signaling in wild-type MCF-7 cells, but not in PI4KIIα knockout MCF-7 cells, indicating that PI-273 is highly selective for PI4KIIα. Mutant analysis revealed a role of palmitoylation insertion in the selectivity of PI-273 for PI4KIIα. In addition, PI-273 treatment retarded cell proliferation by blocking cells in G 2 -M, inducing cell apoptosis and suppressing colony-forming ability. Importantly, PI-273 significantly inhibited MCF-7 cell-induced breast tumor growth without toxicity. PI-273 is the first substrate-competitive, subtype-specific inhibitor of PI4KIIα, the use of which will facilitate evaluations of PI4KIIα as a cancer therapeutic target. Cancer Res; 77(22); 6253-66. ©2017 AACR . ©2017 American Association for Cancer Research.

Characterization and molecular modeling of Inositol 1,3,4 tris phosphate 5/6 kinase-2 from Glycine max (L) Merr.: comprehending its evolutionary conservancy at functional level.

PubMed

Marathe, Ashish; Krishnan, Veda; Mahajan, Mahesh M; Thimmegowda, Vinutha; Dahuja, Anil; Jolly, Monica; Praveen, Shelly; Sachdev, Archana

2018-01-01

Soybean genome encodes a family of four inositol 1,3,4 trisphosphate 5/6 kinases which belong to the ATP-GRASP group of proteins. Inositol 1,3,4 trisphosphate kinase-2 ( GmItpk2 ), catalyzing the ATP-dependent phosphorylation of Inositol 1,3,4 trisphosphate (IP3) to Inositol 1,3,4,5 tetra phosphate or Inositol 1,3,4,6 tetra phosphate, is a key enzyme diverting the flux of inositol phosphate pool towards phytate biosynthesis. Although considerable research on characterizing genes involved in phytate biosynthesis is accomplished at genomic and transcript level, characterization of the proteins is yet to be explored. In the present study, we report the isolation and expression of single copy Itpk 2 (948 bp) from Glycine max cv Pusa-16 predicted to encode 315 amino acid protein with an isoelectric point of 5.9. Sequence analysis revealed that Gm ITPK2 shared highest similarity (80%) with Phaseolus vulgaris. The predicted 3D model confirmed 12 α helices and 14 β barrel sheets with ATP-binding site close to β sheet present towards the C-terminus of the protein molecule. Spatio-temporal transcript profiling signified GmItpk2 to be seed specific, with higher transcript levels in the early stage of seed development. The present study using various molecular and bio-computational tools could, therefore, help in improving our understanding of this key enzyme and prove to be a potential target towards generating low phytate trait in nutritionally rich crop like soybean.

WAVE2 targeting to phosphatidylinositol 3,4,5-triphosphate mediated by insulin receptor substrate p53 through a complex with WAVE2.

PubMed

Takahashi, Kazuhide; Suzuki, Katsuo

2010-11-01

Membrane targeting of WAVE2 along microtubules to phosphatidylinositol 3,4,5-triphosphate (PIP(3)) in response to an extracellular stimulus requires Rac1, Pak1, stathmin, and EB1. However, whether WAVE2 interacts directly with PIP(3) or not remains unclear. We demonstrate that insulin-like growth factor I (IGF-I) induces WAVE2 membrane targeting, accompanied by phosphorylation of Pak1 at serine 199/204 (Ser199/204) and stathmin at Ser38 in the inner cytoplasmic region. This is spatially independent of the membrane region where the IGF-I receptor (IGF-IR) is locally activated. WAVE2, phosphorylated Pak1, and phosphorylated stathmin located at the microtubule ends began to accumulate at the leading edge of cells in close proximity to PIP(3) that was produced in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. The PIP(3)-beads binding assay revealed that insulin receptor substrate p53 (IRSp53) and actin rather than WAVE2 bound to PIP(3). IRSp53 constitutively associated with WAVE2 and these two proteins colocalized with PIP(3) at the leading edge after IGF-I stimulation. Suppression of IRSp53 expression by two independent small interfering RNAs (siRNAs) completely inhibited IGF-I-induced membrane targeting and local accumulation of WAVE2 at the leading edge of cells. We propose that IRSp53 constitutively forms a complex with WAVE2 and is crucial for membrane targeting followed by local accumulation of WAVE2 at the leading edge of cells through linking WAVE2 to PIP(3) that is produced near locally activated IGF-IR in response to IGF-I. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Mechanisms of aldehyde-induced adenosinetriphosphatase activities of kinases.

PubMed

Rendina, A R; Cleland, W W

1984-10-23

Aldehyde analogues of the normal alcohol substrates induce ATPase activities by glycerokinase (D-glyceraldehyde), fructose-6-phosphate kinase (2,5-anhydromannose 6-phosphate), fructokinase (2,5-anhydromannose or 2,5-anhydrotalose), hexokinase (D-gluco-hexodialdose), choline kinase (betaine aldehyde), and pyruvate kinase (glyoxylate). Since purified deuterated aldehydes give V and V/K isotope effects near 1.0 for glycerokinase, fructokinase with 2,5-anhydro[1-2H]talose, hexokinase, choline kinase, and pyruvate kinase, the hydrates of these almost fully hydrated aldehydes are the activators of the ATPase reactions. Fructose-6-phosphate kinase and fructokinase with 2,5-anhydro[1-2H]mannose show V/K deuterium isotope effects of 1.10 and 1.22, respectively, suggesting either that both hydrate and free aldehyde may be activators (predicted values are 1.37 if only the free aldehyde activates the ATPase) or, more likely, that the phosphorylated hydrate breaks down in a rate-limiting step on the enzyme while MgADP is still present and the back-reaction to yield free hydrate in solution is still possible. 18O was transferred from the aldehyde hydrate to phosphate during the ATPase reactions of glycerokinase, fructose-6-phosphate kinase, fructokinase, and hexokinase but not with choline kinase or pyruvate kinase. Thus, direct phosphorylation of the hydrates by the first four enzymes gives the phosphate adduct of the aldehyde, which decomposes nonenzymatically, while with choline kinase and pyruvate kinase the hydrates induce transfer to water (metal-bound hydroxide or water with pyruvate kinase on the basis of pH profiles). Observation of a lag in the release of phosphate from the glycerokinase ATPase reaction at 15 degrees C supports the existence of a phosphorylated hydrate intermediate with a rate constant for breakdown of 0.035-0.043 s-1 at this temperature. Kinases that phosphorylate creatine, 3-phosphoglycerate, and acetate did not exhibit ATPase activities in the presence of keto or aldehyde analogues (N-methylhydantoic acid, D-glyceraldehyde 3-phosphate, and acetaldehyde, respectively), possibly because of the absence of an acid-base catalytic group in the latter two cases. These analogues were competitive inhibitors vs. the normal substrates, and in the latter case, the hydrate of acetaldehyde was shown to be the inhibitory species on the basis of the deuterium isotope effect on the inhibition constant.

Cigarette smoke inhibits efferocytosis via deregulation of sphingosine kinase signaling: reversal with exogenous S1P and the S1P analogue FTY720.

PubMed

Tran, Hai B; Barnawi, Jameel; Ween, Miranda; Hamon, Rhys; Roscioli, Eugene; Hodge, Greg; Reynolds, Paul N; Pitson, Stuart M; Davies, Lorena T; Haberberger, Rainer; Hodge, Sandra

2016-07-01

Alveolar macrophages from chronic obstructive pulmonary disease patients and cigarette smokers are deficient in their ability to phagocytose apoptotic bronchial epithelial cells (efferocytosis). We hypothesized that the defect is mediated via inhibition of sphingosine kinases and/or their subcellular mislocalization in response to cigarette smoke and can be normalized with exogenous sphingosine-1-phosphate or FTY720 (fingolimod), a modulator of sphingosine-1-phosphate signaling, which has been shown to be clinically useful in multiple sclerosis. Measurement of sphingosine kinase 1/2 activities by [(32)P]-labeled sphingosine-1-phosphate revealed a 30% reduction of sphingosine kinase 1 (P < 0.05) and a nonsignificant decrease of sphingosine kinase 2 in THP-1 macrophages after 1 h cigarette smoke extract exposure. By confocal analysis macrophage sphingosine kinase 1 protein was normally localized to the plasma membrane and cytoplasm and sphingosine kinase 2 to the nucleus and cytoplasm but absent at the cell surface. Cigarette smoke extract exposure (24 h) led to a retraction of sphingosine kinase 1 from the plasma membrane and sphingosine kinase 1/2 clumping in the Golgi domain. Selective inhibition of sphingosine kinase 2 with 25 µM ABC294640 led to 36% inhibition of efferocytosis (P < 0.05); 10 µM sphingosine kinase inhibitor/5C (sphingosine kinase 1-selective inhibitor) induced a nonsignificant inhibition of efferocytosis, but its combination with ABC294640 led to 56% inhibition (P < 0.01 vs. control and < 0.05 vs. single inhibitors). Cigarette smoke-inhibited efferocytosis was significantly (P < 0.05) reversed to near-control levels in the presence of 10-100 nM exogenous sphingosine-1-phosphate or FTY720, and FTY720 reduced cigarette smoke-induced clumping of sphingosine kinase 1/2 in the Golgi domain. These data strongly support a role of sphingosine kinase 1/2 in efferocytosis and as novel therapeutic targets in chronic obstructive pulmonary disease. © Society for Leukocyte Biology.

Differential 14-3-3 affinity capture reveals new downstream targets of phosphatidylinositol 3-kinase signaling.

PubMed

Dubois, Fanny; Vandermoere, Franck; Gernez, Aurélie; Murphy, Jane; Toth, Rachel; Chen, Shuai; Geraghty, Kathryn M; Morrice, Nick A; MacKintosh, Carol

2009-11-01

We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d(0)/d(4)) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d(0)/d(4) values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d(0)/d(4) scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser(19) of ZNRF2 (RTRAYpS(19)GS), phospho-Ser(90) of SASH1 (RKRRVpS(90)QD), and phospho- Ser(493) of lipolysis-stimulated lipoprotein receptor (RPRARpS(493)LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways.

Differential 14-3-3 Affinity Capture Reveals New Downstream Targets of Phosphatidylinositol 3-Kinase Signaling*

PubMed Central

Dubois, Fanny; Vandermoere, Franck; Gernez, Aurélie; Murphy, Jane; Toth, Rachel; Chen, Shuai; Geraghty, Kathryn M.; Morrice, Nick A.; MacKintosh, Carol

2009-01-01

We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d0/d4) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d0/d4 values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d0/d4 scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser19 of ZNRF2 (RTRAYpS19GS), phospho-Ser90 of SASH1 (RKRRVpS90QD), and phospho- Ser493 of lipolysis-stimulated lipoprotein receptor (RPRARpS493LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways. PMID:19648646

Deficient tyrosine phosphorylation of c-Cbl and associated proteins in phorbol ester-resistant EL4 mouse thymoma cells.

PubMed

Luo, X; Sando, J J

1997-05-02

Two tyrosine phosphoproteins in phorbol ester-sensitive EL4 (S-EL4) mouse thymoma cells have been identified as the p120 c-Cbl protooncogene product and the p85 subunit of phosphatidylinositol 3-kinase. Tyrosine phosphorylation of p120 and p85 increased rapidly after phorbol ester stimulation. Phorbol ester-resistant EL4 (R-EL4) cells expressed comparable amounts of c-Cbl and phosphatidylinositol 3-kinase protein but greatly diminished tyrosine phosphorylation. Co-immunoprecipitation experiments revealed complexes of c-Cbl with p85, and of p85 with the tyrosine kinase Lck in phorbol ester-stimulated S-EL4 but not in unstimulated S-EL4 or in R-EL4 cells. In vitro binding of c-Cbl with Lck SH2 or SH3 domains was detected in both S-EL4 and R-EL4 cells, suggesting that c-Cbl, p85, and Lck may form a ternary complex. In vitro kinase assays revealed phosphorylation of p85 by Lck only in phorbol ester-stimulated S-EL4 cells. Collectively, these results suggest that Cbl-p85 and Lck-p85 complexes may form in unstimulated S-EL4 and R-EL4 cells but were not detected due to absence of tyrosine phosphorylation of p85. Greatly decreased tyrosine phosphorylation of c-Cbl and p85 in the complexes may contribute to the failure of R-EL4 cells to respond to phorbol ester.

An Autoinhibitory Role for the Pleckstrin Homology Domain of Interleukin-2-Inducible Tyrosine Kinase and Its Interplay with Canonical Phospholipid Recognition.

PubMed

Devkota, Sujan; Joseph, Raji E; Boyken, Scott E; Fulton, D Bruce; Andreotti, Amy H

2017-06-13

Pleckstrin homology (PH) domains are well-known as phospholipid binding modules, yet evidence that PH domain function extends beyond lipid recognition is mounting. In this work, we characterize a protein binding function for the PH domain of interleukin-2-inducible tyrosine kinase (ITK), an immune cell specific signaling protein that belongs to the TEC family of nonreceptor tyrosine kinases. Its N-terminal PH domain is a well-characterized lipid binding module that localizes ITK to the membrane via phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) binding. Using a combination of nuclear magnetic resonance spectroscopy and mutagenesis, we have mapped an autoregulatory protein interaction site on the ITK PH domain that makes direct contact with the catalytic kinase domain of ITK, inhibiting the phospho-transfer reaction. Moreover, we have elucidated an important interplay between lipid binding by the ITK PH domain and the stability of the autoinhibitory complex formed by full length ITK. The ITK activation loop in the kinase domain becomes accessible to phosphorylation to the exogenous kinase LCK upon binding of the ITK PH domain to PIP 3 . By clarifying the allosteric role of the ITK PH domain in controlling ITK function, we have expanded the functional repertoire of the PH domain generally and opened the door to alternative strategies to target this specific kinase in the context of immune cell signaling.

Inositol polyphosphate multikinase is a coactivator for serum response factor-dependent induction of immediate early genes

PubMed Central

Kim, Eunha; Tyagi, Richa; Lee, Joo-Young; Park, Jina; Kim, Young-ran; Beon, Jiyoon; Chen, Po Yu; Cha, Jiyoung Y.; Snyder, Solomon H.; Kim, Seyun

2013-01-01

Inositol polyphosphate multikinase (IPMK) is a notably pleiotropic protein. It displays both inositol phosphate kinase and phosphatidylinositol kinase catalytic activities. Noncatalytically, IPMK stabilizes the mammalian target of rapamycin complex 1 and acts as a transcriptional coactivator for CREB-binding protein/E1A binding protein p300 and tumor suppressor protein p53. Serum response factor (SRF) is a major transcription factor for a wide range of immediate early genes. We report that IPMK, in a noncatalytic role, is a transcriptional coactivator for SRF mediating the transcription of immediate early genes. Stimulation by serum of many immediate early genes is greatly reduced by IPMK deletion. IPMK stimulates expression of these genes, an influence also displayed by catalytically inactive IPMK. IPMK acts by binding directly to SRF and thereby enhancing interactions of SRF with the serum response element of diverse genes. PMID:24248338

Arabidopsis inositol phosphate kinases, IPK1 and ITPK1, constitute a metabolic pathway in maintaining phosphate homeostasis.

PubMed

Kuo, Hui-Fen; Hsu, Yu-Ying; Lin, Wei-Chi; Chen, Kai-Yu; Munnik, Teun; Brearley, Charles A; Chiou, Tzyy-Jen

2018-05-19

Emerging studies have implicated a close link between inositol phosphate (InsP) metabolism and cellular phosphate (P i ) homeostasis in eukaryotes; however, whether a common InsP species is deployed as an evolutionarily conserved metabolic messenger to mediate P i signaling remains unknown. Here, using genetics and InsP profiling combined with P i starvation response (PSR) analysis in Arabidopsis thaliana, we showed that the kinase activity of inositol pentakisphosphate 2-kinase (IPK1), an enzyme required for phytate (inositol hexakisphosphates; InsP 6 ) synthesis, is indispensable for maintaining P i homeostasis under P i -replete conditions, and inositol 1,3,4-trisphosphate 5/6-kinase 1 (ITPK1) plays an equivalent role. Although both ipk1-1 and itpk1 mutants exhibited decreased levels of InsP 6 and diphosphoinositol pentakisphosphate (PP-InsP 5 ; InsP 7 ), disruption of another ITPK family enzyme, ITPK4, which correspondingly caused depletion of InsP 6 and InsP 7 , did not display similar P i -related phenotypes, which precludes these InsP species as effectors. Notably, the level of D/L-Ins(3,4,5,6)P 4 was concurrently elevated in both ipk1-1 and itpk1 mutants, which showed a specific correlation to the misregulated P i phenotypes. However, the level of D/L-Ins(3,4,5,6)P 4 is not responsive to P i starvation that instead manifests a shoot-specific increase in InsP 7 level. This study demonstrates a more nuanced picture of the intersection of InsP metabolism and P i homeostasis and PSR than has previously been elaborated and additionally establishes intermediate steps to phytate biosynthesis in plant vegetative tissues. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Characterization of Runella slithyformis HD-Pnk, a bifunctional DNA/RNA end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase domain.

PubMed

Munir, Annum; Shuman, Stewart

2016-11-28

5' and 3' end healing are key steps in nucleic acid break repair in which 5' -OH ends are phosphorylated by a polynucleotide kinase and 3' -PO 4 or 2',3' -cyclic-PO 4 ends are hydrolyzed by a phosphoesterase to generate the 5' -PO 4 and 3' -OH termini required for sealing by classic polynucleotide ligases. End healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5' -OH polynucleotides (9-mers or longer) in the presence of magnesium and any NTP donor. HD-Pnk dephosphorylates RNA 2',3' -cyclic phosphate, RNA 3' -phosphate, RNA 2' -phosphate, and DNA 3' -phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper or cobalt. HD-Pnkp homologs are present in genera from eleven bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. The present study provides insights to the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnkp as the exemplar of a novel clade of dual 5' and 3' end-healing enzymes that phosphorylate 5' -OH termini and dephosphorylate 2',3' -cyclic-PO 4 , 3' -PO 4 , and 2' -PO 4 ends. The distinctive feature of HD-Pnk is its domain composition: a fusion of an N-terminal HD phosphohydrolase module to a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, domain order, and similar polypeptide size are distributed widely among genera from eleven bacterial phyla. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

ADP-ribosylation factor6 regulates both [3H]-noradrenaline and [14C]-glutamate exocytosis through phosphatidylinositol 4,5-bisphosphate.

PubMed

Zheng, Qian; Bobich, Joseph A

2004-10-01

GTP phosphohydrolase (cell regulating) (EC 3.6.1.47, ADP-ribosylation factor6, ARF6) has been shown to play an important role in different steps of membrane trafficking. It also regulates chromaffin granule exocytosis through phosphatidylcholine phosphatidohydrolase (EC 3.1.4.14, PLD) activation. In this study, the role of ARF6 in neurotransmitter release from both dense-core granules (DCGs) and synaptic vesicles (SVs) in rat brain cortex nerve endings was investigated. We observed that synaptosomal ARF6 is largely particulate but moves to a less easily pelleted compartment upon nerve ending stimulation. We also found that direct inhibition of ARF6 by a specific antibody or interference with ARF6 downstream effects by a myristoylated N-terminal ARF6 peptide both significantly decreased both [3H]-noradrenaline and [14C]-glutamate exocytosis. Addition of phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP2) partially or completely restored exocytosis. These findings suggest that ARF6 plays important regulatory roles for both DCG and SV exocytosis by activating PLD and ATP:1-phosphatidyl-1D-myo-inositol 4-phosphate 5-phosphotransferase (EC 2.7.1.68, PI4P-5K) to enhance PIP2 synthesis and nerve ending membrane trafficking.

Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5 prime -O-(3-thiotriphosphate)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baldassare, J.J.; Henderson, P.A.; Fisher, G.J.

1989-01-10

The effects of thrombin and GTP{gamma}S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous ({sup 3}H)inositol-labeled membranes or with lipid vesicles containing either ({sup 3}H)phosphatidylinositol or ({sup 3}H)phosphatidylinositol 4,5-bisphosphate. GTP{gamma}S (1 {mu}M) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP{sub 3}), inositol bisphosphate (IP{sub 2}), or inositol phosphate (IP) from ({sup 3}H)inositol-labeled membranes. IP{sub 2} and IP{sub 3}, but not IP, from ({sup 3}H)inositol-labeled membranes were, however, stimulated 3-fold by GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). A higher concentration of GTP{gamma}S (100 {mu}M) alone also stimulatedmore » IP{sub 2} and IP{sub 3}, but not IP, release. In the presence of 1 mM calcium, release of IP{sub 2} and IP{sub 3} was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}) by platelet membrane associated PLC was also markedly enhanced by GTP{gamma}S (100 {mu}M) or GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP{sub 2} was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP{gamma}S (100 {mu}M) or calcium (1 mM) dependent PIP{sub 2} breakdown, while TPA inhibited GTP{gamma}S-dependent but not calcium-dependent phospholipase C activity.« less

Phosphatidylinositol 3-phosphates-at the interface between cell signalling and membrane traffic.

PubMed

Marat, Andrea L; Haucke, Volker

2016-03-15

Phosphoinositides (PIs) form a minor class of phospholipids with crucial functions in cell physiology, ranging from cell signalling and motility to a role as signposts of compartmental membrane identity. Phosphatidylinositol 3-phosphates are present at the plasma membrane and within the endolysosomal system, where they serve as key regulators of both cell signalling and of intracellular membrane traffic. Here, we provide an overview of the metabolic pathways that regulate cellular synthesis of PI 3-phosphates at distinct intracellular sites and discuss the mechanisms by which these lipids regulate cell signalling and membrane traffic. Finally, we provide a framework for how PI 3-phosphate metabolism is integrated into the cellular network. © 2016 The Authors.

Diabetes abolishes the cardioprotection induced by sevoflurane postconditioning in the rat heart in vivo: roles of glycogen synthase kinase-3β and its upstream pathways.

PubMed

Tai, Wenjun; Shi, Enyi; Yan, Lihui; Jiang, Xiaojing; Ma, Hong; Ai, Chunyu

2012-11-01

We measured the cardioprotection afforded by sevoflurane postconditioning in streptozotocin-induced diabetic rats (DRs) and determined the roles of glycogen synthase kinase (GSK), phosphatidylinositol-3-kinase/Akt, and extracellular signal-regulated kinase (ERK1/2) in such a procedure. DRs and nondiabetic rats (NDRs) were subjected to a 30-min coronary artery occlusion followed by a 120-min reperfusion. Postconditioning was achieved by inhalation of 1 minimum alveolar concentration sevoflurane at the first 5 min of reperfusion. The infarct size was determined by triphenyltetrazolium chloride staining. Expressions of GSK-3β, Akt, and ERK1/2 were measured using Western blotting. In NDRs, the infarct size was significantly decreased from 53.4% ± 7.6% to 34.9% ± 5.6% by sevoflurane postconditioning (P < 0.01). Such an anti-infarct effect was abolished completely in the DRs, as evidenced by a similar infarct size observed between the sevoflurane-treated and untreated DRs (49.3% ± 8.6% and 49.6% ± 9.3%, respectively, P > 0.05). Direct inhibition of GSK-3β by injection of SB216763 just before the start of reperfusion induced equivalent infarct-sparing effects in both NDRs (37.8% ± 3.9% and 53.4% ± 7.6% in SB216763-treated and untreated NDRs, respectively; P < 0.01) and DRs (38.8% ± 3.2% and 49.3% ± 8.6% in SB216763-treated and untreated DRs, respectively; P < 0.05). Sevoflurane postconditioning remarkably enhanced the phosphorylation of GSK-3β Ser(9), Akt Ser(473), and ERK1/2 in NDRs, which were blocked in DRs. The cardioprotection induced by sevoflurane postconditioning is abolished by diabetes. This might be due to the impairment of phosphorylation of GSK-3β and its upstream signaling pathways of phosphatidylinositol-3-kinase/Akt and ERK1/2 in the presence of diabetes. Copyright © 2012 Elsevier Inc. All rights reserved.

Critical role of PI3-kinase/Akt activation in the PARP inhibitor induced heart function recovery during ischemia-reperfusion.

PubMed

Kovacs, Krisztina; Toth, Ambrus; Deres, Peter; Kalai, Tamas; Hideg, Kalman; Gallyas, Ferenc; Sumegi, Balazs

2006-02-14

Poly(ADP-ribose) polymerase (PARP) inhibitors protect hearts from ischemia-reperfusion (IR)-induced damages by limiting nicotinamide adenine dinucleotide (NAD+) and ATP depletion, and by other, not yet elucidated mechanisms. Our preliminary data suggested that PARP catalyzed ADP-ribosylations may affect signaling pathways in cardiomyocytes. To clarify this possibility, we studied the effect of a well-characterized (4-hydroxyquinazoline) and a novel (carboxaminobenzimidazol-derivative) PARP inhibitor on the activation of phosphatidylinositol-3-kinase (PI3-kinase)/Akt pathway in Langendorff-perfused hearts. PARP inhibitors promoted the restoration of myocardial energy metabolism (assessed by 31P nuclear magnetic resonance spectroscopy) and cardiac function compared to untreated hearts. PARP inhibitors also attenuated the infarct size and reduced the IR-induced lipid peroxidation, protein oxidation and total peroxide concentration. Moreover, PARP inhibitors facilitated Akt phosphorylation and activation, as well as the phosphorylation of its downstream target glycogen synthase kinase-3beta (GSK-3beta) in normoxia and, more robustly, during IR. Blocking PI3-kinase by wortmannin or LY294002 reduced the PARP inhibitor-elicited robust Akt and GSK-3beta phosphorylation upon ischemia-reperfusion, and significantly diminished the recovery of ATP and creatine phosphate showing the importance of Akt activation in the recovery of energy metabolism. In addition, inhibition of PI3-kinase/Akt pathway decreased the protective effect of PARP inhibitors on infarct size and the recovery of heart functions. All these data suggest that contrary to the original view, which considered preservation of NAD+ and consequently ATP pools as the exclusive underlying mechanism for the cytoprotective effect of PARP inhibitors, the activation of PI3-kinase/Akt pathway and related processes are at least equally important in the cardioprotective effects of PARP inhibitors during ischemia-reperfusion.

Phosphatase and Tensin Homolog Is a Growth Repressor of Both Rhizoid and Gametophore Development in the Moss Physcomitrella patens1[OPEN

PubMed Central

Saavedra, Laura; Heilmann, Ingo

2015-01-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase implicated in cellular proliferation and survival. In animal cells, loss of PTEN leads to increased levels of phosphatidylinositol (3,4,5)-trisphosphate, stimulation of glucose and lipid metabolism, cellular growth, and morphological changes (related to adaptation and survival). Intriguingly, in plants, phosphatidylinositol (3,4,5)-trisphosphate has not been detected, and the enzymes that synthesize it were never reported. In this study we performed a genetic, biochemical, and functional characterization of the moss Physcomitrella patens PTEN gene family. P. patens has four PTENs, which are ubiquitously expressed during the entire moss life cycle. Using a knock-in approach, we show that all four genes are expressed in growing tissues, namely caulonemal and rhizoid cells. At the subcellular level, PpPTEN-green fluorescent protein fusions localized to the cytosol and the nucleus. Analysis of single and double knockouts revealed no significant phenotypes at different developmental stages, indicative of functional redundancy. However, compared with wild-type triple and quadruple pten knockouts, caulonemal cells grew faster, switched from the juvenile protonemal stage to adult gametophores earlier, and produced more rhizoids. Furthermore, analysis of lipid content and quantitative real-time polymerase chain reaction data performed in quadruple mutants revealed altered phosphoinositide levels [increase in phosphatidylinositol (3,5)-bisphosphate and decrease in phosphatidylinositol 3-phosphate] and up-regulation of marker genes from the synthesis phase of the cell cycle (e.g. P. patens proliferating cell nuclear antigen, ribonucleotide reductase, and minichromosome maintenance) and of the retinoblastoma-related protein gene P. patens retinoblastoma-related protein1. Together, these results suggest that PpPTEN is a suppressor of cell growth and morphogenic development in plants. PMID:26463087

Phosphatase and Tensin Homolog Is a Growth Repressor of Both Rhizoid and Gametophore Development in the Moss Physcomitrella patens.

PubMed

Saavedra, Laura; Catarino, Rita; Heinz, Tobias; Heilmann, Ingo; Bezanilla, Magdalena; Malhó, Rui

2015-12-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase implicated in cellular proliferation and survival. In animal cells, loss of PTEN leads to increased levels of phosphatidylinositol (3,4,5)-trisphosphate, stimulation of glucose and lipid metabolism, cellular growth, and morphological changes (related to adaptation and survival). Intriguingly, in plants, phosphatidylinositol (3,4,5)-trisphosphate has not been detected, and the enzymes that synthesize it were never reported. In this study we performed a genetic, biochemical, and functional characterization of the moss Physcomitrella patens PTEN gene family. P. patens has four PTENs, which are ubiquitously expressed during the entire moss life cycle. Using a knock-in approach, we show that all four genes are expressed in growing tissues, namely caulonemal and rhizoid cells. At the subcellular level, PpPTEN-green fluorescent protein fusions localized to the cytosol and the nucleus. Analysis of single and double knockouts revealed no significant phenotypes at different developmental stages, indicative of functional redundancy. However, compared with wild-type triple and quadruple pten knockouts, caulonemal cells grew faster, switched from the juvenile protonemal stage to adult gametophores earlier, and produced more rhizoids. Furthermore, analysis of lipid content and quantitative real-time polymerase chain reaction data performed in quadruple mutants revealed altered phosphoinositide levels [increase in phosphatidylinositol (3,5)-bisphosphate and decrease in phosphatidylinositol 3-phosphate] and up-regulation of marker genes from the synthesis phase of the cell cycle (e.g. P. patens proliferating cell nuclear antigen, ribonucleotide reductase, and minichromosome maintenance) and of the retinoblastoma-related protein gene P. patens retinoblastoma-related protein1. Together, these results suggest that PpPTEN is a suppressor of cell growth and morphogenic development in plants. © 2015 American Society of Plant Biologists. All Rights Reserved.

PI3K/Akt signalling pathway and cancer.

PubMed

Fresno Vara, Juan Angel; Casado, Enrique; de Castro, Javier; Cejas, Paloma; Belda-Iniesta, Cristóbal; González-Barón, Manuel

2004-04-01

Phosphatidylinositol-3 kinases, PI3Ks, constitute a lipid kinase family characterized by their ability to phosphorylate inositol ring 3'-OH group in inositol phospholipids to generate the second messenger phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P(3)). RPTK activation results in PI(3,4,5)P(3) and PI(3,4)P(2) production by PI3K at the inner side of the plasma membrane. Akt interacts with these phospholipids, causing its translocation to the inner membrane, where it is phosphorylated and activated by PDK1 and PDK2. Activated Akt modulates the function of numerous substrates involved in the regulation of cell survival, cell cycle progression and cellular growth. In recent years, it has been shown that PI3K/Akt signalling pathway components are frequently altered in human cancers. Cancer treatment by chemotherapy and gamma-irradiation kills target cells primarily by the induction of apoptosis. However, the development of resistance to therapy is an important clinical problem. Failure to activate the apoptotic programme represents an important mode of drug resistance in tumor cells. Survival signals induced by several receptors are mediated mainly by PI3K/Akt, hence this pathway may decisively contribute to the resistant phenotype. Many of the signalling pathways involved in cellular transformation have been elucidated and efforts are underway to develop treatment strategies that target these specific signalling molecules or their downstream effectors. The PI3K/Akt pathway is involved in many of the mechanisms targeted by these new drugs, thus a better understanding of this crossroad can help to fully exploit the potential benefits of these new agents.

A novel fluorescence-based biosynthetic trafficking method provides pharmacologic evidence that PI4-kinase IIIα is important for protein trafficking from the endoplasmic reticulum to the plasma membrane.

PubMed

Bryant, Kirsten L; Baird, Barbara; Holowka, David

2015-02-27

Biosynthetic trafficking of receptors and other membrane-associated proteins from the endoplasmic reticulum (ER) to the plasma membrane (PM) underlies the capacity of these proteins to participate in crucial cellular roles. Phosphoinositides have been shown to mediate distinct biological functions in cells, and phosphatidylinositol 4-phosphate (PI4P), in particular, has emerged as a key regulator of biosynthetic trafficking. To investigate the source of PI4P that orchestrates trafficking events, we developed a novel flow cytometry based method to monitor biosynthetic trafficking of transiently transfected proteins. We demonstrated that our method can be used to assess the trafficking of both type-1 transmembrane and GPI-linked proteins, and that it can accurately monitor the pharmacological disruption of biosynthetic trafficking with brefeldin A, a well-documented inhibitor of early biosynthetic trafficking. Furthermore, utilizing our newly developed method, we applied pharmacological inhibition of different isoforms of PI 4-kinase to reveal a role for a distinct pool of PI4P, synthesized by PI4KIIIα, in ER-to-PM trafficking. Taken together, these findings provide evidence that a specific pool of PI4P plays a role in biosynthetic trafficking of two different classes of proteins from the ER to the Golgi complex. Furthermore, our simple, flow cytometry-based biosynthetic trafficking assay can be widely applied to the study of multiple classes of proteins and varied pharmacological and genetic perturbations.

Sustained activation of c-Jun N-terminal and extracellular signal-regulated kinases in port-wine stain blood vessels.

PubMed

Tan, Wenbin; Chernova, Margarita; Gao, Lin; Sun, Victor; Liu, Huaxu; Jia, Wangcun; Langer, Stephanie; Wang, Gang; Mihm, Martin C; Nelson, J Stuart

2014-11-01

Port-wine stain (PWS) is a congenital, progressive vascular malformation but the pathogenesis remains incompletely understood. We sought to investigate the activation status of various kinases, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, AKT, phosphatidylinositol 3-kinase, P70 ribosomal S6 kinase, and phosphoinositide phospholipase C γ subunit, in PWS biopsy tissues. Immunohistochemistry was performed on 19 skin biopsy samples from 11 patients with PWS. c-Jun N-terminal kinase, extracellular signal-regulated kinase, and P70 ribosomal S6 kinase in pediatric and adult PWS blood vessels were consecutively activated. Activation of AKT and phosphatidylinositol 3-kinase was found in many adult hypertrophic PWS blood vessels but not in infants. Phosphoinositide phospholipase C γ subunit showed strong activation in nodular PWS blood vessels. Infantile PWS sample size was small. Our data suggest a subsequent activation profile of various kinases during different stages of PWS: (1) c-Jun N-terminal and extracellular signal-regulated kinases are firstly and consecutively activated in all PWS tissues, which may contribute to both the pathogenesis and progressive development of PWS; (2) AKT and phosphatidylinositol 3-kinase are subsequently activated, and are involved in the hypertrophic development of PWS blood vessels; and (3) phosphoinositide phospholipase C γ subunit is activated in the most advanced stage of PWS and may participate in nodular formation. Copyright © 2014 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

Dioctanoylglycerol stimulates accumulation of [methyl-14C]choline and its incorporation into acetylcholine and phosphatidylcholine in a human cholinergic neuroblastoma cell line

NASA Technical Reports Server (NTRS)

Slack, B. E.; Richardson, U. I.; Nitsch, R. M.; Wurtman, R. J.

1992-01-01

Dioctanoylglycerol, a synthetic diacylglycerol, stimulated [14C]choline uptake in cultured human neuroblastoma (LA-N-2) cells. As this effect has not, to our knowledge, been reported before, it was of interest to characterize it in more detail. In the presence of 500 microM dioctanoylglycerol the levels of [14C]choline attained during a 2 hour labeling period were elevated by 78 +/- 12%, while [14C]acetylcholine and long fatty acyl chain [14C]phosphatidylcholine levels increased by 26 +/- 2% and 19 +/- 5%, respectively (mean +/- S.E.M.). Total (long chain plus dioctanoyl-) [14C]phosphatidylcholine was increased by 198 +/- 33%. Kinetic analysis showed that dioctanoylglycerol reduced the apparent Km for choline uptake to 56 +/- 9% of control (n = 4). The Vmax was not significantly altered. The stimulation of [14C]choline accumulation by dioctanoylglycerol was not dependent on protein kinase C activation; the effect was not mimicked by phorbol ester or by 1-oleoyl-2-acetylglycerol, and was not inhibited by the protein kinase C inhibitors H-7 or staurosporine, or by prolonged pretreatment with phorbol 12-myristate 13-acetate. The effect of dioctanoylglycerol was slightly (but not significantly) reduced by EGTA and strongly inhibited by the cell-permeant calcium chelator bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)ester. Although these results implicate elevated intracellular calcium in the response, dioctanoylglycerol did not increase phosphatidylinositol hydrolysis in LA-N-2 cells, and its effect was not inhibited by the diacylglycerol kinase inhibitor R 59 022 (which blocks the conversion of diacylglycerol to phosphatidic acid, a known stimulator of phosphatidylinositol hydrolysis).(ABSTRACT TRUNCATED AT 250 WORDS).

PI(4)P Promotes Phosphorylation and Conformational Change of Smoothened through Interaction with Its C-terminal Tail

PubMed Central

Zhang, Jie; Li, Xiang-An; Evers, B. Mark; Zhu, Haining; Jia, Jianhang

2016-01-01

In Hedgehog (Hh) signaling, binding of Hh to the Patched-Interference Hh (Ptc-Ihog) receptor complex relieves Ptc inhibition on Smoothened (Smo). A longstanding question is how Ptc inhibits Smo and how such inhibition is relieved by Hh stimulation. In this study, we found that Hh elevates production of phosphatidylinositol 4-phosphate (PI(4)P). Increased levels of PI(4)P promote, whereas decreased levels of PI(4)P inhibit, Hh signaling activity. We further found that PI(4)P directly binds Smo through an arginine motif, which then triggers Smo phosphorylation and activation. Moreover, we identified the pleckstrin homology (PH) domain of G protein-coupled receptor kinase 2 (Gprk2) as an essential component for enriching PI(4)P and facilitating Smo activation. PI(4)P also binds mouse Smo (mSmo) and promotes its phosphorylation and ciliary accumulation. Finally, Hh treatment increases the interaction between Smo and PI(4)P but decreases the interaction between Ptc and PI(4)P, indicating that, in addition to promoting PI(4)P production, Hh regulates the pool of PI(4)P associated with Ptc and Smo. PMID:26863604

Populations of Radial Glial Cells Respond Differently to Reelin and Neuregulin1 in a Ferret Model of Cortical Dysplasia

DTIC Science & Technology

2010-10-28

which underlies many syndromes including mental retardation, epilepsy, schizophrenia, and autism [7,8,9]. In rodents, neurogenesis and neuronal...Jossin Y, Goffinet AM (2007) Reelin signals through phosphatidylinositol 3- kinase and Akt to control cortical development and through mTor to regulate...the PI3K-AKT- mTOR pathway: progress, pitfalls, and promises. Curr Opin Pharmacol 8(4): 393–412. 60. Nadarajah B, Alifragis P, Wong RO, Parnavelas JG

Phosphoinositides Regulate P2X4 ATP-Gated Channels through Direct Interactions

PubMed Central

Bernier, Louis-Philippe; Ase, Ariel R.; Chevallier, Stéphanie; Blais, Dominique; Zhao, Qi; Boué-Grabot, Éric; Logothetis, Diomedes; Séguéla, Philippe

2008-01-01

P2X receptors are ATP-gated nonselective cation channels highly permeable to calcium that contribute to nociception and inflammatory responses. The P2X4 subtype, upregulated in activated microglia, is thought to play a critical role in the development of tactile allodynia following peripheral nerve injury. Posttranslational regulation of P2X4 function is crucial to the cellular mechanisms of neuropathic pain, however it remains poorly understood. Here, we show that the phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), products of phosphorylation by wortmannin-sensitive phosphatidylinositol 4-kinases and phosphatidylinositol 3-kinases, can modulate the function of native and recombinant P2X4 receptor channels. In BV-2 microglial cells, depleting the intracellular levels of PIP2 and PIP3 with wortmannin significantly decreased P2X4 current amplitude and P2X4-mediated calcium entry measured in patch clamp recordings and ratiometric ion imaging, respectively. Wortmannin-induced depletion of phosphoinositides in Xenopus oocytes decreased the current amplitude of P2X4 responses by converting ATP into a partial agonist. It also decreased their recovery from desensitization and affected their kinetics. Injection of phosphoinositides in wortmannin-treated oocytes reversed these effects and application of PIP2 on excised inside-out macropatches rescued P2X4 currents from rundown. Moreover, we report the direct interaction of phospholipids with the proximal C-terminal domain of P2X4 subunit (Cys360-Val375) using an in vitro binding assay. These results demonstrate novel regulatory roles of the major signaling phosphoinositides PIP2 and PIP3 on P2X4 function through direct channel-lipid interactions. PMID:19036987

Domain Organization in Candida glabrata THI6, a Bifunctional Enzyme Required for Thiamin Biosynthesis in Eukaryotes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paul, Debamita; Chatterjee, Abhishek; Begley, Tadhg P.

2010-11-15

THI6 is a bifunctional enzyme found in the thiamin biosynthetic pathway in eukaryotes. The N-terminal domain of THI6 catalyzes the ligation of the thiamin thiazole and pyrimidine moieties to form thiamin phosphate, and the C-terminal domain catalyzes the phosphorylation of 4-methyl-5-hydroxyethylthiazole in a salvage pathway. In prokaryotes, thiamin phosphate synthase and 4-methyl-5-hydroxyethylthiazole kinase are separate gene products. Here we report the first crystal structure of a eukaryotic THI6 along with several complexes that characterize the active sites responsible for the two chemical reactions. THI6 from Candida glabrata is a homohexamer in which the six protomers form a cage-like structure. Eachmore » protomer is composed of two domains, which are structurally homologous to their monofunctional bacterial counterparts. Two loop regions not found in the bacterial enzymes provide interactions between the two domains. The structures of different protein-ligand complexes define the thiazole and ATP binding sites of the 4-methyl-5-hydroxyethylthiazole kinase domain and the thiazole phosphate and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate binding sites of the thiamin phosphate synthase domain. Our structural studies reveal that the active sites of the two domains are 40 {angstrom} apart and are not connected by an obvious channel. Biochemical studies show 4-methyl-5-hydroxyethylthiazole phosphate is a substrate for THI6; however, adenosine diphospho-5{beta}-ethyl-4-methylthiazole-2-carboxylic acid, the product of THI4, is not a substrate for THI6. This suggests that an unidentified enzyme is necessary to produce the substrate for THI6 from the THI4 product.« less

Identification of a new adapter protein that may link the common beta subunit of the receptor for granulocyte/macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 to phosphatidylinositol 3-kinase.

PubMed

Jücker, M; Feldman, R A

1995-11-17

Binding of human granulocyte/macrophage colony-stimulating factor (hGM-CSF) to its receptor induces the rapid activation of phosphatidylinositol-3 kinase (PI 3-kinase). As hGM-CSF receptor (hGMR) does not contain a consensus sequence for binding of PI 3-kinase, hGMR must use a distinct mechanism for its association with and activation of PI 3-kinase. Here, we describe the identification of a tyrosine-phosphorylated protein of 76-85 kDa (p80) that associates with the common beta subunit of hGMR and with the SH2 domains of the p85 subunit of PI 3-kinase in hGM-CSF-stimulated cells. Src/Yes and Lyn were tightly associated with the p80.PI 3-kinase complex, suggesting that p80 and other phosphotyrosyl proteins present in the complex were phosphorylated by Src family kinases. Tyrosine phosphorylation of p80 was only detected in hGM-CSF or human interleukin-3-stimulated cells, suggesting that activation of p80 might be specific for signaling via the common beta subunit. We postulate that p80 functions as an adapter protein that may participate in linking the hGM-CSF receptor to the PI 3-kinase signaling pathway.

Lipid phosphatase SHIP2 functions as oncogene in colorectal cancer by regulating PKB activation.

PubMed

Hoekstra, Elmer; Das, Asha M; Willemsen, Marcella; Swets, Marloes; Kuppen, Peter J K; van der Woude, Christien J; Bruno, Marco J; Shah, Jigisha P; Ten Hagen, Timo L M; Chisholm, John D; Kerr, William G; Peppelenbosch, Maikel P; Fuhler, Gwenny M

2016-11-08

Colorectal cancer (CRC) is the second most common cause of cancer-related death, encouraging the search for novel therapeutic targets affecting tumor cell proliferation and migration. These cellular processes are under tight control of two opposing groups of enzymes; kinases and phosphatases. Aberrant activity of kinases is observed in many forms of cancer and as phosphatases counteract such "oncogenic" kinases, it is generally assumed that phosphatases function as tumor suppressors. However, emerging evidence suggests that the lipid phosphatase SH2-domain-containing 5 inositol phosphatase (SHIP2), encoded by the INPPL1 gene, may act as an oncogene. Just like the well-known tumor suppressor gene Phosphatase and Tensin Homolog (PTEN) it hydrolyses phosphatidylinositol (3,4,5) triphosphate (PI(3,4,5)P3). However, unlike PTEN, the reaction product is PI(3,4)P2, which is required for full activation of the downstream protein kinase B (PKB/Akt), suggesting that SHIP2, in contrast to PTEN, could have a tumor initiating role through PKB activation. In this work, we investigated the role of SHIP2 in colorectal cancer. We found that SHIP2 and INPPL1 expression is increased in colorectal cancer tissue in comparison to adjacent normal tissue, and this is correlated with decreased patient survival. Moreover, SHIP2 is more active in colorectal cancer tissue, suggesting that SHIP2 can induce oncogenesis in colonic epithelial cells. Furthermore, in vitro experiments performed on colorectal cancer cell lines shows an oncogenic role for SHIP2, by enhancing chemoresistance, cell migration, and cell invasion. Together, these data indicate that SHIP2 expression contributes to the malignant potential of colorectal cancer, providing a possible target in the fight against this devastating disease.

Antimitogenic effect of bitter taste receptor agonists on airway smooth muscle cells.

PubMed

Sharma, Pawan; Panebra, Alfredo; Pera, Tonio; Tiegs, Brian C; Hershfeld, Alena; Kenyon, Lawrence C; Deshpande, Deepak A

2016-02-15

Airway remodeling is a hallmark feature of asthma and chronic obstructive pulmonary disease. Clinical studies and animal models have demonstrated increased airway smooth muscle (ASM) mass, and ASM thickness is correlated with severity of the disease. Current medications control inflammation and reverse airway obstruction effectively but have limited effect on remodeling. Recently we identified the expression of bitter taste receptors (TAS2R) on ASM cells, and activation with known TAS2R agonists resulted in ASM relaxation and bronchodilation. These studies suggest that TAS2R can be used as new therapeutic targets in the treatment of obstructive lung diseases. To further establish their effectiveness, in this study we aimed to determine the effects of TAS2R agonists on ASM growth and promitogenic signaling. Pretreatment of healthy and asthmatic human ASM cells with TAS2R agonists resulted in a dose-dependent inhibition of ASM proliferation. The antimitogenic effect of TAS2R ligands was not dependent on activation of protein kinase A, protein kinase C, or high/intermediate-conductance calcium-activated K(+) channels. Immunoblot analyses revealed that TAS2R agonists inhibit growth factor-activated protein kinase B phosphorylation without affecting the availability of phosphatidylinositol 3,4,5-trisphosphate, suggesting TAS2R agonists block signaling downstream of phosphatidylinositol 3-kinase. Furthermore, the antimitogenic effect of TAS2R agonists involved inhibition of induced transcription factors (activator protein-1, signal transducer and activator of transcription-3, E2 factor, nuclear factor of activated T cells) and inhibition of expression of multiple cell cycle regulatory genes, suggesting a direct inhibition of cell cycle progression. Collectively, these findings establish the antimitogenic effect of TAS2R agonists and identify a novel class of receptors and signaling pathways that can be targeted to reduce or prevent airway remodeling as well as bronchoconstriction in obstructive airway disease. Copyright © 2016 the American Physiological Society.

Antimitogenic effect of bitter taste receptor agonists on airway smooth muscle cells

PubMed Central

Sharma, Pawan; Panebra, Alfredo; Pera, Tonio; Tiegs, Brian C.; Hershfeld, Alena; Kenyon, Lawrence C.

2015-01-01

Airway remodeling is a hallmark feature of asthma and chronic obstructive pulmonary disease. Clinical studies and animal models have demonstrated increased airway smooth muscle (ASM) mass, and ASM thickness is correlated with severity of the disease. Current medications control inflammation and reverse airway obstruction effectively but have limited effect on remodeling. Recently we identified the expression of bitter taste receptors (TAS2R) on ASM cells, and activation with known TAS2R agonists resulted in ASM relaxation and bronchodilation. These studies suggest that TAS2R can be used as new therapeutic targets in the treatment of obstructive lung diseases. To further establish their effectiveness, in this study we aimed to determine the effects of TAS2R agonists on ASM growth and promitogenic signaling. Pretreatment of healthy and asthmatic human ASM cells with TAS2R agonists resulted in a dose-dependent inhibition of ASM proliferation. The antimitogenic effect of TAS2R ligands was not dependent on activation of protein kinase A, protein kinase C, or high/intermediate-conductance calcium-activated K+ channels. Immunoblot analyses revealed that TAS2R agonists inhibit growth factor-activated protein kinase B phosphorylation without affecting the availability of phosphatidylinositol 3,4,5-trisphosphate, suggesting TAS2R agonists block signaling downstream of phosphatidylinositol 3-kinase. Furthermore, the antimitogenic effect of TAS2R agonists involved inhibition of induced transcription factors (activator protein-1, signal transducer and activator of transcription-3, E2 factor, nuclear factor of activated T cells) and inhibition of expression of multiple cell cycle regulatory genes, suggesting a direct inhibition of cell cycle progression. Collectively, these findings establish the antimitogenic effect of TAS2R agonists and identify a novel class of receptors and signaling pathways that can be targeted to reduce or prevent airway remodeling as well as bronchoconstriction in obstructive airway disease. PMID:26684251

Reduction of In-Stent Restenosis by Cholesteryl Ester Transfer Protein Inhibition.

PubMed

Wu, Ben J; Li, Yue; Ong, Kwok L; Sun, Yidan; Shrestha, Sudichhya; Hou, Liming; Johns, Douglas; Barter, Philip J; Rye, Kerry-Anne

2017-12-01

Angioplasty and stent implantation, the most common treatment for atherosclerotic lesions, have a significant failure rate because of restenosis. This study asks whether increasing plasma high-density lipoprotein (HDL) levels by inhibiting cholesteryl ester transfer protein activity with the anacetrapib analog, des-fluoro-anacetrapib, prevents stent-induced neointimal hyperplasia. New Zealand White rabbits received normal chow or chow supplemented with 0.14% (wt/wt) des-fluoro-anacetrapib for 6 weeks. Iliac artery endothelial denudation and bare metal steel stent deployment were performed after 2 weeks of des-fluoro-anacetrapib treatment. The animals were euthanized 4 weeks poststent deployment. Relative to control, dietary supplementation with des-fluoro-anacetrapib reduced plasma cholesteryl ester transfer protein activity and increased plasma apolipoprotein A-I and HDL cholesterol levels by 53±6.3% and 120±19%, respectively. Non-HDL cholesterol levels were unaffected. Des-fluoro-anacetrapib treatment reduced the intimal area of the stented arteries by 43±5.6% ( P <0.001), the media area was unchanged, and the arterial lumen area increased by 12±2.4% ( P <0.05). Des-fluoro-anacetrapib treatment inhibited vascular smooth muscle cell proliferation by 41±4.5% ( P <0.001). Incubation of isolated HDLs from des-fluoro-anacetrapib-treated animals with human aortic smooth muscle cells at apolipoprotein A-I concentrations comparable to their plasma levels inhibited cell proliferation and migration. These effects were dependent on scavenger receptor-B1, the adaptor protein PDZ domain-containing protein 1, and phosphatidylinositol-3-kinase/Akt activation. HDLs from des-fluoro-anacetrapib-treated animals also inhibited proinflammatory cytokine-induced human aortic smooth muscle cell proliferation and stent-induced vascular inflammation. Inhibiting cholesteryl ester transfer protein activity in New Zealand White rabbits with iliac artery balloon injury and stent deployment increases HDL levels, inhibits vascular smooth muscle cell proliferation, and reduces neointimal hyperplasia in an scavenger receptor-B1, PDZ domain-containing protein 1- and phosphatidylinositol-3-kinase/Akt-dependent manner. © 2017 American Heart Association, Inc.

Interaction of PDK1 with Phosphoinositides Is Essential for Neuronal Differentiation but Dispensable for Neuronal Survival

PubMed Central

Zurashvili, Tinatin; Cordón-Barris, Lluís; Ruiz-Babot, Gerard; Zhou, Xiangyu; Lizcano, Jose M.; Gómez, Nestor; Giménez-Llort, Lydia

2013-01-01

3-Phosphoinositide-dependent protein kinase 1 (PDK1) operates in cells in response to phosphoinositide 3-kinase activation and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] production by activating a number of AGC kinases, including protein kinase B (PKB)/Akt. Both PDK1 and PKB contain pleckstrin homology (PH) domains that interact with the PtdIns(3,4,5)P3 second messenger. Disrupting the interaction of the PDK1 PH domain with phosphoinositides by expressing the PDK1 K465E knock-in mutation resulted in mice with reduced PKB activation. We explored the physiological consequences of this biochemical lesion in the central nervous system. The PDK1 knock-in mice displayed a reduced brain size due to a reduction in neuronal cell size rather than cell number. Reduced BDNF-induced phosphorylation of PKB at Thr308, the PDK1 site, was observed in the mutant neurons, which was not rate limiting for the phosphorylation of those PKB substrates governing neuronal survival and apoptosis, such as FOXO1 or glycogen synthase kinase 3 (GSK3). Accordingly, the integrity of the PDK1 PH domain was not essential to support the survival of different embryonic neuronal populations analyzed. In contrast, PKB-mediated phosphorylation of PRAS40 and TSC2, allowing optimal mTORC1 activation and brain-specific kinase (BRSK) protein synthesis, was markedly reduced in the mutant mice, leading to impaired neuronal growth and differentiation. PMID:23275438

Characterization of porcine SKIP gene in skeletal muscle development: polymorphisms, association analysis, expression and regulation of cell growth in C2C12 cells.

PubMed

Xiong, Qi; Chai, Jin; Deng, Changyan; Jiang, Siwen; Liu, Yang; Huang, Tao; Suo, Xiaojun; Zhang, Nian; Li, Xiaofeng; Yang, Qianping; Chen, Mingxin; Zheng, Rong

2012-12-01

Skeletal muscle and kidney-enriched inositol phosphatase (SKIP) was identified as a 5'-inositol phosphatase that hydrolyzes phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3) to PI(3,4)P2 and negatively regulates insulin-induced phosphatidylinositol 3-kinase signaling in skeletal muscle. In this study, two new single nucleotide polymorphisms (SNPs) in porcine SKIP introns 1 and 6 were detected. The C1092T locus in intron 1 showed significant associations with some meat traits, whereas the A17G locus in intron 6 showed significant associations with some carcass traits. Expression analysis showed that porcine SKIP is upregulated at d 65 of gestation and Meishan fetuses have higher and prolonged expression of SKIP compared to Large White at d 100 of gestation. Ectopic expression of porcine SKIP decreased insulin-induced cell proliferation and promoted serum starvation-induced cell cycle arrest in G0/G1 phase in C2C12. Our results suggest that SKIP plays a negative regulatory role in skeletal muscle development partly by preventing cell proliferation. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiba, T.; Fisher, S.K.; Park, J.

The potential role of inositol phospholipid turnover in mediating acid secretion was examined in a preparation enriched for isolated canine gastric parietal cells. The stimulatory effects of carbamoylcholine (carbachol) and gastrin on parietal cell uptake of ({sup 14}C)aminopyrine were linked to dose- and time-dependent selective reduction in cellular phosphatidylinositol content, although the specific fatty acid composition of the phosphoinositides was not altered. Analysis of ({sup 3}H)inositol phosphates accumulated in cells prelabeled with ({sup 3}H)inositol revealed an increase in labeled inositol trisphosphate by 5 min of incubation with either carbachol or gastrin. Furthermore, after preincubation of parietal cells in medium containingmore » ({sup 32}P)orthophosphate, the two secretagogues elicited a time-dependent decrease in {sup 32}P labeling of phosphatidylinositol 4,5-bisphosphate and concomitant increase in labeling of phosphatidic acid. These data demonstrate that the acid secretagogue actions of carbachol and gastrin are correlated with turnover of cellular inositol phospholipids in a preparation consisting predominantly of parietal cells.« less

A Screen for Novel Phosphoinositide 3-kinase Effector Proteins*

PubMed Central

Dixon, Miles J.; Gray, Alexander; Boisvert, François-Michel; Agacan, Mark; Morrice, Nicholas A.; Gourlay, Robert; Leslie, Nicholas R.; Downes, C. Peter; Batty, Ian H.

