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TLR2 ligands attenuate cardiac dysfunction in polymicrobial sepsis via a phosphoinositide 3-kinase-dependent mechanism  

PubMed Central

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. In the present study, we examined the effect of Toll-like receptor 2 (TLR2) ligands, peptidoglycan (PGN), and Pam3CSK4 (Pam3) on cardiac function in cecal ligation and puncture (CLP)-induced sepsis in mice. We also investigated whether the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is involved in the effect of TLR2 ligands on cardiac function in CLP mice. PGN was administered to C57B6/L mice 1 h before the induction of CLP. Sham surgically operated mice served as a control. Cardiac function indexes (rate of change in left ventricular pressure, stroke work, cardiac output, and ejection fraction) were examined by a microconductance pressure catheter. Cardiac function was significantly decreased 6 h after CLP-induced sepsis compared with sham-operated control. In contrast, PGN administration attenuated CLP-induced cardiac dysfunction. Importantly, the therapeutic treatment with Pam3 1 h after CLP also significantly attenuated cardiac dysfunction in CLP mice. However, the beneficial effect of TLR2 ligands on cardiac dysfunction in CLP-mice was abolished in TLR2-deficient mice. PGN administration significantly increased the levels of phospho-Akt and phospho-GSK-3? in the myocardium compared with the levels in untreated CLP mice. PI3K inhibition abolished the PGN-induced attenuation of cardiac dysfunction in CLP mice. In conclusion, these data demonstrate that the administration of TLR2 ligands, PGN, or Pam3 attenuates cardiac dysfunction in septic mice via a TLR2/PI3K-dependent mechanism. More significantly, Pam3 therapeutic treatment will have a potential clinical relevance.

Ha, Tuanzhu; Lu, Chen; Liu, Li; Hua, Fang; Hu, Yulong; Kelley, Jim; Singh, Krishna; Kao, Race L.; Kalbfleisch, John; Williams, David L.; Gao, Xiang



The inositol polyphosphate 5-phosphatase, PIPP, Is a novel regulator of phosphoinositide 3-kinase-dependent neurite elongation.  


The spatial activation of phosphoinositide 3-kinase (PI3-kinase) signaling at the axon growth cone generates phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3), which localizes and facilitates Akt activation and stimulates GSK-3beta inactivation, promoting microtubule polymerization and axon elongation. However, the molecular mechanisms that govern the spatial down-regulation of PtdIns(3,4,5)P3 signaling at the growth cone remain undetermined. The inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) and/or PtdIns(3,4,5)P3. We demonstrate here that PIPP, an uncharacterized 5-phosphatase, hydrolyzes PtdIns(3,4,5)P3 forming PtdIns(3,4)P2, decreasing Ser473-Akt phosphorylation. PIPP is expressed in PC12 cells, localizing to the plasma membrane of undifferentiated cells and the neurite shaft and growth cone of NGF-differentiated neurites. Overexpression of wild-type, but not catalytically inactive PIPP, in PC12 cells inhibited neurite elongation. Targeted depletion of PIPP using RNA interference (RNAi) resulted in enhanced neurite differentiation, associated with neurite hyperelongation. Inhibition of PI3-kinase activity prevented neurite hyperelongation in PIPP-deficient cells. PIPP targeted-depletion resulted in increased phospho-Ser473-Akt and phospho-Ser9-GSK-3beta, specifically at the neurite growth cone, and accumulation of PtdIns(3,4,5)P3 at this site, associated with enhanced microtubule polymerization in the neurite shaft. PIPP therefore inhibits PI3-kinase-dependent neurite elongation in PC12 cells, via regulation of the spatial distribution of phospho-Ser473-Akt and phospho-Ser9-GSK-3beta signaling. PMID:16280363

Ooms, Lisa M; Fedele, Clare G; Astle, Megan V; Ivetac, Ivan; Cheung, Vanessa; Pearson, Richard B; Layton, Meredith J; Forrai, Ariel; Nandurkar, Harshal H; Mitchell, Christina A



Phosphoinositide 3-kinase-dependent Ras activation by tauroursodesoxycholate in rat liver.  

PubMed Central

Ursodesoxycholic acid, widely used for the treatment of cholestatic liver disease, causes choleretic, anti-apoptotic and immunomodulatory effects. Here the effects on choleresis of its taurine conjugate tauroursodesoxycholate (TUDC), which is present in the enterohepatic circulation, were correlated with the activation of important elements of intracellular signal transduction in cultured rat hepatocytes and perfused rat liver. TUDC induced a time- and concentration-dependent activation of the small GTP-binding protein Ras and of phosphoinositide 3-kinase (PI 3-kinase) in cultured hepatocytes. Ras activation was dependent on PI 3-kinase activity, without the involvement of protein kinase C- and genistein-sensitive tyrosine kinases. Ras activation by TUDC was followed by an activation of the mitogen-activated protein kinases extracellular-signal-regulated kinase-1 (Erk-1) and Erk-2. In perfused rat liver, PI 3-kinase inhibitors largely abolished the stimulatory effect of TUDC on taurocholate excretion, suggesting an important role for a PI 3-kinase/Ras/Erk pathway in the choleretic effect of TUDC.

Kurz, A K; Block, C; Graf, D; Dahl, S V; Schliess, F; Haussinger, D



The West Nile Virus Capsid Protein Blocks Apoptosis through a Phosphatidylinositol 3-Kinase-Dependent Mechanism  

PubMed Central

West Nile virus (WNV) is a mosquito-transmitted pathogen that can cause serious disease in humans. Our laboratories are focused on understanding how interactions between WNV proteins and host cells contribute to virus replication and pathogenesis. WNV replication is relatively slow, and on the basis of earlier studies, the virus appears to activate survival pathways that delay host cell death during virus replication. The WNV capsid is the first viral protein produced in infected cells; however, its role in virus assembly is not required until after replication of the genomic RNA. Accordingly, from a temporal perspective, it is perfectly suited to block host cell apoptosis during virus replication. In the present study, we provide evidence that the WNV capsid protein blocks apoptosis through a phosphatidylinositol (PI) 3-kinase-dependent pathway. Specifically, expression of this protein in the absence of other viral proteins increases the levels of phosphorylated Akt, a prosurvival kinase that blocks apoptosis through multiple mechanisms. Treatment of cells with the PI 3-kinase inhibitor LY294002 abrogates the protective effects of the WNV capsid protein.

Urbanowski, Matt D.



Aromatase induction in tamoxifen-resistant breast cancer: Role of phosphoinositide 3-kinase-dependent CREB activation.  


Estrogens are important for the development and growth of estrogen receptor (ER)-positive breast cancer, for which anti-estrogen therapy is one of the most effective treatments. However, its efficacy can be limited by either de novo or acquired resistance. Aromatase is a key enzyme for the biosynthesis of estrogens, and inhibition of this enzyme leads to profound hypoestrogenism. Here, we found that the basal expression and activity of aromatase were significantly increased in tamoxifen (TAM)-resistant human breast cancer (TAMR-MCF-7) cells compared to control MCF-7 cells. We further revealed that aromatase immunoreactivity in tumor tissues was increased in recurrence group after TAM therapy compared to non-recurrence group after TAM therapy. Phosphorylation of Akt, extracellular signal-regulated kinase (ERK), and p38 kinase were all increased in TAMR-MCF-7 cells. Inhibition of phosphoinositide 3-kinase (PI3K) suppressed the transactivation of the aromatase gene and its enzyme activity. Furthermore, we have also shown that PI3K/Akt-dependent cAMP-response element binding protein (CREB) activation was required for the enhanced expression of aromatase in TAMR-MCF-7 cells. Our findings suggest that aromatase expression is up-regulated in TAM-resistant breast cancer via PI3K/Akt-dependent CREB activation. PMID:24836190

Phuong, Nguyen Thi Thuy; Lim, Sung Chul; Kim, Young Mi; Kang, Keon Wook



Phosphoinositide 3-kinase-dependent phosphorylation of the dual adaptor for phosphotyrosine and 3-phosphoinositides by the Src family of tyrosine kinase.  

PubMed Central

We recently identified a novel adaptor protein, termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), that possesses a Src homology (SH2) domain and a pleckstrin homology (PH) domain. DAPP1 exhibits a high-affinity interaction with PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2), which bind to the PH domain. In the present study we show that when DAPP1 is expressed in HEK-293 cells, the agonists insulin, insulin-like growth factor-1 and epidermal growth factor induce the phosphorylation of DAPP1 at Tyr(139). Treatment of cells with phosphoinositide 3-kinase (PI 3-kinase) inhibitors or expression of a dominant-negative PI 3-kinase prevent phosphorylation of DAPP1 at Tyr(139), and a PH-domain mutant of DAPP1, which does not interact with PtdIns(3,4,5)P(3) or PtdIns(3,4)P(2), is not phosphorylated at Tyr(139) following agonist stimulation of cells. Overexpression of a constitutively active form of PI 3-kinase induced the phosphorylation of DAPP1 in unstimulated cells. We demonstrated that Tyr(139) of DAPP1 is likely to be phosphorylated in vivo by a Src-family tyrosine kinase, since the specific Src-family inhibitor, PP2, but not an inactive variant of this drug, PP3, prevented the agonist-induced tyrosine phosphorylation of DAPP1. Src, Lyn and Lck tyrosine kinases phosphorylate DAPP1 at Tyr(139) in vitro at similar rates in the presence or absence of PtdIns(3,4,5)P(3), and overexpression of these kinases in HEK-293 cells induces the phosphorylation of Tyr(139). These findings indicate that, following activation of PI 3-kinases, PtdIns(3,4,5)P(3) or PtdIns(3,4)P(2) bind to DAPP1, recruiting it to the plasma membrane where it becomes phosphorylated at Tyr(139) by a Src-family tyrosine kinase.

Dowler, S; Montalvo, L; Cantrell, D; Morrice, N; Alessi, D R



Ephrin reverse signaling controls palate fusion via a PI3 kinase-dependent mechanism  

PubMed Central

Secondary palate fusion requires adhesion and epithelial to mesenchymal transition (EMT) of the epithelial layers on opposing palatal shelves. This EMT requires transforming growth factor ?3 (TGF?3), and its failure results in cleft palate. Ephrins, and their receptors, the Ephs, are responsible for migration, adhesion, and midline closure events throughout development. Ephrins can also act as signal transducing receptors in these processes, with the Ephs serving as ligands (termed "reverse" signaling). We found that activation of ephrin reverse signaling in chicken palates induced fusion in the absence of TGF?3, and that PI3K inhibition abrogated this effect. Further, blockage of reverse signaling inhibited TGF?3-induced fusion in the chicken and natural fusion in the mouse. Thus, ephrin reverse signaling is necessary and sufficient to induce palate fusion independent of TGF?3. These data describe both a novel role for ephrins in palate morphogenesis, and a previously unknown mechanism of ephrin signaling.

Miguel, Symone San; Serrano, Maria J.; Sachar, Ashneet; Henkemeyer, Mark; Svoboda, Kathy K. H.; Benson, M. Douglas



RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism  

SciTech Connect

In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as a Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85{alpha} and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.

Yamaki, Nao [Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Negishi, Manabu [Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Katoh, Hironori [Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501 (Japan)]. E-mail:



Apelin-13 Inhibits Large-Conductance Ca2+-Activated K+ Channels in Cerebral Artery Smooth Muscle Cells via a PI3-Kinase Dependent Mechanism  

PubMed Central

Apelin-13 causes vasoconstriction by acting directly on APJ receptors in vascular smooth muscle (VSM) cells; however, the ionic mechanisms underlying this action at the cellular level remain unclear. Large-conductance Ca2+-activated K+ (BKCa) channels in VSM cells are critical regulators of membrane potential and vascular tone. In the present study, we examined the effect of apelin-13 on BKCa channel activity in VSM cells, freshly isolated from rat middle cerebral arteries. In whole-cell patch clamp mode, apelin-13 (0.001-1 ?M) caused concentration-dependent inhibition of BKCa in VSM cells. Apelin-13 (0.1 µM) significantly decreased BKCa current density from 71.25±8.14 pA/pF to 44.52±7.10 pA/pF (n=14 cells, P<0.05). This inhibitory effect of apelin-13 was confirmed by single channel recording in cell-attached patches, in which extracellular application of apelin-13 (0.1 µM) decreased the open-state probability (NPo) of BKCa channels in freshly isolated VSM cells. However, in inside-out patches, extracellular application of apelin-13 (0.1µM) did not alter the NPo of BKCa channels, suggesting that the inhibitory effect of apelin-13 on BKCa is not mediated by a direct action on BKCa. In whole cell patches, pretreatment of VSM cells with LY-294002, a PI3-kinase inhibitor, markedly attenuated the apelin-13-induced decrease in BKCa current density. In addition, treatment of arteries with apelin-13 (0.1 µM) significantly increased the ratio of phosphorylated-Akt/total Akt, indicating that apelin-13 significantly increases PI3-kinase activity. Taken together, the data suggest that apelin-13 inhibits BKCa channel via a PI3-kinase-dependent signaling pathway in cerebral artery VSM cells, which may contribute to its regulatory action in the control of vascular tone.

O'Rourke, Stephen T.; Sun, Chengwen



Insulin Generates Free Radicals by an NAD(P)H, Phosphatidylinositol 3'-Kinase-Dependent Mechanism in Human Skin Fibroblasts Ex Vivo  

Microsoft Academic Search

Oxidative stress may be involved in the development of vascular complications associated with diabetes; how- ever, the molecular mechanism responsible for in- creased production of free radicals in diabetes remains uncertain. Therefore, we examined whether acute hy- perinsulinemia increases the production of free radicals and whether this condition affects proliferative extra- cellular signalregulated kinase (ERK-1 and -2) signal- ing in

Giulio Ceolotto; Michela Bevilacqua; Italia Papparella; Elisabetta Baritono; Lorenzo Franco; Carlo Corvaja; Martina Mazzoni; Andrea Semplicini; Angelo Avogaro



Transforming Growth Factor ?1 Inhibits Cystic Fibrosis Transmembrane Conductance Regulator-dependent cAMP-stimulated Alveolar Epithelial Fluid Transport via a Phosphatidylinositol 3-Kinase-dependent Mechanism*  

PubMed Central

Exogenous or endogenous ?2-adrenergic receptor agonists enhance alveolar epithelial fluid transport via a cAMP-dependent mechanism that protects the lungs from alveolar flooding in acute lung injury. However, impaired alveolar fluid clearance is present in most of the patients with acute lung injury and is associated with increased mortality, although the mechanisms responsible for this inhibition of the alveolar epithelial fluid transport are not completely understood. Here, we found that transforming growth factor ?1 (TGF-?1), a critical mediator of acute lung injury, inhibits ?2-adrenergic receptor agonist-stimulated vectorial fluid and Cl? transport across primary rat and human alveolar epithelial type II cell monolayers. This inhibition is due to a reduction in the cystic fibrosis transmembrane conductance regulator activity and biosynthesis mediated by a phosphatidylinositol 3-kinase (PI3K)-dependent heterologous desensitization and down-regulation of the ?2-adrenergic receptors. Consistent with these in vitro results, inhibition of the PI3K pathway or pretreatment with soluble chimeric TGF-? type II receptor restored ?2-adrenergic receptor agonist-stimulated alveolar epithelial fluid transport in an in vivo model of acute lung injury induced by hemorrhagic shock in rats. The results demonstrate a novel role for TGF-?1 in impairing the ?- adrenergic agonist-stimulated alveolar fluid clearance in acute lung injury, an effect that could be corrected by using PI3K inhibitors that are safe to use in humans.

Roux, Jeremie; Carles, Michel; Koh, Hidefumi; Goolaerts, Arnaud; Ganter, Michael T.; Chesebro, Brian B.; Howard, Marybeth; Houseman, Benjamin T.; Finkbeiner, Walter; Shokat, Kevan M.; Paquet, Agnes C.; Matthay, Michael A.; Pittet, Jean-Francois



Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling.  

PubMed Central

Insulin plays a key role in regulating a wide range of cellular processes. However, until recently little was known about the signalling pathways that are involved in linking the insulin receptor with downstream responses. It is now apparent that the activation of class 1a phosphoinositide 3-kinase (PI 3-kinase) is necessary and in some cases sufficient to elicit many of insulin's effects on glucose and lipid metabolism. The lipid products of PI 3-kinase act as both membrane anchors and allosteric regulators, serving to localize and activate downstream enzymes and their protein substrates. One of the major ways these lipid products of PI 3-kinase act in insulin signalling is by binding to pleckstrin homology (PH) domains of phosphoinositide-dependent protein kinase (PDK) and protein kinase B (PKB) and in the process regulating the phosphorylation of PKB by PDK. Using mechanisms such as this, PI 3-kinase is able to act as a molecular switch to regulate the activity of serine/threonine-specific kinase cascades important in mediating insulin's effects on endpoint responses.

Shepherd, P R; Withers, D J; Siddle, K



Cholesterol stabilizes fluid phosphoinositide domains.  


Local accumulation of phosphoinositides (PIPs) is an important factor for a broad range of cellular events including membrane trafficking and cell signaling. The negatively charged phosphoinositide headgroups can interact with cations or cationic proteins and this electrostatic interaction has been identified as the main phosphoinositide clustering mechanism. However, an increasing number of reports show that phosphoinositide-mediated signaling events are at least in some cases cholesterol dependent, suggesting other possible contributors to the segregation of phosphoinositides. Using fluorescence microscopy on giant unilamellar vesicles and monolayers at the air/water interface, we present data showing that cholesterol stabilizes fluid phosphoinositide-enriched phases. The interaction with cholesterol is observed for all investigated phosphoinositides (PI(4)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3) as well as phosphatidylinositol. We find that cholesterol is present in the phosphoinositide-enriched phase and that the resulting phase is fluid. Cholesterol derivatives modified at the hydroxyl group (cholestenone, cholesteryl ethyl ether) do not promote formation of phosphoinositide domains, suggesting an instrumental role of the cholesterol hydroxyl group in the observed cholesterol/phosphoinositide interaction. This leads to the hypothesis that cholesterol participates in an intermolecular hydrogen bond network formed among the phosphoinositide lipids. We had previously reported that the intra- and intermolecular hydrogen bond network between the phosphoinositide lipids leads to a reduction of the charge density at the phosphoinositide phosphomonoester groups (Kooijman et al., 2009). We believe that cholesterol acts as a spacer between the phosphoinositide lipids, thereby reducing the electrostatic repulsion, while participating in the hydrogen bond network, leading to its further stabilization. To illustrate the effect of phosphoinositide segregation on protein binding, we show that binding of the tumor suppressor protein PTEN to PI(5)P and PI(4,5)P2 is enhanced in the presence of cholesterol. These results provide new insights into how phosphoinositides mediate important cellular events. PMID:24556334

Jiang, Zhiping; Redfern, Roberta E; Isler, Yasmin; Ross, Alonzo H; Gericke, Arne



Phosphoinositide Phosphatases in Cell Biology and Disease  

PubMed Central

Phosphoinositides are essential signaling molecules linked to a diverse array of cellular processes in eukaryotic cells. The metabolic interconversions of these phospholipids are subject to exquisite spatial and temporal regulation executed by arrays of phosphatidylinositol (PtdIns) and phosphoinositide-metabolizing enzymes. These include PtdIns- and phosphoinositide-kinases that drive phosphoinositide synthesis, and phospholipases and phosphatases that regulate phosphoinositide degradation. In the past decade, phosphoinositide phosphatases have emerged as topics of particular interest. This interest is driven by the recent appreciation that these enzymes represent primary mechanisms for phosphoinositide degradation, and because of their ever-increasing connections with human diseases. Herein, we review the biochemical properties of six major phosphoinositide phosphatases, the functional involvements of these enzymes in regulating phosphoinositide metabolism, the pathologies that arise from functional derangements of individual phosphatases, and recent ideas concerning the involvements of phosphoinositide phosphatases in membrane traffic control.

Liu, Yang; Bankaitis, Vytas A.



Acanthamoeba castellanii Induces Host Cell Death via a Phosphatidylinositol 3-Kinase-Dependent Mechanism  

Microsoft Academic Search

Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and trans- migrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate be- longing to the T1 genotype on human brain microvascular endothelial cells, which constitute

James Sissons; Kwang Sik Kim; Monique Stins; Samantha Jayasekera; Selwa Alsam; Naveed Ahmed Khan



TLR-induced activation of neutrophils promotes histamine production via a PI3 kinase dependent mechanism.  


Histamine is a bioactive amine that exerts immunomodulatory functions, including many allergic symptoms. It is preformed and stored in mast cells and basophils but recent evidence suggests that other cell types produce histamine in an inducible fashion. During infection, it has been suggested that neutrophils may produce histamine. We also observed that histamine is released in a neutrophil-mediated LPS-induced model of acute lung injury. Therefore, we sought to examine whether innate signals promote histamine production by neutrophils. Bone marrow-derived neutrophils stimulated with a range of TLR agonists secreted histamine in response to LPS or R837, suggesting TLR4 or TLR7 are important. LPS-driven histamine was enhanced by coculture with GM-CSF and led to a transient release of histamine that peaked at 8h post stimulation. This was dependent upon de novo synthesis of histamine, since cells derived from histidine decarboxylase (HDC) deficient mice were unable to produce histamine but did generate reactive oxygen species upon stimulation. Using pharmacological inhibitors, we show that histamine production requires PI3 kinase, which has been shown to regulate other neutrophil functions, including activation and selective granule release. However, unlike mast cells, HDC deficiency did not alter the granule structure of neutrophils, suggesting that histamine does not participate in granule integrity in these cells. Consequently, our findings establish that neutrophils generate histamine in response to a select panel of innate immune triggers and that this might contribute to acute lung injury responses. PMID:21903133

Smuda, Craig; Wechsler, Joshua B; Bryce, Paul J



TLR-induced activation of neutrophils promotes histamine production via a PI3 kinase dependent mechanism  

PubMed Central

Histamine is a bioactive amine that exerts immunomodulatory functions, including many allergic symptoms. It is preformed and stored in mast cells and basophils but recent evidence suggests that other cell types produce histamine in an inducible fashion. During infection, it has been suggested that neutrophils may produce histamine. We also observed that histamine is released in a neutrophil-mediated LPS-induced model of acute lung injury. Therefore, we sought to examine whether innate signals promote histamine production by neutrophils. Bone marrow-derived neutrophils stimulated with a range of TLR agonists secreted histamine in response to LPS or R837, suggesting TLR4 or TLR7 are important. LPS-driven histamine was enhanced by coculture with GM-CSF and led to a transient release of histamine that peaked at 8 hours post stimulation. This was dependent upon de novo synthesis of histamine, since cells derived from histidine decarboxylase (HDC) deficient mice were unable to produce histamine but did generate reactive oxygen species upon stimulation. Using pharmacological inhibitors, we show that histamine production requires PI3 kinase, which has been shown to regulate other neutrophil functions, including activation and selective granule release. However, unlike mast cells, HDC deficiency did not alter the granule structure of neutrophils, suggesting that histamine does not participate in granule integrity in these cells. Consequently, our findings establish that neutrophils generate histamine in response to a select panel of innate immune triggers and that this might contribute to acute lung injury responses.

Smuda, Craig; Wechsler, Joshua B; Bryce, Paul J



Preferred orientations of phosphoinositides in bilayers and their implications in protein recognition mechanisms.  


Phosphoinositides (PIPs), phosphorylated derivatives of phosphatidylinositol (PI), are essential regulatory lipids involved in various cellular processes, including signal transduction, membrane trafficking, and cytoskeletal remodeling. To gain insight into the protein-PIPs recognition process, it is necessary to study the inositol ring orientation (with respect to the membrane) of PIPs with different phosphorylation states. In this study, 8 PIPs (3 PIP, 2 PIP2, and 3 PIP3) with different phosphorylation and protonation sites have been separately simulated in two mixed bilayers (one with 20% phosphatidylserine (PS) lipids and another with PS lipids switched to phosphatidylcholine (PC) lipids), which roughly correspond to yeast membranes. Uniformity of the bilayer properties including hydrophobic thickness, acyl chain order parameters, and heavy atom density profiles is observed in both PS-contained and PC-enriched membranes due to the same hydrophobic core composition. The relationship between the inositol ring orientation (tilt and rotation angles) and its solvent-accessible surface area indicates that the orientation is mainly determined by its solvation energy. Different PIPs exhibit a clear preference in the inositol ring rotation angle. Surprisingly, a larger proportion of PIPs inositol rings stay closer to the surface of PS-contained membranes compared to PC-enriched ones. Such a difference is rationalized with the formation of more hydrogen bonds between the PS/PI headgroups and the PIPs inositol rings in PS-contained membranes. This hydrogen bond network could be functionally important; thus, the present results can potentially add important and detailed features into the existing protein-PIPs recognition mechanism. PMID:24689790

Wu, Emilia L; Qi, Yifei; Song, Kevin C; Klauda, Jeffery B; Im, Wonpil



What a Tangled Web We Weave: Emerging Resistance Mechanisms to Inhibition of the Phosphoinositide 3-kinase Pathway  

PubMed Central

The phosphoinositide 3-kinase (PI3K) pathway is one of the most frequently mutated pathways in cancer, and is actively being pursued as a therapeutic target. Despite the importance of the PI3K pathway in cancer, durable responses to PI3K-pathway targeted therapies are uncommon with monotherapy. Several in vitro and xenograft models have elucidated compensatory signaling and genomic changes which may limit the therapeutic effectiveness of PI3K inhibitors in the clinic. Future clinical trials with prospective evaluation of tumor signaling and genomic changes are likely to identify novel resistance mechanisms as well as subsets of patients who may derive maximal benefit from PI3K pathway inhibitors.

Klempner, Samuel J.; Myers, Andrea P.; Cantley, Lewis C.



Leptin increases cardiomyocyte hyperplasia via extracellular signal-regulated kinase- and phosphatidylinositol 3-kinase-dependent signaling pathways.  


Obesity is a major risk factor for the development of heart failure. Importantly, it is now appreciated that a change in the number of myocytes is one of multiple structural and functional alterations (remodeling) leading to heart failure. Here we investigate the effect of leptin, the product of the obese (ob) gene, on proliferation of human and murine cardiomyocytes. Leptin caused a time- and dose-dependent significant increase in proliferation of HL-1 cells that was inhibited by preincubation with PD98059 and LY294002, suggesting that leptin mediated proliferation via extracellular signal-regulated kinase-1/2- and phosphatidylinositol-3-kinase-dependent signaling pathways. We confirmed that leptin activates both extracellular signal-regulated kinase-1/2 phosphorylation and association of phosphatidylinositol-3-kinase (regulatory p85 subunit) with phosphotyrosine immunoprecipitates. We also examined bromodeoxyuridine incorporation as a measure of new DNA synthesis and demonstrated a stimulatory effect of leptin in both HL-1 cells and human cardiomyocytes. Bromodeoxyuridine incorporation in HL-1 cells was inhibited by PD98059 and LY294002. Our results establish a mitogenic effect of leptin in cardiomyocytes and provide additional evidence for a potential direct link between leptin and cardiac remodeling in obesity. PMID:14715711

Tajmir, Panteha; Ceddia, Rolando B; Li, Ren-Ke; Coe, Imogen R; Sweeney, Gary



Mechanism of the regulation of type IB phosphoinositide 3OH-kinase byG-protein betagamma subunits.  

PubMed Central

Type IB phosphoinositide 3OH-kinase (PI3K) is activated by G-protein betagamma subunits (Gbetagammas). The enzyme is soluble and largely cytosolic in vivo. Its substrate, PtdIns(4,5)P(2), and the Gbetagammas are localized at the plasma membrane. We have addressed the mechanism by which Gbetagammas regulate the PI3K using an in vitro approach. We used sedimentation assays and surface plasmon resonance to determine association of type IB PI3K with lipid monolayers and vesicles of varying compositions, some of which had Gbetagammas incorporated. Association and dissociation rate constants were determined. Our results indicated that in an assay situation in vitro the majority of PI3K will be associated with lipid vesicles, irrespective of the presence or absence of Gbetagammas. In line with this, a constitutively active membrane-targeted PI3K construct could still be activated substantially by Gbetagammas in vitro. We conclude that Gbetagammas activate type IB PI3K by a mechanism other than translocation to the plasma membrane.

Krugmann, Sonja; Cooper, Matthew A; Williams, Dudley H; Hawkins, Phillip T; Stephens, Len R



Protein Kinase B Activation and Lamellipodium Formation Are Independent Phosphoinositide 3-Kinase-Mediated Events Differentially Regulated by Endogenous Ras  

PubMed Central

Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while RasAsn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events.

van Weering, David H. J.; de Rooij, Johan; Marte, Barbara; Downward, Julian; Bos, Johannes L.; Burgering, Boudewijn M. T.



Resveratrol modulates the phosphoinositide 3-kinase pathway through an estrogen receptor alpha-dependent mechanism: relevance in cell proliferation.  


Resveratrol (RES), a natural phytoalexin, has antiproliferative activity in human-derived cancer cells and in rodent models of tumor development. We have previously shown that RES induced apoptotic death in estrogen-responsive MCF-7 human breast cancer cells. Recent data have indicated that the estrogen receptor-alpha (ERalpha), through interaction with p85, regulates phosphoinositide 3-kinase (PI3K) activity, revealing a physiologic, nonnuclear function of the ERalpha potentially relevant in cell proliferation and apoptosis. In our study, using MCF-7, we have analyzed the ability of RES to modulate the ERalpha-dependent PI3K pathway. Immunoprecipitation and kinase activity assays showed that RES increased the ERalpha-associated PI3K activity with a maximum stimulatory effect at concentrations close to 10 microM; concentrations >50 microM decreased PI3K activity. Stimulation of PI3K activity by RES was ERalpha-dependent since it could be blocked by the antiestrogen ICI 182,780. RES did not affect p85 protein expression but induced the proteasome-dependent degradation of the ERalpha. Nevertheless, the amount of PI3K immunoprecipitated by the ERalpha remained unchanged in presence of RES, indicating that ERalpha availability was not limiting PI3K activity. Phosphoprotein kinase B (pPKB/AKT) followed the pattern of PI3K activity, whereas RES did not affect total PKB/AKT expression. PKB/AKT downstream target glycogen synthase kinase 3 (GSK3) also showed a phosphorylation pattern that followed PI3K activity. We propose a mechanism through which RES could inhibit survival and proliferation of estrogen-responsive cells by interfering with an ERalpha-associated PI3K pathway, following a process that could be independent of the nuclear functions of the ERalpha. PMID:14750165

Pozo-Guisado, Eulalia; Lorenzo-Benayas, M Jesús; Fernández-Salguero, Pedro M



Mechanisms regulating phosphoinositide 3-kinase signalling in insulin-sensitive tissues.  


A great deal of evidence has accumulated indicating that the activity of PI 3-kinase is necessary, and in some cases sufficient, for a wide range of insulin's actions in the cell. Most biochemical, genetic and pharmacological studies have focused on identifying potential roles for the class-Ia PI 3-kinases which are rapidly activated following insulin stimulation. However, recent evidence indicates the alpha isoform of class-II PI 3-kinase (PI3K-C2alpha) may also play a role as insulin causes a very rapid activation of this as well. The basic mechanisms by which insulin activates the various members of the PI 3-kinase family are increasingly well understood and these studies reveal multiple mechanisms for modulating the activity and functionality of PI 3-kinase and for down regulating the signals they generate. These include inhibitory phosphorylation events, lipid phosphatases such as PTEN and SHIP2 and inhibitor proteins of the suppressors of cytokine signalling (SOCS) family. The current review will focus on these mechanisms and how defects in these might contribute to the development of insulin resistance. PMID:15654916

Shepherd, P R



Targeting the phosphoinositide 3-kinase pathway in hematologic malignancies  

PubMed Central

The phosphoinositide 3-kinase pathway represents an important anticancer target because it has been implicated in cancer cell growth, survival, and motility. Recent studies show that PI3K may also play a role in the development of resistance to currently available therapies. In a broad range of cancers, various components of the phosphoinositide 3-kinase signaling axis are genetically modified, and the pathway can be activated through many different mechanisms. The frequency of genetic alterations in the phosphoinositide 3-kinase pathway, coupled with the impact in oncogenesis and disease progression, make this signaling axis an attractive target in anticancer therapy. A better understanding of the critical function of the phosphoinositide 3-kinase pathway in leukemias and lymphomas has led to the clinical evaluation of novel rationally designed inhibitors in this setting. Three main categories of phosphoinositide 3-kinase inhibitors have been developed so far: agents that target phosphoinositide 3-kinase and mammalian target of rapamycin (dual inhibitors), pan-phosphoinositide 3-kinase inhibitors that target all class I isoforms, and isoform-specific inhibitors that selectively target the ?, -?, -?, or -? isoforms. Emerging data highlight the promise of phosphoinositide 3-kinase inhibitors in combination with other therapies for the treatment of patients with hematologic malignancies. Further evaluation of phosphoinositide 3-kinase inhibitors in first-line or subsequent regimens may improve clinical outcomes. This article reviews the role of phosphoinositide 3-kinase signaling in hematologic malignancies and the potential clinical utility of inhibitors that target this pathway.

Jabbour, Elias; Ottmann, Oliver G.; Deininger, Michael; Hochhaus, Andreas



Interferon-? Activates Transglutaminase 2 via a Phosphatidylinositol-3-Kinase-Dependent Pathway: Implications for Celiac Sprue TherapyS?  

PubMed Central

The mechanism for activation of extracellular transglutaminase 2 (TG2) in the small intestine remains a fundamental mystery in our understanding of celiac sprue pathogenesis. Using the T84 human enterocytic cell line, we show that interferon-? (IFN-?), the predominant cytokine secreted by gluten-reactive T cells in the celiac intestine, activates extracellular TG2 in a dose-dependent manner. IFN-? mediated activation of TG2 requires phosphatidylinositol-3-kinase (PI3K) activity, but is uninfluenced by a number of other kinases reported to be active in T84 cells. Pharmacological inhibition of PI3K in the presence of IFN-? prevents TG2 activation as well as the previously characterized increase in transepithelial permeability. Our findings therefore establish PI3K as an attractive target for celiac sprue therapy, a possibility that is underscored by the encouraging safety profiles of several PI3K inhibitors undergoing human clinical trials.

DiRaimondo, Thomas R.; Klock, Cornelius



Apoptosis Suppression by Raf-1 and MEK1 Requires MEK- and Phosphatidylinositol 3-Kinase-Dependent Signals  

PubMed Central

Two Ras effector pathways leading to the activation of Raf-1 and phosphatidylinositol 3-kinase (PI3K) have been implicated in the survival signaling by the interleukin 3 (IL-3) receptor. Analysis of apoptosis suppression by Raf-1 demonstrated the requirement for mitochondrial translocation of the kinase in this process. This could be achieved either by overexpression of the antiapoptotic protein Bcl-2 or by targeting Raf-1 to the mitochondria via fusion to the mitochondrial protein Mas p70. Mitochondrially active Raf-1 is unable to activate extracellular signal-related kinase 1 (ERK1) and ERK2 but suppresses cell death by inactivating the proapoptotic Bcl-2 family member BAD. However, genetic and biochemical data also have suggested a role for the Raf-1 effector module MEK-ERK in apoptosis suppression. We thus tested for MEK requirement in cell survival signaling using the interleukin 3 (IL-3)-dependent cell line 32D. MEK is essential for survival and growth in the presence of IL-3. Upon growth factor withdrawal the expression of constitutively active MEK1 mutants significantly delays the onset of apoptosis, whereas the presence of a dominant negative mutant accelerates cell death. Survival signaling by MEK most likely results from the activation of ERKs since expression of a constitutively active form of ERK2 was as effective in protecting NIH 3T3 fibroblasts against doxorubicin-induced cell death as oncogenic MEK. The survival effect of activated MEK in 32D cells is achieved by both MEK- and PI3K-dependent mechanisms and results in the activation of PI3K and in the phosphorylation of AKT. MEK and PI3K dependence is also observed in 32D cells protected from apoptosis by oncogenic Raf-1. Additionally, we also could extend these findings to the IL-3-dependent pro-B-cell line BaF3, suggesting that recruitment of MEK is a common mechanism for survival signaling by activated Raf. Requirement for the PI3K effector AKT in this process is further demonstrated by the inhibitory effect of a dominant negative AKT mutant on Raf-1-induced cell survival. Moreover, a constitutively active form of AKT synergizes with Raf-1 in apoptosis suppression. In summary these data strongly suggest a Raf effector pathway for cell survival that is mediated by MEK and AKT.

von Gise, Alexander; Lorenz, Petra; Wellbrock, Claudia; Hemmings, Brian; Berberich-Siebelt, Friederike; Rapp, Ulf R.; Troppmair, Jakob



Channelopathies linked to plasma membrane phosphoinositides  

PubMed Central

The plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) controls the activity of most ion channels tested thus far through direct electrostatic interactions. Mutations in channel proteins that change their apparent affinity to PIP2 can lead to channelopathies. Given the fundamental role that membrane phosphoinositides play in regulating channel activity, it is surprising that only a small number of channelopathies have been linked to phosphoinositides. This review proposes that for channels whose activity is PIP2-dependent and for which mutations can lead to channelopathies, the possibility that the mutations alter channel-PIP2 interactions ought to be tested. Similarly, diseases that are linked to disorders of the phosphoinositide pathway result in altered PIP2 levels. In such cases, it is proposed that the possibility for a concomitant dysregulation of channel activity also ought to be tested. The ever-growing list of ion channels whose activity depends on interactions with PIP2 promises to provide a mechanism by which defects on either the channel protein or the phosphoinositide levels can lead to disease.

Logothetis, Diomedes E.; Petrou, Vasileios I.; Adney, Scott K.; Mahajan, Rahul



Role of phosphoinositides at the neuronal synapse  

PubMed Central

Synaptic transmission is amongst the most sophisticated and tightly controlled biological phenomena in higher eukaryotes. In the past few decades, tremendous progress has been made in our understanding of the molecular mechanisms underlying multiple facets of neurotransmission, both pre- and postsynaptically. Brought under the spotlight by pioneer studies in the areas of secretion and signal transduction, phosphoinositides and their metabolizing enzymes have been increasingly recognized as key protagonists in fundamental aspects of neurotransmission. Not surprisingly, dysregulation of phosphoinositide metabolism has also been implicated in synaptic malfunction associated with a variety of brain disorders. In the present chapter, we summarize current knowledge on the role of phosphoinositides at the neuronal synapse and highlight some of the outstanding questions in this research field.

Frere, Samuel G.; Chang-Ileto, Belle; Di Paolo, Gilbert



Aggregation of Phosphoinositides at Phisiological Calcium Concentrations  

NASA Astrophysics Data System (ADS)

Phosphoinositides play a crucial role in many cellular functions such as calcium signaling, endocytosis, exocytosis and the targeting of proteins to specific membrane sites. To maintain functional specificity, it has been suggested that phosphoinositides are spatially organized in ``pools'' in the cellular plasma membrane. A possible mechanism that could induce and regulate such organization of phosphoinositides is their interaction with Ca2+ ions. Understanding the physicochemical mechanism that can regulate membrane structure is a crucial step in the development of adaptive biomimetic membrane systems. Using Langmuir monolayers, we investigated the effect of bivalent calcium and magnesium cations on the surface pressure-area/lipid isotherm of monolayers of phosphatidylinositol (PI), phosphatidylinositol bisphosphate (PIP2) and dioleoylphosphatidylglycerol (DOPG) and dipalmitoylphosphatidylcholine (DPPC). It is found that the decrease of area per lipid, i.e. the increase in aggregation, is dependent on both the lipid's head group charge, the bivalent cation and temperature. However, electrostatics are not sufficient to account for all experimental observations. Thus additional interactions between ions and phosphoinositides need to be considered.

Kazadi Badiambile, Adolphe; Forstner, Martin B.



Bcl-xL mediates a survival mechanism independent of the phosphoinositide 3-kinase/Akt pathway in prostate cancer cells.  


Among various molecular strategies by which prostate cancer cells evade apoptosis, phosphoinositide 3-kinase (PI3K)/Akt signaling represents a dominant survival pathway. However, different prostate cancer cell lines such as LNCaP and PC-3 display differential sensitivity to the apoptotic effect of PI3K inhibition in serum-free media, reflecting the heterogeneous nature of prostate cancer in apoptosis regulation. Whereas both cell lines are equally susceptible to LY294002-mediated Akt dephosphorylation, only LNCaP cells default to apoptosis, as evidenced by DNA fragmentation and cytochrome c release. In PC-3 cells, Akt deactivation does not lead to cytochrome c release, suggesting that the intermediary signaling pathway is short-circuited by an antiapoptotic factor. This study presents evidence that Bcl-xL overexpression provides a distinct survival mechanism that protects PC-3 cells from apoptotic signals emanating from PI3K inhibition. First, the Bcl-xL/BAD ratio in PC-3 cells is at least an order of magnitude greater than that of LNCaP cells. Second, ectopic expression of Bcl-xL protects LNCaP cells against LY294002-induced apoptosis. Third, antisense down-regulation of Bcl-xL sensitizes PC-3 cells to the apoptotic effect of LY294002. The physiological relevance of this Bcl-xL-mediated survival mechanism is further underscored by the protective effect of serum on LY294002-induced cell death in LNCaP cells, which is correlated with a multifold increase in Bcl-xL expression. In contrast to Bcl-xL, Bcl-2 expression levels are similar in both cells lines, and do not respond to serum stimulation, suggesting that Bcl-2 may not play a physiological role in antagonizing apoptosis signals pertinent to BAD activation in prostate cancer cells. PMID:12738789

Yang, Chih-Cheng; Lin, Ho-Pi; Chen, Chang-Shi; Yang, Ya-Ting; Tseng, Ping-Hui; Rangnekar, Vivek M; Chen, Ching-Shih



Studies on the mechanism of alteration by propranolol and mepacrine of the metabolism of phosphoinositides and other glycerolipids in the rabbit iris muscle.  


We have investigated the effects and mechanism of action of propranolol and mepacrine, two drugs with local anesthetic-like properties, on phospholipid metabolism in rabbit iris and iris microsomal and soluble fractions. In the iris, propranolol, like mepacrine [A. A. Abdel-Latif and J. P. Smith, Biochim, biophys. Acta 711, 478 (1982)], stimulated the incorporation of [14C]arachidonic acid ( [14C]AA) into phosphatidic acid (PA), CDP-diacylglycerol (CDP-DG), phosphatidylinositol (PI), the polyphosphoinositides (poly PI) and DG, and it inhibited that of phosphatidylcholine (PC), phosphatidylethanolamine (PE), triacylglycerol (TG) and the prostaglandins. Similarly, mepacrine, like propranolol [A. A. Abdel-Latif and J. P. Smith, Biochem. Pharmac. 25, 1697 (1976)], altered the incorporation of [14C]oleic acid, [3H]glycerol, 32Pi and [14C]choline into glycerolipids of the iris. Time-course studies in iris muscle prelabeled with [14C]AA showed an initial decrease in the production of DG and a corresponding increase in that of PA by the drugs, followed by an increase in accumulation of DG at longer time intervals (60-90 min). The above findings are in accord with the hypothesis that these drugs redirect glycerolipid synthesis by inhibiting PA phosphohydrolase. Propranolol and mepacrine stimulated the activities of DG kinase and phosphoinositide kinases and inhibited that of DG cholinephosphotransferase. The drugs had little effect on the activity of DG acyltransferase. It is concluded that propranolol and mepacrine redirect glycerolipid metabolism in the iris by exerting multiple effects on the enzymes involved in phospholipid biosynthesis. We suggest that these drugs could exert their local anesthetic-like effects by effecting an increase in the synthesis of the acidic phospholipids (PA, PI and the poly PI) and subsequently the binding of Ca2+- to the cell plasma membrane. PMID:6318773

Abdel-Latif, A A; Smith, J P; Akhtar, R A



Phosphoinositides alter lipid bilayer properties  

PubMed Central

Phosphatidylinositol-4,5-bisphosphate (PIP2), which constitutes ?1% of the plasma membrane phospholipid, plays a key role in membrane-delimited signaling. PIP2 regulates structurally and functionally diverse membrane proteins, including voltage- and ligand-gated ion channels, inwardly rectifying ion channels, transporters, and receptors. In some cases, the regulation is known to involve specific lipid–protein interactions, but the mechanisms by which PIP2 regulates many of its various targets remain to be fully elucidated. Because many PIP2 targets are membrane-spanning proteins, we explored whether the phosphoinositides might alter bilayer physical properties such as curvature and elasticity, which would alter the equilibrium between membrane protein conformational states—and thereby protein function. Taking advantage of the gramicidin A (gA) channels’ sensitivity to changes in lipid bilayer properties, we used gA-based fluorescence quenching and single-channel assays to examine the effects of long-chain PIP2s (brain PIP2, which is predominantly 1-stearyl-2-arachidonyl-PIP2, and dioleoyl-PIP2) on bilayer properties. When premixed with dioleoyl-phosphocholine at 2 mol %, both long-chain PIP2s produced similar changes in gA channel function (bilayer properties); when applied through the aqueous solution, however, brain PIP2 was a more potent modifier than dioleoyl-PIP2. Given the widespread use of short-chain dioctanoyl-phosphoinositides, we also examined the effects of diC8-phosphoinositol (PI), PI(4,5)P2, PI(3,5)P2, PI(3,4)P2, and PI(3,4,5)P3. The diC8 phosphoinositides, except for PI(3,5)P2, altered bilayer properties with potencies that decreased with increasing head group charge. Nonphosphoinositide diC8 phospholipids generally were more potent bilayer modifiers than the polyphosphoinositides. These results show that physiological increases or decreases in plasma membrane PIP2 levels, as a result of activation of PI kinases or phosphatases, are likely to alter lipid bilayer properties, in addition to any other effects they may have. The results further show that exogenous PIP2, as well as structural analogues that differ in acyl chain length or phosphorylation state, alters lipid bilayer properties at the concentrations used in many cell physiological experiments.