2011-01-01

Class I phosphoinositide 3-kinases exert important cellular effects through their two primary lipid products, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). As few molecular targets for PtdIns(3,4)P2 have yet been identified, a screen for PI 3-kinase-responsive proteins that is selective for these is described. This features a tertiary approach incorporating a unique, primary recruitment of target proteins in intact cells to membranes selectively enriched in PtdIns(3,4)P2. A secondary purification of these proteins, optimized using tandem pleckstrin homology domain containing protein-1 (TAPP-1), an established PtdIns(3,4)P2 selective ligand, yields a fraction enriched in proteins of potentially similar lipid binding character that are identified by liquid chromatography-tandem MS. Thirdly, this approach is coupled to stable isotope labeling with amino acids in cell culture using differential isotope labeling of cells stimulated in the absence and presence of the PI 3-kinase inhibitor wortmannin. This provides a ratio-metric readout that distinguishes authentically responsive components from copurifying background proteins. Enriched fractions thus obtained from astrocytoma cells revealed a subset of proteins that exhibited ratios indicative of their initial, cellular responsiveness to PI 3-kinase activation. The inclusion among these of tandem pleckstrin homology domain containing protein-1, three isoforms of Akt, switch associated protein-70, early endosome antigen-1 and of additional proteins expressing recognized lipid binding domains demonstrates the utility of this strategy and lends credibility to the novel candidate proteins identified. The latter encompass a broad set of proteins that include the gene product of TBC1D2A, a putative Rab guanine nucleotide triphosphatase activating protein (GAP) and IQ motif containing GAP1, a potential tumor promoter. A sequence comparison of the former protein indicates the presence of a pleckstrin homology domain whose lipid binding character remains to be established. IQ motif containing GAP1 lacks known lipid interacting components and a preliminary analysis here indicates that this may exemplify a novel class of atypical phosphoinositide (aPI) binding domain. PMID:21263009

Inhibition of the pentose phosphate shunt by 2,3-diphosphoglycerate in erythrocyte pyruvate kinase deficiency.

PubMed

Tomoda, A; Lachant, N A; Noble, N A; Tanaka, K R

1983-07-01

Pentose phosphate shunt activity was studied by the release of 14CO2 from 14C-1-glucose and 14C-2-glucose in the red cells of five patients with pyruvate kinase deficiency and found to be significantly decreased after new methylene blue stimulation when compared to high reticulocyte controls. Incubated Heinz body formation was increased and the ascorbate cyanide test was positive in blood from these patients. The activity of glucose-6-phosphate dehydrogenase (G6PD) as well as that of 6-phosphogluconate dehydrogenase (6PGD) was inhibited to 20% of baseline in normal red cell haemolysate by 4 mM 2,3-diphosphoglycerate at pH 7.1. 2,3-Diphosphoglycerate was a competitive inhibitor with 6-phosphogluconate (Ki=1.05 mM) and a noncompetitive inhibitor with NADP (Ki=3.3 mM) for 6PGD. Since the intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate and NADP are below their Kms for G6PD and 6PGD, the kinetic data suggest that increased concentrations of 2,3-diphosphoglycerate in pyruvate kinase deficient red cells are sufficiently high to suppress pentose phosphate shunt activity. This suppression may be an additional factor contributing to the haemolytic anaemia of pyruvate kinase deficiency, particularly during periods of infection or metabolic stress.

Updating In Vivo and In Vitro Phosphorylation and Methylation Sites of Voltage-Gated Kv7.2 Potassium Channels.

PubMed

Erdem, Fatma Asli; Salzer, Isabella; Heo, Seok; Chen, Wei-Qiang; Jung, Gangsoo; Lubec, Gert; Boehm, Stefan; Yang, Jae-Won

2017-10-01

Voltage-gated Kv7.2 potassium channels regulate neuronal excitability. The gating of these channels is tightly controlled by various mediators and neurotransmitters acting via G protein-coupled receptors; the underlying signaling cascades involve phosphatidylinositol-4,5-bisphosphate (PIP 2 ), Ca 2+ /calmodulin, and phosphorylation. Recent studies found that the PIP 2 sensitivity of Kv7.2 channels is affected by two posttranslational modifications, phosphorylation and methylation, harboured within putative PIP 2 -binding domains. In this study, we updated phosphorylation and methylation sites in Kv7.2 either heterologously expressed in mammalian cells or as GST-fusion proteins exposed to recombinant protein kinases by using LC-MS/MS. In vitro kinase assays revealed that CDK5, protein kinase C (PKC) alpha, PKA, p38 MAPK, CamKIIα, and GSK3β could mediate phosphorylation. Taken together, we provided a comprehensive map of phosphorylation and methylation in Kv7.2 within protein-protein and protein-lipid interaction domains. This may help to interpret the functional roles of individual PTM sites in Kv7.2 channels. All MS data are available via ProteomeXchange with the identifier PXD005567. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

t10,c12-Conjugated linoleic acid stimulates mammary tumor progression in Her2/ErbB2 mice through activation of both proliferative and survival pathways

PubMed Central

Meng, Xiaojing; Shoemaker, Suzanne F.; McGee, Sibel O.; Ip, Margot M.

2008-01-01

The t10,c12 isomer of conjugated linoleic acid (CLA) inhibits rat mammary carcinogenesis, metastasis from a transplantable mouse mammary tumor and angiogenesis; however, it stimulates mammary tumorigenesis in transgenic mice overexpressing ErbB2 in the mammary epithelium (ErbB2 transgenic mice). In the current study, we report that a 4-week supplementation of the diet with 0.5% trans-10, cis-12 conjugated linoleic acid (t10,c12-CLA) stimulated the growth of established ErbB2-overexpressing mammary tumors by 30% and increased the number of new tumors from 11% to 82%. Additionally, when t10,c12-CLA supplementation of ErbB2 transgenic mice was initiated at 21 weeks of age, a time just prior to tumor appearance, overall survival was decreased from 46.4 weeks in the control to 39.0 weeks in the CLA group, and survival after detection of a palpable tumor from 7.5 to 4.6 weeks. Short-term supplementation from 10 to 14 weeks or 21 to 25 weeks of age temporarily accelerated tumor development, but over the long term, there was no significant effect on mammary tumorigenesis. Long term as well as a short 4-week supplementation increased mammary epithelial hyperplasia and lobular development, and altered the mammary stroma; this was reversible in mice returned to the control diet. t10,c12-CLA altered proliferation and apoptosis of the mammary epithelium, although this differed depending on the length of administration and/or the age of the mice. The increased tumor development with t10,c12-CLA was associated with increased phosphorylation of the IGF-I/insulin receptor, as well as increased signaling through the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways; however, neither phospho-ErbB2 nor ErbB2 was altered. PMID:18339686

Glutamine Enhances the Hypoglycemic Effect of Insulin in L6 Cells via Phosphatidylinositol-3-Kinase (PI3K)/Protein Kinase B (AKT)/Glucose Transporter 4 (GLUT4) Signaling Pathway.

PubMed

Wang, Caijuan; Deng, Yujiao; Yue, Yenan; Chen, Wenting; Zhang, Yu; Shi, Guifang; Wu, Zhongming

2018-03-01

BACKGROUND Diabetes mellitus (DM) is characterized by a decreased blood level of glutamine (Gln), which may contribute to the disturbance in the effect of insulin on skeletal muscle. Therefore, it is crucial to study how to improve the effect of insulin on skeletal muscle by increasing Gln. In the present study, we investigated the effect of Gln on the hypoglycemic action of insulin in skeletal muscle L6 cells at high glucose levels through the insulin signaling pathway and glycogen synthesis pathway. MATERIAL AND METHODS The L6 cells were cultured in and stimulated by Gln and insulin. The glutamine analogue, L-Gamma-Glutamyl-p-nitroanilide (GPNA), was used for verifying the effect of Gln. The expression of insulin signaling molecules, including phosphatidylinositol-3-kinase (PI3K), 3-phosphoinositide-dependent protein kinase-1 (PDK1), protein kinase B (AKT), protein kinase C zeta (PKCz), and glucose transporter 4 (GLUT4), were detected by real-time PCR and Western blot analysis, GLUT4 translocation was observed by immunofluorescence staining, glycogen synthase kinase (GSK) was analyzed by Western blotting, and glucose uptake was measured by glucose oxidase method (GOD). RESULTS The results demonstrated that Gln combined with insulin remarkably up-regulated PI3K and PDK1 and also increased AKT and PKCz phosphorylation. The present study shows that Gln enhanced the impact of insulin on GLUT4 and its translocation. The results of glucose uptake and GSK phosphorylation further confirmed the hypoglycemic effect of Gln accompanied with insulin. The hypoglycemic effect of Gln was reversed by GPNA. CONCLUSIONS These findings suggest that Gln enhances the hypoglycemic role of insulin through the PI3K/AKT/GLUT4 signaling pathway and glycogen synthesis pathway.

l-Ascorbic acid 2-phosphate promotes elongation of hair shafts via the secretion of insulin-like growth factor-1 from dermal papilla cells through phosphatidylinositol 3-kinase.

PubMed

Kwack, M H; Shin, S H; Kim, S R; Im, S U; Han, I S; Kim, M K; Kim, J C; Sung, Y K

2009-06-01

l-Ascorbic acid 2-phosphate (Asc 2-P), a derivative of l-ascorbic acid, promotes elongation of hair shafts in cultured human hair follicles and induces hair growth in mice. To investigate whether the promotion of hair growth by Asc 2-P is mediated by insulin-like growth factor-1 (IGF-1) and, if so, to investigate the mechanism of the Asc 2-P-induced IGF-1 expression. Dermal papilla (DP) cells were cultured and IGF-1 level was measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay after Asc 2-P treatment in the absence or presence of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Also, hair shaft elongation in cultured human scalp hair follicles and proliferation of cocultured keratinocytes were examined after Asc 2-P treatment in the absence or presence of neutralizing antibody against IGF-1. In addition, keratinocyte proliferation in cultured hair follicles after Asc 2-P treatment in the absence or presence of LY294002 was examined by Ki-67 immunostaining. IGF-1 mRNA in DP cells was upregulated and IGF-1 protein in the conditioned medium of DP cells was significantly increased after treatment with Asc 2-P. Immunohistochemical staining showed that IGF-1 staining is increased in the DP of cultured human hair follicles by Asc 2-P. The neutralizing antibody against IGF-1 significantly suppressed the Asc 2-P-mediated elongation of hair shafts in hair follicle organ culture and significantly attenuated Asc 2-P-induced growth of cocultured keratinocytes. LY294002 significantly attenuated Asc 2-P-inducible IGF-1 expression and proliferation of follicular keratinocytes in cultured hair follicles. These data show that Asc 2-P-inducible IGF-1 from DP cells promotes proliferation of follicular keratinocytes and stimulates hair follicle growth in vitro via PI3K.

Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

PubMed Central

Munir, Annum

2016-01-01

ABSTRACT 5′- and 3′-end-healing reactions are key steps in nucleic acid break repair in which 5′-OH ends are phosphorylated by a polynucleotide kinase (Pnk) and 3′-PO4 or 2′,3′-cyclic-PO4 ends are hydrolyzed by a phosphoesterase to generate the 5′-PO4 and 3′-OH termini required for sealing by classic polynucleotide ligases. End-healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2′,3′-phosphoesterase HD domain and a C-terminal 5′-OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5′-OH polynucleotides (9-mers or longer) in the presence of magnesium and any nucleoside triphosphate donor. HD-Pnk dephosphorylates RNA 2′,3′-cyclic phosphate, RNA 3′-phosphate, RNA 2′-phosphate, and DNA 3′-phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper, or cobalt. HD-Pnk homologs are present in genera from 11 bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. IMPORTANCE The present study provides insights regarding the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnk as the exemplar of a novel clade of dual 5′- and 3′-end-healing enzymes that phosphorylate 5′-OH termini and dephosphorylate 2′,3′-cyclic-PO4, 3′-PO4, and 2′-PO4 ends. The distinctive feature of HD-Pnk is its domain composition, i.e., a fusion of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, same domain order, and similar polypeptide sizes are distributed widely among genera from 11 bacterial phyla. PMID:27895092

Modulation of Phosphorylation of Tocopherol and Phosphatidylinositol by hTAP1/SEC14L2-Mediated Lipid Exchange

PubMed Central

Zingg, Jean-Marc; Libinaki, Roksan; Meydani, Mohsen; Azzi, Angelo

2014-01-01

The vitamin E derivative, alpha-tocopheryl phosphate (αTP), is detectable in cultured cells, plasma and tissues in small amounts, suggesting the existence of enzyme(s) with α-tocopherol (αT) kinase activity. Here, we characterize the production of αTP from αT and [γ-32P]-ATP in primary human coronary artery smooth muscle cells (HCA-SMC) using separation by thin layer chromatography (TLC) and subsequent analysis by Ultra Performance Liquid Chromatography (UPLC). In addition to αT, although to a lower amount, also γT is phosphorylated. In THP-1 monocytes, γTP inhibits cell proliferation and reduces CD36 scavenger receptor expression more potently than αTP. Both αTP and γTP activate the promoter of the human vascular endothelial growth factor (VEGF) gene with similar potency, whereas αT and γT had no significant effect. The recombinant human tocopherol associated protein 1 (hTAP1, hSEC14L2) binds both αT and αTP and stimulates phosphorylation of αT possibly by facilitating its transport and presentation to a putative αT kinase. Recombinant hTAP1 reduces the in vitro activity of the phosphatidylinositol-3-kinase gamma (PI3Kγ) indicating the formation of a stalled/inactive hTAP1/PI3Kγ heterodimer. The addition of αT, βT, γT, δT or αTP differentially stimulates PI3Kγ, suggesting facilitated egress of sequestered PI from hTAP1 to the enzyme. It is suggested that the continuous competitive exchange of different lipophilic ligands in hTAPs with cell enzymes and membranes may be a way to make these lipophiles more accessible as substrates for enzymes and as components of specific membrane domains. PMID:24983950

Dynamic formation of ER–PM junctions presents a lipid phosphatase to regulate phosphoinositides

PubMed Central

Jensen, Jill B.; Vivas, Oscar; Kruse, Martin; Traynor-Kaplan, Alexis E.; Hille, Bertil

2016-01-01

Endoplasmic reticulum–plasma membrane (ER–PM) contact sites play an integral role in cellular processes such as excitation–contraction coupling and store-operated calcium entry (SOCE). Another ER–PM assembly is one tethered by the extended synaptotagmins (E-Syt). We have discovered that at steady state, E-Syt2 positions the ER and Sac1, an integral ER membrane lipid phosphatase, in discrete ER–PM junctions. Here, Sac1 participates in phosphoinositide homeostasis by limiting PM phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2. Activation of G protein–coupled receptors that deplete PM PI(4,5)P2 disrupts E-Syt2–mediated ER–PM junctions, reducing Sac1’s access to the PM and permitting PM PI(4)P and PI(4,5)P2 to recover. Conversely, depletion of ER luminal calcium and subsequent activation of SOCE increases the amount of Sac1 in contact with the PM, depleting PM PI(4)P. Thus, the dynamic presence of Sac1 at ER–PM contact sites allows it to act as a cellular sensor and controller of PM phosphoinositides, thereby influencing many PM processes. PMID:27044890

Time-resolved Ultrastructural Detection of Phosphatidylinositol 3-phosphate

PubMed Central

Stuffers, Susanne; Malerød, Lene; Schink, Kay Oliver; Corvera, Silvia; Stenmark, Harald; Brech, Andreas

2010-01-01

Phosphatidylinositol 3-phosphate [PtdIns(3)P] plays an important role in recruitment of various effector proteins in the endocytic and autophagic pathways. In an attempt to follow the distribution of PtdIns(3)P at the ultrastructural level, we are using the Fab1, YOTB, Vac1, and EEA1 (FYVE) domain, which is a zinc finger motif specifically binding to PtdIns(3)P. To follow PtdIns(3)P trafficking during a defined time window, here we have used a monomeric dimerizable FYVE probe, which binds with high avidity to PtdIns(3)P only after rapalog-induced dimerization. The probe localized to early and late endocytic compartments according to the time period of dimerization, which indicates that PtdIns(3)P is turned over via the endocytic machinery. In the functional context of epidermal growth factor (EGF) stimulation, we observed that dimerization of the probe led to clustering of mainly early endocytic structures, leaving most of the probe localized to the limiting membrane of endosomes. Interestingly, these clustered endosomes contained coats positive for the PtdIns(3)P-binding protein hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs), indicating that the probe did not displace Hrs binding. We conclude that the dimerizer-inducible probe is useful for the time-resolved detection of PtdIns(3)P at the ultrastructural level, but its effects on endosome morphology after EGF stimulation need to be taken into account. (J Histochem Cytochem 58:1025–1032, 2010) PMID:20713985

Psoralen and Ultraviolet A Light Treatment Directly Affects Phosphatidylinositol 3-Kinase Signal Transduction by Altering Plasma Membrane Packing*

PubMed Central

Van Aelst, Britt; Devloo, Rosalie; Zachée, Pierre; t'Kindt, Ruben; Sandra, Koen; Vandekerckhove, Philippe; Compernolle, Veerle; Feys, Hendrik B.

2016-01-01

Psoralen and ultraviolet A light (PUVA) are used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma, and graft-versus-host disease. DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery, they are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like amphipathic lipid-packing sensor and α-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton's tyrosine kinase effectors following activation of the collagen glycoprotein VI and thrombin protease-activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol 3,4,5-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (effective half-maximal concentration (EC50), 8.4 ± 1.1 versus 4.3 ± 1.1 μm) and glycoprotein VI (EC50, 1.61 ± 0.85 versus 0.26 ± 0.21 μg/ml) but not PAR4 (EC50, 50 ± 1 versus 58 ± 1 μm) signal transduction. Our findings were confirmed in T-cells from graft-versus-host disease patients treated with extracorporeal photopheresis, a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids, thereby inhibiting membrane recruitment of effector kinases. PMID:27687726

Phorbol Ester Effects on Neurotransmission: Interaction with Neurotransmitters and Calcium in Smooth Muscle

NASA Astrophysics Data System (ADS)

Baraban, Jay M.; Gould, Robert J.; Peroutka, Stephen J.; Snyder, Solomon H.

1985-01-01

Stimulation of the phosphatidylinositol cycle by neurotransmitters generates diacylglycerol, an activator of protein kinase C, which may regulate some forms of neurotransmission. Phorbol esters, potent inflammatory and tumorpromoting compounds, also activate protein kinase C. We demonstrate potent and selective effects of phorbol esters on smooth muscle, indicating a role for protein kinase C in neurotransmission. In rat vas deferens and dog basilar artery, phorbol esters synergize with calcium to mimic the contractile effects of neurotransmitters that act through the phosphatidylinositol cycle. In guinea pig ileum and rat uterus, phorbol esters block contractions produced by these neurotransmitters.

Tec-kinase-mediated phosphorylation of fibroblast growth factor 2 is essential for unconventional secretion.

PubMed

Ebert, Antje D; Laussmann, Mareike; Wegehingel, Sabine; Kaderali, Lars; Erfle, Holger; Reichert, Jürgen; Lechner, Johannes; Beer, Hans-Dietmar; Pepperkok, Rainer; Nickel, Walter

2010-06-01

Fibroblast growth factor 2 (FGF2) is a potent mitogen that is exported from cells by an endoplasmic reticulum (ER)/Golgi-independent mechanism. Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes, a process that depends on the phosphoinositide phosphatidylinositol 4,5-biphosphate (PI(4,5)P(2)) at the inner leaflet as well as heparan sulfate proteoglycans at the outer leaflet of plasma membranes; however, additional core and regulatory components of the FGF2 export machinery have remained elusive. Here, using a highly effective RNAi screening approach, we discovered Tec kinase as a novel factor involved in unconventional secretion of FGF2. Tec kinase does not affect FGF2 secretion by an indirect mechanism, but rather forms a heterodimeric complex with FGF2 resulting in phosphorylation of FGF2 at tyrosine 82, a post-translational modification shown to be essential for FGF2 membrane translocation to cell surfaces. Our findings suggest a crucial role for Tec kinase in regulating FGF2 secretion under various physiological conditions and, therefore, provide a new perspective for the development of a novel class of antiangiogenic drugs targeting the formation of the FGF2/Tec complex.

Anti-apoptotic effect of heat shock protein 90 on hypoxia-mediated cardiomyocyte damage is mediated via the phosphatidylinositol 3-kinase/AKT pathway.

PubMed

Wang, Wei; Peng, Yizhi; Wang, Yuanyuan; Zhao, Xiaohui; Yuan, Zhiqiang

2009-09-01

1. Hypoxia-induced cardiomyocyte apoptosis contributes significantly to cardiac dysfunction following trauma, shock and burn injury. There is evidence that heat shock protein (HSP) 90 is anti-apoptotic in cardiomyocytes subjected to a variety of apoptotic stimuli. Because HSP90 acts as an upstream regulator of the serine/threonine protein kinase Akt survival pathway during cellular stress, we hypothesized that HSP90 exerts a cardioprotective effect via the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. 2. Neonatal rat cardiomyocytes were subjected to normoxia or hypoxia in the absence or presence of the HSP90 inhibitor geldanamycin (1 μg/mL). Cardiomyocyte apoptosis was assessed by release of lactate dehydrogenase (LDH), terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) staining and caspase 3 activity. Expression of HSP90, Akt, Bad and cytochrome c release was determined by western blot analysis. 3. Following exposure of cells to hypoxia, HSP90 was markedly elevated in a time-dependent manner, reaching a peak at 6 h (eightfold increase). Geldanamycin significantly increased hypoxia-induced release of LDH by 114%, the percentage of apoptotic cardiomyocytes by 102% and caspase 3 activity by 78%. Pretreatment of cells with geldanamycin also suppressed phosphorylation of both Akt and its downstream target Bad, but promoted the mitochondrial release of cytochrome c. 4. In conclusion, HSP90 activity is enhanced in cardiomyocytes following hypoxic insult. The anti-apoptotic effect of HSP90 on cardiomyocytes subjected to hypoxia is mediated, at least in part, by the PI3-K/Akt pathway. Key words: apoptosis, cardiomyocyte, heart failure, heat shock protein 90, hypoxia, phosphatidylinositol 3-kinase/Akt signalling pathway, serine/threonine protein kinase Akt.

Up-regulated expression of WNT5a increases inflammation and oxidative stress via PI3K/AKT/NF-κB signaling in the granulosa cells of PCOS patients.

PubMed

Zhao, Yue; Zhang, Chunmei; Huang, Ying; Yu, Yang; Li, Rong; Li, Min; Liu, Nana; Liu, Ping; Qiao, Jie

2015-01-01

Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder accompanied by chronic low-grade inflammation, but the molecular mechanism remains unclear. We investigated the action of WNT5a in the development of chronic inflammation in PCOS and the related molecular signaling pathways. This was a prospective study conducted at the Division of Reproduction Center, Peking University Third Hospital. A total of 35 PCOS patients and 87 control women who reported to the clinic for the in vitro procedure and the cause of marital infertility was male azoospermia were included. Mural granulosa cells (GCs) of 35 PCOS patients and 37 controls were collected during oocyte retrieval and gene expression was analyzed. The human KGN cells and mural GCs from 50 control subjects (six to eight samples were pooled together for each experiment) were cultured in vitro. The regulation of inflammation and oxidative stress was confirmed by quantitative PCR, flow-cytometric assay, and dual-luciferase reporter assay after inflammatory stimuli or WNT5a overexpression. Relevant signaling pathways were identified using specific inhibitors. Our data demonstrate significantly elevated WNT5a expression in the mural GCs of PCOS patients compared with the controls. Lipopolysaccharide stimulation increased WNT5a expression in KGN cells and mural GCs, and BAY-117082 and pyrrolidinedithiocarbamic acid [nuclear factor-κB (NF-κB) inhibitor] treatments suppressed WNT5a mRNA below the control level. WNT5a overexpression also enhanced the expression of inflammation-related genes and increased intracellular reactive oxygen species, whereas both BAY-117082 and LY-294002 (phosphatidylinositol 3-kinase inhibitor) significantly inhibited WNT5a-induced inflammation and oxidative stress. WNT5a acts as a proinflammatory factor in human ovarian GCs. The up-regulated expression of WNT5a in PCOS increases inflammation and oxidative stress predominantly via the phosphatidylinositol 3-kinase/AKT/NF-κB signaling pathway. The proinflammatory cytokines induced might further enhance WNT5a expression via NF-κB-dependent regulation, indicating a novel regulatory system for chronic inflammation in PCOS.

Ursolic Acid Increases Glucose Uptake through the PI3K Signaling Pathway in Adipocytes

PubMed Central

He, Yonghan; Li, Wen; Li, Ying; Zhang, Shuocheng; Wang, Yanwen; Sun, Changhao

2014-01-01

Background Ursolic acid (UA), a triterpenoid compound, is reported to have a glucose-lowering effect. However, the mechanisms are not fully understood. Adipose tissue is one of peripheral tissues that collectively control the circulating glucose levels. Objective The objective of the present study was to determine the effect and further the mechanism of action of UA in adipocytes. Methods and Results The 3T3-L1 preadipocytes were induced to differentiate and treated with different concentrations of UA. NBD-fluorescent glucose was used as the tracer to measure glucose uptake and Western blotting used to determine the expression and activity of proteins involved in glucose transport. It was found that 2.5, 5 and 10 µM of UA promoted glucose uptake in a dose-dependent manner (17%, 29% and 35%, respectively). 10 µM UA-induced glucose uptake with insulin stimulation was completely blocked by the phosphatidylinositol (PI) 3-kinase (PI3K) inhibitor wortmannin (1 µM), but not by SB203580 (10 µM), the inhibitor of mitogen-activated protein kinase (MAPK), or compound C (2.5 µM), the inhibitor of AMP-activated kinase (AMPK) inhibitor. Furthmore, the downstream protein activities of the PI3K pathway, phosphoinositide-dependent kinase (PDK) and phosphoinositide-dependent serine/threoninekinase (AKT) were increased by 10 µM of UA in the presence of insulin. Interestingly, the activity of AS160 and protein kinase C (PKC) and the expression of glucose transporter 4 (GLUT4) were stimulated by 10 µM of UA under either the basal or insulin-stimulated status. Moreover, the translocation of GLUT4 from cytoplasm to cell membrane was increased by UA but decreased when the PI3K inhibitor was applied. Conclusions Our results suggest that UA stimulates glucose uptake in 3T3-L1 adipocytes through the PI3K pathway, providing important information regarding the mechanism of action of UA for its anti-diabetic effect. PMID:25329874

PIKfyve mediates the motility of late endosomes and lysosomes in neuronal dendrites.

PubMed

Tsuruta, Fuminori; Dolmetsch, Ricardo E

2015-09-25

The endosome/lysosome system in the nervous system is critically important for a variety of neuronal functions such as neurite outgrowth, retrograde transport, and synaptic plasticity. In neurons, the endosome/lysosome system is crucial for the activity-dependent internalization of membrane proteins and contributes to the regulation of lipid level on the plasma membrane. Although homeostasis of membrane dynamics plays important roles in the properties of central nervous systems, it has not been elucidated how endosome/lysosome system is regulated. Here, we report that phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) mediates the motility of late endosomes and lysosomes in neuronal dendrites. Endosomes and lysosomes are highly motile in resting neurons, however knockdown of PIKfyve led to a significant reduction in late endosomes and lysosomes motility. We also found that vesicle acidification is crucial for their motility and PIKfyve is associated with this process indirectly. These data suggest that PIKfyve mediates vesicle motility through the regulation of vesicle integrity in neurons. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Leukemia Inhibitory Factor Enhances Endogenous Cardiomyocyte Regeneration after Myocardial Infarction

PubMed Central

Kanda, Masato; Nagai, Toshio; Takahashi, Toshinao; Liu, Mei Lan; Kondou, Naomichi; Naito, Atsuhiko T.; Akazawa, Hiroshi; Sashida, Goro; Iwama, Atsushi; Komuro, Issei; Kobayashi, Yoshio

2016-01-01

Cardiac stem cells or precursor cells regenerate cardiomyocytes; however, the mechanism underlying this effect remains unclear. We generated CreLacZ mice in which more than 99.9% of the cardiomyocytes in the left ventricular field were positive for 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) staining immediately after tamoxifen injection. Three months after myocardial infarction (MI), the MI mice had more X-gal-negative (newly generated) cells than the control mice (3.04 ± 0.38/mm2, MI; 0.47 ± 0.16/mm2, sham; p < 0.05). The cardiac side population (CSP) cell fraction contained label-retaining cells, which differentiated into X-gal-negative cardiomyocytes after MI. We injected a leukemia inhibitory factor (LIF)-expression construct at the time of MI and identified a significant functional improvement in the LIF-treated group. At 1 month after MI, in the MI border and scar area, the LIF-injected mice had 31.41 ± 5.83 X-gal-negative cardiomyocytes/mm2, whereas the control mice had 12.34 ± 2.56 X-gal-negative cardiomyocytes/mm2 (p < 0.05). Using 5-ethynyl-2'-deoxyurinide (EdU) administration after MI, the percentages of EdU-positive CSP cells in the LIF-treated and control mice were 29.4 ± 2.7% and 10.6 ± 3.7%, respectively, which suggests that LIF influenced CSP proliferation. Moreover, LIF activated the Janus kinase (JAK)signal transducer and activator of transcription (STAT), mitogen-activated protein kinase/extracellular signal-regulated (MEK)extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)–AKT pathways in CSPs in vivo and in vitro. The enhanced green fluorescent protein (EGFP)-bone marrow-chimeric CreLacZ mouse results indicated that LIF did not stimulate cardiogenesis via circulating bone marrow-derived cells during the 4 weeks following MI. Thus, LIF stimulates, in part, stem cell-derived cardiomyocyte regeneration by activating cardiac stem or precursor cells. This approach may represent a novel therapeutic strategy for cardiogenesis. PMID:27227407

Phosphorylation of Wheat Germ Initiation Factors and Ribosomal Proteins 1

PubMed Central

Browning, Karen S.; Yan, Tyan Fuh J.; Lauer, Stephen J.; Aquino, Lu Ann; Tao, Mariano; Ravel, Joanne M.

1985-01-01

The ability of the wheat germ initiation factors and ribosomes to serve as substrates for a wheat germ protein kinase (Yan and Tao 1982 J Biol Chem 257: 7037-7043) has been investigated. The wheat germ kinase catalyzes the phosphorylation of the 42,000 dalton subunit of eukaryotic initiation factor (eIF)-2 and the 107,000 dalton subunit of eIF-3. Other initiation factors, eIF-4B and eIF-4A, and elongation factors, EF-1 and EF-2, are not phosphorylated by the kinase. Quantitative analysis indicates that the kinase catalyzes the incorporation of about 0.5 to 0.6 mole of phosphate per mole of the 42,000 dalton subunit of eIF-2 and about 6 moles of phosphate per mole of the 107,000 dalton subunit of eIF-3. Three proteins (Mr = 38,000, 14,800, and 12,600) of the 60S ribosomal subunit are phosphorylated by the kinase, but none of the 40S ribosomal proteins are substrates of the kinase. No effects of phosphorylation on the activities of eIF-2, eIF-3, or 60S ribosomal subunits could be demonstrated in vitro. Images Fig. 1 Fig. 3 Fig. 4 PMID:16664060

The role of phosphatidylinositol-3-OH-kinase (PI3-kinase) and respiratory burst enzymes in the [omim][BF4]-mediated toxic mode of action in mussel hemocytes.

PubMed

Belavgeni, Alexia; Dailianis, Stefanos

2017-09-01

The present study investigates the role of phosphatidylinositol-3-OH-kinase (PI3-kinase) and respiratory burst enzymes, NADPH oxidase and NO synthase, in the 1-methyl-3-octylimidazolium tetrafluoroborate ([omim][BF 4 ])-mediated toxic mode of action in mussel hemocytes. Specifically, cell viability (using the neutral red uptake assay) was primarily tested in hemocytes treated with different concentrations of [omim][BF 4 ] (0.1-10 mg L -1 ) and thereafter [omim][BF 4 ]-mediated oxidative (in terms of superoxide anions/O 2 - and nitric oxide/NO generation, as well as the enhancement of lipid peroxidation by-products, in terms of malondialdehyde/MDA) and genotoxic (in terms of DNA damage) effects were determined in hemocytes treated with 1 mg L -1 [omim][BF 4 ]. Moreover, in order to investigate, even indirectly and non-entirely specific, the role of PI3-kinase, NADPH oxidase and NO synthase, the [omim][BF 4 ]-mediated effects were also investigated in hemocytes pre-incubated with wortmannin (50 nM), diphenyleneiodonium chloride (DPI 10 μM) and N G -nitro- l -arginine methyl ester (l-NAME 10 μM), respectively. The results showed that [omim][BF 4 ] ability to enhance O 2 - , NO, MDA and DNA damage, via its interaction with cellular membranes, was significantly attenuated in the presence of each inhibitor in almost all cases. The current findings revealed for the first time that certain signaling molecules, such as PI3-kinase, as well as respiratory burst enzymes activation, such as NADPH oxidase and NO synthase, could merely attribute to the [omim][BF 4 ]-mediated mode of action, thus enriching our knowledge for the molecular mechanisms of ILs toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

PI3Kδ promotes CD4(+) T-cell interactions with antigen-presenting cells by increasing LFA-1 binding to ICAM-1.

PubMed

Garçon, Fabien; Okkenhaug, Klaus

2016-05-01

Activation of T lymphocytes by peptide/major histocompatibility complex on antigen-presenting cells (APCs) involves dynamic contacts between the two cells, during which T cells undergo marked morphological changes. These interactions are facilitated by integrins. Activation of the T cells increases the binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) expressed by T cells to intercellular adhesion molecule (ICAM)-1 and ICAM-2 expressed by APCs. The signalling pathways that control integrin affinities are incompletely defined. The phosphoinositide 3-kinases (PI3Ks) generate second-messenger signalling molecules that control cell growth, proliferation, differentiation and trafficking. Here we show that in T cells, PI3Kδ attenuates the activation of Rac1, but sustains the activation of Rap1. Consequently, PI3Kδ increases LFA-1-dependent adhesion to form stable conjugates with APCs. Increased Rap1 activity and LFA-1 adhesion were only in part mediated by the downstream kinase Akt, suggesting the involvement of additional phosphatidylinositol(3,4,5)P3-binding proteins. These results establish a link between PI3K activity, cytoskeletal changes and integrin binding and help explain the impaired T-cell-dependent immune responses in PI3Kδ-deficient mice.

Exercise Training Stimulates Ischemia-Induced Neovascularization via Phosphatidylinositol 3-Kinase/Akt-Dependent Hypoxia-Induced Factor-1α Reactivation in Mice of Advanced Age

PubMed Central

Cheng, Xian Wu; Kuzuya, Masafumi; Kim, Weon; Song, Haizhen; Hu, Lina; Inoue, Aiko; Nakamura, Kae; Di, Qun; Sasaki, Takeshi; Tsuzuki, Michitaka; Shi, Guo-Ping; Okumura, Kenji; Murohara, Toyoaki

2011-01-01

Background Exercise stimulates the vascular response in pathological conditions, including ischemia; however, the molecular mechanisms by which exercise improves the impaired hypoxia-induced factor (HIF)-1α–mediated response to hypoxia associated with aging are poorly understood. Here, we report that swimming training (ST) modulates the vascular response to ischemia in aged (24-month-old) mice. Methods and Results Aged wild-type mice (MMP-2+/+) that maintained ST (swimming 1 h/d) from day 1 after surgery were randomly assigned to 4 groups that were treated with either vehicle, LY294002, or deferoxamine for 14 days. Mice that were maintained in a sedentary condition served as controls. ST increased blood flow, capillary density, and levels of p-Akt, HIF-1α, vascular endothelial growth factor, Fit-1, and matrix metalloproteinase-2 (MMP-2) in MMP-2+/+ mice. ST also increased the numbers of circulating endothelial progenitor cells and their function associated with activation of HIF-1α. All of these effects were diminished by LY294002, an inhibitor of phosphatidylinositol 3-kinase; enhanced by deferoxamine, an HIF-1α stabilizer; and impaired by knockout of MMP-2. Finally, bone marrow transplantation confirmed that ST enhanced endothelial progenitor cell homing to ischemic sites in aged mice. Conclusions ST can improve neovascularization in response to hypoxia via a phosphatidylinositol 3-kinase–dependent mechanism that is mediated by the HIF-1α/vascular endothelial growth factor/MMP-2 pathway in advanced age. PMID:20679550

Sorting nexin-2 is associated with tubular elements of the early endosome, but is not essential for retromer-mediated endosome-to-TGN transport

PubMed Central

Carlton, Jez G.; Bujny, Miriam V.; Peter, Brian J.; Oorschot, Viola M. J.; Rutherford, Anna; Arkell, Rebecca S.; Klumperman, Judith; McMahon, Harvey T.; Cullen, Peter J.

2006-01-01

Summary Sorting nexins are a large family of phox-homology-domain-containing proteins that have been implicated in the control of endosomal sorting. Sorting nexin-1 is a component of the mammalian retromer complex that regulates retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network. In yeast, retromer is composed of Vps5p (the orthologue of sorting nexin-1), Vps17p (a related sorting nexin) and a cargo selective subcomplex composed of Vps26p, Vps29p and Vps35p. With the exception of Vps17p, mammalian orthologues of all yeast retromer components have been identified. For Vps17p, one potential mammalian orthologue is sorting nexin-2. Here we show that, like sorting nexin-1, sorting nexin-2 binds phosphatidylinositol 3-monophosphate and phosphatidylinositol 3,5-bisphosphate, and possesses a Bin/Amphiphysin/Rvs domain that can sense membrane curvature. However, in contrast to sorting nexin-1, sorting nexin-2 could not induce membrane tubulation in vitro or in vivo. Functionally, we show that endogenous sorting nexin-1 and sorting nexin-2 co-localise on high curvature tubular elements of the 3-phosphoinositide-enriched early endosome, and that suppression of sorting nexin-2 does not perturb the degradative sorting of receptors for epidermal growth factor or transferrin, nor the steady-state distribution of the cation-independent mannose 6-phosphate receptor. However, suppression of sorting nexin-2 results in a subtle alteration in the kinetics of cation-independent mannose 6-phosphate receptor retrieval. These data suggest that although sorting nexin-2 may be a component of the retromer complex, its presence is not essential for the regulation of endosome-to-trans Golgi network retrieval of the cation-independent mannose 6-phosphate receptor. PMID:16179610

Idelalisib, a selective inhibitor of phosphatidylinositol 3-kinase-δ, as therapy for previously treated indolent non-Hodgkin lymphoma

PubMed Central

Kahl, Brad S.; Leonard, John P.; Furman, Richard R.; Brown, Jennifer R.; Byrd, John C.; Wagner-Johnston, Nina D.; Coutre, Steve E.; Benson, Don M.; Peterman, Sissy; Cho, Yoonjin; Webb, Heather K.; Johnson, David M.; Yu, Albert S.; Ulrich, Roger G.; Godfrey, Wayne R.; Miller, Langdon L.; Spurgeon, Stephen E.

2014-01-01

Idelalisib (GS-1101, CAL-101), an oral inhibitor of phosphatidylinositol 3-kinase-δ, was evaluated in a phase I study in 64 patients with relapsed indolent non-Hodgkin lymphoma (iNHL). Patients had a median (range) age of 64 (32-91) years, 34 (53%) had bulky disease (≥1 lymph nodes ≥5 cm), and 37 (58%) had refractory disease. Patients had received a median (range) of 4 (1-10) prior therapies. Eight dose regimens of idelalisib were evaluated; idelalisib was taken once or twice daily continuously at doses ranging from 50 to 350 mg. After 48 weeks, patients still benefitting (n = 19; 30%) enrolled into an extension study. Adverse events (AEs) occurring in 20% or more patients (total%/grade ≥3%) included diarrhea (36/8), fatigue (36/3), nausea (25/3), rash (25/3), pyrexia (20/3), and chills (20/0). Laboratory abnormalities included neutropenia (44/23), anemia (31/5), thrombocytopenia (25/11), and serum transaminase elevations (48/25). Twelve (19%) patients discontinued therapy due to AEs. Idelalisib induced disease regression in 46/54 (85%) of evaluable patients achieving an overall response rate of 30/64 (47%), with 1 patient having a complete response (1.6%). Median duration of response was 18.4 months, median progression-free survival was 7.6 months. Idelalisib is well tolerated and active in heavily pretreated, relapsed/refractory patients with iNHL. These trials were registered at clinicaltrials.gov as NCT00710528 and NCT01090414. PMID:24615776

Atrial natriuretic peptide administered just prior to reperfusion limits infarction in rabbit hearts.

PubMed

Yang, Xi-Ming; Philipp, Sebastian; Downey, James M; Cohen, Michael V

2006-07-01

We investigated whether atrial natriuretic peptide (ANP) given just prior to reperfusion reduces infarction in rabbit hearts and whether protection is related to activation of protein kinase G (PKG). Isolated rabbit hearts were subjected to a 30-min period of regional ischemia; treated hearts received a 20-min infusion of ANP (0.1 microM) starting 5 min before 2 h of reperfusion. ANP infusion decreased infarction from 31.5+/-2.4% of the risk zone in untreated hearts to 12.5+/-2.0% (P<0.001). To explore mechanisms of protection ischemic hearts were treated simultaneously with ANP and isatin, a blocker of the natriuretic peptide receptor, shortly before reperfusion. ANP's protective effect was aborted (36.8+/-2.9% infarction). There is no acceptable blocker of protein kinase G that can be used in intact organs. However, 8-(4-chlorophenylthio)-guanosine 3', 5'-cyclic monophosphate (10 microM), a cell-permeable cGMP analog that directly activates PKG, was infused from 5 min before to 15 min after reperfusion. The PKG activator mimicked ANP's protection with only 18.2+/-3.6% infarction (P<0.001). 5-Hydroxyde-canoate (5-HD), a putative mitochondrial KATP channel (mKATP) inhibitor, abrogated ANP's protection (34.4+/-2.6% infarction). Unexpectedly, 1H-[1,2,4]oxadiazole- [4,3-a]quinoxalin-1-one (ODQ), a blocker of soluble guanylyl cyclase also prevented ANP's infarct-sparing effect. It is unclear whether this observation implicated participation of soluble guanylyl cyclase in the mechanism or simply a lack of selectivity of ODQ. Finally the reperfusion injury salvage kinases (RISK), phosphatidylinositol 3-kinase and extracellular signal-regulated kinase, were implicated in ANP's mechanism since either wortmannin or PD98059 infused at reperfusion prevented ANP's infarct-sparing effect. ANP administered just prior to reperfusion protects hearts against infarction, likely by activation of PKG, opening of mKATP, and stimulation of downstream kinases.

τ kinases in the rat heat shock model: Possible implications for Alzheimer disease

PubMed Central

Shanavas, Alikunju; Papasozomenos, Sozos Ch.

2000-01-01

We have previously shown, by using the phosphate-dependent anti-τ antibodies Tau-1 and PHF-1, that heat shock induces rapid dephosphorylation of τ followed by hyperphosphorylation in female rats. In this study, we analyzed in forebrain homogenates from female Sprague–Dawley rats the activities of extracellular signal regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), glycogen synthase kinase-3β (GSK-3β), cyclin-dependent kinase 5 (Cdk5), cAMP-dependent protein kinase A (PKA), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) at 0 (n = 5), 3 (n = 4), 6 (n = 5), and 12 (n = 5) h after heat shock and in non-heat-shocked controls (n = 5). Immunoprecipitation kinase assays at 0 h showed suppression of the activities of all kinases except of GSK-3β, which showed increased activity. At 3–6 h, the activities of ERK1/2, JNK, Cdk5, and GSK-3β toward selective substrates were increased; however, only JNK, Cdk5, and GSK-3β but not ERK1/2 were overactivated toward purified bovine τ. At 3–6 h, kinase assays specific for PKA and CaMKII showed no increased activity toward either τ or selective substrates. All of eight anti-τ antibodies tested showed dephosphorylation at 0 h and hyperphosphorylation at 3–6 h, except for 12E8, which showed hyperphosphorylation also at 0 h. Immunoblot analysis using activity-dependent antibodies against ERK1/2, JNK, and GSK-3β confirmed the above data. Increased activation and inhibition of kinases after heat shock were statistically significant in comparison with controls. Because τ is hyperphosphorylated in Alzheimer disease these findings suggest that JNK, GSK-3β, and Cdk5 may play a role in its pathogenesis. PMID:11121021

The p110α and p110β Isoforms of Class I Phosphatidylinositol 3-Kinase Are Involved in Toll-Like Receptor 5 Signaling in Epithelial Cells

PubMed Central

Ivison, Sabine M.; Khan, Mohammed A. S.; Graham, Nicholas R.; Shobab, Leila A.; Yao, Yu; Kifayet, Arnawaz; Sly, Laura M.; Steiner, Theodore S.

2010-01-01

Background. Bacterial flagellin triggers inflammation in mammalian cells via Toll-like receptor (TLR) 5. Release of the chemokine IL-8 in response to flagellin involves NF-κB, p38 MAP kinase, and phosphatidylinositol 3-kinase (PI3K). However, PI3K has been reported to be either pro- or anti-inflammatory in different model systems. We hypothesized that this could be due to different activities of the p110α and β isoforms of PI3K. Results. PI3K and Akt were rapidly activated in Caco-2 colon carcinoma cells by flagellin. Using a plasmid-based shRNA delivery system and novel p110 isoform-specific inhibitors, we found that flagellin-induced IL-8 production was dependent on both p110α and p110β. However in the mouse, inhibition of p110β but not p110α reduced the increase of serum IL-6 levels induced by intraperitoneal injection of flagellin. Conclusions. These data demonstrate that the p110α and β isoforms of class IA PI3K are both required for the proinflammatory response to flagellin. PMID:20953381

mGluR5 stimulating Homer–PIKE formation initiates icariin induced cardiomyogenesis of mouse embryonic stem cells by activating reactive oxygen species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Limin; Huang, Yujie; Zhang, Yingying

2013-06-10

Icariin (ICA) has been reported to facilitate cardiac differentiation of mouse embryonic stem (ES) cells; however, the mechanism by which ICA induced cardiomyogenesis has not been fully elucidated yet. Here, an underlying signaling network including metabotropic glutamate receptor 5 (mGluR5), Homer, phosphatidylinositol 3-Kinase Enhancer (PIKE), phosphatidylinositol 3-Kinase (PI3K), reactive oxygen species (ROS) and nuclear factor-kappaB (NF-κB) was investigated in ICA induced cardiomyogenesis. Our results showed that the co-expression of mGluR5 together with α-actinin or Troponin T in embryoid bodies (EBs) treated with ICA was elevated to 10.86% and 9.62%, compared with the case in the control (4.04% and 3.45%, respectively).more » Exposure of EBs to ICA for 2 h remarkably increased the dimeric form of mGluR5, which was inhibited by small interfering RNA targeting mGluR5 (si-mGluR5). Moreover, the extracellular glutamate concentration in ICA treatment medium was elevated to 28.9±3.5 μM. Furthermore, the activation of mGluR5 by ICA triggered the formation of Homer–PIKE complex and activated PI3K, stimulating ROS generation and NF-κB nuclear translocation. Knockdown of mGluR5 or inhibition of PI3K by LY294002 blocked ICA induced cardiomyogenesis via repressing mGluR5 pathway, reducing ROS and NF-κB activation. These results revealed that the inducible mechanisms of ICA were related to activate mGluR5 pathway. -- Highlights: • ICA increased mGluR5 expression in cardiac differentiation of ES cells. • ICA enhanced the glutamate level and the receptor mGluR5 dimerization, stimulating the formation of Homer–PIKE complex. • Knockdown of mGluR5 or inhibition of PI3K by LY294002 inhibited ICA induced ROS generation and NF-κB nuclear translocation.« less

AMPK regulates autophagy by phosphorylating BECN1 at threonine 388.

PubMed

Zhang, Deyi; Wang, Wei; Sun, Xiujie; Xu, Daqian; Wang, Chenyao; Zhang, Qian; Wang, Huafei; Luo, Wenwen; Chen, Yan; Chen, Huaiyong; Liu, Zhixue

2016-09-01

Macroautophagy/autophagy is a conserved catabolic process that recycles cytoplasmic material during low energy conditions. BECN1/Beclin1 (Beclin 1, autophagy related) is an essential protein for function of the class 3 phosphatidylinositol 3-kinase (PtdIns3K) complexes that play a key role in autophagy nucleation and elongation. Here, we show that AMP-activated protein kinase (AMPK) regulates autophagy by phosphorylating BECN1 at Thr388. Phosphorylation of BECN1 is required for autophagy upon glucose withdrawal. BECN1(T388A), a phosphorylation defective mutant, suppresses autophagy through decreasing the interaction between PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3) and ATG14 (autophagy-related 14). The BECN1(T388A) mutant has a higher affinity for BCL2 than its wild-type counterpart; the mutant is more prone to dimer formation. Conversely, a BECN1 phosphorylation mimic mutant, T388D, has stronger binding to PIK3C3 and ATG14, and promotes higher autophagy activity than the wild-type control. These findings uncover a novel mechanism of autophagy regulation.

The EphA8 Receptor Regulates Integrin Activity through p110γ Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

PubMed Central

Gu, Changkyu; Park, Soochul

2001-01-01

Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via α5β1- or β3 integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110γ isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110γ. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110γ mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110γ PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion. PMID:11416136

Identification of Small Molecule Inhibitors of Phosphatidylinositol 3-Kinase and Autophagy*

PubMed Central

Farkas, Thomas; Daugaard, Mads; Jäättelä, Marja

2011-01-01

Macroautophagy (hereafter autophagy) is a lysosomal catabolic pathway that controls cellular homeostasis and survival. It has recently emerged as an attractive target for the treatment of a variety of degenerative diseases and cancer. The targeting of autophagy has, however, been hampered by the lack of specific small molecule inhibitors. Thus, we screened two small molecule kinase inhibitor libraries for inhibitors of rapamycin-induced autophagic flux. The three most potent inhibitors identified conferred profound inhibition of autophagic flux by inhibiting the formation of autophagosomes. Notably, the autophagy inhibitory effects of all three compounds were independent of their established kinase targets, i.e. ataxia telangiectasia mutated for KU55933, protein kinase C for Gö6976, and Janus kinase 3 for Jak3 inhibitor VI. Instead, we identified phosphatidylinositol 3-kinase (PtdIns3K) as a direct target of KU55933 and Gö6976. Importantly, and in contrast to the currently available inhibitors of autophagosome formation (e.g. 3-methyladenine), none of the three compounds inhibited the cell survival promoting class I phosphoinositide 3-kinase-Akt signaling at the concentrations required for effective autophagy inhibition. Accordingly, they proved to be valuable tools for investigations of autophagy-associated cell death and survival. Employing KU55399, we demonstrated that autophagy protects amino acid-starved cells against both apoptosis and necroptosis. Taken together, our data introduce new possibilities for the experimental study of autophagy and can form a basis for the development of clinically relevant autophagy inhibitors. PMID:21930714

The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth.

PubMed

Ashcroft, M; Stephens, R M; Hallberg, B; Downward, J; Kaplan, D R

1999-08-12

The Trk/Nerve Growth Factor receptor mediates the rapid activation of a number of intracellular signaling proteins, including phosphatidylinositol 3-kinase (PI 3-kinase). Here, we describe a novel, NGF-inducible system that we used to specifically address the signaling potential of endogenous PI 3-kinase in NGF-mediated neuronal survival and differentiation processes. This system utilizes a Trk receptor mutant (Trk(def)) lacking sequences Y490, Y785 and KFG important for the activation of the major Trk targets; SHC, PLC-gammal, Ras, PI 3-kinase and SNT. Trk(def) was kinase active but defective for NGF-induced responses when stably expressed in PC12nnr5 cells (which lack detectable levels of TrkA and are non-responsive to NGF). The PI 3-kinase consensus binding site, YxxM (YVPM), was introduced into the insert region within the kinase domain of Trk(def). NGF-stimulated tyrosine phosphorylation of the Trk(def)+PI 3-kinase addback receptor, resulted in the direct association and selective activation of PI 3-kinase in vitro and the production of PI(3,4)P2 and PI(3,4,5)P3 in vivo (comparable to wild-type). PC12nnr5 cells stably expressing Trk(def) + PI 3-kinase, initiated neurite outgrowth but failed to stably extend and maintain these neurites in response to NGF as compared to PC12 parental cells, or PC12nnr5 cells overexpressing wild-type Trk. However, Trk(def) + PI 3-kinase was fully competent in mediating NGF-induced survival processes. We propose that while endogenous PI 3-kinase can contribute in part to neurite initiation processes, its selective activation and subsequent signaling to downstream effectors such as Akt, functions mainly to promote cell survival in the PC12 system.

The potential signalling pathways which regulate surface changes induced by phytohormones in the potato cyst nematode (Globodera rostochiensis).

PubMed

Akhkha, A; Curtis, R; Kennedy, M; Kusel, J

2004-05-01

It has been demonstrated that the surface lipophilicity of the plant-parasitic nematode Globodera rostochiensis decreases when infective larvae are exposed to the phytohormones indole-3-acetic acid (auxin) or kinetin (cytokinin). In the present study, it was shown that inhibition of phospholipase C (PLC) or phosphatidylinositol 3 kinase (PI3-kinase) reversed the effect of phytohormones on surface lipophilicity. The signalling pathway(s) involved in surface modification were investigated using 'caged' signalling molecules and stimulators or inhibitors of different signalling enzymes. Photolysis of the 'caged' signalling molecules, NPE-caged Ins 1,4,5-P3, NITR-5/AM or caged-cAMP to liberate IP3, Ca2+ or cAMP respectively, decreased the surface lipophilicity. Activation of adenylate cyclase also decreased the surface lipophilicity. In contrast, inhibition of PI3-kinase using Wortmannin, LY-294002 or Quercetin, and inhibition of PLC using U-73122 all increased the surface lipophilicity. Two possible signalling pathways involved in phytohormone-induced surface modification are proposed.

Implication of matrix metalloproteinases 2 and 9 in ceramide 1-phosphate-stimulated macrophage migration.

PubMed

Ordoñez, Marta; Rivera, Io-Guané; Presa, Natalia; Gomez-Muñoz, Antonio

2016-08-01

Cell migration is a complex biological function involved in both physiologic and pathologic processes. Although this is a subject of intense investigation, the mechanisms by which cell migration is regulated are not completely understood. In this study we show that the bioactive sphingolipid ceramide 1-phosphate (C1P), which is involved in inflammatory responses, causes upregulation of metalloproteinases (MMP) -2 and -9 in J774A.1 macrophages. This effect was shown to be dependent on stimulation of phosphatidylinositol 3-kinase (PI3K) and extracellularly regulated kinases 1-2 (ERK1-2) as demonstrated by treating the cells with specific siRNA to knockdown the p85 regulatory subunit of PI3K, or ERK1-2. Inhibition of MMP-2 or MMP-9 pharmacologically or with specific siRNA to silence the genes encoding these MMPs abrogated C1P-stimulated macrophage migration. Also, C1P induced actin polymerization and potently increased phosphorylation of the focal adhesion protein paxillin, which are essential factors in the regulation of cell migration. As expected, blockade of paxillin activation with specific siRNA significantly reduced actin polymerization. In addition, inhibition of actin polymerization with cytochalasin D completely blocked C1P-induced MMP-2 and -9 expression as well as C1P-stimulated macrophage migration. It was also observed that pertussis toxin (Ptx) inhibited Akt, ERK1-2, and paxillin phosphorylation, and completely blocked cell migration. The latter findings support the notion that C1P-stimulated macrophage migration is a receptor mediated effect, and point to MMP-2 and -9 as possible therapeutic targets to control inflammation. Copyright © 2016 Elsevier Inc. All rights reserved.

Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4+ T Cell Proliferation.

PubMed

Celada, Lindsay J; Rotsinger, Joseph E; Young, Anjuli; Shaginurova, Guzel; Shelton, Debresha; Hawkins, Charlene; Drake, Wonder P

2017-01-01

Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4 + T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4 + T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1 + CD4 + T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P < 0.001). There was a negative correlation with PD-1 expression and proliferative capacity (r = -0.70, P < 0.001). Expression of key mediators of cell cycle progression, including PI3K and AKT, were significantly decreased. Gene and protein expression levels reverted to healthy control levels after PD-1 pathway blockade. Reduction in sarcoidosis CD4 + T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression.

Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4+ T Cell Proliferation

PubMed Central

Celada, Lindsay J.; Rotsinger, Joseph E.; Young, Anjuli; Shaginurova, Guzel; Shelton, Debresha; Hawkins, Charlene

2017-01-01

Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4+ T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4+ T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1+ CD4+ T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P < 0.001). There was a negative correlation with PD-1 expression and proliferative capacity (r = −0.70, P < 0.001). Expression of key mediators of cell cycle progression, including PI3K and AKT, were significantly decreased. Gene and protein expression levels reverted to healthy control levels after PD-1 pathway blockade. Reduction in sarcoidosis CD4+ T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression. PMID:27564547

Phosphoinositide 5-phosphatase activities control cell motility in glioblastoma: Two phosphoinositides PI(4,5)P2 and PI(3,4)P2 are involved.

PubMed

Ramos, Ana Raquel; Elong Edimo, William's; Erneux, Christophe

2018-01-01

Inositol polyphosphate 5-phosphatases or phosphoinositide 5-phosphatases (PI 5-phosphatases) are enzymes that can act on soluble inositol phosphates and/or phosphoinositides (PIs). Several PI 5-phosphatases have been linked to human genetic diseases, in particular the Lowe protein or OCRL which is mutated in the Lowe syndrome. There are 10 different members of this family and 9 of them can use PIs as substrate. One of these substrates, PI(3,4,5)P3 binds to specific PH domains and recruits as effectors specific proteins to signaling complexes. Protein kinase B is one target protein and activation of the kinase will have a major impact on cell proliferation, survival and cell metabolism. Two other PIs, PI(4,5)P2 and PI(3,4)P2, are produced or used as substrates of PI 5-phosphatases (OCRL, INPP5B, SHIP1/2, SYNJ1/2, INPP5K, INPP5J, INPP5E). The inositol lipids may influence many aspects of cytoskeletal organization, lamellipodia formation and F-actin polymerization. PI 5-phosphatases have been reported to control cell migration, adhesion, polarity and cell invasion particularly in cancer cells. In glioblastoma, reducing SHIP2 expression can positively or negatively affect the speed of cell migration depending on the glioblastoma cell type. The two PI 5-phosphatases SHIP2 or SKIP could be localized at the plasma membrane and can reduce either PI(3,4,5)P3 or PI(4,5)P2 abundance. In the glioblastoma 1321 N1 cells, SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synergistic activation of G protein-gated inwardly rectifying potassium channels by cholesterol and PI(4,5)P2.

PubMed

Bukiya, Anna N; Rosenhouse-Dantsker, Avia

2017-07-01

G-protein gated inwardly rectifying potassium (GIRK or Kir3) channels play a major role in the control of the heart rate, and require the membrane phospholipid phosphatidylinositol-bis-phosphate (PI(4,5)P 2 ) for activation. Recently, we have shown that the activity of the heterotetrameric Kir3.1/Kir3.4 channel that underlies atrial K ACh currents was enhanced by cholesterol. Similarly, the activities of both the Kir3.4 homomer and its active pore mutant Kir3.4* (Kir3.4_S143T) were also enhanced by cholesterol. Here we employ planar lipid bilayers to investigate the crosstalk between PI(4,5)P 2 and cholesterol, and demonstrate that these two lipids act synergistically to activate Kir3.4* currents. Further studies using the Xenopus oocytes heterologous expression system suggest that PI(4,5)P 2 and cholesterol act via distinct binding sites. Whereas PI(4,5)P 2 binds to the cytosolic domain of the channel, the putative binding region of cholesterol is located at the center of the transmembrane domain overlapping the central glycine hinge region of the channel. Together, our data suggest that changes in the levels of two key membrane lipids - cholesterol and PI(4,5)P 2 - could act in concert to provide fine-tuning of Kir3 channel function. Copyright © 2017 Elsevier B.V. All rights reserved.

Phosphoinositides play differential roles in regulating phototropin1- and phototropin2-mediated chloroplast movements in Arabidopsis.

PubMed

Aggarwal, Chhavi; Labuz, Justyna; Gabryś, Halina

2013-01-01

Phototropins are UVA/blue-light receptors involved in controlling the light-dependent physiological responses which serve to optimize the photosynthetic activity of plants and promote growth. The phototropin-induced phosphoinositide (PI) metabolism has been shown to be essential for stomatal opening and phototropism. However, the role of PIs in phototropin-induced chloroplast movements remains poorly understood. The aim of this work is to determine which PI species are involved in the control of chloroplast movements in Arabidopsis and the nature of their involvement. We present the effects of the inactivation of phospholipase C (PLC), PI3-kinase (PI3K) and PI4-kinase (PI4K) on chloroplast relocations in Arabidopsis. The inhibition of the phosphatidylinositol 4,5-bisphospahte [PI(4,5)P2]-PLC pathway, using neomycin and U73122, suppressed the phot2-mediated chloroplast accumulation and avoidance responses, without affecting movement responses controlled by phot1. On the other hand, PI3K and PI4K activities are more restricted to phot1- and phot2-induced weak-light responses. The inactivation of PI3K and PI4K by wortmannin and LY294002 severely affected the weak blue-light-activated accumulation response but had little effect on the strong blue-light-activated avoidance response. The inhibitory effect observed with PI metabolism inhibitors is, at least partly, due to a disturbance in Ca(2+) ((c)) signaling. Using the transgenic aequorin system, we show that the application of these inhibitors suppresses the blue-light-induced transient Ca(2+) ((c)) rise. These results demonstrate the importance of PIs in chloroplast movements, with the PI(4,5)P2-PLC pathway involved in phot2 signaling while PI3K and PI4K are required for the phot1- and phot2-induced accumulation response. Our results suggest that these PIs modulate cytosolic Ca(2+) signaling during movements.

Hexamethylenebisacetamide modulation of thyroglobulin and protein levels in thyroid cells is not mediated by phosphatidylinositol-3-kinase: a study with wortmannin.

PubMed

Aouani, A; Samih, N; Amphoux-Fazekas, T; Hovsépian, S; Fayet, G

1999-04-01

Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action.

Remobilization of Phytol from Chlorophyll Degradation Is Essential for Tocopherol Synthesis and Growth of Arabidopsis

PubMed Central

vom Dorp, Katharina; Hölzl, Georg; Plohmann, Christian; Eisenhut, Marion; Abraham, Marion

2015-01-01

Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate. PMID:26452599

Analysis of molecular markers as predictive factors of lymph node involvement in breast carcinoma.

PubMed

Paula, Luciana Marques; De Moraes, Luis Henrique Ferreira; Do Canto, Abaeté Leite; Dos Santos, Laurita; Martin, Airton Abrahão; Rogatto, Silvia Regina; De Azevedo Canevari, Renata

2017-01-01

Nodal status is the most significant independent prognostic factor in breast cancer. Identification of molecular markers would allow stratification of patients who require surgical assessment of lymph nodes from the large numbers of patients for whom this surgical procedure is unnecessary, thus leading to a more accurate prognosis. However, up to now, the reported studies are preliminary and controversial, and although hundreds of markers have been assessed, few of them have been used in clinical practice for treatment or prognosis in breast cancer. The purpose of the present study was to determine whether protein phosphatase Mg2+/Mn2+ dependent 1D, β-1,3-N-acetylglucosaminyltransferase, neural precursor cell expressed, developmentally down-regulated 9, prohibitin, phosphoinositide-3-kinase regulatory subunit 5 (PIK3R5), phosphatidylinositol-5-phosphate 4-kinase type IIα, TRF1-interacting ankyrin-related ADP-ribose polymerase 2, BCL2 associated agonist of cell death, G2 and S-phase expressed 1 and PAX interacting protein 1 genes, described as prognostic markers in breast cancer in a previous microarray study, are also predictors of lymph node involvement in breast carcinoma Reverse transcription-quantitative polymerase chain reaction analysis was performed on primary breast tumor tissues from women with negative lymph node involvement (n=27) compared with primary tumor tissues from women with positive lymph node involvement (n=23), and was also performed on primary tumors and paired lymph node metastases (n=11). For all genes analyzed, only the PIK3R5 gene exhibited differential expression in samples of primary tumors with positive lymph node involvement compared with primary tumors with negative lymph node involvement (P=0.0347). These results demonstrate that the PIK3R5 gene may be considered predictive of lymph node involvement in breast carcinoma. Although the other genes evaluated in the present study have been previously characterized to be involved with the development of distant metastases, they did not have predictive potential.

Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes.

PubMed

Takatori, Sho; Tatematsu, Tsuyako; Cheng, Jinglei; Matsumoto, Jun; Akano, Takuya; Fujimoto, Toyoshi

2016-02-01

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P2 using freeze-fracture electron microscopy. GST-ATG18-4×FLAG was used to label PtdIns(3,5)P2 and its binding to phosphatidylinositol 3-phosphate (PtdIns(3)P) was blocked by an excess of the p40(phox) PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P2 was concentrated in intramembrane particle (IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5)P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P2 metabolism is defective. In HeLa cells, PtdIns(3,5)P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5)P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P2 -rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate

PubMed Central

Rzepnikowska, Weronika; Flis, Krzysztof; Kaminska, Joanna; Grynberg, Marcin; Urbanek, Agnieszka; Ayscough, Kathryn R.

2017-01-01

Abstract The rare human disorder chorea-acanthocytosis (ChAc) is caused by mutations in hVPS13A gene. The hVps13A protein interacts with actin and regulates the level of phosphatidylinositol 4-phosphate (PI4P) in the membranes of neuronal cells. Yeast Vps13 is involved in vacuolar protein transport and, like hVps13A, participates in PI4P metabolism. Vps13 proteins are conserved in eukaryotes, but their molecular function remains unknown. One of the mutations found in ChAc patients causes amino acids substitution I2771R which affects the localization of hVps13A in skeletal muscles. To dissect the mechanism of pathogenesis of I2771R, we created and analyzed a yeast strain carrying the equivalent mutation. Here we show that in yeast, substitution I2749R causes dysfunction of Vps13 protein in endocytosis and vacuolar transport, although the level of the protein is not affected, suggesting loss of function. We also show that Vps13, like hVps13A, influences actin cytoskeleton organization and binds actin in immunoprecipitation experiments. Vps13-I2749R binds actin, but does not function in the actin cytoskeleton organization. Moreover, we show that Vps13 binds phospholipids, especially phosphatidylinositol 3-phosphate (PI3P), via its SHR_BD and APT1 domains. Substitution I2749R attenuates this ability. Finally, the localization of Vps13-GFP is altered when cellular levels of PI3P are decreased indicating its trafficking within the endosomal membrane system. These results suggest that PI3P regulates the functioning of Vps13, both in protein trafficking and actin cytoskeleton organization. Attenuation of PI3P-binding ability in the mutant hVps13A protein may be one of the reasons for its mislocalization and disrupted function in cells of patients suffering from ChAc. PMID:28334785

Phosphatidylinositol-4,5-bisphosphate is required for KCNQ1/KCNE1 channel function but not anterograde trafficking

PubMed Central

Royal, Alice A.

2017-01-01

The slow delayed-rectifier potassium current (IKs) is crucial for human cardiac action potential repolarization. The formation of IKs requires co-assembly of the KCNQ1 α-subunit and KCNE1 β-subunit, and mutations in either of these subunits can lead to hereditary long QT syndrome types 1 and 5, respectively. It is widely recognised that the KCNQ1/KCNE1 (Q1/E1) channel requires phosphatidylinositol-4,5-bisphosphate (PIP2) binding for function. We previously identified a cluster of basic residues in the proximal C-terminus of KCNQ1 that form a PIP2/phosphoinositide binding site. Upon charge neutralisation of these residues we found that the channel became more retained in the endoplasmic reticulum, which raised the possibility that channel–phosphoinositide interactions could play a role in channel trafficking. To explore this further we used a chemically induced dimerization (CID) system to selectively deplete PIP2 and/or phosphatidylinositol-4-phosphate (PI(4)P) at the plasma membrane (PM) or Golgi, and we subsequently monitored the effects on both channel trafficking and function. The depletion of PIP2 and/or PI(4)P at either the PM or Golgi did not alter channel cell-surface expression levels. However, channel function was extremely sensitive to the depletion of PIP2 at the PM, which is in contrast to the response of other cardiac potassium channels tested (Kir2.1 and Kv11.1). Surprisingly, when using the CID system IKs was dramatically reduced even before dimerization was induced, highlighting limitations regarding the utility of this system when studying processes highly sensitive to PIP2 depletion. In conclusion, we identify that the Q1/E1 channel does not require PIP2 or PI(4)P for anterograde trafficking, but is heavily reliant on PIP2 for channel function once at the PM. PMID:29020060

Phosphatidylinositol-4,5-bisphosphate is required for KCNQ1/KCNE1 channel function but not anterograde trafficking.

PubMed

Royal, Alice A; Tinker, Andrew; Harmer, Stephen C

2017-01-01

The slow delayed-rectifier potassium current (IKs) is crucial for human cardiac action potential repolarization. The formation of IKs requires co-assembly of the KCNQ1 α-subunit and KCNE1 β-subunit, and mutations in either of these subunits can lead to hereditary long QT syndrome types 1 and 5, respectively. It is widely recognised that the KCNQ1/KCNE1 (Q1/E1) channel requires phosphatidylinositol-4,5-bisphosphate (PIP2) binding for function. We previously identified a cluster of basic residues in the proximal C-terminus of KCNQ1 that form a PIP2/phosphoinositide binding site. Upon charge neutralisation of these residues we found that the channel became more retained in the endoplasmic reticulum, which raised the possibility that channel-phosphoinositide interactions could play a role in channel trafficking. To explore this further we used a chemically induced dimerization (CID) system to selectively deplete PIP2 and/or phosphatidylinositol-4-phosphate (PI(4)P) at the plasma membrane (PM) or Golgi, and we subsequently monitored the effects on both channel trafficking and function. The depletion of PIP2 and/or PI(4)P at either the PM or Golgi did not alter channel cell-surface expression levels. However, channel function was extremely sensitive to the depletion of PIP2 at the PM, which is in contrast to the response of other cardiac potassium channels tested (Kir2.1 and Kv11.1). Surprisingly, when using the CID system IKs was dramatically reduced even before dimerization was induced, highlighting limitations regarding the utility of this system when studying processes highly sensitive to PIP2 depletion. In conclusion, we identify that the Q1/E1 channel does not require PIP2 or PI(4)P for anterograde trafficking, but is heavily reliant on PIP2 for channel function once at the PM.

Computational and Pharmacological Evaluation of Ferrocene-Based Acyl Ureas and Homoleptic Cadmium Carboxylate Derivatives for Anti-diabetic Potential.

PubMed

Bano, Shahar; Khan, Arif-Ullah; Asghar, Faiza; Usman, Muhammad; Badshah, Amin; Ali, Saqib

2017-01-01

We investigated possible anti-diabetic effect of ferrocene-based acyl ureas: 4-ferrocenyl aniline (PFA), 1-(4-chlorobenzoyl)-3-(4-ferrocenylphenyl) urea (DPC1), 1-(3-chlorobenzoyl)-3-(4-ferrocenylphenyl) urea (DMC1), 1-(2-chlorobenzoyl)-3-(4-ferrocenylphenyl) urea (DOC1) and homoleptic cadmium carboxylates: bis (diphenylacetato) cadmium (II) (DPAA), bis (4-chlorophenylacetato) cadmium (II) (CPAA), using in silico and in vivo techniques. PFA, DPC1, DMC1, DOC1, DPAA and CPAA exhibited high binding affinities (ACE ≥ -350 Kcal/mol) against targets: aldose reductase, peroxisome proliferator-activated receptor γ, 11β-hydroxysteroid dehydrogenase-1, C-alpha glucosidase and glucokinase, while showed moderate affinities (ACE ≥ -250 Kcal/mol) against N-alpha glucosidase, dipeptidyl peptidase-IV, phosphorylated-Akt, glycogen synthase kinase-3β, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, whereas revealed lower affinities (ACE < -250 Kcal/mol) vs. alpha amylase, protein tyrosine phosphatases 1B, glycogen phosphorylase and phosphatidylinositol 3 kinase. In alloxan (300 mg/Kg)-induced diabetic mice, DPAA and DPC1 (1-10 mg/Kg) at day 1, 5, 10, 15, and 20th decreased blood glucose levels, compared to diabetic control group and improved the treated animals body weight. DPAA (10 mg/Kg) and DPC1 (5 mg/Kg) in time-dependent manner (30-120 min.) enhanced tolerance of oral glucose overload in mice. DPAA and DPCI dose-dependently at 1, 5, and 10 mg/Kg decreased glycosylated hemoglobin levels in diabetic animals, as caused by metformin. These results indicate that aforementioned derivatives of ferrocene and cadmium possess anti-diabetic potential.

Effects of AICAR and exercise on insulin-stimulated glucose uptake, signaling, and GLUT-4 content in rat muscles.

PubMed

Jessen, Niels; Pold, Rasmus; Buhl, Esben S; Jensen, Lasse S; Schmitz, Ole; Lund, Sten

2003-04-01

Physical activity is known to increase insulin action in skeletal muscle, and data have indicated that 5'-AMP-activated protein kinase (AMPK) is involved in the molecular mechanisms behind this beneficial effect. 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) can be used as a pharmacological tool to repetitively activate AMPK, and the objective of this study was to explore whether the increase in insulin-stimulated glucose uptake after either long-term exercise or chronic AICAR administration was followed by fiber-type-specific changes in insulin signaling and/or changes in GLUT-4 expression. Wistar rats were allocated into three groups: an exercise group trained on treadmill for 5 days, an AICAR group exposed to daily subcutaneous injections of AICAR, and a sedentary control group. AMPK activity, insulin-stimulated glucose transport, insulin signaling, and GLUT-4 expression were determined in muscles characterized by different fiber type compositions. Both exercised and AICAR-injected animals displayed a fiber-type-specific increase in glucose transport with the most marked increase in muscles with a high content of type IIb fibers. This increase was accompanied by a concomitant increase in GLUT-4 expression. Insulin signaling as assessed by phosphatidylinositol 3-kinase and PKB/Akt activity was enhanced only after AICAR administration and in a non-fiber-type-specific manner. In conclusion, chronic AICAR administration and long-term exercise both improve insulin-stimulated glucose transport in skeletal muscle in a fiber-type-specific way, and this is associated with an increase in GLUT-4 content.

Myristoylated peptides potentiate the funny current (If) in sinoatrial myocytes

PubMed Central

Liao, Zhandi; St Clair, Joshua R; Larson, Eric D

2011-01-01

The funny current, If, in sinoatrial myocytes is thought to contribute to the sympathetic fight-or-flight increase in heart rate. If is produced by hyperpolarization-activated cyclic nucleotide sensitive-4 (HCN4) channels, and it is widely believed that sympathetic regulation of If occurs via direct binding of cAMP to HCN4, independent of phosphorylation. However, we have recently shown that Protein Kinase A (PKA) activity is required for sympathetic regulation of If, and that PKA can directly phosphorylate HCN4.1 In the present study, we examined the effects of a myristoylated PKA inhibitory peptide (myr-PKI) on If in mouse sinoatrial myocytes. We found that myr-PKI and another myristoylated peptide potently and specifically potentiated If via a mechanism that did not involve PKA inhibition and that was independent of the peptide sequence, Protein Kinase C or phosphatidylinositol-4,5-bisphosphate. The off-target activation of If by myristoylated peptides limits their usefulness for studies of pacemaker mechanisms in sinoatrial myocytes. PMID:21150293

Golgi and plasma membrane pools of PI(4)P contribute to plasma membrane PI(4,5)P2 and maintenance of KCNQ2/3 ion channel current

PubMed Central

Dickson, Eamonn J.; Jensen, Jill B.; Hille, Bertil

2014-01-01

Plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] regulates the activity of many ion channels and other membrane-associated proteins. To determine precursor sources of the PM PI(4,5)P2 pool in tsA-201 cells, we monitored KCNQ2/3 channel currents and translocation of PHPLCδ1 domains as real-time indicators of PM PI(4,5)P2, and translocation of PHOSH2×2, and PHOSH1 domains as indicators of PM and Golgi phosphatidylinositol 4-phosphate [PI(4)P], respectively. We selectively depleted PI(4)P pools at the PM, Golgi, or both using the rapamycin-recruitable lipid 4-phosphatases. Depleting PI(4)P at the PM with a recruitable 4-phosphatase (Sac1) results in a decrease of PI(4,5)P2 measured by electrical or optical indicators. Depleting PI(4)P at the Golgi with the 4-phosphatase or disrupting membrane-transporting motors induces a decline in PM PI(4,5)P2. Depleting PI(4)P simultaneously at both the Golgi and the PM induces a larger decrease of PI(4,5)P2. The decline of PI(4,5)P2 following 4-phosphatase recruitment takes 1–2 min. Recruiting the endoplasmic reticulum (ER) toward the Golgi membranes mimics the effects of depleting PI(4)P at the Golgi, apparently due to the trans actions of endogenous ER Sac1. Thus, maintenance of the PM pool of PI(4,5)P2 appears to depend on precursor pools of PI(4)P both in the PM and in the Golgi. The decrease in PM PI(4,5)P2 when Sac1 is recruited to the Golgi suggests that the Golgi contribution is ongoing and that PI(4,5)P2 production may be coupled to important cell biological processes such as membrane trafficking or lipid transfer activity. PMID:24843134

Reduction of Mitochondria-Endoplasmic Reticulum Interactions by Acetylcholine Protects Human Umbilical Vein Endothelial Cells From Hypoxia/Reoxygenation Injury.

PubMed

He, Xi; Bi, Xue-Yuan; Lu, Xing-Zhu; Zhao, Ming; Yu, Xiao-Jiang; Sun, Lei; Xu, Man; Wier, W Gil; Zang, Wei-Jin

2015-07-01

We explored the role of endoplasmic reticulum (ER)-mitochondria Ca(2+) cross talk involving voltage-dependent anion channel-1 (VDAC1)/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex and mitofusin 2 in endothelial cells during hypoxia/reoxygenation (H/R), and investigated the protective effects of acetylcholine. Acetylcholine treatment during reoxygenation prevented intracellular and mitochondrial Ca(2+) increases and alleviated ER Ca(2+) depletion during H/R in human umbilical vein endothelial cells. Consequently, acetylcholine enhanced mitochondrial membrane potential and inhibited proapoptotic cascades, thereby reducing cell death and preserving endothelial ultrastructure. This effect was likely mediated by the type-3 muscarinic acetylcholine receptor and the phosphatidylinositol 3-kinase/Akt pathway. In addition, interactions among members of the VDAC1/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex were increased after H/R and were associated with mitochondrial Ca(2+) overload and cell death. Inhibition of the partner of the Ca(2+) channeling complex (VDAC1 siRNA) or a reduction in ER-mitochondria tethering (mitofusin 2 siRNA) prevented the increased protein interaction within the complex and reduced mitochondrial Ca(2+) accumulation and subsequent endothelial cell death after H/R. Intriguingly, acetylcholine could modulate ER-mitochondria Ca(2+) cross talk by inhibiting the VDAC1/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex and mitofusin 2 expression. Phosphatidylinositol 3-kinase siRNA diminished acetylcholine-mediated inhibition of mitochondrial Ca(2+) overload and VDAC1/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex formation induced by H/R. Our data suggest that ER-mitochondria interplay plays an important role in reperfusion injury in the endothelium and may be a novel molecular target for endothelial protection. Acetylcholine attenuates both intracellular and mitochondrial Ca(2+) overload and protects endothelial cells from H/R injury, presumably by disrupting the ER-mitochondria interaction. © 2015 American Heart Association, Inc.

Bis(morpholino-1,3,5-triazine) derivatives: potent adenosine 5'-triphosphate competitive phosphatidylinositol-3-kinase/mammalian target of rapamycin inhibitors: discovery of compound 26 (PKI-587), a highly efficacious dual inhibitor.

PubMed

Venkatesan, Aranapakam M; Dehnhardt, Christoph M; Delos Santos, Efren; Chen, Zecheng; Dos Santos, Osvaldo; Ayral-Kaloustian, Semiramis; Khafizova, Gulnaz; Brooijmans, Natasja; Mallon, Robert; Hollander, Irwin; Feldberg, Larry; Lucas, Judy; Yu, Ker; Gibbons, James; Abraham, Robert T; Chaudhary, Inder; Mansour, Tarek S

2010-03-25

The PI3K/Akt signaling pathway is a key pathway in cell proliferation, growth, survival, protein synthesis, and glucose metabolism. It has been recognized recently that inhibiting this pathway might provide a viable therapy for cancer. A series of bis(morpholino-1,3,5-triazine) derivatives were prepared and optimized to provide the highly efficacious PI3K/mTOR inhibitor 1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea 26 (PKI-587). Compound 26 has shown excellent activity in vitro and in vivo, with antitumor efficacy in both subcutaneous and orthotopic xenograft tumor models when administered intravenously. The structure-activity relationships and the in vitro and in vivo activity of analogues in this series are described.

Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins.

PubMed

Helliwell, Emily E; Vega-Arreguín, Julio; Shi, Zi; Bailey, Bryan; Xiao, Shunyuan; Maximova, Siela N; Tyler, Brett M; Guiltinan, Mark J

2016-03-01

The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P-binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P-binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P-binding site, or by a secreted PI4P-binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P-binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P-binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Insulin elicits a ROS-activated and an IP₃-dependent Ca²⁺ release, which both impinge on GLUT4 translocation.

PubMed

Contreras-Ferrat, Ariel; Llanos, Paola; Vásquez, César; Espinosa, Alejandra; Osorio-Fuentealba, César; Arias-Calderon, Manuel; Lavandero, Sergio; Klip, Amira; Hidalgo, Cecilia; Jaimovich, Enrique

2014-05-01

Insulin signaling includes generation of low levels of H2O2; however, its origin and contribution to insulin-stimulated glucose transport are unknown. We tested the impact of H2O2 on insulin-dependent glucose transport and GLUT4 translocation in skeletal muscle cells. H2O2 increased the translocation of GLUT4 with an exofacial Myc-epitope tag between the first and second transmembrane domains (GLUT4myc), an effect additive to that of insulin. The anti-oxidants N-acetyl L-cysteine and Trolox, the p47(phox)-NOX2 NADPH oxidase inhibitory peptide gp91-ds-tat or p47(phox) knockdown each reduced insulin-dependent GLUT4myc translocation. Importantly, gp91-ds-tat suppressed insulin-dependent H2O2 production. A ryanodine receptor (RyR) channel agonist stimulated GLUT4myc translocation and insulin stimulated RyR1-mediated Ca(2+) release by promoting RyR1 S-glutathionylation. This pathway acts in parallel to insulin-mediated stimulation of inositol-1,4,5-trisphosphate (IP3)-activated Ca(2+) channels, in response to activation of phosphatidylinositol 3-kinase and its downstream target phospholipase C, resulting in Ca(2+) transfer to the mitochondria. An inhibitor of IP3 receptors, Xestospongin B, reduced both insulin-dependent IP3 production and GLUT4myc translocation. We propose that, in addition to the canonical α,β phosphatidylinositol 3-kinase to Akt pathway, insulin engages both RyR-mediated Ca(2+) release and IP3-receptor-mediated mitochondrial Ca(2+) uptake, and that these signals jointly stimulate glucose uptake.

The insulin receptor substrate (IRS)-1 pleckstrin homology domain functions in downstream signaling.

PubMed

Vainshtein, I; Kovacina, K S; Roth, R A

2001-03-16

The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets.

Phase geometries of two-dimensional excitable waves govern self-organized morphodynamics of amoeboid cells

PubMed Central

Taniguchi, Daisuke; Ishihara, Shuji; Oonuki, Takehiko; Honda-Kitahara, Mai; Kaneko, Kunihiko; Sawai, Satoshi

2013-01-01

In both randomly moving Dictyostelium and mammalian cells, phosphatidylinositol (3,4,5)-trisphosphate and F-actin are known to propagate as waves at the membrane and act to push out the protruding edge. To date, however, the relationship between the wave geometry and the patterns of amoeboid shape change remains elusive. Here, by using phase map analysis, we show that morphology dynamics of randomly moving Dictyostelium discoideum cells can be characterized by the number, topology, and position of spatial phase singularities, i.e., points that represent organizing centers of rotating waves. A single isolated singularity near the cellular edge induced a rotational protrusion, whereas a pair of singularities supported a symmetric extension. These singularities appeared by strong phase resetting due to de novo nucleation at the back of preexisting waves. Analysis of a theoretical model indicated excitability of the system that is governed by positive feedback from phosphatidylinositol (3,4,5)-trisphosphate to PI3-kinase activation, and we showed experimentally that this requires F-actin. Furthermore, by incorporating membrane deformation into the model, we demonstrated that geometries of competing waves explain most of the observed semiperiodic changes in amoeboid morphology. PMID:23479620

The presence of a protein activator of sarcolemmal polyphosphoinositide phospholipase C in cardiac cytosol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Quist, E.E.; Kriewaldt, S.D.; Powell, P.B.

1989-01-01

To study polyphosphoinositide phospholipase (PL) C, isolated sarcolemmal membranes were preincubated with Mg({sup 32}P)-ATP to label phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-diphosphate (PIP{sub 2}). After washing, PLC activity was determined by measuring the release of {sup 32}P-labeled inositol diphosphate (IP{sub 2}) and/or inositol trisphospate (IP{sub 3}) from membrane PIP and PIP{sub 2} during incubation at 25{degree}C and pH 7.4. Increasing concentrations of Ca{sup 2+} (0-100 {mu}M) increased IP{sub 2} by 100% over the 0 Ca{sup 2+} control levels. Ca{sup 2+} dependent PLC hydrolyzed both PIP and PIP{sub 2} with apparent D{sub A}'s of approximately 0.5 and 70 {mu}M. Addition ofmore » dialyzed cytosol further increased IP{sub 2} release by 250% without affecting the K{sub A}'s for Ca{sup 2+} activation. The cytosolic activator was partially purified by DEAE Sephacel chromatography was heat labile and sensitive to trypsin pretreatment identifying it as a protein. In contrast, 10 mM NaF increased the Ca{sup 2+} affinity for PLC 2-fold. These results show that cardiac sarcolemma possess a membrane bound Ca{sup 2+} dependent PLC activity which is regulated by a cytosolic protein activator and a G protein. The cytosolic activator would potentially amplify the amount of sarcolemmal polyphosphoinositides hydrolyzed by PLC in response to muscarinic receptor activation by acetylcholine. In addition, activation of PLC by NaF or other G protein activators could result from increasing the Ca{sup 2+} affinity of PLC to physiological intracellular Ca{sup 2+} levels.« less

The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

PubMed Central

Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

1996-01-01

We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-alpha and PKC-beta in plasma membranes of rat adipocytes. PMID:8611143

The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

PubMed

Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

1996-02-01

We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-alpha and PKC-beta in plasma membranes of rat adipocytes.

Activation of cGMP/Protein Kinase G Pathway in Postconditioned Myocardium Depends on Reduced Oxidative Stress and Preserved Endothelial Nitric Oxide Synthase Coupling

PubMed Central

Inserte, Javier; Hernando, Victor; Vilardosa, Úrsula; Abad, Elena; Poncelas‐Nozal, Marcos; Garcia‐Dorado, David

2013-01-01

Background The cGMP/protein kinase G (PKG) pathway is involved in the cardioprotective effects of postconditioning (PoCo). Although PKG signaling in PoCo has been proposed to depend on the activation of the phosphatidylinositol 3‐kinase (PI3K)/Akt cascade, recent data bring into question a causal role of reperfusion injury signaling kinase (RISK) in PoCo protection. We hypothesized that PoCo increases PKG activity by reducing oxidative stress–induced endothelial nitric oxide synthase (NOS) uncoupling at the onset of reperfusion. Methods and Results Isolated rat hearts were submitted to 40 minutes of ischemia and reperfusion with and without a PoCo protocol. PoCo reduced infarct size by 48% and cGMP depletion. Blockade of cGMP synthesis (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one) and inhibition of PKG (KT5823) or NOS (l‐NAME) abolished protection, but inhibition of PI3K/Akt cascade (LY294002) did not (n=5 to 7 per group). Phosphorylation of the RISK pathway was higher in PoCo hearts. However, this difference is due to increased cell death in control hearts because in hearts reperfused with the contractile inhibitor blebbistatin, a drug effective in preventing cell death at the onset of reperfusion, RISK phosphorylation increased during reperfusion without differences between control and PoCo groups. In these hearts, PoCo reduced the production of superoxide (O2−) and protein nitrotyrosylation and increased nitrate/nitrite levels in parallel with a significant decrease in the oxidation of tetrahydrobiopterin (BH4) and in the monomeric form of endothelial NOS. Conclusions These results demonstrate that PoCo activates the cGMP/PKG pathway via a mechanism independent of the PI3K/Akt cascade and dependent on the reduction of O2− production at the onset of reperfusion, resulting in attenuated oxidation of BH4 and reduced NOS uncoupling. PMID:23525447

The osmotic shock-induced glucose transport pathway in 3T3-L1 adipocytes is mediated by gab-1 and requires Gab-1-associated phosphatidylinositol 3-kinase activity for full activation.

PubMed

Janez, A; Worrall, D S; Imamura, T; Sharma, P M; Olefsky, J M

2000-09-01

Osmotic shock treatment of 3T3-L1 adipocytes causes an increase in glucose transport activity and translocation of GLUT4 protein similar to that elicited by insulin treatment. Insulin stimulation of GLUT4 translocation and glucose transport activity was completely inhibited by wortmannin, however, activation by osmotic shock was only partially blocked. Additionally, we have found that the newly identified insulin receptor substrate Gab-1 (Grb2-associated binder-1) is tyrosine-phosphorylated following sorbitol stimulation. Treatment of cells with the tyrosine kinase inhibitor genistein inhibited osmotic shock-stimulated Gab-1 phosphorylation as well as shock-induced glucose transport. Furthermore, pretreatment with the selective Src family kinase inhibitor PP2 completely inhibited the ability of sorbitol treatment to cause tyrosine phosphorylation of Gab-1. We have also shown that microinjection of anti-Gab-1 antibody inhibits osmotic shock-induced GLUT4 translocation. Furthermore, phosphorylated Gab-1 binds and activates phosphatidylinositol 3-kinase (PI3K) in response to osmotic shock. The PI3K activity associated with Gab-1 was 82% of that associated with anti-phosphotyrosine antibodies, indicating that Gab-1 is the major site for PI3K recruitment following osmotic shock stimulation. Although wortmannin only causes a partial block of osmotic shock-stimulated glucose uptake, wortmannin completely abolishes Gab-1 associated PI3K activity. This suggests that other tyrosine kinase-dependent pathways, in addition to the Gab-1-PI3K pathway, contribute to osmotic shock-mediated glucose transport. To date, Gab-1 is the first protein identified as a member of the osmotic shock signal transduction pathway.

Deficiency of MIP phosphatase induces a muscle disorder by disrupting Ca2+ homeostasis

PubMed Central

Shen, Jinhua; Yu, Wen-Mei; Brotto, Marco; Scherman, Joseph A.; Guo, Caiying; Stoddard, Christopher; Nosek, Thomas M.; Valdivia, Héctor H.; Qu, Cheng-Kui

2009-01-01

The intracellular Ca2+ ([Ca2+]i) level of skeletal muscles must be rapidly regulated during the excitation-contraction-relaxation process 1. However, the signaling components involved in such rapid Ca2+ movement are not fully understood. Here, we report that mice deficient in the novel phosphatidylinositol phosphate (PIP) phosphatase MIP displayed muscle weakness and fatigue. Muscles isolated from MIP−/− mice produced less contractile force, markedly prolonged relaxation, and exhibited exacerbated fatigue. Further analyses revealed that MIP deficiency resulted in spontaneous Ca2+ leak from the internal store — the sarcoplasmic reticulum (SR). This was attributed to the decreased metabolism/dephosphorylation and the subsequent accumulation of MIP substrates, especially PI(3,5)P2 and PI(3,4)P2. Furthermore, we found that PI(3,5)P2 and PI(3,4)P2 bound to and directly activated the Ca2+ release channel/ryanodine receptor (RyR1) of the SR. These studies provide the first evidence that finely controlled PIP levels in muscle cells are essential for maintaining Ca2+ homeostasis and muscle performance. PMID:19465920

Phosphatidylinositol-3-kinase-dependent phosphorylation of SLP-76 by the lymphoma-associated ITK-SYK fusion-protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hussain, Alamdar, E-mail: alamdar.hussain@ki.se; Department of Biosciences, COMSATS Institute of Information Technology, Chak Shazad Campus, Islamabad; Faryal, Rani

Recurrent chromosomal translocations have long been implicated in various types of lymphomas and other malignancies. Novel recurrent t(5;9)(q33;q22) has been recently discovered in un-specified peripheral T-cell lymphoma. To elucidate the role of this translocation, the corresponding fusion construct encoding the N-terminal portion of the ITK kinase and the C-terminal catalytic region of the SYK kinase was generated. We herein show that the ITK-SYK fusion-protein is constitutively active. Moreover, we demonstrate that ITK-SYK is phosphorylated on key tyrosine residues and is capable of potently phosphorylating the related adapter proteins BLNK and SLP-76. In transiently transfected cells, SYK was phosphorylated at Y352more » but not detectably at the activation-loop tyrosines Y525/Y526. In contrast, ITK-SYK was phosphorylated both at Y212 and the activation-loop tyrosines Y385/Y386, corresponding to Y352 and Y525/Y526 in SYK, respectively. In resting primary lymphocytes, ITK-SYK predominantly localizes to the cell surface. In addition, we demonstrate that following stimulation, the ITK-SYK fusion-protein in cell lines translocates to the cell membrane and, moreover, that this phenomenon as well as SLP-76 phosphorylation are blocked upon phosphatidylinositol-3-kinase (PI3-kinase) inhibition.« less

A Rapid Cytoplasmic Mechanism for PI3 Kinase Regulation by the Nuclear Thyroid Hormone Receptor, TRβ, and Genetic Evidence for Its Role in the Maturation of Mouse Hippocampal Synapses In Vivo

PubMed Central

Martin, Negin P.; Fernandez de Velasco, Ezequiel Marron; Mizuno, Fengxia; Scappini, Erica L.; Gloss, Bernd; Erxleben, Christian; Williams, Jason G.; Stapleton, Heather M.; Gentile, Saverio

2014-01-01

Several rapid physiological effects of thyroid hormone on mammalian cells in vitro have been shown to be mediated by the phosphatidylinositol 3-kinase (PI3K), but the molecular mechanism of PI3K regulation by nuclear zinc finger receptor proteins for thyroid hormone and its relevance to brain development in vivo have not been elucidated. Here we show that, in the absence of hormone, the thyroid hormone receptor TRβ forms a cytoplasmic complex with the p85 subunit of PI3K and the Src family tyrosine kinase, Lyn, which depends on two canonical phosphotyrosine motifs in the second zinc finger of TRβ that are not conserved in TRα. When hormone is added, TRβ dissociates and moves to the nucleus, and phosphatidylinositol (3, 4, 5)-trisphosphate production goes up rapidly. Mutating either tyrosine to a phenylalanine prevents rapid signaling through PI3K but does not prevent the hormone-dependent transcription of genes with a thyroid hormone response element. When the rapid signaling mechanism was blocked chronically throughout development in mice by a targeted point mutation in both alleles of Thrb, circulating hormone levels, TRβ expression, and direct gene regulation by TRβ in the pituitary and liver were all unaffected. However, the mutation significantly impaired maturation and plasticity of the Schaffer collateral synapses on CA1 pyramidal neurons in the postnatal hippocampus. Thus, phosphotyrosine-dependent association of TRβ with PI3K provides a potential mechanism for integrating regulation of development and metabolism by thyroid hormone and receptor tyrosine kinases. PMID:24932806

Formation of PI 3-kinase products in platelets by thrombin, but not collagen, is dependent on synergistic autocrine stimulation, particularly through secreted ADP.

PubMed

Selheim, F; Idsøe, R; Fukami, M H; Holmsen, H; Vassbotn, F S

1999-10-05

Platelet activation by thrombin or collagen results in secretion and synthesis of several platelet agonists that enhance the responses to the primary agonists (autocrine stimulation). To disclose the effects of thrombin and collagen on the phosphorylation of 3-phosphoinositides per se we incubated platelets with five inhibitors of platelet autocrine stimulation (IAS) that act extracellularly. We found that IAS almost totally blocked thrombin-induced production of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. In contrast, collagen induced massive production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) in the presence of IAS. When testing the effect of each inhibitor individually we found the strongest inhibition of thrombin-induced PtdIns(3,4)P(2) production with the ADP scavenger system CP/CPK. Furthermore, we found a strong synergistic effect between exogenously added ADP and thrombin on production of PtdIns(3,4)P(2). In contrast to the results from 3-phosphorylated phosphoinositides, CP/CPK had little effect on thrombin-induced protein tyrosine phosphorylation. Our results show the importance of autocrine stimulation in thrombin-induced accumulation of 3-phosphorylated phosphoinositides and raise the question as to whether thrombin by itself is capable of inducing PI 3-K activation. In marked contrast to thrombin, collagen per se appears to be able to trigger increased production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3). Copyright 1999 Academic Press.

Localization of phosphatidylinositol signaling components in rat taste cells: Role in bitter taste transduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, P.M.; Verma, A.; Bredt, D.S.

1990-10-01

To assess the role of phosphatidylinositol turnover in taste transduction we have visualized, in rat tongue, ATP-dependent endoplasmic reticular accumulation of {sup 45}Ca{sup 2+}, inositol 1,4,5-trisphosphate receptor binding sites, and phosphatidylinositol turnover monitored by autoradiography of ({sup 3}H)cytidine diphosphate diacylglycerol formed from ({sup 3}H)cytidine. Accumulated {sup 45}Ca{sup 2+}, inositol 1,4,5-trisphosphate receptors, and phosphatidylinositol turnover are selectively localized to apical areas of the taste buds of circumvallate papillae, which are associated with bitter taste. Further evidence for a role of phosphatidylinositol turnover in bitter taste is our observation of a rapid, selective increase in mass levels of inositol 1,4,5-trisphosphate elicited bymore » low concentrations of denatonium, a potently bitter tastant.« less

Identification of a Kinase in Wheat Germ that Phosphorylates the Large Subunit of Initiation Factor 4F 1

PubMed Central

Humphreys, Jean; Browning, Karen S.; Ravel, Joanne M.

1988-01-01

A kinase has been isolated from wheat (Triticum aestivum) germ that phosphorylates the 220 kilodaltons (kD) subunit of wheat germ initiation factor (eIF) 4F, the 80 kD subunit of eIF-4B (an isozyme form of eIF-4F) and eIF-4G (the functional equivalent to mammalian eIF-4B). The kinase elutes from Sephacryl S-200 slightly in front of ovalbumin. The kinase phosphorylates casein and histone IIA to a small extent, but does not phosphorylate phosvitin. Of the wheat germ initiation factors, elongation factors, and small and large ribosomal subunits, only eIF-4F, eIF-4B, and eIF-4G are phosphorylated to a significant extent. The kinase phosphorylates eIF-4F to the extent of two phosphates per mole of the 220 kD subunit and phosphorylates eIF-4B to the extent of one phosphate per mole of the 80 kD subunit. The 26 kD subunit of eIF-4F and the 28 kD subunit of eIF-4B are not phosphorylated by the kinase. The kinase phosphorylates the 59 kD component of eIF-4G to the extent of 0.25 phosphate per mole of eIF-4G. Phosphorylation of eIF-4F and eIF-4B does not affect their ability to support the binding of mRNA to small ribosomal subunits in vitro. Images Fig. 2 Fig. 3 PMID:16666331

The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p

PubMed Central

Stalder, Danièle; Novick, Peter J.

2016-01-01

Sec2p is a guanine nucleotide exchange factor that activates Sec4p, the final Rab GTPase of the yeast secretory pathway. Sec2p is recruited to secretory vesicles by the upstream Rab Ypt32p acting in concert with phosphatidylinositol-4-phosphate (PI(4)P). Sec2p also binds to the Sec4p effector Sec15p, yet Ypt32p and Sec15p compete against each other for binding to Sec2p. We report here that the redundant casein kinases Yck1p and Yck2p phosphorylate sites within the Ypt32p/Sec15p binding region and in doing so promote binding to Sec15p and inhibit binding to Ypt32p. We show that Yck2p binds to the autoinhibitory domain of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P has been reduced. This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling. PMID:26700316

The hepatocyte growth factor antagonist NK4 inhibits indoleamine-2,3-dioxygenase expression via the c-Met-phosphatidylinositol 3-kinase-AKT signaling pathway

PubMed Central

WANG, DONGDONG; SAGA, YASUSHI; SATO, NAOTO; NAKAMURA, TOSHIKAZU; TAKIKAWA, OSAMU; MIZUKAMI, HIROAKI; MATSUBARA, SHIGEKI; FUJIWARA, HIROYUKI

2016-01-01

Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme involved in tumor malignancy. However, the regulatory mechanism underlying its involvement remains largely uncharacterized. The present study aimed to investigate the hypothesis that NK4, an antagonist of hepatocyte growth factor (HGF), can regulate IDO and to characterize the signaling mechanism involved. Following successful transfection of the human ovarian cancer cell line SKOV-3 (which constitutively expresses IDO) with an NK4 expression vector, we observed that NK4 expression suppressed IDO expression; furthermore, NK4 expression did not suppress cancer cell growth in vitro [in the absence of natural killer (NK) cells], but did influence tumor growth in vivo. In addition, NK4 enhanced the sensitivity of cancer cells to NK cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. In an effort to clarify the mechanisms by which NK4 interacts with IDO, we performed investigations utilizing various biochemical inhibitors. The results of these investigations were as follows. First, c-Met (a receptor of HGF) tyrosine kinase inhibitor PHA-665752, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 both suppress IDO expression. Second, enhanced expression of PTEN (a known tumor suppressor) via negative regulation within a PI3K-AKT pathway, inhibits IDO expression. Conversely, neither the MEK1/2 inhibitor U0126 nor the STAT3 inhibitor WP1066 affects IDO expression. These results suggest that NK4 inhibits IDO expression via a c-Met-PI3K-AKT signaling pathway. PMID:27082119

Estradiol regulates the insulin-like growth factor-I (IGF-I) signalling pathway: A crucial role of phosphatidylinositol 3-kinase (PI 3-kinase) in estrogens requirement for growth of MCF-7 human breast carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernard, Laurence; Legay, Christine; Adriaenssens, Eric

2006-12-01

Estrogens can stimulate the proliferation of estrogen-responsive breast cancer cells by increasing their proliferative response to insulin-like growth factors. With a view to investigating the molecular mechanisms implicated, we studied the effect of estradiol on the expression of proteins implicated in the insulin-like growth factor signalling pathway. Estradiol dose- and time-dependently increased the expression of insulin receptor substrate-1 and the p85/p110 subunits of phosphatidylinositol 3-kinase but did not change those of ERK2 and Akt/PKB. ICI 182,780 did not inhibit estradiol-induced IRS-1 and p85 expression. Moreover, two distinct estradiol-BSA conjugate compounds were as effective as estradiol in inducing IRS-1 and p85/p110more » expression indicating the possible implication of an estradiol membrane receptor. Comparative analysis of steroids-depleted and steroids-treated cells showed that IGF-I only stimulates cell growth in the latter condition. Nevertheless, expression of a constitutively active form of PI 3-kinase in steroid-depleted cells triggers proliferation. These results demonstrate that estradiol positively regulates essential proteins of the IGF signalling pathway and put in evidence that phosphatidylinositol 3-kinase plays a central role in the synergistic pro-proliferative action of estradiol and IGF-I.« less

Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C.

PubMed

Disatnik, M H; Hernandez-Sotomayor, S M; Jones, G; Carpenter, G; Mochly-Rosen, D

1994-01-18

Phospholipase C-gamma 1 (PLC-gamma 1; EC 3.1.4.11) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We have previously shown that activated PKC binds to specific receptor proteins, receptors for activated C kinase, or RACKs, of approximately 30 kDa. Here, we show that PLC-gamma 1 bound to these RACKs and inhibited subsequent PKC binding to RACKs. However, unlike PKC, the binding of PLC-gamma 1 to RACKs did not require phospholipids and calcium. After epidermal growth factor treatment of intact A-431 cells, the binding of PLC-gamma 1 to RACKs increased as compared with PLC-gamma 1 from control cells. This increase in PLC-gamma 1 binding to RACKs was due to the phosphorylation of PLC-gamma 1. Additional data indicated that PLC-gamma 1 binds to RACKs in solution; epidermal growth factor receptor-dependent PLC-gamma 1 phosphorylation and activation decreased in the presence of RACKs. It is possible that, in vivo, PLC-gamma 1 associates with RACKs or with other PLC-gamma 1-specific anchoring proteins in the particulate cell fraction. Since a PKC C2 homologous region is present in PLC-gamma 1, the C2 region may mediate the activation-induced translocation of the enzyme to the cell particulate fraction and the anchoring protein-PLC-gamma 1 complex may be the active translocated form of PLC-gamma 1.

Arginine Transcriptional Response Does Not Require Inositol Phosphate Synthesis*

PubMed Central

Bosch, Daniel; Saiardi, Adolfo

2012-01-01

Inositol phosphates are key signaling molecules affecting a large variety of cellular processes. Inositol-polyphosphate multikinase (IPMK) is a central component of the inositol phosphate biosynthetic routes, playing essential roles during development. IPMK phosphorylates inositol 1,4,5-trisphosphate to inositol tetrakisphosphate and subsequently to inositol pentakisphosphate and has also been described to function as a lipid kinase. Recently, a catalytically inactive mammalian IPMK was reported to be involved in nutrient signaling by way of mammalian target of rapamycin and AMP-activated protein kinase. In yeast, the IPMK homologue, Arg82, is the sole inositol-trisphosphate kinase. Arg82 has been extensively studied as part of the transcriptional complex regulating nitrogen sensing, in particular arginine metabolism. Whether this role requires Arg82 catalytic activity has long been a matter of contention. In this study, we developed a novel method for the real time study of promoter strength in vivo and used it to demonstrate that catalytically inactive Arg82 fully restored the arginine-dependent transcriptional response. We also showed that expression in yeast of catalytically active, but structurally very different, mammalian or plant IPMK homologue failed to restore arginine regulation. Our work indicates that inositol phosphates do not regulate arginine-dependent gene expression. PMID:22992733

Coordination of Receptor Tyrosine Kinase Signaling and Interfacial Tension Dynamics Drives Radial Intercalation and Tube Elongation. | Office of Cancer Genomics

Cancer.gov

We sought to understand how cells collectively elongate epithelial tubes. We first used 3D culture and biosensor imaging to demonstrate that epithelial cells enrich Ras activity, phosphatidylinositol (3,4,5)-trisphosphate (PIP3), and F-actin to their leading edges during migration within tissues. PIP3 enrichment coincided with, and could enrich despite inhibition of, F-actin dynamics, revealing a conserved migratory logic compared with single cells. We discovered that migratory cells can intercalate into the basal tissue surface and contribute to tube elongation.

Insulin-like growth factor-1 (IGF-1) promotes primordial follicle growth and reduces DNA fragmentation through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signalling pathway.

PubMed

Bezerra, Maria É S; Barberino, Ricássio S; Menezes, Vanúzia G; Gouveia, Bruna B; Macedo, Taís J S; Santos, Jamile M S; Monte, Alane P O; Barros, Vanessa R P; Matos, Maria H T

2018-05-30

We investigated the effects of insulin-like growth factor 1 (IGF-1) on the morphology and follicular activation of ovine preantral follicles cultured in situ and whether the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway is involved in IGF-1 action in the sheep ovary. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) analyses (fresh control) or cultured in supplemented alpha-minimum essential medium (α-MEM+; control) or α-MEM+ with IGF-1 (1, 10, 50, 100 or 200ngmL-1) for 7 days. Follicles were classified as normal or atretic, primordial or growing and the oocyte and follicle diameters were measured. DNA fragmentation was evaluated by TUNEL assay. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was performed on the fresh control, α-MEM+ and 100ngmL-1 IGF-1 samples. Inhibition of PI3K activity was performed through pretreatment with the PI3K inhibitor LY294002 and phosphorylated AKT (pAKT) expression was analysed after culture in the absence or presence of LY294002. IGF-1 at 100ngmL-1 increased (P<0.05) follicular activation compared with α-MEM+ and decreased TUNEL-positive cells (P<0.05) compared with other treatments. PCNA-positive cells also increased (P<0.05) in 100ngmL-1 IGF-1. LY294002 significantly inhibited follicular activation stimulated by α-MEM+ and 100ngmL-1 IGF-1 and reduced pAKT expression in follicles. Overall, IGF-1 at 100ngmL-1 promoted primordial follicle activation, cell proliferation and reduced DNA fragmentation after in situ culture through the PI3K/AKT pathway.

The identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer .

PubMed

Folkes, Adrian J; Ahmadi, Khatereh; Alderton, Wendy K; Alix, Sonia; Baker, Stewart J; Box, Gary; Chuckowree, Irina S; Clarke, Paul A; Depledge, Paul; Eccles, Suzanne A; Friedman, Lori S; Hayes, Angela; Hancox, Timothy C; Kugendradas, Arumugam; Lensun, Letitia; Moore, Pauline; Olivero, Alan G; Pang, Jodie; Patel, Sonal; Pergl-Wilson, Giles H; Raynaud, Florence I; Robson, Anthony; Saghir, Nahid; Salphati, Laurent; Sohal, Sukhjit; Ultsch, Mark H; Valenti, Melanie; Wallweber, Heidi J A; Wan, Nan Chi; Wiesmann, Christian; Workman, Paul; Zhyvoloup, Alexander; Zvelebil, Marketa J; Shuttleworth, Stephen J

2008-09-25

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.

PKI-179: an orally efficacious dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor.

PubMed

Venkatesan, Aranapakam M; Chen, Zecheng; dos Santos, Osvaldo; Dehnhardt, Christoph; Santos, Efren Delos; Ayral-Kaloustian, Semiramis; Mallon, Robert; Hollander, Irwin; Feldberg, Larry; Lucas, Judy; Yu, Ker; Chaudhary, Inder; Mansour, Tarek S

2010-10-01

A series of mono-morpholino 1,3,5-triazine derivatives (8a-8q) bearing a 3-oxa-8-azabicyclo[3.2.1]octane were prepared and evaluated for PI3-kinase/mTOR activity. Replacement of one of the bis-morpholines in lead compound 1 (PKI-587) with 3-oxa-8-azabicyclo[3.2.1]octane and reduction of the molecular weight yielded 8m (PKI-179), an orally efficacious dual PI3-kinase/mTOR inhibitor. The in vitro activity, in vivo efficacy, and PK properties of 8m are discussed. Copyright © 2010. Published by Elsevier Ltd.

Glut-1 translocation in FRTL-5 thyroid cells: role of phosphatidylinositol 3-kinase and N-glycosylation.

PubMed

Samih, N; Hovsepian, S; Aouani, A; Lombardo, D; Fayet, G

2000-11-01

It was previously demonstrated that insulin or TSH treatment of FRTL-5 cells resulted in an elevation of glucose transport and in an increase of cell surface expression of the glucose transporter Glut-1. However, the signaling mechanisms leading to the insulin or TSH-induced increase in the cell surface expression of Glut-1 were not investigated. In the present study, we demonstrated that wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), interfere both in the signaling pathways of insulin and TSH leading to glucose consumption enhancement and Glut-1 translocation. Two hours after insulin treatment, TSH or cAMP analog (Bu)2cAMP stimulation, glucose transport was increased and most of the intracellular Glut-1 pool was translocated to plasma membranes. Wortmannin or LY294002 blocked the insulin, (Bu)2cAMP, and the TSH-induced translocation of Glut-1. Wortmannin or LY294002 alone did not alter the basal ratio between intracellular and cell surface Glut-1 molecules. These results suggest that in FRTL-5 cells wortmannin and LY294002 inhibited the insulin, (Bu)2cAMP and TSH events leading to Glut-1 translocation from an intracellular compartment to the plasma membrane. Likewise, (Bu)2cAMP effects on glucose transport and Glut-1 translocation to plasma membrane were repressed by PI3-kinase inhibitors but not by the protein kinase A (PKA) inhibitor H89. We suggest that (Bu)2cAMP stimulates Glut-1 translocation to plasma membrane through PI3-kinase-dependent and PKA-independent signaling pathways. To further elucidate mechanisms that regulate the translocation of Glut-1 to cell membrane, we extended this study to the role played by the N-glycosylation in the translocation and in the biological activity of Glut-1 in FRTL-5 cells. For this purpose we used tunicamycin, an inhibitor of the N-glycosylation. Our experiments with tunicamycin clearly showed that both the glycosylated and unglycosylated forms of the transporter reached the cell surface. Likewise, a decrease in glucose consumption (-50%) after treatment of cells with tunicamycin was accompanied by a decrease (-70% vs. control) in the membrane expression of a 50-kDa form of Glut-1 and an increase in its unglycosylated 41-kDa form. These results suggest that carbohydrate moiety is essential for the biological activity of glucose transport but is not required for the translocation of Glut-1 from the intracellular membrane pool to the plasma membrane.

Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

PubMed

Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

1993-06-25

Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

Regulation of the endothelin-1 transmembrane signaling pathway: the potential role of agonist-induced desensitization in the coronary artery of the rapid ventricular pacing-overdrive dog model of heart failure.