Hobart, E. Ashley; Koeppe, Roger E.; Andersen, Olaf S.



Phosphoinositides and Plant Cell Wall Synthesis  

Microsoft Academic Search

\\u000a Phosphoinositides\\u000a are lipid second messengers known to be important for many cellular processes in yeast, including actin cytoskeletal organization,\\u000a vesicle transport, and cell wall assembly. In plant cells, studies on phosphoinositide phosphatases and kinases suggest that\\u000a phosphoinositides are involved in the regulation of actin cytoskeletal organization, cell wall synthesis, and cell morphogenesis.\\u000a It is hypothesized that phosphoinositides may regulate the

Ruiqin Zhong; Ryan L. McCarthy; Zheng-Hua Ye


Regulation of Akt\\/PKB by phosphatidylinositol 3-kinase-dependent and -independent pathways in B-cell chronic lymphocytic leukemia cells: role of protein kinase C  

Microsoft Academic Search

Apoptosis of B cell chronic lympho- cytic leukemia (B-CLL) cells is regulated by the PI-3K-Akt pathway. In the present work, we have analyzed the mechanisms of Akt phosphorylation in B-CLL cells. Freshly isolated cells present basal Akt phosphorylation, which is PI-3K-dependent, as incubation with the PI-3K inhibitor LY294002 de- creased Ser-473 and Thr-308 phosphorylation in most samples analyzed (seven out

Montserrat Barragan; Mercede Frias; Daniel Iglesias-Serret; Clara Campas; Esther Castano; Antonio F. Santidrian; L. Coll-Mulet; A. M. Cosialls; A. Domingo; G. Pons; J. Gil



Gold nanoparticle supported phospholipid membranes as a biomimetic biosensor platform for phosphoinositide signaling detection.  


Enzyme mediated phosphoinositide signaling plays important regulatory roles in diverse cellular processes and has close implication in human diseases. However, detection of phosphoinositide enzymes remains a challenge because of the difficulty in discriminating the phosphorylation patterns of phosphoinositide. Here we develop a novel enzyme-activated gold nanoparticles (AuNPs) assembly strategy as a homogeneous colorimetric biosensor for activity detection of phosphoinositide kinases and phosphatases. This strategy utilizes a biomimetic mechanism of phosphoinositide signaling, in which AuNP supported phospholipid membranes are constructed to mimic the cellular membrane substrate, and AuNPs modified with the pleckstrin homology (PH) domain of cytosolic proteins are designed for specific, multivalent recognition of phosphorylated phosphoinositides. This biomimetic strategy enables efficient enzymatic reactions of the substrate and highly selective detection of target enzyme. The biosensor is demonstrated for the detection of phosphoinositide 3-kinase (PI3K) and phosphatase with tensin homology (PTEN). The results revealed that it allows sensitive, rapid visual detection of the enzymes with pM detection limits and four-decade wide dynamic ranges, and is capable of detecting enzyme activities in complex cell lysate samples. This biosensor might provide a general biosensor platform for high-throughput detection of phosphoinositide enzymes with high sensitivity and selectivity in biomedical research and clinical diagnostics. PMID:24994507

Wen, Qian; Liu, Si-Jia; Tang, Li-Juan; Tang, Ying; Jiang, Jian-Hui



Phosphoinositide metabolism in adipocytes from hypothyroid rats.  


The effect of hypothyroidism on insulin- and epinephrine-stimulated phosphoinositide metabolism was investigated in rat adipocytes. Insulin-mediated phosphoinositide synthesis was enhanced by hypothyroidism (14.5 +/- 1.5% above basal level, control vs. 22.5 +/- 2.0% above basel level, hypothyroid, P less than 0.05). However, insulin did not stimulate hydrolysis of phosphoinositides to inositol phosphates, neither in control nor in hypothyroid rats. The alpha 1-adrenoceptor agonist (e.g. epinephrine) significantly stimulated the incorporation of myo-[3H]inositol into phosphoinositides (P less than 0.01) and hydrolysis of phosphoinositides (P less than 0.01), but this stimulatory action was unaffected by the hypothyroid state. The results suggest that hypothyroidism has differentiated effects on the hormone-regulated phosphoinositide metabolism and that the presumptive G-protein coupled to the alpha 1-adrenoceptor seems to be unaffected by hypothyroidism. PMID:1648503

Andersen, P H; Juhl, H; Pedersen, S B; Richelsen, B



Molecular analysis of protein-phosphoinositide interactions.  


Diverse biological processes including cell growth and survival require transient association of proteins with cellular membranes. A large number of these proteins are drawn to a bilayer through binding of their modular domains to phosphoinositide (PI) lipids. Seven PI isoforms are found to concentrate in distinct pools of intracellular membranes, and this lipid compartmentalization provides an efficient way for recruiting PI-binding proteins to specific cellular organelles. The atomic-resolution structures and membrane docking mechanisms of a dozen PI effectors have been elucidated in the last decade, offering insight into the molecular basis for regulation of the PI-dependent signaling pathways. In this chapter, I summarize the mechanistic aspects of deciphering the 'PI code' by the most common PI-recognizing domains and discuss similarities and differences in the membrane anchoring mechanisms. PMID:23086416

Kutateladze, Tatiana G



The Phosphoinositide 3-Kinase Pathway  

NSDL National Science Digital Library

Phosphorylated lipids are produced at cellular membranes during signaling events and contribute to the recruitment and activation of various signaling components. The role of phosphoinositide 3-kinase (PI3K), which catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate, in cell survival pathways; the regulation of gene expression and cell metabolism; and cytoskeletal rearrangements are highlighted. The PI3K pathway is implicated in human diseases including diabetes and cancer, and understanding the intricacies of this pathway may provide new avenues for therapuetic intervention.

Lewis Cantley (Beth Israel Deaconess Medical Center;Department of Cell Biology, Harvard Medical School and Division of Signal Transduction)



Phosphoinositide 3-kinases-a historical perspective.  


The phosphoinositide 3-kinase (PI 3-K) signal relay pathway represents arguably one of the most intensely studied mechanisms by which extracellular signals elicit cellular responses through the generation of second messengers that are associated with cell growth and transformation. This chapter reviews the many landmark discoveries in the PI 3-K signaling pathway in biology and disease, from the identification of a novel phosphoinositide kinase activity associated with transforming oncogenes in the 1980s, to the identification of oncogenic mutations in the catalytic subunit of PI 3-K in the mid 2000s. Two and a half decades of intense research have provided clear evidence that the PI 3-K pathway controls virtually all aspects of normal cellular physiology, and that deregulation of one or more proteins that regulate or transduce the PI 3-K signal ultimately leads to human pathology. The most recent efforts have focused on the development of specific PI 3-K inhibitors that are currently being evaluated in clinical trials for a range of disease states.This chapter is devoted to a historical review of the landmark findings in the PI 3-K from its relatively humble beginnings in the early to mid 1980s up until the present day. When considering the key findings in the history of PI 3-K, it is essential to recognize the landmark studies by Lowell and Mabel Hokin in the 1950s who were the first to describe that extracellular agonists such as acetylcholine could stimulate the incorporation of radiolabeled phosphate into phospholipids (Hokin and Hokin 1953). Their work initiated an entirely new field of lipid signaling, and subsequent studies in the 1970s by Michell and Lapetina who linked phosphoinositide turnover to membrane-associated receptors that initiate intracellular calcium mobilization (Lapetina and Michell 1973). Later studies revealed that the phospholipase-mediated breakdown of the same minor membrane phospholipids such as PtdIns-4,5-P(2) (phosphatidylinositol-4,5-bisphosphate) is responsible for the release of two additional key second messengers, diacylglycerol (DG) and IP(3) (inositol-1,4,5-trisphosphate) (Kirk et al. 1981; Berridge 1983; Berridge et al. 1983). Berridge, Irvine and Schulz then revealed that one of the byproducts of this lipid signal relay pathway is the release of calcium from intracellular stores such as the endoplasmic reticulum (Streb et al. 1983). Finally, pioneering studies by Nishizuka in the late 1970s identified PKC (protein kinase C) as a phospholipid and diacylglycerol-activated serine/threonine protein kinase (Inoue et al. 1977; Takai et al. 1977). At this point, it probably seemed to most at the time that the story was complete, such that hydrolysis of phosphoinositides such as PtdIns-4,5-P(2) and PtdIns-4-P would account for the major mechanisms of agonist-stimulated lipid signaling leading to physiological responses. On the contrary, the story was far from complete and was about to become a lot more complex. PMID:22403075

Toker, Alex



A Dual Polybasic Motif Determines Phosphoinositide Binding and Regulation in the P2X Channel Family  

PubMed Central

Phosphoinositides modulate the function of several ion channels, including most ATP-gated P2X receptor channels in neurons and glia, but little is known about the underlying molecular mechanism. We identified a phosphoinositide-binding motif formed of two clusters of positively charged amino acids located on the P2X cytosolic C-terminal domain, proximal to the second transmembrane domain. For all known P2X subtypes, the specific arrangement of basic residues in these semi-conserved clusters determines their sensitivity to membrane phospholipids. Neutralization of these positive charges disrupts the functional properties of the prototypical phosphoinositide-binding P2X4 subtype, mimicking wortmannin-induced phosphoinositide depletion, whereas adding basic residues at homologous positions to the natively insensitive P2X5 subtype establishes de novo phosphoinositide-mediated regulation. Moreover, biochemical evidence of in vitro P2X subunit-phospholipid interaction and functional intracellular phosphoinositide-binding assays demonstrate that the dual polybasic cluster is necessary and sufficient for regulation of P2X signaling by phospholipids.

Bernier, Louis-Philippe; Blais, Dominique; Boue-Grabot, Eric; Seguela, Philippe



Phosphoinositides and PDZ domain scaffolds.  


The discovery that PSD-95/Discs large/ZO-1 (PDZ) domains can function as lipid-binding modules, in particular interacting with phosphoinositides (PIs), was made more than 10 years ago (Mol Cell 9(6): 1215-1225, 2002). Confirmatory studies and a series of functional follow-ups established PDZ domains as dual specificity modules displaying both peptide and lipid binding, and prompted a rethinking of the mode of action of PDZ domains in the control of cell signaling. In this chapter, after introducing PDZ domains, PIs and methods for studying protein-lipid interactions, we focus on (i) the prevalence and the specificity of PDZ-PIs interactions, (ii) the molecular determinants of PDZ-PIs interactions, (iii) the integration of lipid and peptide binding by PDZ domains, (iv) the common features of PIs interacting PDZ domains and (v) the regulation and functional significance of PDZ-PIs interactions. PMID:23775690

Wawrzyniak, Anna Maria; Kashyap, Rudra; Zimmermann, Pascale



Single-Molecule Adhesion Forces and Attachment Lifetimes of Myosin-I Phosphoinositide Interactions  

PubMed Central

Phosphoinositides regulate the activities and localization of many cytoskeletal proteins involved in crucial biological processes, including membrane-cytoskeleton adhesion. Yet little is known about the mechanics of protein-phosphoinositide interactions, or about the membrane-attachment mechanics of any peripheral membrane proteins. Myosin-Ic (myo1c) is a molecular motor that links membranes to the cytoskeleton via phosphoinositide binding, so it is particularly important to understand the mechanics of its membrane attachment. We used optical tweezers to measure the strength and attachment lifetime of single myo1c molecules as they bind beads coated with a bilayer of 2% phosphatidylinositol 4,5-bisphosphate and 98% phosphatidylcholine. Adhesion forces measured under ramp-load ranged between 5.5 and 16 pN at loading rates between 250 and 1800 pN/s. Dissociation rates increased linearly with constant force (0.3–2.5 pN), with rates exceeding 360 s?1 at 2.5 pN. Attachment lifetimes calculated from adhesion force measurements were loading-rate-dependent, suggesting nonadiabatic behavior during pulling. The adhesion forces of myo1c with phosphoinositides are greater than the motors stall forces and are within twofold of the force required to extract a lipid molecule from the membrane. However, attachment durations are short-lived, suggesting that phosphoinositides alone do not provide the mechanical stability required to anchor myo1c to membranes during multiple ATPase cycles.

Pyrpassopoulos, Serapion; Shuman, Henry; Ostap, E. Michael



Mutations in Phosphoinositide Metabolizing Enzymes and Human Disease  

NSDL National Science Digital Library

Phosphoinositides are implicated in the regulation of a wide variety of cellular functions. Their importance in cellular and organismal physiology is underscored by the growing number of human diseases linked to perturbation of kinases and phosphatases that catalyze interconversion from one phosphoinositide to another. Many such enzymes are attractive targets for therapeutic interventions. Here, we review diseases linked to inheritable or somatic mutations of these enzymes. Phosphatidylinositol (PtdIns), a membrane phospholipid, can be reversibly phosphorylated at the 3, 4, and 5 positions of the inositol ring to generate seven phosphoinositides [PI3P, PI4P, PI5P, PI(3,4)P2, PI(4,5)P2, PI(3,5)P2, and PI(3,4,5)P3] (FIGURE 1A). The importance of this metabolism in cell regulation was first established in the context of studies on stimulus-secretion coupling. It was found that many stimuli that trigger secretion also trigger enhanced turnover of PtdIns and phosphoinositides (42). Subsequently, it became clear that phospholipase C-dependent hydrolysis of PI(4,5)P2 to generate the second messenger molecules diacyl glycerol and Ins(1,4,5)P3 (IP3) is a mechanism through which many cell surface receptors, including many receptors that stimulate secretion, transduce their signals (10). Diacyl glycerol binds and regulates protein kinase C and a variety of other effectors, whereas IP3 triggers calcium release from the endoplasmic reticulum (10, 42). In another signal transduction pathway, PI(4,5)P2 is cleaved by phospholipase A2 to generate arachidonic acid, a precursor of many signaling molecules.

Heather J. McCrea (Yale University); Pietro De Camilli (Yale University School of Medicine)



Increased levels of phosphoinositides cause neurodegeneration in a Drosophila model of amyotrophic lateral sclerosis  

PubMed Central

The Vesicle-associated membrane protein (VAMP)-Associated Protein B (VAPB) is the causative gene of amyotrophic lateral sclerosis 8 (ALS8) in humans. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective death of motor neurons leading to spasticity, muscle atrophy and paralysis. VAP proteins have been implicated in various cellular processes, including intercellular signalling, synaptic remodelling, lipid transport and membrane trafficking and yet, the molecular mechanisms underlying ALS8 pathogenesis remain poorly understood. We identified the conserved phosphoinositide phosphatase Sac1 as a Drosophila VAP (DVAP)-binding partner and showed that DVAP is required to maintain normal levels of phosphoinositides. Downregulating either Sac1 or DVAP disrupts axonal transport, synaptic growth, synaptic microtubule integrity and the localization of several postsynaptic components. Expression of the disease-causing allele (DVAP-P58S) in a fly model for ALS8 induces neurodegeneration, elicits synaptic defects similar to those of DVAP or Sac1 downregulation and increases phosphoinositide levels. Consistent with a role for Sac1-mediated increase of phosphoinositide levels in ALS8 pathogenesis, we found that Sac1 downregulation induces neurodegeneration in a dosage-dependent manner. In addition, we report that Sac1 is sequestered into the DVAP-P58S-induced aggregates and that reducing phosphoinositide levels rescues the neurodegeneration and suppresses the synaptic phenotypes associated with DVAP-P58S transgenic expression. These data underscore the importance of DVAP–Sac1 interaction in controlling phosphoinositide metabolism and provide mechanistic evidence for a crucial role of phosphoinositide levels in VAP-induced ALS.

Forrest, Stuart; Chai, Andrea; Sanhueza, Mario; Marescotti, Manuela; Parry, Katherine; Georgiev, Atanas; Sahota, Virender; Mendez-Castro, Raquel; Pennetta, Giuseppa



Ceramide Disables 3-Phosphoinositide Binding to the Pleckstrin Homology Domain of Protein Kinase B (PKB)/Akt by a PKC?-Dependent Mechanism  

PubMed Central

Ceramide is generated in response to numerous stress-inducing stimuli and has been implicated in the regulation of diverse cellular responses, including cell death, differentiation, and insulin sensitivity. Recent evidence indicates that ceramide may regulate these responses by inhibiting the stimulus-mediated activation of protein kinase B (PKB), a key determinant of cell fate and insulin action. Here we show that inhibition of this kinase involves atypical PKC?, which physically interacts with PKB in unstimulated cells. Insulin reduces the PKB-PKC? interaction and stimulates PKB. However, dissociation of the kinase complex and the attendant hormonal activation of PKB were prevented by ceramide. Under these circumstances, ceramide activated PKC?, leading to phosphorylation of the PKB-PH domain on Thr34. This phosphorylation inhibited phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding to PKB, thereby preventing activation of the kinase by insulin. In contrast, a PKB-PH domain with a T34A mutation retained the ability to bind PIP3 even in the presence of a ceramide-activated PKC? and, as such, expression of PKB T34A mutant in L6 cells was resistant to inhibition by ceramide treatment. Inhibitors of PKC? and a kinase-dead PKC? both antagonized the inhibitory effect of ceramide on PKB. Since PKB confers a prosurvival signal and regulates numerous pathways in response to insulin, suppressing its activation by a PKC?-dependent process may be one mechanism by which ceramide promotes cell death and induces insulin resistance.

Powell, Darren J.; Hajduch, Eric; Kular, Gursant; Hundal, Harinder S.



Determining Selectivity of Phosphoinositide-Binding Domains  

PubMed Central

The burgeoning of phosphoinositide-binding domains and proteins in cellular signaling and trafficking has drawn laboratories from a wide variety of fields into the study of lipid interactions with peripheral membrane proteins. Many different approaches have been developed to assess phosphoinositide binding, some of which are more problematic than others, and some of which can be quantitated more readily than others. With a focus on the methods used in our laboratory, we describe here the considerations that need to be taken into account when establishing – and quantitating – the specific binding of a protein or domain to phosphoinositides in membranes. We also discuss briefly a few examples in which no clear consensus has yet been reached as to the specificity of a given domain or protein because of discrepancies between different commonly-used approaches.

Narayan, Kartik; Lemmon, Mark A.



Decoding the role of phosphoinositides in phototropin signaling involved in chloroplast movements.  


In angiosperms, light-dependent chloroplast movements are exclusively mediated by UVA/blue light receptors - phototropins. The two photoreceptors of Arabidopsis thaliana, phot1 and phot2, have overlapping roles in the control of these movements. Experiments performed in different plant species point to the participation of phosphoinositides in blue light-controlled chloroplast relocations. Here, we report a summary of recent findings presenting the involvement of phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 3- and 4-phosphates in weak blue light-mediated (accumulation) and strong blue light-mediated (avoidance) responses of chloroplasts. The blue light-activated alterations in phosphoinositide concentration are partly responsible for cytosolic Ca (2+) changes. Ca (2+) influx from apoplast does not seem to be involved in the mechanism of movement responses. In summary, interplay between phosphoinositides and intracellular Ca (2+) regulates chloroplast redistribution in response to blue light in higher plants. PMID:23733070

Aggarwal, Chhavi; Labuz, Justyna; Gabry?, Halina



Decoding the role of phosphoinositides in phototropin signaling involved in chloroplast movements  

PubMed Central

In angiosperms, light-dependent chloroplast movements are exclusively mediated by UVA/blue light receptors - phototropins. The two photoreceptors of Arabidopsis thaliana, phot1 and phot2, have overlapping roles in the control of these movements. Experiments performed in different plant species point to the participation of phosphoinositides in blue light-controlled chloroplast relocations. Here, we report a summary of recent findings presenting the involvement of phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 3- and 4-phosphates in weak blue light-mediated (accumulation) and strong blue light-mediated (avoidance) responses of chloroplasts. The blue light-activated alterations in phosphoinositide concentration are partly responsible for cytosolic Ca2+ changes. Ca2+ influx from apoplast does not seem to be involved in the mechanism of movement responses. In summary, interplay between phosphoinositides and intracellular Ca2+ regulates chloroplast redistribution in response to blue light in higher plants.

Aggarwal, Chhavi; Labuz, Justyna; Gabrys, Halina



Serotonin-stimulated phosphoinositide turnover: mediation by the S2 binding site in rat cerebral cortex but not in subcortical regions  

SciTech Connect

In rat cerebral cortex, serotonin (5-HT) stimulates phosphoinositide turnover with an EC50 of 1 microM in the presence of pargyline. The EC50 is 16-fold higher in the absence of pargyline. Selective S2 antagonists inhibit 5-HT-stimulated phosphoinositide turnover. Schild analysis of the blockade by ketanserin of the 5-HT effect gives an estimated Kd of ketanserin for the phosphoinositide-linked receptor of 11.7 nM, which agrees with the Kd (3.5 nM) of (/sup 3/H)ketanserin for the S2 site. Furthermore, MK-212, 5-HT and 5-fluorotryptamine stimulate phosphoinositide turnover with potencies that resemble their potencies at the S2 but not the S1 binding site. Of 11 agonists tested, the tryptamine derivatives tend to be more efficacious than the piperazine derivatives. The selective S1 agonist 8-hydroxy-2-(di-N-propylamino)tetralin is inactive at stimulating phosphoinositide turnover. No significant relationship exists between the regional distributions of 5-HT-stimulated phosphoinositide turnover and S2 binding sites. Furthermore, the S2 antagonist ketanserin is less potent and less efficacious in hippocampus and limbic forebrain than in cerebral cortex. These data suggest that 5-HT-stimulated phosphoinositide turnover is linked to the S2 binding site in rat cerebral cortex. However, 5-HT increases phosphoinositide turnover in subcortical regions by mechanisms other than stimulation of the S2 receptor.

Conn, P.J.; Sanders-Bush, E.



Phosphoinositide metabolism in Drosophila phototransduction: a coffee break discussion leads to 30 years of history.  


The aim of this review is to summarize the history of Dr. Yoshiki Hotta and his collaborators' contributions to the research field of Drosophila phototransduction. The electroretinogram-defective mutants reported in 1970 by Dr. Hotta and Dr. Seymour Benzer in the article entitled "Genetic dissection of the Drosophila nervous system by means of mosaics" have attracted the interest of many researchers, and have been used as a great tool to dissect the mechanisms underlying phototransduction. The early collaboration of Dr. Hotta with the group of Dr. Tohru Yoshioka, who was studying the roles of phosphoinositides in the nervous system biochemically, combined biochemical and genetic approaches to phototransduction-defective no receptor potential A (norpA) and retinal degeneration A (rdgA) mutants, which led to the hypothesis that phosphoinositide metabolism regulates phototransduction in Drosophila. This was proven later by the identification of the norpA and rdgA mutant genes, which encode phospholipase C and diacylglycerol kinase, respectively. Thus the collaboration of Dr. Hotta and Dr. Yoshioka laid the foundation of our understanding of the role of phosphoinositide metabolism in Drosophila phototransduction. In addition, a collaboration carried out with the group of Dr. Kazushige Hirosawa on the ultrastructural analyses of retinal degeneration mutants, rdgA and rdgB, led to the discovery of the subcellular membrane organelle called submicrovillar cisternae, which is involved in the phosphoinositide metabolism. In this review, the authors will summarize these results, which were inspired by Dr. Hotta's insights. PMID:22283140

Suzuki, Emiko; Masai, Ichiro; Inoue, Hiroko



Brain-Derived Neurotrophic Factor (BDNF) Induces Dendritic Targeting of BDNF and Tyrosine Kinase B mRNAs in Hippocampal Neurons through a Phosphatidylinositol3 Kinase-Dependent Pathway  

Microsoft Academic Search

This study aims to understand the mechanisms of dendritic targeting of brain-derived neurotrophic factor (BDNF) and ty- rosine kinase B (TrkB) mRNAs. We show that brief depolariza- tions are sufficient to induce accumulation of BDNF and TrkB mRNAs in dendrites of hippocampal neurons. Endogenous BDNF, secreted during the KCl stimulation, contributes signifi- cantly to the dendritic accumulation of BDNF-TrkB mRNAs.

Massimo Righi; Enrico Tongiorgi; Antonino Cattaneo



Histamine-stimulated phosphoinositide hydrolysis in developing rat brain.  


Histamine stimulation of phosphoinositide breakdown has been studied in brain cortex from developing rats by measuring the accumulation of [3H]inositol monophosphate in lithium-treated slices prelabeled with [3H]inositol. Histamine stimulated phosphoinositide hydrolysis as early as day 3 after birth. The maximal response increased about 3-fold up to day 15 and remained essentially at the same level until adulthood. This developmental pattern contrasted with the 8-fold increase in [3H]mepyramine binding to histamine H1 receptors which takes place from the first to the end of the fourth week of life. Nonlinear regression analysis of concentration-effect curves for histamine generated at 6, 10, and 15 days of age revealed the presence of two components in the response, whereas a single component was found in adults. A high potency component showed EC50 values increasing from 6.4 microM at day 6 to 26.5 microM at day 15, and its contribution to the maximal response augmented from 65.8% to 78.9% during the same period. In 6-day-old animals, the H1 receptor agonists N alpha-methylhistamine and N alpha,N alpha-dimethylhistamine produced single-component responses with maximal effects similar to that estimated for the high potency component for histamine, and their relative potencies were consistent with an H1 receptor-mediated response. Inhibition constants for mepyramine, calculated from the parallel displacement of the concentration-response curves for N alpha-methylhistamine and the high potency component for histamine (6.3 nM and 10.8 nM, respectively) closely agreed with the reported affinity of rat brain H1 receptors for this compound. The low potency component of the response (EC50 = 175 microM at day 6 and EC50 = 260 microM at day 15) did not involve histamine-H2, serotonin 5-HT2, alpha 1-adrenergic, or muscarinic receptors. Pretreatment of 6-day-old cortical slices with the irreversible antagonist phenoxybenzamine, at concentrations that completely abolish [3H]mepyramine binding, left about 25% of the response to 1 mM histamine unaffected. This portion of the response, which was, moreover, insensitive to 1 microM mepyramine, was of a magnitude similar to that of the low potency component. Thus, at early stages of rat brain development, histamine induces phosphoinositide hydrolysis in part through a mechanism different from activation of H1 receptors. The loss of histamine potency and efficacy at H1 receptors throughout development is discussed and the natural occurrence of H1 receptor subsensitivity suggested. PMID:2823091

Claro, E; García, A; Picatoste, F



Effectors of animal and plant pathogens use a common domain to bind host phosphoinositides.  


Bacterial Type III Secretion Systems deliver effectors into host cells to manipulate cellular processes to the advantage of the pathogen. Many host targets of these effectors are found on membranes. Therefore, to identify their targets, effectors often use specialized membrane-localization domains to localize to appropriate host membranes. However, the molecular mechanisms used by many domains are unknown. Here we identify a conserved bacterial phosphoinositide-binding domain (BPD) that is found in functionally diverse Type III effectors of both plant and animal pathogens. We show that members of the BPD family functionally bind phosphoinositides and mediate localization to host membranes. Moreover, NMR studies reveal that the BPD of the newly identified Vibrio parahaemolyticus Type III effector VopR is unfolded in solution, but folds into a specific structure upon binding its ligand phosphatidylinositol-(4,5)-bisphosphate. Thus, our findings suggest a possible mechanism for promoting refolding of Type III effectors after delivery into host cells. PMID:24346350

Salomon, Dor; Guo, Yirui; Kinch, Lisa N; Grishin, Nick V; Gardner, Kevin H; Orth, Kim



PKB\\/Akt: connecting phosphoinositide 3-kinase to cell survival and beyond  

Microsoft Academic Search

PKB\\/Akt is a serine\\/threonine kinase that contains a pleckstrin-homology (PH) domain and is activated in response to growth-factor treatment of cells by a mechanism involving phosphoinositide 3-OH kinase. PKB\\/Akt provides a survival signal that protects cells from apoptosis induced by various stresses, perhaps explaining its discovery as a retroviral oncogene and its amplification in many human tumours.

Barbara M. Marte; Julian Downward



Excitatory amino acid-induced phosphoinositide hydrolysis in Müller glia.  


The presence of excitatory amino acid (EAA) receptors coupled to phosphoinositide metabolism in primary cultures of Müller (glial) cells from the chick retina was established. The order of potency of analogues for stimulating [3H]inositol phosphate (IP) accumulation was quisqualate (QA) > L-glutamate (L-Glu) = kainate (KA) > N-methyl-D-aspartate (NMDA) > L-aspartate (L-Asp) with EC50 in the range of 1-100 microM. 1-Aminocyclopentane-1,3-dicarboxylate (trans-ACPD), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), 2-amino-3-phosphonopropionate (AP3), and 2-amino-4-phosphonobutyrate (AP4) showed no effect either on basal concentration or on stimulated accumulation of [3H]IPs. The effect of EAA was potently inhibited by the ionotropic NMDA receptor antagonists 2-amino-5-phosphonopentanoate (AP5), 3-[(RS)-2-carboxy-piperazin-4-yl)]-propyl-1-phosphonate (CPP), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-10- imine (MK-801); L-Glu antagonists at non-NMDA receptors, the quinoxalines NBQX and DNQX, inhibited weakly the response to L-Glu, KA, and NMDA, and more potently that to QA. The translocation of protein kinase C was also stimulated by EAA with the same pharmacological profile, and was partially inhibited by kynurenate (KYN). L-Glu and KA induced 45Ca2+ influx, which was decreased by KYN and CNQX. EAA-induced [3H]IPs accumulation was decreased by verapamil but not by nifedipine, and slightly diminished by dantrolene. Results demonstrate that EAA-induced phosphoinositide hydrolysis in Müller cells shows pharmacological differences with that in astrocytes and neuronal cells and could be triggered by a different mechanism. PMID:7902338

López-Colomé, A M; Ortega, A; Romo-de-Vivar, M



Ras mediates radioresistance through both phosphatidylinositol 3-kinase-dependent and Raf-dependent but mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-independent signaling pathways.  


Cells transformed by the oncogenic small GTPase, Ras, display a radioresistant phenotype in response to ionizing radiation (IR). To determine the mechanisms by which Ras mediates radioresistance in epithelial cells, we assessed the importance of three major survival pathways that can be activated by Ras [phosphatidylinositol 3-kinase (PI3-K)>Akt, nuclear factor kappaB (NF-kappaB), and Raf>mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)>extracellular signal-regulated kinase] as necessary or sufficient for Ras-mediated radioresistance in matched pairs of RIE-1 rat intestinal epithelial cells expressing oncogenic Ras or empty vector (RIE-Ras and RIE-vector). Inhibiting PI3-K with LY294002 sensitized RIE-1 cells to IR in a dose-dependent manner, indicating that PI3-K is necessary for radioresistance, whereas inhibition of NF-kappaB with the super-repressor IkappaBalpha had little effect on survival. Expression of either the constitutively active catalytic subunit of PI3-K, p110alpha-CAAX, or the Ras effector domain mutant 12V/40C, which retains binding to PI3-K but is impaired in binding to other Ras effectors, was sufficient to confer partial radioresistance. Expression of either a constitutively active form of the serine/threonine kinase Raf-1 or the Ras effector domain mutant 12V/35S, which retains binding to Raf but is impaired in binding to other Ras effectors, was also sufficient to confer partial radioresistance. Surprisingly, however, even complete inhibition of MEK activity by using U0126 resulted in no change in post-IR survival whatsoever. Thus, whereas Raf contributes to Ras-mediated radioresistance, this is accomplished through a MEK-independent pathway. Taken together, these results indicate that multiple pathways, including both PI3-K-dependent and Raf-dependent but MEK-independent signaling, are required for Ras-mediated radioresistance in epithelial cells. Finally, we demonstrate that Ras-mediated radioresistance can be uncoupled from Ras-mediated transformation, in that PI3-K is required for radioresistance but not transformation, whereas MEK and NF-kappaB are required for transformation but not radioresistance in RIE-1 epithelial cells. PMID:12124353

Grana, Theresa M; Rusyn, Elena V; Zhou, Hong; Sartor, Carolyn I; Cox, Adrienne D



The chemical biology of phosphoinositide 3-kinases.  


Since its discovery in the late 1980s, phosphoinositide 3-kinase (PI3K), and its isoforms have arguably reached the forefront of signal transduction research. Regulation of this lipid kinase, its functions, its effectors, in short its entire signaling network, has been extensively studied. PI3K inhibitors are frequently used in biochemistry and cell biology. In addition, many pharmaceutical companies have launched drug-discovery programs to identify modulators of PI3Ks. Despite these efforts and a fairly good knowledge of the PI3K signaling network, we still have only a rudimentary picture of the signaling dynamics of PI3K and its lipid products in space and time. It is therefore essential to create and use novel biological and chemical tools to manipulate the phosphoinositide signaling network with spatial and temporal resolution. In this review, we discuss the current and potential future tools that are available and necessary to unravel the various functions of PI3K and its isoforms. PMID:22965647

Wymann, Matthias P; Schultz, Carsten



Phosphoinositide 5-phosphatases: how do they affect tumourigenesis?  

PubMed Central

The activity of biological molecules is often affected by their phosphorylation state. Regulatory phosphorylation operates as a binary switch and is usually controlled by counteracting kinases and phosphatases. However, phosphatidylinositol (PtdIns) has three phosphorylation sites on its inositol ring. The phosphorylation status of PtdIns is controlled by multiple kinases and phosphatases with distinct substrate specificities, serving as a ‘lipid code’ or ‘phosphoinositide code’. Class I phosphoinositide 3-kinase (PI3K) converts PtdIns(4,5)P2 to PtdIns(3,4,5)P3, which plays a pivotal role in signals controlling glucose uptake, cytoskeletal reorganization, cell proliferation and apoptosis. PI3K is pro-oncogenic, whereas phosphoinositide phosphatases that degrade PtdIns(3,4,5)P3 are not always anti-oncogenic. Recent studies have revealed the unique characteristics of phosphoinositide 5-phosphatases.

Miyazawa, Keiji



Phosphoinositides in the mammalian endo-lysosomal network  

PubMed Central

The endo-lysosomal system is an interconnected tubulo-vesicular network that acts as a sorting station to process and distribute internalised cargo. This network accepts cargoes from both the plasma membrane and the biosynthetic pathway, and directs these cargos either towards the lysosome for degradation, the peri-nuclear recycling endosome for return to the cell surface, or to the trans-Golgi network. These intracellular membranes are variously enriched in different phosphoinositides that help to shape compartmental identity. These lipids act to localise a number of phosphoinositide-binding proteins that function as sorting machineries to regulate endosomal cargo sorting. Herein we discuss regulation of these machineries by phosphoinositides and explore how phosphoinositide-switching contributes toward sorting decisions made at this platform.

Cullen, Peter J.; Carlton, Jeremy G.



Autoradiographic imaging of phosphoinositide turnover in the brain  

SciTech Connect

With ({sup 3}H)cytidine as a precursor, phosphoinositide turnover can be localized in brain slices by selective autoradiography of the product ({sup 3}H)cytidine diphosphate diacylglycerol, which is membrane-bound. In the cerebellum, glutamatergic stimulation elicits an increase of phosphoinositide turnover only in Purkinje cells and the molecular layer. In the hippocampus, both glutamatergic and muscarinic cholinergic stimulation increase phosphoinositide turnover, but with distinct localizations. Cholinergic stimulation affects CA1, CA3, CA4, and subiculum, whereas glutamatergic effects are restricted to the subiculum and CA3. Imaging phosphoinositide turnover in brain slices, which are amenable to electrophysiologic studies, will permit a dynamic localized analysis of regulation of this second messenger in response to synaptic stimulation of specific neuronal pathways.

Hwang, P.M.; Bredt, D.S.; Snyder, S.H. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA))



SH3YL1 regulates dorsal ruffle formation by a novel phosphoinositide-binding domain  

PubMed Central

Reversible interactions between cytosolic proteins and membrane lipids such as phosphoinositides play important roles in membrane morphogenesis driven by actin polymerization. In this paper, we identify a novel lipid-binding module, which we call the SYLF domain (after the SH3YL1, Ysc84p/Lsb4p, Lsb3p, and plant FYVE proteins that contain it), that is highly conserved from bacteria to mammals. SH3YL1 (SH3 domain containing Ysc84-like 1) strongly bound to phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3) and several D5-phosphorylated phosphoinositides through its SYLF domain and was localized to circular dorsal ruffles induced by platelet-derived growth factor stimulation. Interestingly, SHIP2 (the PI(3,4,5)P3 5-phosphatase, src-homology 2–containing inositol-5-phosphatase 2) was identified as a binding partner of SH3YL1, and knockdown of these proteins significantly suppressed dorsal ruffle formation. Phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), which is mainly synthesized from PI(3,4,5)P3 by the action of SHIP2, was enriched in dorsal ruffles, and PI(3,4)P2 synthesis strongly correlated with formation of the circular membrane structure. These results provide new insight into the molecular mechanism of dorsal ruffle formation and its regulation by phosphoinositide metabolism.

Hasegawa, Junya; Tokuda, Emi; Tenno, Takeshi; Tsujita, Kazuya; Sawai, Haruko; Hiroaki, Hidekazu; Takenawa, Tadaomi



PITPs as Targets for Selectively Interfering With Phosphoinositide Signaling in Cells  

PubMed Central

Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production, and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs, nor PITPs in general, have been exploited as targets for chemical inhibition for such purposes. Herein, we validate the first small molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and are effective inhibitors in vitro and in vivo. We further establish Sec14 is the sole essential NPPM target in yeast, that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects, and demonstrate NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof-of-concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies.

Nile, Aaron H.; Tripathi, Ashutosh; Yuan, Peihua; Mousley, Carl J.; Suresh, Sundari; Wallace, Iain Michael; Shah, Sweety D.; Pohlhaus, Denise Teotico; Temple, Brenda; Nislow, Corey; Giaever, Guri; Tropsha, Alexander; Davis, Ronald W.; St Onge, Robert P.; Bankaitis, Vytas A.



Ras and phosphoinositide 3-kinase: partners in development and tumorigenesis.  


Much progress has been made in understanding the myriad of intracellular signaling pathways responsible for control of cell physiology. Signalling downstream of receptor tyrosine kinases (RTKs) is probably the most studied signalling mechanism to date and many of the molecular components and corresponding interactions involved have been delineated. Importantly, deregulation of RTK signalling has been implicated in the formation and maintenance of many human tumours. Two of the pivotal molecular components in RTK signalling, Ras and phosphoinositide 3-kinase (PI 3-kinase), have been shown to bind to each other, leading to the activation of PI 3-kinase. However, in addition to this Ras-PI 3-kinase interaction, first described over a decade ago, several other molecular interactions have more recently been described that appear to mediate the same signal. This has brought into question the physiological relevance of the Ras-PI 3-kinase interaction during RTK signalling. Through disruption of the interaction in a mouse model, we have now confirmed that the interaction is highly functional in vivo both during mammalian development and during Ras-induced tumorigenesis. Many questions still remain: in this Perspective, we explore the remaining uncertainties surrounding the role of this signalling mechanism, as well as the future directions that will likely shed further light on its role within cells. PMID:17993782

Ramjaun, Antoine R; Downward, Julian



Adenosine receptor activation potentiates phosphoinositide hydrolysis and arachidonic acid release in DDT1-MF2 cells: putative interrelations.  


Studies were undertaken in an effort to discern possible mechanisms by which the A1 adenosine receptor agonist cyclopentyladenosine (CPA) enhances the norepinephrine-stimulated (NE-stimulated) hydrolysis of phosphoinositides in DDT1-MF2 cells. Measurements of arachidonic acid release revealed similar behaviours to those observed in measurements of phosphoinositide hydrolysis. In the presence of NE, both second messenger responses were potentiated by the addition of CPA, whereas in the absence of NE, CPA had little or no effect on either second messenger. The stimulation and potentiation of both second messenger responses were enhanced in the presence of extracellular calcium, and in each case these effects were persistent over time. For either second messenger system the stimulation by NE and the potentiation by CPA appeared to utilize separate mechanisms as evidenced by the fact that the potentiations by CPA were selectively antagonized by a cAMP analogue or by pertussis toxin, whereas the stimulations by NE were essentially unaffected by these agents. Inhibition of phospholipase A2 (PLA2) also blocked the potentiation of PLC by CPA, without affecting NE-stimulated phosphoinositide hydrolysis. Furthermore, in the presence of CPA, the exogenous administration of PLA2 was found to stimulate phosphoinositide hydrolysis in these cells. These data are consistent with a hypothesis whereby the apparent potentiation of NE-stimulated phosphoinositide hydrolysis by CPA is actually due to the stimulation by CPA of a second pathway of phospholipase C activity which is additive to that of NE. The activation of PLC and PLA2 by NE produces phospholipid products which may play a permissive role in the pathway coupling adenosine A1 receptors to these phospholipases. The formation of lysophosphatidic acid is suggested as one possible mediator of this permissive effect. PMID:8519595

Schachter, J B; Yasuda, R P; Wolfe, B B



Phosphoinositide breakdown is associated with Fc-gamma RII-mediated activation of 5'-lipoxygenase in murine eosinophils.  


In this report we present data on the ability of murine eosinophils to generate inositol phosphate derivatives, and their relationship with the activation of 5'-lipoxygenase by a Fc-gamma R-dependent mechanism. The addition of anti-IgG F(ab')2 to mouse eosinophils, previously sensitized with IgG, induces inositol phosphate generation after 2 min and after 10 min of stimulation. Maximal generation of inositol tris and inositol tetrakis phosphate has been detected after 15 min of stimulation, and the optimal concentration of anti-IgG F(ab')2 was found to be 25 micrograms. Inositol tris phosphate formation is also observed at 5 min after the addition of the calcium ionophore A23187 (5 microM). We also report that neomycin, an inhibitor of phosphoinositide-phospholipase C, inhibits Fc-gamma R-mediated phosphoinositide breakdown in a dose-dependent manner (88% inhibition at 150 microM of neomycin). The possible involvement of phosphoinositide breakdown in the activation of 5'-lipoxygenase has been investigated. Using streptolysin-O permeabilized cells and different doses of neomycin that inhibit phosphoinositide breakdown, we have demonstrated a parallel decrease in LTC4 released by these cells, using either A23187 (86% inhibition at 200 microM of neomycin) or anti-IgG F(ab')2 (82.4% inhibition at 100 microM of neomycin). [Ca2+]i elevation has been observed by loading the cells with the fluorescent calcium indicator Fura-2 penta-acetoxy methyl ester and after stimulating with the anti-Fc-gamma RII mAb (2.4G2). It is likely that the activation of murine eosinophils by a Fc-gamma R mechanism stimulates phosphoinositide breakdown as a primary step that leads to the activation of murine 5'-lipoxygenase, producing the formation of leukotriene C4. PMID:1847165

De Andres, B; Del Pozo, V; Cardaba, B; Martin, E; Tramon, P; Lopez-Rivas, A; Palomino, P; Lahoz, C



Developmental expression of a Trypanosoma cruzi phosphoinositide-specific phospholipase C in amastigotes and stimulation of host phosphoinositide hydrolysis.  


Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid modified in its N terminus and localizes to the outer surface of the plasma membrane of amastigotes. We show here that TcPI-PLC is developmentally regulated in amastigotes and shows two peaks of surface expression during the developmental cycle of T. cruzi, the first immediately after differentiation of trypomastigotes into amastigotes and the second before differentiation of amastigotes into trypomastigotes. Surface expression of TcPI-PLC coincides with phosphatidylinositol 4,5-bisphosphate (PIP(2)) depletion in the host cell membrane and with an increase in the levels of its product, inositol 1,4,5-trisphosphate. During extracellular differentiation, PI-PLC is secreted into the incubation medium. Maximal early expression of TcPI-PLC on the surface of amastigotes and PIP(2) depletion coincide with host cytoskeletal changes, Ca(2+) signaling, and transcriptional responses described previously. The presence of TcPI-PLC on the outer surface of the plasma membrane of the parasite and the capacity to be secreted and to alter host phospholipids are novel mechanisms of the host-parasite interaction. PMID:20643853

Martins, Vicente de Paulo; Galizzi, Melina; Salto, Maria Laura; Docampo, Roberto; Moreno, Silvia N J



3-phosphoinositides modulate cyclic nucleotide signaling in olfactory receptor neurons.  