PubMed

Calderone, A; Rouleau, J L; de Champlain, J; Bélichard, P; Stewart, D J

1993-08-01

This study examined the potential role of ET-1 and the contribution of protein kinase C (PKC) in the desensitization of the ET-1 transmembrane signaling pathway in the left circumflex coronary artery (CCA) of a dog model of congestive heart failure (CHF). In the CCA of the rapid ventricular pacing-overdrive dog model of CHF, elevated plasma endothelin levels were associated with a decrease in the basal accumulation of inositol phosphates and ET-1 mediated activation of phosphatidylinositol (PI) turnover (P < 0.05). To assess whether elevated plasma ET-1 levels may have contributed to the diminished ET-1 responsiveness in the heart failure dogs, ET-1 generation of inositol phosphates was measured following a one hour pretreatment of normal coronary artery rings with 0.1 nM ET-1. As compared to non-treated rings, ET-1 pretreatment resulted in a 33% decrease of ET-1 (10 nM) production of inositol phosphates. To evaluate the role of PKC in this process, normal coronary rings pretreated for a period of one hour with the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 microM), resulted in a similar attenuation (36%) of ET-1 production of inositol phosphates. In the presence of the protein kinase C inhibitor staurosporine, both the agonist and phorbol ester induced decreases in ET-1 mediated PI turnover were reversed. Staurosporine even potentiated (75%) ET-1 induced PI turnover despite ET-1 and PMA pretreatments. These results suggest that agonist-induced desensitization of ET-1 mediated PI turnover can occur and is at least one of the possible mechanisms contributing to the desensitization of the ET-1 transmembrane signaling pathway in the pacing-overdrive model of heart failure in the dog.

Role of the phosphatidylinositol-3-kinase/Akt/target of rapamycin pathway during ambidensovirus infection of insect cells.

PubMed

Salasc, F; Mutuel, D; Debaisieux, S; Perrin, A; Dupressoir, T; Grenet, A-S Gosselin; Ogliastro, M

2016-01-01

The phosphatidylinositol-3-kinase (PI3K)/Akt/target of rapamycin (TOR) signalling pathway controls cell growth and survival, and is targeted by a number of viruses at different phases of their infection cycle to control translation. Whether and how insect viruses interact with this pathway remain poorly addressed. Here, we investigated the role of PI3K/Akt/TOR signalling during lethal infection of insect cells with an insect parvovirus. Using Junonia coenia densovirus (JcDV; lepidopteran ambidensovirus 1) and susceptible insect cells as experimental models, we first described JcDV cytopathology, and showed that viral infection affects cell size, cell proliferation and survival. We deciphered the role of PI3K/Akt/TOR signalling in the course of infection and found that non-structural (NS) protein expression correlates with the inhibition of TOR and the shutdown of cellular synthesis, concomitant with the burst of viral protein expression. Together, these results suggest that NS proteins control the cellular translational machinery to favour the translation of viral mRNAs at the expense of cellular mRNAs. As a consequence of TOR inhibition, cell autophagy is activated. These results highlight new functions for NS proteins in the course of multiplication of an insect parvovirus.

Identification and classification of genes regulated by phosphatidylinositol 3-kinase- and TRKB-mediated signalling pathways during neuronal differentiation in two subtypes of the human neuroblastoma cell line SH-SY5Y.

PubMed

Nishida, Yuichiro; Adati, Naoki; Ozawa, Ritsuko; Maeda, Aasami; Sakaki, Yoshiyuki; Takeda, Tadayuki

2008-10-28

SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signalling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is unclear. To investigate this mechanism, we compared the gene expression profiles in SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which show a different phenotype during RA-mediated differentiation. SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF) for full differentiation. After exposing cells to a PI3K inhibitor, LY294002, we identified 386 genes and categorised these genes into two clusters dependent on the PI3K signalling pathway during RA-mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster, including 158 neural genes, was greatly reduced in SK-N-SH cells and partially impaired in SH-SY5Y-E cells, which is consistent with a defect in the neuronal phenotype of these cells. Additional stimulation with BDNF induced a set of neural genes that were down-regulated in RA-treated SH-SY5Y-E cells but were abundant in differentiated SH-SY5Y-A cells. We identified gene clusters controlled by PI3K- and TRKB-mediated signalling pathways during the differentiation of two subtypes of SH-SY5Y cells. The TRKB-mediated bypass pathway compensates for impaired neural function generated by defects in several signalling pathways, including PI3K in SH-SY5Y-E cells. Our expression profiling data will be useful for further elucidation of the signal transduction-transcriptional network involving PI3K or TRKB.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Audigier, S.M.P.; Wang, J.K.T.; Greengard, P.

Synaptosomes, purified from rat cerebral cortex, were prelabeled with (/sup 3/H)inositol to study phosphatidylinositol turnover in nerve terminals. Labeled synaptosomes were either depolarized with 40 mM K/sup +/ or exposed to carbamoylcholine (carbachol). K/sup +/ depolarization increased the level of inositol phosphates in a time-dependent manner. The inositol bisphosphate level also increased rapidly, but its elevated level was sustained during continued depolarization. The elevated level of inositol bisphosphate was reversed upon repolarization of the synaptosomes. The level of inositol monophosphate increased slowly to 120-130% of control. These effects of K/sup +/ depolarization depended on the presence of Ca/sup 2 +/more » in the incubation medium. Carbachol stimulated the turnover of phosphatidylinositol in a dose- and time-dependent manner. The level of inositol bisphosphate increased to 210% of control, and this maximal response was seen from 15 to 60 min. Accumulation of inositol monophosphate was larger than that of inositol bisphosphate, but its time course was slower. Atropine and pirenzepine inhibited the carbachol effect with high affinities. These data show that both Ca/sup 2 +/ influx and M/sub 1/ muscarinic receptor activation stimulate phospholipase C activity in synaptosomes, suggesting that phosphatidylinositol turnover may be involved in regulating neurotransmitter release from nerve terminals.« less

Phosphatidylinositol 4-Kinase III Beta Is Essential for Replication of Human Rhinovirus and Its Inhibition Causes a Lethal Phenotype In Vivo

PubMed Central

Spickler, Catherine; Lippens, Julie; Laberge, Marie-Kristine; Desmeules, Sophie; Bellavance, Édith; Garneau, Michel; Guo, Tim; Hucke, Oliver; Leyssen, Pieter; Neyts, Johan; Vaillancourt, Fréderic H.; Décor, Anne; O'Meara, Jeff; Franti, Michael

2013-01-01

Human rhinovirus (HRV) is the predominant cause of the common cold, but more importantly, infection may have serious repercussions in asthmatics and chronic obstructive pulmonary disorder (COPD) patients. A cell-based antiviral screen against HRV was performed with a subset of our proprietary compound collection, and an aminothiazole series with pan-HRV species and enteroviral activity was identified. The series was found to act at the level of replication in the HRV infectious cycle. In vitro selection and sequencing of aminothiazole series-resistant HRV variants revealed a single-nucleotide mutation leading to the amino acid change I42V in the essential HRV 3A protein. This same mutation has been previously implicated in resistance to enviroxime, a former clinical-stage antipicornavirus agent. Enviroxime-like compounds have recently been shown to target the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIβ). A good correlation between PI4KIIIβ activity and HRV antiviral potency was found when analyzing the data over 80 compounds of the aminothiazole series, covering a 750-fold potency range. The mechanism of action through PI4KIIIβ inhibition was further demonstrated by small interfering RNA (siRNA) knockdown of PI4KB, which reduced HRV replication and also increased the potency of the PI4KIIIβ inhibitors. Inhibitors from two different structural classes with promising pharmacokinetic profiles and with very good selectivity for PI4KIIIβ were used to dissociate compound-related toxicity from target-related toxicity. Mortality was seen in all dosing groups of mice treated with either compound, therefore suggesting that short-term inhibition of PI4KIIIβ is deleterious. PMID:23650168

p56Lck and p59Fyn Regulate CD28 Binding to Phosphatidylinositol 3-Kinase, Growth Factor Receptor-Bound Protein GRB-2, and T Cell-Specific Protein-Tyrosine Kinase ITK: Implications for T-Cell Costimulation

NASA Astrophysics Data System (ADS)

Raab, Monika; Cai, Yun-Cai; Bunnell, Stephen C.; Heyeck, Stephanie D.; Berg, Leslie J.; Rudd, Christopher E.

1995-09-01

T-cell activation requires cooperative signals generated by the T-cell antigen receptor ξ-chain complex (TCRξ-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, ξ-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

GCN5 regulates the activation of PI3K/Akt survival pathway in B cells exposed to oxidative stress via controlling gene expressions of Syk and Btk.

PubMed

Kikuchi, Hidehiko; Kuribayashi, Futoshi; Takami, Yasunari; Imajoh-Ohmi, Shinobu; Nakayama, Tatsuo

2011-02-25

Histone acetyltransferase(s) (HATs) are involved in the acetylation of core histones, which is an important event for transcription regulation through alterations in the chromatin structure in eukaryotes. General control non-depressible 5 (GCN5) was first identified as a global coactivator and transcription-related HAT. Here we report that GCN5 regulates the activation of phosphatidylinositol 3-kinase (PI3K)/acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) survival pathway in B cells exposed to oxidative stress via controlling gene expressions of spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk). The GCN5-deficiency remarkably caused apoptotic cell death by treatment with exogenous hydrogen peroxide (H(2)O(2)) in chicken DT40 cells. In GCN5-deficient DT40 cells, gene expressions of Syk and Btk, which are involved in activation of PI3K/Akt survival pathway in DT40 cells exposed to exogenous H(2)O(2), were remarkably decreased compared with those in wild type DT40 cells. In addition, phosphorylation of Akt in H(2)O(2)-treated GCN5-deficient cells was remarkably suppressed as compared to that of DT40. Chromatin immunoprecipitation assay revealed that GCN5 binds to proximal 5'-upstream regions of Syk and Btk genes in vivo. These results suggest that GCN5 takes part in transcriptional regulations of the Syk and Btk genes, and plays a key role in epigenetic regulation of PI3K/Akt survival pathway in B cells exposed to reactive oxygen species such as H(2)O(2). Copyright © 2011 Elsevier Inc. All rights reserved.

Erythropoietin activates telomerase through transcriptional and posttranscriptional regulation in human erythroleukemic JAS-REN-A cells.

PubMed

Akiyama, Masaharu; Kawano, Takeshi; Mikami-Terao, Yoko; Agawa-Ohta, Miyuki; Yamada, Osamu; Ida, Hiroyuki; Yamada, Hisashi

2011-03-01

We evaluated the molecular mechanism of telomerase activation by erythropoietin (EPO) in human erythroleukemic JAS-REN-A cells. Telomerase activity increased 3-4 fold after 3-24h of culture with EPO and was associated with increases in c-myc mRNA after 1-3h, of c-Myc protein after 3-6h, and of human telomerase reverse transcriptase (hTERT) mRNA and hTERT protein after 6-24h. Simultaneously EPO induced phosphorylation of signal transducer activator of transcription 5 (STAT5), AKT, and extracellular signal-regulated kinase (ERK). Telomerase activity induced by EPO was significantly inhibited by AG490, PD98059, and LY294002. AG490 downregulated c-myc and hTERT mRNA expression with inhibited STAT5 and AKT phosphorylation. PD98059 also reduced c-myc and hTERT expression and inhibited ERK phosphorylation. However, LY294002 did not inhibit c-myc or hTERT mRNA expression despite inhibiting STAT5 and AKT phosphorylation. These results suggest that EPO activates telomerase in JAS-REN-A cells through dual regulation: hTERT gene transcription by Janus tyrosine kinase 2/STAT5/c-Myc and hTERT protein phosphorylation by phosphatidylinositol 3'-kinase/AKT. Copyright © 2010 Elsevier Ltd. All rights reserved.

Expression of human choline kinase in NIH 3T3 fibroblasts increases the mitogenic potential of insulin and insulin-like growth factor I.

PubMed

Chung, T; Huang, J S; Mukherjee, J J; Crilly, K S; Kiss, Z

2000-05-01

In mammalian cells, growth factors, oncogenes, and carcinogens stimulate phosphocholine (PCho) synthesis by choline kinase (CK), suggesting that PCho may regulate cell growth. To validate the role of PCho in mitogenesis, we determined the effects of insulin, insulin-like growth factor I (IGF-I), and other growth factors on DNA synthesis in NIH 3T3 fibroblast sublines highly expressing human choline kinase (CK) without increasing phosphatidylcholine synthesis. In serum-starved CK expressor cells, insulin and IGF-I stimulated DNA synthesis, p70 S6 kinase (p70 S6K) activity, phosphatidylinositol 3-kinase (PI3K) activity, and activating phosphorylation of p42/p44 mitogen-activated protein kinases (MAPK) to greater extents than in the corresponding vector control cells. Furthermore, the CK inhibitor hemicholinium-3 (HC-3) inhibited insulin- and IGF-I-induced DNA synthesis in the CK overexpressors, but not in the vector control cells. The results indicate that high cellular levels of PCho potentiate insulin- and IGF-I-induced DNA synthesis by MAPK- and p70 S6K-regulated mechanisms.

Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

PubMed Central

Han, Shi-Chong; Guo, Hui-Chen; Sun, Shi-Qi; Jin, Ye; Wei, Yan-Quan; Feng, Xia; Yao, Xue-Ping; Cao, Sui-Zhong; Xiang Liu, Ding; Liu, Xiang-Tao

2016-01-01

Virus entry is an attractive target for therapeutic intervention. Here, using a combination of electron microscopy, immunofluorescence assay, siRNA interference, specific pharmacological inhibitors, and dominant negative mutation, we demonstrated that the entry of foot-and-mouth disease virus (FMDV) triggered a substantial amount of plasma membrane ruffling. We also found that the internalization of FMDV induced a robust increase in fluid-phase uptake, and virions internalized within macropinosomes colocalized with phase uptake marker dextran. During this stage, the Rac1-Pak1 signaling pathway was activated. After specific inhibition on actin, Na+/H+ exchanger, receptor tyrosine kinase, Rac1, Pak1, myosin II, and protein kinase C, the entry and infection of FMDV significantly decreased. However, inhibition of phosphatidylinositol 3-kinase (PI3K) did not reduce FMDV internalization but increased the viral entry and infection to a certain extent, implying that FMDV entry did not require PI3K activity. Results showed that internalization of FMDV exhibited the main hallmarks of macropinocytosis. Moreover, intracellular trafficking of FMDV involves EEA1/Rab5-positive vesicles. The present study demonstrated macropinocytosis as another endocytic pathway apart from the clathrin-mediated pathway. The findings greatly expand our understanding of the molecular mechanisms of FMDV entry into cells, as well as provide potential insights into the entry mechanisms of other picornaviruses. PMID:26757826

Plasma membrane proteins Slm1 and Slm2 mediate activation of the AGC kinase Ypk1 by TORC2 and sphingolipids in S. cerevisiae.

PubMed

Niles, Brad J; Powers, Ted

2012-10-15

The PH domain-containing proteins Slm1 and Slm2 were originally identified as substrates of the rapamycin-insensitive TOR complex 2 (TORC2) and as mediators of signaling by the lipid second messenger phosphatidyl-inositol-4,5-bisphosphate (PI4,5P2) in budding yeast S. cerevisiae. More recently, these proteins have been identified as critical effectors that facilitate phosphorylation and activation of the AGC kinases Ypk1 and Ypk2 by TORC2. Here, we review the molecular basis for this regulation as well as place it within the context of recent findings that have revealed Slm1/2 and TORC2-dependent phosphorylation of Ypk1 is coupled to the biosynthesis of complex sphingolipids and to their levels within the plasma membrane (PM) as well as other forms of PM stress. Together, these studies reveal the existence of an intricate homeostatic feedback mechanism, whereby the activity of these signaling components is linked to the biosynthesis of PM lipids according to cellular need.

Synthesis and SAR of 1-acetanilide-4-aminopyrazole-substituted quinazolines: selective inhibitors of Aurora B kinase with potent anti-tumor activity.

PubMed

Foote, Kevin M; Mortlock, Andrew A; Heron, Nicola M; Jung, Frédéric H; Hill, George B; Pasquet, Georges; Brady, Madeleine C; Green, Stephen; Heaton, Simon P; Kearney, Sarah; Keen, Nicholas J; Odedra, Rajesh; Wedge, Stephen R; Wilkinson, Robert W

2008-03-15

A new class of 1-acetanilide-4-aminopyrazole-substituted quinazoline Aurora kinase inhibitors has been discovered possessing highly potent cellular activity. Continuous infusion into athymic mice bearing SW620 tumors of the soluble phosphate derivative 2 led to dose-proportional exposure of the des-phosphate compound 8 with a high-unbound fraction. The combination of potent cell activity and high free-drug exposure led to pharmacodynamic changes in the tumor at low doses, indicative of Aurora B-kinase inhibition and a reduction in tumor volume.

Chromium picolinate enhances skeletal muscle cellular insulin signaling in vivo in obese, insulin-resistant JCR:LA-cp rats.

PubMed

Wang, Zhong Q; Zhang, Xian H; Russell, James C; Hulver, Matthew; Cefalu, William T

2006-02-01

Chromium is one of the few trace minerals for which a specific cellular mechanism of action has not been identified. Recent in vitro studies suggest that chromium supplementation may improve insulin sensitivity by enhancing insulin receptor signaling, but this has not been demonstrated in vivo. We investigated the effect of chromium supplementation on insulin receptor signaling in an insulin-resistant rat model, the JCR:LA-corpulent rat. Male JCR:LA-cp rats (4 mo of age) were randomly assigned to receive chromium picolinate (CrPic) (obese n=6, lean n=5) or vehicle (obese n=5, lean n=5) for 3 mo. The CrPic was provided in the water, and based on calculated water intake, rats randomized to CrPic received 80 microg/(kg.d). At the end of the study, skeletal muscle (vastus lateralis) biopsies were obtained at baseline and at 5, 15, and 30 min postinsulin stimulation to assess insulin signaling. Obese rats treated with CrPic had significantly improved glucose disposal rates and demonstrated a significant increase in insulin-stimulated phosphorylation of insulin receptor substrate (IRS)-1 and phosphatidylinositol (PI)-3 kinase activity in skeletal muscle compared with obese controls. The increase in cellular signaling was not associated with increased protein levels of the IRS proteins, PI-3 kinase or Akt. However, protein tyrosine phosphatase 1B (PTP1B) levels were significantly lower in obese rats administered CrPic than obese controls. When corrected for protein content, PTP1B activity was also significantly lower in obese rats administered CrPic than obese controls. Our data suggest that chromium supplementation of obese, insulin-resistant rats may improve insulin action by enhancing intracellular signaling.

Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

PubMed Central

McNulty, Shannon; Bornmann, William; Schriewer, Jill; Werner, Chas; Smith, Scott K.; Olson, Victoria A.; Damon, Inger K.; Buller, R. Mark; Heuser, John; Kalman, Daniel

2010-01-01

Poxvirus morphogenesis is a complex process that involves the successive wrapping of the virus in host cell membranes. We screened by plaque assay a focused library of kinase inhibitors for those that caused a reduction in viral growth and identified several compounds that selectively inhibit phosphatidylinositol 3-kinase (PI3K). Previous studies demonstrated that PI3Ks mediate poxviral entry. Using growth curves and electron microscopy in conjunction with inhibitors, we show that that PI3Ks additionally regulate morphogenesis at two distinct steps: immature to mature virion (IMV) transition, and IMV envelopment to form intracellular enveloped virions (IEV). Cells derived from animals lacking the p85 regulatory subunit of Type I PI3Ks (p85α−/−β−/−) presented phenotypes similar to those observed with PI3K inhibitors. In addition, VV appear to redundantly use PI3Ks, as PI3K inhibitors further reduce plaque size and number in p85α−/−β−/− cells. Together, these data provide evidence for a novel regulatory mechanism for virion morphogenesis involving phosphatidylinositol dynamics and may represent a new therapeutic target to contain poxviruses. PMID:20526370

Interactions of polyomavirus middle T with the SH2 domains of the pp85 subunit of phosphatidylinositol-3-kinase.

PubMed Central

Yoakim, M; Hou, W; Liu, Y; Carpenter, C L; Kapeller, R; Schaffhausen, B S

1992-01-01

The binding of phosphatidylinositol-3-kinase to the polyomavirus middle T antigen is facilitated by tyrosine phosphorylation of middle T on residue 315. The pp85 subunit of phosphatidylinositol-3-kinase contains two SH2 domains, one in the middle of the molecule and one at the C terminus. When assayed by blotting with phosphorylated middle T, the more N-terminal SH2 domain is responsible for binding to middle T. When assayed in solution with glutathione S transferase fusions, both SH2s are capable of binding phosphorylated middle T. While both SH2 fusions can compete with intact pp85 for binding to middle T, the C-terminal SH2 is the more efficient of the two. Interaction between pp85 or its SH2 domains and middle T can be blocked by a synthetic peptide comprising the tyrosine phosphorylation sequence around middle T residue 315. Despite the fact that middle T can interact with both SH2s, these domains are not equivalent. Only the C-terminal SH2-middle T interaction was blocked by anti-SH2 antibody; the two SH2 fusions also interact with different cellular proteins. Images PMID:1380095

Atypical Protein Kinase C Activity in the Hypothalamus Is Required for Lipopolysaccharide-Mediated Sickness Responses

PubMed Central

Thaler, Joshua P.; Choi, Sun Ju; Sajan, Mini P.; Ogimoto, Kayoko; Nguyen, Hong T.; Matsen, Miles; Benoit, Stephen C.; Wisse, Brent E.; Farese, Robert V.; Schwartz, Michael W.

2009-01-01

By activating the Toll-like receptor 4-nuclear factor-κB signal transduction pathway, the bacterial endotoxin lipopolysaccharide (LPS) induces anorexia, weight loss, fever, and other components of the sickness response. By comparison, the hormones leptin and insulin cause anorexia without sickness via a central mechanism involving the phosphatidylinositol-3 kinase signaling pathway. In the current study, we investigated whether a common Toll-like receptor 4 and phosphatidylinositol-3 kinase signaling intermediate, atypical protein kinase Cζ/λ (aPKC), contributes to changes of energy balance induced by these stimuli. Immunohistochemistry analysis revealed that aPKC is expressed in the arcuate and paraventricular nuclei of the hypothalamus, key sites of leptin, insulin, and LPS action. Although administration of LPS, insulin, and leptin each acutely increased hypothalamic aPKC activity at doses that also reduce food intake, LPS treatment caused over 10-fold greater activation of hypothalamic a PKC signaling than that induced by leptin or insulin. Intracerebroventricular pretreatment with an aPKC inhibitor blocked anorexia induced by LPS but not insulin or leptin. Similarly, LPS-induced hypothalamic inflammation (as judged by induction of proinflammatory cytokine gene expression) and neuronal activation in the paraventricular nucleus (as judged by c-fos induction) were reduced by central aPKC inhibition. Although intracerebroventricular aPKC inhibitor administration also abolished LPS-induced fever, it had no effect on sickness-related hypoactivity or weight loss. We conclude that although hypothalamic aPKC signaling is not required for food intake inhibition by insulin or leptin, it plays a key role in inflammatory anorexia and fever induced by LPS. PMID:19819945

CB1 Cannabinoid Receptors Modulate Kinase and Phosphatase Activity during Extinction of Conditioned Fear in Mice

ERIC Educational Resources Information Center

Kamprath, Kornelia; Hermann, Heike; Lutz, Beat; Marsicano, Giovanni; Cannich, Astrid; Wotjak, Carsten T.

2004-01-01

Cannabinoid receptors type 1 (CB1) play a central role in both short-term and long-term extinction of auditory-cued fear memory. The molecular mechanisms underlying this function remain to be clarified. Several studies indicated extracellular signal-regulated kinases (ERKs), the phosphatidylinositol 3-kinase with its downstream effector AKT, and…

Voltage-sensing phosphatase modulation by a C2 domain.

PubMed

Castle, Paul M; Zolman, Kevin D; Kohout, Susy C

2015-01-01

The voltage-sensing phosphatase (VSP) is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD), the inter-domain linker, the cytosolic catalytic domain, and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate (PIP) lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (Kalli et al., 2014). Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5)P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry (VCF) were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5)P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in VSP function.

Voltage-sensing phosphatase modulation by a C2 domain

PubMed Central

Castle, Paul M.; Zolman, Kevin D.; Kohout, Susy C.

2015-01-01

The voltage-sensing phosphatase (VSP) is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD), the inter-domain linker, the cytosolic catalytic domain, and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate (PIP) lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (Kalli et al., 2014). Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5)P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry (VCF) were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5)P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in VSP function. PMID:25904865

Echinacea purpurea root extract inhibits TNF release in response to Pam3Csk4 in a phosphatidylinositol-3-kinase dependent manner.

PubMed

Fast, David J; Balles, John A; Scholten, Jeffrey D; Mulder, Timothy; Rana, Jatinder

2015-10-01

Polysaccharides derived from Echinacea have historically been shown to be immunostimulatory. We describe in this work however the anti-inflammatory effect of a water extract of Echinacea purpurea roots (EPRW) that inhibited Pam3Csk4 stimulated production of TNFα by human monocytic THP-1 cells. The polyphenols and alkylamides typically found in Echinacea extracts were absent in EPRW suggesting that the anti-inflammatory component(s) was a polysaccharide. This anti-inflammatory activity was shown to be mediated by the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway as chemical inhibition of PI3K abolished the EPRW anti-inflammatory effect. Demonstration of phosphorylation of Akt and ribosomal S6 proteins, downstream targets of PI3K confirmed EPRW-mediated activation of this pathway. In conclusion, this observation suggests that non-alkylamide/non-polyphenolic phytochemicals from Echinacea may contribute in part to some of the anti-inflammatory therapeutic effects such as reduced severity of symptoms that have been observed in vivo in the treatment of upper respiratory tract infections with Echinacea. Copyright © 2015 Elsevier Inc. All rights reserved.

Pharmacokinetic-pharmacodynamic modeling of tumor growth inhibition and biomarker modulation by the novel phosphatidylinositol 3-kinase inhibitor GDC-0941.

PubMed

Salphati, Laurent; Wong, Harvey; Belvin, Marcia; Bradford, Delia; Edgar, Kyle A; Prior, Wei Wei; Sampath, Deepak; Wallin, Jeffrey J

2010-09-01

The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation. Its deregulation, by activation or transforming mutations of the p110alpha subunit, is associated with the development of many cancers. 2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) is a novel small molecule inhibitor of PI3K currently being evaluated in the clinic as an anticancer agent. The objectives of these studies were to characterize the relationships between GDC-0941 plasma concentrations and tumor reduction in MCF7.1 breast cancer xenografts and to evaluate the association between the tumor pharmacodynamic biomarker [phosphorylated (p) Akt and phosphorylated proline-rich Akt substrate of 40 kDa (pPRAS40)] responses and antitumor efficacy. MCF7.1 tumor-bearing mice were treated for up to 3 weeks with GDC-0941 at various doses (12.5-200 mg/kg) and dosing schedules (daily to weekly). An indirect response model fitted to tumor growth data indicated that the GDC-0941 plasma concentration required for tumor stasis was approximately 0.3 muM. The relationship between GDC-0941 plasma concentrations and inhibition of pAkt and pPRAS40 in tumor was also investigated after a single oral dose of 12.5, 50, or 150 mg/kg. An indirect response model was fitted to the inhibition of Akt and PRAS40 phosphorylation data and provided IC(50) estimates of 0.36 and 0.29 muM for pAkt and pPRAS40, respectively. The relationship between pAkt inhibition and tumor volume was further explored using an integrated pharmacokinetic biomarker tumor growth model, which showed that a pAkt inhibition of at least 30% was required to achieve stasis after GDC-0941 treatment of the MCF7.1 xenograft.

Infectious bursal disease virus activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by interaction of VP5 protein with the p85{alpha} subunit of PI3K

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei Li; Hou Lei; Zhu Shanshan

Phosphatidylinositol 3-kinase (PI3K)/Akt signaling is commonly activated upon virus infection and has been implicated in the regulation of diverse cellular functions such as proliferation and apoptosis. The present study demonstrated for the first time that infectious bursal disease virus (IBDV), the causative agent of a highly contagious disease in chickens, can induce Akt phosphorylation in cultured cells, by a mechanism that is dependent on PI3K. Inhibition of PI3K activation greatly enhanced virus-induced cytopathic effect and apoptotic cell death as evidenced by cleavage of poly-ADP ribose polymerase and activation of caspase-3. Investigations into the mechanism of PI3K/Akt activation revealed that IBDVmore » activates PI3K/Akt signaling through binding of the non-structural protein VP5 to regulatory subunit p85{alpha} of PI3K resulting in the suppression of premature apoptosis and improved virus growth after infection. The results presented here provide a basis for understanding molecular mechanism of IBDV infection.« less

Direct modification and regulation of a nuclear receptor-PIP2 complex by the nuclear inositol-lipid kinase IPMK

PubMed Central

Blind, Raymond D.; Suzawa, Miyuki; Ingraham, Holly A.

2012-01-01

Phosphatidylinositol (4,5)-bisphosphate (PIP2) is best known as a plasma membrane-bound regulatory lipid. While PIP2 and phosphoinositide-modifying enzymes coexist in the nucleus, their roles in the nucleus remain unclear. Here we show that the nuclear inositol polyphosphate multikinase (IPMK), which functions both as an inositol- and a PI3-kinase, interacts with the nuclear receptor SF-1 (NR5A1) and phosphorylates its bound ligand, PIP2. IPMK failed to recognize SF-1/PIP2 after blocking or displacing PIP2 from SF-1’s large hydrophobic pocket. In contrast to IPMK, p110 catalytic subunits of type 1 PI3-kinases were inactive on SF-1/PIP2. These and other in vitro analyses demonstrated specificity of IPMK for the SF-1/PIP2 protein/lipid complex. Once generated, SF-1/PIP3 is readily dephosphorylated by the lipid phosphatase PTEN. Importantly, decreasing IPMK or increasing PTEN expression greatly reduced SF-1 transcriptional activity. This ability of lipid kinases and phosphatases to alter the activity and directly remodel a non-membrane protein/lipid complex such SF-1/PIP2, establishes a new pathway for promoting lipid-mediated signaling in the nucleus. PMID:22715467

Combined metabolic and transcriptional profiling identifies pentose phosphate pathway activation by HSP27 phosphorylation during cerebral ischemia.

PubMed

Imahori, Taichiro; Hosoda, Kohkichi; Nakai, Tomoaki; Yamamoto, Yusuke; Irino, Yasuhiro; Shinohara, Masakazu; Sato, Naoko; Sasayama, Takashi; Tanaka, Kazuhiro; Nagashima, Hiroaki; Kohta, Masaaki; Kohmura, Eiji

2017-05-04

The metabolic pathophysiology underlying ischemic stroke remains poorly understood. To gain insight into these mechanisms, we performed a comparative metabolic and transcriptional analysis of the effects of cerebral ischemia on the metabolism of the cerebral cortex using middle cerebral artery occlusion (MCAO) rat model. Metabolic profiling by gas-chromatography/mass-spectrometry analysis showed clear separation between the ischemia and control group. The decreases of fructose 6-phosphate and ribulose 5-phosphate suggested enhancement of the pentose phosphate pathway (PPP) during cerebral ischemia (120-min MCAO) without reperfusion. Transcriptional profiling by microarray hybridization indicated that the Toll-like receptor and mitogen-activated protein kinase (MAPK) signaling pathways were upregulated during cerebral ischemia without reperfusion. In relation to the PPP, upregulation of heat shock protein 27 (HSP27) was observed in the MAPK signaling pathway and was confirmed through real-time polymerase chain reaction. Immunoblotting showed a slight increase in HSP27 protein expression and a marked increase in HSP27 phosphorylation at serine 85 after 60-min and 120-min MCAO without reperfusion. Corresponding upregulation of glucose 6-phosphate dehydrogenase (G6PD) activity and an increase in the NADPH/NAD + ratio were also observed after 120-min MCAO. Furthermore, intracerebroventricular injection of ataxia telangiectasia mutated (ATM) kinase inhibitor (KU-55933) significantly reduced HSP27 phosphorylation and G6PD upregulation after MCAO, but that of protein kinase D inhibitor (CID755673) did not affect HSP27 phosphorylation. Consequently, G6PD activation via ischemia-induced HSP27 phosphorylation by ATM kinase may be part of an endogenous antioxidant defense neuroprotection mechanism during the earliest stages of ischemia. These findings have important therapeutic implications for the treatment of stroke. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

NF-κB Is the Transcription Factor for FGF-2 That Causes Endothelial Mesenchymal Transformation in Cornea

PubMed Central

Lee, Jeong Goo

2012-01-01

Purpose. To determine the role of nuclear factor-κB (NF-κB) during FGF-2–mediated endothelial mesenchymal transformation (EMT) in response to interleukin (IL)-1β stimulation in corneal endothelial cells (CECs). Methods. Expression and/or activation of IL-1 receptor–associated protein kinase (IRAK), TNF receptor–associated factor 6 (TRAF6), phosphatidylinositol 3-kinase (PI 3-kinase), IκB kinase (IKK), IκB, NF-κB, and FGF-2 were analyzed by immunoblot analysis. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. NF-κB activity was measured by NF-κB ELISA kit, while binding of NF-κB to the promoter region of FGF-2 gene was determined by chromatin immunoprecipitation. Results. Brief stimulation of CECs with IL-1β upregulated expression of IRAK and TRAF6 and activated PI 3-kinase; expression of IRAK and TRAF6 reached maximum within 60 minutes, after which the expression disappeared, while PI 3-kinase activity was observed up to 4 hours after IL-1β stimulation. Use of specific inhibitor to PI 3-kinase or IRAK demonstrated that IRAK activates PI 3-kinase, the signaling of which phosphorylates IKKα/β and degrades IκB, subsequently leading to activation of NF-κB. The induction of FGF-2 by IL-1β was completely blocked by inhibitors to NF-κB activation (sulfasalazine) or PI 3-kinase (LY294002), and both inhibitors greatly blocked cell proliferation of CECs. Chromatin immunoprecipitation further demonstrated that NF-κB is the transcription factor of FGF-2 as NF-κB binds the putative NF-κB binding site of the FGF-2 promoter. Conclusions. These data suggest that IL-1β signaling combines the canonical pathway and the PI 3-kinase signaling to upregulate FGF-2 production through NF-κB, which plays a key role as a transcription factor of FGF-2 gene. PMID:22323467

Crystal structures of Salmonella typhimurium propionate kinase and its complex with Ap4A: evidence for a novel Ap4A synthetic activity.

PubMed

Simanshu, Dhirendra K; Savithri, H S; Murthy, M R N

2008-03-01

Propionate kinase catalyses the last step in the anaerobic breakdown of L-threonine to propionate in which propionyl phosphate and ADP are converted to propionate and ATP. Here we report the structures of propionate kinase (TdcD) in the native form as well as in complex with diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) by X-ray crystallography. Structure of TdcD obtained after cocrystallization with ATP showed Ap4A bound to the active site pocket suggesting the presence of Ap4A synthetic activity in TdcD. Binding of Ap4A to the enzyme was confirmed by the structure determination of a TdcD-Ap4A complex obtained after cocrystallization of TdcD with commercially available Ap4A. Mass spectroscopic studies provided further evidence for the formation of Ap4A by propionate kinase in the presence of ATP. In the TdcD-Ap4A complex structure, Ap4A is present in an extended conformation with one adenosine moiety present in the nucleotide binding site and other in the proposed propionate binding site. These observations tend to support direct in-line transfer of phosphoryl group during the kinase reaction. 2007 Wiley-Liss, Inc.

The dynamics of plasma membrane PtdIns(4,5)P(2) at fertilization of mouse eggs.

PubMed

Halet, Guillaume; Tunwell, Richard; Balla, Tamas; Swann, Karl; Carroll, John

2002-05-15

A series of intracellular Ca2+ oscillations are responsible for triggering egg activation and cortical granule exocytosis at fertilization in mammals. These Ca2+ oscillations are generated by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], which results from the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)]. Using confocal imaging to simultaneously monitor Ca2+ and plasma membrane PtdIns(4,5)P(2) in single living mouse eggs we have sought to establish the relationship between the kinetics of PtdIns(4,5)P(2) metabolism and the Ca2+ oscillations at fertilization. We report that there is no detectable net loss of plasma membrane PtdIns(4,5)P(2) either during the latent period or during the subsequent Ca2+ oscillations. When phosphatidylinositol 4-kinase is inhibited with micromolar wortmannin a limited decrease in plasma membrane PtdIns(4,5)P(2) is detected in half the eggs studied. Although we were unable to detect a widespread loss of PtdIns(4,5)P(2), we found that fertilization triggers a net increase in plasma membrane PtdIns(4,5)P(2) that is localized to the vegetal cortex. The fertilization-induced increase in PtdIns(4,5)P(2) follows the increase in Ca2+, is blocked by Ca2+ buffers and can be mimicked, albeit with slower kinetics, by photoreleasing Ins(1,4,5)P(3). Inhibition of Ca2+-dependent exocytosis of cortical granules, without interfering with Ca2+ transients, inhibits the PtdIns(4,5)P(2) increase. The increase appears to be due to de novo synthesis since it is inhibited by micromolar wortmannin. Finally, there is no increase in PtdIns(4,5)P(2) in immature oocytes that are not competent to extrude cortical granules. These studies suggest that fertilization does not deplete plasma membrane PtdIns(4,5)P(2) and that one of the pathways for increasing PtdIns(4,5)P(2) at fertilization is invoked by exocytosis of cortical granules.

In vitro glucose uptake activity of Aegles marmelos and Syzygium cumini by activation of Glut-4, PI3 kinase and PPARgamma in L6 myotubes.

PubMed

Anandharajan, R; Jaiganesh, S; Shankernarayanan, N P; Viswakarma, R A; Balakrishnan, A

2006-06-01

The purpose of the present study is to investigate the effect of methanolic extracts of Aegles marmelos and Syzygium cumini on a battery of targets glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPARgamma) and phosphatidylinositol 3' kinase (PI3 kinase) involved in glucose transport. A. marmelos and S. cumini are anti-diabetic medicinal plants being used in Indian traditional medicine. Different solvent extracts extracted sequentially were analysed for glucose uptake activity at each step and methanol extracts were found to be significantly active at 100ng/ml dose comparable with insulin and rosiglitazone. Elevation of Glut-4, PPARgamma and PI3 kinase by A. marmelos and S. cumini in association with glucose transport supported the up-regulation of glucose uptake. The inhibitory effect of cycloheximide on A. marmelos- and S. cumini-mediated glucose uptake suggested that new protein synthesis is required for the elevated glucose transport. Current observation concludes that methanolic extracts of A. marmelos and S. cumini activate glucose transport in a PI3 kinase-dependent fashion.

Gene expression of tumour necrosis factor and insulin signalling-related factors in subcutaneous adipose tissue during the dry period and in early lactation in dairy cows.

PubMed

Sadri, H; Bruckmaier, R M; Rahmani, H R; Ghorbani, G R; Morel, I; van Dorland, H A

2010-10-01

Gene expression of adipose factors, which may be part of the mechanisms that underlie insulin sensitivity, were studied in dairy cows around parturition. Subcutaneous fat biopsies and blood samples were taken from 27 dairy cows in week 8 antepartum (a.p.), on day 1 postpartum (p.p.) and in week 5 p.p. In the adipose tissue samples, mRNA was quantified by real-time reverse transcription polymerase chain reaction for tumour necrosis factor alpha (TNFα), insulin-independent glucose transporter (GLUT1), insulin-responsive glucose transporter (GLUT4), insulin receptor, insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), regulatory subunit of phosphatidylinositol-3 kinase (p85) and catalytic subunit of phosphatidylinositol-3 kinase. Blood plasma was assayed for concentrations of glucose, β-hydroxybutyric acid, non-esterified fatty acids (NEFA) and insulin. Plasma parameters followed a pattern typically observed in dairy cows. Gene expression changes were observed, but there were no changes in TNFα concentrations, which may indicate its local involvement in catabolic adaptation of adipose tissue. Changes in GLUT4 and GLUT1 mRNA abundance may reflect their involvement in reduced insulin sensitivity and in sparing glucose for milk synthesis in early lactation. Unchanged gene expression of IRS1, IRS2 and p85 over time may imply a lack of their involvement in terms of insulin sensitivity dynamics. Alternatively, it may indicate that post-transcriptional modifications of these factors came into play and may have concealed an involvement. © 2010 Blackwell Verlag GmbH.

Phosphatidylinositol 3-Kinase Plays a Vital Role in Regulation of Rice Seed Vigor via Altering NADPH Oxidase Activity

PubMed Central

Liu, Jian; Zhou, Jun; Xing, Da

2012-01-01

Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination. PMID:22448275

Milrinone-induced postconditioning reduces hepatic ischemia-reperfusion injury in rats: the roles of phosphatidylinositol 3-kinase and nitric oxide.

PubMed

Toyoda, Tomomi; Tosaka, Shinya; Tosaka, Reiko; Maekawa, Takuji; Cho, Sungsam; Eguchi, Susumu; Nakashima, Masahiro; Sumikawa, Koji

2014-01-01

Ischemic postconditioning (PostC) protects the liver against ischemia-reperfusion (IR) injury. Milrinone, a phosphodiesterase 3 inhibitor, has been reported to exhibit preconditioning properties against hepatic IR injury; however, its PostC properties remain unknown. This study investigated whether milrinone has PostC properties against hepatic IR injury and the roles of phosphatidylinositol 3-kinase (PI3K) and nitric oxide synthase (NOS). Male Wistar rats were separated into six groups: (1) group S: animals that underwent sham operation without ischemia, (2) group C: ischemia followed by reperfusion with no other intervention, (3) group M: milrinone administered immediately after reperfusion, (4) group MW: wortmannin, a PI3K inhibitor, injected before milrinone administration, (5) group MN: l-NAME, a NOS inhibitor, injected before milrinone administration, and (6) group MD, milrinone administered 30 min after reperfusion. Except for group S, all groups underwent 1 h of warm ischemia of median and left lateral lobes, followed by 5 h of reperfusion. Biochemical liver function analysis and histologic examination were performed. Serum aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase levels, histologic damage scores, and apoptotic rate in group M were significantly lower than those in group C. The inhibition of PI3K or NOS prevented this protective effect. Milrinone administered 30 min after reperfusion did not show obvious protective effects. Milrinone-induced PostC protects against hepatic IR injury when it is administered immediately after reperfusion, and PI3K and NOS may play an important role in this protective effect. Copyright © 2014 Elsevier Inc. All rights reserved.

Multifunctional Role of ATM/Tel1 Kinase in Genome Stability: From the DNA Damage Response to Telomere Maintenance

PubMed Central

2014-01-01

The mammalian protein kinase ataxia telangiectasia mutated (ATM) is a key regulator of the DNA double-strand-break response and belongs to the evolutionary conserved phosphatidylinositol-3-kinase-related protein kinases. ATM deficiency causes ataxia telangiectasia (AT), a genetic disorder that is characterized by premature aging, cerebellar neuropathy, immunodeficiency, and predisposition to cancer. AT cells show defects in the DNA damage-response pathway, cell-cycle control, and telomere maintenance and length regulation. Likewise, in Saccharomyces cerevisiae, haploid strains defective in the TEL1 gene, the ATM ortholog, show chromosomal aberrations and short telomeres. In this review, we outline the complex role of ATM/Tel1 in maintaining genomic stability through its control of numerous aspects of cellular survival. In particular, we describe how ATM/Tel1 participates in the signal transduction pathways elicited by DNA damage and in telomere homeostasis and its importance as a barrier to cancer development. PMID:25247188

Zonda is a novel early component of the autophagy pathway in Drosophila

PubMed Central

Melani, Mariana; Valko, Ayelén; Romero, Nuria M.; Aguilera, Milton O.; Acevedo, Julieta M.; Bhujabal, Zambarlal; Perez-Perri, Joel; de la Riva-Carrasco, Rocío V.; Katz, Maximiliano J.; Sorianello, Eleonora; D’Alessio, Cecilia; Juhász, Gabor; Johansen, Terje; Colombo, María I.; Wappner, Pablo

2017-01-01

Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. Here we report that a novel Drosophila immunophilin, which we have named Zonda, is critically required for starvation-induced autophagy. We show that Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions and, soon after starvation, nucleates in endoplasmic reticulum–associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13, and Atg17 but does not require Vps34, Vps15, Atg6, or Atg14. Zonda interacts physically with Atg1 through its kinase domain, as well as with Atg6 and Vps34. We propose that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation. PMID:28904211

Targeting Phosphatidylinositol 4-Kinase IIIα for Radiosensitization: A Potential Model of Drug Repositioning Using an Anti-Hepatitis C Viral Agent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Jeanny; Kim, Dan Hyo; Park, Ji Min

Purpose: To investigate which isotype of phosphatidylinositol 4-kinase (PI4K) may affect radiosensitivity and examine whether anti–hepatitis C viral (HCV) agents, some of which have been shown to inhibit PI4K IIIα activity, could be repositioned as a radiosensitizer in human cancer cells. Methods and Materials: U251, BT474, and HepG2 cell lines and normal human astrocyte were used. Ribonucleic acid interference, clonogenic assays, Western blotting, immunofluorescence, annexin V assay, lysotracker staining, and β-galactosidase assay were performed. Results: Of the 4 PI4K isotypes, specific inhibition of IIIα increased radiosensitivity. For pharmacologic inhibition of PI4K IIIα, we screened 9 anti-HCV agents by half-maximal inhibitorymore » concentration assay. Simeprevir was selected, and its inhibition of PI4K IIIα activity was confirmed. Combination of simeprevir treatment and radiation significantly attenuated expression of phospho-phospho-PKC and phospho-Akt and increased radiation-induced cell death in tested cell lines. Pretreatment with simeprevir prolonged γH2AX foci formation and down-regulation of phospho-DNA-PKcs, indicating impairment of nonhomologous end-joining repair. Cells pretreated with simeprevir exhibited mixed modes of cell death, including apoptosis and autophagy. Conclusion: These data demonstrate that targeting PI4K IIIα using an anti-HCV agent is a viable approach to enhance the therapeutic efficacy of radiation therapy in various human cancers, such as glioma, breast, and hepatocellular carcinoma.« less

Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

PubMed

Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

2016-07-01

Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application.

Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines

PubMed Central

Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

2016-01-01

Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334

Insulin-sensitive phospholipid signaling systems and glucose transport. Update II.

PubMed

Farese, R V

2001-04-01

Insulin provokes rapid changes in phospholipid metabolism and thereby generates biologically active lipids that serve as intracellular signaling factors that regulate glucose transport and glycogen synthesis. These changes include: (i) activation of phosphatidylinositol 3-kinase (PI3K) and production of PIP3; (ii) PIP3-dependent activation of atypical protein kinase Cs (PKCs); (iii) PIP3-dependent activation of PKB; (iv) PI3K-dependent activation of phospholipase D and hydrolysis of phosphatidylcholine with subsequent increases in phosphatidic acid (PA) and diacylglycerol (DAG); (v) PI3K-independent activation of glycerol-3-phosphate acylytansferase and increases in de novo synthesis of PA and DAG; and (vi) activation of DAG-sensitive PKCs. Recent findings suggest that atypical PKCs and PKB serve as important positive regulators of insulin-stimulated glucose metabolism, whereas mechanisms that result in the activation of DAG-sensitive PKCs serve mainly as negative regulators of insulin signaling through PI3K. Atypical PKCs and PKB are rapidly activated by insulin in adipocytes, liver, skeletal muscles, and other cell types by a mechanism requiring PI3K and its downstream effector, 3-phosphoinositide-dependent protein kinase-1 (PDK-1), which, in conjunction with PIP3, phosphorylates critical threonine residues in the activation loops of atypical PKCs and PKB. PIP3 also promotes increases in autophosphorylation and allosteric activation of atypical PKCs. Atypical PKCs and perhaps PKB appear to be required for insulin-induced translocation of the GLUT 4 glucose transporter to the plasma membrane and subsequent glucose transport. PKB also appears to be the major regulator of glycogen synthase. Together, atypical PKCs and PKB serve as a potent, integrated PI3K/PDK-1-directed signaling system that is used by insulin to regulate glucose metabolism.

Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane.

PubMed

Hirama, Takashi; Das, Raibatak; Yang, Yanbo; Ferguson, Charles; Won, Amy; Yip, Christopher M; Kay, Jason G; Grinstein, Sergio; Parton, Robert G; Fairn, Gregory D

2017-08-25

Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (Cavs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled Cav1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveola formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable Cav1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of Cav1 and cavin1 was diminished, and cavin1 degradation was increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns(4,5)P 2 has minimal impact on caveola assembly but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper Cav clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane

PubMed Central

Hirama, Takashi; Das, Raibatak; Yang, Yanbo; Ferguson, Charles; Won, Amy; Yip, Christopher M.; Kay, Jason G.; Grinstein, Sergio; Parton, Robert G.; Fairn, Gregory D.

2017-01-01

Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (Cavs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled Cav1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveola formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable Cav1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of Cav1 and cavin1 was diminished, and cavin1 degradation was increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns(4,5)P2 has minimal impact on caveola assembly but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper Cav clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability. PMID:28698382

Detection of PIK3CA gene mutations with HRM analysis and association with IGFBP-5 expression levels in breast cancer.

PubMed

Dirican, Ebubekir; Kaya, Zehra; Gullu, Gokce; Peker, Irem; Ozmen, Tolga; Gulluoglu, Bahadir M; Kaya, Handan; Ozer, Ayse; Akkiprik, Mustafa

2014-01-01

Breast cancer is the second most common cancer and second leading cause of cancer deaths in women. Phosphatidylinositol-3-kinase (PI3K)/AKT pathway mutations are associated with cancer and phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene mutations have been observed in 25-45% of breast cancer samples. Insulin growth factor binding protein-5 (IGFBP-5) can show different effects on apoptosis, cell motility and survival in breast cancer. We here aimed to determine the association between PIK3CA gene mutations and IGFBP-5 expressions for the first time in breast cancer patients. Frozen tumor samples from 101 Turkish breast cancer patients were analyzed with high resolution melting (HRM) for PIK3CA mutations (exon 9 and exon 20) and 37 HRM positive tumor samples were analyzed by DNA sequencing, mutations being found in 31. PIK3CA exon 9 mutations (Q546R, E542Q, E545K, E542K and E545D) were found in 10 tumor samples, exon 20 mutations (H1047L, H1047R, T1025T and G1049R) in 21, where only 1 tumor sample had two exon 20 mutations (T1025T and H1047R). Moreover, we detected one sample with both exon 9 (E542Q) and exon 20 (H1047R) mutations. 35% of the tumor samples with high IGFBP-5 mRNA expression and 29.4% of the tumor samples with low IGFBP-5 mRNA expression had PIK3CA mutations (p=0.9924). This is the first study of PIK3CA mutation screening results in Turkish breast cancer population using HRM analysis. This approach appears to be a very effective and reliable screening method for the PIK3CA exon 9 and 20 mutation detection. Further analysis with a greater number of samples is needed to clarify association between PIK3CA gene mutations and IGFBP-5 mRNA expression, and also clinical outcome in breast cancer patients.

Assessment of tris (1, 3-dichloro-2-propyl) phosphate toxicology in PC12 cells by using digital gene expression profiling.

PubMed

Li, Li; Jiang, Shuai; Li, Kang; Lin, Bencheng; Wang, Ziyu; Zhang, Zhiqing; Fang, Yanjun

2017-09-01

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP), one of the most universally used organophosphate flame retardants (OPFRs), is an environmental pollutant. However, limited information is available regarding its toxicity and environmental health risk. In the present study, PC12 cells provided a useful model for the evaluation of the toxic effects of TDCIPP. Exposure to 7.5, 15, 30, or 60 μM TDCIPP for 72 h inhibited cell viability, and enhanced cellular apoptosis and oxidative stress. To further explore the underlying mechanisms, digital gene expression (DGE) technology was used to identify early transcriptional changes following TDCIPP exposure. Expression of the transcripts of 161 genes was significantly altered upon treatment with TDCIPP. Functional and pathway analysis of the transcriptional profile demonstrated that genes showing significant TDCIPP-associated changes in expression were involved in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, extracellular matrix-receptor interactions, protein digestion and absorption, and microRNAs in cancer. Using quantitative real-time PCR, we validated the differential expression of selected genes. These results showed that the expression profiles of cells exposed to 60 μM TDCIPP were consistent with the DGE data. Furthermore, western blotting showed that treatment with TDCIPP reduced the Bcl-2/Bax ratio and attenuated PI3K/Akt/Myc signaling. Taken together, these data suggest that TDCIPP exposure can reduce cell viability and induce apoptosis in PC12 cells by inhibiting activation of the PI3K/Akt/Myc signaling pathway. These observations provide valuable preliminary information regarding the mechanisms of TDCIPP-induced toxicity in PC12 cells and indicate that further study of the toxicity of other environmental OPFRs is warranted. Copyright © 2017 Elsevier Ltd. All rights reserved.

p110α Hot Spot Mutations E545K and H1047R Exert Metabolic Reprogramming Independently of p110α Kinase Activity.

PubMed

Chaudhari, Aditi; Krumlinde, Daniel; Lundqvist, Annika; Akyürek, Levent M; Bandaru, Sashidhar; Skålén, Kristina; Ståhlman, Marcus; Borén, Jan; Wettergren, Yvonne; Ejeskär, Katarina; Rotter Sopasakis, Victoria

2015-10-01

The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Deleting the DAG Kinase Dgk1 Augments Yeast Vacuole Fusion Through In-creased Ypt7 Activity and Altered Membrane Fluidity

PubMed Central

Miner, Gregory E.; Starr, Matthew L.; Hurst, Logan R.; Fratti, Rutilio A.

2017-01-01

Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P2, and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1. Previously we found that pah1Δ vacuoles were fragmented, blocked in SNARE priming and showed arrested endosomal maturation. In other pathways the effects of deleting PAH1 can be compensated for by additionally deleting DGK1, however deleting both genes did not rescue the pah1Δ vacuolar defects. Deleting DGK1 alone caused a marked increase in vacuole fusion that was attributed to elevated DAG levels. This was accompanied by a gain in resistance to the inhibitory effects of PA as well as inhibitors of Ypt7 activity. Together these data show that Dgk1 function can act as a negative regulator of vacuole fusion through the production of PA at the cost of depleting DAG and reducing Ypt7 activity. PMID:28276191

Galangin Induces Apoptosis in MCF-7 Human Breast Cancer Cells Through Mitochondrial Pathway and Phosphatidylinositol 3-Kinase/Akt Inhibition.

PubMed

Liu, Dan; You, Pengtao; Luo, Yan; Yang, Min; Liu, Yanwen

2018-06-07

The study aimed to investigate the molecular mechanism of inhibition of proliferation and apoptosis induction by galangin against MCF-7 human breast cancer cells. Cell Counting Kit-8 assay was used to assess cell viability and flow cytometry was used to detect cell apoptosis. The expression level of apoptosis-related proteins (cleaved-caspase-9, cleaved-caspase-8, cleaved-caspase-3, Bad, cleaved-Bid, Bcl-2, Bax, p-phosphatidylinositol 3-kinase [PI3K], and p-Akt) and cell cycle-related proteins (cyclin D3, cyclin B1, cyclin-dependent kinases CDK1, CDK2, CDK4, p21, p27, p53) were evaluated by Western blotting. Galangin increased the expression of Bax and decreased the expression of Bcl-2 in a concentration-dependent manner, inhibited cell viability, and induced apoptosis. Meanwhile, the expression of cleavage of caspase-9, caspase-8, caspase-3, Bid, and Bad increased significantly while the expression of p-PI3K and p-Akt proteins decreased. In addition, the protein levels of cyclin D3, cyclin B1, CDK1, CDK2, and CDK4 were downregulated while the expression levels of p21, p27, and p53 were upregulated significantly. Galangin could suppress the viability of MCF-7 cells and induce cell apoptosis via the mitochondrial pathway and PI3K/Akt inhibition as well as cell cycle arrest. © 2018 S. Karger AG, Basel.

Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prashek, Jennifer; Bouyain, Samuel; Fu, Mingui

De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two domains. In this studymore » we show that isolated PH and START domains interact with each other. The crystal structure of a PH–START complex revealed that the START domain binds to the PH domain at the same site for PtdIns(4)P-binding, suggesting that the START domain competes with PtdIns(4)P for association with the PH domain. We further report that mutations disrupting the PH–START interaction increase both PtdIns(4)P-binding affinity and ceramide transfer activity of a CERT-serine–rich phosphorylation mimic. We also found that these mutations increase the Golgi localization of CERT inside the cell, consistent with enhanced PtdIns(4)P binding of the mutant. Collectively, our structural, biochemical, and cellular investigations provide important structural insight into the regulation of CERT function and localization.« less

The structure of myristoylated Mason-Pfizer monkey virus matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in its membrane binding.

PubMed

Prchal, Jan; Srb, Pavel; Hunter, Eric; Ruml, Tomáš; Hrabal, Richard

2012-10-26

We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C(8) fatty acid chains was monitored by observation of concentration-dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in (31)P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein-phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a (13)C-filtered/(13)C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P(2) binding was not strong enough for triggering of the myristoyl-switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein. Copyright © 2012. Published by Elsevier Ltd.

PCI-24781 down-regulates EZH2 expression and then promotes glioma apoptosis by suppressing the PIK3K/Akt/mTOR pathway.

PubMed

Zhang, Wei; Lv, Shengqing; Liu, Jun; Zang, Zhenle; Yin, Junyi; An, Ning; Yang, Hui; Song, Yechun

2014-10-01

PCI-24781 is a novel histone deacetylase inhibitor that inhibits tumor proliferation and promotes cell apoptosis. However, it is unclear whether PCI-24781 inhibits Enhancer of Zeste 2 (EZH2) expression in malignant gliomas. In this work, three glioma cell lines were incubated with various concentrations of PCI-24781 (0, 0.25, 0.5, 1, 2.5 and 5 μM) and analyzed for cell proliferation by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and colony formation, and cell cycle and apoptosis were assessed by flow cytometry. The expression of EZH2 and apoptosis-related proteins was assessed by western blotting. Malignant glioma cells were also transfected with EZH2 siRNA to examine how PCI-24781 suppresses tumor cells. EZH2 was highly expressed in the three glioma cell lines. Incubation with PCI-24781 reduced cell proliferation and increased cell apoptosis by down-regulating EZH2 in a concentration-dependent manner. These effects were simulated by EZH2 siRNA. In addition, PCI-24781 or EZH2 siRNA accelerated cell apoptosis by down-regulating the expression of AKT, mTOR, p70 ribosomal protein S6 kinase (p70s6k), glycogen synthase kinase 3A and B (GSK3a/b) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). These data suggest that PCI-24781 may be a promising therapeutic agent for treating gliomas by down-regulating EZH2 which promotes cell apoptosis by suppressing the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway.

TGF-β directs trafficking of the epithelial sodium channel ENaC which has implications for ion and fluid transport in acute lung injury.

PubMed

Peters, Dorothea M; Vadász, István; Wujak, Lukasz; Wygrecka, Malgorzata; Olschewski, Andrea; Becker, Christin; Herold, Susanne; Papp, Rita; Mayer, Konstantin; Rummel, Sebastian; Brandes, Ralph P; Günther, Andreas; Waldegger, Siegfried; Eickelberg, Oliver; Seeger, Werner; Morty, Rory E

2014-01-21

TGF-β is a pathogenic factor in patients with acute respiratory distress syndrome (ARDS), a condition characterized by alveolar edema. A unique TGF-β pathway is described, which rapidly promoted internalization of the αβγ epithelial sodium channel (ENaC) complex from the alveolar epithelial cell surface, leading to persistence of pulmonary edema. TGF-β applied to the alveolar airspaces of live rabbits or isolated rabbit lungs blocked sodium transport and caused fluid retention, which--together with patch-clamp and flow cytometry studies--identified ENaC as the target of TGF-β. TGF-β rapidly and sequentially activated phospholipase D1, phosphatidylinositol-4-phosphate 5-kinase 1α, and NADPH oxidase 4 (NOX4) to produce reactive oxygen species, driving internalization of βENaC, the subunit responsible for cell-surface stability of the αβγENaC complex. ENaC internalization was dependent on oxidation of βENaC Cys(43). Treatment of alveolar epithelial cells with bronchoalveolar lavage fluids from ARDS patients drove βENaC internalization, which was inhibited by a TGF-β neutralizing antibody and a Tgfbr1 inhibitor. Pharmacological inhibition of TGF-β signaling in vivo in mice, and genetic ablation of the nox4 gene in mice, protected against perturbed lung fluid balance in a bleomycin model of lung injury, highlighting a role for both proximal and distal components of this unique ENaC regulatory pathway in lung fluid balance. These data describe a unique TGF-β-dependent mechanism that regulates ion and fluid transport in the lung, which is not only relevant to the pathological mechanisms of ARDS, but might also represent a physiological means of acutely regulating ENaC activity in the lung and other organs.

The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

PubMed

Ho, Cheuk Y; Choy, Christopher H; Wattson, Christina A; Johnson, Danielle E; Botelho, Roberto J

2015-04-10

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole*

PubMed Central

Ho, Cheuk Y.; Choy, Christopher H.; Wattson, Christina A.; Johnson, Danielle E.; Botelho, Roberto J.

2015-01-01

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes. PMID:25713145

Molecular Dynamics Simulations of PIP2 and PIP3 in Lipid Bilayers: Determination of Ring Orientation, and the Effects of Surface Roughness on a Poisson-Boltzmann Description

PubMed Central

Li, Zheng; Venable, Richard M.; Rogers, Laura A.; Murray, Diana; Pastor, Richard W.

2009-01-01

Abstract Molecular dynamics (MD) simulations of phosphatidylinositol (4,5)-bisphosphate (PIP2) and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) bilayers indicate that the inositol rings are tilted ∼40° with respect to the bilayer surface, as compared with 17° for the P-N vector of POPC. Multiple minima were obtained for the ring twist (analogous to roll for an airplane). The phosphates at position 1 of PIP2 and PIP3 are within an Ångström of the plane formed by the phosphates of POPC; lipids in the surrounding shell are depressed by 0.5–0.8 Å, but otherwise the phosphoinositides do not substantially perturb the bilayer. Finite size artifacts for ion distributions are apparent for systems of ∼26 waters/lipid, but, based on simulations with a fourfold increase of the aqueous phase, the phosphoinositide positions and orientations do not show significant size effects. Electrostatic potentials evaluated from Poisson-Boltzmann (PB) calculations show a strong dependence of potential height and ring orientation, with the maxima on the −25 mV surfaces (17.1 ± 0.1 Å for PIP2 and 19.4 ± 0.3 Å for PIP3) occurring near the most populated orientations from MD. These surfaces are well above the background height of 10 Å estimated for negatively charged cell membranes, as would be expected for lipids involved in cellular signaling. PB calculations on microscopically flat bilayers yield similar maxima as the MD-based (microscopically rough) systems, but show less fine structure and do not clearly indicate the most probable regions. Electrostatic free energies of interaction with pentalysine are also similar for the rough and flat systems. These results support the utility of a rigid/flat bilayer model for PB-based studies of PIP2 and PIP3 as long as the orientations are judiciously chosen. PMID:19580753

Evaluation of type 2 diabetic mellitus animal models via interactions between insulin and mitogen‑activated protein kinase signaling pathways induced by a high fat and sugar diet and streptozotocin.

PubMed

Zhuo, Juncheng; Zeng, Qiaohuang; Cai, Dake; Zeng, Xiaohui; Chen, Yuxing; Gan, Haining; Huang, Xuejun; Yao, Nan; Huang, Dane; Zhang, Chengzhe

2018-04-01

Type 2 diabetic mellitus (T2DM), which is characterized by insulin resistance (IR), hyperglycemia and hyperlipidemia, is a comprehensive dysfunction of metabolism. The insulin receptor (INSR)/phosphoinositide 3‑kinase (PI3K)/AKT signaling pathway is well acknowledged as a predominant pathway associated with glucose uptake; however, the effect of streptozotocin (STZ) plus a high fat and sugar diet (HFSD) on the proteins associated with this pathway requires further elucidation. In order to explore this effect, a T2DM rat model was constructed to investigate T2DM pathogenesis and potential therapeutic advantages. Rats were randomly divided into control and model groups, including normal diet (ND) and HFSD types. ND types were administered intraperitoneal (IP) injections of STZ (35 mg/kg) or a combination of STZ and alloxan monohydrate (AON) (40 mg/kg), whereas HFSD types were composed of HFSD pre‑given, post‑given and simul‑given groups, and were modeled as follows: IP or intramuscular (IM) injection of STZ (35 mg/kg) or a combination of STZ and AON (40 mg/kg). Results indicated that, compared with controls, blood glucose, insulin, homeostatic model assessment‑insulin resistance and total triglyceride were significantly elevated in groups with HFSD and modeling agents (P<0.05 or P<0.01), whereas total cholesterol and low‑density lipoprotein levels were significantly elevated in groups simultaneously administered HFSD and modeling agents (P<0.05 or P<0.01), in addition to downregulation of the expression of insulin signaling pathway proteins in the liver, including INSR, PI3K, AKT1, phosphatidylinositol-5-phosphate 4‑kinase type‑2α (PIP5Kα) and glucose transporter (GLUT)2, and increased expression of inflammatory factors, including p38, tumor necrosis factor (TNF)α and interleukin (IL)6. Furthermore, compared with other two HFSD types including pre‑given and post‑given group, the simul‑given group that received IM injection with STZ exhibited decreased expression levels of major insulin signal pathway proteins INSR, PI3K, AKT1, PIP5Kα, GLUT2 or GLUT4 in the liver and pancreas (P<0.05 or P<0.01), whereas the opposite was observed in the skeletal muscle. In addition, the protein expression levels of phosphorylated‑p38, p38, IL6 and TNFα in the simul‑given group that received IM injection with STZ were increased (P<0.05 or P<0.01), and histopathology also indicated inflammation in pancreas and liver. The present findings suggest that a low dose of STZ may partially impair the β cells of the pancreas, whereas long‑term excess intake of HFSD may increase lipid metabolites, inhibit the insulin signaling pathway and activate the mitogen‑activated protein kinase p38 signaling pathway. The combined action of STZ and AON may result in insulin resistance, which ultimately results in abnormalities in glucose and lipid metabolism. The present model, analogue to T2DM onset of humans, evaluated the medical effect on metabolic dysfunction and provides an insight into the underlining mechanism of IR.

Association between mutations of critical pathway genes and survival outcomes according to the tumor location in colorectal cancer.

PubMed

Lee, Dae-Won; Han, Sae-Won; Cha, Yongjun; Bae, Jeong Mo; Kim, Hwang-Phill; Lyu, Jaemyun; Han, Hyojun; Kim, Hyoki; Jang, Hoon; Bang, Duhee; Huh, Iksoo; Park, Taesung; Won, Jae-Kyung; Jeong, Seung-Yong; Park, Kyu Joo; Kang, Gyeong Hoon; Kim, Tae-You

2017-09-15

Colorectal cancer (CRC) develops through the alteration of several critical pathways. This study was aimed at evaluating the influence of critical pathways on survival outcomes for patients with CRC. Targeted next-generation sequencing of 40 genes included in the 5 critical pathways of CRC (WNT, P53, RTK-RAS, phosphatidylinositol-4,5-bisphosphate 3-kinase [PI3K], and transforming growth factor β [TGF-β]) was performed for 516 patients with stage III or high-risk stage II CRC treated with surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy. The associations between critical pathway mutations and relapse-free survival (RFS) and overall survival were analyzed. The associations were further analyzed according to the tumor location. The mutation rates for the WNT, P53, RTK-RAS, PI3K, and TGF-β pathways were 84.5%, 69.0%, 60.7%, 30.0%, and 28.9%, respectively. A mutation in the PI3K pathway was associated with longer RFS (adjusted hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.36-0.99), whereas a mutation in the RTK-RAS pathway was associated with shorter RFS (adjusted HR, 1.60; 95% CI, 1.01-2.52). Proximal tumors showed a higher mutation rate than distal tumors, and the mutation profile was different according to the tumor location. The mutation rates of Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), and B-Raf proto-oncogene serine/threonine kinase (BRAF) were higher in proximal tumors, and the mutation rates of adenomatous polyposis coli (APC), tumor protein 53 (TP53), and neuroblastoma RAS viral oncogene homolog (NRAS) were higher in distal tumors. The better RFS with the PI3K pathway mutation was significant only for proximal tumors, and the worse RFS with the RTK-RAS pathway mutation was significant only for distal tumors. A PI3K pathway mutation was associated with better RFS for CRC patients treated with adjuvant chemotherapy, and an RTK-RAS pathway mutation was associated with worse RFS. The significance of the prognostic impact differed according to the tumor location. Cancer 2017;123:3513-23. © 2017 American Cancer Society. © 2017 American Cancer Society.

Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1.

PubMed

Zhou, Yixing; Wu, Shilan; Wang, Huanchen; Hayakawa, Yoichi; Bird, Gary S; Shears, Stephen B

2012-12-01

Using immortalized [3H]inositol-labelled S3 cells, we demonstrated in the present study that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides). HPLC analysis revealed that dGBP (Drosophila GBP) elevated Ins(1,4,5)P3 levels 9-fold. By using fluorescent Ca2+ probes, we determined that dGBP initially mobilized Ca2+ from intracellular pools; the ensuing depletion of intracellular Ca2+ stores by dGBP subsequently activated a Ca2+ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P3 receptor almost completely eliminated mobilization of intracellular Ca2+ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promoted increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P4, Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. We therefore created a cell-line (S3ITPK1) in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. We found that dGBP-stimulated S3ITPK1 cells did not synthesize Ins(3,4,5,6)P4, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P5+Ins(1,3,4)P3→Ins(3,4,5,6)P4+Ins(1,3,4,6)P4] is driven solely by the laws of mass action [Chamberlain, Qian, Stiles, Cho, Jones, Lesley, Grabau, Shears and Spraggon (2007) J. Biol. Chem. 282, 28117-28125]. This conclusion represents a fundamental breach in our understanding of ITPK1 signalling.

Role of nongenomic activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase 1/2 pathways in 1,25D3-mediated apoptosis in squamous cell carcinoma cells.

PubMed

Ma, Yingyu; Yu, Wei-Dong; Kong, Rui-Xian; Trump, Donald L; Johnson, Candace S

2006-08-15

Vitamin D is a steroid hormone that regulates calcium homeostasis and bone metabolism. The active form of vitamin D [1 alpha,25-dihydroxyvitamin D(3) (1,25D3)] acts through both genomic and nongenomic pathways. 1,25D3 has antitumor effects in a variety of cancers, including colorectal, prostate, breast, ovarian, and skin cancers. 1,25D3 exerts growth-inhibitory effects in cancer cells through the induction of apoptosis, cell cycle arrest, and differentiation. The mechanisms regulating 1,25D3-induced apoptosis remain unclear. We investigated the role of nongenomic signaling in 1,25D3-mediated apoptosis in squamous cell carcinoma (SCC) cells. 1,25D3 induced rapid and sustained activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2 pathways in SCC cells. These effects were nongenomic: they occurred rapidly and were not inhibited by cycloheximide or actinomycin D. To examine whether the nongenomic activation of Akt and ERK1/2 plays a role in 1,25D3-mediated apoptosis, the expression of Akt or ERK1/2 was reduced by small interfering RNA (siRNA). siRNA-Akt significantly enhanced 1,25D3-induced apoptosis as indicated by increased levels of Annexin V-positive cells and increased sub-G(1) population and DNA fragmentation. In contrast, siRNA-ERK1/2 had no effects on 1,25D3-induced apoptosis. In addition, siRNA-Akt transfection followed by 1,25D3 treatment induced apoptosis much sooner than 1,25D3 alone. siRNA-Akt and 1,25D3 induced caspase-10 activation, suppressed the expression of c-IAP1 and XIAP, and promoted 1,25D3-induced caspase-3 activation. These results support a link between 1,25D3-induced nongenomic signaling and apoptosis. 1,25D3 induces the activation of phosphatidylinositol 3-kinase/Akt, which suppresses 1,25D3-mediated apoptosis and prolongs the survival of SCC cells.

Structural basis for interdomain communication in SHIP2 providing high phosphatase activity.

PubMed

Le Coq, Johanne; Camacho-Artacho, Marta; Velázquez, José Vicente; Santiveri, Clara M; Gallego, Luis Heredia; Campos-Olivas, Ramón; Dölker, Nicole; Lietha, Daniel

2017-08-09

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P 3 ) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P 3 . Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions.

LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun Haipeng; Xu Beibei; Sheveleva, Elena

2008-10-01

Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression.more » LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca{sup 2+} concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes.« less

IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85.

PubMed Central

Myers, M G; Backer, J M; Sun, X J; Shoelson, S; Hu, P; Schlessinger, J; Yoakim, M; Schaffhausen, B; White, M F

1992-01-01

IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains. Images PMID:1332046

Gastric Inhibitory Peptide Controls Adipose Insulin Sensitivity via Activation of cAMP-response Element-binding Protein and p110β Isoform of Phosphatidylinositol 3-Kinase*

PubMed Central

Mohammad, Sameer; Ramos, Lavoisier S.; Buck, Jochen; Levin, Lonny R.; Rubino, Francesco; McGraw, Timothy E.

2011-01-01

Gastric inhibitory peptide (GIP) is an incretin hormone secreted in response to food intake. The best known function of GIP is to enhance glucose-dependent insulin secretion from pancreatic β-cells. Extra-pancreatic effects of GIP primarily occur in adipose tissues. Here, we demonstrate that GIP increases insulin-dependent translocation of the Glut4 glucose transporter to the plasma membrane and exclusion of FoxO1 transcription factor from the nucleus in adipocytes, establishing that GIP has a general effect on insulin action in adipocytes. Stimulation of adipocytes with GIP alone has no effect on these processes. Using pharmacologic and molecular genetic approaches, we show that the effect of GIP on adipocyte insulin sensitivity requires activation of both the cAMP/protein kinase A/CREB signaling module and p110β phosphoinositol-3′ kinase, establishing a novel signal transduction pathway modulating insulin action in adipocytes. This insulin-sensitizing effect is specific for GIP because isoproterenol, which elevates adipocyte cAMP and activates PKA/CREB signaling, does not affect adipocyte insulin sensitivity. The insulin-sensitizing activity points to a more central role for GIP in intestinal regulation of peripheral tissue metabolism, an emerging feature of inter-organ communication in the control of metabolism. PMID:22027830

Xenograft tumors derived from malignant pleural effusion of the patients with non-small-cell lung cancer as models to explore drug resistance.

PubMed

Xu, Yunhua; Zhang, Feifei; Pan, Xiaoqing; Wang, Guan; Zhu, Lei; Zhang, Jie; Wen, Danyi; Lu, Shun

2018-05-09

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) fusions show dramatic responses to specific tyrosine kinase inhibitors (TKIs); however, after 10-12 months, secondary mutations arise that confer resistance. We generated a murine xenograft model using patient-derived NSCLC cells isolated from the pleural fluid of two patients with NSCLC to investigate the mechanisms of resistance against the ALK- and EGFR-targeted TKIs crizotinib and osimertinib, respectively. Genotypes of patient biopsies and xenograft tumors were determined by whole exome sequencing (WES), and patients and xenograft-bearing mice received targeted treatment (crizotinib or osimertinib) accordingly. Xenograft mice were also treated for prolonged periods to identify whether the development of drug resistance and/or treatment responses were associated with tumor size. Finally, the pathology of patients biopsies and xenograft tumors were compared histologically. The histological characteristics and chemotherapy responses of xenograft tumors were similar to the actual patients. WES showed that the genotypes of the xenograft and patient tumors were similar (an echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) gene fusion (patient/xenograft: CTC15035 EML4-ALK ) and EGFR L858R and T790M mutations (patient/xenograft: CTC15063 EGFR L858R, T790M )). After continuous crizotinib or osimertinib treatment, WES data suggested that acquired ALK E1210K mutation conferred crizotinib resistance in the CTC15035 EML4-ALK xenograft, while decreased frequencies of EGFR L858R and T790M mutations plus the appearance of v-RAF murine sarcoma viral oncogene homolog B (BRAF) G7V mutations and phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha (PIK3C2A) A86fs frame shift mutations led to osimertinib resistance in the CTC15063 EGFR L858R, T790M xenografts. We successfully developed a new method of generating drug resistance xenograft models from liquid biopsies using microfluidic technology, which might be a useful tool to investigate the mechanisms of drug resistance in NSCLC.

Dysregulation of mTOR signaling in fragile X syndrome.

PubMed

Sharma, Ali; Hoeffer, Charles A; Takayasu, Yukihiro; Miyawaki, Takahiro; McBride, Sean M; Klann, Eric; Zukin, R Suzanne

2010-01-13

Fragile X syndrome, the most common form of inherited mental retardation and leading genetic cause of autism, is caused by transcriptional silencing of the Fmr1 gene. The fragile X mental retardation protein (FMRP), the gene product of Fmr1, is an RNA binding protein that negatively regulates translation in neurons. The Fmr1 knock-out mouse, a model of fragile X syndrome, exhibits cognitive deficits and exaggerated metabotropic glutamate receptor (mGluR)-dependent long-term depression at CA1 synapses. However, the molecular mechanisms that link loss of function of FMRP to aberrant synaptic plasticity remain unclear. The mammalian target of rapamycin (mTOR) signaling cascade controls initiation of cap-dependent translation and is under control of mGluRs. Here we show that mTOR phosphorylation and activity are elevated in hippocampus of juvenile Fmr1 knock-out mice by four functional readouts: (1) association of mTOR with regulatory associated protein of mTOR; (2) mTOR kinase activity; (3) phosphorylation of mTOR downstream targets S6 kinase and 4E-binding protein; and (4) formation of eukaryotic initiation factor complex 4F, a critical first step in cap-dependent translation. Consistent with this, mGluR long-term depression at CA1 synapses of FMRP-deficient mice is exaggerated and rapamycin insensitive. We further show that the p110 subunit of the upstream kinase phosphatidylinositol 3-kinase (PI3K) and its upstream activator PI3K enhancer PIKE, predicted targets of FMRP, are upregulated in knock-out mice. Elevated mTOR signaling may provide a functional link between overactivation of group I mGluRs and aberrant synaptic plasticity in the fragile X mouse, mechanisms relevant to impaired cognition in fragile X syndrome.

Regulating Prostate Cancer Sensitivity to Chemotherapy through Translational Control of CCAAT/Enhancer Binding Proteins

DTIC Science & Technology

2016-10-01

DISTRIBUTION STATEMENT: Approved for public release; distribution unlimited The views, opinions and/or findings contained in this report are those of...1. Introduction: Central to the survival of prostate cancer (PCa) are the androgen receptor ( AR ) and phosphatidylinositol-3 kinase (PI3K)-AKT...and tensin homolog (PTEN). AR gene amplification is also frequently observed in hormone refractory prostate cancer (HRPC). Combined blockade of PI3K

The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells.

PubMed

Lucato, Christina M; Halls, Michelle L; Ooms, Lisa M; Liu, Heng-Jia; Mitchell, Christina A; Whisstock, James C; Ellisdon, Andrew M

2015-08-21

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP3 and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP3/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP3 and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

d-myo-Inositol-3-Phosphate Affects Phosphatidylinositol-Mediated Endomembrane Function in Arabidopsis and Is Essential for Auxin-Regulated Embryogenesis[W][OA

PubMed Central

Luo, Yu; Qin, Genji; Zhang, Jun; Liang, Yuan; Song, Yingqi; Zhao, Meiping; Tsuge, Tomohiko; Aoyama, Takashi; Liu, Jingjing; Gu, Hongya; Qu, Li-Jia

2011-01-01

In animal cells, myo-inositol is an important regulatory molecule in several physiological and biochemical processes, including signal transduction and membrane biogenesis. However, the fundamental biological functions of myo-inositol are still far from clear in plants. Here, we report the genetic characterization of three Arabidopsis thaliana genes encoding d-myo-inositol-3-phosphate synthase (MIPS), which catalyzes the rate-limiting step in de novo synthesis of myo-inositol. Each of the three MIPS genes rescued the yeast ino1 mutant, which is defective in yeast MIPS gene INO1, and they had different dynamic expression patterns during Arabidopsis embryo development. Although single mips mutants showed no obvious phenotypes, the mips1 mips2 double mutant and the mips1 mips2 mips3 triple mutant were embryo lethal, whereas the mips1 mips3 and mips1 mips2+/− double mutants had abnormal embryos. The mips phenotypes resembled those of auxin mutants. Indeed, the double and triple mips mutants displayed abnormal expression patterns of DR5:green fluorescent protein, an auxin-responsive fusion protein, and they had altered PIN1 subcellular localization. Also, membrane trafficking was affected in mips1 mips3. Interestingly, overexpression of PHOSPHATIDYLINOSITOL SYNTHASE2, which converts myo-inositol to membrane phosphatidylinositol (PtdIns), largely rescued the cotyledon and endomembrane defects in mips1 mips3. We conclude that myo-inositol serves as the main substrate for synthesizing PtdIns and phosphatidylinositides, which are essential for endomembrane structure and trafficking and thus for auxin-regulated embryogenesis. PMID:21505066

Regulation of Manganese Antioxidants by Nutrient Sensing Pathways in Saccharomyces cerevisiae

PubMed Central

Reddi, Amit R.; Culotta, Valeria C.

2011-01-01

In aerobic organisms, protection from oxidative damage involves the combined action of enzymatic and nonproteinaceous cellular factors that collectively remove harmful reactive oxygen species. One class of nonproteinaceous antioxidants includes small molecule complexes of manganese (Mn) that can scavenge superoxide anion radicals and provide a backup for superoxide dismutase enzymes. Such Mn antioxidants have been identified in diverse organisms; however, nothing regarding their physiology in the context of cellular adaptation to stress was known. Using a molecular genetic approach in Bakers’ yeast, Saccharomyces cerevisiae, we report that the Mn antioxidants can fall under control of the same pathways used for nutrient sensing and stress responses. Specifically, a serine/threonine PAS-kinase, Rim15p, that is known to integrate phosphate, nitrogen, and carbon sensing, can also control Mn antioxidant activity in yeast. Rim15p is negatively regulated by the phosphate-sensing kinase complex Pho80p/Pho85p and by the nitrogen-sensing Akt/S6 kinase homolog, Sch9p. We observed that loss of either of these upstream kinase sensors dramatically inhibited the potency of Mn as an antioxidant. Downstream of Rim15p are transcription factors Gis1p and the redundant Msn2/Msn4p pair that typically respond to nutrient and stress signals. Both transcription factors were found to modulate the potency of the Mn antioxidant but in opposing fashions: loss of Gis1p was seen to enhance Mn antioxidant activity whereas loss of Msn2/4p greatly suppressed it. Our observed roles for nutrient and stress response kinases and transcription factors in regulating the Mn antioxidant underscore its physiological importance in aerobic fitness. PMID:21926297

Dysregulation of the Phosphatidylinositol 3-kinase Pathway in Thyroid Neoplasia

PubMed Central

Paes, John E.; Ringel, Matthew D.

2008-01-01

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is an important regulator of many cellular events, including apoptosis, proliferation, and motility. Enhanced activation of this pathway can occur through several mechanisms, such as inactivation of its negative regulator, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and activating mutations and gene amplification of the gene encoding the catalytic subunit of PI3K (PIK3CA). These genetic abnormalities have been particularly associated with follicular thyroid neoplasia and anaplastic thyroid cancer, suggesting an important role for PI3K signaling in these disorders. In this review, the role of PI3K pathway activation in thyroid cancer will be discussed, with a focus on recent advances. PMID:18502332

Activation of phosphatidylinositol-3 kinase by nerve growth factor involves indirect coupling of the trk proto-oncogene with src homology 2 domains.

PubMed

Ohmichi, M; Decker, S J; Saltiel, A R

1992-10-01

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including the p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase. The activation of this enzyme by growth factors is evaluated in PC12 pheochromocytoma cells and NIH 3T3 fibroblasts expressing the pp140c-trk nerve growth factor (NGF) receptor (3T3-c-trk). NGF causes the rapid stimulation of PI-3 kinase activity detected in anti-phosphotyrosine, but not in anti-trk, immunoprecipitates. This effect coincides with the tyrosine phosphorylation of two proteins, with molecular masses of of 100 kd and 110 kd, that coimmunoprecipitate with p85. Similar phosphorylation patterns are induced when an immobilized fusion protein containing the amino-terminal SH2 domain of p85 is used to precipitate tyrosine-phosphorylated proteins. Thus, although NGF produces the rapid activation of PI-3 kinase through a mechanism that involves tyrosine phosphorylation, there is no evidence for tyrosine phosphorylation of p85, or for its ligand-dependent association with the NGF receptor. Perhaps another phosphoprotein may link the NGF receptor to this enzyme.

Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

PubMed

Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

2015-11-01

Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P 2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

Genetic Evidence for the Physiological Significance of the d-Tagatose 6-Phosphate Pathway of Lactose and d-Galactose Degradation in Staphylococcus aureus1

PubMed Central

Bissett, Donald L.; Anderson, Richard L.

1974-01-01

Mutants of Staphylococcus aureus were isolated which were unable to utilize d-galactose or lactose, but which were able to utilize all other carbohydrates tested. Growth of the mutants on a peptone-containing medium was inhibited by d-galactose. Of those mutants selected for further study, one (tagI2) was missing d-galactose 6-phosphate isomerase, one (tagK3) was missing d-tagatose 6-phosphate kinase, and one (tagA4) was missing d-tagatose 1, 6-diphosphate aldolase. Each of these mutants accumulated the substrate of the missing enzyme intracellularly. Spontaneous revertants of each of the mutants simultaneously regained their ability to utilize d-galactose and lactose, lost their sensitivity to d-galactose, regained the missing enzymatic activities, and no longer accumulated intermediates of the d-tagatose 6-phosphate pathway. These data support our previous contention that the physiologically significant route for the metabolism of d-galactose and the d-galactosyl moiety of lactose in S. aureus is the d-tagatose 6-phosphate pathway. Furthermore, a mutant constitutive for all three enzymes of this pathway was isolated, indicating that the products of the tagI, tagK, and tagA genes are under common genetic control. This conclusion was supported by the demonstration that d-galactose 6-phosphate isomerase, d-tagatose 6-phosphate kinase, and d-tagatose 1, 6-diphosphate aldolase are coordinately induced in the parental strain. PMID:4277494

A single ataxia telangiectasia gene with a product similar to PI-3 kinase.

PubMed

Savitsky, K; Bar-Shira, A; Gilad, S; Rotman, G; Ziv, Y; Vanagaite, L; Tagle, D A; Smith, S; Uziel, T; Sfez, S; Ashkenazi, M; Pecker, I; Frydman, M; Harnik, R; Patanjali, S R; Simmons, A; Clines, G A; Sartiel, A; Gatti, R A; Chessa, L; Sanal, O; Lavin, M F; Jaspers, N G; Taylor, A M; Arlett, C F; Miki, T; Weissman, S M; Lovett, M; Collins, F S; Shiloh, Y

1995-06-23

A gene, ATM, that is mutated in the autosomal recessive disorder ataxia telangiectasia (AT) was identified by positional cloning on chromosome 11q22-23. AT is characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, cancer predisposition, radiation sensitivity, and cell cycle abnormalities. The disease is genetically heterogeneous, with four complementation groups that have been suspected to represent different genes. ATM, which has a transcript of 12 kilobases, was found to be mutated in AT patients from all complementation groups, indicating that it is probably the sole gene responsible for this disorder. A partial ATM complementary DNA clone of 5.9 kilobases encoded a putative protein that is similar to several yeast and mammalian phosphatidylinositol-3' kinases that are involved in mitogenic signal transduction, meiotic recombination, and cell cycle control. The discovery of ATM should enhance understanding of AT and related syndromes and may allow the identification of AT heterozygotes, who are at increased risk of cancer.

Activation of the Ca2+-sensing receptors increases currents through inward rectifier K+ channels via activation of phosphatidylinositol 4-kinase.

PubMed

Liu, Chung-Hung; Chang, Hsueh-Kai; Lee, Sue-Ping; Shieh, Ru-Chi

2016-11-01

Inward rectifier K + channels are important for maintaining normal electrical function in many cell types. The proper function of these channels requires the presence of membrane phosphoinositide 4,5-bisphosphate (PIP 2 ). Stimulation of the Ca 2+ -sensing receptor CaR, a pleiotropic G protein-coupled receptor, activates both G q/11 , which decreases PIP 2 , and phosphatidylinositol 4-kinase (PI-4-K), which, conversely, increases PIP 2 . How membrane PIP 2 levels are regulated by CaR activation and whether these changes modulate inward rectifier K + are unknown. In this study, we found that activation of CaR by the allosteric agonist, NPSR568, increased inward rectifier K + current (I K1 ) in guinea pig ventricular myocytes and currents mediated by Kir2.1 channels exogenously expressed in HEK293T cells with a similar sensitivity. Moreover, using the fluorescent PIP 2 reporter tubby-R332H-cYFP to monitor PIP 2 levels, we found that CaR activation in HEK293T cells increased membrane PIP 2 concentrations. Pharmacological studies showed that both phospholipase C (PLC) and PI-4-K are activated by CaR stimulation with the latter played a dominant role in regulating membrane PIP 2 and, thus, Kir currents. These results provide the first direct evidence that CaR activation upregulates currents through inward rectifier K + channels by accelerating PIP 2 synthesis. The regulation of I K1 plays a critical role in the stability of the electrical properties of many excitable cells, including cardiac myocytes and neurons. Further, synthetic allosteric modulators that increase CaR activity have been used to treat hyperparathyroidism, and negative CaR modulators are of potential importance in the treatment of osteoporosis. Thus, our results provide further insight into the roles played by CaR in the cardiovascular system and are potentially valuable for heart disease treatment and drug safety.

BDNF regulates the translation of a select group of mRNAs by a mammalian target of rapamycin-phosphatidylinositol 3-kinase-dependent pathway during neuronal development.

PubMed

Schratt, Gerhard M; Nigh, Elizabeth A; Chen, Wen G; Hu, Linda; Greenberg, Michael E

2004-08-18

Local regulation of mRNA translation plays an important role in axon guidance, synaptic development, and neuronal plasticity. Little is known, however, regarding the mechanisms that control translation in neurons, and only a few mRNAs have been identified that are locally translated within axon and dendrites. Using Affymetrix gene arrays to identify mRNAs that are newly associated with polysomes after exposure to BDNF, we identified subsets of mRNAs for which translation is enhanced in neurons at different developmental stages. In mature neurons, many of these mRNAs encode proteins that are known to function at synapses, including CamKIIalpha, NMDA receptor subunits, and the postsynaptic density (PSD) scaffolding protein Homer2. BDNF regulates the translation of Homer2 locally in the synaptodendritic compartment by activating translational initiation via a mammalian target of rapamycin-phosphatidylinositol 3-kinase-dependent pathway. These findings suggest that BDNF likely regulates synaptic function by inducing the local synthesis of numerous synaptic proteins. The local translation of the cytoskeleton-associated protein Homer2 in particular might have important implications for growth cone dynamics and dendritic spine development.

Cellular Energetic Status Supervises the Synthesis of Bis-Diphosphoinositol Tetrakisphosphate Independently of AMP-Activated Protein Kinase

PubMed Central

Choi, Kuicheon; Mollapour, Elahe; Choi, Jae H.; Shears, Stephen B.

2009-01-01

Cells aggressively defend adenosine nucleotide homeostasis; intracellular biosensors detect variations in energetic status and communicate with other cellular networks to initiate adaptive responses. Here, we demonstrate some new elements of this communication process, and we show that this networking is compromised by off-target, bioenergetic effects of some popular pharmacological tools. Treatment of cells with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), so as to simulate elevated AMP levels, reduced the synthesis of bis-diphosphoinositol tetrakisphosphate ([PP]2-InsP4), an intracellular signal that phosphorylates proteins in a kinase-independent reaction. This was a selective effect; levels of other inositol phosphates were unaffected by AICAR. By genetically manipulating cellular AMP-activated protein kinase activity, we showed that it did not mediate these effects of AICAR. Instead, we conclude that the simulation of deteriorating adenosine nucleotide balance itself inhibited [PP]2-InsP4 synthesis. This conclusion is consistent with our demonstrating that oligomycin elevated cellular [AMP] and selectively inhibited [PP]2-InsP4 synthesis without affecting other inositol phosphates. In addition, we report that the short-term increases in [PP]2-InsP4 levels normally seen during hyperosmotic stress were attenuated by 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (PD184352). The latter is typically considered an exquisitely specific mitogen-activated protein kinase kinase (MEK) inhibitor, but small interfering RNA against MEK or extracellular signal-regulated kinase revealed that this mitogen-activated protein kinase pathway was not involved. Instead, we demonstrate that [PP]2-InsP4 synthesis was inhibited by PD184352 through its nonspecific effects on cellular energy balance. Two other MEK inhibitors, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2′-amino-3′-methoxyflavone (PD98059), had similar off-target effects. We conclude that the levels and hence the signaling strength of [PP]2-InsP4 is supervised by cellular adenosine nucleotide balance, signifying a new link between signaling and bioenergetic networks. PMID:18460607

Protein Phosphatase 2A Signaling in Human Prostate Cancer

DTIC Science & Technology

2013-06-01

family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 2001;353:417-39. (6) Eto M, Bennouna J, Hunter OC...phosphatidylinositol 3’-kinase and Akt/protein kinase B. Cancer Res 1999;59:1449-53. (14) Grethe S, Porn -Ares MI. p38 MAPK regulates phosphorylation of Bad

Astragaloside IV Attenuated 3,4-Benzopyrene-Induced Abdominal Aortic Aneurysm by Ameliorating Macrophage-Mediated Inflammation.

PubMed

Wang, Jiaoni; Zhou, Yingying; Wu, Shaoze; Huang, Kaiyu; Thapa, Saroj; Tao, Luyuan; Wang, Jie; Shen, Yigen; Wang, Jinsheng; Xue, Yangjing; Ji, Kangting

2018-01-01

Abdominal aortic aneurysm (AAA), characterized by macrophage infiltration-mediated inflammation and oxidative stress, is a potentially fatal disease. Astragaloside IV (AS-IV) has been acknowledged to exhibit antioxidant and anti-inflammatory properties. This study was designed to investigate the protective effect of AS-IV against AAA formation induced by 3,4-benzopyrene (Bap) and angiotensin II (Ang II), and to explore probable mechanisms. Results showed that AS-IV decreased AAA formation, and reduced macrophage infiltration and expression of matrix metalloproteinase. Furthermore, AS-IV abrogated Bap-/Ang II-induced NF-κB activation and oxidative stress. In vitro , AS-IV inhibition of macrophage activation and NF-κB was correlated with increased phosphorylation of phosphatidylinositol 3-kinase (PI3-K)/AKT. Together, our findings suggest that AS-IV has potential as an intervention in the formation of AAA. (1)The protective effect of Astragaloside IV (AS-IV) on abdominal aortic aneurysm (AAA) is associated with its suppressing effects on inflammation in the aortic wall.(2)AS-IV abrogated 3,4-benzopyrene (Bap)/angiotensin II (Ang II)-induced nuclear factor-κB (NF-κB) activation and oxidative stress.(3)AS-IV inhibited Bap-induced RAW264.7 macrophage cells activation by inhibiting oxidative stress and NF-κB activation through phosphatidylinositol 3-kinase (PI3-K)/AKT pathway.AS-IV is a potential preventive agent for cigarette smoking-related AAA.

DMT efficiently inhibits hepatic gluconeogenesis by regulating the Gαq signaling pathway.

PubMed

Zhou, Ting-Ting; Ma, Fei; Shi, Xiao-Fan; Xu, Xin; Du, Te; Guo, Xiao-Dan; Wang, Gai-Hong; Yu, Liang; Rukachaisirikul, Vatcharin; Hu, Li-Hong; Chen, Jing; Shen, Xu

2017-08-01

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease with complicated pathogenesis and targeting gluconeogenesis inhibition is a promising strategy for anti-diabetic drug discovery. G protein-coupled receptors (GPCRs) are classified as distinct families by heterotrimeric G proteins, primarily including Gαs, Gαi and Gαq. Gαs-coupled GPCRs function potently in the regulation of hepatic gluconeogenesis by activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and Gαi-coupled GPCRs exhibit inhibitory effect on adenylyl cyclase and reduce intracellular cAMP level. However, little is known about the regulation of Gαq-coupled GPCRs in hepatic gluconeogenesis. Here, small-molecule 2-(2,4-dimethoxy-3-methylphenyl)-7-(thiophen-2-yl)-9-(trifluoromethyl)-2,3-dihydropyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4( 1H )-one (DMT) was determined to suppress hepatic glucose production and reduce mRNA levels of gluconeogenic genes. Treatment of DMT in db/db mice decreased fasting blood glucose and hemoglobin A1C (HbA1c) levels, while improved glucose tolerance and pyruvate tolerance. Mechanism study demonstrated that DMT-inhibited gluconeogenesis by regulating the Gαq/phospholipase C (PLC)/inositol-1,4,5-triphosphate receptor (IP3R)-mediated calcium (Ca 2+ )/calmodulin (CaM)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT)/forkhead box protein O1 (FOXO1) signaling pathway. To our knowledge, DMT might be the first reported small molecule able to suppress hepatic gluconeogenesis by regulating Gαq signaling, and our current work has also highlighted the potential of DMT in the treatment of T2DM. © 2017 Society for Endocrinology.

The Human T-Cell Leukemia Virus Type 1 Oncoprotein Tax Controls Forkhead Box O4 Activity through Degradation by the Proteasome▿

PubMed Central

Oteiza, Alexandra; Mechti, Nadir

2011-01-01

Activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway by the viral Tax oncoprotein plays a pivotal role in clonal expansion of human T-cell leukemia virus type 1 (HTLV-1)-infected cells. As the Forkhead box O (FoxO) tumor suppressors act as downstream effectors of PI3K/Akt, they represent good candidate targets whose dysregulation by Tax might be involved in HTLV-1-mediated activation and transformation of infected cells. In this report, we provide evidence showing that Tax induces a dose-dependent degradation of FoxO4 by the ubiquitin-proteasome pathway. Consistent with that, we demonstrate that Tax expression increases the interaction between FoxO4 and Mdm2 E3 ligase, leading to a strong FoxO4 polyubiquitination. These processes require the phosphorylation of FoxO4 by Akt, since a mutant of FoxO4 with mutations on its three Akt phosphorylation sites appears to be resistant to Tax-mediated degradation and ubiquitination. In addition, we show that Tax expression is associated with degradation and phosphorylation of endogenous FoxO4 in Jurkat T cells. Finally, we demonstrate that Tax represses FoxO4 transcriptional activity. Our study demonstrates that Tax can control FoxO4 protein stability and transcriptional activity and provides new insight into the subversion of cell signaling pathways during HTLV-1 infection. PMID:21525355

Stimulating the GPR30 estrogen receptor with a novel tamoxifen analogue activates SF-1 and promotes endometrial cell proliferation.

PubMed

Lin, Benjamin C; Suzawa, Miyuki; Blind, Raymond D; Tobias, Sandra C; Bulun, Serdar E; Scanlan, Thomas S; Ingraham, Holly A

2009-07-01

Estrogens and selective estrogen receptor (ER) modulators such as tamoxifen are known to increase uterine cell proliferation. Mounting evidence suggests that estrogen signaling is mediated not only by ERalpha and ERbeta nuclear receptors, but also by GPR30 (GPER), a seven transmembrane (7TM) receptor. Here, we report that primary human endometriotic H-38 cells express high levels of GPR30 with no detectable ERalpha or ERbeta. Using a novel tamoxifen analogue, STX, which activates GPR30 but not ERs, significant stimulation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways was observed in H-38 cells and in Ishikawa endometrial cancer cells expressing GPR30; a similar effect was observed in JEG3 choriocarcinoma cells. STX treatment also increased cellular pools of phosphatidylinositol (3,4,5) triphosphate, a proposed ligand for the nuclear hormone receptor SF-1 (NR5A1). Consistent with these findings, STX, tamoxifen, and the phytoestrogen genistein were able to increase SF-1 transcription, promote Ishikawa cell proliferation, and induce the SF-1 target gene aromatase in a GPR30-dependent manner. Our findings suggest a novel signaling paradigm that is initiated by estrogen activation of the 7TM receptor GPR30, with signal transduction cascades (PI3K and MAPK) converging on nuclear hormone receptors (SF-1/LRH-1) to modulate their transcriptional output. We propose that this novel GPR30/SF-1 pathway increases local concentrations of estrogen, and together with classic ER signaling, mediate the proliferative effects of synthetic estrogens such as tamoxifen, in promoting endometriosis and endometrial cancers.

WNK1 Promotes PIP2 Synthesis to Coordinate Growth Factor and GPCR-Gq Signaling

PubMed Central

An, Sung-Wan; Cha, Seung-Kuy; Yoon, Joonho; Chang, Seungwoo; Ross, Elliott M.; Huang, Chou-Long

2011-01-01

Summary Background PLC-β signaling is generally thought to be mediated by allosteric activation by G proteins and Ca2+. While availability of the PIP2 substrate is limiting in some cases, its production has not been shown to be independently regulated as a signaling mechanism. WNK1 protein kinase is known to regulate ion homeostasis and cause hypertension when expression is increased by gene mutations. However, its signaling functions remain largely elusive. Results Using diacylglycerol-stimulated TRPC6 and inositol trisphosphate-mediated Ca2+ transients as cellular biosensors, we show that WNK1 stimulates PLC-β signaling in cells by promoting the synthesis of PIP2 via stimulation of phosphatidylinositol 4-kinase IIIα. WNK1 kinase activity is not required. Stimulation of PLC-β by WNK1 and by Gαq are synergistic; WNK1 activity is essential for regulation of PLC-β signaling by Gq-coupled receptors and basal input from Gq is necessary for WNK1 signaling via PLC-β. WNK1 further amplifies PLC-β signaling when it is phosphorylated by Akt kinase in response to insulin-like growth factor. Conclusions WNK1 is a novel regulator of PLC-β that acts by controlling substrate availability. WNK1 thereby coordinates signaling between G protein and Akt kinase pathways. Because PIP2 is itself a signaling molecule, regulation of PIP2 synthesis by WNK1 also allows the cell to initiate PLC signaling while independently controlling the effects of PIP2 on other targets. These findings describe a new signaling pathway for Akt-activating growth factors, a mechanism for G protein-growth factor crosstalk and a means to independently control PLC signaling and PIP2 availability. PMID:22119528

Effects of inorganic polyphosphate on bone sialoprotein gene expression.

PubMed

Wang, Zhitao; Li, Xinyue; Li, Zhengyang; Yang, Li; Sasaki, Yoko; Wang, Shuang; Zhou, Liming; Araki, Shouta; Mezawa, Masaru; Takai, Hideki; Ogata, Yorimasa

2010-03-01

Inorganic polyphosphate (poly(P)) is a biopolymer existing in almost all cells and tissues. The biological functions of poly(P) in micro-organisms have been extensively investigated in studies of poly(P) in eukaryotic cells, especially osteoblasts, and are increasing. Bone sialoprotein (BSP) is thought to function in bone mineralization, and is selectively expressed by differentiated osteoblasts. In this study, application of sodium phosphate glass type 25 (SPG25, 12.5 and 125 microM) increased BSP mRNA levels at 12 h in osteoblast-like ROS 17/2.8 cells. In transient transfection assay, 12.5 and 125 microM SPG25 increased luciferase activities of the constructs pLUC3 (-116 to +60), pLUC4 (-425 to +60), pLUC5 (-801 to +60) and pLUC6 (-938 to +60). Introduction of 2 bp mutations to the luciferase constructs showed that the effects of SPG25 were mediated by a FGF2 response element (FRE) and a homeodomain protein binding site (HOX). Luciferase activities induced by SPG25 were blocked by tyrosine kinase inhibitor herbimycine A, MAP kinase kinase inhibitor U0126, PI3-kinase/Akt inhibitor LY249002 and inorganic phosphate transport inhibitor foscarnet. Gel shift analyses showed that both 12.5 and 125 microM SPG25 increased nuclear protein binding to FRE and HOX elements. These studies demonstrate that SPG25 stimulates BSP transcription by targeting FRE and HOX elements in the proximal promoter of the rat BSP gene. Copyright 2010 Elsevier B.V. All rights reserved.

Light-regulation of enzyme activity in anacystis nidulans (Richt.).

PubMed

Duggan, J X; Anderson, L E

1975-01-01

The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

Association of p21ras with phosphatidylinositol 3-kinase.

PubMed Central

Sjölander, A; Yamamoto, K; Huber, B E; Lapetina, E G

1991-01-01

In mammalian cells, ras genes code for 21-kDa GTP-binding proteins. Increased expression and mutations in specific amino acids have been closely linked to alterations of normal cell morphology, growth, and differentiation and, in particular, to neoplastic transformation. The signal transduction induced by these p21ras proteins is largely unknown; however, the signaling pathways of several growth factors have been reported to involve phosphatidylinositol (PtdIns) 3-kinase. In the present study of a Ha-ras-transformed epithelial cell line, we demonstrated increased PtdIns 3-kinase activity in anti-phosphotyrosine and anti-receptor (insulin and hybrid insulin-like growth factor I) immunoprecipitates of cells that had been stimulated with insulin or insulin-like growth factor I. The PtdIns 3-kinase activity was also immunoprecipitated in these experiments by the anti-Ras monoclonal antibody Y13-259. The specificity of this association with p21ras was ascertained by the neutralizing effect of the antigen peptide and the absence of PtdIns 3-kinase activity in Y13-259 immunoprecipitates from cells in which the ras gene was turned off. These data indicate that PtdIns 3-kinase activity is an important step in the cascade of reactions in p21ras signal transduction, suggesting that the alterations of the cytoskeleton and growth in ras-transformed cells could be mediated by PtdIns 3-kinase activity. Images PMID:1716764

Regulation of Cdk7 activity through a phosphatidylinositol (3)-kinase/PKC-ι-mediated signaling cascade in glioblastoma

PubMed Central

Desai, Shraddha R.; Pillai, Prajit P.; Patel, Rekha S.; McCray, Andrea N.; Win-Piazza, Hla Y.; Acevedo-Duncan, Mildred E.

2012-01-01

The objective of this research was to study the potential function of protein kinase C (PKC)-ι in cell cycle progression and proliferation in glioblastoma. PKC-ι is highly overexpressed in human glioma and benign and malignant meningioma; however, little is understood about its role in regulating cell proliferation of glioblastoma. Several upstream molecular aberrations and/or loss of PTEN have been implicated to constitutively activate the phosphatidylinositol (PI) (3)-kinase pathway. PKC-ι is a targeted mediator in the PI (3)-kinase signal transduction repertoire. Results showed that PKC-ι was highly activated and overexpressed in glioma cells. PKC-ι directly associated and phosphorylated Cdk7 at T170 in a cell cycle-dependent manner, phosphorylating its downstream target, cdk2 at T160. Cdk2 has a major role in inducing G1–S phase progression of cells. Purified PKC-ι phosphorylated both endogenous and exogenous Cdk7. PKC-ι downregulation reduced Cdk7 and cdk2 phosphorylation following PI (3)-kinase inhibition, phosphotidylinositol-dependent kinase 1 knockdown as well as PKC-ι silencing (by siRNA treatment). It also diminished cdk2 activity. PKC-ι knockdown inhibited overall proliferation rates and induced apoptosis in glioma cells. These findings suggest that glioma cells may be proliferating through a novel PI (3)-kinase-/PKC-ι/Cdk7/cdk2-mediated pathway. PMID:22021906

Regulation of Cdk7 activity through a phosphatidylinositol (3)-kinase/PKC-ι-mediated signaling cascade in glioblastoma.

PubMed

Desai, Shraddha R; Pillai, Prajit P; Patel, Rekha S; McCray, Andrea N; Win-Piazza, Hla Y; Acevedo-Duncan, Mildred E

2012-01-01

The objective of this research was to study the potential function of protein kinase C (PKC)-ι in cell cycle progression and proliferation in glioblastoma. PKC-ι is highly overexpressed in human glioma and benign and malignant meningioma; however, little is understood about its role in regulating cell proliferation of glioblastoma. Several upstream molecular aberrations and/or loss of PTEN have been implicated to constitutively activate the phosphatidylinositol (PI) (3)-kinase pathway. PKC-ι is a targeted mediator in the PI (3)-kinase signal transduction repertoire. Results showed that PKC-ι was highly activated and overexpressed in glioma cells. PKC-ι directly associated and phosphorylated Cdk7 at T170 in a cell cycle-dependent manner, phosphorylating its downstream target, cdk2 at T160. Cdk2 has a major role in inducing G(1)-S phase progression of cells. Purified PKC-ι phosphorylated both endogenous and exogenous Cdk7. PKC-ι downregulation reduced Cdk7 and cdk2 phosphorylation following PI (3)-kinase inhibition, phosphotidylinositol-dependent kinase 1 knockdown as well as PKC-ι silencing (by siRNA treatment). It also diminished cdk2 activity. PKC-ι knockdown inhibited overall proliferation rates and induced apoptosis in glioma cells. These findings suggest that glioma cells may be proliferating through a novel PI (3)-kinase-/PKC-ι/Cdk7/cdk2-mediated pathway.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takuwa, Y.; Takuwa, N.; Rasmussen, H.

The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of (/sup 3/H)inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in (/sup 3/H)inositol radioactivitymore » from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of (/sup 3/H)inositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca/sup 2 +/ and does not appear to be secondary to an increase in intracellular Ca/sup 2 +/. These results indicate that carbachol causes phospholipase C-mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2-diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,2-diacylglycerol.« less

Overexpression of protein kinase C ɛ improves retention and survival of transplanted mesenchymal stem cells in rat acute myocardial infarction.

PubMed

He, H; Zhao, Z-H; Han, F-S; Liu, X-H; Wang, R; Zeng, Y-J

2016-01-21

We assessed the effects of protein kinase C ɛ (PKCɛ) for improving stem cell therapy for acute myocardial infarction (AMI). Primary mesenchymal stem cells (MSCs) were harvested from rat bone marrow. PKCɛ-overexpressed MSCs and control MSCs were transplanted into infarct border zones in a rat AMI model. MSCs and PKCɛ distribution and expression of principal proteins involved in PKCɛ signaling through the stromal cell-derived factor 1 (SDF-1)/CXC chemokine receptor type 4 (CXCR4) axis and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway were analyzed by immunofluorescence and western blot 1 day after transplantation. Echocardiographic measurements and histologic studies were performed at 4 weeks after transplantation, and MSC survival, expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor β (TGFβ), cardiac troponin I (cTnI), von Willebrand factor (vWF), smooth muscle actin (SMA) and factor VIII and apoptosis in infarct border zones were assessed. Rat heart muscles retained more MSCs and SDF-1, CXCR4, PI3K and phosphorylated AKT increased with PKCɛ overexpression 1 day after transplantation. MSC survival and VEGF, bFGF, TGFβ, cTnI, vWF, SMA and factor VIII expression increased in animals with PKCɛ-overexpressed MSCs at 4 weeks after transplantation and cardiac dysfunction and remodeling improved. Infarct size and apoptosis decreased as well. Inhibitory actions of CXCR4 or PI3K partly attenuated the effects of PKCɛ. Activation of PKCɛ may improve retention, survival and differentiation of transplanted MSCs in myocardia. Augmentation of PKCɛ expression may enhance the therapeutic effects of stem cell therapy for AMI.

The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation

PubMed Central

Guerrero-Mendiola, Carlos; García-Trejo, José J.; Encalada, Rusely; Saavedra, Emma

2017-01-01

In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium. PMID:28686591

Dietary TiO2 particles modulate expression of hormone-related genes in Bombyx mori.

PubMed

Shi, Guofang; Zhan, Pengfei; Jin, Weiming; Fei, JianMing; Zhao, Lihua

2017-08-01

Silkworm (Bombyx mori) is an economically beneficial insect. Its growth and development are regulated by endogenous hormones. In the present study, we found that feeding titanium dioxide nanoparticles (TiO 2 NP) caused a significant increase of body size. TiO 2 NP stimulated the transcription of several genes, including the insulin-related hormone bombyxin, PI3K/Akt/TOR (where PI3K is phosphatidylinositol 3-kinase and TOR is target of rapamycin), and the adenosine 5'-monophosphateactivated protein kinase (AMPK)/target of rapamycin (TOR) pathways. Differentially expressed gene (DEG) analysis documented 26 developmental hormone signaling related genes that were differentially expressed following dietary TiO 2 NP treatment. qPCR analysis confirmed the upregulation of insulin/ecdysteroid signaling genes, such as bombyxin B-1, bombyxin B-4, bombyxin B-7, MAPK, P70S6K, PI3k, eIF4E, E75, ecdysteroid receptor (EcR), and insulin-related peptide binding protein precursor 2 (IBP2). We infer from the upregulated expression of bombyxins and the signaling network that they act in bombyxin-stimulated ecdysteroidogenesis. © 2017 Wiley Periodicals, Inc.

Studies of ATM Kinase Activity Using Engineered ATM Sensitive to ATP Analogues (ATM-AS).