Phosphatidylinositol 3-kinase (PI3K)-dependent phosphoinositide signaling has been implicated in diverse cellular systems coupled to receptors for many different ligands, but the extent to which it functions in sensory transduction is yet to be determined. We now report that blocking PI3K activity increases odorant-evoked, cyclic nucleotide-dependent elevation of [Ca(2+)](i) in acutely dissociated rat olfactory receptor neurons and does so in an odorant-specific manner. These findings imply that 3-phosphoinositide signaling acts in vertebrate olfactory transduction to inhibit cyclic nucleotide-dependent excitation of the cells and that the interaction of the two signaling pathways is important in odorant coding, indicating that 3-phosphoinositide signaling can play a role in sensory transduction. PMID:11879650

Spehr, Marc; Wetzel, Christian H; Hatt, Hanns; Ache, Barry W



Regulation of Hippo pathway by mitogenic growth factors via phosphoinositide 3-kinase and phosphoinositide-dependent kinase-1  

PubMed Central

The Hippo signaling pathway inhibits cell growth and regulates organ size through a kinase cascade that leads to the phosphorylation and nuclear exclusion of the growth-promoting transcriptional coactivator Yes-associated protein (YAP)/Yorkie. It mediates contact inhibition of cell growth downstream of cadherin adhesion molecules and other cell surface proteins. Contact inhibition is often antagonized by mitogenic growth factor signaling. We report an important mechanism for this antagonism, inhibition of Hippo pathway signaling by mitogenic growth factors. EGF treatment of immortalized mammary cells triggers the rapid translocation of YAP into the nucleus along with YAP dephosphorylation, both of which depend on Lats, the terminal kinase in the Hippo pathway. A small-molecule inhibitor screen of downstream effector pathways shows that EGF receptor inhibits the Hippo pathway through activation of PI3-kinase (PI3K) and phosphoinositide-dependent kinase (PDK1), but independent of AKT activity. The PI3K-PDK1 pathway also mediates YAP nuclear translocation downstream of lysophosphatidic acid and serum as a result of constitutive oncogenic activation of PI3K. PDK1 associates with the core Hippo pathway-kinase complex through the scaffold protein Salvador. The entire Hippo core complex dissociates in response to EGF signaling in a PI3K-PDK1–dependent manner, leading to inactivation of Lats, dephosphorylation of YAP, and YAP nuclear accumulation and transcriptional activation of its target gene, CTGF. These findings show that an important activity of mitogenic signaling pathways is to inactivate the growth-inhibitory Hippo pathway and provide a mechanism for antagonism between contact inhibition and growth factor action.

Fan, Run; Kim, Nam-Gyun; Gumbiner, Barry M.



Synthesis and Function of Membrane Phosphoinositides in Budding Yeast, Saccharomyces cerevisiae  

PubMed Central

It is now well appreciated that derivatives of phosphatidylinositol (PtdIns) are key regulators of many cellular processes in eukaryotes. Of particular interest are phosphoinositides (mono- and polyphosphorylated adducts to the inositol ring in PtdIns), which are located at the cytoplasmic face of cellular membranes. Phosphoinositides serve both a structural and a signaling role via their recruitment of proteins that contain phosphoinositide-binding domains. Phosphoinositides also have a role as precursors of several types of second messengers for certain intracellular signaling pathways. Realization of the importance of phosphoinositides has brought increased attention to characterization of the enzymes that regulate their synthesis, interconversion, and turnover. Here we review the current state of our knowledge about the properties and regulation of the ATP-dependent lipid kinases responsible for synthesis of phosphoinositides and also the additional temporal and spatial controls exerted by the phosphatases and a phospholipase that act on phosphoinositides in yeast.

Strahl, Thomas; Thorner, Jeremy



Two structural components in CNGA3 support regulation of cone CNG channels by phosphoinositides  

PubMed Central

Cyclic nucleotide-gated (CNG) channels in retinal photoreceptors play a crucial role in vertebrate phototransduction. The ligand sensitivity of photoreceptor CNG channels is adjusted during adaptation and in response to paracrine signals, but the mechanisms involved in channel regulation are only partly understood. Heteromeric cone CNGA3 (A3) + CNGB3 (B3) channels are inhibited by membrane phosphoinositides (PIPn), including phosphatidylinositol 3,4,5-triphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2), demonstrating a decrease in apparent affinity for cyclic guanosine monophosphate (cGMP). Unlike homomeric A1 or A2 channels, A3-only channels paradoxically did not show a decrease in apparent affinity for cGMP after PIPn application. However, PIPn induced an ?2.5-fold increase in cAMP efficacy for A3 channels. The PIPn-dependent change in cAMP efficacy was abolished by mutations in the C-terminal region (R643Q/R646Q) or by truncation distal to the cyclic nucleotide-binding domain (613X). In addition, A3-613X unmasked a threefold decrease in apparent cGMP affinity with PIPn application to homomeric channels, and this effect was dependent on conserved arginines within the N-terminal region of A3. Together, these results indicate that regulation of A3 subunits by phosphoinositides exhibits two separable components, which depend on structural elements within the N- and C-terminal regions, respectively. Furthermore, both N and C regulatory modules in A3 supported PIPn regulation of heteromeric A3+B3 channels. B3 subunits were not sufficient to confer PIPn sensitivity to heteromeric channels formed with PIPn-insensitive A subunits. Finally, channels formed by mixtures of PIPn-insensitive A3 subunits, having complementary mutations in N- and/or C-terminal regions, restored PIPn regulation, implying that intersubunit N–C interactions help control the phosphoinositide sensitivity of cone CNG channels.

Dai, Gucan; Peng, Changhong; Liu, Chunming



The Skap-hom Dimerization and PH Domains Comprise a 3?-Phosphoinositide-Gated Molecular Switch  

PubMed Central

Summary PH domains, by binding to phosphoinositides, often serve as membrane-targeting modules. Using crystallographic, biochemical and cell biological approaches, we have uncovered a mechanism that the integrin-signaling adaptor Skap-hom uses to mediate cytoskeletal interactions. Skap-hom is a homodimer containing an N-terminal four-helix-bundle dimerization domain, against which its two PH domains pack in a conformation incompatible with phosphoinositide binding. The isolated PH domains bind PI[3,4,5]P3, and mutations targeting the dimerization domain or the PH domain’s PI[3,4,5]P3-binding pocket prevent Skap-hom localization to ruffles. Targeting is retained when the PH domain is deleted or by combined mutation of the PI[3,4,5]P3-binding pocket and the PH/dimerization domain interface. Thus, the dimerization and PH domain form a novel PI[3,4,5]P3-responsive molecular switch that controls Skap-hom function.

Swanson, Kenneth D.; Tang, Yong; Ceccarelli, Derek F.; Poy, Florence; Sliwa, Jan P.; Neel, Benjamin G.; Eck, Michael J.



Treponema denticola major outer sheath protein impairs the cellular phosphoinositide balance that regulates neutrophil chemotaxis.  


The major outer sheath protein (Msp) of Treponema denticola inhibits neutrophil polarization and directed chemotaxis together with actin dynamics in vitro in response to the chemoattractant N-formyl-methionine-leucine-phenylanine (fMLP). Msp disorients chemotaxis through inhibition of a Rac1-dependent signaling pathway, but the upstream mechanisms are unknown. We challenged murine bone marrow neutrophils with enriched native Msp to determine the role of phospholipid modifying enzymes in chemotaxis and actin assembly downstream of fMLP-stimulation. Msp modulated cellular phosphoinositide levels through inhibition of phosphatidylinositol 3-kinase (PI3-kinase) together with activation of the lipid phosphatase, phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Impaired phosphatidylinositol[(3,4,5)]-triphosphate (PIP3) levels prevented recruitment and activation of the downstream mediator Akt. Release of the actin capping proteins gelsolin and CapZ in response to fMLP was also inhibited by Msp exposure. Chemical inhibition of PTEN restored PIP3 signaling, as measured by Akt activation, Rac1 activation, actin uncapping, neutrophil polarization and chemotaxis in response to fMLP-stimulation, even in the presence of Msp. Transduction with active Rac1 also restored fMLP-mediated actin uncapping, suggesting that Msp acts at the level of PIP3 in the hierarchical feedback loop of PIP3 and Rac1 activation. Taken together, Msp alters the phosphoinositide balance in neutrophils, impairing the cell "compass", which leads to inhibition of downstream chemotactic events. PMID:23755300

Visser, Michelle B; Sun, Chun-Xiang; Koh, Adeline; Ellen, Richard P; Glogauer, Michael



Sequential Activities of Phosphoinositide 3-Kinase, PKB/Akt, and Rab7 during Macropinosome Formation in Dictyostelium  

PubMed Central

Macropinocytosis plays an important role in the internalization of antigens by dendritic cells and is the route of entry for many bacterial pathogens; however, little is known about the molecular mechanisms that regulate the formation or maturation of macropinosomes. Like dendritic cells, Dictyostelium amoebae are active in macropinocytosis, and various proteins have been identified that contribute to this process. As described here, microscopic analysis of null mutants have revealed that the class I phosphoinositide 3-kinases, PIK1 and PIK2, and the downstream effector protein kinase B (PKB/Akt) are important in regulating completion of macropinocytosis. Although actin-rich membrane protrusions form in these cell lines, they recede without forming macropinosomes. Imaging of cells expressing green fluorescent protein (GFP) fused to the pleckstrin homology domain (PH) of PKB (GFP-PHPKB) indicates that D3 phosphoinositides are enriched in the forming macropinocytic cup and remain associated with newly formed macropinosomes for <1 minute. A fusion protein, consisting of GFP fused to an F-actin binding domain, overlaps with GFP-PHPKB in the timing of association with forming macropinosomes. Although macropinocytosis is reduced in cells expressing dominant negative Rab7, microscopic imaging studies reveal that GFP-Rab7 associates only with formed macropinosomes at approximately the time that F-actin and D3 phosphoinositide levels decrease. These results support a model in which F-actin modulating proteins and vesicle trafficking proteins coordinately regulate the formation and maturation of macropinosomes.

Rupper, Adam; Lee, Kyung; Knecht, David; Cardelli, James



Soman-induced seizures impair norepinephrine-stimulated phosphoinositide turnover  

SciTech Connect

Seizure activity increases turnover of phosphoinositide bisphosphate (PIP2). Turnover of PIP2 is thought to be modulated by neurotransmitter interactions. The effect of soman-induced seizures on neurotransmitter-stimulated PIP 2 turnover was examined in rats. Thirty minutes after induction of seizure activity, rats were euthanized and slices prepared from the hippocampus or cerebral cortex were incubated with myo-(2-3H) inositol for incorporation into phospholipids. Hydrolysis of phosphoinositides was determined by measuring the accumulation of (3H) inositol-l-phosphate (IP1) in the presence of LiCl. Carbachol, norepinephrine (NE) and high K+ increased accumulation of IP1 in slices from control rats. GABA was without effect on IP1 accumulation but potentiated the stimulation of PIP, hydrolysis by NE. NE-stimulated IP1 accumulation in slices from rats undergoing seizures was significantly reduced. GABA potentiation of the NE-stimulated hydrolysis was also reduced.

Filbert, M.G.; Phann, S.; Forster, J.; Ballough, G.P.; Cann, F.J.



Essential Role of Phosphoinositide Metabolism in Synaptic Vesicle Recycling  

Microsoft Academic Search

Growing evidence suggests that phosphoinositides play an important role in membrane traffic. A polyphosphoinositide phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1–deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich

Ottavio Cremona; Gilbert Di Paolo; Markus R Wenk; Anita Lüthi; Warren T Kim; Kohji Takei; Laurie Daniell; Yasuo Nemoto; Stephen B Shears; Richard A Flavell; David A McCormick; Pietro De Camilli



Signalling through the lipid products of phosphoinositide-3OH kinase  

Microsoft Academic Search

When a stimulatory agonist molecule binds at the exterior of the cell membrane, a second messenger transduces the signal to the interior of the cell. Second messengers can be derived from phospholipids in the membrane by the action of the enzymes phospholipase C or phosphoinositide-3-OH kinase (PI(3)K). PI(3)K is a key player in many cellular responses, including the movement of

Alex Toker; Lewis C. Cantley



The role of the phosphoinositides at the Golgi complex.  


Eukaryotic cells are organized into a complex system of subcompartments, each with its distinct protein and lipid composition. A continuous flux of membranes crosses these compartments, and in some cases direct connections exist between the different organelles. It is thus surprising that they can maintain their individual identities. Small GTPases and the phosphoinositides have emerged as the key regulators in the maintenance of the identity of the Golgi complex. This property is due to their ability to act either alone or, more often, in combination, as cues directing and controlling the recruitment of proteins that possess phosphoinositide-binding domains. Among these many proteins there are the lipid transfer proteins, which can transfer ceramide, oxysterol, cholesterol and possibly glucosylceramide. By regulating these lipid transfer proteins in this way, this binomial combination of the small GTPases and the phosphoinositides acquires a further important role: control of the synthesis and/or distribution of other important integral constituents of cell organelles, such as the sphingolipids and cholesterol. This role is particularly relevant at the level of the Golgi complex, a key organelle in the biosynthesis, transport and sorting of both lipids and proteins that is located at the intersection of the secretory and endocytic pathways. PMID:17233584

De Matteis, Maria Antonietta; D'Angelo, Giovanni



Phosphoinositides as regulators of membrane trafficking in health and disease.  


Membrane trafficking is crucial in the homeostasis of the highly compartmentalized eukaryotic cells. This compartmentalization occurs both at the organelle level, with distinct organelles maintaining their identities while also intensely interchanging components, and at a sub-organelle level, with adjacent subdomains of the same organelle containing different sets of lipids and proteins. A central question in the field is thus how this compartmentalization is established and maintained despite the intense exchange of components and even physical continuities within the same organelle. The phosphorylated derivatives of phosphatidylinositol, known as the phosphoinositides, have emerged as key components in this context, both as regulators of membrane trafficking and as finely tuned spatial and temporal landmarks for organelle and sub-organelle domains. The central role of the phosphoinositides in cell homeostasis is highlighted by the severe consequences of the derangement of their metabolism caused by genetic deficiencies of the enzymes involved, and from the systematic hijacking of phosphoinositide metabolism that pathogens operate to promote their entry and/or survival in host cells. PMID:18726176

Vicinanza, M; D'Angelo, G; Di Campli, A; De Matteis, M A



Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation  

PubMed Central

Phosphoinositides (PIs) make up only a small fraction of cellular phospholipids, yet they control almost all aspects of a cell's life and death. These lipids gained tremendous research interest as plasma membrane signaling molecules when discovered in the 1970s and 1980s. Research in the last 15 years has added a wide range of biological processes regulated by PIs, turning these lipids into one of the most universal signaling entities in eukaryotic cells. PIs control organelle biology by regulating vesicular trafficking, but they also modulate lipid distribution and metabolism via their close relationship with lipid transfer proteins. PIs regulate ion channels, pumps, and transporters and control both endocytic and exocytic processes. The nuclear phosphoinositides have grown from being an epiphenomenon to a research area of its own. As expected from such pleiotropic regulators, derangements of phosphoinositide metabolism are responsible for a number of human diseases ranging from rare genetic disorders to the most common ones such as cancer, obesity, and diabetes. Moreover, it is increasingly evident that a number of infectious agents hijack the PI regulatory systems of host cells for their intracellular movements, replication, and assembly. As a result, PI converting enzymes began to be noticed by pharmaceutical companies as potential therapeutic targets. This review is an attempt to give an overview of this enormous research field focusing on major developments in diverse areas of basic science linked to cellular physiology and disease.



Eukaryotic virulence determinants utilize phosphoinositides at the ER and host cell surface  

PubMed Central

Similar to bacteria, eukaryotic pathogens may also utilize common strategies of pathogenic secretion, as effector proteins from the oomycete Phytophthora infestans and virulence determinants from the human malaria parasite Plasmodium falciparum share a functionally equivalent host-cell targeting motif (RxLR-dEER in P. infestans and RxLxE/D/Q in P. falciparum). Here we summarize recent studies that reveal that the malarial motif may function differently than previously envisioned. Rather, binding the lipid phosphatidylinositol 3-phosphate [PI(3)P] is a critical step in accessing the host for both pathogens, but occurs in different locations. Nanomolar affinity for PI(3)P by these short amino acid motifs, suggests a new mechanism of phosphoinositide binding unexpectedly in secretory locations that has been exploited for virulence by diverse eukaryotic pathogens.

Jiang, Rays H.Y.; Stahelin, Robert V.; Bhattacharjee, Souvik; Haldar, Kasturi



Cytotoxic amphiphiles and phosphoinositides bind to two discrete sites on the Akt1 PH domain.  


The mechanism of binding of two promising anticancer agents (the cytotoxic alkylphospholipids perifosine and miltefosine) to the Akt PH domain is investigated by high-resolution field-cycling (31)P nuclear magnetic resonance (NMR) spectroscopy using a spin-labeled recombinant PH domain. These results strongly indicate that there are two discrete amphiphile binding sites on the domain: (i) the cationic site that binds phosphoinositides and some alkylphospholipids and (ii) a second site that is occupied by only the alkylphospholipids. The identification of this second site for amphiphiles on the Akt1 PH domain provides a new target for drug development as well as insights into the regulation of the activity of the intact Akt1 protein. The field-cycling NMR methodology could be used to define discrete phospholipid or amphiphile binding sites on a wide variety of peripheral membrane proteins. PMID:24383815

Gradziel, Cheryl S; Wang, Yanling; Stec, Boguslaw; Redfield, Alfred G; Roberts, Mary F



3'-phosphoinositide-dependent kinase-1 (PDK-1) in PI 3-kinase signaling.  


The recently discovered 3'-phosphoinositide-dependent kinase-1 (PDK-1) is a serine/threonine protein kinase which phosphorylates several members of the conserved AGC kinase superfamily (comprising the prototypes protein kinases A (PKA), G (PKG) and C (PKC)). Phosphorylation of a threonine or serine residue in the activation loop (also known as the T-loop) of these kinases is a critical step in their activation, and is typically accompanied by additional phosphorylations elsewhere in the molecule. Phosphorylation of the activation loop is a common regulatory mechanism shared by most serine/threonine as well as tyrosine kinases as it facilitates alignment of amino acid residues in the active site which are involved in the phosphotransferase reaction. Therefore the discovery of PDK-1 as the enzyme which mediates this event in many protein kinases introduced a new and important step in signaling pathways which regulate numerous important cellular processes including cellular survival, glucose transport and metabolism, tumor progression as well as protein translation. Moreover, the finding that PDK-1 function is mediated in part by the phosphoinositide 3'-OH-kinase (PI 3-K) pathway also provided an explanation as to how the lipid products of PI 3-K, namely phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol-3,4-5-trisphosphate (PtdIns-3,4,5-P3) stimulate the activation of protein kinase-dependent signaling pathways. These initial landmark observations were followed by many important studies which provided additional mechanistic insight into both PDK-1 regulation as well as the role of this kinase in cellular function. This review will focus on the regulation of PDK-1 and the various mechanisms which it uses to contribute to the activation of target kinases. PMID:11897568

Storz, Peter; Toker, Alex



Phorbol Esters Inhibit Alpha-Adrenergic Receptor-Stimulated Phosphoinositide Hydrolysis and Contraction in Rat Aorta: Evidence for a Link between Vascular Contraction and Phosphoinositide Turnover.  

National Technical Information Service (NTIS)

We investigated the actions of two biologically active phorbol esters, phorbol dibutyrate (PDB) and phorbol myristate acetate (PMA), on receptor-stimulated phosphoinositide hydrolysis in rat aorta. We found both PDB and PMA potently inhibited norepinephri...

M. McMillan B. Chernow B. L. Roth



Studies on phosphoinositide metabolism in retinal rod outer segments  

SciTech Connect

The present work was undertaken to determine whether isolated mammalian photoreceptors (bovine rod outer segments, ROS) were capable of synthesizing and hydrolyzing phosphoinositides, and if so, how synthesis and hydrolysis might be regulated. Synthesis of phosphoinositides was demonstrated by the incorporation of radioactively labeled precursors. ROS incubated with ({gamma}{sup 32}P)ATP produced labeled phosphatidic acid (PA), phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP{sub 2}). When Mn{sup 2+}, CTP and inositol were added, labeled phosphatidylinositol (PI) was also produced. ({sup 3}H)Inositol was incorporated into PI, PIP and PIP{sub 2}, although prolonged incubation was required for detectable incorporation into PIP{sub 2}. Incorporation of ({sup 3}H)inositol was dependent on CTP, indicating that labeling proceeded via synthesis, not base exchange. Incubation with ({alpha}-{sup 32}P)CTP produced labeled CDP-diacylglycerol, an intermediate in PI synthesis. Incorporation of labeled precursors was stimulated by Mg{sup 2+}, Mn{sup 2+} and spermine, but unaffected by light. Phosphoinositide hydrolysis was measured using exogenous {sup 3}H-labeled substrates. PI, PIP, and PIP{sub 2} were all hydrolyzed but most attention was devoted to PIP{sub 2}. Crude enzyme preparations contained an endogenous inhibitor whose effects were Ca{sup 2+}-dependently relieved by calmodulin antagonists. This inhibitor does not appear to be calmodulin but may be a novel Ca{sup 2+}-binding regulatory protein. No evidence for regulation of PLC activity by light or G-proteins was obtained. The effects on PLC activity of Mg{sup 2+}, Mn{sup 2+}, spermine, pH and detergents were also characterized.

Gehm, B.D.



Quantitation of adenosine-5'-triphosphate used for phosphoinositide metabolism in human erythrocytes  

SciTech Connect

The human erythrocyte actively phosphorylates and dephosphorylates phosphatidylinositol present in the membrane in an apparent ''futile cycle.'' Recent reports have proposed that this phosphorylation/dephosphorylation cycle is a significant consumer of adenosine-5'-triphosphate (ATP) in the erythrocyte. This study details two independent techniques for quantitating the ATP consumed by this phosphoinositide futile cycle. With the first technique a quasi-steady-state labeling of erythrocyte ATP with TSP-phosphate was obtained, and the rate of synthesis of TSP-phosphoinositides was then monitored. The second technique used a novel labeling strategy that allowed only ATP to be labeled with TSP; the transfer of TSP from ATP to phosphoinositides was then an independent measure of the ATP consumed for phosphoinositide synthesis. These two techniques documented that 0.5% to 1.0% of net ATP produced by the erythrocyte is used for phosphoinositide synthesis.

Dale, G.L.



Signalling through the lipid products of phosphoinositide-3-OH kinase.  


When a stimulatory agonist molecule binds at the exterior of the cell membrane, a second messenger transduces the signal to the interior of the cell. Second messengers can be derived from phospholipids in the membrane by the action of the enzymes phospholipase C or phosphoinositide-3-OH kinase (PI(3)K). PI(3)K is a key player in many cellular responses, including the movement of organelle membranes, shape alteration through rearrangement of cytoskeletal actin, transformation and chemotaxis. But how PI(3)K mediates these responses is only now becoming clear. PMID:9192891

Toker, A; Cantley, L C



The nuclear localization of 3'-phosphoinositide-dependent kinase-1 is dependent on its association with the protein tyrosine phosphatase SHP1  

Microsoft Academic Search

3'-Phosphoinositide-dependent protein kinase-1 (PDK1), the direct upstream kinase of Akt, can localize to the nucleus during specific signalling events. The mechanism used for its import into the nucleus, however, remains unresolved as it lacks a canonical nuclear localization signal (NLS). Expression of activated Src kinase in C6 glioblastoma cells promotes the association of tyrosylphosphorylated PDK1 with the NLS-containing tyrosine phosphatase

C. F. Sephton; D. Zhang; T. M. Lehmann; P. R. Pennington; M. P. Scheid; D. D. Mousseau



Phosphoinositide 3-kinase ? regulates chromosome segregation in mitosis  

PubMed Central

Class IA phosphoinositide 3-kinases (PI3K) are enzymes composed of a p85 regulatory and a p110 catalytic subunit that control formation of 3-poly-phosphoinositides (PIP3). The PI3K pathway regulates cell survival, migration, and division, and is mutated in approximately half of human tumors. For this reason, it is important to define the function of the ubiquitous PI3K subunits, p110? and p110?. Whereas p110? is activated at G1-phase entry and promotes protein synthesis and gene expression, p110? activity peaks in S phase and regulates DNA synthesis. PI3K activity also increases at the onset of mitosis, but the isoform activated is unknown; we have examined p110? and p110? function in mitosis. p110? was activated at mitosis entry and regulated early mitotic events, such as PIP3 generation, prometaphase progression, and spindle orientation. In contrast, p110? was activated near metaphase and controlled dynein/dynactin and Aurora B activities in kinetochores, chromosome segregation, and optimal function of the spindle checkpoint. These results reveal a p110? function in preserving genomic stability during mitosis.

Silio, Virginia; Redondo-Munoz, Javier; Carrera, Ana C.



Alcohol induced changes in phosphoinositide signaling system in rat brain  

SciTech Connect

Agonist-induced phosphoinositide break down functions as a signal generating system in a manner similar to the C-AMP system. In order to examine if the changes produced by chronic ethanol treatment on membrane lipid composition and metabolism effect the cellular functions of the neuron, the authors have examined the effect of chronic ethanol exposure on norepinephrine (NE) serotonin (5HT) and calcium ionophore (CI) stimulated phosphoinositide (PI) hydrolysis in rat cortical slices. Rats were maintained on liber-decarli diet alcohol and control liquid diet containing isocaloric sucrose substitute for two months. They were then sacrificed and brain was removed for determination of PI turnover. 5HT stimulated {sup 3}H- inositol monophosphate ({sup 3}H-IPI) formation was significantly lower in the cortex of alcohol treated rats as compared to control rats. However, neither CI nor NE stimulated IP1 formation was significantly different from control rats. The results thus indicate that chronic exposure to ethanol decreases 5HT induced PI breakdown in rat cortex. In order to examine if this decrease is related to a decrease in 5HT2 receptors, or decreased in coupling of receptor to the effector pathway, the authors are currently determining the number and affinity of 5HT2 receptors in alcohol treated rats.

Pandey, S.; Piano, M.; Schwertz, D.; Davis, J.; Pandey, G. (Univ. of Illinois, Chicago (United States))



Phosphoinositide Kinases Play Key Roles in Norepinephrine- and Angiotensin II-induced Increase in Phosphatidylinositol 4,5-Bisphosphate and Modulation of Cardiac Function.  


The seemly paradoxical Gq agonist-stimulated phosphoinositide production has long been known, but the underlying mechanism and its physiological significance are not known. In this study, we studied cardiac phosphoinositide levels in both cells and whole animals under the stimulation of norepinephrine (NE), angiotensin II (Ang II), and other physiologically relevant interventions. The results demonstrated that activation of membrane receptors related to NE or Ang II caused an initial increase and a later fall in phosphatidylinositol 4,5-bisphosphate (PIP2) levels in the primary cultured cardiomyocytes from adult rats. The possible mechanism underlying this increase in PIP2 was found to be through an enhanced activity of phosphatidylinositol 4-kinase III?, which was mediated by an up-regulated interaction between phosphatidylinositol 4-kinase III? and PKC; the increased activity of phosphatidylinositol 4-phosphate 5-kinase ? was also involved for NE-induced increase of PIP2. When the systolic functions of the NE/Ang II-treated cells were measured, a maintained or failed contractility was found to be correlated with a rise or fall in corresponding PIP2 levels. In two animal models of cardiac hypertrophy, PIP2 levels were significantly reduced in hypertrophic hearts induced by isoprenaline but not in those induced by swimming exercise. This study describes a novel mechanism for phosphoinositide metabolism and modulation of cardiac function. PMID:24448808

Xu, Jia-Xi; Si, Man; Zhang, Hui-Ran; Chen, Xing-Juan; Zhang, Xi-Dong; Wang, Chuan; Du, Xiao-Na; Zhang, Hai-Lin



Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity  

SciTech Connect

The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).

Morrison, W.J.



Phosphoinositide kinases and the synthesis of polyphosphoinositides in higher plant cells  

NASA Technical Reports Server (NTRS)

Phosphoinositides are a family of inositol-containing phospholipids which are present in all eukaryotic cells. Although in most cells these lipids, with the exception of phosphatidylinositol, constitute only a very minor proportion of total cellular lipids, they have received immense attention by researchers in the past 15-20 years. This is due to the discovery that these lipids, rather than just having structural functions, play key roles in a wide range of important cellular processes. Much less is known about the plant phosphoinositides than about their mammalian counterparts. However, it has been established that a functional phosphoinositide system exists in plant cells and it is becoming increasingly clear that inositol-containing lipids are likely to play many important roles throughout the life of a plant. It is not our intention to give an exhaustive overview of all aspects of the field, but rather we focus on the phosphoinositide kinases responsible for the synthesis of all phosphorylated forms of phosphatidylinositol. Also, we mention some of the aspects of current phosphoinositide research which, in our opinion, are most likely to provide a suitable starting point for further research into the role of phosphoinositides in plants.

Drobak, B. K.; Dewey, R. E.; Boss, W. F.; Davies, E. (Principal Investigator)



Kinetics of the Interaction of myo1c with Phosphoinositides*  

PubMed Central

myo1c is a single-headed myosin that dynamically links membranes to the actin cytoskeleton. A putative pleckstrin homology domain has been identified in the myo1c tail that binds phosphoinositides and soluble inositol phosphates with high affinity. However, the kinetics of association and dissociation and the influence of phospholipid composition on the kinetics have not been determined. Stopped-flow spectroscopy was used to measure the binding and dissociation of a recombinant myo1c construct containing the tail and regulatory domains (myo1cIQ-tail) to and from 100-nm diameter large unilamellar vesicles (LUVs). We found the time course of association of myo1cIQ-tail with LUVs containing 2% phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) followed a two-exponential time course, and the rate of the predominant fast phase depended linearly upon the total lipid concentration. The apparent second-order rate constant was approximately diffusion-limited. Increasing the molar ratio of anionic phospholipid by adding phosphatidylserine, additional PtdIns(4,5)P2, or by situating PtdIns(4,5)P2 in a more physiologically relevant lipid background increased the apparent association rate constant less than 2-fold. myo1cIQ-tail dissociated from PtdIns(4,5)P2 at a slower rate (2.0 s?1) than the pleckstrin homology domain of phospholipase C-? (13 s?1). The presence of additional anionic phospholipid reduced the myo1cIQ-tail dissociation rate constant >50-fold but marginally changed the dissociation rate of phospholipase C-?, suggesting that additional electrostatic interactions in myo1cIQ-tail help to stabilize binding. Remarkably, high concentrations of soluble inositol phosphates induce dissociation of myo1cIQ-tail from LUVs, suggesting that phosphoinositides are able to bind to and dissociate from myo1cIQ-tail as it remains bound to the membrane.

McKenna, Jennine M. Dawicki; Ostap, E. Michael



Multiple roles for Plasmodium berghei phosphoinositide-specific phospholipase C in regulating gametocyte activation and differentiation.  


Critical events in the life cycle of malaria parasites are controlled by calcium-dependent signalling cascades, yet the molecular mechanisms of calcium release remain poorly understood. The synchronized development of Plasmodium berghei gametocytes relies on rapid calcium release from internal stores within 10?s of gametocytes being exposed to mosquito-derived xanthurenic acid (XA). Here we addressed the function of phosphoinositide-specific phospholipase C (PI-PLC) for regulating gametocyte activation. XA triggered the hydrolysis of PIP(2) and the production of the secondary messenger IP(3) in gametocytes. Both processes were selectively blocked by a PI-PLC inhibitor, which also reduced the early Ca(2+) signal. However, microgametocyte differentiation into microgametes was blocked even when the inhibitor was added up to 5?min after activation, suggesting a requirement for PI-PLC beyond the early mobilization of calcium. In contrast, inhibitors of calcium release through ryanodine receptor channels were active only during the first minute of gametocyte activation. Biochemical determination of PI-PLC activity was confirmed using transgenic parasites expressing a fluorescent PIP(2) /IP(3) probe that translocates from the parasite plasmalemma to the cytosol upon cell activation. Our study revealed a complex interdependency of Ca(2+) and PI-PLC activity, with PI-PLC being essential throughout gamete formation, possibly explaining the irreversibility of this process. PMID:21518218

Raabe, Andreas C; Wengelnik, Kai; Billker, Oliver; Vial, Henri J



The Sac domain-containing phosphoinositide phosphatases: structure, function, and disease  

PubMed Central

Phosphoinositides (PIs) have long been known to have an essential role in cell physiology. Their intracellular localization and concentration must be tightly regulated for their proper function. This spatial and temporal regulation is achieved by a large number of PI kinases and phosphatases that are present throughout eukaryotic species. One family of these enzymes contains a conserved PI phosphatase domain termed Sac. Although the Sac domain is homologous among different Sac domain-containing proteins, all appear to exhibit varied substrate specificity and subcellular localization. Dysfunctions in several members of this family are implicated in a range of human diseases such as cardiac hypertrophy, bipolar disorder, Down’s syndrome, Charcot-Marie-Tooth disease (CMT) and Amyotrophic Lateral Sclerosis (ALS). In plant, several Sac domain-containing proteins have been implicated in the stress response, chloroplast function and polarized secretion. In this review, we focus on recent findings in the family of Sac domain-containing PI phosphatases in yeast, mammal and plant, including the structural analysis into the mechanism of enzymatic activity, cellular functions, and their roles in disease pathophysiology.

HSU, FoSheng; MAO, Yuxin



The Phosphoinositide Kinase PIKfyve Mediates EGF Receptor Trafficking to the Nucleus  

PubMed Central

ErbB receptor tyrosine kinases can transit to nuclei in tumor cells, where they have been shown to regulate gene expression as components of transcriptional complexes. Quantitative analysis of a human bladder cancer tissue microarray identified nuclear EGFR in tumor cells and also showed an increased frequency of this histologic feature in cancer relative to normal tissues. This observation suggests a potential role for nuclear EGFR in bladder cancer. We confirmed that EGFR could be induced to transit to nuclei in cultured human bladder cancer cells in response to the urothelial cell growth factor and EGFR ligand, HB-EGF. Mass spectrometric analysis of EGFR immune complexes from a transitional carcinoma cell line (TCCSUP) identified the phosphoinositide kinase, PIKfyve, as a potential component of the EGFR trafficking mechanism. RNA silencing indicated that PIKfyve is a mediator of HB-EGF-stimulated EGFR nuclear trafficking, EGFR binding to the cyclin D1 promoter, and cell cycle progression. These results identify a novel mediator of the EGFR transcription function and further suggest that nuclear EGFR and the lipid kinase PIKfyve may play a role in bladder oncogenesis.

Kim, Jayoung; Jahng, Wan Jin; Di Vizio, Dolores; Lee, Julie S.; Jhaveri, Raj; Rubin, Mark A.; Shisheva, Assia; Freeman, Michael R.



The phosphoinositide kinase PIKfyve mediates epidermal growth factor receptor trafficking to the nucleus.  


ErbB receptor tyrosine kinases can transit to nuclei in tumor cells, where they have been shown to regulate gene expression as components of transcriptional complexes. Quantitative analysis of a human bladder cancer tissue microarray identified nuclear epidermal growth factor receptor (EGFR) in tumor cells and also showed an increased frequency of this histologic feature in cancer relative to normal tissues. This observation suggests a potential role for nuclear EGFR in bladder cancer. We confirmed that EGFR could be induced to transit to nuclei in cultured human bladder cancer cells in response to the urothelial cell growth factor and EGFR ligand heparin-binding EGF-like growth factor (HB-EGF). Mass spectrometric analysis of EGFR immune complexes from a transitional carcinoma cell line (TCCSUP) identified the phosphoinositide kinase, PIKfyve, as a potential component of the EGFR trafficking mechanism. RNA silencing indicated that PIKfyve is a mediator of HB-EGF-stimulated EGFR nuclear trafficking, EGFR binding to the cyclin D1 promoter, and cell cycle progression. These results identify a novel mediator of the EGFR transcription function and further suggest that nuclear EGFR and the lipid kinase PIKfyve may play a role in bladder oncogenesis. PMID:17909029

Kim, Jayoung; Jahng, Wan Jin; Di Vizio, Dolores; Lee, Julie S; Jhaveri, Raj; Rubin, Mark A; Shisheva, Assia; Freeman, Michael R



The tyrosine phosphatase CD148 interacts with the p85 regulatory subunit of phosphoinositide 3-kinase.  


CD148 is a transmembrane tyrosine phosphatase that has been implicated in the regulation of cell growth and transformation. However, the signalling mechanisms of CD148 are incompletely understood. To identify the specific intracellular molecules involved in CD148 signalling, we carried out a modified yeast two-hybrid screening assay. Using the substrate-trapping mutant form of CD148 (CD148 D/A) as bait, we recovered the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase). CD148 D/A, but not catalytically active CD148, interacted with p85 in a phosphorylation-dependent manner in vitro and in intact cells. Growth factor receptor and PI3K activity were also trapped by CD148 D/A via p85 from pervanadate-treated cell lysates. CD148 prominently and specifically dephosphorylated p85 in vitro. Co-expression of CD148 reduced p85 phosphorylation induced by active Src, and attenuated the increases in PI3K activity, yet CD148 did not alter the basal PI3K activity. Finally, CD148 knock-down by siRNA (short interfering RNA) increased PI3K activity on serum stimulation. Taken together, these results demonstrate that CD148 may interact with and dephosphorylate p85 when it is phosphorylated and modulate the magnitude of PI3K activity. PMID:18348712

Tsuboi, Nobuo; Utsunomiya, Tadahiko; Roberts, Richard L; Ito, Hideyuki; Takahashi, Keiko; Noda, Masaharu; Takahashi, Takamune



Oxysterol-binding proteins: sterol and phosphoinositide sensors coordinating transport, signaling and metabolism.  


Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of sterol and phosphoinositide binding proteins conserved in eukaryotes. The mechanisms of ORP function have remained incompletely understood. However, several ORPs are present at membrane contact sites and control the activity of enzymatic effectors or assembly of protein complexes, with impacts on signaling, vesicle transport, and lipid metabolism. An increasing number of protein interaction partners of ORPs have been identified, providing clues of their involvement in multiple aspects of cell regulation. The functions assigned for mammalian ORPs include coordination of sterol and sphingolipid metabolism and mitogenic signaling (OSBP), control of ER-late endosome (LE) contacts and LE motility (ORP1L), neutral lipid metabolism (ORP2), cell adhesion (ORP3), cholesterol eggress from LE (ORP5), macrophage lipid homeostasis, migration and high-density lipoprotein metabolism (ORP8), apolipoprotein B-100 secretion (ORP10), and adipogenesis (ORP11). The anti-proliferative ORPphilin compounds target OSBP and ORP4, revealing a function of ORPs in cell proliferation and survival. The Saccharomyces cerevisiae OSBP homologue (Osh) proteins execute multifaceted functions in sterol and sphingolipid homeostasis, post-Golgi vesicle transport, as well as phosphatidylinositol-4-phosphate and target of rapamycin complex 1 (TORC1) signaling. These observations identify ORPs as coordinators of lipid signals with an unforeseen variety of cellular processes. PMID:23830809

Olkkonen, Vesa M; Li, Shiqian



Corneal Cell Survival in Adenovirus Type 19 Infection Requires Phosphoinositide 3-Kinase/Akt Activation  

PubMed Central

Adenovirus type 19 is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of phosphoinositide 3-kinase (PI3K) and Akt and their downstream targets in adenovirus infection, and here we report the novel finding that adenovirus type 19 utilizes the PI3K/Akt pathway to maintain corneal fibroblast viability in acute infection. We demonstrate phosphorylation of GSK-3? and nuclear translocation of the p65 subunit of NF-?B, both downstream targets of the PI3K/Akt pathway, in adenovirus-infected corneal fibroblasts in a PI3K-dependent manner. Inhibition of PI3K had no effect on early viral gene expression, suggesting normal viral internalization, but pretreatment with the PI3K inhibitor LY294002 or overexpression of dominant negative Akt induced early cytopathic effect and caspase-mediated cell death in adenovirus-infected cells. Early cell death could be circumvented despite LY294002 by overexpression of constitutively active Akt. Furthermore, we show an interaction between cSrc and the p85 regulatory subunit of PI3K in infected cells through a phosphorylation-dependent mechanism. The results presented in this paper provide the first direct evidence that PI3K-mediated Akt activation in adenovirus-infected corneal cells may contribute to viral pathogenesis by the prolongation of cell viability.

Rajala, Maitreyi S.; Rajala, Raju V. S.; Astley, Roger A.; Butt, Amir L.; Chodosh, James



Phosphoinositide 3-kinase mediated signaling in lobster olfactory receptor neurons  

PubMed Central

In vertebrates and some invertebrates, odorant molecules bind to G protein-coupled receptors (GPCRs) on olfactory receptor neurons (ORNs) to initiate signal transduction. Phosphoinositide 3-kinase (PI3K) activity has been implicated physiologically in olfactory signal transduction, suggesting a potential role for a GPCR-activated class I PI3K. Using isoform-specific antibodies, we identified a protein in the olfactory signal transduction compartment of lobster ORNs that is antigenically similar to mammalian PI3K? and cloned a gene for a PI3K with amino acid homology with PI3K?. The lobster olfactory PI3K co-immunoprecipitates with the G protein ? and ? subunits, and an odorant-evoked increase in phosphatidylinositol (3,4,5)-trisphosphate can be detected in the signal transduction compartment of the ORNs. PI3K? and ? isoform-specific inhibitors reduce the odorant-evoked output of lobster ORNs in vivo. Collectively, these findings provide evidence that PI3K is indeed activated by odorant receptors in lobster ORNs and further support the potential involvement of G protein activated PI3K signaling in olfactory transduction.

Corey, Elizabeth A.; Bobkov, Yuriy; Pezier, Adeline; Ache, Barry W.



Phosphoinositides Differentially Regulate Protrudin Localization through the FYVE Domain*  

PubMed Central

Protrudin is a FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain-containing protein involved in transport of neuronal cargoes and implicated in the onset of hereditary spastic paraplegia. Our image-based screening of the lipid binding domain library revealed novel plasma membrane localization of the FYVE domain of protrudin unlike canonical FYVE domains that are localized to early endosomes. The membrane binding study by surface plasmon resonance analysis showed that this FYVE domain preferentially binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) unlike canonical FYVE domains that specifically bind phosphatidylinositol 3-phosphate (PtdIns(3)P). Furthermore, we found that these phosphoinositides (PtdInsP) differentially regulate shuttling of protrudin between endosomes and plasma membrane via its FYVE domain. Protrudin mutants with reduced PtdInsP-binding affinity failed to promote neurite outgrowth in primary cultured hippocampal neurons. These results suggest that novel PtdInsP selectivity of the protrudin-FYVE domain is critical for its cellular localization and its role in neurite outgrowth.

Gil, Jung-Eun; Kim, Eui; Kim, Il-Shin; Ku, Bonsu; Park, Wei Sun; Oh, Byung-Ha; Ryu, Sung Ho; Cho, Wonhwa; Heo, Won Do



Cholinergic stimulation of phosphoinositide hydrolysis in rabbit kidney slices  

SciTech Connect

The release of inositol phosphates (IP) from phosphoinositides (PI) by carbachol was studied in the tissue slices from cortex (C), outer medulla (OM) and inner medulla (IM) of rabbit kidneys. The method involved the incubation of the slices with (/sup 3/H)inositol for its incorporation into the PI and measurement of the release of IP in presence of lithium which prevents dephosphorylation of IP. The results of (/sup 3/H)IP formation are expressed as % of total (/sup 3/H)inositol incorporation in the tissue. No significant effect of carbachol was found on the release of IP in the C. The drug produced a 48% increase in IP release in the OM. In the IM, carbachol produced a concentration dependent increase in IP release with a maximum of 772% at 1 mM. The release of IP in the IM by 1 mM carbachol was completely blocked by 1 atropine. Our results indicate that IP release by carbachol is due to activation of muscarinic receptors in the IM of the rabbit kidney.

Garg, L.C.; McArdle, S.; Crews, F.T.



Identification and structural characterization of a Legionella phosphoinositide phosphatase.  


Bacterial pathogen Legionella pneumophila is the causative agent of Legionnaires' disease, which is associated with intracellular replication of the bacteria in macrophages of human innate immune system. Recent studies indicate that pathogenic bacteria can subvert host cell phosphoinositide (PI) metabolism by translocated virulence effectors. However, in which manner Legionella actively exploits PI lipids to benefit its infection is not well characterized. Here we report that L. pneumophila encodes an effector protein, named SidP, that functions as a PI-3-phosphatase specifically hydrolyzing PI(3)P and PI(3,5)P2 in vitro. This activity of SidP rescues the growth phenotype of a yeast strain defective in PI(3)P phosphatase activity. Crystal structure of SidP orthologue from Legionella longbeachae reveals that this unique PI-3-phosphatase is composed of three distinct domains: a large catalytic domain, an appendage domain that is inserted into the N-terminal portion of the catalytic domain, and a C-terminal ?-helical domain. SidP has a small catalytic pocket that presumably provides substrate specificity by limiting the accessibility of bulky PIs with multiple phosphate groups. Together, our identification of a unique family of Legionella PI phosphatases highlights a common scheme of exploiting host PI lipids in many intracellular bacterial pathogen infections. PMID:23843460

Toulabi, Leila; Wu, Xiaochun; Cheng, Yanshu; Mao, Yuxin



Specific interactions among transmembrane 4 superfamily (TM4SF) proteins and phosphoinositide 4-kinase.  

PubMed Central

In earlier work we established that phosphoinositide 4-kinase (PI 4-kinase) may associate with transmembrane 4 superfamily (TM4SF, tetraspanin) proteins, but critical specificity issues were not addressed. Here we demonstrate that at least five different TM4SF proteins (CD9, CD63, CD81, CD151 and A15/TALLA1) can associate with a similar or identical 55 kDa type II PI 4-kinase. These associations were specific, since we found no evidence for other phosphoinositide kinases (e.g. phosphoinositide 3-kinase and phosphoinositide-4-phosphate 5-kinase) associating with TM4SF proteins, and many other TM4SF proteins (including CD82 and CD53) did not associate with PI 4-kinase. CD63-PI 4-kinase complexes were almost entirely intracellular, and thus are distinct from other TM4SF-PI 4-kinase complexes (e.g. involving CD9), which are largely located in the plasma membrane. These results suggest that a specific subset of TM4SF proteins may recruit PI 4-kinase to specific membrane locations, and thereby influence phosphoinositide-dependent signalling.

Yauch, R L; Hemler, M E



Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity.  


3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287-292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased approximately 6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinase in vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family. PMID:11481331

Park, J; Hill, M M; Hess, D; Brazil, D P; Hofsteenge, J; Hemmings, B A



Endocytosed transferrin receptors recycle via distinct dynamin and phosphatidylinositol 3-kinase-dependent pathways.  


Recycling of endocytosed membrane proteins involves passage through early endosomes and recycling endosomes. Previously, we demonstrated a role for clathrin-coated vesicles in transferrin receptor recycling. These clathrin-coated vesicles are formed from recycling endosomes in a process that was inhibited in dynamin-1(G273D)-overexpressing cells. Here we show a second transferrin recycling pathway, which requires phosphatidylinositol 3-kinase activity. Two unrelated phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, retained endocytosed transferrin in early endosomes but did not affect transfer through recycling endosomes. The inhibitory effects of LY294002 and dynamin-1(G273D) on transferrin recycling were additive. In combination with brefeldin A, a drug that prevents the formation of clathrin-coated buds at recycling endosomes, LY294002 inhibited transferrin recycling synergistically. Collectively, these data indicate two distinct recycling pathways. One pathway involves transfer from early endosomes to recycling endosomes, from where clathrin/dynamin-coated vesicles provide for further transport, whereas the other route bypasses recycling endosomes and requires phosphatidylinositol 3-kinase activity. PMID:12372835

van Dam, Ellen M; Ten Broeke, Toine; Jansen, Karen; Spijkers, Patricia; Stoorvogel, Willem



Phosphoinositide 3-Kinase Signaling Mediates ?-Catenin Activation in Intestinal Epithelial Stem and Progenitor Cells in Colitis  

PubMed Central

Background & Aims Mechanisms responsible for crypt architectural distortion in chronic ulcerative colitis (CUC) are not well understood. Data indicate that Akt signaling cooperates with Wnt to activate ?-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-?-catenin552). We investigated whether phosphoinositide 3- kinase (PI3K) is required for Akt-mediated activation of ?-catenin during intestinal inflammation. Methods The class IA subunit of PI3K was conditionally deleted from intestinal epithelial cells in mice. Acute inflammation was induced in these mice (I-pik3r1KO) and their intestines were analyzed by biochemical and histological methods. The effects of chemically blocking PI3K in colitic IL-10?/? mice were examined. Biopsy samples from patients were examined. Results Compared to wild type mice, I-pik3r1KO mice had reduced T-cell–mediated Akt and ?-catenin signaling in intestinal stem and progenitor cells and limited crypt epithelial proliferation. Biochemical analyses indicated that PI3K–Akt signaling increased nuclear total ?-catenin and P-?-catenin552 levels and reduced phosphorylation of N-terminal ?-catenin, which is associated with degradation. PI3K inhibition in IL-10?/? mice impaired colitis-induced epithelial Akt and ?-catenin activation, reduced progenitor cell expansion, and prevented dysplasia. Human samples had increased numbers of progenitor cells with P-?-catenin552 throughout expanded crypts and increased mRNA expression of ?-catenin target genes in CUC, colitis-associated cancer, tubular adenomas, and sporadic colorectal cancer, compared with control samples. Conclusions PI3K–Akt signaling cooperates with Wnt to increase ?-catenin signaling during inflammation. PI3K-induced and Akt-mediated ?-catenin signaling are required for progenitor cell activation during the progression from CUC to CAC; these factors might be used as biomarkers of dysplastic transformation in the colon.

Lee, Goo; Goretsky, Tatiana; Managlia, Elizabeth; Dirisina, Ramanarao; Singh, Ajay Pal; Brown, Jeffrey B; May, Randal; Yang, Guang-Yu; Ragheb, Josette William; Evers, B Mark; Weber, Christopher R.; Turner, Jerrold R; He, Xi C; Katzman, Rebecca B.; Li, Linheng; Barrett, Terrence A



Triggering actin comets versus membrane ruffles: distinctive effects of phosphoinositides on actin reorganization.  


A limited set of phosphoinositide membrane lipids regulate diverse cellular functions including proliferation, differentiation, and migration. We developed two techniques based on rapamycin-induced protein dimerization to rapidly change the concentration of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. First, using a membrane-recruitable form of PI(4)P 5-kinase, we increased PI(4,5)P(2) synthesis from phosphatidylinositol 4-phosphate [PI(4)P] and found that COS-7, HeLa, and human embryonic kidney 293 cells formed bundles of motile actin filaments known as actin comets. In contrast, a second technique that increased the concentration of PI(4,5)P(2) without consuming PI(4)P induced membrane ruffles. These distinct phenotypes were mediated by dynamin-mediated vesicular trafficking and mutually inhibitory crosstalk between the small guanosine triphosphatases Rac and RhoA. Our results indicate that the effect of PI(4,5)P(2) on actin reorganization depends on the abundance of other phosphoinositides, such as PI(4)P. Thus, combinatorial regulation of phosphoinositide concentrations may contribute to the diversity of phosphoinositide functions. PMID:22169478

Ueno, Tasuku; Falkenburger, Björn H; Pohlmeyer, Christopher; Inoue, Takanari




EPA Science Inventory

Recent evidence indicates that the neurotoxic metal aluminum interferes with the phosphoinositide second messenger system in adult rats both in vitro and in vivo. e have examined the age-related effects of aluminum chloride (AlCl3) on receptor-stimulated inositol phosphate (IP) a...


A class II phosphoinositide 3-kinase plays an indispensable role in hepatitis C virus replication.  


Phosphoinositides function as fundamental signaling molecules and play roles in diverse cellular processes. Certain types of viruses may employ host cell phosphoinositide signaling systems to facilitate their replication cycles. Here we demonstrate that the ? isoform of class II PI3K (PI3K-C2?) plays an indispensable role in hepatitis C virus (HCV) propagation in human hepatocellular carcinoma cells. Knockdown of PI3K-C2? abrogated HCV propagation in the cell. Using an HCV replicon system, we found that knockdown of PI3K-C2? substantially repressed the full-genome replication, while showing relatively small reductions in sub-genome replication, in which structural proteins including core protein were deleted. We also found that HCV core protein showed the binding activity towards D4-phosphorylated phosphoinositides and overlapped localization with phosphatidylinositol 3,4-bisphosphate in the cell. These results suggest that the phosphoinositide generated by PI3K-C2? plays an indispensable role in the HCV replication cycle through the binding to HCV core protein. PMID:24055031

Maehama, Tomohiko; Fukasawa, Masayoshi; Date, Tomoko; Wakita, Takaji; Hanada, Kentaro



Corneal Cell Survival in Adenovirus Type 19 Infection Requires Phosphoinositide 3Kinase\\/Akt Activation  

Microsoft Academic Search

Received 26 March 2005\\/Accepted 13 July 2005 Adenovirus type 19 is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of phosphoinositide 3-kinase (PI3K) and Akt and their downstream targets in adenovirus infection, and here we report the novel finding that adenovirus type 19 utilizes the

Maitreyi S. Rajala; Raju V. S. Rajala; Roger A. Astley; Amir L. Butt; James Chodosh




EPA Science Inventory

The effects of age on cholinergic markers and receptor-stimulated phosphoinositide hydrolysis was dined in the frontal cortex and striatum of male Fischer-344 rats. holine acetyltransferase activity was decreased 27% in the striatum of aged (24 month) rats cared to young (3 month...


Corequirement of Specific Phosphoinositides and Small GTP-binding Protein Cdc42 in Inducing Actin Assembly in Xenopus Egg Extracts  

PubMed Central

Both phosphoinositides and small GTP-binding proteins of the Rho family have been postulated to regulate actin assembly in cells. We have reconstituted actin assembly in response to these signals in Xenopus extracts and examined the relationship of these pathways. We have found that GTP?S stimulates actin assembly in the presence of endogenous membrane vesicles in low speed extracts. These membrane vesicles are required, but can be replaced by lipid vesicles prepared from purified phospholipids containing phosphoinositides. Vesicles containing phosphatidylinositol (4,5) bisphosphate or phosphatidylinositol (3,4,5) trisphosphate can induce actin assembly even in the absence of GTP?S. RhoGDI, a guanine-nucleotide dissociation inhibitor for the Rho family, inhibits phosphoinositide-induced actin assembly, suggesting the involvement of the Rho family small G proteins. Using various dominant mutants of these G proteins, we demonstrate the requirement of Cdc42 for phosphoinositide-induced actin assembly. Our results suggest that phosphoinositides may act to facilitate GTP exchange on Cdc42, as well as to anchor Cdc42 and actin nucleation activities. Hence, both phosphoinositides and Cdc42 are required to induce actin assembly in this cell-free system.

Le Ma; Cantley, Lewis C.; Janmey, Paul A.; Kirschner, Marc W.



Atg18 phosphoregulation controls organellar dynamics by modulating its phosphoinositide-binding activity  

PubMed Central

The PROPPIN family member Atg18 is a phosphoinositide-binding protein that is composed of a seven ?-propeller motif and is part of the conserved autophagy machinery. Here, we report that the Atg18 phosphorylation in the loops in the propellar structure of blade 6 and blade 7 decreases its binding affinity to phosphatidylinositol 3,5-bisphosphate in the yeast Pichia pastoris. Dephosphorylation of Atg18 was necessary for its association with the vacuolar membrane and caused septation of the vacuole. Upon or after dissociation from the vacuolar membrane, Atg18 was rephosphorylated, and the vacuoles fused and formed a single rounded structure. Vacuolar dynamics were regulated according to osmotic changes, oxidative stresses, and nutrient conditions inducing micropexophagy via modulation of Atg18 phosphorylation. This study reveals how the phosphoinositide-binding activity of the PROPPIN family protein Atg18 is regulated at the membrane association domain and highlights the importance of such phosphoregulation in coordinated intracellular reorganization.

Tamura, Naoki; Oku, Masahide; Ito, Moemi; Noda, Nobuo N.; Inagaki, Fuyuhiko




PubMed Central

Summary Inositol lipids and calcium signaling has been inseparable twins during the 1980s when the molecular details of phospholipase C-mediated generation of inositol 1,4,5-trisphosphate (InsP3) and its Ca2+ mobilizing action were discovered. Since then, both the Ca2+- and inositol lipid signaling fields have hugely expanded and the tools allowing dissection of the finest details of their molecular organization also followed closely. Although phosphoinositides regulate many cell functions unrelated to Ca2+ signaling there are still many open questions even in the Ca2+ field that would benefit from single cell monitoring of PtdIns(4,5)P2 or InsP3 changes during agonist stimulation. This chapter is designed to provide practical guidance as well as some theoretical background on measurements of phosphoinositides in live cells using protein domain-GFP chimeras that could be also useful for people working on calcium signaling.

Varnai, Peter; Balla, Tamas



Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p  

PubMed Central

Coupling of Rab GTPase activation and SNARE complex assembly during membrane fusion is poorly understood. The homotypic fusion and vacuole protein sorting (HOPS) complex links these two processes: it is an effector for the vacuolar Rab GTPase Ypt7p and is required for vacuolar SNARE complex assembly. We now report that pure, active HOPS complex binds phosphoinositides and the PX domain of the vacuolar SNARE protein Vam7p. These binding interactions support HOPS complex association with the vacuole and explain its enrichment at the same microdomains on docked vacuoles as phosphoinositides, Ypt7p, Vam7p, and the other SNARE proteins. Concentration of the HOPS complex at these microdomains may be a key factor for coupling Rab GTPase activation to SNARE complex assembly.

Stroupe, Christopher; Collins, Kevin M; Fratti, Rutilio A; Wickner, William



Phosphoinositide 3-kinase\\/Akt signaling pathway and its therapeutical implications for human acute myeloid leukemia  

Microsoft Academic Search

The phosphoinositide 3-kinase (PI3K)\\/Akt signaling pathway is crucial to many aspects of cell growth, survival and apoptosis, and its constitutive activation has been implicated in the both the pathogenesis and the progression of a wide variety of neoplasias. Hence, this pathway is an attractive target for the development of novel anticancer strategies. Recent studies showed that PI3K\\/Akt signaling is frequently

A M Martelli; M Nyĺkern; G Tabellini; R Bortul; P L Tazzari; C Evangelisti; L Cocco



Probing the phosphoinositide 4,5- bisphosphate binding site of human profilin I  

Microsoft Academic Search

Background: Profilin is a widely and highly expressed 14 kDa protein that binds actin monomers, poly(L-proline) and polyp hosphoinositol lipids. It participates in regulating actin-filament dynamics that are essential for many types of cell motility. We sought to investigate the site of interaction of profilin with phosphoinositides.Results: Human profilin I was covalently modified using three tritium-labeled 4-benzoyldihydrocinnamoyl (BZDC)-containing photoaffinity analogs

Anu Chaudhary; Jian Chen; Qu-Ming Gu; Walter Witke; David J. Kwiatkowski; Glenn D. Prestwich



Structural Determinants of Phosphoinositide 3Kinase Inhibition by Wortmannin, LY294002, Quercetin, Myricetin, and Staurosporine  

Microsoft Academic Search

The specific phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 have been invaluable tools for elucidating the roles of these enzymes in signal transduction pathways. The X-ray crystallographic structures of PI3K? bound to these lipid kinase inhibitors and to the broad-spectrum protein kinase inhibitors quercetin, myricetin, and staurosporine reveal how these compounds fit into the ATP binding pocket. With a nanomolar

Edward H. Walker; Michael E. Pacold; Olga Perisic; Len Stephens; Philip T. Hawkins; Matthias P. Wymann; Roger L. Williams



Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance  

Microsoft Academic Search

Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc). This tandem system serves as a nutrient sensor to couple systemic metabolic status to cellular regulation of signal transduction, transcription, and protein degradation. Here we show that O-GlcNAc transferase (OGT) harbours a previously unrecognized type of phosphoinositide-binding domain. After

Xiaoyong Yang; Pat P. Ongusaha; Philip D. Miles; Joyce C. Havstad; Fengxue Zhang; W. Venus So; Jeffrey E. Kudlow; Robert H. Michell; Jerrold M. Olefsky; Seth J. Field; Ronald M. Evans



Learning Deficits and Agenesis of Synapses and Myelinated Axons in Phosphoinositide3 Kinase-Deficient Mice  

Microsoft Academic Search

Although previous studies have reported a role for phosphoinositide-3 kinase (PI3K) in axonal definition and growth in vitro, it is not clear whether PI3K regulates axonal formation and synaptogenesis in vivo. The goal of the present study was to clarify the role of PI3K in behavioral functions and some underlying neuroanatomical structures. Immunohistochemistry, an electron-microscopic analysis and behavioral tests were

Chihiro Tohda; Ruiko Nakanishi; Makoto Kadowaki



The Phosphoinositide 3Kinase\\/Akt1\\/Par4 Axis: A Cancer-Selective Therapeutic Target  

Microsoft Academic Search

Activation of the phosphoinositide 3-kinase (PI3K)\\/Akt cell survival pathway in many cancers makes it an appealing target for therapeutic development. However, because this pathway also has an important role in the survival of normal cells, tactics to achieve cancer selectivity may prove impor- tant. We recently showed that the cancer-selective proapop- totic protein Par-4 is a key target for inactivation

Anindya Goswami


Phospholipase C-? and Phosphoinositide 3Kinase Mediate Cytoplasmic Signaling in Nerve Growth Cone Guidance  

Microsoft Academic Search

Expression of rat TrkA in Xenopus spinal neurons confers responsiveness of these neurons to nerve growth factor (NGF) in assays of neuronal survival and growth cone chemotropism. Mutational analysis indicates that coactivation of phospholipase C-? (PLC-?) and phosphoinositide 3-kinase (PI3-kinase) by specific cytoplasmic domains of TrkA is essential for triggering chemoattraction of the growth cone in an NGF gradient. Uniform

Guo-li Ming; Hong-jun Song; Benedikt Berninger; Naoyuki Inagaki; Marc Tessier-Lavigne; Mu-ming Poo



Phosphoinositide 3Kinase ? Is an Essential Amplifier of Mast Cell Function  

Microsoft Academic Search

Mast cells are key regulators in allergy and inflammation, and release histamine upon clustering of their IgE receptors. Here we demonstrate that murine mast cell responses are exacerbated in vitro and in vivo by autocrine signals through G protein-coupled receptors (GPCRs) and require functional phosphoinositide 3-kinase ? (PI3K?). Adenosine, acting through the A3 adenosine receptor (A3AR) as well as other

Muriel Laffargue; Ronan Calvez; Peter Finan; Alexandre Trifilieff; Maryse Barbier; Fiorella Altruda; Emilio Hirsch; Matthias P Wymann



Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B ?  

Microsoft Academic Search

Background: Protein kinase B (PKB), also known as c-Akt, is activated rapidly when mammalian cells are stimulated with insulin and growth factors, and much of the current interest in this enzyme stems from the observation that it lies ‘downstream’ of phosphoinositide 3-kinase on intracellular signalling pathways. We recently showed that insulin or insulin-like growth factor 1 induce the phosphorylation of

Dario R. Alessi; Stephen R. James; C. Peter Downes; Andrew B. Holmes; Piers R. J. Gaffney; Colin B. Reese; Philip Cohen



Thrombin-induced phosphoinositide hydrolysis in platelets. Receptor occupancy and desensitization.  

PubMed Central

The relationship between occupancy of thrombin receptors on platelets and enhanced phosphoinositide hydrolysis was analysed by examination of the dose-response relationship, the effects of thrombin inhibitors and the contribution of secondary effects. Washed human platelets were labelled with [3H]inositol, and agonist-induced accumulation of labelled inositol phosphates was measured. The dose-response curves and the time courses for alpha-thrombin- or gamma-thrombin-induced accumulation of inositol phosphates were similar to those for dense-granule secretion. Addition of the thrombin inhibitor hirudin to thrombin-activated platelets revealed that the continuous presence of active thrombin was required to maintain the accumulation of labelled inositol phosphates; the total production of inositol phosphates increased with longer periods of exposure to thrombin, reaching a maximum between 5 and 10 min. After activation with thrombin, the ability of a second, greater, addition of thrombin to induce additional phosphoinositide hydrolysis decreased with time; it was absent within 10 min after the first addition. The failure to sustain accumulation of labelled inositol phosphates or to respond to a second addition of thrombin beyond 10 min was not due to depletion of the pool of labelled precursors, because the platelets retained their ability to respond to collagen. Addition of ADP-consuming enzymes decreased sensitivity to thrombin, but inhibition of cyclo-oxygenase with indomethacin did not impair the thrombin-induced hydrolysis of phosphoinositides. It was concluded that thrombin-induced hydrolysis of phosphoinositides has characteristics consistent with mediation by a receptor that is similar to that that triggers dense-granule secretion, requires continuous presence of active thrombin to be maintained, is mediated by a receptor that displays thrombin-induced desensitization, and is only partially enhanced by secondary agents.

Huang, E M; Detwiler, T C



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

PubMed Central

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.

Zurashvili, Tinatin; Cordon-Barris, Lluis; Ruiz-Babot, Gerard; Zhou, Xiangyu; Lizcano, Jose M.; Gomez, Nestor; Gimenez-Llort, Lydia



Sequential breakdown of 3-phosphorylated phosphoinositides is essential for the completion of macropinocytosis  

PubMed Central

Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and solutes are internalized into cells. Macropinocytosis starts with the formation of membrane ruffles at the plasma membrane and ends with their closure. The transient and sequential emergence of phosphoinositides PI(3,4,5)P3 and PI(3,4)P2 in the membrane ruffles is essential for macropinocytosis. By making use of information in the Caenorhabditis elegans mutants defective in fluid-phase endocytosis, we found that mammalian phosphoinositide phosphatase MTMR6 that dephosphorylates PI(3)P to PI, and its binding partner MTMR9, are required for macropinocytosis. INPP4B, which dephosphorylates PI(3,4)P2 to PI(3)P, was also found to be essential for macropinocytosis. These phosphatases operate after the formation of membrane ruffles to complete macropinocytosis. Finally, we showed that KCa3.1, a Ca2+-activated K+ channel that is activated by PI(3)P, is required for macropinocytosis. We propose that the sequential breakdown of PI(3,4,5)P3 ? PI(3,4)P2 ? PI(3)P ? PI controls macropinocytosis through specific effectors of the intermediate phosphoinositides.

Maekawa, Masashi; Terasaka, Shimpei; Mochizuki, Yasuhiro; Kawai, Katsuhisa; Ikeda, Yuka; Araki, Nobukazu; Skolnik, Edward Y.; Taguchi, Tomohiko; Arai, Hiroyuki



Interplay between phosphoinositide lipids and calcium signals at the leading edge of chemotaxing ameboid cells.  


The chemotactic migration of eukaryotic ameboid cells up concentration gradients is among the most advanced forms of cellular behavior. Chemotaxis is controlled by a complex network of signaling proteins bound to specific lipids on the cytoplasmic surface of the plasma membrane at the front of the cell, or the leading edge. The central lipid players in this leading edge signaling pathway include the phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), both of which play multiple roles. The products of PI(4,5)P2 hydrolysis, diacylglycerol (DAG) and Ins(1,4,5)P3 (IP3), are also implicated as important players. Together, these leading edge phosphoinositides and their degradation products, in concert with a local Ca(2+) signal, control the recruitment and activities of many peripheral membrane proteins that are crucial to the leading edge signaling network. The present critical review summarizes the current molecular understanding of chemotactic signaling at the leading edge, including newly discovered roles of phosphoinositide lipids and Ca(2+), while highlighting key questions for future research. PMID:24451847

Falke, Joseph J; Ziemba, Brian P



Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods  

PubMed Central

We characterized phosphoinositide binding of the S. cerevisiae PROPPIN Hsv2 qualitatively with density flotation assays and quantitatively through isothermal titration calorimetry (ITC) measurements using liposomes. We discuss the design of these experiments and show with liposome flotation assays that Hsv2 binds with high specificity to both PtdIns3P and PtdIns(3,5)P2. We propose liposome flotation assays as a more accurate alternative to the commonly used PIP strips for the characterization of phosphoinositide-binding specificities of proteins. We further quantitatively characterized PtdIns3P binding of Hsv2 with ITC measurements and determined a dissociation constant of 0.67 µM and a stoichiometry of 2:1 for PtdIns3P binding to Hsv2. PtdIns3P is crucial for the biogenesis of autophagosomes and their precursors. Besides the PROPPINs there are other PtdIns3P binding proteins with a link to autophagy, which includes the FYVE-domain containing proteins ZFYVE1/DFCP1 and WDFY3/ALFY and the PX-domain containing proteins Atg20 and Snx4/Atg24. The methods described could be useful tools for the characterization of these and other phosphoinositide-binding proteins.

Busse, Ricarda A.; Scacioc, Andreea; Hernandez, Javier M.; Krick, Roswitha; Stephan, Milena; Janshoff, Andreas; Thumm, Michael; Kuhnel, Karin



Stimulation of phosphoinositide breakdown in brain synaptoneurosomes by agents that activate sodium influx: antagonism by tetrodotoxin, saxitoxin, and cadmium.  


Agents that increase intracellular concentrations of Na+ stimulate phosphoinositide breakdown in guinea pig cerebral cortical synaptoneurosomes. When combined, these agents did not have additive effects on phosphoinositide breakdown but did have additive or greater than additive effects with carbamylcholine. Scorpion venom (Leiurus quinquestriatus) and pumiliotoxin B, which induce small increases in influx of 22Na+ in synaptoneurosomes, stimulate phosphoinositide breakdown by about 6- and 3-fold, respectively; both effects are inhibited by tetrodotoxin (TTX). Batrachotoxin (BTX) and veratridine, which cause a large increase in influx of 22Na+ through activation of voltage-dependent sodium channels, induce a 5- to 6-fold dose-dependent increase in phosphoinositide breakdown, which appears competitively inhibited by 5 microM TTX. BTX- and veratridine-elicited influx of 22Na+ into synaptoneurosomes is virtually completely blocked by 5 microM TTX. Agents that block voltage-dependent calcium channels, such as D-600, nifedipine, and Co2+, do not inhibit either influx of 22Na+ or stimulation of phosphoinositide breakdown elicited by scorpion venom, pumiliotoxin B, or BTX. Cadmium ions (200 microM), which are known to block TTX-resistant sodium channels, block phosphoinositide breakdown induced by agents that activate sodium influx through sodium channels. Cadmium blocks BTX-induced phosphoinositide breakdown with an IC50 value of 48 microM, while blocking BTX-induced 22Na+ influx in synaptoneurosomes with a 13-fold lower potency (IC50, 610 microM). In the presence of 0.5 microM TTX, the IC50 for Cd2+ inhibition of BTX-induced 22Na+ influx is now 430 microM. Neither TTX nor Cd2+ antagonize neurotransmitter- or monensin-induced phosphoinositide breakdown. It appears that BTX-induced phosphoinositide breakdown in guinea pig synaptoneurosomes is dependent primarily on activation of TTX-resistant, Cd2+-sensitive sodium channels that account for only a small fraction of the total sodium influx induced by BTX in synaptoneurosomes. However, cadmium also may in some way inhibit phosphoinositide breakdown elicited by sodium channel agents at a point subsequent to sodium influx. PMID:2444871

Gusovsky, F; McNeal, E T; Daly, J W



Phosphoinositide 3-Kinase C2? Regulates RhoA and the Actin Cytoskeleton through an Interaction with Dbl  

PubMed Central

The regulation of cell morphology is a dynamic process under the control of multiple protein complexes acting in a coordinated manner. Phosphoinositide 3-kinases (PI3K) and their lipid products are widely involved in cytoskeletal regulation by interacting with proteins regulating RhoGTPases. Class II PI3K isoforms have been implicated in the regulation of the actin cytoskeleton, although their exact role and mechanism of action remain to be established. In this report, we have identified Dbl, a Rho family guanine nucleotide exchange factor (RhoGEF) as an interaction partner of PI3KC2?. Dbl was co-immunoprecipitated with PI3KC2? in NIH3T3 cells and cancer cell lines. Over-expression of Class II phosphoinositide 3-kinase PI3KC2? in NIH3T3 fibroblasts led to increased stress fibres formation and cell spreading. Accordingly, we found high basal RhoA activity and increased serum response factor (SRF) activation downstream of RhoA upon serum stimulation. In contrast, the dominant-negative form of PI3KC2? strongly reduced cell spreading and stress fibres formation, as well as SRF response. Platelet-derived growth factor (PDGF) stimulation of wild-type PI3KC2? over-expressing NIH3T3 cells strongly increased Rac and c-Jun N-terminal kinase (JNK) activation, but failed to show similar effect in the cells with the dominant-negative enzyme. Interestingly, epidermal growth factor (EGF) and PDGF stimulation led to increased extracellular signal-regulated kinase (Erk) and Akt pathway activation in cells with elevated wild-type PI3KC2? expression. Furthermore, increased expression of PI3KC2? protected NIH3T3 from detachment-dependent death (anoikis) in a RhoA-dependent manner. Taken together, these findings suggest that PI3KC2? modulates the cell morphology and survival through a specific interaction with Dbl and the activation of RhoA.

Leitner, Laura; Uth, Kristin; Posern, Guido; Arcaro, Alexandre



T-cell function is partially maintained in the absence of class IA phosphoinositide 3-kinase signaling  

PubMed Central

The class IA subgroup of phosphoinositide 3-kinase (PI3K) is activated downstream of antigen receptors, costimulatory molecules, and cytokine receptors on lymphocytes. Targeted deletion of individual genes for class IA regulatory subunits severely impairs the development and function of B cells but not T cells. Here we analyze conditional mutant mice in which thymocytes and T cells lack the major class IA regulatory subunits p85?, p55?, p50?, and p85?. These cells exhibit nearly complete loss of PI3K signaling downstream of the T-cell receptor (TCR) and CD28. Nevertheless, T-cell development is largely unperturbed, and peripheral T cells show only partial impairments in proliferation and cytokine production in vitro. Both genetic and pharmacologic experiments suggest that class IA PI3K signaling plays a limited role in T-cell proliferation driven by TCR/CD28 clustering. In vivo, class IA–deficient T cells provide reduced help to B cells but show normal ability to mediate antiviral immunity. Together these findings provide definitive evidence that class IA PI3K regulatory subunits are essential for a subset of T-cell functions while challenging the notion that this signaling mechanism is a critical mediator of costimulatory signals downstream of CD28.

Deane, Jonathan A.; Kharas, Michael G.; Oak, Jean S.; Stiles, Linda N.; Luo, Ji; Moore, Travis I.; Ji, Hong; Rommel, Christian; Cantley, Lewis C.; Lane, Thomas E.



TbFRP, a novel FYVE-domain containing phosphoinositide-binding Ras-like GTPase from trypanosomes.  


Ras-like small GTPases are regulatory proteins that control multiple aspects of cellular function, and are particularly prevalent in vesicular transport. A proportion of GTPase paralogs appear restricted to certain eukaryote lineages, suggesting roles specific to a restricted lineage, and hence potentially reflecting adaptation to individual lifestyles or ecological niche. Here we describe the role of a GTPase, TbFRP, a FYVE domain N-terminally fused to a Ras-like GTPase, originally identified in Trypanosoma brucei. As FYVE-domains specifically bind phosphoinositol 3-phosphate (PI3P), which associates with endosomes, we suggest that TbFRP may unite phosphoinositide and small G protein endosomal signaling in trypanosomatids. TbFRP orthologs are present throughout the Euglenazoa suggesting that FRP has functions throughout the group. We show that the FYVE domain of TbFRP is functional in PI3P-dependent membrane targeting and localizes at the endosomal region. Further, while TbFRP is apparently non-essential, knockdown and immunochemical evidence indicates that TbFRP is rapidly cleaved upon synthesis, releasing the GTPase and FYVE-domains. Finally, TbFRP expression at both mRNA and protein levels is cell density-dependent. Together, these data suggest that TbFRP is an endocytic GTPase with a highly unusual mechanism of action that involves proteolysis of the nascent protein and membrane targeting via PI3P. PMID:23220323

Adung'a, Vincent O; Field, Mark C



TbFRP, a novel FYVE-domain containing phosphoinositide-binding Ras-like GTPase from trypanosomes  

PubMed Central

Ras-like small GTPases are regulatory proteins that control multiple aspects of cellular function, and are particularly prevalent in vesicular transport. A proportion of GTPase paralogs appear restricted to certain eukaryote lineages, suggesting roles specific to a restricted lineage, and hence potentially reflecting adaptation to individual lifestyles or ecological niche. Here we describe the role of a GTPase, TbFRP, a FYVE domain N-terminally fused to a Ras-like GTPase, originally identified in Trypanosoma brucei. As FYVE-domains specifically bind phosphoinositol 3-phosphate (PI3P), which associates with endosomes, we suggest that TbFRP may unite phosphoinositide and small G protein endosomal signaling in trypanosomatids. TbFRP orthologs are present throughout the Euglenazoa suggesting that FRP has functions throughout the group. We show that the FYVE domain of TbFRP is functional in PI3P-dependent membrane targeting and localizes at the endosomal region. Further, while TbFRP is apparently non-essential, knockdown and immunochemical evidence indicates that TbFRP is rapidly cleaved upon synthesis, releasing the GTPase and FYVE-domains. Finally, TbFRP expression at both mRNA and protein levels is cell density-dependent. Together, these data suggest that TbFRP is an endocytic GTPase with a highly unusual mechanism of action that involves proteolysis of the nascent protein and membrane targeting via PI3P.

Adung'a, Vincent O.; Field, Mark C.



Phorbol esters inhibit alpha/sub 1/-adrenergic receptor stimulated phosphoinositide hydrolysis and contraction in rat aorta  

SciTech Connect

The mechanisms of pharmacomechanical coupling in vascular tissue are at the present time unclear. The authors and others have proposed that receptor-induced activation of phosphoinositide (PI) hydrolysis may be involved. To investigate this possibility they studied the actions of two biologically active phorbol esters: phorbol dibutyrate (PDB) and phorbol myristate diacetate (PMA) on receptor-stimulated PI hydrolysis in rat aortic rings. They found both PDB (IC/sub 5//sup 0/ approx. 5nM) and PMA (IC/sub 50/ approx. 30 nM) but not 4-..cap alpha..-phorbol (IC32%/sub 0/ > 10,000 nM) inhibited norepinephrine-stimulated PI hydrolysis. In the presence of the calcium channel antagonist nitrendipine, PDB potently inhibited both the phasic and tonic components of norepinephrine-induced vascular contraction. In the presence of 10/sup -7/M nitrendipine, PDB had an IC/sub 50/ for contraction of approximately 10nM. The results thus suggest a functional coupling between ..cap alpha../sub 1/-adrenergic receptor-stimulated PI hydrolysis and vascular contraction. The findings further imply a mode of feed-back regulation in vascular tissue involving phorbol ester and receptor-stimulated PI hydrolysis.

Not Available



Spatial analysis of 3' phosphoinositide signaling in living fibroblasts, III: influence of cell morphology and morphological Polarity.  


Activation of phosphoinositide (PI) 3-kinase is a required signaling pathway in fibroblast migration directed by platelet-derived growth factor. The pattern of 3' PI lipids in the plasma membrane, integrating local PI 3-kinase activity as well as 3' PI diffusion and turnover, influences the spatiotemporal regulation of the cytoskeleton. In fibroblasts stimulated uniformly with platelet-derived growth factor, visualized using total internal reflection fluorescence microscopy, we consistently observed localized regions with significantly higher or lower 3' PI levels than adjacent regions (hot and cold spots, respectively). A typical cell contained multiple hot spots, coinciding with apparent leading edge structures, and at most one cold spot at the rear. Using a framework for finite-element modeling with actual cell contact area geometries, we find that although the 3' PI pattern is affected by irregular contact area shape, cell morphology alone cannot explain the presence of hot or cold spots. Our results and analysis instead suggest that these regions reflect different local 3' PI dynamics, specifically through a combination of mechanisms: enhanced PI 3-kinase activity, reduced 3' PI turnover, and possibly slow/constrained 3' PI diffusion. The morphological polarity of the cell may thus bias 3' PI signaling to promote persistent migration in fibroblasts. PMID:15923219

Schneider, Ian C; Parrish, Elizabeth M; Haugh, Jason M



Momordica charantia (bitter melon) reduces plasma apolipoprotein B-100 and increases hepatic insulin receptor substrate and phosphoinositide-3 kinase interactions.  


Aqueous extracts or juice from unripened fruit of Momordica charantia (bitter melon) has traditionally been used in the treatment of diabetes and its complications. Insulin resistance is characterized by significant down-regulation of hepatic insulin signalling as documented by attenuated phosphorylation of insulin receptor (IR), IR substrates 1 and 2, phosphoinositide-3 kinase, protein kinase B, and over-expression of phosphotyrosine phosphatase 1B. We recently demonstrated that bitter melon juice (BMJ) is a potent inhibitor of apoB secretion and TAG synthesis and secretion in human hepatoma cells, HepG2, that may be involved in plasma lipid- and VLDL-lowering effects observed in animal studies. The aim of this study was to evaluate the effects of BMJ on plasma apoB levels and hepatic insulin signalling cascade in mice fed high-fat diet (HFD). Female C57BL/6 mice (4-6 weeks old) were randomized into three groups receiving regular rodent chow, HFD and HFD+BMJ. The data indicate that BMJ not only improves glucose and insulin tolerance but also lowers plasma apoB-100 and apoB-48 in HFD-fed mice as well as modulates the phosphorylation status of IR and its downstream signalling molecules. Investigating the biochemical and molecular mechanisms involved in amelioration of diabetic dyslipidaemia by BMJ may lead to identification of new molecular targets for dietary/alternative therapies. PMID:18321395

Nerurkar, Pratibha V; Lee, Yun Kyung; Motosue, Megan; Adeli, Khosrow; Nerurkar, Vivek R



Spatial Regulation of Membrane Fusion Controlled by Modification of Phosphoinositides  

Microsoft Academic Search

Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG) facilitate fusion. However, the specific role of lipids

Fabrice Dumas; Richard D. Byrne; Ben Vincent; Tina M. C. Hobday; Dominic L. Poccia; Banafshé Larijani; Richard Steinhardt



The role of phosphoinositide 3-kinase and phosphatidic acid in the regulation of mammalian target of rapamycin following eccentric contractions  

PubMed Central

Resistance exercise induces a hypertrophic response in skeletal muscle and recent studies have begun to shed light on the molecular mechanisms involved in this process. For example, several studies indicate that signalling by the mammalian target of rapamycin (mTOR) is necessary for a hypertrophic response. Furthermore, resistance exercise has been proposed to activate mTOR signalling through an upstream pathway involving the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB); however, this hypothesis has not been thoroughly tested. To test this hypothesis, we first evaluated the temporal pattern of signalling through PI3K–PKB and mTOR following a bout of resistance exercise with eccentric contractions (EC). Our results indicated that the activation of signalling through PI3K–PKB is a transient event (<15 min), while the activation of mTOR is sustained for a long duration (>12 h). Furthermore, inhibition of PI3K–PKB activity did not prevent the activation of mTOR signalling by ECs, indicating that PI3K–PKB is not part of the upstream regulatory pathway. These observations led us to investigate an alternative pathway for the activation of mTOR signalling involving the synthesis of phosphatidic acid (PA) by phospholipase D (PLD). Our results demonstrate that ECs induce a sustained elevation in [PA] and inhibiting the synthesis of PA by PLD prevented the activation of mTOR. Furthermore, we determined that similar to ECs, PA activates mTOR signalling through a PI3K–PKB-independent mechanism. Combined, the results of this study indicate that the activation of mTOR following eccentric contractions occurs through a PI3K–PKB-independent mechanism that requires PLD and PA.

O'Neil, T K; Duffy, L R; Frey, J W; Hornberger, T A



Characterization of cholinergic muscarinic receptor-stimulated phosphoinositide metabolism in brain from immature rats  

SciTech Connect

Hydrolysis of phosphoinositides elicited by stimulation of cholinergic muscarinic receptors has been studied in brain from neonatal (7-day-old) rats in order to determine: (1) whether the neonatal rat could provide a good model system to study this signal-transduction pathway; and (2) whether potential differences with adult nerve tissue would explain the differential, age-related effects of cholinergic agonists. Accumulation of (3H) inositol phosphates in (3H)inositol prelabeled slices from neonatal and adult rats was measured as an index of phosphoinositide metabolism. Full (acetylcholine, methacholine, carbachol) and partial (oxotremorine, bethanechol) agonists had qualitatively similar, albeit quantitatively different, effects in neonatal and adult rats. Atropine and pirenzepine effectively blocked the carbachol-induced response with inhibition constants of 1.2 and 20.7 nM, respectively. In all brain areas, response to all agonists was higher in neonatal than adult rats, and in hippocampus and cerebral cortex the response was higher than in cerebellum or brainstem. The relative intrinsic activity of partial agonists was higher in the latter two areas (0.6-0.7) than in the former two (0.3-0.4). Carbachol-stimulated phosphoinositide metabolism in brain areas correlated well with the binding of (3H)QNB (r2 = 0.627) and, particularly, with (3H)pirenzepine (r2 = 0.911). In cerebral cortex the effect of carbachol was additive to that of norepinephrine and glutamate. The presence of calcium (250-500 microM) was necessary for maximal response to carbachol to be elicited; the EC50 value for Ca2+ was 65.4 microM. Addition of EDTA completely abolished the response. Removal of sodium ions from the incubation medium reduced the response to carbachol by 50%.

Balduini, W.; Murphy, S.D.; Costa, L.G. (Univ. of Washington, Seattle (USA))



Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells  

PubMed Central

Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3K? (phosphoinositide 3-kinase ?) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3K? in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3K? is pre-formed as a 1:1 p110?:p85? complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3K? was recruited to the PM, but oncogenic forms of PI3K? were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3K?. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity.

Layton, Meredith J.; Rynkiewicz, Natalie K.; Ivetac, Ivan; Horan, Kristy A.; Mitchell, Christina A.; Phillips, Wayne A.



Membrane Targeting by APPL1 and APPL2: Dynamic Scaffolds that Oligomerize and Bind Phosphoinositides  

PubMed Central

Human adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1) and adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2) are homologous effectors of the small guanosine triphosphatase RAB5 that interact with a diverse set of receptors and signaling proteins and are proposed to function in endosome-mediated signaling. Herein, we investigated the membrane-targeting properties of the APPL1 and APPL2 Bin/Amphiphysin/Rvs (BAR), pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains. Coimmunoprecipitation and yeast two-hybrid studies demonstrated that full-length APPL proteins formed homooligomers and heterooligomers and that the APPL minimal BAR domains were necessary and sufficient for mediating APPL-APPL interactions. When fused to a fluorescent protein and overexpressed, all three domains (minimal BAR, PH and PTB) were targeted to cell membranes. Furthermore, full-length APPL proteins bound to phosphoinositides, and the APPL isolated PH or PTB domains were sufficient for in vitro phosphoinositide binding. Live cell imaging showed that full-length APPL-yellow fluorescent protein (YFP) fusion proteins associated with cytosolic membrane structures that underwent movement, fusion and fission events. Overexpression of full-length APPL-YFP fusion proteins was sufficient to recruit endogenous RAB5 to enlarged APPL-associated membrane structures, although APPL1 was not necessary for RAB5 membrane targeting. Taken together, our findings suggest a role for APPL proteins as dynamic scaffolds that modulate RAB5-associated signaling endosomal membranes by their ability to undergo domain-mediated oligomerization, membrane targeting and phosphoinositide binding.

Chial, Heidi J.; Wu, Ruping; Ustach, Carolyn V.; McPhail, Linda C.; Mobley, William C.; Chen, Yong Q.



Phosphoinositide-mediated oligomerization of a defensin induces cell lysis  

PubMed Central

Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique ‘cationic grip’ configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism. DOI:

Poon, Ivan KH; Baxter, Amy A; Lay, Fung T; Mills, Grant D; Adda, Christopher G; Payne, Jennifer AE; Phan, Thanh Kha; Ryan, Gemma F; White, Julie A; Veneer, Prem K; van der Weerden, Nicole L; Anderson, Marilyn A; Kvansakul, Marc; Hulett, Mark D



Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation.  