PubMed

Enari, Masato; Matsushima-Hibiya, Yuko; Miyazaki, Makoto; Otomo, Ryo

2017-01-01

Ataxia-telangiectasia mutated (ATM) protein is a member of the phosphatidylinositol 3-phosphate kinase (PI3-K)-related protein kinase (PIKK) family and is implicated in the initiation of signaling pathways following DNA double strand breaks (DSBs) elicited by exposure to ionizing irradiation (IR) or radiomimetic compounds. Loss of function of the ATM gene product results in the human genetic disorder ataxia-telangiectasia (A-T) characterized by neurodegeneration, immunodeficiency, genomic instability, and cancer predisposition. In response to DSBs, ATM is activated and phosphorylates Ser/Thr-Gln (S/T-Q) sequences on numerous proteins participating in DNA-damage responses. Among these proteins, phosphorylation of the tumor suppressor p53 at Ser15 is known as a target for ATM, which leads to the dissociation of MDM2, an E3 ubiquitin ligase, from p53 to prevent MDM2-dependent p53 degradation. Ser46 on p53 is phosphorylated in response to DSBs and contributes to the preferential transactivation of pro-apoptotic genes, such as p53AIP1, Noxa, and PUMA, to prevent tumor formation. Our group have shown that not only ATM preferentially phosphorylates S/T-Q sequences, but also Ser46, which is a noncanonical site with an S-P sequence for ATM. Ser46 on p53 is directly phosphorylated by ATM in a p53 conformation-dependent manner using the ATP analogue-accepting ATM mutant (ATM-AS) system. This protocol summarizes an approach to identify direct numerous targets for ATM kinase and is used to elucidate ATM signaling pathways in the DNA damage responses.

Hydrolysis of membrane phospholipids by phospholipases of rat liver lysosomes

PubMed Central

Richards, Donald E.; Irvine, Robin F.; Dawson, Rex M. C.

1979-01-01

(1) The hydrolysis of 32P- or myo-[2-3H]inositol-labelled rat liver microsomal phospholipids by rat liver lysosomal enzymes has been studied. (2) The relative rates of hydrolysis of phospholipids at pH4.5 are: sphingomyelin>phosphatidylethanolamine>phosphatidylcholine> phosphatidylinositol. (3) The predominant products of phosphatidylcholine and phosphatidylethanolamine hydrolysis are their corresponding lyso-compounds, indicating a slow rate of total deacylation. (4) Ca2+ inhibits the hydrolysis of all phospholipids, though only appreciably at high (>5mm) concentration. The hydrolysis of sphingomyelin is considerably less sensitive to Ca2+ than that of glycerophospholipids. (5) Analysis of the water-soluble products of phosphatidylinositol hydrolysis (by using myo-[3H]inositol-labelled microsomal fraction as a substrate) produced evidence that more than 95% of the product is phosphoinositol, which was derived by direct cleavage from phosphatidylinositol, rather than by hydrolysis of glycerophosphoinositol. (6) This production of phosphoinositol, allied with negligible lysophosphatidylinositol formation and a detectable accumulation of diacylglycerol, indicates that lysosomes hydrolyse membrane phosphatidylinositol almost exclusively in a phospholipase C-like manner. (7) Comparisons are drawn between the hydrolysis by lysosomal enzymes of membrane substrates and that of pure phospholipid substrates, and also the possible role of phosphatidylinositol-specific lysosomal phospholipase C in cellular phosphatidylinositol catabolism is discussed. PMID:508301

Synergistic Cytotoxicity of Bendamustine and the BTK Inhibitor in a Mantle Cell Lymphoma Cell Line.

PubMed

Hagiwara, Kazumi; Tokunaga, Takashi; Iida, Hiroatsu; Nagai, Hirokazu

2015-12-01

Bendamustine is effective in B-cell malignancies, including mantle cell lymphoma (MCL), alone and in combination with other agents. This study investigated the combination effect of bendamustine and the Bruton tyrosine kinase (BTK) inhibitor PCI-32765 on MCL cell death and the underlying mechanisms. Cytotoxicity was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MIT) assay. Apoptosis was assessed by annexin V/propidium iodide staining and protein expression was analyzed by western blotting. When combined with bendamustine, PCI-32765 showed a synergistic effect on growth inhibition of the MCL cell line Jeko-1. Cleavage of caspase-3 and poly-(ADP-ribose) polymerase was increased, indicating enhanced apoptosis induction. In addition, this combination decreased the protein expression of cyclin D1. Phosphorylated v-akt murine thymoma viral oncogene homolog 1 (AKT) (Ser473) was also down-regulated, suggesting a suppression of the phosphatidylinositol 3-kinase/AKT signaling pathway. Combination treatment with bendamustine and a BTK inhibitor may be effective in MCL therapy. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Molecular insights into the binding of phosphoinositides to the TH domain region of TIPE proteins.

PubMed

Antony, Priya; Baby, Bincy; Vijayan, Ranjit

2016-11-01

Phosphatidylinositols and their phosphorylated derivatives, phosphoinositides, play a central role in regulating diverse cellular functions. These phospholipids have been shown to interact with the hydrophobic TH domain of the tumor necrosis factor (TNF)-α-induced protein 8 (TIPE) family of proteins. However, the precise mechanism of interaction of these lipids is unclear. Here we report the binding mode and interactions of these phospholipids in the TH domain, as elucidated using molecular docking and simulations. Results indicate that phosphoinositides bind to the TH domain in a similar way by inserting their lipid tails in the hydrophobic cavity. The exposed head group is stabilized by interactions with critical positively charged residues on the surface of these proteins. Further MD simulations confirmed the dynamic stability of these lipids in the TH domain. This computational analysis thus provides insight into the binding mode of phospholipids in the TH domain of the TIPE family of proteins. Graphical abstract A phosphoinositide (phosphatidylinositol 4-phosphate; PtdIns4P) docked to TIPE2.

Native and reconstituted HDL activate Stat3 in ventricular cardiomyocytes via ERK1/2: role of sphingosine-1-phosphate.

PubMed

Frias, Miguel A; James, Richard W; Gerber-Wicht, Christine; Lang, Ursula

2009-05-01

High-density lipoprotein (HDL) has been reported to have cardioprotective properties independent from its cholesterol transport activity. The influence of native HDL and reconstituted HDL (rHDL) on Stat3, the transcription factor playing an important role in myocardium adaptation to stress, was analysed in neonatal rat ventricular cardiomyocytes. We have investigated modulating the composition of rHDL as a means of expanding its function and potential cardioprotective effects. Stat3 phosphorylation and activation were determined by western blotting and electrophoretic mobility shift assay (EMSA). In ventricular cardiomyocytes, HDL and the HDL constituent sphingosine-1-phosphate (S1P) induce a concentration- and time-dependent increase in Stat3 activation. They also enhance extracellular signal-regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. U0126, a specific inhibitor of MEK1/2, the upstream activator of ERK1/2, abolishes HDL- and S1P-induced Stat3 activation, whereas the p38 MAPK blocker SB203580 has no significant effect. Inhibition of the tyrosine kinase family Src (Src) caused a significant reduction of Stat3 activation, whereas inhibition of phosphatidylinositol 3-kinase (PI3K) had no effect. S1P and rHDL containing S1P have a similar strong stimulatory action on Stat3, ERK1/2, and p38 MAPK comparable to native HDL. S1P-free rHDL has a much weaker effect. Experiments with agonists and antagonists of the S1P receptor subtypes indicate that HDL and S1P activate Stat3 mainly through the S1P2 receptor. In ventricular cardiomyocytes, addition of S1P to rHDL enhances its therapeutic potential by improving its capacity to activate Stat3. Activation of Stat3 occurs mainly via the S1P constituent and the lipid receptor S1P2 requiring stimulation of ERK1/2 and Src but not p38 MAPK or PI3K. The study underlines the therapeutic potential of tailoring rHDL to confront particular clinical situations.

Rapid desensitization of vasopressin-stimulated phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine hydrolysis questions the role of these pathways in sustained diacylglycerol formation in A10 vascular-smooth-muscle cells.

PubMed

Plevin, R; Wakelam, M J

1992-08-01

The kinetics of vasopressin-stimulated PtdIns(4,5)P2 and phosphatidylcholine (PtdCho) hydrolysis in relation to sustained diacylglycerol (DAG) formation was investigated in A10 vascular-smooth-muscle cells in culture. Vasopressin stimulated a transient increase in Ins(1,4,5)P3 mass formation, which was mirrored by a decrease in PtdIns(4,5)P2 mass levels. Vasopressin stimulated sustained accumulation of total [3H]inositol phosphates ([3H]IP) in the presence of Li+; however, this was significantly decreased by adding a vasopressin-receptor antagonist at different times after initial stimulation. Vasopressin-stimulated phospholipase D (PLD) activity was found to be a transient phenomenon lasting approx. 2 min. Experiments involving agonist preincubation with subsequent addition of butanol confirmed that vasopressin-stimulated PLD activity was desensitized. Vasopressin stimulated an increase in formation of choline, but not of phosphocholine, suggesting that PLD was the major catalytic route of PtdCho hydrolysis in this cell line. The roles of choline and inositol phospholipid hydrolysis in the prolonged phase of DAG formation was examined by comparing vasopressin-stimulated changes in DAG levels in the presence of butanol, the protein kinase C inhibitor Ro-31-8220 or a V1a-receptor antagonist. Vasopressin-stimulated DAG formation was decreased by 40-50% in the presence of butanol between 1 and 10 min; however, during more prolonged stimulation butanol was without significant effect. In cells pretreated with Ro-31-8220, vasopressin-stimulated DAG formation was decreased by approx. 30% at 2 min, but was significantly potentiated at later times. This coincided with an enhancement of vasopressin-stimulated [3H]IP accumulation. In cells exposed to the V1a-receptor antagonist 5 min after addition of vasopressin, subsequent DAG formation was significantly decreased, indicating that sustained formation of DAG, like [3H]IP accumulation, was dependent on continual agonist receptor activation. The results are discussed in terms of different phospholipid-hydrolytic pathways providing DAG generation.

Regulation of the putative TRPV1t salt taste receptor by phosphatidylinositol 4,5-bisphosphate.

PubMed

Lyall, Vijay; Phan, Tam-Hao T; Ren, ZuoJun; Mummalaneni, Shobha; Melone, Pamela; Mahavadi, Sunila; Murthy, Karnam S; DeSimone, John A

2010-03-01

Regulation of the putative amiloride and benzamil (Bz)-insensitive TRPV1t salt taste receptor by phosphatidylinositol 4,5-bisphosphate (PIP(2)) was studied by monitoring chorda tympani (CT) taste nerve responses to 0.1 M NaCl solutions containing Bz (5 x 10(-6) M; a specific ENaC blocker) and resiniferatoxin (RTX; 0-10 x 10(-6) M; a specific TRPV1 agonist) in Sprague-Dawley rats and in wildtype (WT) and TRPV1 knockout (KO) mice. In rats and WT mice, RTX elicited a biphasic effect on the NaCl + Bz CT response, increasing the CT response between 0.25 x 10(-6) and 1 x 10(-6) M. At concentrations >1 x 10(-6) M, RTX inhibited the CT response. An increase in PIP(2) by topical lingual application of U73122 (a phospholipase C blocker) or diC8-PIP(2) (a short chain synthetic PIP(2)) inhibited the control NaCl + Bz CT response and decreased its sensitivity to RTX. A decrease in PIP(2) by topical lingual application of phenylarsine oxide (a phosphoinositide 4 kinase blocker) enhanced the control NaCl + Bz CT response, increased its sensitivity to RTX stimulation, and inhibited the desensitization of the CT response at RTX concentrations >1 x 10(-6) M. The ENaC-dependent NaCl CT responses were not altered by changes in PIP(2). An increase in PIP(2) enhanced CT responses to sweet (0.3 M sucrose) and bitter (0.01 M quinine) stimuli. RTX produced the same increase in the Bz-insensitive Na(+) response when present in salt solutions containing 0.1 M NaCl + Bz, 0.1 M monosodium glutamate + Bz, 0.1 M NaCl + Bz + 0.005 M SC45647, or 0.1 M NaCl + Bz + 0.01 M quinine. No effect of RTX was observed on CT responses in WT mice and rats in the presence of the TRPV1 blocker N-(3-methoxyphenyl)-4-chlorocinnamide (1 x 10(-6) M) or in TRPV1 KO mice. We conclude that PIP(2) is a common intracellular effector for sweet, bitter, umami, and TRPV1t-dependent salt taste, although in the last case, PIP(2) seems to directly regulate the taste receptor protein itself, i.e., the TRPV1 ion channel or its taste receptor variant, TRPV1t.

A Balanced Chromosomal Translocation Disrupting ARHGEF9 Is Associated With Epilepsy, Anxiety, Aggression, and Mental Retardation

PubMed Central

Kalscheuer, Vera M.; Musante, Luciana; Fang, Cheng; Hoffmann, Kirsten; Fuchs, Celine; Carta, Eloisa; Deas, Emma; Venkateswarlu, Kanamarlapudi; Menzel, Corinna; Ullmann, Reinhard; Tommerup, Niels; Dalprà, Leda; Tzschach, Andreas; Selicorni, Angelo; Lüscher, Bernhard; Ropers, Hans-Hilger; Harvey, Kirsten; Harvey, Robert J.

2013-01-01

Clustering of inhibitory γ-aminobutyric acidA (GABAA) and glycine receptors at synapses is thought to involve key interactions between the receptors, a “scaffolding” protein known as gephyrin and the RhoGEF collybistin. We report the identification of a balanced chromosomal translocation in a female patient presenting with a disturbed sleep-wake cycle, late-onset epileptic seizures, increased anxiety, aggressive behavior, and mental retardation, but not hyperekplexia. Fine mapping of the breakpoint indicates disruption of the collybistin gene (ARHGEF9) on chromosome Xq11, while the other breakpoint lies in a region of 18q11 that lacks any known or predicted genes. We show that defective collybistin transcripts are synthesized and exons 7–10 are replaced by cryptic exons from chromosomes X and 18. These mRNAs no longer encode the pleckstrin homology (PH) domain of collybistin, which we now show binds phosphatidylinositol-3-phosphate (PI3P/ PtdIns-3-P), a phosphoinositide with an emerging role in membrane trafficking and signal transduction, rather than phosphatidylinositol 3,4,5-trisphosphate (PIP3/PtdIns-3,4,5-P) as previously suggested in the “membrane activation model” of gephyrin clustering. Consistent with this finding, expression of truncated collybistin proteins in cultured neurons interferes with synaptic localization of endogenous gephyrin and GABAA receptors. These results suggest that collybistin has a key role in membrane trafficking of gephyrin and selected GABAA receptor subtypes involved in epilepsy, anxiety, aggression, insomnia, and learning and memory. PMID:18615734

Effects of inositol trisphosphate on calcium mobilization in high-voltage and saponin-permeabilized platelets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gear, A.R.L.; Hallam, T.J.

1986-03-01

Interest in phosphatidylinositol metabolism has been greatly stimulated by the findings that diglyceride and inositol phosphates may serve as second messengers in modulating cellular function. Formation of 1,4,5-inositol trisphosphate (IP/sub 3/), in particular, has been linked to mobilization of intracellular calcium in a number of cell types. The authors have examined the ability of IP/sub 3/ to mobilize calcium in human platelets permeabilized by either saponin or high-voltage discharge. Saponin at 15 ..mu..g/ml effectively permeabilized platelets to exogenous inositol 1,4,5-trisphosphate which released bound (/sup 45/Ca) within 1 min and with a Ka of 7.4 +/- 4.1 ..mu..M. A small (25%)more » azide-sensitive pool was also responsive to inositol trisphosphate. The calcium pools were completely discharged by A-23187 and the ATP-dependent uptake was prevented by dinitrophenol. In contrast to the result with saponin, platelets accessed by high-voltage discharge were insensitive to challenge by inositol 1,4,5-trisphosphate. The data suggest that while inositol 1,4,5-trisphosphate can rapidly mobilize platelet calcium, the ability to demonstrate this depends on the method of permeabilization.« less

Ultrafine particulate matter impairs mitochondrial redox homeostasis and activates phosphatidylinositol 3-kinase mediated DNA damage responses in lymphocytes.

PubMed

Bhargava, Arpit; Tamrakar, Shivani; Aglawe, Aniket; Lad, Harsha; Srivastava, Rupesh Kumar; Mishra, Dinesh Kumar; Tiwari, Rajnarayan; Chaudhury, Koel; Goryacheva, Irina Yu; Mishra, Pradyumna Kumar

2018-03-01

Particulate matter (PM), broadly defined as coarse (2.5-10 μm), fine (0.1-2.5 μm) and ultrafine particles (≤0.1 μm), is a major constituent of ambient air pollution. Recent studies have linked PM exposure (coarse and fine particles) with several human diseases including cancer. However, the molecular mechanisms underlying ultrafine PM exposure induced cellular and sub-cellular repercussions are ill-defined. Since mitochondria are one of the major targets of different environmental pollutants, we herein aimed to understand the molecular repercussion of ultrafine PM exposure on mitochondrial machinery in peripheral blood lymphocytes. Upon comparative analysis, a significantly higher DCF fluorescence was observed in ultrafine PM exposed cells that confirmed the strong pro-oxidant nature of these particles. In addition, the depleted activity of antioxidant enzymes, glutathione reductase and superoxide dismutase suggested the strong association of ultrafine PM with oxidative stress. These results further coincided with mitochondrial membrane depolarization, altered mitochondrial respiratory chain enzyme activity and decline in mtDNA copy number. Moreover, the higher accumulation of DNA damage response proteins (γH2AX, pATM, p-p53), suggested that exposure to ultrafine PM induces DNA damage and triggers phosphatidylinositol 3 kinase mediated response pathway. Further, the alterations in mitochondrial machinery and redox balance among ultrafine PM exposed cells were accompanied by a considerably elevated pro-inflammatory cytokine response. Interestingly, the lower apoptosis levels observed in ultrafine particle treated cells suggest the possibility that the marked alterations may lead to the impairment of mitochondrial-nuclear cross talk. Together, our results showed that ultrafine PM, because of their smaller size possesses significant ability to disturb mitochondrial redox homeostasis and activates phosphatidylinositol 3 kinase mediated DNA damage response pathway, an unknown molecular paradigm of ultrafine PM exposure. Our findings also indicate that maneuvering through the mitochondrial function might be a viable, indirect method to modulate lymphocyte homeostasis in air pollution associated immune disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

Streptococcus mutans serotype c tagatose 6-phosphate pathway gene cluster.

PubMed Central

Jagusztyn-Krynicka, E K; Hansen, J B; Crow, V L; Thomas, T D; Honeyman, A L; Curtiss, R

1992-01-01

DNA cloned into Escherichia coli K-12 from a serotype c strain of Streptococcus mutans encodes three enzyme activities for galactose utilization via the tagatose 6-phosphate pathway: galactose 6-phosphate isomerase, tagatose 6-phosphate kinase, and tagatose-1,6-bisphosphate aldolase. The genes coding for the tagatose 6-phosphate pathway were located on a 3.28-kb HindIII DNA fragment. Analysis of the tagatose proteins expressed by recombinant plasmids in minicells was used to determine the sizes of the various gene products. Mutagenesis of these plasmids with transposon Tn5 was used to determine the order of the tagatose genes. Tagatose 6-phosphate isomerase appears to be composed of 14- and 19-kDa subunits. The sizes of the kinase and aldolase were found to be 34 and 36 kDa, respectively. These values correspond to those reported previously for the tagatose pathway enzymes in Staphylococcus aureus and Lactococcus lactis. Images PMID:1328153

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

PubMed Central

McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

1992-01-01

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

Insulin and IGF-1 activate Kir4.1/5.1 channels in cortical collecting duct principal cells to control basolateral membrane voltage.

PubMed

Zaika, Oleg; Palygin, Oleg; Tomilin, Viktor; Mamenko, Mykola; Staruschenko, Alexander; Pochynyuk, Oleh

2016-02-15

Potassium Kir4.1/5.1 channels are abundantly expressed at the basolateral membrane of principal cells in the cortical collecting duct (CCD), where they are thought to modulate transport rates by controlling transepithelial voltage. Insulin and insulin-like growth factor-1 (IGF-1) stimulate apically localized epithelial sodium channels (ENaC) to augment sodium reabsorption in the CCD. However, little is known about their actions on potassium channels localized at the basolateral membrane. In this study, we implemented patch-clamp analysis in freshly isolated murine CCD to assess the effect of these hormones on Kir4.1/5.1 at both single channel and cellular levels. We demonstrated that K(+)-selective conductance via Kir4.1/5.1 is the major contributor to the macroscopic current recorded from the basolateral side in principal cells. Acute treatment with 10 μM amiloride (ENaC blocker), 100 nM tertiapin-Q (TPNQ; ROMK inhibitor), and 100 μM ouabain (Na(+)-K(+)-ATPase blocker) failed to produce a measurable effect on the macroscopic current. In contrast, Kir4.1 inhibitor nortriptyline (100 μM), but not fluoxetine (100 μM), virtually abolished whole cell K(+)-selective conductance. Insulin (100 nM) markedly increased the open probability of Kir4.1/5.1 and nortriptyline-sensitive whole cell current, leading to significant hyperpolarization of the basolateral membrane. Inhibition of the phosphatidylinositol 3-kinase cascade with LY294002 (20 μM) abolished action of insulin on Kir4.1/5.1. IGF-1 had similar stimulatory actions on Kir4.1/5.1-mediated conductance only when applied at a higher (500 nM) concentration and was ineffective at 100 nM. We concluded that both insulin and, to a lesser extent, IGF-1 activate Kir4.1/5.1 channel activity and open probability to hyperpolarize the basolateral membrane, thereby facilitating Na(+) reabsorption in the CCD. Copyright © 2016 the American Physiological Society.

The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization.

PubMed

Sawicka, Marta; Wanrooij, Paulina H; Darbari, Vidya C; Tannous, Elias; Hailemariam, Sarem; Bose, Daniel; Makarova, Alena V; Burgers, Peter M; Zhang, Xiaodong

2016-06-24

The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

P38 AND EGF RECEPTOR KINASE-MEDIATED ACTIVATION OF THE PHOSPHATIDYLINOSITOL 3-KINASE/AKT PATHWAY IS REQUIRED FOR ZN2+INDUCED CYCLOOXYGENASE-2 EXPRESSION

EPA Science Inventory

Cyclooxygenase 2 (COX-2) expression is induced by physiological and inflammatory stimuli. Regulation of COX-2 expression is stimulus- and cell type-specific. Exposure to Zn2+ has been associated with activation of multiple intracellular signaling pathways as well as the induction...

Phosphatidylinositol 3-kinase is a key mediator of central sensitization in painful inflammatory conditions

PubMed Central

Pezet, Sophie; Marchand, Fabien; D'Mello, Richard; Grist, John; Clark, Anna K.; Malcangio, Marzia; Dickenson, Anthony H.; Williams, Robert J.; McMahon, Stephen B.

2010-01-01

Here we show that phosphatidylinositol 3-kinase (PI3K) is a key player in the establishment of central sensitization, the spinal cord phenomenon associated with persistent afferent inputs and contributing to chronic pain states. We demonstrated electrophysiologically that PI3K is required for the full expression of spinal neuronal wind-up. In an inflammatory pain model, intrathecal administration of LY294002, a potent PI3K inhibitor, dose-dependently inhibited pain related behavior. This effect was correlated with a reduction of the phosphorylation of extracellular signal-regulated kinase (ERK) and CaMKinase II. In addition, we observed a significant decrease in the phosphorylation of the NMDA receptor subunit NR2B, decreased translocation to the plasma membrane of the GluR1 AMPA receptor subunit in the spinal cord and a reduction of evoked neuronal activity as measured using c-Fos immunohistochemistry. Our study suggests that PI3K is a major factor in the expression of central sensitization after noxious inflammatory stimuli. PMID:18417706

Nuclear PTEN deficiency causes microcephaly with decreased neuronal soma size and increased seizure susceptibility.

PubMed

Igarashi, Atsushi; Itoh, Kie; Yamada, Tatsuya; Adachi, Yoshihiro; Kato, Takashi; Murata, Daisuke; Sesaki, Hiromi; Iijima, Miho

2018-06-15

Defects in phosphatase and tensin homolog (PTEN) are associated with neurological disorders and tumors. PTEN functions at two primary intracellular locations: the plasma membrane and the nucleus. At the membrane, PTEN functions as a phosphatidylinositol (3,4,5)-trisphosphate phosphatase and suppresses PI 3-kinase signaling that drives cell growth and tumorigenesis. However, the in vivo function of nuclear PTEN is unclear. Here, using CRISPR/Cas9, we generated a mouse model in which PTEN levels in the nucleus are decreased. Nuclear PTEN-deficient mice were born with microcephaly and maintained a small brain during adulthood. The size of neuronal soma was significantly smaller in the cerebellum, cerebral cortex, and hippocampus. Also, these mice were prone to seizure. No changes in PI 3-kinase signaling were observed. By contrast, the size of other organs was unaffected. Therefore, nuclear PTEN is essential for the health of the brain by promoting the growth of neuronal soma size during development. © 2018 Igarashi et al.

Synergism between thrombin and adrenaline (epinephrine) in human platelets. Marked potentiation of inositol phospholipid metabolism.

PubMed Central

Steen, V M; Tysnes, O B; Holmsen, H

1988-01-01

We have studied synergism between adrenaline (epinephrine) and low concentrations of thrombin in gel-filtered human platelets prelabelled with [32P]Pi. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure changes in the levels of 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidate (PA), aggregation and dense-granule secretion after stimulation. Adrenaline alone (3.5-4.0 microM) did not cause a change in any parameter (phosphoinositide metabolism, aggregation and dense-granule secretion), but markedly enhanced the thrombin-induced responses over a narrow range of thrombin concentrations (0.03-0.08 units/ml). The thrombin-induced hydrolysis of inositol phospholipids by phospholipase C, which was measured as the formation of [32P]PA, was potentiated by adrenaline, as was the increase in the levels of [32P]PIP2 and [32P]PIP. The presence of adrenaline caused a shift to the left for the thrombin-induced changes in the phosphoinositide metabolism, without affecting the maximal levels of 32P-labelled compounds obtained. A similar shift by adrenaline in the dose-response relationship was previously demonstrated for thrombin-induced aggregation and dense-granule secretion. Also, the narrow range of concentrations of thrombin over which adrenaline potentiates thrombin-induced platelet responses is the same for changes in phosphoinositide metabolism and physiological responses (aggregation and dense-granule secretion). Our observations clearly indicate that adrenaline directly or indirectly influences thrombin-induced changes in phosphoinositide metabolism. PMID:2845924

Ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry.

PubMed

Qiu, Shirley; Leung, Anders; Bo, Yuxia; Kozak, Robert A; Anand, Sai Priya; Warkentin, Corina; Salambanga, Fabiola D R; Cui, Jennifer; Kobinger, Gary; Kobasa, Darwyn; Côté, Marceline

2018-01-01

For entry, Ebola virus (EBOV) requires the interaction of its viral glycoprotein with the cellular protein Niemann-Pick C1 (NPC1) which resides in late endosomes and lysosomes. How EBOV is trafficked and delivered to NPC1 and whether this is positively regulated during entry remain unclear. Here, we show that the PIKfyve-ArPIKfyve-Sac3 cellular complex, which is involved in the metabolism of phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P 2 ), is critical for EBOV infection. Although the expression of all subunits of the complex was required for efficient entry, PIKfyve kinase activity was specifically critical for entry by all pathogenic filoviruses. Inhibition of PIKfyve prevented colocalization of EBOV with NPC1 and led to virus accumulation in intracellular vesicles with characteristics of early endosomes. Importantly, genetically-encoded phosphoinositide probes revealed an increase in PtdIns(3,5)P 2 -positive vesicles in cells during EBOV entry. Taken together, our studies suggest that EBOV requires PtdIns(3,5)P 2 production in cells to promote efficient delivery to NPC1. Copyright © 2017 Elsevier Inc. All rights reserved.

Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate

PubMed Central

Soldovieri, Maria Virginia; Ambrosino, Paolo; Mosca, Ilaria; De Maria, Michela; Moretto, Edoardo; Miceli, Francesco; Alaimo, Alessandro; Iraci, Nunzio; Manocchio, Laura; Medoro, Alessandro; Passafaro, Maria; Taglialatela, Maurizio

2016-01-01

Kv7.2 and Kv7.3 subunits underlie the M-current, a neuronal K+ current characterized by an absolute functional requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). Kv7.2 gene mutations cause early-onset neonatal seizures with heterogeneous clinical outcomes, ranging from self-limiting benign familial neonatal seizures to severe early-onset epileptic encephalopathy (Kv7.2-EE). In this study, the biochemical and functional consequences prompted by a recurrent variant (R325G) found independently in four individuals with severe forms of neonatal-onset EE have been investigated. Upon heterologous expression, homomeric Kv7.2 R325G channels were non-functional, despite biotin-capture in Western blots revealed normal plasma membrane subunit expression. Mutant subunits exerted dominant-negative effects when incorporated into heteromeric channels with Kv7.2 and/or Kv7.3 subunits. Increasing cellular PIP2 levels by co-expression of type 1γ PI(4)P5-kinase (PIP5K) partially recovered homomeric Kv7.2 R325G channel function. Currents carried by heteromeric channels incorporating Kv7.2 R325G subunits were more readily inhibited than wild-type channels upon activation of a voltage-sensitive phosphatase (VSP), and recovered more slowly upon VSP switch-off. These results reveal for the first time that a mutation-induced decrease in current sensitivity to PIP2 is the primary molecular defect responsible for Kv7.2-EE in individuals carrying the R325G variant, further expanding the range of pathogenetic mechanisms exploitable for personalized treatment of Kv7.2-related epilepsies. PMID:27905566

Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate.

PubMed

Soldovieri, Maria Virginia; Ambrosino, Paolo; Mosca, Ilaria; De Maria, Michela; Moretto, Edoardo; Miceli, Francesco; Alaimo, Alessandro; Iraci, Nunzio; Manocchio, Laura; Medoro, Alessandro; Passafaro, Maria; Taglialatela, Maurizio

2016-12-01

Kv7.2 and Kv7.3 subunits underlie the M-current, a neuronal K + current characterized by an absolute functional requirement for phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Kv7.2 gene mutations cause early-onset neonatal seizures with heterogeneous clinical outcomes, ranging from self-limiting benign familial neonatal seizures to severe early-onset epileptic encephalopathy (Kv7.2-EE). In this study, the biochemical and functional consequences prompted by a recurrent variant (R325G) found independently in four individuals with severe forms of neonatal-onset EE have been investigated. Upon heterologous expression, homomeric Kv7.2 R325G channels were non-functional, despite biotin-capture in Western blots revealed normal plasma membrane subunit expression. Mutant subunits exerted dominant-negative effects when incorporated into heteromeric channels with Kv7.2 and/or Kv7.3 subunits. Increasing cellular PIP 2 levels by co-expression of type 1γ PI(4)P5-kinase (PIP5K) partially recovered homomeric Kv7.2 R325G channel function. Currents carried by heteromeric channels incorporating Kv7.2 R325G subunits were more readily inhibited than wild-type channels upon activation of a voltage-sensitive phosphatase (VSP), and recovered more slowly upon VSP switch-off. These results reveal for the first time that a mutation-induced decrease in current sensitivity to PIP 2 is the primary molecular defect responsible for Kv7.2-EE in individuals carrying the R325G variant, further expanding the range of pathogenetic mechanisms exploitable for personalized treatment of Kv7.2-related epilepsies.

Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor

PubMed Central

Aurass, Philipp; Oates, Clare V.; Tate, Edward W.; Hartland, Elizabeth L.; Flieger, Antje

2015-01-01

Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo. PMID:26216420

Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor.

PubMed

Schroeder, Gunnar N; Aurass, Philipp; Oates, Clare V; Tate, Edward W; Hartland, Elizabeth L; Flieger, Antje; Frankel, Gad

2015-10-01

Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Strawberry Phytochemicals Inhibit Azoxymethane/Dextran Sodium Sulfate-Induced Colorectal Carcinogenesis in Crj: CD-1 Mice

PubMed Central

Shi, Ni; Clinton, Steven K.; Liu, Zhihua; Wang, Yongquan; Riedl, Kenneth M.; Schwartz, Steven J.; Zhang, Xiaoli; Pan, Zui; Chen, Tong

2015-01-01

Human and experimental colon carcinogenesis are enhanced by a pro-inflammatory microenvironment. Pharmacologically driven chemopreventive agents and dietary variables are hypothesized to have future roles in the prevention of colon cancer by targeting these processes. The current study was designed to determine the ability of dietary lyophilized strawberries to inhibit inflammation-promoted colon carcinogenesis in a preclinical animal model. Mice were given a single i.p. injection of azoxymethane (10 mg kg−1 body weight). One week after injection, mice were administered 2% (w/v) dextran sodium sulfate in drinking water for seven days and then an experimental diet containing chemically characterized lyophilized strawberries for the duration of the bioassay. Mice fed control diet, or experimental diet containing 2.5%, 5.0% or 10.0% strawberries displayed tumor incidence of 100%, 64%, 75% and 44%, respectively (p < 0.05). The mechanistic studies demonstrate that strawberries reduced expression of proinflammatory mediators, suppressed nitrosative stress and decreased phosphorylation of phosphatidylinositol 3-kinase, Akt, extracellular signal-regulated kinase and nuclear factor kappa B. In conclusion, strawberries target proinflammatory mediators and oncogenic signaling for the preventive efficacies against colon carcinogenesis in mice. This works supports future development of fully characterized and precisely controlled functional foods for testing in human clinical trials for this disease. PMID:25763529

A method for the 32P labeling of peptides or peptide nucleic acid oligomers

NASA Technical Reports Server (NTRS)

Kozlov, I. A.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

1998-01-01

A novel approach to the radioactive labeling of peptides and PNA oligomers is described. It is based on the conjugation of a deoxynucleoside 3'-phosphate with the terminal amine of the substrate, followed by phosphorylation of the 5'-hydroxyl group of the nucleotide using T4 polynucleotide kinase and [gamma-32P]ATP.

Neurotoxicity induced by dexamethasone in the human neuroblastoma SH-SY5Y cell line can be prevented by folic acid.

PubMed

Budni, J; Romero, A; Molz, S; Martín-de-Saavedra, M D; Egea, J; Del Barrio, L; Tasca, C I; Rodrigues, A L S; López, M G

2011-09-08

Folic acid (folate) is a vitamin of the B-complex group that is essential for cell replication. Folate is a major determinant of one-carbon metabolism, in which S-adenosylmethionine donates methyl groups that are crucial for neurological function. Many roles for folic acid have been reported, including neuroprotective and antidepressant properties. On the other hand, increased concentrations of corticoids have proven neurotoxic effects and hypersecretion of glucocorticoids has been linked to different mood disorders. The purpose of this study was to investigate the potential protective effect of folic acid on dexamethasone-induced cellular death in SH-SY5Y neuroblastoma cell line and the possible intracellular signaling pathway involved in such effect. Exposure to 1 mM dexamethasone for 48 h caused a significant reduction of cell viability measured as 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) reduction. Exposure of SH-SY5Y cells for 72 h to increasing concentrations of folate (1-300 μM) was not cytotoxic. However, pretreatment with folate (10-300 μM) reduced dexamethasone-induced toxicity in a significant manner. To explore the putative intracellular signaling pathways implicated in the protective effect of folate we used different protein kinase inhibitors. The protective effect of folic acid on dexamethasone-induced neurotoxicity was reversed by the phosphatidylinositol-3 kinase/Akt (PI3K/Akt, LY294002), Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII, KN-93), and protein kinase A (PKA, H-89) inhibitors, but not the mitogen-activated protein/extracellular signal-regulated kinase (MEK1/2, PD98059) and protein kinase C (PKC, chelerythrine) inhibitors. In conclusion, the results of this study show that folic acid can protect against dexamethasone-induced neurotoxicity and its protective mechanism is related to a signaling pathway that involves PI3K/Akt, CaMKII, and PKA. Copyright © 2011. Published by Elsevier Ltd.

Blueberry Phytochemicals Inhibit Growth and Metastatic Potential of MDA-MB-231 Breast Cancer Cells Through Modulation of the Phosphatidylinositol 3-Kinase Pathway

PubMed Central

Adams, Lynn S.; Phung, Sheryl; Yee, Natalie; Seeram, Navindra P.; Li, Liya; Chen, Shiuan

2010-01-01

Dietary phytochemicals are known to exhibit a variety of anti-carcinogenic properties. This study investigated the chemopreventive activity of blueberry extract in triple negative breast cancer cell lines in vitro and in vivo. Blueberry decreased cell proliferation in HCC38, HCC1937 and MDA-MB-231 cells with no effect on the non-tumorigenic MCF-10A cell line. Decreased metastatic potential of MDA-MB-231 cells by blueberry was shown through inhibition of cell motility using wound healing assays and migration through a PET membrane. Blueberry treatment decreased the activity of matrix metalloproteinase 9 and the secretion of urokinase-type plasminogen activator while increasing tissue inhibitor of metalloproteinase-1 and plasminogen activator inhibitor-1 secretion in MDA-MB-231 conditioned medium as shown by western blotting. Cell signaling pathways that control the expression/activation of these processes were investigated via western blotting and reporter gene assay. Treatment with blueberry decreased phosphatidylinositol 3-kinase (PI3K)/AKT and nuclear factor kappa-B (NFκB) activation in MDA-MB-231 cells where protein kinase C (PKC) and extracellular regulated kinase (ERK) were not affected. In vivo, the efficacy of blueberry to inhibit triple negative breast tumor growth was evaluated using the MDA-MB-231 xenograft model. Tumor weight and proliferation (Ki-67 expression) were decreased in blueberry treated mice, where apoptosis (caspase-3 expression) was increased compared to controls. Immunohistochemical analysis of tumors from blueberry-fed mice showed decreased activation of AKT and p65 NFκB signaling proteins with no effect on the phosphorylation of ERK. These data illustrate the inhibitory effect of blueberry phytochemicals on the growth and metastatic potential of MDA-MB-231 cells through modulation of the PI3K/AKT/NFκB pathway. PMID:20388778

LRP1 regulates architecture of the vascular wall by controlling PDGFRbeta-dependent phosphatidylinositol 3-kinase activation.

PubMed

Zhou, Li; Takayama, Yoshiharu; Boucher, Philippe; Tallquist, Michelle D; Herz, Joachim

2009-09-09

Low density lipoprotein receptor-related protein 1 (LRP1) protects against atherosclerosis by regulating the activation of platelet-derived growth factor receptor beta (PDGFRbeta) in vascular smooth muscle cells (SMCs). Activated PDGFRbeta undergoes tyrosine phosphorylation and subsequently interacts with various signaling molecules, including phosphatidylinositol 3-kinase (PI3K), which binds to the phosphorylated tyrosine 739/750 residues in mice, and thus regulates actin polymerization and cell movement. In this study, we found disorganized actin in the form of membrane ruffling and enhanced cell migration in LRP1-deficient (LRP1-/-) SMCs. Marfan syndrome-like phenotypes such as tortuous aortas, disrupted elastic layers and abnormally activated transforming growth factor beta (TGFbeta) signaling are present in smooth muscle-specific LRP1 knockout (smLRP1-/-) mice. To investigate the role of LRP1-regulated PI3K activation by PDGFRbeta in atherogenesis, we generated a strain of smLRP1-/- mice in which tyrosine 739/750 of the PDGFRbeta had been mutated to phenylalanines (PDGFRbeta F2/F2). Spontaneous atherosclerosis was significantly reduced in the absence of hypercholesterolemia in these mice compared to smLRP1-/- animals that express wild type PDGFR. Normal actin organization was restored and spontaneous SMC migration as well as PDGF-BB-induced chemotaxis was dramatically reduced, despite continued overactivation of TGFbeta signaling, as indicated by high levels of nuclear phospho-Smad2. Our data suggest that LRP1 regulates actin organization and cell migration by controlling PDGFRbeta-dependent activation of PI3K. TGFbeta activation alone is not sufficient for the expression of the Marfan-like vascular phenotype. Thus, regulation of PI3 Kinase by PDGFRbeta is essential for maintaining vascular integrity, and for the prevention of atherosclerosis as well as Marfan syndrome.

Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Huanchen; Falck, J.R.; Hall, Traci M. Tanaka

2012-01-11

Inositol pyrophosphates (such as IP7 and IP8) are multifunctional signaling molecules that regulate diverse cellular activities. Inositol pyrophosphates have 'high-energy' phosphoanhydride bonds, so their enzymatic synthesis requires that a substantial energy barrier to the transition state be overcome. Additionally, inositol pyrophosphate kinases can show stringent ligand specificity, despite the need to accommodate the steric bulk and intense electronegativity of nature's most concentrated three-dimensional array of phosphate groups. Here we examine how these catalytic challenges are met by describing the structure and reaction cycle of an inositol pyrophosphate kinase at the atomic level. We obtained crystal structures of the kinase domainmore » of human PPIP5K2 complexed with nucleotide cofactors and either substrates, product or a MgF{sub 3}{sup -} transition-state mimic. We describe the enzyme's conformational dynamics, its unprecedented topological presentation of nucleotide and inositol phosphate, and the charge balance that facilitates partly associative in-line phosphoryl transfer.« less

LPIAT1 regulates arachidonic acid content in phosphatidylinositol and is required for cortical lamination in mice

PubMed Central

Lee, Hyeon-Cheol; Inoue, Takao; Sasaki, Junko; Kubo, Takuya; Matsuda, Shinji; Nakasaki, Yasuko; Hattori, Mitsuharu; Tanaka, Fumiharu; Udagawa, Osamu; Kono, Nozomu; Itoh, Toshiki; Ogiso, Hideo; Taguchi, Ryo; Arita, Makoto; Sasaki, Takehiko; Arai, Hiroyuki

2012-01-01

Dietary arachidonic acid (AA) has roles in growth, neuronal development, and cognitive function in infants. AA is remarkably enriched in phosphatidylinositol (PI), an important constituent of biological membranes in mammals; however, the physiological significance of AA-containing PI remains unknown. In an RNA interference–based genetic screen using Caenorhabditis elegans, we recently cloned mboa-7 as an acyltransferase that selectively incorporates AA into PI. Here we show that lysophosphatidylinositol acyltransferase 1 (LPIAT1, also known as MBOAT7), the closest mammalian homologue, plays a crucial role in brain development in mice. Lpiat1−/− mice show almost no LPIAT activity with arachidonoyl-CoA as an acyl donor and show reduced AA contents in PI and PI phosphates. Lpiat1−/− mice die within a month and show atrophy of the cerebral cortex and hippocampus. Immunohistochemical analysis reveals disordered cortical lamination and delayed neuronal migration in the cortex of E18.5 Lpiat1−/− mice. LPIAT1 deficiency also causes disordered neuronal processes in the cortex and reduced neurite outgrowth in vitro. Taken together, these results demonstrate that AA-containing PI/PI phosphates play an important role in normal cortical lamination during brain development in mice. PMID:23097495

Phosphatidylinositol 3-Kinase: A Link Between Inflammation and Pancreatic Cancer

PubMed Central

Birtolo, Chiara; Go, Vay Liang W.; Ptasznik, Andrzej; Eibl, Guido; Pandol, Stephen J.

2016-01-01

Even though a strong association between inflammation and cancer has been widely accepted, the underlying precise molecular mechanisms are still largely unknown. A complex signaling network between tumor and stromal cells is responsible for the infiltration of inflammatory cells into the cancer micro-environment. Tumor stromal cells such as pancreatic stellate cells (PSCs) and immune cells create a microenvironment that protects cancer cells through a complex interaction, ultimately facilitating their local proliferation and their migration to different sites. Furthermore, PSCs have multiple functions related to local immunity, angiogenesis, inflammation and fibrosis. Recently, many studies have shown that members of the phosphoinositol-3-phosphate kinase (PI3K) family are activated in tumor cells, PSCs and tumor infiltrating inflammatory cells to promote cancer growth. Pro-inflammatory cytokines and chemokines secreted by immune cells and fibroblasts within the tumor environment can activate the PI3K pathway both in cancer and inflammatory cells. In this review, we focus on the central role of the PI3K pathway in regulating the cross-talk between immune/stromal cells and cancer cells. Understanding the role of the PI3K pathway in the development of chronic pancreatitis and cancer is crucial for the discovery of novel and efficacious treatment options. PMID:26658038

Ischemic postconditioning: from receptor to end-effector.

PubMed

Cohen, Michael V; Downey, James M

2011-03-01

Ischemic preconditioning, a robust cardioprotective intervention, has limited clinical efficacy because it must be initiated before myocardial ischemia. Conversely, ischemic postconditioning, repeated brief reocclusions of a coronary artery after release of prolonged coronary occlusion, provides cardioprotection in clinically feasible settings, that is, coronary angioplasty. Ischemic postconditioning's signaling is being investigated to identify pharmacological triggers that could be used without angioplasty. In initial minutes of reperfusion H(+) washes out of previously ischemic cells. pH rises enabling mitochondrial permeability transition pores (MPTPs) to form leading to cessation of ATP production and cell necrosis. Coronary reocclusions maintain sufficient acidosis to keep MPTP closed while signaling is initiated that can generate endogenous antagonists of MPTP formation even after cellular pH normalizes. Reintroduction of oxygen generates reactive oxygen species that activate protein kinase C to increase sensitivity of adenosine A(2b) receptors allowing adenosine released from ischemic cells to bind leading to activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2. Phosphatidylinositol 3-kinase activation results in phosphorylation of Akt promoting activation of nitric oxide synthase and nitric oxide production, which inhibits glycogen synthase kinase-3β, perhaps the final cytosolic signaling step before inhibition of MPTP formation. Interference with MPTP may be the final step that determines cell salvage.

An In Vitro TORC1 Kinase Assay That Recapitulates the Gtr-Independent Glutamine-Responsive TORC1 Activation Mechanism on Yeast Vacuoles

PubMed Central

Tanigawa, Mirai

2017-01-01

ABSTRACT Evolutionarily conserved target of rapamycin (TOR) complex 1 (TORC1) responds to nutrients, especially amino acids, to promote cell growth. In the yeast Saccharomyces cerevisiae, various nitrogen sources activate TORC1 with different efficiencies, although the mechanism remains elusive. Leucine, and perhaps other amino acids, was reported to activate TORC1 via the heterodimeric small GTPases Gtr1-Gtr2, the orthologues of the mammalian Rag GTPases. More recently, an alternative Gtr-independent TORC1 activation mechanism that may respond to glutamine was reported, although its molecular mechanism is not clear. In studying the nutrient-responsive TORC1 activation mechanism, the lack of an in vitro assay hinders associating particular nutrient compounds with the TORC1 activation status, whereas no in vitro assay that shows nutrient responsiveness has been reported. In this study, we have developed a new in vitro TORC1 kinase assay that reproduces, for the first time, the nutrient-responsive TORC1 activation. This in vitro TORC1 assay recapitulates the previously predicted Gtr-independent glutamine-responsive TORC1 activation mechanism. Using this system, we found that this mechanism specifically responds to l-glutamine, resides on the vacuolar membranes, and involves a previously uncharacterized Vps34-Vps15 phosphatidylinositol (PI) 3-kinase complex and the PI-3-phosphate [PI(3)P]-binding FYVE domain-containing vacuolar protein Pib2. Thus, this system was proved to be useful for dissecting the glutamine-responsive TORC1 activation mechanism. PMID:28483912

House dust mite-specific immunotherapy alters the basal expression of T regulatory and FcεRI pathway genes.

PubMed

Pevec, Branko; Radulovic Pevec, Mira; Stipic Markovic, Asja; Batista, Irena; Rijavec, Matija; Silar, Mira; Kosnik, Mitja; Korosec, Peter

2012-01-01

Regulatory T (Treg) cells and IgE-mediated signaling pathways could play important roles in the induction of allergen tolerance during house dust mite-specific subcutaneous immunotherapy (HDM-SCIT). Our aim was to compare the basal expression levels of Treg, T helper 1 (Th1) and Th2 transcription factors and components involved in IgE-mediated signaling in healthy subjects with those in HDM-allergic patients both untreated and successfully treated with HDM-SCIT. Thirty-nine HDM-allergic patients who completed a 3- to 5-year course of mite extract SCIT, 20 mite-allergic controls and 25 healthy controls participated in this study. The efficacy of SCIT was monitored using skin-prick tests (SPTs), total immunoglobulin E (tIgE), specific IgE (sIgE), sIgG(4), nasal challenge and visual analog scale (VAS) scores at several time points. The mRNA levels of forkhead box protein 3 (FOXP3), T-BET, GATA-3, FcεRI, spleen tyrosine kinase (Syk), phosphatidylinositol 3 kinase (PI3K) and SH2 domain-containing inositol phosphatase (SHIP) were quantified by real-time RT-PCR using nonstimulated whole blood samples. Decreased wheal sizes and VAS scores, negative challenges and increased sIgG(4) levels indicated that SCIT was effective in the treated patients. Basal expression levels of FOXP3 and GATA-3 decreased and T-BET levels increased in both treated patients and in healthy controls compared to untreated patients. The IgE-mediated pathway kinases Syk and PI3K exhibited reduced expression, whereas SHIP phosphatase levels were elevated in both treated patients and healthy controls relative to untreated patients. The expression levels of FcεRI were not significantly altered. Immunotherapy using HDM extracts results in a modification of the basal expression levels of several IgE-related signaling factors and induces a highly significant upregulation of Th1-response and downregulation of Th2-response transcription factors. Interestingly, this therapy also appears to reduce the basal expression of FOXP3. Copyright © 2012 S. Karger AG, Basel.

Determination of 6-thioguanosine diphosphate and triphosphate and nucleoside diphosphate kinase activity in erythrocytes: novel targets for thiopurine therapy?

PubMed

Karner, Susanne; Shi, Shaojun; Fischer, Christine; Schaeffeler, Elke; Neurath, Markus F; Herrlinger, Klaus R; Hofmann, Ute; Schwab, Matthias

2010-04-01

6-Thioguanine nucleotides are the sum of 6-thioguanosine 5'-monophosphate (TGMP), -diphosphate (TGDP), and -triphosphate (TGTP) representing essential metabolites involved in drug action of thiopurines. Elevated levels of TGDP have been associated with poor response to azathioprine therapy in patients with inflammatory bowel disease. The conversion of TGDP to TGTP is supposed to be catalyzed by nucleoside diphosphate kinase (NDPK). The aim of this work was to investigate simultaneously individual 6-thioguanosine phosphate levels and NDPK activity in red blood cells (RBCs) of patients on azathioprine therapy. Ion-pair high-performance liquid chromatography methods with fluorescence and ultraviolet detection were applied to quantify individual levels of 6-thioguanosine 5'-phosphates and NDPK activity, respectively, in RBCs. Recombinantly expressed NDPK isoforms A and B were unequivocally identified to catalyze the formation of TGTP (30.6 +/- 3.88 nmol x min x mg for NDPK A versus 41.2 +/- 1.05 nmol x min x mg for NDPK B). Comprehensive analyses on the stability of TGMP, TGDP, and TGTP and the reproducibility of NDPK activity in RBCs were performed to provide a reliable sampling protocol for clinical practice. Of note, isolation of RBCs within 6 hours followed by immediate storage at -80 degrees C is crucial for prevention of degradation of 5'-phosphates. In a clinical study of 37 patients on azathioprine, TGTP was the predominant 6-thioguanosine phosphate in RBCs. In contrast, three patients showed TGTP/(TGDP + TGTP) ratios of 57.2%, 64.3%, and 66% corresponding to elevated TGDP levels. NDPK activity ranged from 4.1 to 11.3 nmol x min x mg hemoglobin. No correlation between NDPK activity and the 6-thioguanosine phosphate levels was found. The question whether interindividual variability of NDPK activity may explain differences in 6-thioguanosine 5'-phosphates levels has to be investigated in a prospective large-scale study.

Oleanolic acid supplement attenuates liquid fructose-induced adipose tissue insulin resistance through the insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt signaling pathway in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Ying; Wang, Jianwei, E-mail: wangjianwei1968@gmail.com; Gu, Tieguang

Oleanolic acid, a triterpenoid contained in more than 1620 plants including various fruits and foodstuffs, has numerous metabolic effects, such as hepatoprotection. However, its underlying mechanisms remain poorly understood. Adipose tissue insulin resistance (Adipo-IR) may contribute to the development and progress of metabolic abnormalities through release of excessive free fatty acids from adipose tissue. This study investigated the effect of oleanolic acid on Adipo-IR. The results showed that supplement with oleanolic acid (25 mg/kg, once daily, by oral gavage) over 10 weeks attenuated liquid fructose-induced increase in plasma insulin concentration and the homeostasis model assessment of insulin resistance (HOMA-IR) indexmore » in rats. Simultaneously, oleanolic acid reversed the increase in the Adipo-IR index and plasma non-esterified fatty acid concentrations during the oral glucose tolerance test assessment. In white adipose tissue, oleanolic acid enhanced mRNA expression of the genes encoding insulin receptor, insulin receptor substrate (IRS)-1 and phosphatidylinositol 3-kinase. At the protein level, oleanolic acid upregulated total IRS-1 expression, suppressed the increased phosphorylated IRS-1 at serine-307, and restored the increased phosphorylated IRS-1 to total IRS-1 ratio. In contrast, phosphorylated Akt to total Akt ratio was increased. Furthermore, oleanolic acid reversed fructose-induced decrease in phosphorylated-Akt/Akt protein to plasma insulin concentration ratio. However, oleanolic acid did not affect IRS-2 mRNA expression. Therefore, these results suggest that oleanolic acid supplement ameliorates fructose-induced Adipo-IR in rats via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway. Our findings may provide new insights into the mechanisms of metabolic actions of oleanolic acid. - Highlights: • Adipose insulin resistance (Adipo-IR) contributes to metabolic abnormalities. • We investigated the effect of oleanolic acid (OA) on adipo-IR in fructose-fed rats. • OA attenuated fructose-induced increase in Adipo-IR index and NEFA concentrations. • OA modulated adipose IRS-1/phosphatidylinositol 3-kinase/Akt signaling. • OA ameliorates Adipo-IR via the IRS-1/PI3K/Akt signaling pathway in rats.« less

Pib2 and the EGO complex are both required for activation of TORC1.

PubMed

Varlakhanova, Natalia V; Mihalevic, Michael J; Bernstein, Kara A; Ford, Marijn G J

2017-11-15

The TORC1 complex is a key regulator of cell growth and metabolism in Saccharomyces cerevisiae The vacuole-associated EGO complex couples activation of TORC1 to the availability of amino acids, specifically glutamine and leucine. The EGO complex is also essential for reactivation of TORC1 following rapamycin-induced growth arrest and for its distribution on the vacuolar membrane. Pib2, a FYVE-containing phosphatidylinositol 3-phosphate (PI3P)-binding protein, is a newly discovered and poorly characterized activator of TORC1. Here, we show that Pib2 is required for reactivation of TORC1 following rapamycin-induced growth arrest. Pib2 is required for EGO complex-mediated activation of TORC1 by glutamine and leucine as well as for redistribution of Tor1 on the vacuolar membrane. Therefore, Pib2 and the EGO complex cooperate to activate TORC1 and connect phosphoinositide 3-kinase (PI3K) signaling and TORC1 activity. © 2017. Published by The Company of Biologists Ltd.

Viral Membrane Fusion and Nucleocapsid Delivery into the Cytoplasm are Distinct Events in Some Flaviviruses

PubMed Central

Nour, Adel M.; Li, Yue; Wolenski, Joseph; Modis, Yorgo

2013-01-01

Flaviviruses deliver their genome into the cell by fusing the viral lipid membrane to an endosomal membrane. The sequence and kinetics of the steps required for nucleocapsid delivery into the cytoplasm remain unclear. Here we dissect the cell entry pathway of virions and virus-like particles from two flaviviruses using single-particle tracking in live cells, a biochemical membrane fusion assay and virus infectivity assays. We show that the virus particles fuse with a small endosomal compartment in which the nucleocapsid remains trapped for several minutes. Endosomal maturation inhibitors inhibit infectivity but not membrane fusion. We propose a flavivirus cell entry mechanism in which the virus particles fuse preferentially with small endosomal carrier vesicles and depend on back-fusion of the vesicles with the late endosomal membrane to deliver the nucleocapsid into the cytoplasm. Virus entry modulates intracellular calcium release and phosphatidylinositol-3-phosphate kinase signaling. Moreover, the broadly cross-reactive therapeutic antibody scFv11 binds to virus-like particles and inhibits fusion. PMID:24039574

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation

PubMed Central

Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E. M.; Jenkins, Jermaine L.; Heimiller, Chelsea; Maines, Mahin D.

2016-01-01

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1–3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T308 before S473 autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S473 independent of hBVR’s kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S473 independent of hBVR’s kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S230 in hBVR 225RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR’s PDK1 binding 161RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.—Miralem, T., Lerner-Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation. PMID:27166089

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

PubMed

Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E M; Jenkins, Jermaine L; Heimiller, Chelsea; Maines, Mahin D

2016-08-01

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner-Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation. © FASEB.

Suppression of transforming growth factor-beta-induced apoptosis through a phosphatidylinositol 3-kinase/Akt-dependent pathway.