Platelet stimulation by thrombin leads to the activation of phosphoinositide 3-kinase (PI 3K) and to the production of the D3 phosphoinositides, phosphatidylinositol 3,4-bisphosphate (PdtIns-3,4P2) and 3,4,5-trisphosphate (PdtIns-3,4,5-P3). Because changes in the levels of these phosphoinositides correlate with the kinetics of actin assembly, they have been proposed to mediate actin assembly, causing cell shape changes. Wortmannin and LY294002, two unrelated inhibitors of PI 3-K, were used to investigate the role of PI 3-K in platelet actin assembly and aggregation. Both PI 3-K inhibitors abrogated the production of PdtIns-3,4-P2 and PdtIns-3,4,5-P3 in thrombin receptor-activating peptide (TRAP)-stimulated cells. However, neither wortmannin nor LY294002 altered the kinetics of actin assembly or the exposure of nucleation sites in TRAP-stimulated cells. In contrast, PI 3-K inhibitors showed a specific inhibitory pattern of cell aggregation, characterized by a primary phase of aggregation followed by progressive disaggregation. Flow cytometry analysis with the PAC1 monoclonal antibody or with FITC-labeled fibrinogen indicated that wortmannin inhibited the maintenance of the platelet integrin GPIIb-IIIa in its active state. Wortmannin also inhibited, in a dose-dependent manner, platelet aggregation induced by the binding of the monoclonal antibodies P256 and LIBS-6 to GPIIb-IIIa. LIBS Fab-induced aggregation also led to the production of PdtIns-3,4-P2. Platelet secretion, as evidenced by the release of preloaded 14C-5-hydroxy-tryptamine secretion or P-selectin up-regulation, was not affected by PI 3-K inhibition. These results demonstrate that the generation of D3 phosphoinositides is not required for actin assembly in TRAP-activated platelets. However, PI 3-K stimulation is necessary for prolonged GPIIb-IIIa activation and irreversible platelet aggregation. PI 3-K stimulation downstream of GPIIb-IIIa engagement may provide positive feedback required to sustain active GPIIb-IIIa. PMID:7744773

Kovacsovics, T J; Bachelot, C; Toker, A; Vlahos, C J; Duckworth, B; Cantley, L C; Hartwig, J H



Suppression of Phosphoinositide 3-Kinase Signaling and Alteration of Multiple Ion Currents in Drug-Induced Long QT Syndrome  

PubMed Central

Many drugs, including some commonly used medications, can cause abnormal heart rhythms and sudden death, as manifest by a prolonged QT interval in the electrocardiogram. Cardiac arrhythmias caused by drug-induced long QT syndrome are thought to result mainly from reductions in the delayed rectifier potassium ion (K+) current IKr. Here, we report a mechanism for drug-induced QT prolongation that involves changes in multiple ion currents caused by a decrease in phosphoinositide 3-kinase (PI3K) signaling. Treatment of canine cardiac myocytes with inhibitors of tyrosine kinases or PI3Ks caused an increase in action potential duration that was reversed by intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate. The inhibitors decreased the delayed rectifier K+ currents IKr and IKs, the L-type calcium ion (Ca2+) current ICa,L, and the peak sodium ion (Na+) current INa and increased the persistent Na+ current INaP. Computer modeling of the canine ventricular action potential showed that the drug-induced change in any one current accounted for less than 50% of the increase in action potential duration. Mouse hearts lacking the PI3K p110? catalytic subunit exhibited a prolonged action potential and QT interval that were at least partly a result of an increase in INaP. These results indicate that down-regulation of PI3K signaling directly or indirectly via tyrosine kinase inhibition prolongs the QT interval by affecting multiple ion channels. This mechanism may explain why some tyrosine kinase inhibitors in clinical use are associated with increased risk of life-threatening arrhythmias.

Lu, Zhongju; Wu, Chia-Yen C.; Jiang, Ya-Ping; Ballou, Lisa M.; Clausen, Chris; Cohen, Ira S.; Lin, Richard Z.



Spatial Regulation of Membrane Fusion Controlled by Modification of Phosphoinositides  

PubMed Central

Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG) facilitate fusion. However, the specific role of lipids in membrane fusion of natural membranes is not well established. Nuclear envelope (NE) assembly was used as a model for membrane fusion. A natural membrane population highly enriched in the enzyme and substrate needed to produce DAG has been isolated and is required for fusions leading to nuclear envelope formation, although it contributes only a small amount of the membrane eventually incorporated into the NE. It was postulated to initiate and regulate membrane fusion. Here we use a multidisciplinary approach including subcellular membrane purification, fluorescence spectroscopy and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM) to demonstrate that initiation of vesicle fusion arises from two unique sites where these vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic reticulum-derived membranes that make up the bulk of the NE to ultimately enclose the chromatin. We show how initiation of multiple vesicle fusions can be controlled by localised production of DAG and propagated bidirectionally. Phospholipase C (PLC?), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P2) are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism.

Dumas, Fabrice; Byrne, Richard D.; Vincent, Ben; Hobday, Tina M. C.; Poccia, Dominic L.; Larijani, Banafshe



Activation of Muscarinic and Serotonergic Receptors Results in Phosphoinositide Hydrolysis but Not in Mobilization of Calcium in Sympathetic Neurons  

Microsoft Academic Search

The effects of various neurotransmitters on phosphoinositide hydrolysis, mobilization of Ca2+ and release of [3H]-norepinephrine ([3H]-NE) were studied in cultures of sympathetic neurons of chick embryos. [3H]-inositol-1,4,5-triphosphate ([3H]-IP3) was increased in sympathetic neurons by acetylcholine (ACh), muscarine and serotonin (5-HT). Dopamine and norepinephrine did not stimulate phosphoinositide hydrolysis. Intracellular concentration of free Ca2+ ([Ca2+]i) was measured in Indo-1-loaded sympathetic neurons

Taruna D. Wakade; Anjali S. Bhave; Sanjiv V. Bhave; Arun R. Wakade



Electrostatics of phosphoinositide bilayer membranes. Theoretical and experimental results.  

PubMed Central

We made fluorescence, electron paramagnetic resonance (EPR), electrophoretic mobility, and ionizing electrode measurements to study the effect of the monovalent lipid phosphatidylinositol (PI) and the trivalent lipid phosphatidylinositol 4,5-bisphosphate (PIP2) on the electrostatic potential adjacent to bilayer membranes. When the membranes were formed from mixtures of PI and the zwitterionic lipid phosphatidylcholine (PC), the Gouy-Chapman-Stern (GCS) theory described adequately the dependence of potential on distance (0, 1, 2 nm) from the membrane, mole % negative lipid, and [KCI]. Furthermore, all EPR and fluorescence probes reported identical surface potentials with a PC/PI membrane. With PC/PIP2 membranes, however, the anionic (coion) probes reported less negative potentials than the cationic (counterion) probes; the deviations from the GCS theory were greater for the coions than the counterions. Discreteness-of-charge theories based on the Poisson-Boltzmann equation incorrectly predict that deviations from the GCS theory should be greater for counterions than for coions. We discuss a consistent statistical mechanical theory that takes into account three effects ignored in the GCS theory: the finite size of the ions in the double layer, the electrical interaction between pairs of ions (correlation effects), and the mobile discrete nature of the surface charges. This theory correctly predicts that deviations from GCS theory should be negligible for monovalent lipids, significant for trivalent lipids, and greater for coions than for counterions.

Langner, M; Cafiso, D; Marcelja, S; McLaughlin, S



5-Hydroxytryptamine receptor-mediated phosphoinositide hydrolysis in canine cultured tracheal smooth muscle cells.  

PubMed Central

1. 5-Hydroxytryptamine (5-HT) has been shown to induce contraction of tracheal smooth muscle. However, the mechanisms of action of 5-HT are not known. We therefore investigated the effects of 5-HT on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and its regulation in canine cultured tracheal smooth muscle cells (TSMCs) labelled with [3H]-inositol. 5-HT-induced inositol phosphates (IPs) accumulation was time- and dose-dependent with a half-maximal response (EC50) and a maximal response at 0.38 +/- 0.05 and 10 microM, respectively. 2. Ketanserin and mianserin (10 and 100 nM), 5-HT2 receptor antagonists, were equipotent in blocking the 5-HT-induced IPs accumulation with pKB values of 8.46 and 8.21, respectively. In contrast, the dose-response curves of 5-HT-induced IPs accumulation were not shifted until the concentrations of NAN-190 and metoclopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased up to 10 microM. 3. Pretreatment of TSMCs with pertussis toxin or cholera toxin did not inhibit the 5-HT-induced IPs accumulation, but partially inhibited the AlF(4-)-induced IPs response. 4. Stimulation of IPs accumulation by 5-HT required the presence of external Ca2+ and was blocked by EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. A further Ca(2+)-dependent increase in IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphoshate) (GTP gamma S) or 5-HT. The combination of GTP gamma S and 5-HT elicited an additive effect on IPs accumulation. 5. Treatment with phorbol 12-myristate 13-acetate (PMA, 1 microM, 30 min) abolished the 5-HT-induced IPs accumulation. The concentrations of PMA that gave a half-maximal and maximal inhibition of 5-HT-induced IPs accumulation were 2.2 +/- 0.4 nM and 1 microM, n = 3, respectively. The protein kinase C (PKC) activator, 4 alpha-phorbol 12,13-didecanoate, at 1 microM, did not influence this response. The inhibitory effect of PMA was reversed by staurosporine, a PKC inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 6. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the effect of PMA is distal to the 5-HT receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Yang, C. M.; Yo, Y. L.; Hsieh, J. T.; Ong, R.



Expression and evolution of the phosphoinositide-specific phospholipase C gene family in Arabidopsis thaliana.  


Phosphoinositide-specific phospholipase C cleaves the substrate phosphatidylinositol 4,5-bisphosphate and generates inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, both of which are second messengers in the phosphoinositide signal transduction pathways operative in animal cells. Five PI-PLC isoforms, beta, gamma, delta, epsilon and zeta, have been identified in mammals. Plant PI-PLCs are structurally close to the mammalian PI-PLC-zeta isoform. The Arabidopsis genome contains nine AtPLC genes. Expression patterns of all nine genes in different organs and in response to various environmental stimuli were studied by applying a quantitative RT-PCR approach. Multiple members of the gene family were differentially expressed in Arabidopsis organs, suggesting putative roles for this enzyme in plant development, including tissue and organ differentiation. This study also shows that a majority of the AtPLC genes are induced in response to various environmental stimuli, including cold, salt, nutrients Murashige-Skoog salts, dehydration, and the plant hormone abscisic acid. Results of this and previous studies strongly suggest that transcriptional activation of the PI-PLC gene family is important for adapting plants to stress environments. Expression patterns and phylogenetic relationships indicates that AtPLC gene members probably evolved through multiple rounds of gene duplication events, with AtPLC4 and AtPLC5 and AtPLC8 and AtPLC9 being duplicated in tandem in recent times. PMID:18534862

Tasma, I Made; Brendel, Volker; Whitham, Steven A; Bhattacharyya, Madan K



Actin and phosphoinositide binding by the ActA protein of the bacterial pathogen Listeria monocytogenes.  


The surface protein ActA of the pathogenic bacterium Listeria monocytogenes induces actin-driven movement of bacteria in the cytoplasm of infected host cells and serves as a model for actin-based motility in general. We generated and purified soluble recombinant fragments of ActA and assessed their ability to interact with the acidic phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, both implicated in the regulation of actin polymerization. Purified ActA consisted of biologically active, elongated molecules with an alpha-helix and beta-sheet content of 11 and 32%, respectively. In the presence of either phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate, but not phosphatidylcholine, ActA molecules underwent a structural change that raised the alpha-helix content to 19% and lowered the beta-sheet content to 27%. Co-sedimentation experiments with phosphatidylcholine vesicles containing different acidic phospholipids demonstrated that ActA binds preferentially to D-3 phosphoinositides. The D-3 phosphoinositide binding activity was mapped to a small subregion in the N-terminal domain of ActA. This subregion comprised 19 amino acids and showed homology to cecropins. In addition, we found that amino acids 33 to 74 of ActA mediated actin binding by the whole, folded ActA molecule. These findings shed new light on ActA function. PMID:10559250

Cicchetti, G; Maurer, P; Wagener, P; Kocks, C



Phosphoinositides: Minor Lipids Make a Major Impact on Photoreceptor Cell Functions  

PubMed Central

Activation of the phosphoinositide (PI) cycle generates the second messengers that control various aspects of cellular signaling. We have previously shown that two PI cycle enzymes, type II phosphatidylinositol 5-phosphate 4-kinase (PIPK II?) and phosphoinositide 3-kinase (PI3K), are activated through light stimulation. In our earlier studies, we measured enzyme activities, instead of directly measuring the products, due to lack of sensitive analytical techniques. Cells have very low levels of PIs, compared to other lipids, so special techniques and sensitive analytical instruments are necessary for their identification and quantification. There are also other considerations, such as different responses in different cell types, which may complicate quantification of PIs. For example, although light activated PIPK II?, there was no increase in PI-4,5-P2 measured by liquid chromatography–mass spectrometry (LC/MS) This discrepancy is due to the heterogeneous nature of the retina, which is composed of various cell types. In this study, we examined PI generation in situ using immunohistochemistry with specific PI antibodies. PIs were generated in specific retinal cell layers, suggesting that analyzing PIs from the total retina by LC/MS underscores the significance. This suggests that PI-specific antibodies are useful tools to study the cell-specific regulation of PIs in the retina.

Rajala, Raju V. S.; Rajala, Ammaji; Morris, Andrew J.; Anderson, Robert E.



Phosphoinositide Binding by the Toll Adaptor dMyD88 Controls Antibacterial Responses in Drosophila  

PubMed Central

The cell biological principles that govern innate immune responses in Drosophila are unknown. Here, we report that Toll signaling in flies was dictated by the subcellular localization of the adaptor protein dMyD88. dMyD88 was located at the plasma membrane by a process dependent on a C-terminal phosphoinositide-binding domain. In vivo analysis revealed that lipid binding by dMyD88 was necessary for its antimicrobial and developmental functions, as well as for the recruitment of the downstream cytosolic adaptor Tube to the cell surface. These data are reminiscent of the interactions between the mammalian Toll adaptors MyD88 and TIRAP with one major exception. In the mammalian system, MyD88 is the cytosolic adaptor that depends on the phosphoinositide-binding protein TIRAP for its recruitment to the cell surface. We therefore propose that dMyD88 is the functional homologue of TIRAP, and that both proteins function as sorting adaptors to recruit downstream signaling adaptors to activated receptors.

Marek, Lorri R.; Kagan, Jonathan C.



Energetics and Location of Phosphoinositide Binding in Human Kir2.1 Channels*  

PubMed Central

Kir2.1 channels are uniquely activated by phosphoinositide 4,5-bisphosphate (PI(4,5)P2) and can be inhibited by other phosphoinositides (PIPs). Using biochemical and computational approaches, we assess PIP-channel interactions and distinguish residues that are energetically critical for binding from those that alter PIP sensitivity by shifting the open-closed equilibrium. Intriguingly, binding of each PIP is disrupted by a different subset of mutations. In silico ligand docking indicates that PIPs bind to two sites. The second minor site may correspond to the secondary anionic phospholipid site required for channel activation. However, 96–99% of PIP binding localizes to the first cluster, which corresponds to the general PI(4,5)P2 binding location in recent Kir crystal structures. PIPs can encompass multiple orientations; each di- and triphosphorylated species binds with comparable energies and is favored over monophosphorylated PIPs. The data suggest that selective activation by PI(4,5)P2 involves orientational specificity and that other PIPs inhibit this activation through direct competition.

D'Avanzo, Nazzareno; Lee, Sun-Joo; Cheng, Wayland W. L.; Nichols, Colin G.



Chronic exposure to paclitaxel diminishes phosphoinositide signaling by calpain-mediated neuronal calcium sensor-1 degradation  

PubMed Central

Paclitaxel (Taxol) is a well established chemotherapeutic agent for the treatment of solid tumors, but it is limited in its usefulness by the frequent induction of peripheral neuropathy. We found that prolonged exposure of a neuroblastoma cell line and primary rat dorsal root ganglia with therapeutic concentrations of Taxol leads to a reduction in inositol trisphosphate (InsP3)-mediated Ca2+ signaling. We also observed a Taxol-specific reduction in neuronal calcium sensor 1 (NCS-1) protein levels, a known modulator of InsP3 receptor (InsP3R) activity. This reduction was also found in peripheral neuronal tissue from Taxol treated animals. We further observed that short hairpin RNA-mediated NCS-1 knockdown had a similar effect on phosphoinositide-mediated Ca2+ signaling. When NCS-1 protein levels recovered, so did InsP3-mediated Ca2+ signaling. Inhibition of the Ca2+-activated protease ?-calpain prevented alterations in phosphoinositide-mediated Ca2+ signaling and NCS-1 protein levels. We also found that NCS-1 is readily degraded by ?-calpain in vitro and that ?-calpain activity is increased in Taxol but not vehicle-treated cells. From these results, we conclude that prolonged exposure to Taxol activates ?-calpain, which leads to the degradation of NCS-1, which, in turn, attenuates InsP3mediated Ca2+ signaling. These findings provide a previously undescribed approach to understanding and treating Taxol-induced peripheral neuropathy.

Boehmerle, Wolfgang; Zhang, Kun; Sivula, Michael; Heidrich, Felix M.; Lee, Yashang; Jordt, Sven-Eric; Ehrlich, Barbara E.



Energetics and location of phosphoinositide binding in human Kir2.1 channels.  


Kir2.1 channels are uniquely activated by phosphoinositide 4,5-bisphosphate (PI(4,5)P2) and can be inhibited by other phosphoinositides (PIPs). Using biochemical and computational approaches, we assess PIP-channel interactions and distinguish residues that are energetically critical for binding from those that alter PIP sensitivity by shifting the open-closed equilibrium. Intriguingly, binding of each PIP is disrupted by a different subset of mutations. In silico ligand docking indicates that PIPs bind to two sites. The second minor site may correspond to the secondary anionic phospholipid site required for channel activation. However, 96-99% of PIP binding localizes to the first cluster, which corresponds to the general PI(4,5)P2 binding location in recent Kir crystal structures. PIPs can encompass multiple orientations; each di- and triphosphorylated species binds with comparable energies and is favored over monophosphorylated PIPs. The data suggest that selective activation by PI(4,5)P2 involves orientational specificity and that other PIPs inhibit this activation through direct competition. PMID:23564459

D'Avanzo, Nazzareno; Lee, Sun-Joo; Cheng, Wayland W L; Nichols, Colin G



Specificity and commonality of the phosphoinositide-binding proteome analyzed by quantitative mass spectrometry.  


Phosphoinositides (PIPs) play key roles in signaling and disease. Using high-resolution quantitative mass spectrometry, we identified PIP-interacting proteins and profiled their binding specificities toward all seven PIP variants. This analysis revealed 405 PIP-binding proteins, which is greater than the total number of phospho- or ubiquitin-binding domains. Translocation and inhibitor assays of identified PIP-binding proteins confirmed that our methodology targets direct interactors. The PIP interactome encompasses proteins from diverse cellular compartments, prominently including the nucleus. Our data set revealed a consensus motif for PI(3,4,5)P3-interacting pleckstrin homology (PH) domains, which enabled in silico identification of phosphoinositide interactors. Members of the dedicator of cytokinesis family C exhibited specificity toward both PI(3,4,5)P3 and PI(4,5)P2. Structurally, this dual specificity is explained by a decreased number of positively charged residues in the L1 subdomain compared with DOCK1. The presented PIP-binding proteome and its specificity toward individual PIPs should be a valuable resource for the community. PMID:24462288

Jungmichel, Stephanie; Sylvestersen, Kathrine B; Choudhary, Chunaram; Nguyen, Steve; Mann, Matthias; Nielsen, Michael L



Structural Basis for Activation and Inhibition of Class I Phosphoinositide 3-Kinases  

NSDL National Science Digital Library

Phosphoinositide 3-kinases (PI3Ks) phosphorylate a hydroxyl group on phosphoinositide lipids. The 3-phosphorylated inositol lipids act as membrane-resident second messengers, recruiting downstream signaling components that control cell growth, proliferation, differentiation, survival, and motility. The best studied of the PI3Ks, the class I enzymes, are heterodimers with a catalytic and a regulatory subunit and have been implicated in many human diseases. Class I PI3Ks can be stimulated downstream of receptor tyrosine kinases and heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors as well as small G proteins of the Ras superfamily. Both the catalytic and regulatory subunits have a multidomain organization. Crystal structures, biochemical analysis, and oncogenic mutations in PI3Ks have shown that interdomain interactions are not static but undergo regulated conformational cycles, resulting in enzyme activation or inhibition. This Review, which contains 7 figures and 104 references, highlights the molecular details of how their regulatory partners selectively inhibit and activate PI3K isoforms.

Oscar Vadas (Cambridge;Laboratory of Molecular Biology REV); John E. Burke (Singapore;Nanyang Technological University REV); Xuxiao Zhang (Singapore;Nanyang Technological University REV); Alex Berndt (Cambridge;Laboratory of Molecular Biology REV); Roger L. Williams (Cambridge;Laboratory of Molecular Biology REV)



Localization of Myosin 1b to Actin Protrusions Requires Phosphoinositide Binding*  

PubMed Central

Myosin 1b (Myo1b), a class I myosin, is a widely expressed, single-headed, actin-associated molecular motor. Transient kinetic and single-molecule studies indicate that it is kinetically slow and responds to tension. Localization and subcellular fractionation studies indicate that Myo1b associates with the plasma membrane and certain subcellular organelles such as endosomes and lysosomes. Whether Myo1b directly associates with membranes is unknown. We demonstrate here that full-length rat Myo1b binds specifically and with high affinity to phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-triphosphate (PIP3), two phosphoinositides that play important roles in cell signaling. Binding is not Ca2+-dependent and does not involve the calmodulin-binding IQ region in the neck domain of Myo1b. Furthermore, the binding site is contained entirely within the C-terminal tail region, which contains a putative pleckstrin homology domain. Single mutations in the putative pleckstrin homology domain abolish binding of the tail domain of Myo1b to PIP2 and PIP3 in vitro. These same mutations alter the distribution of Myc-tagged Myo1b at membrane protrusions in HeLa cells where PIP2 localizes. In addition, we found that motor activity is required for Myo1b localization in filopodia. These results suggest that binding of Myo1b to phosphoinositides plays an important role in vivo by regulating localization to actin-enriched membrane projections.

Komaba, Shigeru; Coluccio, Lynne M.



Phosphoinositides: minor lipids make a major impact on photoreceptor cell functions.  


Activation of the phosphoinositide (PI) cycle generates the second messengers that control various aspects of cellular signaling. We have previously shown that two PI cycle enzymes, type II phosphatidylinositol 5-phosphate 4-kinase (PIPK II?) and phosphoinositide 3-kinase (PI3K), are activated through light stimulation. In our earlier studies, we measured enzyme activities, instead of directly measuring the products, due to lack of sensitive analytical techniques. Cells have very low levels of PIs, compared to other lipids, so special techniques and sensitive analytical instruments are necessary for their identification and quantification. There are also other considerations, such as different responses in different cell types, which may complicate quantification of PIs. For example, although light activated PIPK II?, there was no increase in PI-4,5-P2 measured by liquid chromatography-mass spectrometry (LC/MS) This discrepancy is due to the heterogeneous nature of the retina, which is composed of various cell types. In this study, we examined PI generation in situ using immunohistochemistry with specific PI antibodies. PIs were generated in specific retinal cell layers, suggesting that analyzing PIs from the total retina by LC/MS underscores the significance. This suggests that PI-specific antibodies are useful tools to study the cell-specific regulation of PIs in the retina. PMID:24964953

Rajala, Raju V S; Rajala, Ammaji; Morris, Andrew J; Anderson, Robert E



Somatostatin inhibits vasopressin-stimulated phosphoinositide hydrolysis and influx of extracellular calcium in clonal hamster beta (HIT) cells.  

PubMed Central

Vasopressin (VP) stimulates insulin secretion and inositol phosphate (InsP) production in clonal hamster beta cells (HIT) via a cyclic AMP-independent V1-receptor-mediated signal-transduction pathway. Somatostatin (SRIF) inhibited VP-stimulated insulin secretion, and the effects of SRIF were abolished by pretreatment with pertussis toxin. The Ca(2+)-channel blockers verapamil and nifedipine also inhibited VP-stimulated insulin secretion during 20 min incubations, but verapamil was ineffective at 2 min, and the effects of SRIF and nifedipine together were not addictive. SRIF failed to inhibit further the attenuated insulin response to VP in Ca(2+)-free medium. VP-stimulated InsP production was also inhibited by SRIF in a pertussis-toxin-sensitive manner. Whereas VP-stimulated insulin secretion was almost completely inhibited by SRIF at an equimolar concentration, VP-stimulated InsP production was much less sensitive to inhibition by SRIF, even at a 100-fold excess concentration. VP increased cytosolic Ca2+ in HIT cells loaded with fura 2, the fluorescent Ca2+ indicator. The increase was biphasic, with an initial rapid spike increase followed by a prolonged second phase. Both SRIF, at a concentration which inhibited VP-stimulated insulin secretion but not InsP production, and verapamil failed to inhibit the rapid spike increase in intracellular Ca2+, but did inhibit the second phase. We conclude that VP induces biphasic changes in cytosolic Ca2+, secondary to mobilization of intracellular Ca2+ and influx of extracellular Ca2+. SRIF inhibits insulin secretion by interrupting influx of extracellular Ca2+, likely by inhibiting Gi-subunit activity. Inhibition of VP-stimulated phosphoinositide hydrolysis, which is also pertussis-toxin-sensitive, may represent an additional mechanism of action of SRIF.

Richardson, S B; Laya, T; Gibson, M; Eyler, N; Van Ooy, M



The yeast VAP homolog Scs2p has a phosphoinositide-binding ability that is correlated with its activity  

SciTech Connect

The yeast VAMP-associated protein (VAP) homolog Scs2p is an endoplasmic reticulum (ER)/nuclear membrane protein that binds to an FFAT (diphenylalanine in an acidic tract) motif found in various lipid-metabolic proteins, including Opi1p, a negative regulator of phospholipid biosynthesis. Here, we show that Scs2p is a novel phosphoinositide-binding protein that can bind to phosphatidylinositol monophosphates and bisphosphates in vitro. The phosphoinositide-binding domain was assigned to the N-terminal major sperm protein (MSP) domain which also contains the FFAT-binding domain. When several lysine residues in the MSP domain were substituted for alanine, the resulting mutant Scs2 proteins lost the phosphoinositide-binding ability and failed to complement the inositol auxotrophy of an scs2 deletion strain. However, the mutant proteins still localized in the ER/nuclear membrane, in a similar manner to wild-type Scs2p. These results suggest the possibility that Scs2p activity is regulated by phosphoinositides to coordinate phospholipid biosynthesis in response to changes in phospholipid composition.

Kagiwada, Satoshi [Department of Biological Sciences, Faculty of Science, Nara Women's University, Nara 630-8506 (Japan)], E-mail:; Hashimoto, Misa [Department of Biological Sciences, Faculty of Science, Nara Women's University, Nara 630-8506 (Japan)



Identification of putative phosphoinositide-specific phospholipase C genes in filamentous fungi.  


Five putative phosphoinositide-specific phospholipases C (PLC) genes were identified in three species of filamentous fungi. Using polymerase chain reaction with degenerate oligonucleotide primers, gene fragments encoding amino acid sequences homologous to PLCs of mammals and other organisms were amplified: one sequence from Botryotinia fuckeliana, one from Aspergillus nidulans, and three from Neurospora crassa. The molecular cloning and sequencing of a putative PLC gene (BCPLC1) from B. fuckeliana showed that it encoded a polypeptide containing X and Y domains, the two conserved regions found in all known PLCs. The hypothetical gene product of BCPLC1 was of delta type in its primary structural organization. The identification of three PLC genes in N. crassa shows that multiple PLC isozymes also occur in microbial eukaryotes. PMID:9163731

Jung, O J; Lee, E J; Kim, J W; Chung, Y R; Lee, C W



The phosphoinositide 3-kinase/Akt1/Par-4 axis: a cancer-selective therapeutic target.  


Activation of the phosphoinositide 3-kinase (PI3K)/Akt cell survival pathway in many cancers makes it an appealing target for therapeutic development. However, because this pathway also has an important role in the survival of normal cells, tactics to achieve cancer selectivity may prove important. We recently showed that the cancer-selective proapoptotic protein Par-4 is a key target for inactivation by PI3K/Akt signaling. Additionally, we found that Par-4 participates in mediating apoptosis by PTEN, the tumor suppressor responsible for blocking PI3K/Akt signaling. As a central player in cancer cell survival, Par-4 may provide a useful focus for the development of cancer-selective therapeutics. PMID:16540633

Goswami, Anindya; Ranganathan, Padhma; Rangnekar, Vivek M



Wortmannin, a widely used phosphoinositide 3-kinase inhibitor, also potently inhibits mammalian polo-like kinase.  


Polo-like kinases (PLKs) play critical roles throughout mitosis. Here, we report that wortmannin, which was previously thought to be a highly selective inhibitor of phosphoinositide (PI) 3-kinases, is a potent inhibitor of mammalian PLK1. Observation of the wortmannin-PLK1 interaction was enabled by a tetramethylrhodamine-wortmannin conjugate (AX7503) that permits rapid detection of PLK1 activity and expression in complex proteomes. Importantly, we show that wortmannin inhibits PLK1 activity in an in vitro kinase assay with an IC(50) of 24 nM and when incubated with intact cells. Taken together, our results indicate that, at the concentrations of wortmannin commonly used to inhibit PI 3-kinases, PLK1 is also significantly inhibited. PMID:15664519

Liu, Yongsheng; Shreder, Kevin R; Gai, Wenzhi; Corral, Sergio; Ferris, Douglas K; Rosenblum, Jonathan S



Homotypic Vacuole Fusion Requires VTI11 and Is Regulated by Phosphoinositides.  


Most plant cells contain a large central vacuole that is essential to maintain cellular turgor. We report a new mutant allele of VTI11 that implicates the SNARE protein VTI11 in homotypic fusion of protein storage and lytic vacuoles. Fusion of the multiple vacuoles present in vti11 mutants could be induced by treatment with Wortmannin and LY294002, which are inhibitors of Phosphatidylinositol 3-Kinase (PI3K). We provide evidence that Phosphatidylinositol 3-Phosphate (PtdIns(3)P) regulates vacuole fusion in vti11 mutants, and that fusion of these vacuoles requires intact microtubules and actin filaments. Finally, we show that Wortmannin also induced the fusion of guard cell vacuoles in fava beans, where vacuoles are naturally fragmented after ABA-induced stomata closure. These results suggest a ubiquitous role of phosphoinositides in vacuole fusion, both during the development of the large central vacuole and during the dynamic vacuole remodeling that occurs as part of stomata movements. PMID:24569132

Zheng, Jiameng; Han, Sang Won; Rodriguez-Welsh, Maria Fernanda; Rojas-Pierce, Marcela



Phosphoinositide Binding Differentially Regulates NHE1 Na+/H+ Exchanger-dependent Proximal Tubule Cell Survival*  

PubMed Central

Tubular atrophy predicts chronic kidney disease progression, and is caused by proximal tubular epithelial cell (PTC) apoptosis. The normally quiescent Na+/H+ exchanger-1 (NHE1) defends against PTC apoptosis, and is regulated by PI(4,5)P2 binding. Because of the vast array of plasma membrane lipids, we hypothesized that NHE1-mediated cell survival is dynamically regulated by multiple anionic inner leaflet phospholipids. In membrane overlay and surface plasmon resonance assays, the NHE1 C terminus bound phospholipids with low affinity and according to valence (PIP3 > PIP2 > PIP = PA > PS). NHE1-phosphoinositide binding was enhanced by acidic pH, and abolished by NHE1 Arg/Lys to Ala mutations within two juxtamembrane domains, consistent with electrostatic interactions. PI(4,5)P2-incorporated vesicles were distributed to apical and lateral PTC domains, increased NHE1-regulated Na+/H+ exchange, and blunted apoptosis, whereas NHE1 activity was decreased in cells enriched with PI(3,4,5)P3, which localized to basolateral membranes. Divergent PI(4,5)P2 and PI(3,4,5)P3 effects on NHE1-dependent Na+/H+ exchange and apoptosis were confirmed by selective phosphoinositide sequestration with pleckstrin homology domain-containing phospholipase C? and Akt peptides, PI 3-kinase, and Akt inhibition in wild-type and NHE1-null PTCs. The results reveal an on-off switch model, whereby NHE1 toggles between weak interactions with PI(4,5)P2 and PI(3,4,5)P3. In response to apoptotic stress, NHE1 is stimulated by PI(4,5)P2, which leads to PI 3-kinase activation, and PI(4,5)P2 phosphorylation. The resulting PI(3,4,5)P3 dually stimulates sustained, downstream Akt survival signaling, and dampens NHE1 activity through competitive inhibition and depletion of PI(4,5)P2.

Abu Jawdeh, Bassam G.; Khan, Shenaz; Deschenes, Isabelle; Hoshi, Malcolm; Goel, Monu; Lock, Jeffrey T.; Shinlapawittayatorn, Krekwit; Babcock, Gerald; Lakhe-Reddy, Sujata; DeCaro, Garren; Yadav, Satya P.; Mohan, Maradumane L.; Naga Prasad, Sathyamangla V.; Schilling, William P.; Ficker, Eckhard; Schelling, Jeffrey R.



Excitatory amino acid receptor-stimulated phosphoinositide turnover in primary cerebrocortical cultures.  

PubMed Central

1. Characterization of excitatory amino acid-induced accumulation of [3H]-phosphoinositides was carried out in primary cerebrocortical cultures isolated from foetal rats. 2. All of the excitatory amino acid receptor agonists examined caused concentration-dependent enhancement of phosphoinositide (PI) formation. The most potent excitatory amino acid receptor agonists were quisqualate, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD), ibotenate and glutamate with mean EC50 values of 0.9 +/- 0.4 microM, 15 +/- 5 microM, 15 +/- 3 microM and 41 +/- 8 microM respectively. 3. The selective ionotropic receptor antagonists kynurenic acid (1 mM), 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX, 10 microM) and (+/-)-4-(3-phosphonopropyl)-2 piperazinecarboxylic acid (CPP, 100 microM), failed to block responses to quisqualate, (1S,3R)-ACPD or glutamate. D,L-2-Amino-3-phosphonopropionate (D,L-AP3) did not block 1S,3R-ACPD or quisqualate-induced PI turnover, but had an additive effect with quisqualate or (1S,3R)-ACPD. 4. Exposure of cultures to agonists in the absence of added extracellular calcium reduced the maximal quisqualate response by approximately 45%, revealing a two-component concentration-response curve. Concentration-response curves to ibotenate and glutamate became flattened by omission of extracellular calcium, whereas (1S,3R)-ACPD-stimulated PI turnover was unaffected. 5. Pretreatment of cultures with pertussis toxin markedly inhibited PI responses evoked by (1S,3R)-ACPD. 6. These results suggest that excitatory amino acid-stimulated PI turnover in cerebrocortical cultures is independent of ionotropic receptor activation and is mediated via specific G-protein-linked metabotropic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Birrell, G. J.; Marcoux, F. W.



Alternative Splicing Governs Cone Cyclic Nucleotide-gated (CNG) Channel Sensitivity to Regulation by Phosphoinositides.  


Precursor mRNA encoding CNGA3 subunits of cone photoreceptor cyclic nucleotide-gated (CNG) channels undergoes alternative splicing, generating isoforms differing in the N-terminal cytoplasmic region of the protein. In humans, four variants arise from alternative splicing, but the functional significance of these changes has been a persistent mystery. Heterologous expression of the four possible CNGA3 isoforms alone or with CNGB3 subunits did not reveal significant differences in basic channel properties. However, inclusion of optional exon 3, with or without optional exon 5, produced heteromeric CNGA3 + CNGB3 channels exhibiting an ?2-fold greater shift in K1/2,cGMP after phosphatidylinositol 4,5-biphosphate or phosphatidylinositol 3,4,5-trisphosphate application compared with channels lacking the sequence encoded by exon 3. We have previously identified two structural features within CNGA3 that support phosphoinositides (PIPn) regulation of cone CNG channels: N- and C-terminal regulatory modules. Specific mutations within these regions eliminated PIPn sensitivity of CNGA3 + CNGB3 channels. The exon 3 variant enhanced the component of PIPn regulation that depends on the C-terminal region rather than the nearby N-terminal region, consistent with an allosteric effect on PIPn sensitivity because of altered N-C coupling. Alternative splicing of CNGA3 occurs in multiple species, although the exact variants are not conserved across CNGA3 orthologs. Optional exon 3 appears to be unique to humans, even compared with other primates. In parallel, we found that a specific splice variant of canine CNGA3 removes a region of the protein that is necessary for high sensitivity to PIPn. CNGA3 alternative splicing may have evolved, in part, to tune the interactions between cone CNG channels and membrane-bound phosphoinositides. PMID:24675082

Dai, Gucan; Sherpa, Tshering; Varnum, Michael D



Distinctive changes in plasma membrane phosphoinositides underlie differential regulation of TRPV1 in nociceptive neurons.  


Transient Receptor Potential Vanilloid 1 (TRPV1) is a polymodal, Ca(2+)-permeable cation channel crucial to regulation of nociceptor responsiveness. Sensitization of TRPV1 by G-protein coupled receptor (GPCR) agonists to its endogenous activators, such as low pH and noxious heat, is a key factor in hyperalgesia during tissue injury as well as pathological pain syndromes. Conversely, chronic pharmacological activation of TRPV1 by capsaicin leads to calcium influx-induced adaptation of the channel. Paradoxically, both conditions entail activation of phospholipase C (PLC) enzymes, which hydrolyze phosphoinositides. We found that in sensory neurons PLC? activation by bradykinin led to a moderate decrease in phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), but no sustained change in the levels of its precursor PI(4)P. Preventing this selective decrease in PI(4,5)P2 inhibited TRPV1 sensitization, while selectively decreasing PI(4,5)P2 independently of PLC potentiated the sensitizing effect of protein kinase C (PKC) on the channel, thereby inducing increased TRPV1 responsiveness. Maximal pharmacological TRPV1 stimulation led to a robust decrease of both PI(4,5)P2 and its precursor PI(4)P in sensory neurons. Attenuating the decrease of either lipid significantly reduced desensitization, and simultaneous reduction of PI(4,5)P2 and PI(4)P independently of PLC inhibited TRPV1. We found that, on the mRNA level, the dominant highly Ca(2+)-sensitive PLC isoform in dorsal root ganglia is PLC?4. Capsaicin-induced desensitization of TRPV1 currents was significantly reduced, whereas capsaicin-induced nerve impulses in the skin-nerve preparation increased in mice lacking this isoform. We propose a comprehensive model in which differential changes in phosphoinositide levels mediated by distinct PLC isoforms result in opposing changes in TRPV1 activity. PMID:23843517

Lukacs, Viktor; Yudin, Yevgen; Hammond, Gerald R; Sharma, Esseim; Fukami, Kiyoko; Rohacs, Tibor



Phosphoinositide-signaling is one component of a robust plant defense response  

PubMed Central

The phosphoinositide pathway and inositol-1,4,5-triphosphate (InsP3) have been implicated in plant responses to many abiotic stresses; however, their role in response to biotic stress is not well characterized. In the current study, we show that both basal defense and systemic acquired resistance responses are affected in transgenic plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase) which have greatly reduced InsP3 levels. Flagellin induced Ca2+-release as well as the expressions of some flg22 responsive genes were attenuated in the InsP 5-ptase plants. Furthermore, the InsP 5-ptase plants were more susceptible to virulent and avirulent strains of Pseudomonas syringae pv. tomato (Pst) DC3000. The InsP 5-ptase plants had lower basal salicylic acid (SA) levels and the induction of SAR in systemic leaves was reduced and delayed. Reciprocal exudate experiments showed that although the InsP 5-ptase plants produced equally effective molecules that could trigger PR-1 gene expression in wild type plants, exudates collected from either wild type or InsP 5-ptase plants triggered less PR-1 gene expression in InsP 5-ptase plants. Additionally, expression profiles indicated that several defense genes including PR-1, PR-2, PR-5, and AIG1 were basally down regulated in the InsP 5-ptase plants compared with wild type. Upon pathogen attack, expression of these genes was either not induced or showed delayed induction in systemic leaves. Our study shows that phosphoinositide signaling is one component of the plant defense network and is involved in both basal and systemic responses. The dampening of InsP3-mediated signaling affects Ca2+ release, modulates defense gene expression and compromises plant defense responses.

Hung, Chiu-Yueh; Aspesi Jr, Peter; Hunter, Melissa R.; Lomax, Aaron W.; Perera, Imara Y.



Pharmacological characterization of the phosphoinositide second messenger system in the rabbit kidney  

SciTech Connect

The cellular response to hormones and neurotransmitters is a result of receptor activation of a second messenger system to initiate the intracellular cascade. In several tissues, such as brain and liver, one of the second messenger systems involves the hydrolysis of phosphoinositides (PIs) for the formation of inositol phosphate and diacylglycerol as the intracellular messengers. In the present study, they examined the effect of various agents on the hydrolysis of PIs in the rabbit kidney. In the kidney, the effect of the various hormones and neurotransmitters was region specific. Hydrolysis of PIs was stimulated in the inner medulla by (arg{sup 8})-vasopressin, angiotensin II, and atriopeptin I, and in the outer medulla by histamine, adenosine, and secretin. Only carbachol was able to stimulate the hydrolysis of PIs in both the inner and outer medulla. None of the substances tested were able to stimulate this response in the cortex. The following agents did not have an effect in any of the three zones of the kidney: norepinephrine, dopamine, atriopeptins II, and III. They have directly demonstrated the presence of a high affinity saturable binding site on inner medullary collecting duct (IMCD) cells with studies of binding characteristics of the radiolabelled muscarinic antagonist, 1-quinuclidinyl (phenyl-4-{sup 3}H) benzilate (({sup 3}H)QNB). The K{sub d} of 0.27 nM and the B{sub max} of 27.5 fmol/mg protein were determined from Scatchard analysis of the saturation data. In summary, they have demonstrated that cholinergic muscarinic receptors are present in the rabbit kidney, specifically in the IMCD cells. These receptors, which are coupled to the hydrolysis of phosphoinositides, may be involved in the vasodilatory and/or diuretic effects of cholinergic agents.

McArdle, S.



Phosphoinositide-signaling is one component of a robust plant defense response.  


The phosphoinositide pathway and inositol-1,4,5-triphosphate (InsP3) have been implicated in plant responses to many abiotic stresses; however, their role in response to biotic stress is not well characterized. In the current study, we show that both basal defense and systemic acquired resistance responses are affected in transgenic plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase) which have greatly reduced InsP3 levels. Flagellin induced Ca(2+)-release as well as the expressions of some flg22 responsive genes were attenuated in the InsP 5-ptase plants. Furthermore, the InsP 5-ptase plants were more susceptible to virulent and avirulent strains of Pseudomonas syringae pv. tomato (Pst) DC3000. The InsP 5-ptase plants had lower basal salicylic acid (SA) levels and the induction of SAR in systemic leaves was reduced and delayed. Reciprocal exudate experiments showed that although the InsP 5-ptase plants produced equally effective molecules that could trigger PR-1 gene expression in wild type plants, exudates collected from either wild type or InsP 5-ptase plants triggered less PR-1 gene expression in InsP 5-ptase plants. Additionally, expression profiles indicated that several defense genes including PR-1, PR-2, PR-5, and AIG1 were basally down regulated in the InsP 5-ptase plants compared with wild type. Upon pathogen attack, expression of these genes was either not induced or showed delayed induction in systemic leaves. Our study shows that phosphoinositide signaling is one component of the plant defense network and is involved in both basal and systemic responses. The dampening of InsP3-mediated signaling affects Ca(2+) release, modulates defense gene expression and compromises plant defense responses. PMID:24966862

Hung, Chiu-Yueh; Aspesi, Peter; Hunter, Melissa R; Lomax, Aaron W; Perera, Imara Y



Signaling via Class IA Phosphoinositide 3-Kinases (PI3K) in Human, Breast-Derived Cell Lines  

PubMed Central

We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (?, ?, ? and ?) and p50–101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110?, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110?>>?>? with undetectable p110?. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110?-, but not ?- or ?- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110?, but not ?- or ?- activity. In the presence of single, endogenous alleles of onco-mutant p110? (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110? inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN?/? cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110?, but not ?- or ?- activity; in PTEN?/? MCF10a it remained, like the parental cells, p110?-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110? is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110? augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110? function in the absence of PTEN.

Juvin, Veronique; Malek, Mouhannad; Anderson, Karen E.; Dion, Carine; Chessa, Tamara; Lecureuil, Charlotte; Ferguson, G. John; Cosulich, Sabina



Leptin Increases Cardiomyocyte Hyperplasia via Extracellular Signal-Regulated Kinase and Phosphatidylinositol 3-KinaseDependent Signaling Pathways  

Microsoft Academic Search

Obesity is a major risk factor for the development of heart failure. Importantly, it is now appreciated that a change in the number of myocytes is one of multiple structural and func- tional alterations (remodeling) leading to heart failure. Here we investigate the effect of leptin, the product of the obese (ob) gene, on proliferation of human and murine cardiomyocytes.




Tauroursodeoxycholic Acid Prevents Amyloid-? Peptide-Induced Neuronal Death Via a Phosphatidylinositol 3-Kinase-Dependent Signaling Pathway  

Microsoft Academic Search

Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates cell death by interrupting classic pathways of apoptosis. Amyloid-? (A?) peptide has been implicated in the pathogenesis of Alzheimer's disease, where a significant loss of neuronal cells is thought to occur by apoptosis. In this study, we explored the cell death pathway and signaling mecha- nisms involved in A?-induced toxicity and further




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

SciTech Connect

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 Y352 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.

Hussain, Alamdar, E-mail: [Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, SE-141 86 Huddinge (Sweden) [Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, SE-141 86 Huddinge (Sweden); Department of Biosciences, COMSATS Institute of Information Technology, Chak Shazad Campus, Islamabad (Pakistan); Faryal, Rani [Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, SE-141 86 Huddinge (Sweden) [Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, SE-141 86 Huddinge (Sweden); Department of Biosciences, COMSATS Institute of Information Technology, Chak Shazad Campus, Islamabad (Pakistan); Nore, Beston F.; Mohamed, Abdalla J. [Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, SE-141 86 Huddinge (Sweden)] [Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, SE-141 86 Huddinge (Sweden); Smith, C.I. Edvard, E-mail: [Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, SE-141 86 Huddinge (Sweden)] [Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, SE-141 86 Huddinge (Sweden)



Effects of glucose on sorbitol pathway activation, cellular redox, and metabolism of myo-inositol, phosphoinositide, and diacylglycerol in cultured human retinal pigment epithelial cells.  

PubMed Central

Sorbitol (aldose reductase) pathway flux in diabetes perturbs intracellular metabolism by two putative mechanisms: reciprocal osmoregulatory depletion of other organic osmolytes e.g., myo-inositol, and alterations in NADPH/NADP+ and/or NADH/NAD+. The "osmolyte" and "redox" hypotheses predict secondary elevations in CDP-diglyceride, the rate-limiting precursor for phosphatidylinositol synthesis, but through different mechanisms: the "osmolyte" hypothesis via depletion of intracellular myo-inositol (the cosubstrate for phosphatidylinositol-synthase) and the "redox" hypothesis through enhanced de novo synthesis from triose phosphates. The osmolyte hypothesis predicts diminished phosphoinositide-derived arachidonyl-diacylglycerol, while the redox hypothesis predicts increased total diacylglycerol and phosphatidic acid. In high aldose reductase expressing retinal pigment epithelial cells, glucose-induced, aldose reductase inhibitor-sensitive CDP-diglyceride accumulation and inhibition of 32P-incorporation into phosphatidylinositol paralleled myo-inositol depletion (but not cytoplasmic redox, that was unaffected by glucose) and depletion of arachidonyl-diacylglycerol. 3 mM pyruvate added to the culture medium left cellular redox unaltered, but stimulated Na(+)-dependent myo-inositol uptake, accumulation, and incorporation into phosphatidylinositol. These results favor myo-inositol depletion rather than altered redox as the primary cause of glucose-induced aldose reductase-related defects in phospholipid metabolism in cultured retinal pigment epithelial cells. Images

Thomas, T P; Porcellati, F; Kato, K; Stevens, M J; Sherman, W R; Greene, D A



Effects of acetylcholine and other agents on /sup 32/P-prelabeled phosphoinositides and phosphatidate in crude synaptosomal preparations  

SciTech Connect

Experimental conditions are described which permit effects of various agents on polyphosphoinositides and phosphatidic acid (PA) to be evaluated simultaneously in crude nerve-ending preparations from rat brain. Acetylcholine (3-100 microM) or carbachol (30-1,000 microM) induced the hydrolysis of prelabeled polyphosphoinositides and, at the same time, stimulated the net label incorporated in phosphatidic acid. All muscarinic effects were blocked by atropine or pirenzepine. Non-muscarinic agonists (glutamate, adenosine, norepinephrine) stimulated polyphosphoinositide hydrolysis in this preparation, but of these only norepinephrine affected phosphatidic acid turnover. A potentiation of acetylcholine-induced phosphoinositide turnover by KCl was observed, as well as an apparent selective inhibition of PIP2 hydrolysis by LiCl. Acetylcholine-stimulated turnover of PA was not necessarily coupled to phosphoinositide hydrolysis.

White, H.L.



The PHD Finger of the Chromatin-Associated Protein ING2 Functions as a Nuclear Phosphoinositide Receptor  

Microsoft Academic Search

Phosphoinositides (PtdInsPs) play critical roles in cytoplasmic signal transduction pathways. However, their functions in the nucleus are unclear, as specific nuclear receptors for PtdInsPs have not been identified. Here, we show that ING2, a candidate tumor suppressor protein, is a nuclear PtdInsP receptor. ING2 contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. We find that

Gozani; Philip Karuman; David R. Jones; Dmitri Ivanov; James Cha; Alexey A. Lugovskoy; Cheryl L. Baird; Hong Zhu; Seth J. Field; Stephen L. Lessnick; Jennifer Villasenor; Bharat Mehrotra; Jian Chen; Vikram R. Rao; Joan S. Brugge; Colin G. Ferguson; Bernard Payrastre; David G. Myszka; Lewis C. Cantley; Gerhard Wagner; Nullin Divecha; Glenn D. Prestwich; Junying Yuan



Wortmannin Inactivates Phosphoinositide 3Kinase by Covalent Modification of Lys802, a Residue Involved in the Phosphate Transfer Reaction  

Microsoft Academic Search

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA- dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortman- nin on



Depolarization activates the phosphoinositide phosphatase Ci-VSP, as detected in Xenopus oocytes coexpressing sensors of PIP2  

PubMed Central

Voltage-evoked signals play critical roles in neural activities, muscle contraction and exocytosis. Ciona voltage-sensor containing phosphatase (Ci-VSP) consists of the transmembrane voltage sensor domain (VSD) and a cytoplasmic domain of phosphoinositide phosphatase, homologous to phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Previous experiments utilizing potassium channels as the sensor for phosphoinositides have demonstrated that phosphatase activities of Ci-VSP are voltage dependent. However, it still remained unclear whether enzyme activity is activated by depolarization or hyperpolarization. Further, a large gap in voltage dependency was found between the charge movement of the VSD and potassium channel-reporting phosphatase activities. In this study, voltage-dependent dynamics of phosphoinositides mediated by Ci-VSP were examined by confocal imaging and electrical measurements in Xenopus oocytes. Imaging of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) using green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains from phospholipase C ? subunit (PLC-?) showed that PtdIns(4,5)P2 concentration is reduced during depolarization. In the presence of Ci-VSP, IRK1 channels with higher sensitivity to phosphoinositide than GIRK2 channels decreased their magnitude during depolarization over 0 mV, indicating that the PtdIns(4,5)P2 level is reduced upon depolarization. KCNQ2/3 channels coexpressed with Ci-VSP exhibited voltage-dependent decay of the outward current that became sharper with higher depolarization in a voltage range up to 100 mV. These results indicate that Ci-VSP has an activity that depletes PtdIns(4,5)P2 unlike PTEN and that depolarization-activated voltage sensor movement is translated into activation of phosphatase activity.

Murata, Yoshimichi; Okamura, Yasushi



Lead optimization of a dihydropyrrolopyrimidine inhibitor against phosphoinositide 3-kinase (PI3K) to improve the phenol glucuronic acid conjugation.  


Our lead compound for a phosphoinositide 3-kinase (PI3K) inhibitor (1) was metabolically unstable because of rapid glucuronidation of the phenol moiety. Based on structure-activity relationship (SAR) information and a FlexSIS docking simulation score, aminopyrimidine was identified as a bioisostere of phenol. An X-ray structure study revealed a hydrogen bonding pattern of aminopyrimidine derivatives. Finally, aminopyrimidine derivatives 33 showed strong tumor growth inhibition against a KPL-4 breast cancer xenograft model in vivo. PMID:23265889

Kawada, Hatsuo; Ebiike, Hirosato; Tsukazaki, Masao; Nakamura, Mitsuaki; Morikami, Kenji; Yoshinari, Kiyoshi; Yoshida, Miyuki; Ogawa, Kotaro; Shimma, Nobuo; Tsukuda, Takuo; Ohwada, Jun



Constitutive Macropinocytosis in Oncogene transformed Fibroblasts Depends on Sequential Permanent Activation of Phosphoinositide 3Kinase and Phospholipase C  

Microsoft Academic Search

Macropinocytosis results from the closure of lamellipodia generated by membrane ruffling, thereby reflecting cortical actin dynamics. Both transformation of Rat-1 fibroblasts by v-Src or K-Ras and stable transfection for expression of dominant-positive, wild-type phosphoinositide 3-kinase (PI3K) regulatory subunit p85a constitutively led to stress fiber disruption, cortical actin recruitment, extensive ruffling, and macropinosome formation, as measured by a selective accel- eration

Mustapha Amyere; Bernard Payrastre; Ulrike Krause; Patrick Van Der Smissen; Alex Veithen; Pierre J. Courtoy


The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells  

Microsoft Academic Search

Background: Protein kinase B (PKB), and the p70 and p90 ribosomal S6 kinases (p70 S6 kinase and p90 Rsk, respectively), are activated by phosphorylation of two residues, one in the ‘T-loop’ of the kinase domain and, the other, in the hydrophobic motif carboxy terminal to the kinase domain. The 3-phosphoinositide-dependent protein kinase 1 (PDK1) activates many AGC kinases in vitro

Michayla R. Williams; J. Simon C. Arthur; Anudharan Balendran; Jeroen van der Kaay; Valeria Poli; Philip Cohen; Dario R. Alessi



Phosphoinositide3-kinase inhibition induces sodium\\/iodide symporter expression in rat thyroid cells and human papillary thyroid cancer cells  

Microsoft Academic Search

TSH stimulation of sodium iodide symporter (NIS) expression in thyroid cancer promotes radioiodine uptake and is required to deliver an effective treatment dose. Activation of the insulin\\/phosphoinositide-3-kinase (PI3K) signaling pathway in TSH-stimulated thyroid cells reduces NIS expression at the transcriptional level. We, therefore, investigated the effects of PI3K pathway inhibition on iodide uptake and NIS expression in rat thyroid cell

Takahiko Kogai; Saima Sajid-Crockett; Lynell S Newmarch; Yan-Yun Liu; Gregory A Brent



Effects of the Metabotropic Glutamate Receptor Antagonist MCPG on Phosphoinositide Turnover and Synaptic Plasticity in Visual Cortex  

Microsoft Academic Search

The neurotransmitter glutamate, in addition to activating ligand- gated ion channels, also stimulates phosphoinositide (PI) hydro- lysis in neurons by activating a group of G-protein-coupled metabotropic glutamate receptors (mGluRs). A role for mGluRs in synaptic plasticity originally was hypothesized based on the ob- servation that the developmental decline in glutamate-stimulated PI turnover is well correlated with the decline in experience-

Kimberly M. Huber; Nathaniel B. Sawtell; Mark F. Bear



Ontogeny of phosphoinositide 3-kinase signaling in developing heart: effect of acute beta-adrenergic stimulation.  


Signaling pathways underlying transition of cardiomyocyte growth from hyperplasia in fetal/newborn to hypertrophy in postnatal/adult hearts are not well understood. We have shown that beta-adrenergic receptor (beta-AR)-mediated regulation of neonatal cardiomyocyte proliferation involves p70 ribosomal protein S6 kinase (p70S6K). Here we examined the ontogeny of phosphoinositide 3-kinase (PI3K)/p70S6K signaling pathway in rat hearts and investigated the influence of beta-AR on this pathway during development. Cardiac PI3K and p70S6K1 activities were high in the embryonic day 20 fetus, decreased gradually postnatally, and were low in the adult. In contrast, p70S6K2 was barely detectable. Phosphorylation of p70S6K1, Akt, and phosphoinositide-dependent protein kinase 1 were markedly increased in late gestation and early postnatal life but not in adult hearts. Phosphatase and tensin homolog on chromosome 10 (PTEN), a negative regulator of PI3K, was highly expressed in adult hearts but only at low levels and mostly in the phosphorylated (inactivated) form in the fetus. Beta-AR stimulation resulted in increased cardiac p70S6K1 activity only in animals > or = 2 wk old, whereas Akt level was increased in all developmental stages tested. These increases were accompanied by increased Bcl-2 associated death promoter (Ser136) phosphorylation without changes in PTEN level. Thus there is globally high input of cardiac PI3K signaling during the fetal-neonatal transition period. Inactivation of PTEN may in part contribute to the high activity of PI3K signaling, which coincides with the period of high cardiomyocyte proliferation. Beta-AR stimulation activates cardiac p70S6K1 and Akt in postnatal animals and may activate cardiac survival signals. These data provide further evidence for the importance of beta-AR and PI3K signaling in the regulation of cardiac growth during development. PMID:16006545

Tseng, Yi-Tang; Yano, Naohiro; Rojan, Adam; Stabila, Joan P; McGonnigal, Bethany G; Ianus, Vlad; Wadhawan, Rajan; Padbury, James F



Characterization of testis-specific serine-threonine kinase 3 and its activation by phosphoinositide-dependent kinase-1-dependent signalling.  


The family of testis-specific serine-threonine kinases (TSSKs) consists of four members whose expression is confined almost exclusively to testis. Very little is known about their physiological role and mechanisms of action. We cloned human and mouse TSSK3 and analysed the biochemical properties, substrate specificity and in vitro activation. In vitro TSSK3 exhibited the ability to autophosphorylate and to phosphorylate test substrates such as histones, myelin basic protein and casein. Interestingly, TSSK3 showed maximal in vitro kinase activity at 30 degrees C, in keeping with it being testis specific. Sequence comparison indicated the existence of a so-called 'T-loop' within the TSSK3 catalytic domain, a structure present in the AGC family of protein kinases. To test if this T-loop is engaged in TSSK3 regulation, we mutated the critical threonine residue within the T-loop to alanine (T168A) which resulted in inactivation of TSSK3 kinase. Furthermore, Thr168 is phosphorylated in vitro by the T-loop kinase phosphoinositide-dependent protein kinase-1 (PDK1). PDK1-induced phosphorylation increased in vitro TSSK3 kinase activity, suggesting that TSSK3 can be regulated in the same way as AGC kinase family members. Analysis of peptide sequences identifies the peptide sequence RRSSSY containing Ser5 that is a target for TSSK3 phosphorylation, as an efficient and specific substrate for TSSK3. PMID:16336268

Bucko-Justyna, Marta; Lipinski, Leszek; Burgering, Boudewijn M Th; Trzeciak, Lech



Neuroglobin overexpression in cultured human neuronal cells protects against hydrogen peroxide insult via activating phosphoinositide-3 kinase and opening the mitochondrial K(ATP) channel.  


Cultured neurons tolerate low H(2)O(2) concentrations (< or =50 microM) through the activity of constitutive antioxidant response elements (ARE). At H(2)O(2) levels (> or =100 microM), neurons increase expression of the gene encoding for inducible hemoxygenase-1 while superoxide dismutase-2 and catalase remain unchanged. Despite this adaptive response, the endogenous antioxidant systems are overwhelmed, leading to decreased viability. Elevating the neuronal cell content of human neuroglobin (Ngb) prior to insult with 100 or 200 microM H(2)O(2) enhanced cell viability and this resulted in a significant decrease in oxidative stress and an increase in the intracellular ATP concentration, whereas in parental cells exposed to the same H(2)O(2)-insult, oxidative stress and ATP increased and decreased, respectively. The mechanism for this increase in ATP involves sustained activation of the mito-K(ATP) channel and an increase in phosphoinositide-3 kinase (PI3K)-mediated phosphorylation of Akt. Pharmacological inhibitors directed toward PI3K (wortmannin and LY294002), or the mito-K(ATP) channel (glybenclamide) inhibited the H(2)O(2)-mediated increase in ATP in cells overexpressing human Ngb and consequently cell viability decreased. Neuroglobin's ability to bolster the intracellular pool of ATP in response to added H(2)O(2) is central to the preservation of cytoskeletal integrity and cell viability. PMID:20367258

Antao, Shane T; Duong, T T Hong; Aran, Roshanak; Witting, Paul K



The structure of the inter-SH2 domain of class IA phosphoinositide 3-kinase determined by site-directed spin labeling EPR and homology modeling  

PubMed Central

Phosphoinositide (PI) 3-kinases catalyze the phosphorylation of the D3 position of the inositol ring of PI, and its phosphorylated derivatives and play important roles in many intracellular signal transducing pathways. Class IA PI3-kinases contain distinct regulatory (p85) and catalytic (p110) subunits. p110 is stabilized and inhibited by constitutive association with p85, and is disinhibited when the SH2 domains of p85 bind to tyrosyl-phosphorylated proteins. Because the two subunits do not dissociate, disinhibition of p110 presumably occurs by an allosteric mechanism. To explore the means by which p85 regulates the activity of p110, structures of the inter-SH2 domain of p85 were determined with and without phosphopeptide by using a combination of site directed spin labeling EPR and homology modeling and molecular dynamics. The inter-SH2 domain is assigned as a rigid anti-parallel coiled-coil whose primary function is to bind p110, facilitating inhibition of p110 by the N-terminal SH2 domain of p85.

Fu, Zheng; Aronoff-Spencer, Eliah; Backer, Jonathan M.; Gerfen, Gary J.



Osteopontin is a myosphere-derived secretory molecule that promotes angiogenic progenitor cell proliferation through the phosphoinositide 3-kinase/Akt pathway  

SciTech Connect

We have reported that skeletal myosphere-derived progenitor cells (MDPCs) can differentiate into vascular cells, and that MDPC transplantation into cardiomyopathic hearts improves cardiac function. However, the autocrine/paracrine molecules and underlying mechanisms responsible for MDPC growth have not yet been determined. To explore the molecules enhancing the proliferation of MDPCs, we performed serial analysis of gene expression and signal sequence trap methods using RNA isolated from MDPCs. We identified osteopontin (OPN), a secretory molecule, as one of most abundant molecules expressed in MDPCs. OPN provided a proliferative effect for MDPCs. MDPCs treated with OPN showed Akt activation, and inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway repressed the proliferative effect of OPN. Furthermore, OPN-pretreated MDPCs maintained their differentiation potential into endothelial and vascular smooth muscle cells. These findings indicate an important role of OPN as an autocrine/paracrine molecule in regulating the proliferative growth of muscle-derived angiogenic progenitor cells via the PI3K/Akt pathway.

Ogata, Takehiro [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Ueyama, Tomomi [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan)]. E-mail:; Nomura, Tetsuya [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Asada, Satoshi [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Tagawa, Masashi [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Nakamura, Tomoyuki [Department of Pharmacology, Kansai Medical University, Moriguchi, Osaka 570-8507 (Japan); Takahashi, Tomosaburo [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Matsubara, Hiroaki [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan); Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto 602-8566 (Japan); Oh, Hidemasa [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507 (Japan)]. E-mail:



Receptor regulation of phosphoinositide 3-hydroxykinase in the NG115-401L-C3 neuronal cell line: stimulation by insulin-like growth factor-I.  

PubMed Central

The activation of phosphoinositide 3-hydroxykinase (P13K) is currently believed to represent the critical regulatory event which leads to the production of a novel intracellular signal. We have examined the control of this pathway by a number of cell-surface receptors in NG115-401L-C3 neuronal cells. Insulin-like growth factor-I stimulated the accumulation of 3-phosphorylated inositol lipids in intact cells and the appearance of P13K in antiphosphotyrosine-antibody-directed immunoprecipitates prepared from lysed cells, suggesting that P13K had been activated by a mechanism involving a protein tyrosine kinase. In contrast, P13K in these cells was not regulated by a variety of G-protein-coupled receptors, nerve growth factor acting via a low affinity receptor, or receptors for transforming growth factor-beta and interleukin-1. The receptor-specificity of P13K activation in these cells places significant constraints on the possible physiological function(s) of this pathway.

Poyner, D R; Hanley, M R; Jackson, T R; Hawkins, P T



The phosphoinositide-3-kinase-Akt-mTOR pathway as a therapeutic target in breast cancer.  


The phosphoinositide-3-kinase (PI3-kinase)-Akt-mTOR pathway is a central signal transduction pathway that regulates many critical aspects of normal and cancer physiology, including cell proliferation, apoptosis, cell morphology and migration, protein synthesis, and integration of metabolism. In breast cancer, somatic mutations that activate the pathway occur in more than 50% of tumors, underscoring the potentially broad impact of targeting the pathway for therapy. A vast body of preclinical data demonstrates the efficacy of pathway inhibition on tumor growth, and evidence also shows that activation of the pathway occurs in models of acquired resistance to hormonal therapy. This preclinical work led to the investigation of allosteric mTOR inhibitors, everolimus and temsirolimus, in metastatic hormone receptor-positive breast cancer. The recent BOLERO-2 trial comparing everolimus plus exemestane versus placebo plus exemestane in women with resistance to nonsteroidal aromatase inhibitors demonstrated a 6-month improvement in progression-free survival and led to FDA approval of everolimus for this indication in the United States. This landmark trial is the first demonstration of significant clinical benefit using drugs targeting this pathway in breast cancer. Many questions remain about the role of everolimus and other pathway-targeting drugs in clinical development in breast cancer treatment. This article reviews the role of the PI3-kinase-Akt-mTOR pathway in breast cancer biology and the clinical trial evidence available to date. PMID:23744866

Lauring, Josh; Park, Ben Ho; Wolff, Antonio C



Changes in phosphoinositide metabolism with days in culture affect signal transduction pathways in galdieria sulphuraria  


The metabolism of phosphatidylinositol-4,5-bisphosphate (PIP2) changed during the culture period of the thermoacidophilic red alga Galdieria sulphuraria. Seven days after inoculation, the amount of PIP2 in the cells was 910 +/- 100 pmol g-1 fresh weight; by 12 d, PIP2 levels increased to 1200 +/- 150 pmol g-1 fresh weight. In vitro assays indicated that phosphatidylinositol monophosphate (PIP) kinase specific activity increased from 75 to 230 pmol min-1 mg-1 protein between d 7 and 12. When G. sulphuraria cells were osmostimulated, transient increases of up to 4-fold could be observed in inositol-1,4,5-trisphosphate (IP3) levels within 90 s, regardless of the age of the cells. In d-12 cells, the increase in IP3 was preceded by a transient increase of up to 5-fold in specific PIP kinase activity, whereas no such increase was detected after osmostimulation of d-7 cells. The increase in PIP kinase activity before IP3 signaling in d-12 cells indicates that there is an additional pathway for regulation of phosphoinositide metabolism after stimulation other than an initial activation of phospholipase C. Also, the rapid activation of PIP2 biosynthesis in cells with already-high PIP2 levels suggests that the PIP2 present was not available for signal transduction. By comparing the response of the cells at d 7 and 12, we have identified two potentially distinct pools of PIP2. PMID:10198092

Heilmann; Perera; Gross; Boss



TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration  

PubMed Central

Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is frequently overexpressed in carcinomas, suggesting a specific role in cancer. Although TRAF4 protein is predominantly found at tight junctions (TJs) in normal mammary epithelial cells (MECs), it accumulates in the cytoplasm of malignant MECs. How TRAF4 is recruited and functions at TJs is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)-binding domain crucial for its recruitment to TJs. Of interest, this property is shared by the other members of the TRAF protein family. Indeed, the TRAF domain of all TRAF proteins (TRAF1 to TRAF6) is a bona fide PIP-binding domain. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer that binds up to three lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts as a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJs and favoring cell migration.

Rousseau, Adrien; McEwen, Alastair G.; Poussin-Courmontagne, Pierre; Rognan, Didier; Nomine, Yves; Rio, Marie-Christine; Tomasetto, Catherine; Alpy, Fabien



Activation of phosphoinositide 3-kinase by D2 receptor prevents apoptosis in dopaminergic cell lines.  

PubMed Central

Whereas dopamine agonists are known to provide symptomatic benefits for Parkinson's disease, recent clinical trials suggest that they might also be neuroprotective. Laboratory studies demonstrate that dopamine agonists can provide neuroprotective effects in a number of model systems, but the role of receptor-mediated signalling in these effects is controversial. We find that dopamine agonists have robust, concentration-dependent anti-apoptotic activity in PC12 cells that stably express human D(2L) receptors from cell death due to H(2)O(2) or trophic withdrawal and that the protective effects are abolished in the presence of D(2)-receptor antagonists. D(2) agonists are also neuroprotective in the nigral dopamine cell line SN4741, which express endogenous D(2) receptors, whereas no anti-apoptotic activity is observed in native PC12 cells, which do not express detectable D(2) receptors. Notably, the agonists studied differ in their relative efficacy to mediate anti-apoptotic effects and in their capacity to stimulate [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding, an indicator of G-protein activation. Studies with inhibitors of phosphoinositide 3-kinase (PI 3-kinase), extracellular-signal-regulated kinase or p38 mitogen-activated protein kinase indicate that the PI 3-kinase pathway is required for D(2) receptor-mediated cell survival. These studies indicate that certain dopamine agonists can complex with D(2) receptors to preferentially transactivate neuroprotective signalling pathways and to mediate increased cell survival.

Nair, Venugopalan D; Olanow, C Warren; Sealfon, Stuart C



Structure-based Analyses Reveal Distinct Binding Sites for Atg2 and Phosphoinositides in Atg18*  

PubMed Central

Autophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by an autophagosome and delivered to a lysosome/vacuole. Atg18 plays a critical role in autophagosome formation as a complex with Atg2 and phosphatidylinositol 3-phosphate (PtdIns(3)P). However, little is known about the structure of Atg18 and its recognition mode of Atg2 or PtdIns(3)P. Here, we report the crystal structure of Kluyveromyces marxianus Hsv2, an Atg18 paralog, at 2.6 ? resolution. The structure reveals a seven-bladed ?-propeller without circular permutation. Mutational analyses of Atg18 based on the K. marxianus Hsv2 structure suggested that Atg18 has two phosphoinositide-binding sites at blades 5 and 6, whereas the Atg2-binding region is located at blade 2. Point mutations in the loops of blade 2 specifically abrogated autophagy without affecting another Atg18 function, the regulation of vacuolar morphology at the vacuolar membrane. This architecture enables Atg18 to form a complex with Atg2 and PtdIns(3)P in parallel, thereby functioning in the formation of autophagosomes at autophagic membranes.

Watanabe, Yasunori; Kobayashi, Takafumi; Yamamoto, Hayashi; Hoshida, Hisashi; Akada, Rinji; Inagaki, Fuyuhiko; Ohsumi, Yoshinori; Noda, Nobuo N.



Calcium mobilization and phosphoinositide turnover in fluoride-activated human neutrophils  

SciTech Connect

Fluoride ion, at concentrations above 10 mM, has been found to activate a superoxide production response in human neutrophils which is strongly dependent on the presence of extracellular calcium. In an attempt to further explore the calcium requirement of fluoride-induced neutrophil activation, intracellular calcium concentrations were monitored through use of the fluorescent calcium probe, Quin 2. Fluoride ion, at concentrations between 10 and 20 mM, was found to elicit a rise in intracellular calcium levels which was characterized by a lag period of 4 to 10 min and a prolonged duration of action (greater than 20 min). In contrast, the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (FMLP), induced a rise in intracellular calcium concentration which peaked within 1 min. Preincubation of cells with 1 pertussis toxin resulted in inhibition of the FMLP-induced response, but not that elicited by fluoride. Furthermore, anion exchange chromatography indicated that inositol phosphate accumulation occurred in fluoride-treated cells in association with calcium mobilization. Recent evidence suggests that the FMLP receptor is coupled to phospholipase C and phosphoinositide turnover through a guanine nucleotide binding protein susceptible to inhibition by pertussis toxin. Present results suggest that fluoride ion may serve to activate this protein in a manner resistant to inhibition by pertussis toxin.

Strnad, C.F.; Wong, K.



The Phosphoinositide-3-Kinase-Akt-mTOR Pathway as a Therapeutic Target in Breast Cancer  

PubMed Central

The phosphoinositide-3-kinase (PI3-kinase)-Akt-mTOR pathway is a central signal transduction pathway that regulates many critical aspects of normal and cancer physiology, including cell proliferation, apoptosis, cell morphology and migration, protein synthesis, and integration of metabolism. In breast cancer, somatic mutations that activate the pathway occur in more than 50% of tumors, underscoring the potentially broad impact of targeting the pathway for therapy. A vast body of preclinical data demonstrates the efficacy of pathway inhibition on tumor growth, and evidence also shows that activation of the pathway occurs in models of acquired resistance to hormonal therapy. This preclinical work led to the investigation of allosteric mTOR inhibitors, everolimus and temsirolimus, in metastatic hormone receptor–positive breast cancer. The recent BOLERO-2 trial comparing everolimus plus exemestane versus placebo plus exemestane in women with resistance to nonsteroidal aromatase inhibitors demonstrated a 6-month improvement in progression-free survival and led to FDA approval of everolimus for this indication in the United States. This landmark trial is the first demonstration of significant clinical benefit using drugs targeting this pathway in breast cancer. Many questions remain about the role of everolimus and other pathway-targeting drugs in clinical development in breast cancer treatment. This article reviews the role of the PI3-kinase-Akt-mTOR pathway in breast cancer biology and the clinical trial evidence available to date.

Lauring, Josh; Park, Ben Ho; Wolff, Antonio C.



Nuclear but Not Cytosolic Phosphoinositide 3-Kinase Beta Has an Essential Function in Cell Survival ?  

PubMed Central

Class IA phosphoinositide 3-kinases (PI3Ks) are heterodimeric enzymes composed of a p85 regulatory and a p110 catalytic subunit that induce the formation of 3-polyphosphoinositides, which mediate cell survival, division, and migration. There are two ubiquitous PI3K isoforms p110? and p110? that have nonredundant functions in embryonic development and cell division. However, whereas p110? concentrates in the cytoplasm, p110? localizes to the nucleus and modulates nuclear processes such as DNA replication and repair. At present, the structural features that determine p110? nuclear localization remain unknown. We describe here that association with the p85? regulatory subunit controls p110? nuclear localization. We identified a nuclear localization signal (NLS) in p110? C2 domain that mediates its nuclear entry, as well as a nuclear export sequence (NES) in p85?. Deletion of p110? induced apoptosis, and complementation with the cytoplasmic C2-NLS p110? mutant was unable to restore cell survival. These studies show that p110? NLS and p85? NES regulate p85?/p110? nuclear localization, supporting the idea that nuclear, but not cytoplasmic, p110? controls cell survival.

Kumar, Amit; Redondo-Munoz, Javier; Perez-Garcia, Vicente; Cortes, Isabel; Chagoyen, Monica; Carrera, Ana C.



Requirement for Class II Phosphoinositide 3-Kinase C2? in Maintenance of Glomerular Structure and Function?  

PubMed Central

An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2? (PI3KC2?) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2?-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2? deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2? deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2? is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function.

Harris, David P.; Vogel, Peter; Wims, Marie; Moberg, Karen; Humphries, Juliane; Jhaver, Kanchan G.; DaCosta, Christopher M.; Shadoan, Melanie K.; Xu, Nianhua; Hansen, Gwenn M.; Balakrishnan, Sanjeevi; Domin, Jan; Powell, David R.; Oravecz, Tamas



Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl.  


Like several Rho GDP/GTP exchange factors (GEFs), Kalirin7 (Kal7) contains an N-terminal Sec14 domain and multiple spectrin repeats. A natural splice variant of Kalrn lacking the Sec14 domain and four spectrin repeats is unable to increase spine formation; our goal was to understand the function of the Sec14 and spectrin repeat domains. Kal7 lacking its Sec14 domain still increased spine formation, but the spines were short. Strikingly, Kal7 truncation mutants containing only the Sec14 domain and several spectrin repeats increased spine formation. The Sec14 domain bound phosphoinositides, a minor but crucial component of cellular membranes, and binding was increased by a phosphomimetic mutation. Expression of KalSec14-GFP in nonneuronal cells impaired receptor-mediated endocytosis, linking Kal7 to membrane trafficking. Consistent with genetic studies placing Abl, a non-receptor tyrosine kinase, and the Drosophila orthologue of Kalrn into the same signaling pathway, Abl1 phosphorylated two sites in the fourth spectrin repeat of Kalirin, increasing its sensitivity to calpain-mediated degradation. Treating cortical neurons of the wild-type mouse, but not the Kal7(KO) mouse, with an Abl inhibitor caused an increase in linear spine density. Phosphorylation of multiple sites in the N-terminal Sec14/spectrin region of Kal7 may allow coordination of the many signaling pathways contributing to spine morphogenesis. PMID:24600045

Ma, Xin-Ming; Miller, Megan B; Vishwanatha, K S; Gross, Maegan J; Wang, Yanping; Abbott, Thomas; Lam, Tukiet T; Mains, Richard E; Eipper, Betty A



TRAF4 is a novel phosphoinositide-binding protein modulating tight junctions and favoring cell migration.  


Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is frequently overexpressed in carcinomas, suggesting a specific role in cancer. Although TRAF4 protein is predominantly found at tight junctions (TJs) in normal mammary epithelial cells (MECs), it accumulates in the cytoplasm of malignant MECs. How TRAF4 is recruited and functions at TJs is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)-binding domain crucial for its recruitment to TJs. Of interest, this property is shared by the other members of the TRAF protein family. Indeed, the TRAF domain of all TRAF proteins (TRAF1 to TRAF6) is a bona fide PIP-binding domain. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer that binds up to three lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts as a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJs and favoring cell migration. PMID:24311986

Rousseau, Adrien; McEwen, Alastair G; Poussin-Courmontagne, Pierre; Rognan, Didier; Nominé, Yves; Rio, Marie-Christine; Tomasetto, Catherine; Alpy, Fabien



Evaluation of variation in the phosphoinositide-3-kinase catalytic subunit alpha oncogene and breast cancer risk  

PubMed Central

Background: Somatic mutations in phosphoinositide-3-kinase catalytic subunit alpha (PIK3CA) are frequent in breast tumours and have been associated with oestrogen receptor (ER) expression, human epidermal growth factor receptor-2 overexpression, lymph node metastasis and poor survival. The goal of this study was to evaluate the association between inherited variation in this oncogene and risk of breast cancer. Methods: A single-nucleotide polymorphism from the PIK3CA locus that was associated with breast cancer in a study of Caucasian breast cancer cases and controls from the Mayo Clinic (MCBCS) was genotyped in 5436 cases and 5280 controls from the Cancer Genetic Markers of Susceptibility (CGEMS) study and in 30?949 cases and 29?788 controls from the Breast Cancer Association Consortium (BCAC). Results: Rs1607237 was significantly associated with a decreased risk of breast cancer in MCBCS, CGEMS and all studies of white Europeans combined (odds ratio (OR)=0.97, 95% confidence interval (CI) 0.95–0.99, P=4.6 × 10?3), but did not reach significance in the BCAC replication study alone (OR=0.98, 95% CI 0.96–1.01, P=0.139). Conclusion: Common germline variation in PIK3CA does not have a strong influence on the risk of breast cancer

Stevens, K N; Garcia-Closas, M; Fredericksen, Z; Kosel, M; Pankratz, V S; Hopper, J L; Dite, G S; Apicella, C; Southey, M C; Schmidt, M K; Broeks, A; Van 't Veer, L J; Tollenaar, R A E M; Fasching, P A; Beckmann, M W; Hein, A; Ekici, A B; Johnson, N; Peto, J; dos Santos Silva, I; Gibson, L; Sawyer, E; Tomlinson, I; Kerin, M J; Chanock, S; Lissowska, J; Hunter, D J; Hoover, R N; Thomas, G D; Milne, R L; Perez, JI Arias; Gonzalez-Neira, A; Benitez, J; Burwinkel, B; Meindl, A; Schmutzler, R K; Bartrar, C R; Hamann, U; Ko, Y D; Bruning, T; Chang-Claude, J; Hein, R; Wang-Gohrke, S; Dork, T; Schurmann, P; Bremer, M; Hillemanns, P; Bogdanova, N; Zalutsky, J V; Rogov, Y I; Antonenkova, N; Lindblom, A; Margolin, S; Mannermaa, A; Kataja, V; Kosma, V-M; Hartikainen, J; Chenevix-Trench, G; Chen, X; Peterlongo, P; Bonanni, B; Bernard, L; Manoukian, S; Wang, X; Cerhan, J; Vachon, C M; Olson, J; Giles, G G; Baglietto, L; McLean, C A; Severi, G; John, E M; Miron, A; Winqvist, R; Pylkas, K; Jukkola-Vuorinen, A; Grip, M; Andrulis, I; Knight, J A; Glendon, G; Mulligan, A M; Cox, A; Brock, I W; Elliott, G; Cross, S S; Pharoah, P P; Dunning, A M; Pooley, K A; Humphreys, M K; Wang, J; Kang, D; Yoo, K-Y; Noh, D-Y; Sangrajrang, S; Gabrieau, V; Brennan, P; McKay, J; Anton-Culver, H; Ziogas, A; Couch, F J; Easton, D F



Effects of Novel Isoform-Selective Phosphoinositide 3-Kinase Inhibitors on Natural Killer Cell Function  

PubMed Central

Phosphoinositide 3-kinases (PI3Ks) are promising targets for therapeutic development in cancer. The class I PI3K isoform p110? has received considerable attention in oncology because the gene encoding p110? (PIK3CA) is frequently mutated in human cancer. However, little is known about the function of p110? in lymphocyte populations that modulate tumorigenesis. We used recently developed investigational inhibitors to compare the function of p110? and other isoforms in natural killer (NK) cells, a key cell type for immunosurveillance and tumor immunotherapy. Inhibitors of all class I isoforms (pan-PI3K) significantly impaired NK cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity against tumor cells, whereas p110?-selective inhibitors had no effect. In NK cells stimulated through NKG2D, p110? inhibition modestly reduced PI3K signaling output as measured by AKT phosphorylation. Production of IFN-? and NK cell-derived chemokines was blocked by a pan-PI3K inhibitor and partially reduced by a p110?inhibitor, with lesser effects of p110? inhibitors. Oral administration of mice with MLN1117, a p110? inhibitor in oncology clinical trials, had negligible effects on NK subset maturation or terminal subset commitment. Collectively, these results support the targeting of PIK3CA mutant tumors with selective p110? inhibitors to preserve NK cell function.

Yea, Sung Su; So, Lomon; Mallya, Sharmila; Lee, Jongdae; Rajasekaran, Kamalakannan; Malarkannan, Subramaniam; Fruman, David A.



A steep phosphoinositide bis-phosphate gradient forms during fungal filamentous growth  

PubMed Central

Membrane lipids have been implicated in many critical cellular processes, yet little is known about the role of asymmetric lipid distribution in cell morphogenesis. The phosphoinositide bis-phosphate PI(4,5)P2 is essential for polarized growth in a range of organisms. Although an asymmetric distribution of this phospholipid has been observed in some cells, long-range gradients of PI(4,5)P2 have not been observed. Here, we show that in the human pathogenic fungus Candida albicans a steep, long-range gradient of PI(4,5)P2 occurs concomitant with emergence of the hyphal filament. Both sufficient PI(4)P synthesis and the actin cytoskeleton are necessary for this steep PI(4,5)P2 gradient. In contrast, neither microtubules nor asymmetrically localized mRNAs are critical. Our results indicate that a gradient of PI(4,5)P2, crucial for filamentous growth, is generated and maintained by the filament tip–localized PI(4)P-5-kinase Mss4 and clearing of this lipid at the back of the cell. Furthermore, we propose that slow membrane diffusion of PI(4,5)P2 contributes to the maintenance of such a gradient.

Vernay, Aurelia; Schaub, Sebastien; Guillas, Isabelle; Bassilana, Martine



Applying conformational selection theory to improve crossdocking efficiency in 3-phosphoinositide dependent protein kinase-1.  


The emerging picture of biomolecular recognition is that of conformational selection followed by induced-fit. Conformational selection theory states that binding partners exist in various conformations in solution, with binding involving a "selection" between complementary conformers. In this study, we devise a docking protocol that mimics conformational selection in protein-ligand binding and demonstrate that it significantly enhances crossdocking accuracy over Glide's flexible docking protocol, which is widely used in the pharmaceutical industry. Our protocol uses a pregenerated conformational ensemble to simulate ligand flexibility. The ensemble was generated by thorough conformational sampling coupled with conformer minimization. The generated conformers were then rigidly docked in the active site of the protein along with a postdocking minimization step that allows limited induced fit effects to be modeled for the ligand. We illustrate the improved performance of our protocol through crossdocking of 31 ligands to cocomplexed proteins of the kinase 3-phosphoinositide dependent protein kinase-1 extracted from the crystal structures 1H1W (ATP bound), 1OKY (staurosporine bound) and 3QD0 (bound to a potent inhibitor). Consistent with conformational selection theory, the performance of our protocol was the best for crossdocking to the cognate protein bound to the natural ligand, ATP. PMID:23999908

Kotasthane, Anuja; Mulakala, Chandrika; Viswanadhan, Vellarkad N



Class I and class III phosphoinositide 3-kinases are required for actin polymerization that propels phagosomes  

PubMed Central

Actin polymerization drives the extension of pseudopods that trap and engulf phagocytic targets. The polymerized actin subsequently dissociates as the phagocytic vacuole seals and detaches from the plasma membrane. We found that phagosomes formed by engagement of integrins that serve as complement receptors (CR3) undergo secondary waves of actin polymerization, leading to the formation of “comet tails” that propel the vacuoles inside the cells. Actin tail formation was accompanied by and required de novo formation of PI(3,4)P2 and PI(3,4,5)P3 on the phagosomal membrane by class I phosphoinositide 3-kinases (PI3Ks). Although the phosphatidylinositide phosphatase Inpp5B was recruited to nascent phagosomes, it rapidly detached from the membrane after phagosomes sealed. Detachment of Inpp5B required the formation of PI(3)P. Thus, class III PI3K activity was also required for the accumulation of PI(4,5)P2 and PI(3,4,5)P3 and for actin tail formation. These experiments reveal a new PI(3)P-sensitive pathway leading to PI(3,4)P2 and PI(3,4,5)P3 formation and signaling in endomembranes.

Bohdanowicz, Michal; Cosio, Gabriela; Backer, Jonathan M.



Unique serotonin receptor (5HT-1C) in choroid plexus is linked to phosphoinositide hydrolysis  

SciTech Connect

The binding of /sup 125/I-LSD to the 5HT-1C site and of /sup 3/H-ket-anserin to the 5HT-2 site was determined in choroid plexus and cerebral cortex of male Sprague-Dawley rats, respectively. As an index of phosphoinositide (PI) hydrolysis, whole choroid plexus and cerebral cortex slices were prelabelled with /sup 3/H-inositol and serotonin (5HT) stimulated release of /sup 3/H-inositol-1-phosphate was measured. 5HT stimulated PI hydrolysis in choroid plexus (6-fold) and in cerebral cortex (2.5-fold). 5HT was more potent in choroid plexus (EC/sub 50/ = 46 nM) consistent with the involvement of the 5HT-1C site. 5HT antagonists, ketanserin, mianserin and spiperone, inhibited the response to 5HT with different potencies in the two tissues. In cerebral cortex all 3 antagonists had nM affinities and a rank order (spiperone > ketanserin > mianserin) consistent with the 5HT-2 site. In choroid plexus, however, the rank order (mianserin > ketanserin > spiperone) and absolute potencies agreed with binding to the 5HT-1C site. These data suggest that the 5HT-1C site is a functional receptor which utilizes PI hydrolysis as its biochemical effector system.

Sanders-Bush, E.; Conn, P.J.; Hoffman, B.J.; Hartig, P.R.



Phosphoinositide 3-kinase ? gene mutation predisposes to respiratory infection and airway damage  

PubMed Central

Genetic mutations cause primary immunodeficiencies (PIDs), which predispose to infections. Here we describe Activated PI3K-? Syndrome (APDS), a PID associated with a dominant gain-of-function mutation E1021K in the p110? protein, the catalytic subunit of phosphoinositide 3-kinase ? (PI3K?), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3,346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased IgM and reduced IgG2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110?. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110? inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, suggesting a therapeutic approach for patients with APDS.

Angulo, Ivan; Vadas, Oscar; Garcon, Fabien; Banham-Hall, Edward; Plagnol, Vincent; Leahy, Timothy R.; Baxendale, Helen; Coulter, Tanya; Curtis, James; Wu, Changxin; Blake-Palmer, Katherine; Perisic, Olga; Smyth, Deborah; Maes, Mailis; Fiddler, Christine; Juss, Jatinder; Cilliers, Deirdre; Markelj, Gasper; Chandra, Anita; Farmer, George; Kielkowska, Anna; Clark, Jonathan; Kracker, Sven; Debre, Marianne; Picard, Capucine; Pellier, Isabelle; Jabado, Nada; Morris, James A.; Barcenas-Morales, Gabriela; Fischer, Alain; Stephens, Len; Hawkins, Phillip; Barrett, Jeffrey C.; Abinun, Mario; Clatworthy, Menna; Durandy, Anne; Doffinger, Rainer; Chilvers, Edwin; Cant, Andrew J.; Kumararatne, Dinakantha; Okkenhaug, Klaus; Williams, Roger L.; Condliffe, Alison; Nejentsev, Sergey



Effect of albumin-bound DHA on phosphoinositide phosphorylation in collagen stimulated human platelets  

SciTech Connect

The effect of exogenous albumin-bound docosahexaenoic acid (22:6n-3) (DHA), arachidonic acid (20:4n-6) (AA), and eicosapendaenoic acid (20:5n-3) (EPA) on phosphoinositide metabolism following collagen stimulation was studied using (3H)inositol prelabelled platelets. Collagen stimulation (3 min, 1.8 micrograms/ml) increased the labelling of both phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-biphosphate (PIP2). Of the fatty acids tested, only pre-incubation (2 min) with DHA (20 microM) significantly attenuated the collagen-induced increased PIP and PIP2 labelling; EPA was without effect, while AA enhanced PIP labelling. Forty microM DHA was less effective at attenuating the increased PIP and PIP2 labelling even though this concentration of DHA resulted in greater inhibition of platelet aggregation. Neither concentration of DHA attenuated the increased polyphosphoinositide labelling resulting from stimulation by the endoperoxide analogue U46619, or the phorbol ester, PMA. These data suggest that the effect of DHA on attenuating the increased PIP and PIP2 labelling following collagen stimulation likely occurs before thromboxane receptor occupancy, may not occur at the level of protein kinase C activation, and could be mediated in part via a lessened synthesis of thromboxane A2.