PubMed

Chen, R H; Su, Y H; Chuang, R L; Chang, T Y

1998-10-15

Insulin and insulin receptor substrate 1 (IRS-1) are capable of protecting liver cells from apoptosis induced by transforming growth factor-beta1 (TGF-beta). The Ras/mitogen-activated protein kinase (MAP kinase) and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways are both activated upon insulin stimulation and can protect against apoptosis under certain circumstances. We investigated which of these pathways is responsible for the protective effect of insulin on TGF-beta-induced apoptosis. An activated Ras, although elicited a strong mitogenic effect, could not protect Hep3B cells from TGF-beta-induced apoptosis. Furthermore, PD98059, a selective inhibitor of MEK, did not suppress the antiapoptotic effect of insulin. In contrast, the PI 3-kinase inhibitor, LY294002, efficiently blocked the effect of insulin. Protection against TGF-beta-induced apoptosis conferred by PI 3-kinase was further verified by stable transfection of an activated PI 3-kinase. Downstream targets of PI 3-kinase involved in this protection was further investigated. An activated Akt mimicked the antiapoptotic effect of insulin, whereas a dominant-negative Akt inhibited such effect. However, rapamycin, the p70S6 kinase inhibitor, had no effect on the protectivity of insulin against TGF-beta-induced apoptosis, suggesting that the antiapoptotic target of PI 3-kinase/Akt pathway is independent or lies upstream of the p70S6 kinase. The mechanism by which PI 3-kinase/Akt pathway interferes with the apoptotic signaling of TGF-beta was explored. Activation of PI 3-kinase did not lead to a suppression of Smad hetero-oligomerization or nuclear translocation but blocked TGF-beta-induced caspase-3-like activity. In summary, the PI 3-kinase/Akt pathway, but not the Ras/MAP kinase pathway, protects against TGF-beta-induced apoptosis by inhibiting a step downstream of Smad but upstream of caspase-3.

Leptin modulates the expression of catabolic genes in rat nucleus pulposus cells through the mitogen-activated protein kinase and Janus kinase 2/signal transducer and activator of transcription 3 pathways.

PubMed

Miao, Daoyi; Zhang, Lingzhou

2015-08-01

Obesity has been demonstrated to be involved in the progress of intervertebral disc degeneration (IDD). However, the associated mechanisms remain to be elucidated. The purpose the present study was to examine the effect of leptin on the expression of degeneration-associated genes in rat nucleus pulposus (NP) cells, and determine the possible mechanism. Normal NP cells, obtained from Sprague Dawley rats, were identified using immunocytochemistry for the expression of collagen II and CA125, and treated with leptin and/or interleukin (IL)-β. Subsequently, the mRNA expression levels of matrix metalloproteinase (MMP)-1, MMP-3, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5, aggrecan and COL2A1 were detected by reverse transcription-quantitative polymerase chain reaction (RT-q-PCR). Alcian staining and immunocytochemistry were used to examine the expression levels of proteoglycan and collagen II. The pathway activation was investigated using western blotting, and inhibitors of the pathways were used to reveal the effect of these pathways on the NP cells. The results of the RT-qPCR demonstrated that leptin alone upregulated the mRNA expression levels of MMP-1, MMP-13, ADAMTS-4, ADAMTS-5 and COL2A1. Synergy of leptin and IL-β was found in the increased expression levels of MMP-1, MMP-3 and ADAMTS-5. The leptin-treated NP cells exhibited decreased expression of collagen II. The mitrogen-activated protein kinase (MAPK) pathway (c-Jun-N-terminal kinase, phosphorylated extracellular signal-regulated kinase and p38), phosphatidylinositol 3-kinase (PI3K)/Akt pathway and Janus kinase (JAK)2/signal transducer and activator of transcription 3 pathway were all activated by leptin, however, inhibitors of all the pathways, with the exception of the PI3K/Akt pathway, reversed the expression levels of MMP-1 and MMP-13. These results suggested that leptin promoted catabolic metabolism in the rat NP cells via the MAPK and JAK2/STAT3 pathways, which may be the mechanism mediating the association between obesity and IDD.

Leptin modulates the expression of catabolic genes in rat nucleus pulposus cells through the mitogen-activated protein kinase and Janus kinase 2/signal transducer and activator of transcription 3 pathways

PubMed Central

MIAO, DAOYI; ZHANG, LINGZHOU

2015-01-01

Obesity has been demonstrated to be involved in the progress of intervertebral disc degeneration (IDD). However, the associated mechanisms remain to be elucidated. The purpose the present study was to examine the effect of leptin on the expression of degeneration-associated genes in rat nucleus pulposus (NP) cells, and determine the possible mechanism. Normal NP cells, obtained from Sprague Dawley rats, were identified using immunocytochemistry for the expression of collagen II and CA125, and treated with leptin and/or interleukin (IL)-β. Subsequently, the mRNA expression levels of matrix metalloproteinase (MMP)-1, MMP-3, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5, aggrecan and COL2A1 were detected by reverse transcription-quantitative polymerase chain reaction (RT-q-PCR). Alcian staining and immunocytochemistry were used to examine the expression levels of proteoglycan and collagen II. The pathway activation was investigated using western blotting, and inhibitors of the pathways were used to reveal the effect of these pathways on the NP cells. The results of the RT-qPCR demonstrated that leptin alone upregulated the mRNA expression levels of MMP-1, MMP-13, ADAMTS-4, ADAMTS-5 and COL2A1. Synergy of leptin and IL-β was found in the increased expression levels of MMP-1, MMP-3 and ADAMTS-5. The leptin-treated NP cells exhibited decreased expression of collagen II. The mitrogen-activated protein kinase (MAPK) pathway (c-Jun-N-terminal kinase, phosphorylated extracellular signal-regulated kinase and p38), phosphatidylinositol 3-kinase (PI3K)/Akt pathway and Janus kinase (JAK)2/signal transducer and activator of transcription 3 pathway were all activated by leptin, however, inhibitors of all the pathways, with the exception of the PI3K/Akt pathway, reversed the expression levels of MMP-1 and MMP-13. These results suggested that leptin promoted catabolic metabolism in the rat NP cells via the MAPK and JAK2/STAT3 pathways, which may be the mechanism mediating the association between obesity and IDD. PMID:25892402

Structural basis for interdomain communication in SHIP2 providing high phosphatase activity

PubMed Central

Le Coq, Johanne; Camacho-Artacho, Marta; Velázquez, José Vicente; Santiveri, Clara M; Gallego, Luis Heredia; Campos-Olivas, Ramón; Dölker, Nicole; Lietha, Daniel

2017-01-01

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P3. Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions. DOI: http://dx.doi.org/10.7554/eLife.26640.001 PMID:28792888

Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors.

PubMed

Blake, James F; Xu, Rui; Bencsik, Josef R; Xiao, Dengming; Kallan, Nicholas C; Schlachter, Stephen; Mitchell, Ian S; Spencer, Keith L; Banka, Anna L; Wallace, Eli M; Gloor, Susan L; Martinson, Matthew; Woessner, Richard D; Vigers, Guy P A; Brandhuber, Barbara J; Liang, Jun; Safina, Brian S; Li, Jun; Zhang, Birong; Chabot, Christine; Do, Steven; Lee, Leslie; Oeh, Jason; Sampath, Deepak; Lee, Brian B; Lin, Kui; Liederer, Bianca M; Skelton, Nicholas J

2012-09-27

The discovery and optimization of a series of 6,7-dihydro-5H-cyclopenta[d]pyrimidine compounds that are ATP-competitive, selective inhibitors of protein kinase B/Akt is reported. The initial design and optimization was guided by the use of X-ray structures of inhibitors in complex with Akt1 and the closely related protein kinase A. The resulting compounds demonstrate potent inhibition of all three Akt isoforms in biochemical assays and poor inhibition of other members of the cAMP-dependent protein kinase/protein kinase G/protein kinase C extended family and block the phosphorylation of multiple downstream targets of Akt in human cancer cell lines. Biological studies with one such compound, 28 (GDC-0068), demonstrate good oral exposure resulting in dose-dependent pharmacodynamic effects on downstream biomarkers and a robust antitumor response in xenograft models in which the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway is activated. 28 is currently being evaluated in human clinical trials for the treatment of cancer.

Modulatory effects of bombyxin on ecdysteroidogenesis in Bombyx mori prothoracic glands.

PubMed

Gu, Shi-Hong; Chen, Chien-Hung; Hsieh, Yun-Chin; Lin, Pei-Ling; Young, Shun-Chieh

2015-01-01

In the present study, we investigated the modulatory effects of ecdysteroidogenesis of prothoracic glands (PGs) by bombyxin, an endogenous insulin-like peptide in the silkworm, Bombyx mori. The results showed that bombyxin stimulated ecdysteroidogenesis during a long-term incubation period and in a dose-dependent manner. Moreover, the injection of bombyxin into day 4-last instar larvae increased ecdysteroidogenesis 24h after the injection, indicating its possible in vivo function. Phosphorylation of the insulin receptor and Akt, and the target of rapamycin (TOR) signaling were stimulated by bombyxin, and stimulation of Akt phosphorylation and TOR signaling appeared to be dependent on phosphatidylinositol 3-kinase (PI3K). Bombyxin inhibited the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK), and the inhibition appeared to be PI3K-independent. Bombyxin-stimulated ecdysteroidogenesis was blocked by either an inhibitor of PI3K (LY294002) or a chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, AICAR), indicating involvement of the PI3K/Akt and AMPK signaling pathway. Bombyxin did not stimulate extracellular signal-regulated kinase (ERK) signaling of PGs. Bombyxin, but not prothoracicotropic hormone (PTTH) stimulated cell viability of PGs. In addition, bombyxin treatment also affected mRNA expression levels of insulin receptor, Akt, AMPKα, -β, and -γ in time-dependent manners. These results suggest that bombyxin modulates ecdysteroidogenesis in B. mori PGs during development. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rho-kinase inhibitor and nicotinamide adenine dinucleotide phosphate oxidase inhibitor prevent impairment of endothelium-dependent cerebral vasodilation by acute cigarette smoking in rats.

PubMed

Iida, Hiroki; Iida, Mami; Takenaka, Motoyasu; Fukuoka, Naokazu; Dohi, Shuji

2008-06-01

We previously reported that acute cigarette smoking can cause a dysfunction of endothelium-dependent vasodilation in cerebral vessels, and that blocking the angiotensin II (Ang II) type 1 (AT1) receptor with valsartan prevented this impairment. Our aim was to investigate the effects of a Rho-kinase inhibitor (fasudil) and a Nicotinamide Adenine Dinucleotide PHosphate (NADPH) oxidase inhibitor (apocynin) on smoking-induced endothelial dysfunction in cerebral arterioles. In Sprague-Dawley rats, we used a closed cranial window preparation to measure changes in pial vessel diameters following topical acetylcholine (ACh) before smoking. After one-minute smoking, we again examined the arteriolar responses to ACh. Finally, after intravenous fasudil or apocynin pre-treatment we re-examined the vasodilator responses to topical ACh (before and after cigarette smoking). Under control conditions, cerebral arterioles were dose-dependently dilated by topical ACh (10(-6) M and 10(-5) M). One hour after a one-minute smoking (1 mg-nicotine cigarette), 10(-5) M ACh constricted cerebral arterioles. However, one hour after a one-minute smoking, 10(-5) M ACh dilated cerebral pial arteries both in the fasudil pre-treatment and the apocynin pre-treatment groups, responses that were significantly different from those obtained without fasudil or apocynin pre-treatment. Thus, inhibition of Rho-kinase and NADPH oxidase activities may prevent the above smoking-induced impairment of endothelium-dependent vasodilation.

Cooperation between STAT5 and phosphatidylinositol 3-kinase in the IL-3-dependent survival of a bone marrow derived cell line.

PubMed

Rosa Santos, S C; Dumon, S; Mayeux, P; Gisselbrecht, S; Gouilleux, F

2000-02-24

Cytokine-dependent activation of distinct signaling pathways is a common scheme thought to be required for the subsequent programmation into cell proliferation and survival. The PI 3-kinase/Akt, Ras/MAP kinase, Ras/NFIL3 and JAK/STAT pathways have been shown to participate in cytokine mediated suppression of apoptosis in various cell types. However the relative importance of these signaling pathways seems to depend on the cellular context. In several cases, individual inhibition of each pathway is not sufficient to completely abrogate cytokine mediated cell survival suggesting that cooperation between these pathways is required. Here we showed that individual inhibition of STAT5, PI 3-kinase or MEK activities did not or weakly affected the IL-3 dependent survival of the bone marrow derived Ba/F3 cell line. However, the simultaneous inhibition of STAT5 and PI 3-kinase activities but not that of STAT5 and MEK reduced the IL-3 dependent survival of Ba/F3. Analysis of the expression of the Bcl-2 members indicated that phosphorylation of Bad and Bcl-x expression which are respectively regulated by the PI 3-kinase/Akt pathway and STAT5 probably explain this cooperation. Furthermore, we showed by co-immunoprecipitation studies and pull down experiments with fusion proteins encoding the GST-SH2 domains of p85 that STAT5 in its phosphorylated form interacts with the p85 subunit of the PI 3-kinase. These results indicate that the activations of STAT5 and the PI 3-kinase by IL-3 in Ba/F3 cells are tightly connected and cooperate to mediate IL-3-dependent suppression of apoptosis by modulating Bad phosphorylation and Bcl-x expression.

High concentrations of landiolol, a beta(1)-adrenoceptor antagonist, stimulate smooth muscle contraction of the rat trachea through the Rho-kinase pathway.

PubMed

Shibata, Osamu; Nishioka, Kenji; Yamaguchi, Masakazu; Makita, Tetsuji; Sumikawa, Koji

2008-01-01

Gradually progressing contraction of airway smooth muscle is suggested to be due to the Rho-kinase signaling pathway. In our preliminary study in rat tracheas, landiolol, a beta(1)-adrenoceptor antagonist, at high doses caused gradually progressing contraction, and this contraction reached a plateau after 20 min. Therefore, this study was carried out to clarify whether landiolol could stimulate the Rho-kinase pathway or the phosphatidylinositol (PI) response in the rat trachea. Seventy-eight male Wistar rats weighing 250-350 g were used for the experiments. Their tracheas were cut into 3-mm-wide ring segments or 1-mm-wide slices. Measurements of isometric tension and [(3)H] inositol monophosphate (IP(1)) production were conducted, using these tracheal rings or slices. Data values are expressed as means +/- SD, and statistical significance (P < 0.05) was determined using analysis of variance (ANOVA). Landiolol (700 microM)-induced contraction was completely inhibited by fasudil at 30 microM, while the landiolol-induced contraction was not inhibited by 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP), ketanserin, or nicardipine. Landiolol did not stimulate IP(1) production. These results suggest that high concentrations of landiolol could cause airway smooth muscle contraction through the Rho-kinase pathway, but not through the PI response coupled with muscarinic M(3) receptors, 5-HT receptors or the activation of L-type Ca(2+) channels.

Sitagliptin decreases ventricular arrhythmias by attenuated glucose-dependent insulinotropic polypeptide (GIP)-dependent resistin signalling in infarcted rats.

PubMed

Lee, Tsung-Ming; Chen, Wei-Ting; Chang, Nen-Chung

2016-01-25

Myocardial infarction (MI) was associated with insulin resistance, in which resistin acts as a critical mediator. We aimed to determine whether sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, can attenuate arrhythmias by regulating resistin-dependent nerve growth factor (NGF) expression in postinfarcted rats. Normoglycaemic male Wistar rats after ligating coronary artery were randomized to either vehicle or sitagliptin for 4 weeks starting 24 h after operation. Post-infarction was associated with increased myocardial noradrenaline [norepinephrine (NE)] levels and sympathetic hyperinnervation. Compared with vehicle, sympathetic innervation was blunted after administering sitagliptin, as assessed by immunofluorescent analysis of tyrosine hydroxylase, growth-associated factor 43 and neurofilament and western blotting and real-time quantitative RT-PCR of NGF. Arrhythmic scores in the sitagliptin-treated infarcted rats were significantly lower than those in vehicle. Furthermore, sitagliptin was associated with reduced resistin expression and increased Akt activity. Ex vivo studies showed that glucose-dependent insulinotropic polypeptide (GIP) infusion, but not glucagon-like peptide-1 (GLP-1), produced similar reduction in resistin levels to sitagliptin in postinfarcted rats. Furthermore, the attenuated effects of sitagliptin on NGF levels can be reversed by wortmannin (a phosphatidylinositol 3-kinase antagonist) and exogenous resistin infusion. Sitagliptin protects ventricular arrhythmias by attenuating sympathetic innervation in the non-diabetic infarcted rats. Sitagliptin attenuated resistin expression via the GIP-dependent pathway, which inhibited sympathetic innervation through a signalling pathway involving phosphatidylinositol 3-kinase (PI3K) and Akt protein. © 2016 Authors.

Both Creatine and Its Product Phosphocreatine Reduce Oxidative Stress and Afford Neuroprotection in an In Vitro Parkinson’s Model

PubMed Central

Martín-de-Saavedra, Maria D.; Romero, Alejandro; Egea, Javier; Ludka, Fabiana K.; Tasca, Carla I.; Farina, Marcelo; Rodrigues, Ana Lúcia S.; López, Manuela G.

2014-01-01

Creatine is the substrate for creatine kinase in the synthesis of phosphocreatine (PCr). This energetic system is endowed of antioxidant and neuroprotective properties and plays a pivotal role in brain energy homeostasis. The purpose of this study was to investigate the neuroprotective effect of creatine and PCr against 6-hydroxydopamine (6-OHDA)-induced mitochondrial dysfunction and cell death in rat striatal slices, used as an in vitro Parkinson’s model. The possible involvement of the signaling pathway mediated by phosphatidylinositol-3 kinase (PI3K), protein kinase B (Akt), and glycogen synthase kinase-3β (GSK3β) was also evaluated. Exposure of striatal slices to 6-OHDA caused a significant disruption of the cellular homeostasis measured as 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction, lactate dehydrogenase release, and tyrosine hydroxylase levels. 6-OHDA exposure increased the levels of reactive oxygen species and thiobarbituric acid reactive substances production and decreased mitochondrial membrane potential in rat striatal slices. Furthermore, 6-OHDA decreased the phosphorylation of Akt (Serine473) and GSK3β (Serine9). Coincubation with 6-OHDA and creatine or PCr reduced the effects of 6-OHDA toxicity. The protective effect afforded by creatine or PCr against 6-OHDA-induced toxicity was reversed by the PI3K inhibitor LY294002. In conclusion, creatine and PCr minimize oxidative stress in striatum to afford neuroprotection of dopaminergic neurons. PMID:25424428

Autophagic kinases SmVPS34 and SmVPS15 are required for viability in the filamentous ascomycete Sordaria macrospora.

PubMed

Voigt, Oliver; Herzog, Britta; Jakobshagen, Antonia; Pöggeler, Stefanie

2014-01-01

Autophagy is a tightly controlled degradation process of all eukaryotes. It includes the sequestration of cytoplasmic contents and organelles within a double-membraned autophagosome. Autophagy involves core autophagy related (atg) genes as well as genes regulating vesicle trafficking. Previously, we analyzed the impact of proteins of the core autophagic machinery SmATG7, SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. While deletion of Smatg8 and Smatg4 abolished fruiting-body formation and impaired vegetative growth, Smatg7 is required for viability. In yeast, the phosphatidylinositol 3-kinase vacuolar protein sorting 34 (Vps34) and its myristoylated membrane targeting unit, the protein kinase Vps15 have been shown to be important regulators of autophagy and vacuolar protein sorting. However, their exact role in filamentous ascomycetes remains elusive. To determine the function of Smvps34 and Smvps15 we isolated genes with high sequence similarity to Saccharomyces cerevisiae VPS34 and VPS15. For both genes we were not able to generate a homokaryotic knockout mutant in S. macrospora, suggesting that Smvps34 and Smvps15 are required for viability. Furthermore, we analyzed the repertoire of vps genes encoded by S. macrospora and could identify putative homologs of nearly all of the 61 VPS genes of S. cerevisiae. Copyright © 2013 Elsevier GmbH. All rights reserved.

Scoparone attenuates RANKL-induced osteoclastic differentiation through controlling reactive oxygen species production and scavenging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Sang-Hyun; Jang, Hae-Dong, E-mail: haedong@hnu.kr

Scoparone, one of the bioactive components of Artemisia capillaris Thunb, has various biological properties including immunosuppressive, hepatoprotective, anti-allergic, anti-inflammatory, and antioxidant effects. This study aims at evaluating the anti-osteoporotic effect of scoparone and its underlying mechanism in vitro. Scoparone demonstrated potent cellular antioxidant capacity. It was also found that scoparone inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and suppressed cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression via c-jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)/p38-mediated c-Fos–nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway. During osteoclast differentiation, the production of general reactive oxygen speciesmore » (ROS) and superoxide anions was dose-dependently attenuated by scoparone. In addition, scoparone diminished NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) expression and activation via the tumor necrosis factor receptor-associated factor 6 (TRAF6)–cSrc–phosphatidylinositol 3-kinase (PI3k) signaling pathway and prevented the disruption of mitochondrial electron transport chain system. Furthermore, scoparone augmented the expression of superoxide dismutase 1 (SOD1) and catalase (CAT). The overall results indicate that the inhibitory effect of scoparone on RANKL-induced osteoclast differentiation is attributed to the suppressive effect on ROS and superoxide anion production by inhibiting Nox1 expression and activation and protecting the mitochondrial electron transport chain system and the scavenging effect of ROS resulting from elevated SOD1 and CAT expression. - Highlights: • Scoparone dose-dependently inhibited RANKL-induced osteoclast differentiation. • Scoparone diminished general ROS and superoxide anions in a dose-dependent manner. • Scoparone inhibited Nox1 expression and activation. • Scoparone prevented the disruption of mitochondrial electron transport chain system. • Scoparone augmented superoxide dismutase and catalase expression.« less

Small G proteins in insulin action: Rab and Rho families at the crossroads of signal transduction and GLUT4 vesicle traffic.

PubMed

Ishikura, S; Koshkina, A; Klip, A

2008-01-01

Insulin stimulates glucose uptake into muscle and adipose tissues through glucose transporter 4 (GLUT4). GLUT4 cycles between the intracellular compartments and the plasma membrane. GLUT4 traffic-regulating insulin signals are largely within the insulin receptor-insulin receptor substrate-phosphatidylinositol 3-kinase (IR-IRS-PI3K) axis. In muscle cells, insulin signal bifurcates downstream of the PI3K into one arm leading to the activation of the Ser/Thr kinases Akt and atypical protein kinase C, and another leading to the activation of Rho family protein Rac1 leading to actin remodelling. Activated Akt inactivates AS160, a GTPase-activating protein for Rab family small G proteins. Here we review the roles of Rab and Rho proteins, particularly Rab substrates of AS160 and Rac1, in insulin-stimulated GLUT4 traffic. We discuss: (1) how distinct steps in GLUT4 traffic may be regulated by discrete Rab proteins, and (2) the importance of Rac1 activation in insulin-induced actin remodelling in muscle cells, a key element for the net gain in surface GLUT4.

Mutant JAK3 phosphoproteomic profiling predicts synergism between JAK3 inhibitors and MEK/BCL2 inhibitors for the treatment of T-cell acute lymphoblastic leukemia

PubMed Central

Degryse, S; de Bock, C E; Demeyer, S; Govaerts, I; Bornschein, S; Verbeke, D; Jacobs, K; Binos, S; Skerrett-Byrne, D A; Murray, H C; Verrills, N M; Van Vlierberghe, P; Cools, J; Dun, M D

2018-01-01

Mutations in the interleukin-7 receptor (IL7R) or the Janus kinase 3 (JAK3) kinase occur frequently in T-cell acute lymphoblastic leukemia (T-ALL) and both are able to drive cellular transformation and the development of T-ALL in mouse models. However, the signal transduction pathways downstream of JAK3 mutations remain poorly characterized. Here we describe the phosphoproteome downstream of the JAK3(L857Q)/(M511I) activating mutations in transformed Ba/F3 lymphocyte cells. Signaling pathways regulated by JAK3 mutants were assessed following acute inhibition of JAK1/JAK3 using the JAK kinase inhibitors ruxolitinib or tofacitinib. Comprehensive network interrogation using the phosphoproteomic signatures identified significant changes in pathways regulating cell cycle, translation initiation, mitogen-activated protein kinase and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signaling, RNA metabolism, as well as epigenetic and apoptotic processes. Key regulatory proteins within pathways that showed altered phosphorylation following JAK inhibition were targeted using selumetinib and trametinib (MEK), buparlisib (PI3K) and ABT-199 (BCL2), and found to be synergistic in combination with JAK kinase inhibitors in primary T-ALL samples harboring JAK3 mutations. These data provide the first detailed molecular characterization of the downstream signaling pathways regulated by JAK3 mutations and provide further understanding into the oncogenic processes regulated by constitutive kinase activation aiding in the development of improved combinatorial treatment regimens. PMID:28852199

The cytomegalovirus homolog of interleukin-10 requires phosphatidylinositol 3-kinase activity for inhibition of cytokine synthesis in monocytes.

PubMed

Spencer, Juliet V

2007-02-01

Human cytomegalovirus (CMV) has evolved numerous strategies for evading host immune defenses, including piracy of cellular cytokines. A viral homolog of interleukin-10, designated cmvIL-10, binds to the cellular IL-10 receptor and effects potent immune suppression. The signaling pathways employed by cmvIL-10 were investigated, and the classic IL-10R/JAK1/Stat3 pathway was found to be activated in monocytes. However, inhibition of JAK1 had little effect on cmvIL-10-mediated suppression of tumor necrosis factor alpha (TNF-alpha) production. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway had a more significant impact on TNF-alpha levels but did not completely relieve the immune suppression, demonstrating that cmvIL-10 stimulates multiple signaling pathways to modulate cell function.

Over-expression of Rififylin, a new RING finger and FYVE-like domain-containing protein, inhibits recycling from the endocytic recycling compartment.

PubMed

Coumailleau, Franck; Das, Vincent; Alcover, Andres; Raposo, Graça; Vandormael-Pournin, Sandrine; Le Bras, Stéphanie; Baldacci, Patricia; Dautry-Varsat, Alice; Babinet, Charles; Cohen-Tannoudji, Michel

2004-10-01

Endocytosed membrane components are recycled to the cell surface either directly from early/sorting endosomes or after going through the endocytic recycling compartment (ERC). Studying recycling mechanisms is difficult, in part due to the fact that specific tools to inhibit this process are scarce. In this study, we have characterized a novel widely expressed protein, named Rififylin (Rffl) for RING Finger and FYVE-like domain-containing protein, that, when overexpressed in HeLa cells, induced the condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region. Internalized transferrin was able to access these condensed endosomes but its exit from this compartment was delayed. Using deletion mutants, we show that the carboxy-terminal RING finger of Rffl is dispensable for its action. In contrast, the amino-terminal domain of Rffl, which shows similarities with the phosphatidylinositol-3-phosphate-binding FYVE finger, is critical for the recruitment of Rffl to recycling endocytic membranes and for the inhibition of recycling, albeit in a manner that is independent of PtdIns(3)-kinase activity. Rffl overexpression represents a novel means to inhibit recycling that will help to understand the mechanisms involved in recycling from the ERC to the plasma membrane.

A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling.

PubMed

Denis, Deborah; Rouleau, Cecile; Schaffhausen, Brian S

2017-01-15

Middle T antigen (MT), the principal oncoprotein of murine polyomavirus, transforms by association with cellular proteins. Protein phosphatase 2A (PP2A), YAP, Src family tyrosine kinases, Shc, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLCγ1) have all been implicated in MT transformation. Mutant dl1015, with deletion of residues 338 to 347 in the C-terminal region, has been an enigma, because the basis for its transformation defect has not been apparent. This work probes the dl1015 region of MT. Because the region is proline rich, the hypothesis that it targets Src homology domain 3 (SH3) domains was tested, but mutation of the putative SH3 binding motif did not affect transformation. During this work, two point mutants, W348R and E349K, were identified as transformation defective. Extensive analysis of the E349K mutant is described here. Similar to wild-type MT, the E349K mutant associates with PP2A, YAP, tyrosine kinases, Shc, PI3 kinase, and PLCγ1. The E349K mutant was examined to determine the mechanism for its transformation defect. Assays of cell localization and membrane targeting showed no obvious difference in localization. Src association was normal as assayed by in vitro kinase and MT phosphopeptide mapping. Shc activation was confirmed by its tyrosine phosphorylation. Association of type 1 PI3K with MT was demonstrated by coimmunoprecipitation, showing both PI3K subunits and in vitro activity. Nonetheless, expression of the mutants failed to lead to the activation of two known downstream targets of PI3K, Akt and Rac-1. Strikingly, despite normal association of the E349K mutant with PI3K, cells expressing the mutant failed to elevate phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in mutant-expressing cells. These results indicate a novel unsuspected aspect to PI3K control. The gene coding for middle T antigen (MT) is the murine polyomavirus oncogene most responsible for tumor formation. Its study has a history of uncovering novel aspects of mammalian cell regulation. The importance of PI3K activity and tyrosine phosphorylation are two examples of insights coming from MT. This study describes new mutants unable to transform like the wild type that point to novel regulation of PI3K signaling. Previous mutants were defective in PI3K because they failed to bind the enzyme and bring the activity to the membrane. These mutants recruit PI3K activity like the wild type, but fail to elevate the cellular level of PIP3, the product used to signal downstream of PI3K. As a result, they fail to activate either Akt or Rac1, explaining the transformation defect. Copyright © 2017 American Society for Microbiology.

The Phospholipase C Isozymes and Their Regulation

PubMed Central

Gresset, Aurelie; Sondek, John

2013-01-01

The physiological effects of many extracellular neurotransmitters, hormones, growth factors, and other stimuli are mediated by receptor-promoted activation of phospholipase C (PLC) and consequential activation of inositol lipid signaling pathways. These signaling responses include the classically described conversion of phosphatidylinositol(4,5)P2 to the Ca2+-mobilizing second messenger inositol(1,4,5)P3 and the protein kinase C-activating second messenger diacylglycerol as well as alterations in membrane association or activity of many proteins that harbor phosphoinositide binding domains. The 13 mammalian PLCs elaborate a minimal catalytic core typified by PLC-δ to confer multiple modes of regulation of lipase activity. PLC-β isozymes are activated by Gαq- and Gβγ-subunits of heterotrimeric G proteins, and activation of PLC-γ isozymes occurs through phosphorylation promoted by receptor and non-receptor tyrosine kinases. PLC-ε and certain members of the PLC-β and PLC-γ subclasses of isozymes are activated by direct binding of small G proteins of the Ras, Rho, and Rac subfamilies of GTPases. Recent high resolution three dimensional structures together with biochemical studies have illustrated that the X/Y linker region of the catalytic core mediates autoinhibition of most if not all PLC isozymes. Activation occurs as a consequence of removal of this autoinhibition. PMID:22403074

Growth regulation in tip-growing cells that develop on the epidermis.

PubMed

Honkanen, Suvi; Dolan, Liam

2016-12-01

Plants develop tip-growing extensions-root hairs and rhizoids-that initiate as swellings on the outer surface of individual epidermal cells. A conserved genetic mechanism controls the earliest stages in the initiation of these swellings. The same mechanism controls the formation of multicellular structures that develop from swellings on epidermal cells in early diverging land plants. Details of the molecular events that regulate the positioning of the swellings involve sterols and phosphatidylinositol phosphates. The final length of root hairs is determined by the intensity of a pulse of transcription factor synthesis. Genes encoding similar transcription factors control root hair development in cereals and are potential targets for crop improvement. Copyright © 2016. Published by Elsevier Ltd.

Inhibition profiles of phosphatidylinositol 3-kinase inhibitors against PI3K superfamily and human cancer cell line panel JFCR39.

PubMed

Kong, Dexin; Dan, Shingo; Yamazaki, Kanami; Yamori, Takao

2010-04-01

As accumulating evidences suggest close involvement of phosphatidylinositol 3-kinase (PI3K) in various diseases particularly cancer, considerable competition occurs in development of PI3K inhibitors. Consequently, novel PI3K inhibitors such as ZSTK474, GDC-0941 and NVP-BEZ235 have been developed. Even though all these inhibitors were reported to inhibit class I PI3K but not dozens of protein kinases, whether they have different molecular targets remained unknown. To investigate such molecular target specificity, we have determined the inhibitory effects of these novel inhibitors together with classical PI3K inhibitor LY294002 on PI3K superfamily (including classes I, II, and III PI3Ks, PI4K and PI3K-related kinases) by using several novel non-radioactive biochemical assays. As a result, ZSTK474 and GDC-0941 indicated highly similar inhibition profiles for PI3K superfamily, with class I PI3K specificity much higher than NVP-BEZ235 and LY294002. We further investigated their growth inhibition effects on JFCR39, a human cancer cell line panel which we established for molecular target identification, and analysed their cell growth inhibition profiles (fingerprints) by using COMPARE analysis programme. Interestingly, we found ZSTK474 exhibited a highly similar fingerprint with GDC-0941 (r=0.863), more similar than with that of either NVP-BEZ235 or LY294002, suggesting that ZSTK474 shares more in molecular targets with GDC-0941 than with either of the other two PI3K inhibitors, consistent with the biochemical assay result. The biological implication of the difference in molecular target specificity of these PI3K inhibitors is under investigation. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Cardiac effects of 3-iodothyronamine: a new aminergic system modulating cardiac function.

PubMed

Chiellini, Grazia; Frascarelli, Sabina; Ghelardoni, Sandra; Carnicelli, Vittoria; Tobias, Sandra C; DeBarber, Andrea; Brogioni, Simona; Ronca-Testoni, Simonetta; Cerbai, Elisabetta; Grandy, David K; Scanlan, Thomas S; Zucchi, Riccardo

2007-05-01

3-Iodothyronamine T1AM is a novel endogenous thyroid hormone derivative that activates the G protein-coupled receptor known as trace anime-associated receptor 1 (TAAR1). In the isolated working rat heart and in rat cardiomyocytes, T1AM produced a reversible, dose-dependent negative inotropic effect (e.g., 27+/-5, 51+/-3, and 65+/-2% decrease in cardiac output at 19, 25, and 38 microM concentration, respectively). An independent negative chronotropic effect was also observed. The hemodynamic effects of T1AM were remarkably increased in the presence of the tyrosine kinase inhibitor genistein, whereas they were attenuated in the presence of the tyrosine phosphatase inhibitor vanadate. No effect was produced by inhibitors of protein kinase A, protein kinase C, calcium-calmodulin kinase II, phosphatidylinositol-3-kinase, or MAP kinases. Tissue cAMP levels were unchanged. In rat ventricular tissue, Western blot experiments with antiphosphotyrosine antibodies showed reduced phosphorylation of microsomal and cytosolic proteins after perfusion with synthetic T1AM; reverse transcriptase-polymerase chain reaction experiments revealed the presence of transcripts for at least 5 TAAR subtypes; specific and saturable binding of [125I]T1AM was observed, with a dissociation constant in the low micromolar range (5 microM); and endogenous T1AM was detectable by tandem mass spectrometry. In conclusion, our findings provide evidence for the existence of a novel aminergic system modulating cardiac function.

Adaptations of the aging animal to exercise: role of daily supplementation with melatonin.

PubMed

Mendes, Caroline; Lopes, Ana Maria de Souza; do Amaral, Fernanda Gaspar; Peliciari-Garcia, Rodrigo A; Turati, Ariane de Oliveira; Hirabara, Sandro M; Scialfa Falcão, Julieta H; Cipolla-Neto, José

2013-10-01

The pineal gland, through melatonin, seems to be of fundamental importance in determining the metabolic adaptations of adipose and muscle tissues to physical training. Evidence shows that pinealectomized animals fail to develop adaptive metabolic changes in response to aerobic exercise and therefore do not exhibit the same performance as control-trained animals. The known prominent reduction in melatonin synthesis in aging animals led us to investigate the metabolic adaptations to physical training in aged animals with and without daily melatonin replacement. Male Wistar rats were assigned to four groups: sedentary control (SC), trained control (TC), sedentary treated with melatonin (SM), and trained treated with melatonin (TM). Melatonin supplementation lasted 16 wk, and the animals were subjected to exercise during the last 8 wk of the experiment. After euthanasia, samples of liver, muscle, and adipose tissues were collected for analysis. Trained animals treated with melatonin presented better results in the following parameters: glucose tolerance, physical capacity, citrate synthase activity, hepatic and muscular glycogen content, body weight, protein expression of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and protein kinase activated by adenosine monophosphate (AMPK) in the liver, as well as the protein expression of the glucose transporter type 4 (GLUT4) and AMPK in the muscle. In conclusion, these results demonstrate that melatonin supplementation in aging animals is of great importance for the required metabolic adaptations induced by aerobic exercise. Adequate levels of circulating melatonin are, therefore, necessary to improve energetic metabolism efficiency, reducing body weight and increasing insulin sensitivity. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

SP6616 as a new Kv2.1 channel inhibitor efficiently promotes β-cell survival involving both PKC/Erk1/2 and CaM/PI3K/Akt signaling pathways.

PubMed

Zhou, T T; Quan, L L; Chen, L P; Du, T; Sun, K X; Zhang, J C; Yu, L; Li, Y; Wan, P; Chen, L L; Jiang, B H; Hu, L H; Chen, J; Shen, X

2016-05-05

Kv2.1 as a voltage-gated potassium (Kv) channel subunit has a pivotal role in the regulation of glucose-stimulated insulin secretion (GSIS) and pancreatic β-cell apoptosis, and is believed to be a promising target for anti-diabetic drug discovery, although the mechanism underlying the Kv2.1-mediated β-cell apoptosis is obscure. Here, the small molecular compound, ethyl 5-(3-ethoxy-4-methoxyphenyl)-2-(4-hydroxy-3-methoxybenzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate (SP6616) was discovered to be a new Kv2.1 inhibitor. It was effective in both promoting GSIS and protecting β cells from apoptosis. Evaluation of SP6616 on either high-fat diet combined with streptozocin-induced type 2 diabetic mice or db/db mice further verified its efficacy in the amelioration of β-cell dysfunction and glucose homeostasis. SP6616 treatment efficiently increased serum insulin level, restored β-cell mass, decreased fasting blood glucose and glycated hemoglobin levels, and improved oral glucose tolerance. Mechanism study indicated that the promotion of SP6616 on β-cell survival was tightly linked to its regulation against both protein kinases C (PKC)/extracellular-regulated protein kinases 1/2 (Erk1/2) and calmodulin(CaM)/phosphatidylinositol 3-kinase(PI3K)/serine/threonine-specific protein kinase (Akt) signaling pathways. To our knowledge, this may be the first report on the underlying pathway responsible for the Kv2.1-mediated β-cell protection. In addition, our study has also highlighted the potential of SP6616 in the treatment of type 2 diabetes.

Interkinase domain of kit contains the binding site for phosphatidylinositol 3' kinase.

PubMed Central

Lev, S; Givol, D; Yarden, Y

1992-01-01

Our previous analysis of the signal transduction pathway used by the c-kit-encoded receptor for the stem cell factor (SCF) indicated efficient coupling to the type I phosphatidylinositol 3' kinase (PI3K). In an attempt to localize the receptor's site of interaction with PI3K, we separately deleted either the noncatalytic 68-amino-acid-long interkinase domain or the carboxyl-terminal portion distal to the catalytic sequences. Loss of ligand-induced association of PI3K with the former deletion mutant and retention of the PI3K association by the carboxyl-terminally deleted receptor implied interactions of PI3K with the kinase insert. This was further supported by partial inhibition of the association by an anti-peptide antibody directed against the kinase insert and lack of effect of an antibody directed to the carboxyl tail of the SCF receptor. A bacterially expressed kinase insert domain was used as a fusion protein to directly test its presumed function as a PI3K association site. This protein bound PI3K from cell lysate as demonstrated by PI3K activity and by an associated phosphoprotein of 85 kDa. The association was dependent on phosphorylation of the tyrosine residues on the expressed kinase insert. On the basis of these observations, we conclude that the kinase insert domain of the SCF receptor selectively interacts with the p85 regulatory subunit of PI3K and that this association requires phosphorylation of tyrosine residues in the kinase insert region, with apparently no involvement of the bulk cytoplasmic structure or tyrosine kinase function of the receptor. Images PMID:1370584

[THE SOMATIC MUTATIONS AND ABERRANT METHYLATION AS POTENTIAL GENETIC MARKERS OF URINARY BLADDER CANCER].

PubMed

Mikhailenko, D S; Kushlinskii, N E

2016-02-01

All around the world, more than 330 thousands cases of bladder cancer are registered annually hence representing actual problem of modern oncology. Still in demand are search and characteristic of new molecular markers of bladder cancer detecting in tumor cells from urinary sediment and having high diagnostic accuracy. The studies of last decade, especially using methods of genome-wide sequencing, permitted to receive a large amount of experimental data concerning development and progression of bladder cancer The review presents systematic analysis of publications available in PubMed data base mainly of last five years. The original studies of molecular genetic disorders under bladder cancer and meta-analyzes were considered This approach permitted to detected the most common local alterations of DNA under bladder cancer which can be detected using routine genetic methods indifferent clinical material and present prospective interest for development of test-systems. The molecular genetic markers of disease can be activating missense mutations in 7 and 10 exons of gene of receptor of growth factor of fibroblasts 3 (FGFR3), 9 and 20 exons of gene of Phosphatidylinositol-4,5-bi-phosphate-3-kinase (PIK3CA) and mutation in -124 and -146 nucleotides in promoter of gene of catalytic subunit telomerase (TERT). The development of test-systems on the basis of aberrant methylation of CpG-islets of genes-suppressors still is seemed as a difficult task because of differences in pattern of methylation of different primary tumors at various stages of clonal evolution of bladder cancer though they can be considered as potential markers.

Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners.

PubMed

Rowland, Meng M; Bostic, Heidi E; Gong, Denghuang; Speers, Anna E; Lucas, Nathan; Cho, Wonhwa; Cravatt, Benjamin F; Best, Michael D

2011-12-27

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P₃ that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P₃ headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P₃ headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P₃-binding proteins.

Phosphatidylinositol (3,4,5)-Trisphosphate Activity Probes for the Labeling and Proteomic Characterization of Protein Binding Partners

PubMed Central

Rowland, Meng M.; Bostic, Heidi E.; Gong, Denghuang; Speers, Anna E.; Lucas, Nathan; Cho, Wonhwa; Cravatt, Benjamin F.; Best, Michael D.

2013-01-01

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3), regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane-association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P3 that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins as well as a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by on-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P3 headgroup analog as well as through protein denaturation, indicating specific labeling. In addition, probes featuring different linker lengths between the PI(3,4,5)P3 headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts, labeled proteins were observed by in-gel detection and characterized using post-labeling with biotin, affinity chromatography and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P3-binding proteins. PMID:22074223

Neuronal suppressor of cytokine signaling-3 deficiency enhances hypothalamic leptin-dependent phosphatidylinositol 3-kinase signaling.

PubMed

Metlakunta, Anantha S; Sahu, Maitrayee; Yasukawa, Hideo; Dhillon, Sandeep S; Belsham, Denise D; Yoshimura, Akihiko; Sahu, Abhiram

2011-05-01

Suppressor of cytokine signaling-3 (SOCS3) is thought to be involved in the development of central leptin resistance and obesity by inhibiting STAT3 pathway. Because phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in transducing leptin action in the hypothalamus, we examined whether SOCS3 exerted an inhibition on this pathway. We first determined whether leptin sensitivity in the hypothalamic PI3K pathway was increased in brain-specific Socs3-deficient (NesKO) mice. In NesKO mice, hypothalamic insulin receptor substrate-1 (IRS1)-associated PI3K activity was significantly increased at 30 min and remained elevated up to 2 h after leptin intraperitoneal injection, but in wild-type (WT) littermates, the significant increase was only at 30 min. Hypothalamic p-STAT3 levels were increased up to 5 h in NesKO as opposed to 2 h in WT mice. In food-restricted WT mice with reduced body weight, leptin increased hypothalamic PI3K activity only at 30 min, and p-STAT3 levels at 30-120 min postinjection. These results suggest increased leptin sensitivity in both PI3K and STAT3 pathways in the hypothalamus of NesKO mice, which was not due to a lean phenotype. In the next experiment with a clonal hypothalamic neuronal cell line expressing proopiomelanocortin, we observed that whereas leptin significantly increased IRS1-associated PI3K activity and p-JAK2 levels in cells transfected with control vector, it failed to do so in SOCS3-overexpressed cells. Altogether, these results imply a SOCS3 inhibition of the PI3K pathway of leptin signaling in the hypothalamus, which may be one of the mechanisms behind the development of central leptin resistance and obesity.

Neuronal suppressor of cytokine signaling-3 deficiency enhances hypothalamic leptin-dependent phosphatidylinositol 3-kinase signaling

PubMed Central

Metlakunta, Anantha S.; Sahu, Maitrayee; Yasukawa, Hideo; Dhillon, Sandeep S.; Belsham, Denise D.; Yoshimura, Akihiko

2011-01-01

Suppressor of cytokine signaling-3 (SOCS3) is thought to be involved in the development of central leptin resistance and obesity by inhibiting STAT3 pathway. Because phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in transducing leptin action in the hypothalamus, we examined whether SOCS3 exerted an inhibition on this pathway. We first determined whether leptin sensitivity in the hypothalamic PI3K pathway was increased in brain-specific Socs3-deficient (NesKO) mice. In NesKO mice, hypothalamic insulin receptor substrate-1 (IRS1)-associated PI3K activity was significantly increased at 30 min and remained elevated up to 2 h after leptin intraperitoneal injection, but in wild-type (WT) littermates, the significant increase was only at 30 min. Hypothalamic p-STAT3 levels were increased up to 5 h in NesKO as opposed to 2 h in WT mice. In food-restricted WT mice with reduced body weight, leptin increased hypothalamic PI3K activity only at 30 min, and p-STAT3 levels at 30–120 min postinjection. These results suggest increased leptin sensitivity in both PI3K and STAT3 pathways in the hypothalamus of NesKO mice, which was not due to a lean phenotype. In the next experiment with a clonal hypothalamic neuronal cell line expressing proopiomelanocortin, we observed that whereas leptin significantly increased IRS1-associated PI3K activity and p-JAK2 levels in cells transfected with control vector, it failed to do so in SOCS3-overexpressed cells. Altogether, these results imply a SOCS3 inhibition of the PI3K pathway of leptin signaling in the hypothalamus, which may be one of the mechanisms behind the development of central leptin resistance and obesity. PMID:21325649

Activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) increases the expression of prostaglandin E2 receptor subtype EP4. The roles of phosphatidylinositol 3-kinase and CCAAT/enhancer-binding protein beta.

PubMed

Han, ShouWei; Ritzenthaler, Jeffrey D; Wingerd, Byron; Roman, Jesse

2005-09-30

The prostaglandin E2 receptor subtype EP4 has been implicated in the growth and progression of human non-small cell lung carcinoma (NSCLC). However, the factors that control its expression have not been entirely elucidated. Our studies show that NSCLC cells express peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) protein and that treatment with a selective PPARbeta/delta agonist (GW501516) increases EP4 mRNA and protein levels. GW501516 induced NSCLC cell proliferation, and this effect was prevented by PPARbeta/delta antisense or EP4 short interfering RNA (siRNA). GW501516 increased the phosphorylation of Akt and decreased PTEN expression. The selective inhibitor of phosphatidylinositol 3-kinase (PI3-K), wortmannin, and PPARbeta/delta antisense, abrogated the effect of GW501516 on EP4 expression, whereas that of the inhibitor of Erk did not. GW501516 also increased EP4 promoter activity through effects on the region between -1555 and -992 bp in the EP4 promoter, and mutation of the CCAAT/enhancer-binding protein (C/EBP) site in this region abrogated the effect of GW501516. GW501516 increased not only the binding activity of C/EBP to the NF-IL6 site in the EP4 promoter, which was prevented by the inhibitor of PI3-K, but also increased C/EBPbeta protein in a dose- and PPARbeta/delta-dependent manner. The effect of GW501516 on EP4 protein was eliminated in the presence of C/EBPbeta siRNA. Finally, we showed that pretreatment of NSCLC with GW501516 further increased NSCLC cell proliferation in response to exogenous dimethyl-prostaglandin E2 (PGE2) that was diminished in the presence of PPARbeta/delta antisense and EP4 siRNA. Taken together, these findings suggest that activation of PPARbeta/delta induces PGE2 receptor subtype EP4 expression through PI3-K signals and increases human lung carcinoma cell proliferation in response to PGE2. The increase in transcription of the EP4 gene by PPARbeta/delta agonist was associated with increased C/EBP binding activity in the NF-IL6 site of EP4 promoter region and C/EBPbeta protein expression that were mediated through both PI3-K/Akt and PPARbeta/delta signaling pathways.

Crosstalk between apoptosis and autophagy within the Beclin 1 interactome.

PubMed

Maiuri, Maria Chiara; Criollo, Alfredo; Kroemer, Guido

2010-02-03

Although the essential genes for autophagy (Atg) have been identified, the molecular mechanisms through which Atg proteins control 'self eating' in mammalian cells remain elusive. Beclin 1 (Bec1), the mammalian orthologue of yeast Atg6, is part of the class III phosphatidylinositol 3-kinase (PI3K) complex that induces autophagy. The first among an increasing number of Bec1-interacting proteins that has been identified is the anti-apoptotic protein Bcl-2. The dissociation of Bec1 from Bcl-2 is essential for its autophagic activity, and Bcl-2 only inhibits autophagy when it is present in the endoplasmic reticulum (ER). A paper in this issue of the EMBO Journal has identified a novel protein, NAF-1 (nutrient-deprivation autophagy factor-1), that binds Bcl-2 at the ER. NAF-1 is a component of the inositol-1,4,5 trisphosphate (IP3) receptor complex, which contributes to the interaction of Bcl-2 with Bec1 and is required for Bcl-2 to functionally antagonize Bec1-mediated autophagy. This work provides mechanistic insights into how autophagy- and apoptosis-regulatory molecules crosstalk at the ER.

Metabolism of d-Arabinose: Origin of a d-Ribulokinase Activity in Escherichia coli1

PubMed Central

LeBlanc, Donald J.; Mortlock, Robert P.

1971-01-01

The kinase responsible for the phosphorylation of d-ribulose was purified 45.5-fold from a strain of Escherichia coli K-12 capable of growth on d-arabinose with no separation of d-ribulo- or l-fuculokinase activities. Throughout the purification, the ratios of activities remained essentially constant. A nonadditive effect of combining both substrates in an assay mixture; identical Km values for adenosine triphosphate with either l-fuculose or d-ribulose as substrate; and, the irreversible loss of activity on both substrates, after removal of magnesium ions from the enzyme preparation, suggest that the dual activity is due to the same enzyme. A fourfold greater affinity of the enzyme for l-fuculose than for d-ribulose, as well as a higher relative activity on l-fuculose, suggest that the natural substrate for this enzyme is l-fuculose. The product of the purified enzyme, with d-ribulose as substrate, was prepared. The ratio of total phosphorous to ribulose phosphate was 1.01:1, indicating that the product was ribulose monophosphate. The behavior of the kinase product in the cysteine-carbazole and orcinol reactions, as well as the results of periodate oxidation assays, provided evidence that it was not d-ribulose-5-phosphate. Reaction of this compound with a cell-free extract of E. coli possessing l-fuculose-l-phosphate aldolase activity resulted in the production of dihydroxyacetone phosphate and glycolaldehyde. The kinase product failed to reduce 2,3,5-triphenyltetrazolium and possessed a half-life of approximately 1.5 min in the presence of 1 n HCl at 100 C. These properties suggested that the phosphate group was attached to carbon atom 1 of d-ribulose. PMID:4323967

Quantitative properties and receptor reserve of the IP3 and calcium branch of Gq-coupled receptor signaling

PubMed Central

Dickson, Eamonn J.; Falkenburger, Björn H.

2013-01-01

Gq-coupled plasma membrane receptors activate phospholipase C (PLC), which hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This leads to calcium release, protein kinase C (PKC) activation, and sometimes PIP2 depletion. To understand mechanisms governing these diverging signals and to determine which of these signals is responsible for the inhibition of KCNQ2/3 (KV7.2/7.3) potassium channels, we monitored levels of PIP2, IP3, and calcium in single living cells. DAG and PKC are monitored in our companion paper (Falkenburger et al. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210887). The results extend our previous kinetic model of Gq-coupled receptor signaling to IP3 and calcium. We find that activation of low-abundance endogenous P2Y2 receptors by a saturating concentration of uridine 5′-triphosphate (UTP; 100 µM) leads to calcium release but not to PIP2 depletion. Activation of overexpressed M1 muscarinic receptors by 10 µM Oxo-M leads to a similar calcium release but also depletes PIP2. KCNQ2/3 channels are inhibited by Oxo-M (by 85%), but not by UTP (<1%). These differences can be attributed purely to differences in receptor abundance. Full amplitude calcium responses can be elicited even after PIP2 was partially depleted by overexpressed inducible phosphatidylinositol 5-phosphatases, suggesting that very low amounts of IP3 suffice to elicit a full calcium release. Hence, weak PLC activation can elicit robust calcium signals without net PIP2 depletion or KCNQ2/3 channel inhibition. PMID:23630337

Quantitative properties and receptor reserve of the IP(3) and calcium branch of G(q)-coupled receptor signaling.

PubMed

Dickson, Eamonn J; Falkenburger, Björn H; Hille, Bertil

2013-05-01

Gq-coupled plasma membrane receptors activate phospholipase C (PLC), which hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This leads to calcium release, protein kinase C (PKC) activation, and sometimes PIP2 depletion. To understand mechanisms governing these diverging signals and to determine which of these signals is responsible for the inhibition of KCNQ2/3 (KV7.2/7.3) potassium channels, we monitored levels of PIP2, IP3, and calcium in single living cells. DAG and PKC are monitored in our companion paper (Falkenburger et al. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210887). The results extend our previous kinetic model of Gq-coupled receptor signaling to IP3 and calcium. We find that activation of low-abundance endogenous P2Y2 receptors by a saturating concentration of uridine 5'-triphosphate (UTP; 100 µM) leads to calcium release but not to PIP2 depletion. Activation of overexpressed M1 muscarinic receptors by 10 µM Oxo-M leads to a similar calcium release but also depletes PIP2. KCNQ2/3 channels are inhibited by Oxo-M (by 85%), but not by UTP (<1%). These differences can be attributed purely to differences in receptor abundance. Full amplitude calcium responses can be elicited even after PIP2 was partially depleted by overexpressed inducible phosphatidylinositol 5-phosphatases, suggesting that very low amounts of IP3 suffice to elicit a full calcium release. Hence, weak PLC activation can elicit robust calcium signals without net PIP2 depletion or KCNQ2/3 channel inhibition.

Insulin-Like growth factor-II (IGF-II) prevents proinflammatory cytokine-induced apoptosis and significantly improves islet survival after transplantation.

PubMed

Hughes, Amy; Mohanasundaram, Daisy; Kireta, Svjetlana; Jessup, Claire F; Drogemuller, Chris J; Coates, P Toby H

2013-03-15

The early loss of functional islet mass (50-70%) due to apoptosis after clinical transplantation contributes to islet allograft failure. Insulin-like growth factor (IGF)-II is an antiapoptotic protein that is highly expressed in β-cells during development but rapidly decreases in postnatal life. We used an adenoviral (Ad) vector to overexpress IGF-II in isolated rat islets and investigated its antiapoptotic action against exogenous cytokines interleukin-1β- and interferon-γ-induced islet cell death in vitro. Using an immunocompromised marginal mass islet transplant model, the ability of Ad-IGF-II-transduced rat islets to restore euglycemia in nonobese diabetic/severe combined immunodeficient diabetic recipients was assessed. Ad-IGF-II transduction did not affect islet viability or function. Ad-IGF-II cytokine-treated islets exhibited decreased cell death (40% ± 2.8%) versus Ad-GFP and untransduced control islets (63.2% ± 2.5% and 53.6% ± 2.3%, respectively). Ad-IGF-II overexpression during cytokine treatment resulted in a marked reduction in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells (8.3% ± 1.4%) versus Ad-GFP control (41% ± 4.2%) and untransduced control islets (46.5% ± 6.2%). Western blot analysis confirmed that IGF-II inhibits apoptosis via activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. Transplantation of IGF-II overexpressing islets under the kidney capsule of diabetic mice restored euglycemia in 77.8% of recipients compared with 18.2% and 47.5% of Ad-GFP and untransduced control islet recipients, respectively (P<0.05, log-rank [Mantel-Cox] test). Antiapoptotic IGF-II decreases apoptosis in vitro and significantly improved islet transplant outcomes in vivo. Antiapoptotic gene transfer is a potentially powerful tool to improve islet survival after transplantation.

Inhibition of muscarinic receptor-induced inositol phospholipid hydrolysis by caffeine, beta-adrenoceptors and protein kinase C in intestinal smooth muscle.