Gaudette, D.C.; Holub, B.J. (Univ. of Guelph, Ontario (Canada))



Ablation of phosphoinositide-3-kinase class II alpha suppresses hepatoma cell proliferation  

SciTech Connect

Cancer such as hepatocellular carcinoma (HCC) is characterized by complex perturbations in multiple signaling pathways, including the phosphoinositide-3-kinase (PI3K/AKT) pathways. Herein we investigated the role of PI3K catalytic isoforms, particularly class II isoforms in HCC proliferation. Among the siRNAs tested against the eight known catalytic PI3K isoforms, specific ablation of class II PI3K alpha (PIK3C2{alpha}) was the most effective in impairing cell growth and this was accompanied by concomitant decrease in PIK3C2{alpha} mRNA and protein levels. Colony formation ability of cells deficient for PIK3C2{alpha} was markedly reduced and growth arrest was associated with increased caspase 3 levels. A small but significant difference in gene dosage and expression levels was detected between tumor and non-tumor tissues in a cohort of 19 HCC patients. Taken together, these data suggest for the first time that in addition to class I PI3Ks in cancer, class II PIK3C2{alpha} can modulate HCC cell growth.

Ng, Stanley K.L. [Singapore Immunology Network A-STAR (Singapore)] [Singapore Immunology Network A-STAR (Singapore); Neo, Soek-Ying, E-mail: [Singapore Immunology Network A-STAR (Singapore)] [Singapore Immunology Network A-STAR (Singapore); Yap, Yann-Wan [Singapore Immunology Network A-STAR (Singapore)] [Singapore Immunology Network A-STAR (Singapore); Karuturi, R. Krishna Murthy; Loh, Evelyn S.L. [Genome Institute of Singapore A-STAR (Singapore)] [Genome Institute of Singapore A-STAR (Singapore); Liau, Kui-Hin [Department of General Surgery, Tan Tock Seng Hospital (Singapore)] [Department of General Surgery, Tan Tock Seng Hospital (Singapore); Ren, Ee-Chee, E-mail: [Singapore Immunology Network A-STAR (Singapore) [Singapore Immunology Network A-STAR (Singapore); Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore (Singapore)



Identification of Novel Piperazinylquinoxaline Derivatives as Potent Phosphoinositide 3-Kinase (PI3K) Inhibitors  

PubMed Central

Background Development of small-molecule inhibitors targeting phosphoinositide 3-kinase (PI3K) has been an appealing strategy for the treatment of various types of cancers. Methodology/Principal Finding Our approach was to perform structural modification and optimization based on previously identified morpholinoquinoxaline derivative WR1 and piperidinylquinoxaline derivative WR23 with a total of forty-five novel piperazinylquinoxaline derivatives synthesized. Most target compounds showed low micromolar to nanomolar antiproliferative potency against five human cancer cell lines using MTT method. Selected compounds showed potent PI3K? inhibitory activity in a competitive fluorescent polarization assay, such as compound 22 (IC50 40 nM) and 41 (IC50: 24 nM), which induced apoptosis in PC3 cells. Molecular docking analysis was performed to explore possible binding modes between target compounds and PI3K. Conclusions/Significance The identified novel piperazinylquinoxaline derivatives that showed potent PI3K? inhibitory activity and cellular antiproliferative potency may be promising agents for potential applications in cancer treatment.

Wu, Peng; Su, Yi; Guan, Xianghong; Liu, Xiaowen; Zhang, Jiankang; Dong, Xiaowu; Huang, Wenhai; Hu, Yongzhou



Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl  

PubMed Central

Like several Rho GDP/GTP exchange factors (GEFs), Kalirin7 (Kal7) contains an N-terminal Sec14 domain and multiple spectrin repeats. A natural splice variant of Kalrn lacking the Sec14 domain and four spectrin repeats is unable to increase spine formation; our goal was to understand the function of the Sec14 and spectrin repeat domains. Kal7 lacking its Sec14 domain still increased spine formation, but the spines were short. Strikingly, Kal7 truncation mutants containing only the Sec14 domain and several spectrin repeats increased spine formation. The Sec14 domain bound phosphoinositides, a minor but crucial component of cellular membranes, and binding was increased by a phosphomimetic mutation. Expression of KalSec14-GFP in nonneuronal cells impaired receptor-mediated endocytosis, linking Kal7 to membrane trafficking. Consistent with genetic studies placing Abl, a non–receptor tyrosine kinase, and the Drosophila orthologue of Kalrn into the same signaling pathway, Abl1 phosphorylated two sites in the fourth spectrin repeat of Kalirin, increasing its sensitivity to calpain-mediated degradation. Treating cortical neurons of the wild-type mouse, but not the Kal7KO mouse, with an Abl inhibitor caused an increase in linear spine density. Phosphorylation of multiple sites in the N-terminal Sec14/spectrin region of Kal7 may allow coordination of the many signaling pathways contributing to spine morphogenesis.

Ma, Xin-Ming; Miller, Megan B.; Vishwanatha, K. S.; Gross, Maegan J.; Wang, Yanping; Abbott, Thomas; Lam, TuKiet T.; Mains, Richard E.; Eipper, Betty A.



Molecular mechanism of membrane targeting by the GRP1 PH domain,boxs  

Microsoft Academic Search

Abstract,The general,receptor,for phosphoinositides iso- form,1 (GRP1) is recruited,to the plasma,membrane,in re- sponse,to activation,of phosphoinositide,3-kinases and accumulation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]. GRP1?s pleckstrin homology (PH) do- main recognizes PtdIns(3,4,5)P3 with high specificity and af- finity, however, the precise mechanism of its association with membranes remains unclear. Here, we detail the mo- lecular basis of membrane,anchoring,by the GRP1 PH do- main. Our

Ju He; Rachel M. Haney; Mohsin Vora; Vladislav V. Verkhusha; Robert V. Stahelin; Tatiana G. Kutateladze



Cyclic AMP differentiates two separate but interacting pathways of phosphoinositide hydrolysis in the DDT1-MF2 smooth muscle cell line.  


The activation of adenosine A1 receptors in DDT1-MF2 smooth muscle cells resulted in both the inhibition of agonist-stimulated cAMP accumulation and the potentiation of norepinephrine-stimulated phosphoinositide hydrolysis. Pharmacological analysis indicated the involvement of an A1 adenosine receptor subtype in both of these responses. In the absence of norepinephrine, the activation of the adenosine receptor did not directly stimulate phosphoinositide hydrolysis. The adenosine receptor-mediated augmentation of norepinephrine-stimulated phosphoinositide hydrolysis was pertussis toxin sensitive and was selectively antagonized by agents that mimicked cAMP (8-bromo-cAMP) or raised cellular cAMP levels (forskolin). This initially suggested that cAMP might partially regulate the magnitude of the phospholipase C response to norepinephrine and that adenosine agonists might enhance the phospholipase C response by reducing cAMP levels. However, neither the reduction of cellular cAMP levels by other agents nor the inhibition of cAMP-dependent protein kinase was sufficient to replicate the action of adenosine receptor activation on phosphoinositide hydrolysis. Thus, in the presence of norepinephrine, adenosine receptor agonists appear to stimulate phosphoinositide hydrolysis via a pathway that is separate from, but dependent upon, that of norepinephrine. This second pathway can be distinguished from that which is stimulated by norepinephrine on the basis of its sensitivity to inhibition by both cAMP and pertussis toxin. PMID:1312218

Schachter, J B; Wolfe, B B



Promiscuous activation of transient receptor potential vanilloid 1 (TRPV1) channels by negatively charged intracellular lipids: the key role of endogenous phosphoinositides in maintaining channel activity.  


The regulation of the heat- and capsaicin-activated transient receptor potential vanilloid 1 (TRPV1) channels by phosphoinositides is controversial. Data in cellular systems support the dependence of TRPV1 activity on phosphoinositides. The purified TRPV1, however, was recently shown to be fully functional in artificial liposomes in the absence of phosphoinositides. Here, we show that several other negatively charged phospholipids, including phosphatidylglycerol, can also support TRPV1 activity in excised patches at high concentrations. When we incorporated TRPV1 into planar lipid bilayers consisting of neutral lipids, capsaicin-induced activity depended on phosphatidylinositol 4,5-bisphosphate. We also found that TRPV1 activity in excised patches ran down and that MgATP reactivated the channel. Inhibition of phosphatidylinositol 4-kinases or enzymatic removal of phosphatidylinositol abolished this effect of MgATP, suggesting that it activated TRPV1 by generating endogenous phosphoinositides. We conclude that endogenous phosphoinositides are positive cofactors for TRPV1 activity. Our data highlight the importance of specificity in lipid regulation of ion channels and may reconcile discordant data obtained in various experimental settings. PMID:24158445

Lukacs, Viktor; Rives, Jan-Michael; Sun, Xiaohui; Zakharian, Eleonora; Rohacs, Tibor



Internalin B activates nuclear factor-kappa B via Ras, phosphoinositide 3-kinase, and Akt.  


Internalin B (InlB), a 630-amino acid protein loosely attached to the surface of Listeria monocytogenes, participates in the entry of the bacterium into mammalian cells. This process requires the activation of phosphoinositide (PI) 3-kinase by InlB. Previously, we demonstrated that InlB activates the transcription factor Nuclear Factor-kappaB in murine J774 macrophage-like cells, an event that also requires PI 3-kinase. Here we have further investigated this phenomenon. InlB activated the small G-protein Ras in J774 cells. Inhibition of Ras with the farnesyltransferase inhibitor manumycin A inhibited NF-kappaB activation and the recruitment of the p85 subunit of PI 3-kinase, implying that Ras is required for PI 3-kinase activation. InlB also activated the PI 3-kinase downstream effector, Akt, as assessed by increased phosphorylation of Akt on serine 473. Transfection of Hep2 cells with dominant negative Ras N17 or dominant negative Akt inhibited the induction of a reporter gene linked to the interleukin-8 promoter by InlB. Furthermore, the Ras inhibitor manumycin A, the PI 3-kinase inhibitor LY294002, and an Akt inhibitor all blocked the induction of interleukin-8 by InlB. Our study is the first report of a bacterial product activating a pathway involving Ras, PI 3-kinase, and Akt, which leads to NF-kappaB activation. This process could be involved in host defense or the inhibition of apoptosis during infection. PMID:11571285

Mansell, A; Khelef, N; Cossart, P; O'Neill, L A



Corticosteroid insensitivity is reversed by formoterol via phosphoinositide-3-kinase inhibition  

PubMed Central

BACKGROUND AND PURPOSE Patients with chronic obstructive pulmonary disease (COPD) show a poor response to corticosteroids, which has been linked to oxidative stress. Here we show that the long-acting ?2-agonist formoterol (FM) reversed corticosteroid insensitivity under oxidative stress via inhibition of phosphoinositide-3-kinase (PI3K) signalling. EXPERIMENTAL APPROACH Responsiveness to corticosteroids dexamethasone (Dex), budesonide (Bud) and fluticasone propionate (FP) was determined, as IC50 values on TNF-?-induced interleukin 8 release, in U937 monocytic cell line treated with hydrogen peroxide (H2O2) or peripheral blood mononuclear cells (PBMCs) from patients with COPD or severe asthma. KEY RESULTS PBMCs from severe asthma and COPD were less sensitive to Dex compared with those from healthy subjects. Both FM (10?9 M) and salmeterol (SM, 10?8 M) reversed Dex insensitivity in severe asthma, but only FM restored Dex sensitivity in COPD. Although H2O2 exposure decreased steroid sensitivity in U937 cells, FM restored responsiveness to Bud and FP while the effects of SM were weaker. Additionally, FM, but not SM, partially inhibited H2O2-induced PI3K?-dependent (PKB) phosphorylation. H2O2 decreased SM-induced cAMP production in U937 cells, but did not significantly affect the response to FM. The reduction of SM effects by H2O2 was reversed by pretreatment with LY294002, a PI3K inhibitor, or IC87114, a PI3K? inhibitor. CONCLUSION AND IMPLICATIONS FM reversed oxidative stress-induced corticosteroid insensitivity and decreased ?2 adrenoceptor-dependent cAMP production via inhibition of PI3K? signalling. FM will be more effective than SM, when combined with corticosteroids, for the treatment of respiratory diseases under conditions of high oxidative stress, such as in COPD.

Rossios, C; To, Y; Osoata, G; Ito, M; Barnes, PJ; Ito, K



p50?/p55? Phosphoinositide 3-Kinase Knockout Mice Exhibit Enhanced Insulin Sensitivity  

PubMed Central

Class Ia phosphoinositide (PI) 3-kinases are heterodimers composed of a regulatory and a catalytic subunit and are essential for the metabolic actions of insulin. In addition to p85? and p85?, insulin-sensitive tissues such as fat, muscle, and liver express the splice variants of the pik3r1 gene, p50? and p55?. ?? define the role of these variants, we have created mice with a deletion of p50? and p55? by using homologous recombination. These mice are viable, grow normally, and maintain normal blood glucose levels but have lower fasting insulin levels. Results of an insulin tolerance test indicate that p50?/p55? knockout mice have enhanced insulin sensitivity in vivo, and there is an increase in insulin-stimulated glucose transport in isolated extensor digitorum longus muscle tissues and adipocytes. In muscle, loss of p50?/p55? results in reduced levels of insulin-stimulated insulin receptor substrate 1 (IRS-1) and phosphotyrosine-associated PI 3-kinase but enhanced levels of IRS-2-associated PI 3-kinase and Akt activation, whereas in adipocytes levels of both insulin-stimulated PI 3-kinase and Akt are unchanged. Despite this, adipocytes of the knockout mice are smaller and have increased glucose uptake with altered glucose metabolic pathways. When treated with gold thioglucose, p50?/p55? knockout mice become hyperphagic like their wild-type littermates. However, they accumulate less fat and become mildly less hyperglycemic and markedly less hyperinsulinemic. Taken together, these data indicate that p50? and p55? play an important role in insulin signaling and action, especially in lipid and glucose metabolism.

Chen, Dong; Mauvais-Jarvis, Franck; Bluher, Matthias; Fisher, Simon J.; Jozsi, Alison; Goodyear, Laurie J.; Ueki, Kohjiro; Kahn, C. Ronald



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


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. PMID:23405144

Aggarwal, Chhavi; Labuz, Justyna; Gabry?, Halina



Endosomal Maturation, Rab7 GTPase and Phosphoinositides in African Swine Fever Virus Entry  

PubMed Central

Here we analyzed the dependence of African swine fever virus (ASFV) infection on the integrity of the endosomal pathway. Using confocal immunofluorescence with antibodies against viral capsid proteins, we found colocalization of incoming viral particles with early endosomes (EE) during the first minutes of infection. Conversely, viral capsid protein was not detected in acidic late endosomal compartments, multivesicular bodies (MVBs), late endosomes (LEs) or lysosomes (LY). Using an antibody against a viral inner core protein, we found colocalization of viral cores with late compartments from 30 to 60 minutes postinfection. The absence of capsid protein staining in LEs and LYs suggested that virus desencapsidation would take place at the acid pH of these organelles. In fact, inhibitors of intraluminal acidification of endosomes caused retention of viral capsid staining virions in Rab7 expressing endosomes and more importantly, severely impaired subsequent viral protein production. Endosomal acidification in the first hour after virus entry was essential for successful infection but not thereafter. In addition, altering the balance of phosphoinositides (PIs) which are responsible of the maintenance of the endocytic pathway impaired ASFV infection. Early infection steps were dependent on the production of phosphatidylinositol 3-phosphate (PtdIns3P) which is involved in EE maturation and multivesicular body (MVB) biogenesis and on the interconversion of PtdIns3P to phosphatidylinositol 3, 5-biphosphate (PtdIns(3,5)P2). Likewise, GTPase Rab7 activity should remain intact, as well as processes related to LE compartment physiology, which are crucial during early infection. Our data demonstrate that the EE and LE compartments and the integrity of the endosomal maturation pathway orchestrated by Rab proteins and PIs play a central role during early stages of ASFV infection.

Cuesta-Geijo, Miguel A.; Galindo, Inmaculada; Hernaez, Bruno; Quetglas, Jose Ignacio; Dalmau-Mena, Inmaculada; Alonso, Covadonga



Initiation of human astrovirus type 1 infection was blocked by inhibitors of phosphoinositide 3-kinase  

PubMed Central

Background Upon initial contact with a virus, host cells activate a series of cellular signaling cascades that facilitate viral entry and viral propagation within the cell. Little is known about how the human astrovirus (HAstV) exploits signaling cascades to establish an infection in host cells. Recent studies showed that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is important for HAstV infection, though the involvement of other signaling cascades remains unclear. Methods A panel of kinase blockers was used to search for cellular signaling pathways important for HAstV1 infection. To determine their impact on the infectious process, we examined viral gene expression, RNA replication, and viral RNA and capsid protein release from host cells. Results Inhibitors of phosphoinositide 3-kinase (PI3K) activation interfered with the infection, independent of their effect on ERK 1/2 activation. Activation of the PI3K signaling cascade occurred at an early phase of the infection, judging from the timeframe of Akt phosphorylation. PI3K inhibition at early times, but not at later times, blocked viral gene expression. However, inhibiting the downstream targets of PI3K activation, Akt and Rac1, did not block infection. Inhibition of protein kinase A (PKA) activation was found to block a later phase of HAstV1 production. Conclusions Our results reveal a previously unknown, essential role of PI3K in the life cycle of HAstV1. PI3K participates in the early stage of infection, possibly during the viral entry process. Our results also reveal the role of PKA in viral production.



A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages  

PubMed Central

Phosphoinositide 3-kinase (PI 3-kinase) has been implicated in growth factor signal transduction and vesicular membrane traffic. It is thought to mediate the earliest steps leading from ligation of cell surface receptors to increased cell surface ruffling. We show here that inhibitors of PI 3-kinase inhibit endocytosis in macrophages, not by interfering with the initiation of the process but rather by preventing its completion. Consistent with earlier studies, the inhibitors wortmannin and LY294002 inhibited fluid-phase pinocytosis and Fc receptor-mediated phagocytosis, but they had little effect on the receptor-mediated endocytosis of diI-labeled, acetylated, low density lipoprotein. Large solute probes of endocytosis reported greater inhibition by wortmannin than smaller probes did, indicating that macropinocytosis was affected more than micropinocytosis. Since macropinocytosis and phagocytosis are actin-mediated processes, we expected that their inhibition by wortmannin resulted from deficient signaling from macrophage colony-stimulating factor (M-CSF) receptors or Fc receptors to the actin cytoskeleton. However, video microscopy showed cell surface ruffling in wortmannin-treated cells, and increased ruffling after addition of M-CSF or phorbol myristate acetate. Quantitative measurements of video data reported slightly diminished ruffling in wortmannin-treated cells. Remarkably, the ruffles that formed in wortmannin-treated macrophages all receded into the cytoplasm without closing into macropinosomes. Similarly, wortmannin and LY294002 did not inhibit the extension of actin-rich pseudopodia along IgG- opsonized sheep erythrocytes, but instead prevented them from closing into phagosomes. These findings indicate that PI 3-kinase is not necessary for receptor-mediated stimulation of pseudopod extension, but rather functions in the closure of macropinosomes and phagosomes into intracellular organelles.



Vasopressin stimulates phosphoinositide hydrolysis in LLC-PK sub 1 cells  

SciTech Connect

LLC-PK{sub 1} cells have been shown to possess vasopressin (VP) receptors (V{sub 2} type) that are coupled to adenyl cyclase to generate adenosine 3,5{prime}-cyclic monophosphate (cAMP). To determine whether VP also stimulates phosphoinositide (PI) hydrolysis to generate inositol phosphate (IP) and diacylglycerol (DAG) messenger system in LLC-PK{sub 1} cells, the authors measured the release of IP in LLC-PK{sub 1} cells in the absence and presence of various concentrations of VP. In addition, the authors also determined the effect of an increase in osmolality of the incubation medium on VP-stimulated PI hydrolysis in LLC-PK{sub 1} cells. The methods involved the incubation of LLC-PK{sub 1} cells with ({sup 3}H)inositol for its incorporation into membrane PI and the measurement of the release of ({sup 3}H)IP in the presence of LiCl which prevents dephosphorylation. The osmolality of the incubation media was increased from 300 to 600, 900, and 1,200 mosmol/kgH{sub 2}O by the addition of NaCl and urea. In an isosmotic incubation medium, VP (10{sup {minus}8} M) produced a 100% increase in PI hydrolysis in LLC-PK{sub 1} cells. The effect was much greater at higher concentrations of the hormone. The results suggest that in LLC-PK{sub 1} cells, VP stimulates PI hydrolysis probably through VP receptors that are coupled to phospholipase C. Furthermore, VP-stimulated PI messenger system in LLC-PK{sub 1} cells is influenced by osmolality of the extracellular fluid.

Garg, L.C.; Kapturczak, E.; Steiner, M.; Phillips, M.I. (Univ. of Florida College of Medicine, Gainesville (USA))



IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold  

SciTech Connect

Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.

Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M.F.; Downes, C. Peter; Batty, Ian H. (Toronto); (Dundee)



3-Phosphoinositide-dependent protein kinase-1 as an emerging target in the management of breast cancer  

PubMed Central

It should be noted that 3-phosphoinositide-dependent protein kinase-1 (PDK1) is a protein encoded by the PDPK1 gene, which plays a key role in the signaling pathways activated by several growth factors and hormones. PDK1 is a crucial kinase that functions downstream of phosphoinositide 3-kinase activation and activates members of the AGC family of protein kinases, such as protein kinase B (Akt), protein kinase C (PKC), p70 ribosomal protein S6 kinases, and serum glucocorticoid-dependent kinase, by phosphorylating serine/threonine residues in the activation loop. AGC kinases are known to play crucial roles in regulating physiological processes relevant to metabolism, growth, proliferation, and survival. Changes in the expression and activity of PDK1 and several AGC kinases have been linked to human diseases including cancer. Recent data have revealed that the alteration of PDK1 is a critical component of oncogenic phosphoinositide 3-kinase signaling in breast cancer, suggesting that inhibition of PDK1 can inhibit breast cancer progression. Indeed, PDK1 is highly expressed in a majority of human breast cancer cell lines and both PDK1 protein and messenger ribonucleic acid are overexpressed in a majority of human breast cancers. Furthermore, overexpression of PDK1 is sufficient to transform mammary epithelial cells. PDK1 plays an essential role in regulating cell migration, especially in the context of phosphatase and tensin homologue deficiency. More importantly, downregulation of PDK1 levels inhibits migration and experimental metastasis of human breast cancer cells. Thus, targeting PDK1 may be a valuable anticancer strategy that may improve the efficacy of chemotherapeutic strategies in breast cancer patients. In this review, we summarize the evidence that has been reported to support the idea that PDK1 may be a key target in breast cancer management.

Fyffe, Chanse; Falasca, Marco



Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation  

PubMed Central

Protein–phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47phox, a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47phox with phosphoinositides. Although the isolated phox homology (PX) domain of p47phox can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47phox is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47phox to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47phox, because protein kinase C treatment of the wild-type p47phox but not of a mutant protein with the S303/304/328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47phox translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47phox nor lipid-binding-defective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47phox enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47phox from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.

Ago, Tetsuro; Kuribayashi, Futoshi; Hiroaki, Hidekazu; Takeya, Ryu; Ito, Takashi; Kohda, Daisuke; Sumimoto, Hideki



The novel molecule 2-[5-(2-chloroethyl)-2-acetoxy-benzyl]-4-(2-chloroethyl)-phenyl acetate inhibits phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signalling through JNK activation in cancer cells.  


Screening a compound library of compound 48/80 analogues, we identified 2-[5-(2-chloroethyl)-2-acetoxy-benzyl]-4-(2-chloroethyl)-phenyl acetate (E1) as a novel inhibitor of the phosphoinositide 3-kinase/Akt pathway. In order to determine the mechanism of action of E1, we analysed the effect of E1 on components of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. E1 demonstrated dose-dependent and time-dependent repression of Akt and mTOR activity in prostate and breast cancer cell lines, PC-3 and MCF-7, respectively. Inhibition of Akt and mTOR activity by E1 also coincided with increased c-Jun NH2-terminal kinase (JNK) phosphorylation. However, the mode of action of E1 is different from that of the mTOR inhibitor rapamycin. Proliferation and cell cycle analysis revealed that E1 induced cell cycle arrest and cell death in PC-3 and MCF-7 cells. Moreover, pretreatment of cancer cells with the JNK inhibitor SP600125 abolished the repression of Akt and mTOR activity by E1, indicating that the inhibition of Akt and mTOR by E1 is mediated through JNK activation. Consistently, E1 repressed Akt and mTOR activity in wild-type and p38-null mouse embryonic fibroblasts (MEFs), but not in MEFs lacking JNK1/2, and JNK-null MEFs were less sensitive to the antiproliferative effects of E1. We further showed that E1 can function cooperatively with suboptimal concentrations of paclitaxel to induce cell death in PC-3 and MCF-7 cells. Taken together, these data suggest that E1 induces cancer cell death through the JNK-dependent repression of Akt and mTOR activity and may provide a valuable compound for further development and research. PMID:19549188

Ho, Ka-Kei; Rosivatz, Evelyn; Gunn, Richard M; Smith, Mark E B; Stavropoulou, Alexandra V; Rosivatz, Erika; Numbere, Macba G; Wong, John B; Lafitte, Valerie G H; Behrendt, Jonathan M; Myatt, Stephen S; Hailes, Helen C; Woscholski, Rüdiger; Lam, Eric W-F



Common mechanisms of PIKK regulation  

PubMed Central

Kinases in the phosphoinositide 3-kinase related kinase (PIKK) family include ATM (ataxia-telangiectasia mutated), ATR (ATM and Rad3-related), DNA-PKcs (DNA-dependent protein kinase catalytic subunit, mTOR (mammalian target of rapamycin), and SMG1 (suppressor with morphological effect on genitalia family member). These atypical protein kinases regulate DNA damage responses, nutrient-dependent signaling, and nonsense-mediated mRNA decay. This review focuses on the mechanisms regulating the PIKK family with a strong emphasis on the DNA damage regulated kinases. We outline common regulatory themes and suggest how discoveries about the regulation of one PIKK can be informative for the other family members.

Lovejoy, Courtney A.; Cortez, David



Pharmacologic profiling of phosphoinositide 3-kinase inhibitors as mitigators of ionizing radiation-induced cell death.  


Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated ?IR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)], administered 4 hours after a lethal dose of ?IR, statistically significantly (P < 0.02) enhanced in vivo survival. Because cell cycle checkpoints are important regulators of cell survival after IR, we examined cell cycle distribution in NCCIT cells after ?IR and PI3K inhibitor treatment. LY294002 and PX-867 treatment of nonirradiated cells produced a marked decrease in S phase cells with a concomitant increase in the G1 population. In irradiated cells, LY294002 and PX-867 treatment also decreased S phase and increased the G1 and G2 populations. Treatment with LY294002 or PX-867 decreased ?IR-induced DNA damage as measured by ?H2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after IR abrogated cell death. PMID:24068833

Lazo, John S; Sharlow, Elizabeth R; Epperly, Michael W; Lira, Ana; Leimgruber, Stephanie; Skoda, Erin M; Wipf, Peter; Greenberger, Joel S



The Phosphoinositide 3-Kinase Inhibitor PI-103 Downregulates Choline Kinase ? Leading to Phosphocholine and Total Choline Decrease Detected by Magnetic Resonance Spectroscopy  

PubMed Central

The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus (31P)- and proton (1H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103–induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase ? (ChoK?), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r2 = 0.9, P = 0.009) was found only between PC concentrations and ChoK? expression but not with FAS or pACL. This study identified inhibition of ChoK? as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of 1H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with 31P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors.

Al-Saffar, Nada M. S.; Jackson, L. Elizabeth; Raynaud, Florence I.; Clarke, Paul A.; de Molina, Ana Ramirez; Lacal, Juan C.; Workman, Paul; Leach, Martin O.



Role of phosphoinositide 3-kinase and the Cbl adaptor protein in coupling the alpha4beta1 integrin to mitogen-activated protein kinase signalling.  

PubMed Central

Cell adhesion mediated by beta1 integrin receptors leads to the initiation of intracellular signals that affect cell differentiation and survival. Here we have analysed the mechanism by which the alpha4beta1 integrin activates the mitogen-activated protein kinase pathway in HL60 cells, a myelomonocytic cell line that lacks the expression of focal adhesion kinase. A role for phosphoinositide 3-kinase (PI-3K) in alpha4 integrin-mediated activation of extracellular signal-regulated protein kinase 2 (ERK2) is suggested by the ability of PI-3K inhibitors and a dominant-negative form of the p85 subunit of PI-3K to block the activation of ERK2 by integrin. Stimulation of alpha4beta1 integrins on HL60 cells also leads to increased tyrosine phosphorylation of the 120 kDa adaptor protein Cbl. PI-3K activity associated with Cbl also increases on the stimulation of alpha4beta1 integrins, although immunodepletion experiments suggest that Cbl-associated PI-3K does not account for all of the PI-3K activity induced on the stimulation of integrins in these cells. The expression of wild-type Cbl or the 70Z/3 Cbl mutant enhances basal ERK2 activity in transfectants with a minimal effect on alpha4 integrin-mediated ERK2 activity. In contrast, overexpression of the Hut Cbl truncation mutant, which does not associate with p85, has no effect on the ERK2 pathway. These results suggest that PI-3K has a major role in coupling alpha4beta1 integrins to ERK2 activation in myeloid cells and that the Cbl adaptor protein has a role in basal, but not alpha4beta1 integrin-mediated, activation of ERK2.

Finkelstein, L D; Shimizu, Y



Non-genomic action of resveratrol on androgen and oestrogen receptors in prostate cancer: modulation of the phosphoinositide 3-kinase pathway.  


Prostate cancer represents a major concern in human oncology and the phytoalexin resveratrol (RES) inhibits growth and proliferation of prostate cancer cells through the induction of apoptosis. In addition, previous data indicate that in oestrogen-responsive human breast cancer cells, RES induces apoptosis by inhibition of the phosphoinositide-3-kinase (PI3K) pathway. Here, using androgen receptor (AR)-positive LNCaP and oestrogen receptor alpha (ERalpha)-expressing PC-3 prostate tumour cells, we have analysed whether the antiproliferative activity of RES takes place by inhibition of the AR- or ERalpha-dependent PI3K pathway. Although RES treatment (up to 150 microM) decreased AR and ERalpha protein levels, it did not affect AR and ERalpha interaction with p85-PI3K. Immunoprecipitation and kinase assays showed that RES inhibited AR- and ERalpha-dependent PI3K activities in LNCaP and PC-3, respectively. Consistently, lower PI3K activities correlated with decreased phosphorylation of downstream targets protein kinase B/AKT (PKB/AKT) and glycogen synthase kinase-3 (GSK-3). GSK-3 dephosphorylation could be responsible for the decreased cyclin D1 levels observed in both cell lines. Importantly, RES markedly decreased PKB/AKT phosphorylation in primary cultures from human prostate tumours, suggesting that the mechanism proposed here could take place in vivo. Thus, RES could have antitumoral activity in androgen-sensitive and androgen-non-sensitive human prostate tumours by inhibiting survival pathways such as that mediated by PI3K. PMID:17486135

Benitez, D A; Pozo-Guisado, E; Clementi, M; Castellón, E; Fernandez-Salguero, P M



Mutation of SAC1, an Arabidopsis SAC Domain Phosphoinositide Phosphatase, Causes Alterations in Cell Morphogenesis, Cell Wall Synthesis, and Actin Organization  

Microsoft Academic Search

SAC (for suppressor of actin) domain proteins in yeast and animals have been shown to modulate the levels of phosphoinositides, thereby regulating several cellular activities such as signal transduction, actin cytoskeleton organiza- tion, and vesicle trafficking. Nine genes encoding SAC domain-containing proteins are present in the Arabidopsis thaliana genome, but their roles in plant cellular functions and plant growth and

Ruiqin Zhong; David H. Burk; C. Joseph Nairn; Alicia Wood-Jones; W. Herbert Morrison III; Zheng-Hua Yea



Differences in agonist and antagonist activities for two indices of metabotropic glutamate receptor-stimulated phosphoinositide turnover.  

PubMed Central

1. The abilities of the four diastereoisomers of 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) to stimulate, and the metabotropic glutamate receptor (mGluR) antagonist (+/-)-alpha-methylcarboxyphenylglycine (MCPG) to inhibit, phosphoinositide turnover in neonatal rat cerebral cortex have been studied. Two indices of phosphoinositide cycle activity were assessed; inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation, and total inositol phosphate [3H]-InsPx accumulation (in the presence of Li+) in myo-[3H]-inositol prelabelled slices. 2. The diastereoisomers of ACPD stimulated each response with a rank order of potency of 1S, 3R > 1R, 3R > 1S, 3S >> 1R, 3S. The response to 1R, 3R-ACPD was largely prevented by pre-addition of the NMDA-receptor antagonist, MK-801, or omission of extracellular Ca2+, suggesting that this isomer acts indirectly on phosphoinositide responses through activation of NMDA-type ionotropic glutamate receptors. In contrast, the responses to 1S, 3R- and 1S, 3S-ACPD were unaffected by prior addition of MK-801, but were blocked by MCPG. 3. The concentration of 1S, 3R-ACPD required to half-maximally stimulate the Ins(1,4,5)P3 response (-log EC50 (M), -4.09 +/- 0.10) was significantly higher than that required to exert a similar effect on [3H]-InsPx accumulation (-log EC50 (M), -4.87 +/- 0.07; P < 0.01; n = 4). A similar marked 8-9 fold discrepancy between these two values was observed for the 1S, 3S isomer, which elicited similar maximal responses to those caused by 1S, 3R-ACPD. 4. Significant differences were also observed with respect to the ability of (+/-)-MCPG (1 mM) to cause a rightward shift in the concentration-response relationships for 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 (5.59 +/- 0.24 fold shift) and [3H]-InsPx (3.04 +/- 0.34 fold shift; P < 0.01; n = 4) responses, giving rise to Kd values of 218 and 490 microM for (+/-)-MCPG antagonism of the respective responses. 5. The potency difference between the 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 and [3H]-InsPx responses was reduced when experiments were performed in nominally calcium-free medium ([Ca2+]e = 2 - 5 microM) and EC50 values were almost identical when extracellular calcium was reduced further by EGTA addition ([Ca2+]e < or = 100 nM). Similarly, the Kd value for (+/-)-MCPG antagonism of the 1S, 3R-ACPD-stimulated [3H]-InsPx response decreased under [Ca2+]e-free conditions, approaching those obtained for the 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 response in the presence of normal [Ca2+]e. 6. These data suggest that estimates of the activities of mGluR agonists and antagonists, derived by measuring phosphoinositide turnover, can differ significantly depending on whether Ins(1,4,5)P3 mass or [3H]-InsPx responses are measured. In particular, the possibility that the mGluR-mediated [3H]-InsPx response may not simply reflect direct receptor/G protein/phosphoinositidase C (PIC) activation, but may also be the consequence of stimulation of a facilitatory Ca2+-influx pathway is discussed.

Mistry, R.; Challiss, R. A.



Phosphoinositide 3-kinase pathway mediates early aldosterone action on morphology and epithelial sodium channel in Mammalian renal epithelia.  


Involvement of phosphoinositide 3-kinases (PI3Ks) in early aldosterone action on epithelial sodium channel (ENaC) in mammalian renal epithelia was investigated by hopping probe ion conductance microscopy combined with patch-clamping in this study. Aldosterone treatment enlarged the cell volume and elevated the apical membrane of renal mpkCCDc14 epithelia, which resulted in enhancing the open probability of ENaC. Inhibition of PI3K pathway by LY294002 obviously suppressed these aldosterone-induced changes in both cell morphology and ENaC activity. These results indicated the important role of PI3K pathway in early aldosterone action and the close relationship between cell morphology and ENaC activity in mammalian renal epithelia. PMID:24723072

Zhou, Yuan; Chen, Xuewei; Liu, Xiao; Lu, Hujie; Li, Ying; Zhu, Hui; An, Gaihong; Zhang, Na; Zhang, Jianning; Ma, Qiang; Zhang, Yanjun



Reversible Ser/Thr SHIP phosphorylation: a new paradigm in phosphoinositide signalling?: Targeting of SHIP1/2 phosphatases may be controlled by phosphorylation on Ser and Thr residues.  


Phosphoinositide (PI) phosphatases such as the SH2 domain-containing inositol 5-phosphatases 1/2 (SHIP1 and 2) are important signalling enzymes in human physiopathology. SHIP1/2 interact with a large number of immune and growth factor receptors. Tyrosine phosphorylation of SHIP1/2 has been considered to be the determining regulatory modification. However, here we present a hypothesis, based on recent key publications, highlighting the determining role of Ser/Thr phosphorylation in regulating several key properties of SHIP1/2. Since a subunit of the Ser/Thr phosphatase PP2A has been shown to interact with SHIP2, a putative mechanism for reversing SHIP2 Ser/Thr phosphorylation can be anticipated. PI phosphatases are potential target molecules in human diseases, particularly, but not exclusively, in cancer and diabetes. Therefore, this novel regulatory mechanism deserves further attention in the hunt for discovering novel or complementary therapeutic strategies. This mechanism may be more broadly involved in regulating PI signalling in the case of synaptojanin1 or the phosphatase, tensin homolog, deleted on chromosome TEN. PMID:22641604

Edimo, William's Elong; Janssens, Veerle; Waelkens, Etienne; Erneux, Christophe



Nuclear envelope assembly is promoted by phosphoinositide-specific phospholipase C with selective recruitment of phosphatidylinositol-enriched membranes  

PubMed Central

Nuclear envelope (NE) formation in a cell-free egg extract proceeds by precursor membrane vesicle binding to chromatin in an ATP-dependent manner, followed by a GTP-induced NE assembly step. The requirement for GTP in the latter step of this process can be mimicked by addition of bacterial PI-PLC [phosphoinositide (PtdIns)-specific phospholipase C]. The NE assembly process is here dissected in relation to the requirement for endogenous phosphoinositide metabolism, employing recombinant eukaryotic PI-PLC, inhibitors and direct phospholipid analysis using ESI-MS (electrospray ionization mass spectrometry). PtdIns (phosphatidylinositol) species analysis by ESI-MS indicates that the chromatin-bound NE precursor vesicles are enriched for specific PtdIns species. Moreover, during GTP-induced precursor vesicle fusion, the membrane vesicles become partially depleted of the PtdIns 18:0/20:4 species. These data indicate that eukaryotic PI-PLC can support NE formation, and the sensitivity to exogenous recombinant PtdIns-5-phosphatases shows that the endogenous PLC hydrolyses a 5-phosphorylated species. It is shown further that the downstream target of this DAG (diacylglycerol) pathway does not involve PKC (protein kinase C) catalytic function, but is mimicked by phorbol esters, indicating a possible engagement of one of the non-PKC phorbol ester receptors. The results show that ESI-MS can be used as a sensitive means to measure the lipid composition of biological membranes and their changes during, for example, membrane fusogenic events. We have exploited this and the intervention studies to illustrate a pivotal role for PI-PLC and its product DAG in the formation of NEs.



Xanomeline compared to other muscarinic agents on stimulation of phosphoinositide hydrolysis in vivo and other cholinomimetic effects.  