PubMed Central

Prestwich, S A; Bolton, T B

1995-01-01

1. The effects of caffeine, isoprenaline, dibutyryl cyclic AMP, isobutylmethylxanthine (IBMX), 12-O-tetradecanoylphorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG), (protein kinase C (PKC) activators), 2-methoxy verapamil (D600), thapsigargin and ryanodine on muscarinic acetylcholine receptor (AChR)-stimulated inositol phospholipid hydrolysis were studied in smooth muscle fragments from the longitudinal layer of the small intestine of the guinea-pig. 2. Incubation of the fragments with the muscarinic agonist, carbachol (CCh) (100 microM) resulted in rapid increases in the levels of all the inositol phosphate isomers with maximal increases in the [3H]-inositol (1,4,5) trisphosphate ([3H]-Ins(1,4,5)P3) isomer occurring 10 s following incubation. 3. The beta-adrenoceptor agonist, isoprenaline (10 microM) and dibutyryl cyclic AMP (10 microM), a membrane permeant analogue of cyclic AMP both reduced the CCh stimulation, but not the basal levels of [3H]-inositol phosphates. This inhibition by dibutyryl cyclic AMP was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. CCh inhibited the isoprenaline-induced increases in the levels of cyclic AMP and this was via a pertussi toxin (PTX)-sensitive G-protein mechanism. 4. TPA (1 microM) and OAG (100 microM) a 1,2-diacylglycerol (DAG) analogue both reduced the CCh-induced increases in [3H]-inositol phosphates levels but neither affected basal values nor the basal levels of cyclic AMP. 5. D600 (10 microM), which blocks voltage-dependent Ca2+ channels, also reduced the CCh-stimulated levels of [3H]-inositol phosphates suggesting that some of the agonist-induced increases are due to a potentiating effect of Ca2+ entering the cell. 6. Caffeine (0.5-30 mM) significantly inhibited both the basal and CCh-induced increases in all the [3H]-inositol phosphate isomers. Its inhibitory action was not due to increases in cyclic AMP since caffeine had no effect on the levels of cyclic AMP at concentrations up to 30 mM. 7. Incubation with thapsigargin (1 microM) and ryanodine (10 microM) had no effect on either basal or CCh-induced inositol phospholipid hydrolysis or cyclic AMP levels. 8. The results indicate a reciprocal inhibition by beta-adrenoceptors and muscarinic AChRs of their effects on cyclic AMP and inositol phosphate levels respectively. Ca2+ entering the cell (but not the action of ryanodine or thapsigargin) potentiates while caffeine inhibits muscarinic AChR-induced rises in inositol phosphate levels. Diacylglycerols may exert a negative feedback inhibition on inositol phosphate production. PMID:7537591

5'-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates.

PubMed

Parmar, Rubina; Willoughby, Jennifer L S; Liu, Jingxuan; Foster, Donald J; Brigham, Benjamin; Theile, Christopher S; Charisse, Klaus; Akinc, Akin; Guidry, Erin; Pei, Yi; Strapps, Walter; Cancilla, Mark; Stanton, Matthew G; Rajeev, Kallanthottathil G; Sepp-Lorenzino, Laura; Manoharan, Muthiah; Meyers, Rachel; Maier, Martin A; Jadhav, Vasant

2016-06-02

Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of the Sterol and Phosphatidylinositol 4-Phosphate Binding Properties of Golgi-Associated OSBP-Related Protein 9 (ORP9)

PubMed Central

Liu, Xinwei; Ridgway, Neale D.

2014-01-01

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus. PMID:25255026

Characterization of the sterol and phosphatidylinositol 4-phosphate binding properties of Golgi-associated OSBP-related protein 9 (ORP9).

PubMed

Liu, Xinwei; Ridgway, Neale D

2014-01-01

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.

Magnolol suppresses vascular endothelial growth factor-induced angiogenesis by inhibiting Ras-dependent mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt signaling pathways.

PubMed

Kim, Ki Mo; Kim, No Soo; Kim, Jinhee; Park, Jong-Shik; Yi, Jin Mu; Lee, Jun; Bang, Ok-Sun

2013-01-01

Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, has been reported to possess anticancer activity. Recent studies have also demonstrated that magnolol inhibits cell growth and induces the apoptosis of cancer cells. However, the effects of magnolol on vascular endothelial growth factor (VEGF)-induced angiogenesis in endothelial cells have not been studied. In the present study, we have used human umbilical vein endothelial cells (HUVECs) to investigate the antiangiogenic effect and molecular mechanism of magnolol. Magnolol inhibited the VEGF-induced proliferation, chemotactic motility and tube formation of HUVECs in vitro as well as the vessel sprouting of the aorta ex vivo. Furthermore, magnolol inhibited VEGF-induced Ras activation and subsequently suppressed extracellular signal-regulated kinase (ERK), phosphatidylinositol-3-kinase (PI3K)/Akt and p38, but not Src and focal adhesion kinase (FAK). Interestingly, the knockdown of Ras by short interfering RNA produced inhibitory effects that were similar to the effects of magnolol on VEGF-induced angiogenic signaling events, such as ERK and Akt/eNOS activation, and resulted in the inhibition of proliferation, migration, and vessel sprouting in HUVECs. In combination, these results demonstrate that magnolol is an inhibitor of angiogenesis and suggest that this compound could be a potential candidate in the treatment of angiogenesis-related diseases.

SAR405, a PIK3C3/Vps34 inhibitor that prevents autophagy and synergizes with MTOR inhibition in tumor cells.

PubMed

Pasquier, Benoit

2015-04-03

Autophagy plays an important role in cancer and it has been suggested that it functions not only as a tumor suppressor pathway to prevent tumor initiation, but also as a prosurvival pathway that helps tumor cells endure metabolic stress and resist death triggered by chemotherapeutic agents. We recently described the discovery of inhibitors of PIK3C3/Vps34 (phosphatidylinositol 3-kinase, catalytic subunit type 3), the lipid kinase component of the class III phosphatidylinositol 3-kinase (PtdIns3K). This PtdIns3K isoform has attracted significant attention in recent years because of its role in autophagy. Following chemical optimization we identified SAR405, a low molecular mass kinase inhibitor of PIK3C3, highly potent and selective with regard to other lipid and protein kinases. We demonstrated that inhibiting the catalytic activity of PIK3C3 disrupts vesicle trafficking from late endosomes to lysosomes. SAR405 treatment also inhibits autophagy induced either by starvation or by MTOR (mechanistic target of rapamycin) inhibition. Finally our results show that combining SAR405 with everolimus, the FDA-approved MTOR inhibitor, results in a significant synergy on the reduction of cell proliferation using renal tumor cells. This result indicates a potential therapeutic application for PIK3C3 inhibitors in cancer.

Neuregulin 1-ErbB4-PI3K signaling in schizophrenia and phosphoinositide 3-kinase-p110δ inhibition as a potential therapeutic strategy.

PubMed

Law, Amanda J; Wang, Yanhong; Sei, Yoshitatsu; O'Donnell, Patricio; Piantadosi, Patrick; Papaleo, Francesco; Straub, Richard E; Huang, Wenwei; Thomas, Craig J; Vakkalanka, Radhakrishna; Besterman, Aaron D; Lipska, Barbara K; Hyde, Thomas M; Harrison, Paul J; Kleinman, Joel E; Weinberger, Daniel R

2012-07-24

Neuregulin 1 (NRG1) and ErbB4, critical neurodevelopmental genes, are implicated in schizophrenia, but the mediating mechanisms are unknown. Here we identify a genetically regulated, pharmacologically targetable, risk pathway associated with schizophrenia and with ErbB4 genetic variation involving increased expression of a PI3K-linked ErbB4 receptor (CYT-1) and the phosphoinositide 3-kinase subunit, p110δ (PIK3CD). In human lymphoblasts, NRG1-mediated phosphatidyl-inositol,3,4,5 triphosphate [PI(3,4,5)P3] signaling is predicted by schizophrenia-associated ErbB4 genotype and PIK3CD levels and is impaired in patients with schizophrenia. In human brain, the same ErbB4 genotype again predicts increased PIK3CD expression. Pharmacological inhibition of p110δ using the small molecule inhibitor, IC87114, blocks the effects of amphetamine in a mouse pharmacological model of psychosis and reverses schizophrenia-related phenotypes in a rat neonatal ventral hippocampal lesion model. Consistent with these antipsychotic-like properties, IC87114 increases AKT phosphorylation in brains of treated mice, implicating a mechanism of action. Finally, in two family-based genetic studies, PIK3CD shows evidence of association with schizophrenia. Our data provide insight into a mechanism of ErbB4 association with schizophrenia; reveal a previously unidentified biological and disease link between NRG1-ErbB4, p110δ, and AKT; and suggest that p110δ is a previously undescribed therapeutic target for the treatment of psychiatric disorders.

Prevotella intermedia induces prostaglandin E2 via multiple signaling pathways.

PubMed

Guan, S-M; Fu, S-M; He, J-J; Zhang, M

2011-01-01

Prostaglandin E(2) (PGE(2)) plays important roles in the bone resorption of inflammatory diseases such as rheumatoid arthritis and periodontitis via specific prostaglandin receptors (i.e., EP1-EP4). In this study, the authors examined whether Prevotella intermedia regulates PGE(2) production and EP expression in human periodontal ligament fibroblasts (hPDLs); they also explored the potential signaling pathways involved in PGE(2) production. P. intermedia induced PGE(2) production and cyclooxygenase-2 (COX-2) expression in a dose- and time-dependent manner. Indomethacin and NS-398 completely abrogated the P. intermedia-induced PGE(2) production without modulating COX-2 expression. Specific inhibitors of extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38, phosphatidylinositol 3-kinase, and protein kinase C--but not c-AMP and protein kinase A--significantly attenuated the P. intermedia-induced COX-2 and PGE(2) expression. P. intermedia reduced EP1 expression in a concentration- and time-dependent manner. The results indicate that the COX-2-dependent induction of PGE(2) by P. intermedia in hPDLs is mediated by multiple signaling pathways.

Blockage of PI3K/mTOR Pathways Inhibits Laser-Induced Choroidal Neovascularization and Improves Outcomes Relative to VEGF-A Suppression Alone.

PubMed

Ma, Jie; Sun, Yu; López, Francisco J; Adamson, Peter; Kurali, Edit; Lashkari, Kameran

2016-06-01

Choroidal neovascularization (CNV) is a major cause of visual loss with age-related macular degeneration (AMD). We evaluated whether blockade of phosphatidyl-inositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR), by impairing VEGF-A and other growth factor receptors like platelet-derived growth factor (PDGF), would reduce laser-induced CNV in mice. Choroidal neovascularization lesions were induced in C57BL/6 mice. Two groups of mice received oral GSK2126458 (3 mg/kg) or vehicle for 14 days following laser, whereas three groups were treated with GSK2126458 (6 μg/eye), aflibercept (2 μL/eye), or vehicle intravitreally on days 0 and 7 after laser. Vascular leakage was measured by fluorescein angiography (FA) on day 14. Choroidal neovascularization membranes were evaluated on choroidal flat mounts following FITC-dextran perfusion, as well as ED1 and isolectin B4 (IB4) immunohistochemistry. Oral and intravitreal (IVT) GSK2126458 reduced leakage and area of CNV lesions. Greater probability of leaking lesions (∼60%; P < 0.05) was observed in both vehicle groups. Fluorescein isothiocyanate-dextran-labeled total CNV burden area (total lesion area/eye) was reduced ∼67% (P < 0.05) and 35% (P = 0.0528) after oral and IVT GSK2126458 administration. GSK2126458 treatment reduced lesion size by ∼80% (P < 0.05) and 50% (P < 0.05) for oral and IVT control groups. Aflibercept did not alter lesion size (∼27% reduction). Phosphatidyl-inositol-3-kinase/mTOR is involved in laser-induced CNV angiogenic processes. GSK2126458 effectively reduces CNV size and leakage. Choroidal neovascularization size following IVT GSK2126458 was smaller than after oral administration. Therefore, inhibition of PI3K/mTOR pathways may be more effective due to blockade of action of multiple growth factors.

Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

PubMed

Roskoski, Robert

2005-11-11

Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of interaction with several enzymes and adaptor proteins.

Regulatory effects of GRK2 on GPCRs and non-GPCRs and possible use as a drug target (Review).

PubMed

Han, Chen-Chen; Ma, Yang; Li, Yifan; Wang, Yang; Wei, Wei

2016-10-01

G protein-coupled receptor kinase 2 (GRK2) is a key member of the G protein-coupled receptor kinase (GRK) family. GRK2 activity is regulated by the C-terminus of GRK2 which contains a plekstrin homology domain and the N-terminus of GRK2 which contains the RGS homology domain with binding sites for several proteins and lipids such as G protein-coupled receptors (GPCRs), G protein, phospholipase C, phosphatidylinositol 4,5-bisphosphate, extracellular signal‑regulated kinase, protein kinase A and Gβγ. GRK2 phosphorylates the GPCR and enhances the affinity of binding to arrestins, which uncouple the receptors from G proteins, and target the receptors for desensitization and internalization. GRK2 also regulates non‑GPCR desensitization and internalization by phosphorylation, and is important in maintaining the balance between the receptors and signal transduction. Previous findings have indicated that the upregulation of GRK2 in heart failure enhances dysfunctional adrenergic signaling and myocyte death. Collagen-induced arthritis induces the upregulation of GRK2 expression in fibroblast-like synoviocytes. In this review, we discussed the evidence for the association between altered GRK2 levels and various diseases, which suggests that GRK2 may be an effective drug target for preventing and treating heart failure, hypertension and inflammatory disease.

A Regulatory Role for Src Homology 2 Domain–Containing Inositol 5′-Phosphatase (Ship) in Phagocytosis Mediated by Fcγ Receptors and Complement Receptor 3 (αMβ2; Cd11b/Cd18)

PubMed Central

Cox, Dianne; Dale, Benjamin M.; Kashiwada, Masaki; Helgason, Cheryl D.; Greenberg, Steven

2001-01-01

The Src homology 2 domain–containing inositol 5′-phosphatase (SHIP) is recruited to immunoreceptor tyrosine-based inhibition motif (ITIM)–containing proteins, thereby suppressing phosphatidylinositol 3-kinase (PI 3-kinase)–dependent pathways. The role of SHIP in phagocytosis, a PI 3-kinase–dependent pathway, is unknown. Overexpression of SHIP in macrophages led to an inhibition of phagocytosis mediated by receptors for the Fc portion of IgG (FcγRs). In contrast, macrophages expressing catalytically inactive SHIP or lacking SHIP expression demonstrated enhanced phagocytosis. To determine whether SHIP regulates phagocytosis mediated by receptors that are not known to recruit ITIMs, we determined the effect of SHIP expression on complement receptor 3 (CR3; CD11b/CD18; αMβ2)–dependent phagocytosis. Macrophages overexpressing SHIP demonstrated impaired CR3-mediated phagocytosis, whereas macrophages expressing catalytically inactive SHIP demonstrated enhanced phagocytosis. CR3-mediated phagocytosis in macrophages derived from SHIP−/− mice was up to 2.5 times as efficient as that observed in macrophages derived from littermate controls. SHIP was localized to FcγR- and CR3-containing phagocytic cups and was recruited to the cytoskeleton upon clustering of CR3. In a transfected COS cell model of activation-independent CR3-mediated phagocytosis, catalytically active but not inactive SHIP also inhibited phagocytosis. We conclude that PI 3-kinase(s) and SHIP regulate multiple forms of phagocytosis and that endogenous SHIP plays a role in modulating β2 integrin outside-in signaling. PMID:11136821

Protein-tyrosine-phosphatase-mediated epidermal growth factor (EGF) receptor transinactivation and EGF receptor-independent stimulation of mitogen-activated protein kinase by bradykinin in A431 cells.

PubMed Central

Graness, A; Hanke, S; Boehmer, F D; Presek, P; Liebmann, C

2000-01-01

Transactivation of the epidermal growth factor (EGF) receptor (EGFR) has been proposed to represent an essential link between G-protein-coupled receptors and the mitogen-activated protein kinase (MAPK) pathway in various cell types. In the present work we report, in contrast, that in A431 cells bradykinin transinactivates the EGFR and stimulates MAPK activity independently of EGFR tyrosine phosphorylation. Both effects of bradykinin are mediated by a pertussis-toxin-insensitive G-protein. Three lines of evidence suggest the activation of a protein tyrosine phosphatase (PTP) by bradykinin: (i) treatment of A431 cells with bradykinin decreases both basal and EGF-induced EGFR tyrosine phosphorylation, (ii) this effect of bradykinin can be blocked by two different PTP inhibitors, and (iii) bradykinin significantly increased the PTP activity in total A431 cell lysates when measured in vitro. The transmembrane receptor PTP sigma was identified as a putative mediator of bradykinin-induced downregulation of EGFR autophosphorylation. Activation of MAPK in response to bradykinin was insensitive towards AG 1478, a specific inhibitor of EGFR tyrosine kinase, but was blocked by wortmannin or bisindolylmaleimide, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and protein kinase C (PKC) respectively. These results also suggest that the bradykinin-induced activation of MAPK is independent of EGFR and indicate a pathway involving PI3-K and PKC. In addition, bradykinin evokes a rapid and transient increase in Src kinase activity. Although Src does not participate in bradykinin-induced stimulation of PTP activity, inhibition of Src by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine leads to an increase in MAPK activation by bradykinin. Our results suggest that in A431 cells the G(q/11)-protein-coupled bradykinin B(2) receptor may stimulate PTP activity and thereby transinactivate the EGFR, and may simultaneously activate MAPK by an alternative signalling pathway which can bypass EGFR. PMID:10749673

Inhibition of phosphatidylinositol 3-kinase causes apoptosis in retinoic acid differentiated hl-60 leukemia cells.

PubMed

Ma, Jin; Liu, Qiang; Zeng, Yi-Xin

2004-01-01

Phosphatidylinositol 3-kinase (PI3-K) signaling may inhibit apoptosis in neoplastic cells. The PI-3K inhibitor wortmannin renders cells apoptosis-prone. Inducers of differentiation may also cause apoptosis. To detect the effect of wortmannin on the survival of differentiated human acute promyeloid leukemia cells, HL-60 cells were induced to differentiation with treatment of all trans-retinoic acid (ATRA) followed by treatment with wortmannin. Results showed that apoptosis occurred in cells that underwent differentiation, but not in undifferentiated HL-60 cells. The pro-apoptotic molecule, Bad, played a role in this apoptotic mechanism. Thus, the survival of differentiated HL-60 cells induced by ATRA depends on the ability of the PI3-K pathway to transduce survival signals; the PI3-K inhibitor, wortmannin, can induce apoptosis of differentiated HL-60 cells. These results may indicate a novel method for treating cancer with differentiation induction and signal pathway regulation.

Reactive oxygen species enhance insulin sensitivity

PubMed Central

Loh, Kim; Deng, Haiyang; Fukushima, Atsushi; Cai, Xiaochu; Boivin, Benoit; Galic, Sandra; Bruce, Clinton; Shields, Benjamin J.; Skiba, Beata; Ooms, Lisa M.; Stepto, Nigel; Wu, Ben; Mitchell, Christina A.; Tonks, Nicholas K.; Watt, Matthew J.; Febbraio, Mark A.; Crack, Peter J.; Andrikopoulos, Sofianos; Tiganis, Tony

2010-01-01

SUMMARY Chronic reactive oxygen species (ROS) production by mitochondria may contribute to the development of insulin resistance, a primary feature of type 2 diabetes. In recent years it has become apparent that ROS generation in response to physiological stimuli such as insulin may also facilitate signaling by reversibly oxidizing and inhibiting protein tyrosine phosphatases (PTPs). Here we report that mice lacking one of the key enzymes involved in the elimination of physiological ROS, glutathione peroxidase 1 (Gpx1), were protected from high fat diet-induced insulin resistance. The increased insulin sensitivity in Gpx1−/− mice was attributed to insulin-induced phosphatidylinositol-3-kinase/Akt signaling and glucose uptake in muscle and could be reversed by the anti-oxidant N-acetylcysteine. Increased insulin signaling correlated with enhanced oxidation of the PTP family member PTEN, which terminates signals generated by phosphatidylinositol-3-kinase. These studies provide causal evidence for the enhancement of insulin signaling by ROS in vivo. PMID:19808019

The TryPIKinome of five human pathogenic trypanosomatids: Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Leishmania braziliensis and Leishmania infantum--new tools for designing specific inhibitors.

PubMed

Bahia, Diana; Oliveira, Luciana Márcia; Lima, Fabio Mitsuo; Oliveira, Priscila; Silveira, José Franco da; Mortara, Renato Arruda; Ruiz, Jerônimo Conceição

2009-12-18

Phosphatidylinositol (PI) kinases are at the heart of one of the major pathways of intracellular signal transduction. Herein, we present the first report on a survey made by similarity searches against the five human pathogenic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Leishmania braziliensis and Leishmania infantum genomes available to date for phosphatidylinositol- and related-kinases (TryPIKs). In addition to generating a panel called "The TryPIKinome", we propose a model of signaling pathways for these TryPIKs. The involvement of TryPIKs in fundamental pathways, such as intracellular signal transduction and host invasion processes, makes the study of TryPIKs an important area for further inquiry. New subtype-specific inhibitors are expected to work on individual members of the PIK family and, therefore, can presumably neutralize trypanosomatid invasion processes.

Reorganization of Actin Cytoskeleton by the Phosphoinositide Metabolite Glycerophosphoinositol 4-Phosphate

PubMed Central

Mancini, Raffaella; Piccolo, Enza; Mariggio', Stefania; Filippi, Beatrice Maria; Iurisci, Cristiano; Pertile, Paolo; Berrie, Christopher P.; Corda, Daniela

2003-01-01

Glycerophosphoinositol 4-phosphate (GroPIns-4P) is a biologically active, water-soluble phospholipase A metabolite derived from phosphatidylinositol 4-phosphate, whose cellular concentrations have been reported to increase in Ras-transformed cells. It is therefore important to understand its biological activities. Herein, we have examined whether GroPIns-4P can regulate the organization of the actin cytoskeleton, because this could be a Ras-related function involved in cell motility and metastatic invasion. We find that in serum-starved Swiss 3T3 cells, exogenously added GroPIns-4P rapidly and potently induces the formation of membrane ruffles, and, later, the formation of stress fibers. These actin structures can be regulated by the small GTPases Cdc42, Rac, and Rho. To analyze the mechanism of action of GroPIns-4P, we selectively inactivated each of these GTPases. GroPIns-4P requires active Rac and Rho, but not Cdc42, for ruffle and stress fiber formation, respectively. Moreover, GroPIns-4P induces a rapid translocation of the green fluorescent protein-tagged Rac into ruffles, and increases the fraction of GTP-bound Rac, in intact cells. The activation of Rac by GroPIns-4P was near maximal and long-lasting. Interestingly, this feature seems to be critical in the induction of actin ruffles by GroPIns-4P. PMID:12589050

Hypoxia increases the heterogeneity of melanoma cell populations and affects the response to vemurafenib.

PubMed

Pucciarelli, Daniela; Lengger, Nina; Takáčová, Martina; Csaderova, Lucia; Bartosova, Maria; Breiteneder, Heimo; Pastorekova, Silvia; Hafner, Christine

2016-04-01

A hypoxic microenvironment is one of the predominant reasons for incomplete response to melanoma treatment. Vemurafenib, which targets the mutated BRAF-V600 kinase, improves melanoma patient survival, however, resistance invariably develops. The present study evaluated the effect of hypoxia on three BRAF-V600E mutant melanoma cell lines, M14, A375 and 518A2, treated with vemurafenib. Compared with the other two cell lines, hypoxic vemurafenib-treated A375 cells exhibited an enhanced cell proliferation rate and migratory capacity compared with normoxic vemurafenib-treated A375 cells. Immunoblotting analyses revealed that the expression levels of hypoxia inducible factor (HIF)1α and carbonic anhydrase IX were reduced in vemurafenib‑treated M14 and 518A2 cells, however, not in A375 cells. The expression levels of the mitogen‑activated protein kinase, Janus kinase-signal transducer and activator of transcription, and phosphatidylinositol-4,5-bisphosphate 3‑kinase signaling pathway proteins revealed a cell‑type specific response to vemurafenib and hypoxia. Knockdown experiments of HIF1α performed in hypoxic A375 cells decreased the expression of phosphorylated (p‑)protein kinase B, which was restored following vemurafenib treatment, and increased the expression of p‑extracellular‑signal‑regulated kinases. Therefore, three melanoma cell lines responded to vemurafenib under hypoxia in a cell type‑specific manner, suggesting that a subset of cells provides a treatment-resistant pool, from which disease relapse may originate. These data confirmed that vemurafenib may be successful in treating the proliferating cells, whereas the non‑proliferating subpopulation must be addressed by a combination of vemurafenib with other treatment strategies.

[PSI+] prion propagation is controlled by inositol polyphosphates

PubMed Central

Wickner, Reed B.; Kelly, Amy C.; Bezsonov, Evgeny E.; Edskes, Herman K.

2017-01-01

The yeast prions [PSI+] and [URE3] are folded in-register parallel β-sheet amyloids of Sup35p and Ure2p, respectively. In a screen for antiprion systems curing [PSI+] without protein overproduction, we detected Siw14p as an antiprion element. An array of genetic tests confirmed that many variants of [PSI+] arising in the absence of Siw14p are cured by restoring normal levels of the protein. Siw14p is a pyrophosphatase specifically cleaving the β phosphate from 5-diphosphoinositol pentakisphosphate (5PP-IP5), suggesting that increased levels of this or some other inositol polyphosphate favors [PSI+] propagation. In support of this notion, we found that nearly all variants of [PSI+] isolated in a WT strain were lost upon loss of ARG82, which encodes inositol polyphosphate multikinase. Inactivation of the Arg82p kinase by D131A and K133A mutations (preserving Arg82p’s nonkinase transcription regulation functions) resulted the loss of its ability to support [PSI+] propagation. The loss of [PSI+] in arg82Δ is independent of Hsp104’s antiprion activity. [PSI+] variants requiring Arg82p could propagate in ipk1Δ (IP5 kinase), kcs1Δ (IP6 5-kinase), vip1Δ (IP6 1-kinase), ddp1Δ (inositol pyrophosphatase), or kcs1Δ vip1Δ mutants but not in ipk1Δ kcs1Δ or ddp1Δ kcs1Δ double mutants. Thus, nearly all [PSI+] prion variants require inositol poly-/pyrophosphates for their propagation, and at least IP6 or 5PP-IP4 can support [PSI+] propagation. PMID:28923943

Distribution of the phosphatidylinositol 3-kinase inhibitors Pictilisib (GDC-0941) and GNE-317 in U87 and GS2 intracranial glioblastoma models-assessment by matrix-assisted laser desorption ionization imaging.

PubMed

Salphati, Laurent; Shahidi-Latham, Sheerin; Quiason, Cristine; Barck, Kai; Nishimura, Merry; Alicke, Bruno; Pang, Jodie; Carano, Richard A; Olivero, Alan G; Phillips, Heidi S

2014-07-01

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and the limited available treatment options have not meaningfully impacted patient survival in the past decades. Such poor outcomes can be at least partly attributed to the inability of most drugs tested to cross the blood-brain barrier and reach all areas of the glioma. The objectives of these studies were to visualize and compare by matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry the brain and tumor distribution of the phosphatidylinositol 3-kinase (PI3K) inhibitors pictilisib (GDC-0941, 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine) and GNE-317 [5-(6-(3-methoxyoxetan-3-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-2-yl)pyrimidin-2-amine] in U87 and GS2 orthotopic models of GBM, models that exhibit differing blood-brain barrier characteristics. Following administration to tumor-bearing mice, pictilisib was readily detected within tumors of the contrast-enhancing U87 model whereas it was not located in tumors of the nonenhancing GS2 model. In both GBM models, pictilisib was not detected in the healthy brain. In contrast, GNE-317 was uniformly distributed throughout the brain in the U87 and GS2 models. MALDI imaging revealed also that the pictilisib signal varied regionally by up to 6-fold within the U87 tumors whereas GNE-317 intratumor levels were more homogeneous. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses of the nontumored half of the brain showed pictilisib had brain-to-plasma ratios lower than 0.03 whereas they were greater than 1 for GNE-317, in agreement with their brain penetration properties. These results in orthotopic models representing either the contrast-enhancing or invasive areas of GBM clearly demonstrate the need for whole-brain distribution to potentially achieve long-term efficacy in GBM. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR.

PubMed

Russell, R R; Bergeron, R; Shulman, G I; Young, L H

1999-08-01

Insulin increases glucose uptake through the translocation of GLUT-4 via a pathway mediated by phosphatidylinositol 3-kinase (PI3K). In contrast, myocardial glucose uptake during ischemia and hypoxia is stimulated by the translocation of GLUT-4 to the surface of cardiac myocytes through a PI3K-independent pathway that has not been characterized. AMP-activated protein kinase (AMPK) activity is also increased by myocardial ischemia, and we examined whether AMPK stimulates glucose uptake and GLUT-4 translocation. In isolated rat ventricular papillary muscles, 5-aminoimidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), an activator of AMPK, as well as cyanide-induced chemical hypoxia and insulin, increased 2-[(3)H]deoxyglucose uptake two- to threefold. Wortmannin, a PI3K inhibitor, did not affect either the AICAR- or the cyanide-stimulated increase in deoxyglucose uptake but eliminated the insulin-stimulated increase in deoxyglucose uptake. Immunofluorescence studies demonstrated translocation of GLUT-4 to the myocyte sarcolemma in response to stimulation with AICAR, cyanide, or insulin. Preincubation of papillary muscles with the kinase inhibitor iodotubercidin or adenine 9-beta-D-arabinofuranoside (araA), a precursor of araATP (a competitive inhibitor of AMPK), decreased AICAR- and cyanide-stimulated glucose uptake but did not affect basal or insulin-stimulated glucose uptake. In vivo infusion of AICAR caused myocardial AMPK activation and GLUT-4 translocation in the rat. We conclude that AMPK activation increases cardiac muscle glucose uptake through translocation of GLUT-4 via a pathway that is independent of PI3K. These findings suggest that AMPK activation may be important in ischemia-induced translocation of GLUT-4 in the heart.

A Retrospective Evaluation of Vemurafenib as Treatment for BRAF-Mutant Melanoma Brain Metastases.

PubMed

Harding, James J; Catalanotti, Federica; Munhoz, Rodrigo R; Cheng, Donavan T; Yaqubie, Amin; Kelly, Nicole; McDermott, Gregory C; Kersellius, Romona; Merghoub, Taha; Lacouture, Mario E; Carvajal, Richard D; Panageas, Katherine S; Berger, Michael F; Rosen, Neal; Solit, David B; Chapman, Paul B

2015-07-01

RAF inhibitors are an effective therapy for patients with BRAF-mutant melanoma and brain metastasis. Efficacy data are derived from clinical studies enriched with physiologically fit patients; therefore, it is of interest to assess the real-world experience of vemurafenib in this population. Tumor-specific genetic variants that influence sensitivity to RAF kinase inhibitors also require investigation. Records of patients with BRAF-mutant melanoma and brain metastases who were treated with vemurafenib were reviewed. Clinical data were extracted to determine extracranial and intracranial objective response rates, progression-free survival (PFS), overall survival (OS), and safety. A bait-capture, next-generation sequencing assay was used to identify mutations in pretreatment tumors that could explain primary resistance to vemurafenib. Among patients with intracranial disease treated with vemurafenib, 27 were included in survival analyses and 22 patients were assessable for response. The extracranial and intracranial objective response rates were 71% and 50%, respectively. Discordant responses were observed between extracranial and intracranial metastatic sites in 4 of 19 evaluable patients. Median PFS was 4.1 months (95% confidence interval [CI]: 2.6-7.9); median intracranial PFS was 4.6 months (95% CI: 2.7-7.9), median OS was 7.5 months (95% CI: 4.3-not reached), with a 30.4% 1-year OS rate. Outcomes were influenced by performance status. Vemurafenib was tolerable, although radiation-induced dermatitis occurred in some patients who received whole-brain radiotherapy. Adequate samples for next-generation sequencing analysis were available for seven patients. Melanomas categorized as "poorly sensitive" (≥20% tumor growth, new lesions, or ≤50% shrinkage for <4 months) harbored co-occurring mutations in genes predicted to activate the phosphatidylinositol 3-kinase-AKT (PI3K-AKT) pathway. Vemurafenib is highly active in BRAF-mutant melanoma brain metastases but has limited activity in patients with poor performance status. The safety and efficacy of concurrent radiotherapy and RAF inhibition requires careful clinical evaluation. Combination strategies blocking the MAPK and PI3K-AKT pathway may be warranted in a subset of patients. Vemurafenib is active for BRAF-mutant intracranial melanoma metastases in an unselected patient population typical of routine oncologic practice. Patients with poor performance status appear to have poor outcomes despite vemurafenib therapy. Preliminary data indicate that co-occurring or secondary alterations in the phosphatidylinositol 3-kinase-AKT (PI3K-AKT) pathway are involved in resistance to RAF inhibition, thus providing a rationale for dual MAPK and PI3K-AKT pathway inhibition in this patient population. ©AlphaMed Press.

Phosphatidylinositol 3-Kinase (PI3K) δ blockade increases genomic instability in B cells

PubMed Central

Compagno, Mara; Wang, Qi; Pighi, Chiara; Cheong, Taek-Chin; Meng, Fei-Long; Poggio, Teresa; Yeap, Leng-Siew; Karaca, Elif; Blasco, Rafael B.; Langellotto, Fernanda; Ambrogio, Chiara; Voena, Claudia; Wiestner, Adrian; Kasar, Siddha N.; Brown, Jennifer R.; Sun, Jing; Wu, Catherine J.; Gostissa, Monica; Alt, Frederick W.; Chiarle, Roberto

2017-01-01

Activation-induced cytidine deaminase (AID) is a B-cell specific enzyme that targets immunoglobulin (Ig) genes to initiate class switch recombination (CSR) and somatic hypermutation (SHM)1. Through off-target activity, however, AID has a much broader impact on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in lymphoma development and progression2. AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation3. The phosphatidylinositol 3-kinase (PI3K) δ pathway plays a key role in AID regulation by suppressing its expression in B cells4. Novel drugs for leukemia or lymphoma therapy such as idelalisib, duvelisib or ibrutinib block PI3Kδ activity directly or indirectly5–8, potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation (SHM) and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both these effects were completely abrogated in AID deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumors in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IgH and AID off-target sites in human chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased SHM in AID off-targets. In summary, we show that PI3Kδ or BTK inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism, an effect that should be carefully considered as such inhibitors are administered for years to patients. PMID:28199309

A retrospective study of proteinuria in dogs receiving toceranib phosphate.

PubMed

Piscoya, Sindy L; Hume, Kelly R; Balkman, Cheryl E

2018-06-01

The incidence of proteinuria in humans receiving tyrosine kinase inhibitors has been well-documented. Reports of proteinuria with this class of drugs are limited in veterinary medicine. This retrospective study describes the incidence, severity, and progression of proteinuria in 55 dogs treated with toceranib phosphate, with or without concurrent glucocorticoid or NSAID (non-steroidal anti-inflammatory drug). Six dogs were proteinuric at baseline. Twelve of the 49 dogs that were not proteinuric at baseline developed proteinuria while receiving toceranib phosphate. Median urine protein:creatinine (UPC) ratio when proteinuria developed was 0.75 (range: 0.6 to 4.9). There was no association with intermittent glucocorticoid or NSAID use and development of proteinuria ( P = 0.5 and P = 0.7, respectively). Overall duration of toceranib phosphate treatment ranged from 70 to 802 days in proteinuric dogs and 28 to 1285 days in non-proteinuric dogs. Our results indicate a subset of dogs receiving toceranib phosphate may develop proteinuria; careful monitoring with serial UPCs is recommended.

Design and synthesis of imidazopyridine analogues as inhibitors of phosphoinositide 3-kinase signaling and angiogenesis.

PubMed

Kim, Okseon; Jeong, Yujeong; Lee, Hyunseung; Hong, Sun-Sun; Hong, Sungwoo

2011-04-14

Phosphatidylinositol 3-kinase α (PI3Kα) is an important regulator of intracellular signaling pathways, controlling remarkably diverse arrays of physiological processes. Because the PI3K pathway is frequently up-regulated in human cancers, the inhibition of PI3Kα can be a promising approach to cancer therapy. In this study, we have designed and synthesized a new series of imidazo[1,2-a]pyridine derivatives as PI3Kα inhibitors through the fragment-growing strategy. By varying groups at the 3- and 6-positions of imidazo[1,2-a]pyridines, we studied the structure-activity relationships (SAR) profiles and identified a series of potent PI3Kα inhibitors. Representative derivatives showed good activity in cellular proliferation and apoptosis assays. Moreover, these inhibitors exhibited noteworthy antiangiogenic activity.

A Phosphatidylinositol 3-kinase-regulated Akt-independent signaling promotes cigarette smoke-induced FRA-1 expression.

PubMed

Zhang, Qin; Adiseshaiah, Pavan; Kalvakolanu, Dhananjaya V; Reddy, Sekhar P

2006-04-14

The FRA-1 proto-oncogene is overexpressed in a variety of human tumors and is known to up-regulate the expression of genes involved in tumor progression and invasion. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway is also known to regulate these cellular processes. More importantly, respiratory toxicants and carcinogens activate both the PI3K-Akt pathway and FRA-1 expression in human bronchial epithelial (HBE) cells. In this study we investigated a potential link between the PI3K-Akt pathway and the cigarette smoke (CS)-stimulated epidermal growth factor receptor-mediated FRA-1 induction in non-oncogenic HBE cells. Treatment of cells with LY294002, an inhibitor of the PI3K-Akt pathway, completely blocked CS-induced FRA-1 expression. Surprisingly pharmacological inhibition of Akt had no significant effect on CS-induced FRA-1 expression. Likewise the inhibition of protein kinase C zeta, which is a known downstream effector of PI3K, did not alter FRA-1 expression. We found that the PI3K through p21-activated kinase 1 regulates FRA-1 proto-oncogene induction by CS and the subsequent activation of the Elk1 and cAMP-response element-binding protein transcription factors that are bound to the promoter in HBE cells.

The Guareschi Pyridine Scaffold as a Valuable Platform for the Identification of Selective PI3K Inhibitors.

PubMed

Galli, Ubaldina; Ciraolo, Elisa; Massarotti, Alberto; Margaria, Jean Piero; Sorba, Giovanni; Hirsch, Emilio; Tron, Gian Cesare

2015-09-18

A novel series of 4-aryl-3-cyano-2-(3-hydroxyphenyl)-6-morpholino-pyridines have been designed as potential phosphatidylinositol-3-kinase (PI3K) inhibitors. The compounds have been synthesized using the Guareschi reaction to prepare the key 4-aryl-3-cyano-2,6-dihydroxypyridine intermediate. A different selectivity according to the nature of the aryl group has been observed. Compound 9b is a selective inhibitor against the PI3Kα isoform, maintaining a good inhibitory activity. Docking studies were also performed in order to rationalize its profile of selectivity.

Initiative for Molecular Profiling and Advanced Cancer Therapy (IMPACT): An MD Anderson Precision Medicine Study

PubMed Central

Tsimberidou, Apostolia-Maria; Hong, David S.; Ye, Yang; Cartwright, Carrie; Wheler, Jennifer J.; Falchook, Gerald S.; Naing, Aung; Fu, Siqing; Piha-Paul, Sarina; Janku, Filip; Meric-Bernstam, Funda; Hwu, Patrick; Kee, Bryan; Kies, Merrill S.; Broaddus, Russell; Mendelsohn, John; Hess, Kenneth R.; Kurzrock, Razelle

2017-01-01

Purpose Genomic profiling is increasingly used in the management of cancer. We have previously reported preliminary results of our precision medicine program. Here, we present response and survival outcomes for 637 additional patients who were referred for phase I trials and were treated with matched targeted therapy (MTT) when available. Patients and Methods Patients with advanced cancer who underwent tumor genomic analyses were treated with MTT when available. Results Overall, 1,179 (82.1%) of 1,436 patients had one or more alterations (median age, 59.7 years; men, 41.2%); 637 had one or more actionable aberrations and were treated with MTT (n = 390) or non-MTT (n = 247). Patients who were treated with MTT had higher rates of complete and partial response (11% v 5%; P = .0099), longer failure-free survival (FFS; 3.4 v 2.9 months; P = .0015), and longer overall survival (OS; 8.4 v 7.3 months; P = .041) than did unmatched patients. Two-month landmark analyses showed that, for MTT patients, FFS for responders versus nonresponders was 7.6 versus 4.3 months (P < .001) and OS was 23.4 versus 8.5 months (P < .001), whereas for non-MTT patients (responders v nonresponders), FFS was 6.6 versus 4.1 months (P = .001) and OS was 15.2 versus 7.5 months (P = .43). Patients with phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase pathway alterations matched to PI3K/Akt/mammalian target of rapamycin axis inhibitors alone demonstrated outcomes comparable to unmatched patients. Conclusion Our results support the use of genomic matching. Subset analyses indicate that matching patients who harbor a PI3K and mitogen-activated protein kinase pathway alteration to only a PI3K pathway inhibitor does not improve outcome. We have initiated IMPACT2, a randomized trial to compare treatment with and without genomic selection. PMID:29082359

TGF-β directs trafficking of the epithelial sodium channel ENaC which has implications for ion and fluid transport in acute lung injury

PubMed Central

Peters, Dorothea M.; Vadász, István; Wujak, Łukasz; Wygrecka, Małgorzata; Olschewski, Andrea; Becker, Christin; Herold, Susanne; Papp, Rita; Mayer, Konstantin; Rummel, Sebastian; Brandes, Ralph P.; Günther, Andreas; Waldegger, Siegfried; Eickelberg, Oliver; Seeger, Werner; Morty, Rory E.

2014-01-01

TGF-β is a pathogenic factor in patients with acute respiratory distress syndrome (ARDS), a condition characterized by alveolar edema. A unique TGF-β pathway is described, which rapidly promoted internalization of the αβγ epithelial sodium channel (ENaC) complex from the alveolar epithelial cell surface, leading to persistence of pulmonary edema. TGF-β applied to the alveolar airspaces of live rabbits or isolated rabbit lungs blocked sodium transport and caused fluid retention, which—together with patch-clamp and flow cytometry studies—identified ENaC as the target of TGF-β. TGF-β rapidly and sequentially activated phospholipase D1, phosphatidylinositol-4-phosphate 5-kinase 1α, and NADPH oxidase 4 (NOX4) to produce reactive oxygen species, driving internalization of βENaC, the subunit responsible for cell-surface stability of the αβγENaC complex. ENaC internalization was dependent on oxidation of βENaC Cys43. Treatment of alveolar epithelial cells with bronchoalveolar lavage fluids from ARDS patients drove βENaC internalization, which was inhibited by a TGF-β neutralizing antibody and a Tgfbr1 inhibitor. Pharmacological inhibition of TGF-β signaling in vivo in mice, and genetic ablation of the nox4 gene in mice, protected against perturbed lung fluid balance in a bleomycin model of lung injury, highlighting a role for both proximal and distal components of this unique ENaC regulatory pathway in lung fluid balance. These data describe a unique TGF-β–dependent mechanism that regulates ion and fluid transport in the lung, which is not only relevant to the pathological mechanisms of ARDS, but might also represent a physiological means of acutely regulating ENaC activity in the lung and other organs. PMID:24324142

N-WASP and WAVE2 acting downstream of phosphatidylinositol 3-kinase are required for myogenic cell migration induced by hepatocyte growth factor.

PubMed

Kawamura, Kazuhiro; Takano, Kazunori; Suetsugu, Shiro; Kurisu, Shusaku; Yamazaki, Daisuke; Miki, Hiroaki; Takenawa, Tadaomi; Endo, Takeshi

2004-12-24

During skeletal muscle regeneration caused by injury, muscle satellite cells proliferate and migrate toward the site of muscle injury. This migration is mainly induced by hepatocyte growth factor (HGF) secreted by intact myofibers and also released from injured muscle. However, the intracellular machinery for the satellite cell migration has not been elucidated. To examine the mechanisms of satellite cell migration, we utilized satellite cell-derived mouse C2C12 skeletal muscle cells. HGF induced reorganization of actin cytoskeleton to form lamellipodia in C2C12 myoblasts. HGF treatment facilitated both nondirectional migration of the myoblasts in phagokinetic track assay and directional chemotactic migration toward HGF in a three-dimensional migration chamber assay. Endogenous N-WASP and WAVE2 were concentrated in the lamellipodia at the leading edge of the migrating cells. Moreover, exogenous expression of wild-type N-WASP or WAVE2 promoted lamellipodial formation and migration. By contrast, expression of the dominant-negative mutant of N-WASP or WAVE2 and knockdown of N-WASP or WAVE2 expression by the RNA interference prevented the HGF-induced lamellipodial formation and migration. When the cells were treated with LY294002, an inhibitor of phosphatidylinositol 3-kinase, the HGF-induced lamellipodial formation and migration were abrogated. These results imply that both N-WASP and WAVE2, which are activated downstream of phosphati-dylinositol 3-kinase, are required for the migration through the lamellipodial formation of C2C12 cells induced by HGF.

Physical exercise prevents the development of type 2 diabetes mellitus in Psammomys obesus.

PubMed

Heled, Yuval; Shapiro, Yair; Shani, Yoav; Moran, Dani S; Langzam, Lea; Braiman, Liora; Sampson, Sanford R; Meyerovitch, Joseph

2002-02-01

We hypothesized that exercise training might prevent diabetes mellitus in Psammomys obesus. Animals were assigned to three groups: high-energy diet (CH), high-energy diet and exercise (EH), and low-energy diet (CL). The EH group ran on a treadmill 5 days/wk, twice a day. After 4 wk, 93% of the CH group were diabetic compared with only 20% of the EH group. There was no difference in weight gain among the groups. Both EH and CH groups were hyperinsulinemic. Epididymal fat (% of body weight) was higher in the CH group than in either the EH and or the CL group. Protein kinase C (PKC)-delta activity and serine phosphorylation were higher in the EH group. No differences were found in tyrosine phosphorylation of the insulin receptor, insulin receptor substrate-1, and phosphatidylinositol 3-kinase among the groups. We demonstrate for the first time that exercise training effectively prevents the progression of diabetes mellitus type 2 in Psammomys obesus. PKC-delta may be involved in the adaptive effects of exercise in skeletal muscles that lead to the prevention of type 2 diabetes mellitus.

Vasoconstriction induced by G1, a G-protein-coupled oestrogen receptor1 (GPER-1) agonist, in the isolated perfused rat kidney.

PubMed

Kurt, Akif Hakan; Buyukafsar, Kansu

2013-02-28

Vascular effects of the G protein-coupled oestrogen receptor1 (GPER-1) agonist, G1 (10(-7)-5×10(-6) M), the main oestrogenic hormone, 17β-estradiol (10(-9)-10(-4) M), the NR3A1 agonist, PPT (10(-8)-10(-5) M), the NR3A2 agonist DPN (10(-8)-10(-5) M), and the classical oestrogen receptor blocker but also a GPER agonist, ICI-182780 (10(-8)-3×10(-6) M), were investigated on the perfusion pressure in the isolated rat kidney. To seek cellular mechanisms involved in GPER-1-induced signalling we tested several compounds including the inhibitors of Rho-kinase (ROCK) (Y-27632), tyrosine kinase (genistein), p38MAPK (SB203580), p44/42MAPK (PD98059), protein kinase C (PKC) (GF109203X), Jun-kinase (JNK) (SP600125), phosphatidylinositol-3-kinase (PI3K) (LY294002), Ca(2+) channels (nifedipine), GPER-1 (G15) and epidermal growth factor (EGF) receptor kinase (AG-1478). Moreover, the effect of saponin (50mg/ml) that was used for endothelium removal was explored on G1-elicited vascular action. G1, 17β-estradiol and ICI-182780 but not PPT and DPN induced vasoconstrictions in basal renal perfusion pressure. In contrast, G1 promoted vasodilatation when the perfusion pressure was elevated in advance by phenylephrine. G1-elicited vasoconstriction was not modified by endothelial removal; however, it was markedly inhibited by GPER-1 antagonist, G15. The vasoconstrictor response to G1 was also significantly attenuated by Y-27632, PD98059, SB203580, GF109203X, genistein, AG-1478, and nifedipine, but not LY294002 and SP600125. Western blotting indicated the expression of GPER-1 in renal artery, medulla and cortex of rat kidney. In conclusion, GPER-1 could substantially modulate vascular responses through a variety of signalling pathways including ROCK, PKC, p38 MAPK, p42/44 MAPK, tyrosine kinase, EGF receptor kinase and VOCC but not JNK or PI3K in isolated perfused rat kidney. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of aniracetam on phosphatidylinositol transfer protein alpha in cytosolic and plasma membrane fractions of astrocytes subjected to simulated ischemia in vitro.

PubMed

Gabryel, Bozena; Chalimoniuk, Małgorzata; Małecki, Andrzej; Strosznajder, Joanna B

2005-01-01

Brain ischemia affects phosphoinositide metabolism and the level of lipid-derived second messengers. Phosphatidylinositol transfer proteins (PI-PTs) are responsible for the transport of phosphatidylinositol (PI) and other phospholipids through membranes. Isoform of PI-TPs (PI-TPalpha) is an essential component in ensuring substrate supply for phospholipase C (PLC). The current study was conducted to examine potential effect of aniracetam on PI-TPalpha expression and to characterize the PI-TPalpha isoform distribution between membrane and cytosol fractions of astrocytes exposed to simulated ischemia in vitro. After 8 h period of ischemia, the level of PI-TPalpha was significantly higher in cytosol (by about 28%) as well as in membrane fraction (by about 80%) in comparison with control. We have found that aniracetam treatment of astrocytes in normoxia significantly increased the level of PI-TPalpha in membrane fraction with a maximal effect at 0.1 microM concentration of aniracetam (by about 195% of control). In membrane fractions of ischemic cells, aniracetam increased PI-TPalpha expression in a concentration-dependent manner. In ischemic cells, aniracetam (10 microM) has elevated PI-TPalpha expression up to 155% and 428% in cytosolic and membrane fractions in comparison with ischemic untreated cells, respectively. The study has shown that aniracetam significantly activates PI-TPalpha in cell membrane fraction and this effect might be connected with previously described activation of MAP kinase cascade.

Pterostilbene protects against UVB-induced photo-damage through a phosphatidylinositol-3-kinase-dependent Nrf2/ARE pathway in human keratinocytes.

PubMed

Li, Huaping; Jiang, Na; Liang, Bihua; Liu, Qing; Zhang, Erting; Peng, Liqian; Deng, Huiyan; Li, Runxiang; Li, Zhenjie; Zhu, Huilan

2017-11-01

Ultraviolet B (UVB) irradiation is the initial etiological factor for various skin disorders, including erythema, sunburn, photoaging, and photocarcinogenesis. Pterostilbene (Pter) displayed remarkable antioxidant, anti-inflammatory, and anticarcinogenic activities. This study aimed to investigate the effective mechanism of Pter against UVB-induced photodamage in immortalized human keratinocytes. Human keratinocytes were pretreated with Pter (5 and 10 μM) for 24 h prior to UVB irradiation (300 mJ/cm 2 ). Harvested cells were analyzed by MTT, DCFH-DA, comet, western blotting, luciferase promoter, small interference RNA transfection, and quantitative real-time polymerase chain reaction assay. Pter significantly attenuated UVB-induced cell death and reactive oxygen species (ROS) generation, and effectively increased nuclear translocation of NF-E2-related factor-2 (Nrf2), expression of Nrf2-dependent antioxidant enzymes, and DNA repair activity. Moreover, the protective effects of Pter were abolished by small interference RNA-mediated Nrf2 silencing. Furthermore, Pter was also found to induce the phosphorylation of Nrf2 and the known phosphatidylinositol-3-kinase (PI3K) phosphorylated kinase, Akt. The specific inhibitor of PI3K, LY294002, successfully abrogated Pter-induced Nrf2 phosphorylation, activation of Nrf2-antioxidant response element pathway, ROS scavenging ability, and DNA repair activity. The present study indicated that Pter effectively protected against UVB-induced photodamage by increasing endogenous defense mechanisms, scavenging UVB-induced ROS, and aiding in damaged DNA repair through a PI3K-dependent activation of Nrf2/ARE pathway.

Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities

PubMed Central

Hubbard, Paul A.; Moody, Colleen L.; Murali, Ramachandran

2014-01-01

GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways. PMID:25566081

The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila.

PubMed

Juhász, Gábor; Hill, Jahda H; Yan, Ying; Sass, Miklós; Baehrecke, Eric H; Backer, Jonathan M; Neufeld, Thomas P

2008-05-19

Degradation of cytoplasmic components by autophagy requires the class III phosphatidylinositol 3 (PI(3))-kinase Vps34, but the mechanisms by which this kinase and its lipid product PI(3) phosphate (PI(3)P) promote autophagy are unclear. In mammalian cells, Vps34, with the proautophagic tumor suppressors Beclin1/Atg6, Bif-1, and UVRAG, forms a multiprotein complex that initiates autophagosome formation. Distinct Vps34 complexes also regulate endocytic processes that are critical for late-stage autophagosome-lysosome fusion. In contrast, Vps34 may also transduce activating nutrient signals to mammalian target of rapamycin (TOR), a negative regulator of autophagy. To determine potential in vivo functions of Vps34, we generated mutations in the single Drosophila melanogaster Vps34 orthologue, causing cell-autonomous disruption of autophagosome/autolysosome formation in larval fat body cells. Endocytosis is also disrupted in Vps34(-/-) animals, but we demonstrate that this does not account for their autophagy defect. Unexpectedly, TOR signaling is unaffected in Vps34 mutants, indicating that Vps34 does not act upstream of TOR in this system. Instead, we show that TOR/Atg1 signaling regulates the starvation-induced recruitment of PI(3)P to nascent autophagosomes. Our results suggest that Vps34 is regulated by TOR-dependent nutrient signals directly at sites of autophagosome formation.

Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.

PubMed

Ishikawa-Sasaki, Kumiko; Nagashima, Shigeo; Taniguchi, Koki; Sasaki, Jun

2018-04-15

Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs. IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of viral RNA replication sites for replication. Previously, we demonstrated that Aichi virus (AiV), a picornavirus, forms a complex comprising certain proteins of AiV, the Golgi apparatus protein ACBD3, and the lipid kinase PI4KB to synthesize PI4P lipid at the sites for AiV RNA replication. Here, we confirmed cholesterol accumulation at the AiV RNA replication sites, which are established by hijacking the host cholesterol transfer machinery mediated by a PI4P-binding cholesterol transfer protein, OSBP. We showed that the component proteins of the machinery, OSBP, VAP, SAC1, and PITPNB, are all essential host factors for AiV replication. Importantly, the machinery is directly recruited to the RNA replication sites through previously unknown interactions of VAP/OSBP/SAC1 with the AiV proteins and with ACBD3. Consequently, we propose a specific strategy employed by AiV to efficiently accumulate cholesterol at the RNA replication sites via protein-protein interactions. Copyright © 2018 American Society for Microbiology.

The effect on some enzymes of rat tissue of diets low in fat content.

PubMed

Bartley, W; Dean, B; Taylor, C B; Bailey, E

1967-05-01

1. Rats of two strains were kept on three different diets; one was a commercial diet of rat pellets, one contained about 80% of sucrose and 20% of casein and was supplemented with corn oil, and the third was a similar diet without the corn oil. 2. On the commercial diet, the specific activities of pyruvate kinase, glucose 6-phosphate dehydrogenase and fructose 1,6-diphosphatase in the livers of one strain of rats (strain A) were 1.5-3 times those in the other strain (strain B). When the diet high in sucrose and supplemented with corn oil was given, there were large increases in the specific activity of pyruvate kinase, glucose 6-phosphate dehydrogenase and fructose 1,6-diphosphatase in the livers of strain A rats. With strain B rats the increases were much smaller. Omission of corn oil from the diet caused a threefold increase in the specific activity of glucose 6-phosphate dehydrogenase in strain B rats, but had little effect on other enzymes. 3. The enzymes of the kidneys and hearts of strain A rats were also more active than those of strain B rats. In strain A rats, the specific activities of pyruvate kinase and fructose 1,6-diphosphatase in the kidney increased when the sucrose content of the diet was high, but in the kidneys of strain B rats there was little change. 4. In strain A rats, the specific activity of pyruvate kinase in the heart more than doubled with the high-sucrose-corn oil diet and increased threefold when corn oil was omitted. No changes were seen in strain B rats. 5. In strain A rats, omission of corn oil from the diet increased the ability of the kidneys to synthesize glucose from lactate. 6. In strain B rats, addition of corn oil to the diet resulted in a decrease in the liver in the specific activity of ATP citrate lyase and in the ability to incorporate acetate into lipid.