Activation of muscarinic m1 receptors which are coupled to the phosphoinositide (PI) second messenger transduction system is the initial objective of cholinergic replacement therapy in Alzheimer's disease. Thus, we evaluated the ability of the selective muscarinic receptor agonist (SMRA) xanomeline to stimulate in vivo phosphoinositide (PI) hydrolysis and compared it to a number of direct acting muscarinic agonists, two cholinesterase inhibitors and a putative m1 agonist/muscarinic m2 antagonist. Using a radiometric technique, it was determined that administration of xanomeline robustly stimulated in vivo PI hydrolysis and the effect was blocked by muscarinic antagonists, demonstrating mediation by muscarinic receptors. The non-selective muscarinic agonists pilocarpine, oxotremorine, RS-86, S-aceclidine, but not the less active isomer R-aceclidine, also effectively stimulated PI hydrolysis in mice. Amongst the putative m1 agonists, thiopilocarpine, hexylthio-TZTP as well as xanomeline effectively stimulated PI hydrolysis, but milameline, WAL 2014, SKB 202026 and PD 142505 did not significantly alter PI hydrolysis. Furthermore, WAL 2014 and SKB 202026 inhibited agonist-induced PI stimulation, suggesting that they act as antagonists at PI-coupled receptors in vivo. The cholinesterase inhibitors, tacrine and physostigmine, and the mixed muscarinic m1 agonist/m2 antagonist LU25-109 did not activate in vivo PI hydrolysis. Xanomeline, hexylthio-TZTP and thiopilocarpine were relatively free of cholinergic side effects, whereas milameline, WAL 2014 and SKB 202026 produced non-selective effects. Therefore, these data demonstrate that xanomeline selectively activates in vivo PI hydrolysis, consistent with activation of biochemical processes involved in memory and cognition and xanomeline's beneficial clinical effects on cognition in Alzheimers patients. PMID:9622623

Bymaster, F P; Carter, P A; Peters, S C; Zhang, W; Ward, J S; Mitch, C H; Calligaro, D O; Whitesitt, C A; DeLapp, N; Shannon, H E; Rimvall, K; Jeppesen, L; Sheardown, M J; Fink-Jensen, A; Sauerberg, P



Targeted Expression of the Class II Phosphoinositide 3Kinase in Drosophila melanogaster Reveals Lipid Kinase-Dependent Effects on Patterning and Interactions with Receptor Signaling Pathways  

Microsoft Academic Search

Phosphoinositide 3-kinases (PI3Ks) can be divided into three distinct classes (I, II, and III) on the basis of their domain structures and the lipid signals that they generate. Functions have been assigned to the class I and class III enzymes but have not been established for the class II PI3Ks. We have obtained the first evidence for a biological function

Lindsay K. MacDougall; Mary Elizabeth Gagou; Sally J. Leevers; Ernst Hafen; Michael D. Waterfield



Identification of the Tuberous Sclerosis Complex2 Tumor Suppressor Gene Product Tuberin as a Target of the Phosphoinositide 3Kinase\\/Akt Pathway  

Microsoft Academic Search

The S\\/T-protein kinases activated by phosphoinositide 3-kinase (PI3K) regulate a myriad of cellular processes. Here, we show that an approach using a combination of biochemistry and bioinformatics can identify substrates of these kinases. This approach identifies the tuberous sclerosis complex-2 gene product, tuberin, as a potential target of Akt\\/PKB. We demonstrate that, upon activation of PI3K, tuberin is phosphorylated on

Brendan D. Manning; Andrew R. Tee; M. Nicole Logsdon; John Blenis; Lewis C. Cantley



Involvement of phosphoinositide 3-kinase in insulin stimulation of MAP-kinase and phosphorylation of protein kinase-B in human skeletal muscle: implications for glucose metabolism  

Microsoft Academic Search

Summary   Isolated skeletal muscle from healthy individuals was used to evaluate the role of phosphoinositide 3-kinase (PI 3-kinase)\\u000a in insulin signalling pathways regulating mitogen activated protein kinase (MAP-kinase) and protein kinase-B and to investigate\\u000a whether MAP-kinase was involved in signalling pathways regulating glucose metabolism. Insulin stimulated glycogen synthase\\u000a activity ( ? 1.7 fold), increased 3-o-methylglucose transport into human skeletal muscle

P. R. Shepherd; B. T. Nave; J. Rincon; R. J. Haigh; E. Foulstone; C. Proud; J. R. Zierath; K. Siddle; H. Wallberg-Henriksson



Role of mitogen-activated protein kinase and phosphoinositide 3-kinase in the differentiation of rat pancreatic AR42J cells induced by hepatocyte growth factor  

Microsoft Academic Search

Aims\\/hypothesis. Pancreatic AR42J cells express both exocrine and neuroendocrine properties. When exposed to activin A, approximately 50\\u000a % of the cells die within 3 days by apoptosis. Addition of hepatocyte growth factor prevents apoptosis induced by activin\\u000a A and induces differentiation into insulin-producing cells. The present study was conducted to examine the role of mitogen-activated\\u000a protein kinase and phosphoinositide 3-kinase

M. Furukawa; Y. Q. Zhang; L. Nie; H. Shibata; I. Kojima



The Phosphoinositide Phosphatase Sjl2 Is Recruited to Cortical Actin Patches in the Control of Vesicle Formation and Fission during Endocytosis  

Microsoft Academic Search

The Saccharomyces cerevisiae synaptojanin-like proteins (Sjl1, Sjl2, and Sjl3) are phosphoinositide (PI) phosphatases that regulate PI metabolism in the control of actin organization and membrane trafficking. However, the primary sites of action for each of the yeast synaptojanin-like proteins remain unclear. In this study, we show that Sjl2 is localized to cortical actin patches, sites of endocytosis. Cortical recruitment of

Christopher J. Stefan; Steven M. Padilla; Anjon Audhya; Scott D. Emr



Regulation of imaginal disc cell size, cell number and organ size by Drosophila class I A phosphoinositide 3-kinase and its adaptor  

Microsoft Academic Search

Background: Class IA phosphoinositide 3-kinases (PI 3-kinases) have been implicated in the regulation of several cellular processes including cell division, cell survival and protein synthesis. The size of Drosophila imaginal discs (epithelial structures that give rise to adult organs) is maintained by factors that can compensate for experimentally induced changes in these PI 3-kinase-regulated processes. Overexpression of the gene encoding

David Weinkove; Thomas P. Neufeld; Thomas Twardzik; Michael D. Waterfield; Sally J. Leevers



Mutations of the lutropin\\/choriogonadotropin receptor that do not activate the phosphoinositide cascade allow hCG to induce aromatase expression in immature rat granulosa cells  

Microsoft Academic Search

Using primary cultures of immature rat granulosa cells and adenoviral infections we expressed two mutants of the human lutropin receptor (hLHR) that do not activate the phosphoinositide cascade. One mutant (hLFF) has the extracellular domain of the hLHR and the transmembrane and intracellular domains of the hFSHR. The other (hLHR-L457D) has a leucine to aspartate mutation in residue 457 of

Nebojsa Andric; Mario Ascoli



An Acidic pH and Activation of Phosphoinositide 3Kinase Stimulate Differentiation of Pancreatic Progenitors Into Insulin-Producing Cells  

Microsoft Academic Search

Adult pancreatic nonendocrine cells represent a potential alternative source of insulin-producing tissue for the treatment of diabetes. Differentiation of these cells is regulated by various signaling pathways including the phosphoinositide 3-kinase (PI3K) pathway. Therefore, we evaluated the effect of PI3K on this process. Compared with untreated cells the differentiation of human nonendocrine pancreatic cells into insulin-producing elements was increased after

T. Koblas; K. Zacharovova; Z. Berkova; P. Girman; F. Saudek



Repression of phosphoinositide-dependent protein kinase 1 expression by ciglitazone via Egr-1 represents a new approach for inhibition of lung cancer cell growth  

PubMed Central

Background Peroxisome proliferator-activated receptors gamma (PPAR?) ligands have been shown to inhibit the growth of non-small cell lung cancer (NSCLC) cells.?However, the mechanisms underlying this effect remain incompletely elucidated. Methods Cell proliferation and apoptosis were measured by cell viability, MTT and caspase3/7 activity assays. Phosphorylation/protein expression and gene silence/overexpression of AMPK?, phosphoinositide-dependent protein kinase 1 (PDK1), Egr-1 and PPAR? were performed by Western blot and siRNA/transfection assays. Dual-Luciferase Reporter Kit was used to measure the PPAR response elements (PPRE) reporter and PDK1 promoter activities, and ChIP assay was used to detect the Egr-1 protein binding to the DNA site in the PDK1 gene promoter. Results We found that ciglitazone, one synthetic PPAR? ligand, inhibited growth and induced apoptosis of NSCLC cells through decreased expression of PDK1, which was not blocked by GW9662 (a specific PPAR? antagonist). Overexpression of PDK1 overcame the effect of ciglitazone on cell growth and caspase 3/7 activity. Ciglitazone increased the phosphorylation of AMPK? and c-Jun N-terminal kinase (JNK), and the inhibitor of AMPK (compound C), but not JNK (SP600125), reversed the effect of ciglitazone on PDK1 protein expression. Ciglitazone reduced PDK1 gene promoter activity, which was not observed in cells exposed to compound C, but not silenced of PPAR? siRNA. Combination of ciglitazone and metformin further reduced PDK1 expression and promoter activity.?Furthermore, we showed that ciglitazone induced the protein expression of Egr-1, which was not observed in cells silencing of AMPK?. Moreover, silencing of Egr-1 abrogated the effect of ciglitazone on PDK1 promoter activity and cell growth. On the contrary, overexpression of Egr-1 enhanced the effect of ciglitazone on PDK1 gene promoter activity. ChIP assays demonstrated that ciglitazone induced Egr-1 protein bind to the specific DNA site in the PDK1 gene promoter. Conclusion Collectively, our results demonstrate that ciglitazone inhibits PDK1 expression through AMPK?-mediated induction of Egr-1 and Egr-1 binding to the specific DNA site in the PDK1 gene promoter, which is independent of PPAR?. Activation of AMPK? by metformin enhances the effect of ciglitazone. In turn, this leads to inhibition of NSCLC cell proliferation.



Regulation of the phosphoinositide pathway in cultured Sertoli cells from immature rats: effects of follicle-stimulating hormone and fluoride  

SciTech Connect

Many hormones elicit effects on target cells by stimulating the enzyme phospholipase-C, which catalyzes the hydrolysis of phosphoinositides to the intracellular second messengers diacylglycerol and inositol phosphates. The present study examined the roles of FSH and guanine nucleotide-binding proteins (G-proteins) in regulating the hydrolysis of phosphoinositides in Sertoli cells. Sertoli cell cultures prepared from 16- to 18-day-old rats were incubated for 24 h with myo-(2-3H) inositol to label endogenous phospholipids. Treatment of cells from 0.5-20 min with preparations of ovine FSH ranging in potency from 1-60 times that of NIH FSH S1 did not affect accumulation of inositol phosphates. Levels of total (3H)inositol phosphates ((3H)inositol mono-, di-, and triphosphates (IP, IP2, and IP3)) in FSH-treated cultures was 75-120% the levels in control cultures over the various time intervals studied. Addition of testosterone and the combination of testosterone plus retinoic acid, agents that have been shown to potentiate effects of FSH in other systems, did not affect accumulation of inositol phosphates in response to FSH. In contrast to the lack of effect on accumulation of inositol phosphates, FSH stimulated 4- to 11-fold increases in estradiol secretion over 24 h of culture, indicating that Sertoli cells were viable and responsive to FSH. AIF4- has been shown to activate G-proteins involved in regulation of adenylate cyclase activity. In the present study, AIF4- induced 4- to 5-fold increases in IP, IP2, and IP3 in experiments wherein FSH had no effect. Pretreatment of Sertoli cells with pertussis toxin (100 and 1000 ng/ml) for 24 h inhibited fluoride-induced generation of IP, IP2, and IP3 by 24-51%. Similar treatment with cholera toxin had no effect on basal or fluoride-induced generation of IP2 or IP3, but increased fluoride-induced generation of IP by 20-34%.

Quirk, S.M.; Reichert, L.E. Jr.



Sodium Antimony Gluconate Induces Generation of Reactive Oxygen Species and Nitric Oxide via Phosphoinositide 3-Kinase and Mitogen-Activated Protein Kinase Activation in Leishmania donovani-Infected Macrophages  

PubMed Central

Pentavalent antimony complexes, such as sodium stibogluconate and sodium antimony gluconate (SAG), are still the first choice for chemotherapy against various forms of leishmaniasis, including visceral leishmaniasis, or kala-azar. Although the requirement of a somewhat functional immune system for the antileishmanial action of antimony was reported previously, the cellular and molecular mechanism of action of SAG was not clear. Herein, we show that SAG induces extracellular signal-regulated kinase 1 (ERK-1) and ERK-2 phosphorylation through phosphoinositide 3-kinase (PI3K), protein kinase C, and Ras activation and p38 mitogen-activated protein kinase (MAPK) phosphorylation through PI3K and Akt activation. ERK-1 and ERK-2 activation results in an increase in the production of reactive oxygen species (ROS) 3 to 6 h after SAG treatment, while p38 MAPK activation and subsequent tumor necrosis factor alpha release result in the production of nitric oxide (NO) 24 h after SAG treatment. Thus, this study has provided the first evidence that SAG treatment induces activation of some important components of the intracellular signaling pathway, which results in an early wave of ROS-dependent parasite killing and a stronger late wave of NO-dependent parasite killing. This opens up the possibility of this metalloid chelate being used in the treatment of various diseases either alone or in combination with other drugs and vaccines.

Mookerjee Basu, Jayati; Mookerjee, Ananda; Sen, Prosenjit; Bhaumik, Suniti; Sen, Pradip; Banerjee, Subha; Naskar, Ksudiram; Choudhuri, Soumitra K.; Saha, Bhaskar; Raha, Sanghamitra; Roy, Syamal



Functional significance of beta gamma-subunit carboxymethylation for the activation of phospholipase C and phosphoinositide 3-kinase.  


The gamma subunits of heterotrimeric G proteins are isoprenylated and methylated at their carboxyl-terminal cysteine residues. Since methylation is the only reversible reaction in the isoprenylation pathway, it could be a site of regulation of G protein activity. beta gamma subunits have been shown to activate a number of effectors involved in signal transduction pathways. The methyl group of retinal transducin (T) can be hydrolyzed by an immobilized form of pig liver esterase, allowing for a direct determination of the activities of methylated and demethylated T beta gamma. The abilities of methylated and demethylated T beta gamma to stimulate G protein regulated phosphatidylinositol-specific phospholipase C (PIPLC) and phosphoinositide 3-kinase (PI3K) were determined. It is reported here that there is a strong dependence on methylation for activating both PIPLC and PI3K. Demethylated T beta gamma is at least 10-fold less active than its methylated counterpart. Therefore, methylation may play an important role in the regulation of these effectors and of signal transduction processes in general. PMID:7779819

Parish, C A; Smrcka, A V; Rando, R R



The p85beta regulatory subunit of phosphoinositide 3-kinase has unique and redundant functions in B cells.  


Phosphoinositide kinase (PI3K) is activated by various receptors on lymphocytes and regulates development, activation, and tolerance. Genetic ablation of PI3K function in T cells leads to the appearance of autoimmune disorders. In B cells, loss of the class IA regulatory subunit p85alpha causes a partial defect in B cell development and proliferation, whereas loss of p85beta alone causes no apparent changes in B cell function. Here we investigate further the consequences of p85beta deletion in B cells, in the presence or absence of p85alpha. We demonstrate that p85beta partially compensates for loss of p85alpha in B cell development and peripheral survival, with greater defects observed when both isoforms are absent. BCR-mediated AKT phosphorylation is partially reduced in p85alpha-deficient B cells and further diminished with concomitant loss of p85beta. Unexpectedly, loss of p85beta results in increased BCR-mediated proliferation and ERK phosphorylation. These results indicate that the p85beta regulatory isoform has partially overlapping functions with p85alpha in B cells as well as a unique role in opposing BCR responses. PMID:19811262

Oak, Jean S; Chen, Jing; Peralta, Raechel Q; Deane, Jonathan A; Fruman, David A



Phosphoinositide-3 kinase/protein kinase-b/mammalian target of rapamycin pathway in psoriasis pathogenesis. A potential therapeutic target?  


Psoriasis is a common chronic inflammatory disease of the skin. Its pathogenesis has not been completely elucidated. Phosphoinositide-3 kinase/protein kinase-B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway has been identified as a key signaling pathway for important cellular functions. The data collected in this review suggest that overexpression of the PI3K/Akt/mTOR pathway may play an important role in the pathogenesis of psoriasis by mediating the immune-pathogenesis, the epidermal hyperplasia or/and the angiogenesis in the disease. Advances in understanding the pathogenesis of psoriasis has provided new insight into potential therapeutic targets, including the development of biological therapies, resulting in remarkable clinical responses in patients with severe psoriasis. More recently, small molecule oral preparations targeting intracellular signaling that may prove effective have been developed. Data suggest that PI3K/Akt/mTOR pathway may be a potential target for treatment of psoriasis. PMID:24217655

Huang, Tian; Lin, Xiran; Meng, Xianmin; Lin, Mao



Phosphoinositide-dependent kinase 1 and mTORC2 synergistically maintain postnatal heart growth and heart function in mice.  


The protein kinase Akt plays a critical role in heart function and is activated by phosphorylation of threonine 308 (T308) and serine 473 (S473). While phosphoinositide-dependent kinase 1 (PDK1) is responsible for Akt T308 phosphorylation, the identities of the kinases for Akt S473 phosphorylation in the heart remain controversial. Here, we disrupted mTOR complex 2 (mTORC2) through deletion of Rictor in the heart and found normal heart growth and function. Rictor deletion caused significant reduction of Akt S473 phosphorylation but enhanced Akt T308 phosphorylation, suggesting that a high level of Akt T308 phosphorylation maintains Akt activity and heart function. Deletion of Pdk1 in the heart caused significantly enhanced Akt S473 phosphorylation that was suppressed by removal of Rictor, leading to worsened dilated cardiomyopathy (DCM) and accelerated heart failure in Pdk1-deficient mice. In addition, we found that increasing Akt S473 phosphorylation through deletion of Pten or chemical inhibition of PTEN reversed DCM and heart failure in Pdk1-deficient mice. Investigation of heart samples from human DCM patients revealed changes similar to those in the mouse models. These results demonstrated that PDK1 and mTORC2 synergistically promote postnatal heart growth and maintain heart function in postnatal mice. PMID:24662050

Zhao, Xia; Lu, Shuangshuang; Nie, Junwei; Hu, Xiaoshan; Luo, Wen; Wu, Xiangqi; Liu, Hailang; Feng, Qiuting; Chang, Zai; Liu, Yaoqiu; Cao, Yunshan; Sun, Haixiang; Li, Xinli; Hu, Yali; Yang, Zhongzhou



Acylation-dependent Export of Trypanosoma cruzi Phosphoinositide-specific Phospholipase C to the Outer Surface of Amastigotes*  

PubMed Central

Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid-modified in its N terminus and localizes to the plasma membrane of amastigotes. Here, we show that TcPI-PLC is located onto the extracellular phase of the plasma membrane of amastigotes and that its N-terminal 20 amino acids are necessary and sufficient to target the fused GFP to the outer surface of the parasite. Mutagenesis of the predicted acylated residues confirmed that myristoylation of a glycine residue in the 2nd position and acyl modification of a cysteine in the 4th but not in the 8th or 15th position of the coding sequence are required for correct plasma membrane localization in T. cruzi epimastigotes or amastigotes. Interestingly, mutagenesis of the cysteine at the 8th position increased its flagellar localization. When expressed as fusion constructs with GFP, the N-terminal 6 and 10 amino acids fused to GFP are predominantly located in the cytosol and concentrated in a compartment that co-localizes with a Golgi complex marker. The N-terminal 20 amino acids of TcPI-PLC associate with lipid rafts when dually acylated. Taken together, these results indicate that N-terminal acyl modifications serve as a molecular addressing system for sending TcPI-PLC to the outer surface of the cell.

de Paulo Martins, Vicente; Okura, Michael; Maric, Danijela; Engman, David M.; Vieira, Mauricio; Docampo, Roberto; Moreno, Silvia N. J.



Requirement for class II phosphoinositide 3-kinase C2alpha in maintenance of glomerular structure and function.  


An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2? (PI3KC2?) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2?-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2? deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2? deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2? is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function. PMID:20974805

Harris, David P; Vogel, Peter; Wims, Marie; Moberg, Karen; Humphries, Juliane; Jhaver, Kanchan G; DaCosta, Christopher M; Shadoan, Melanie K; Xu, Nianhua; Hansen, Gwenn M; Balakrishnan, Sanjeevi; Domin, Jan; Powell, David R; Oravecz, Tamas



Different roles of protein kinase C-beta and -delta in arachidonic acid cascade, superoxide formation and phosphoinositide hydrolysis.  

PubMed Central

In contrast with protein kinase C (PKC)-beta, PKC-delta is exclusively detectable in the membrane fraction of liver macrophages. After long-term treatment with phorbol 12-myristate 13-acetate (PMA) PKC-beta is depleted faster (within 3 h) than PKC-delta (> 7h). Simultaneously, pretreatment with PMA for 3 h inhibits the PMA- and zymosan-induced generation of superoxide and the PMA-induced formation of prostaglandin (PG) E2, whereas a preincubation of more than 7 h is required to affect the zymosan-induced release of PGE2 and inositol phosphates. These results support an involvement of PKC-beta in the PMA-induced activation of the arachidonic acid cascade and in superoxide formation and imply an involvement of PKC-delta in zymosan-induced phosphoinositide hydrolysis and PGE2 formation. Two phorbol ester derivates, sapintoxin A (SAPA) and 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA), which have been previously reported to activate preferentially PLC-beta but not PKC-delta in vitro [Ryves, Evans, Olivier, Parker and Evans (1992) FEBS Lett. 288, 5-9], induce the formation of PGE2 and superoxide, down-regulate PKC-delta and potentiate inositol phosphate formation in parallel SAPA, but not DOPPA, down-regulates PKC-beta and inhibits the PMA-induced formation of eicosanoids and superoxide. Images Figure 1 Figure 2 Figure 5

Duyster, J; Schwende, H; Fitzke, E; Hidaka, H; Dieter, P



Phosphoinositide 3-Kinase Catalytic Subunit Deletion and Regulatory Subunit Deletion Have Opposite Effects on Insulin Sensitivity in Mice  

PubMed Central

Studies ex vivo have shown that phosphoinositide 3-kinase (PI3K) activity is necessary but not sufficient for insulin-stimulated glucose uptake. Unexpectedly, mice lacking either of the PI3K regulatory subunits p85? or p85? exhibit increased insulin sensitivity. The insulin hypersensitivity is particularly unexpected in p85??/? p55??/? p50??/? mice, where a decrease in p110? and p110? catalytic subunits was observed in insulin-sensitive tissues. These results raised the possibility that decreasing total PI3K available for stimulation by insulin might circumvent negative feedback loops that ultimately shut off insulin-dependent glucose uptake in vivo. Here we present results arguing against this explanation. We show that p110?+/? p110?+/? mice exhibit mild glucose intolerance and hyperinsulinemia in the fasted state. Unexpectedly, p110?+/? p110?+/? mice showed a ?50% decrease in p85 expression in liver and muscle. Consistent with this in vivo observation, knockdown of p110 by RNA interference in mammalian cells resulted in loss of p85 proteins due to decreased protein stability. We propose that insulin sensitivity is regulated by a delicate balance between p85 and p110 subunits and that p85 subunits mediate a negative role in insulin signaling independent of their role as mediators of PI3K activation.

Brachmann, Saskia M.; Ueki, Kohjiro; Engelman, Jeffrey A.; Kahn, Ronald C.; Cantley, Lewis C.



Phospholipase C ? and Phosphoinositide 3-Kinase Link Cytokines to ERK Activation in Hematopoietic Cells With Normal and Oncogenic Kras  

PubMed Central

Oncogenic K-Ras proteins, such as K-RasG12D, accumulate in the active, guanosine triphosphate (GTP)–bound conformation and stimulate signaling through effector kinases. The presence of the K-RasG12D oncoprotein at a similar abundance to that of endogenous wild-type K-Ras results in only minimal phosphorylation and activation of the canonical Raf–mitogen-activated or extracellular signal–regulated protein kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) and phosphoinositide-3 kinase (PI3K)–Akt–mammalian target of rapamycin (mTOR) signaling cascades in primary hematopoietic cells, and these pathways remain dependent on growth factors for efficient activation. Here, we show that phospholipase C ? (PLC-?), PI3K, and their generated second messengers link activated cytokine receptors to Ras and ERK signaling in differentiated bone marrow cells and in a cell population highly enriched for leukemia stem cells. Cells expressing endogenous oncogenic K-RasG12D remained dependent on the second messenger diacylglycerol for the efficient activation of Ras-ERK signaling. These data raise the unexpected possibility of therapeutically targeting proteins that function upstream of oncogenic Ras in cancer.

Diaz-Flores, Ernesto; Goldschmidt, Hana; Depeille, Philippe; Ng, Victor; Akutagawa, Jon; Krisman, Kimberly; Crone, Michael; Burgess, Michael R.; Williams, Olusegun; Houseman, Benjamin; Shokat, Kevan; Sampath, Deepak; Bollag, Gideon; Roose, Jeroen P.; Braun, Benjamin S.; Shannon, Kevin



Androgen receptor expression is regulated by the phosphoinositide 3-kinase/Akt pathway in normal and tumoral epithelial cells.  

PubMed Central

The androgen receptor (AR) is a ligand-responsive transcription factor known to play a central role in the pathogenesis of prostate cancer. However, the regulation of AR gene expression in the normal and pathological prostate remains poorly understood. This study focuses on the effect of the phosphoinositide 3-kinase (PI 3-kinase)/Akt axis on AR expression in vas deferens epithelial cells (VDEC), a suitable model to study androgen regulation of gene expression, and LNCaP cells (derived from a metastasis at the left supraclavicular lymph node from a 50-year-old patient with a confirmed diagnosis of metastatic prostate carcinoma). Taken together, our data show for the first time that the PI 3-kinase/Akt pathway is required for basal and dihydrotestosterone-induced AR protein expression in both VDEC and LNCaP. Inhibition of the PI 3-kinase/Akt pathway reduced AR expression and the decline in AR protein level correlated with a decrease in AR mRNA in VDEC but not in LNCaP. Since PI 3-kinase/Akt axis is active in prostate cancer, cross-talk between PI 3-kinase/Akt and AR signalling pathways may have implications for endocrine therapy.

Manin, Michele; Baron, Silvere; Goossens, Karine; Beaudoin, Claude; Jean, Claude; Veyssiere, Georges; Verhoeven, Guido; Morel, Laurent



Leukotriene D4 receptor-mediated hydrolysis of phosphoinositide and mobilization of calcium in sheep tracheal smooth muscle cells  

SciTech Connect

A sheep tracheal smooth muscle primary culture cell system was developed to characterize leukotriene D4 (LTD4) receptor-mediated biochemical and pharmacological effects. (/sup 3/H)LTD4 binding to the enriched plasma membrane receptor was specific, stereoselective and saturable. LTE4 and high affinity receptor antagonists bound to the receptors with a rank-order potency that was expected from previous smooth muscle contraction studies. In the (/sup 3/H)myoinositol labeled cells, LTD4 and LTE4 induced phosphoinositide hydrolysis. The biosynthesis of (/sup 3/H)inositol-trisphosphate was rapid and the induction of biosynthesis of (/sup 3/H)inositol-monophosphate by LTs was stereoselective and specific and was inhibited specifically by a receptor antagonist, SKF 104353. In the fura-2 loaded smooth muscle cells, LTD4 and LTE4 induced transient intracellular Ca++ mobilization. The fura-2/Ca++ transient was stereoselective and specific and was inhibited by receptor antagonist, SKF 104353. These results suggest that the cultured sheep tracheal smooth muscle cells have plasma membrane receptors for LTD4. These receptors were coupled to a phospholipase C that, when activated by agonists, induced hydrolysis of inositol containing phospholipids. The hydrolysis products, e.g. diacylglycerol and inositol-trisphosphate, may serve as intracellular messengers that trigger or contribute to the contractile effect in sheep tracheal smooth muscle.

Mong, S.; Miller, J.; Wu, H.L.; Crooke, S.T.



The Type I? Inositol Polyphosphate 4-Phosphatase Generates and Terminates Phosphoinositide 3-Kinase Signals on Endosomes and the Plasma Membrane  

PubMed Central

Endosomal trafficking is regulated by the recruitment of effector proteins to phosphatidylinositol 3-phosphate [PtdIns(3)P] on early endosomes. At the plasma membrane, phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P2] binds the pleckstrin homology (PH) domain-containing proteins Akt and TAPP1. Type I? inositol polyphosphate 4-phosphatase (4-phosphatase) dephosphorylates PtdIns(3,4)P2, forming PtdIns(3)P, but its subcellular localization is unknown. We report here in quiescent cells, the 4-phosphatase colocalized with early and recycling endosomes. On growth factor stimulation, 4-phosphatase endosomal localization persisted, but in addition the 4-phosphatase localized at the plasma membrane. Overexpression of the 4-phosphatase in serum-stimulated cells increased cellular PtdIns(3)P levels and prevented wortmannin-induced endosomal dilatation. Furthermore, mouse embryonic fibroblasts from homozygous Weeble mice, which have a mutation in the type I 4-phosphatase, exhibited dilated early endosomes. 4-Phosphatase translocation to the plasma membrane upon growth factor stimulation inhibited the recruitment of the TAPP1 PH domain. The 4-phosphatase contains C2 domains, which bound PtdIns(3,4)P2, and C2-domain-deletion mutants lost PtdIns(3,4)P2 4-phosphatase activity, did not localize to endosomes or inhibit TAPP1 PH domain membrane recruitment. The 4-phosphatase therefore both generates and terminates phosphoinositide 3-kinase signals at distinct subcellular locations.

Ivetac, Ivan; Munday, Adam D.; Kisseleva, Marina V.; Zhang, Xiang-Ming; Luff, Susan; Tiganis, Tony; Whisstock, James C.; Rowe, Tony; Majerus, Phillip W.; Mitchell, Christina A.



Role of calcium in phosphoinositide metabolism and inhibition of norepinephrine transport into synaptic vesicles by amphetamine analogs  

SciTech Connect

Norepinephrine-(NE) and calcium ionophore A23187-stimulated phosphoinositide (PIn) metabolism in rat brain slices was studied under varying calcium conditions. Tissue was labelled with /sup 3/H-myo-inositol and /sup 3/H-inositol phosphates (IPn), products of PIn metabolism were measured. In the absence of media calcium the response to NE was decreased while that to A23187 was little affected A23187 can release calcium from intracellular stores. Basal and stimulated accumulation of /sup 3/H-IPn was reversibly antagonized with EGTA by addition of calcium. Using calcium buffers, approximately 10/sup -7/ M free calcium was required to support hydrolysis. Free intracellular calcium is maintained at approximately this level. Thus calcium is required for PIn hydrolysis but appears to play a permissive role, basal levels being sufficient to support metabolism. Conformationally-defined (rigid) and -restricted (semi-rigid) analogs of the most stable conformations of amphetamine, antiperiplanar (exo) and gauche (endo), were utilized to probe the conformational requirements of vesicular NE transport. Analogs tested were 2-aminotetralin (2AT), 3-methyltetrahydroisoquinoline, anti- and syn-9-aminobenzobicyclo(2.2.1)heptene, and endo and exo conformers of 2-aminobenzobicyclo(2.2.1)heptene and 2-aminobenzobicyclo(2.2.2)octene.

Knepper, S.M.



Molecular pharmacology in a simple model system: Implicating MAP kinase and phosphoinositide signalling in bipolar disorder  

Microsoft Academic Search

Understanding the mechanisms of drug action has been the primary focus for pharmacological researchers, traditionally using rodent models. However, non-sentient model systems are now increasingly being used as an alternative approach to better understand drug action or targets. One of these model systems, the social amoeba Dictyostelium, enables the rapid ablation or over-expression of genes, and the subsequent use of

Marthe H. R. Ludtmann; Katrina Boeckeler; Robin S. B. Williams



Growth Hormone Inhibition of Glucose Uptake in Adipocytes Occurs without Affecting GLUT4 Translocation through an Insulin Receptor Substrate-2-Phosphatidylinositol 3-Kinase-dependent Pathway*  

PubMed Central

Growth hormone (GH) pretreatment of 3T3-L1 adipocytes resulted in a concentration- and time-dependent inhibition of insulin-stimulated glucose uptake. Surprisingly, this occurred without significant effect on insulin-stimulated glucose transporter (GLUT) 4 translocation or fusion with the plasma membrane. In parallel, the inhibitory actions of chronic GH pretreatment also impaired insulin-dependent activation of phosphatidylinositol (PI) 3-kinase bound to insulin receptor substrate (IRS)-2 but not to IRS-1. In addition, insulin-stimulated Akt phosphorylation was inhibited by GH pretreatment. In contrast, overexpression of IRS-2 or expression of a constitutively active Akt mutant prevented the GH-induced insulin resistance of glucose uptake. Moreover, small interfering RNA-mediated IRS-2 knockdown also inhibited insulin-stimulated Akt activation and glucose uptake without affecting GLUT4 translocation and plasma membrane fusion. Together, these data support a model in which chronic GH stimulation inhibits insulin-dependent activation of phosphatidylinositol 3-kinase through a specific interaction of phosphatidylinositol 3-kinase bound to IRS-2. This inhibition leads to suppression of Akt activation coupled to glucose transport activity but not translocation or plasma membrane fusion of GLUT4.

Sasaki-Suzuki, Naoko; Arai, Kiyoshi; Ogata, Tomomi; Kasahara, Kouhei; Sakoda, Hideyuki; Chida, Kazuhiro; Asano, Tomoichiro; Pessin, Jeffrey E.; Hakuno, Fumihiko; Takahashi, Shin-Ichiro



Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts.  

PubMed Central

Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative P13K p85 subunit. Consistently, Gas6 activates the P13K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks Gas6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for Gas6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited Gas6-stimulated entry into the S phase. Accordingly, Gas6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of Gas6. Taken together, the evidence presented here identifies elements involved in the Gas6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival.

Goruppi, S; Ruaro, E; Varnum, B; Schneider, C



Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation  

Microsoft Academic Search

The initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in initiating translation of mRNAs, including those encoding oncogenic proteins. Therefore, eIF4E is considered a survival protein involved in cell cycle progression, cell transformation, and apoptotic resistance. Phosphorylation of eIF4E (usually at Ser209) increases its binding affinity for the cap of mRNA and may also favor its

Xuerong Wang; Ping Yue; Chi-Bun Chan; Keqiang Ye; Takeshi Ueda; Rie Watanabe-Fukunaga; Rikiro Fukunaga; Haian Fu; Fadlo R. Khuri; Shi-Yong Sun



Norepinephrine Increases Glucose Transport in Brown Adipocytes via  3-Adrenoceptors through a cAMP, PKA, and PI3-Kinase-Dependent Pathway Stimulating Conventional and Novel PKCs  

Microsoft Academic Search

To identify the signaling pathways that mediate the adren- ergic stimulation of glucose uptake in brown adipose tissue, we used mouse brown adipocytes in culture. The endogenous adrenergic neurotransmitter norepinephrine (NE) induced 2-deoxy-D-glucose uptake 3-fold in a concentration-dependent manner (pEC50 6.5). The uptake was abolished by high doses of propranolol. The NE effect was mimicked by isoprenaline (pEC50 6.9), BRL




PI 3-kinase-dependent phosphorylation of Plk1-Ser99 promotes association with 14-3-3? and is required for metaphase-anaphase transition  

PubMed Central

Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1–Thr210 phosphorylation. Plk1–Ser99 phosphorylation creates a docking site for 14-3-3?, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3? or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1–Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1–Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1–Thr210 phosphorylation, but also by Plk1 binding to 14-3-3? following Plk1–Ser99 phosphorylation downstream of the PI3K–Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase.

Kasahara, Kousuke; Goto, Hidemasa; Izawa, Ichiro; Kiyono, Tohru; Watanabe, Nobumoto; Elowe, Sabine; Nigg, Erich A; Inagaki, Masaki



Deletion of the p85? Regulatory Subunit of Phosphoinositide 3-Kinase in Cone Photoreceptor Cells Results in Cone Photoreceptor Degeneration  

PubMed Central

Purpose. Downregulation of the retinal insulin/mTOR pathway in mouse models of retinitis pigmentosa is linked to cone cell death, which can be delayed by systemic administration of insulin. A classic survival kinase linking extracellular trophic/growth factors with intracellular antiapoptotic pathways is phosphoinositide 3-kinase (PI3K), which the authors have shown to protect rod photoreceptors from stress-induced cell death. The role of PI3K in cones was studied by conditional deletion of its p85? regulatory subunit. Methods. Mice expressing Cre recombinase in cones were bred to mice with a floxed pi3k gene encoding the p85? regulatory subunit of the PI3K and were back-crossed to ultimately generate offspring with cone-specific p85? knockout (cKO). Cre expression and cone-specific localization were confirmed by Western blot analysis and immunohistochemistry (IHC), respectively. Cone structural integrity was determined by IHC using peanut agglutinin and an M-opsin–specific antibody. Electroretinography (ERG) was used to assess rod and cone photoreceptor function. Retinal structure was examined by light and electron microscopy. Results. An age-related cone degeneration was found in cKO mice, evidenced by a reduction in photopic ERG amplitudes and loss of cone cells. By 12 months of age, approximately 78% of cones had died, and progressive disorganization of synaptic ultrastructure was noted in surviving cone terminals in cKO retinas. Rod viability was unaffected in p85? cKO mice. Conclusions. The present study suggests that PI3K signaling pathway is essential for cone survival in the mouse retina.

Ivanovic, Ivana; Anderson, Robert E.; Le, Yun Z.; Fliesler, Steven J.; Sherry, David M.



A drug targeting only p110? can block phosphoinositide 3-kinase signalling and tumour growth in certain cell types  

PubMed Central

Genetic alterations in PI3K (phosphoinositide 3-kinase) signalling are common in cancer and include deletions in PTEN (phosphatase and tensin homologue deleted on chromosome 10), amplifications of PIK3CA and mutations in two distinct regions of the PIK3CA gene. This suggests drugs targeting PI3K, and p110? in particular, might be useful in treating cancers. Broad-spectrum inhibition of PI3K is effective in preventing growth factor signalling and tumour growth, but suitable inhibitors of p110? have not been available to study the effects of inhibiting this isoform alone. In the present study we characterize a novel small molecule, A66, showing the S-enantiomer to be a highly specific and selective p110? inhibitor. Using molecular modelling and biochemical studies, we explain the basis of this selectivity. Using a panel of isoform-selective inhibitors, we show that insulin signalling to Akt/PKB (protein kinase B) is attenuated by the additive effects of inhibiting p110?/p110?/p110? in all cell lines tested. However, inhibition of p110? alone was sufficient to block insulin signalling to Akt/PKB in certain cell lines. The responsive cell lines all harboured H1047R mutations in PIK3CA and have high levels of p110? and class-Ia PI3K activity. This may explain the increased sensitivity of these cells to p110? inhibitors. We assessed the activation of Akt/PKB and tumour growth in xenograft models and found that tumours derived from two of the responsive cell lines were also responsive to A66 in vivo. These results show that inhibition of p110? alone has the potential to block growth factor signalling and reduce growth in a subset of tumours.

Jamieson, Stephen; Flanagan, Jack U.; Kolekar, Sharada; Buchanan, Christina; Kendall, Jackie D.; Lee, Woo-Jeong; Rewcastle, Gordon W.; Denny, William A.; Singh, Ripudaman; Dickson, James; Baguley, Bruce C.; Shepherd, Peter R.



Novel phosphoinositide 3-kinase ?,? inhibitor: potent anti-inflammatory effects and joint protection in models of rheumatoid arthritis.  


Phosphoinositide 3-kinases ? and ? (PI3K? and PI3K?) are expressed in rheumatoid arthritis (RA) synovium and regulate innate and adaptive immune responses. We determined the effect of a potent PI3K?,? inhibitor, IPI-145, in two preclinical models of RA. IPI-145 was administered orally in rat adjuvant-induced arthritis (AA) and intraperitoneally in mouse collagen-induced arthritis (CIA). Efficacy was assessed by paw swelling, clinical scores, histopathology and radiography, and microcomputed tomography scanning. Gene expression and Akt phosphorylation in joint tissues were determined by quantitative real-time polymerase chain reaction and Western blot analysis. Serum concentrations of anti-type II collagen (CII) IgG and IgE were measured by immunoassay. T-cell responses to CII were assayed using thymidine incorporation and immunoassay. IPI-145 significantly reduced arthritis severity in both RA models using dosing regimens initiated before onset of clinical disease. Treatment of established arthritis with IPI-145 in AA, but not CIA, significantly decreased arthritis progression. In AA, histology scores, radiographic joint damage, and matrix metalloproteinase (MMP)-13 expression were reduced in IPI-145-treated rats. In CIA, joint histology scores and expression of MMP-3 and MMP-13 mRNA were lower in the IPI-145 early treatment group than in the vehicle group. The ratio of anti-CII IgG2a to total IgG in CIA was modestly reduced. Interleukin-17 production in response to CII was decreased in the IPI-145-treated group, suggesting an inhibitory effect on T-helper cell 17 differentiation. These data show that PI3K?,? inhibition suppresses inflammatory arthritis, as well as bone and cartilage damage, through effects on innate and adaptive immunity and that IPI-145 is a potential therapy for RA. PMID:24244039

Boyle, David L; Kim, Hae-Rim; Topolewski, Katharyn; Bartok, Beatrix; Firestein, Gary S



In utero hypoxic ischemia decreases the cholinergic agonist-stimulated poly-phosphoinositide turnover in the developing rat brain.  


Perinatal hypoxic-ischemic (HI) insult is known to cause cellular and molecular disturbances leading to functional and behavioral abnormalities during brain development. In this study, we examined the effects of an in utero HI insult on poly-phosphoinositide turnover in vivo in the cerebrum and cerebellum as well as cholinergic-stimulated turnover in cortical slices from developing rat brain. In utero HI treatment was carried out by clamping the uterine blood vessels of near-term fetuses for 5, 10 and 15 min followed by resuscitation of the newborn pups. The in vivo protocol for examining poly-PI signaling activity in 2 week-old pup brain involved intracerebral injection of [3H]inositol for 16 hr and subsequent intraperitoneal injection with lithium (8 meq/kg) for 4 hr prior to decapitation. In the control pups, lithium elicited a 2.6 fold increase in labeled inositol phosphate (IP) in the cerebrum as compared to a 1.3 fold increase in the cerebellum. In utero HI insult (5 to 15 min) resulted in a small increase in labeled IP in the cerebrum but not in the cerebellum. Carbachol stimulation of poly-PI turnover was examined in brain slices prelabeled with [3H]inositol in vivo. Incubation of the prelabeled slices with carbachol in the presence of LiCl (10 mM) resulted in a time-, dose- and age-dependent increase in labeled IP. Brain slices from 2 week-old pups that experienced in utero HI-treatment for 10 and 15 min (but not 5 min) showed a significant decrease in carbachol-stimulation of labeled IP as compared with control pups. These results indicate the effects of in utero HI on the choninergic-stimulated poly-PI signaling pathway and its implication on related functional deficits in the developing brain. PMID:8789611

Hersey, K; Hu, Z Y; Zhang, J P; Rhodes, P G; Sun, G Y



An integrin-targeted, pan-isoform, phosphoinositide-3 kinase inhibitor, SF1126, has activity against multiple myeloma in vivo  

PubMed Central

Purpose Multiple reports point to an important role for the phosphoinositide-3 kinase (PI3K) and AKT signaling pathways in tumor survival and chemoresistance in multiple myeloma (MM). The goals of our study were: (1) to generate the preclinical results necessary to justify a Phase I clinical trial of SF1126 in hematopoietic malignancies including multiple myeloma, and (2) to begin combining pan PI-3 kinase inhibitors with other agents to augment antitumor activity of this class of agent in preparation for combination therapy in Phase I/II trials. Methods We determined the in vitro activity of SF1126 with16 human MM cell lines. In vivo tumor growth suppression was determined with human myeloma (MM.1R) xenografts in athymic mice. In addition, we provide evidence that SF1126 has pharmacodynamic activity in the treatment of patients with MM. Results SF1126 was cytotoxic to all tested MM lines and potency was augmented by the addition of bortezomib. SF1126 affected MM.1R cell line signaling in vitro, inhibiting phospho-AKT, phospho-ERK, and the hypoxic stabilization of HIF1?. Tumor growth was 94% inhibited, with a marked decrease in both cellular proliferation (PCNA immunostaining) and angiogenesis (tumor microvessel density via CD31 immunostaining). Our clinical results demonstrate pharmacodynamic knockdown of p-AKT in primary patient derived MM tumor cells in vivo. Conclusions Our results establish three important points: (1) SF1126, a pan PI-3 kinase inhibitor has potent antitumor activity against multiple myeloma in vitro and in vivo, (2) SF1126 displays augmented antimyeloma activity when combined with proteasome inhibitor, bortezomib/Velcade®, and (3) SF1126 blocks the IGF-1 induced activation of AKT in primary MM tumor cells isolated from SF1126 treated patients The results support the ongoing early Phase I clinical trial in MM and suggest a future Phase I trial in combination with bortezomib in hematopoietic malignancies.

De, Pradip; Dey, Nandini; Terakedis, Breanne; Bersagel, Leif; Li, Zhi Hua; Mahadevan, Daruka; Garlich, Joseph R.; Trudel, Suzanne; Makale, Milan T.; Durden, Donald L.



The Arabidopsis DREB2 genetic pathway is constitutively repressed by basal phosphoinositide-dependent phospholipase C coupled to diacylglycerol kinase  

PubMed Central

Phosphoinositide-dependent phospholipases C (PI-PLCs) are activated in response to various stimuli. They utilize substrates provided by type III-Phosphatidylinositol-4 kinases (PI4KIII) to produce inositol triphosphate and diacylglycerol (DAG) that is phosphorylated into phosphatidic acid (PA) by DAG-kinases (DGKs). The roles of PI4KIIIs, PI-PLCs, and DGKs in basal signaling are poorly understood. We investigated the control of gene expression by basal PI-PLC pathway in Arabidopsis thaliana suspension cells. A transcriptome-wide analysis allowed the identification of genes whose expression was altered by edelfosine, 30 ?M wortmannin, or R59022, inhibitors of PI-PLCs, PI4KIIIs, and DGKs, respectively. We found that a gene responsive to one of these molecules is more likely to be similarly regulated by the other two inhibitors. The common action of these agents is to inhibit PA formation, showing that basal PI-PLCs act, in part, on gene expression through their coupling to DGKs. Amongst the genes up-regulated in presence of the inhibitors, were some DREB2 genes, in suspension cells and in seedlings. The DREB2 genes encode transcription factors with major roles in responses to environmental stresses, including dehydration. They bind to C-repeat motifs, known as Drought-Responsive Elements that are indeed enriched in the promoters of genes up-regulated by PI-PLC pathway inhibitors. PA can also be produced by phospholipases D (PLDs). We show that the DREB2 genes that are up-regulated by PI-PLC inhibitors are positively or negatively regulated, or indifferent, to PLD basal activity. Our data show that the DREB2 genetic pathway is constitutively repressed in resting conditions and that DGK coupled to PI-PLC is active in this process, in suspension cells and seedlings. We discuss how this basal negative regulation of DREB2 genes is compatible with their stress-triggered positive regulation.

Djafi, Nabila; Vergnolle, Chantal; Cantrel, Catherine; Wietrzynski, Wojciech; Delage, Elise; Cochet, Francoise; Puyaubert, Juliette; Soubigou-Taconnat, Ludivine; Gey, Delphine; Collin, Sylvie; Balzergue, Sandrine; Zachowski, Alain; Ruelland, Eric



Increasing Plasma Membrane Phosphatidylinositol(4,5)Bisphosphate Biosynthesis Increases Phosphoinositide Metabolism in Nicotiana tabacum[W][OA  

PubMed Central

A genetic approach was used to increase phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] biosynthesis and test the hypothesis that PtdInsP kinase (PIPK) is flux limiting in the plant phosphoinositide (PI) pathway. Expressing human PIPKI? in tobacco (Nicotiana tabacum) cells increased plasma membrane PtdIns(4,5)P2 100-fold. In vivo studies revealed that the rate of 32Pi incorporation into whole-cell PtdIns(4,5)P2 increased >12-fold, and the ratio of [3H]PtdInsP2 to [3H]PtdInsP increased 6-fold, but PtdInsP levels did not decrease, indicating that PtdInsP biosynthesis was not limiting. Both [3H]inositol trisphosphate and [3H]inositol hexakisphosphate increased 3-and 1.5-fold, respectively, in the transgenic lines after 18 h of labeling. The inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] binding assay showed that total cellular Ins(1,4,5)P3/g fresh weight was >40-fold higher in transgenic tobacco lines; however, even with this high steady state level of Ins(1,4,5)P3, the pathway was not saturated. Stimulating transgenic cells with hyperosmotic stress led to another 2-fold increase, suggesting that the transgenic cells were in a constant state of PI stimulation. Furthermore, expressing Hs PIPKI? increased sugar use and oxygen uptake. Our results demonstrate that PIPK is flux limiting and that this high rate of PI metabolism increased the energy demands in these cells.

Im, Yang Ju; Perera, Imara Y.; Brglez, Irena; Davis, Amanda J.; Stevenson-Paulik, Jill; Phillippy, Brian Q.; Johannes, Eva; Allen, Nina S.; Boss, Wendy F.



Wortmannin alters the intracellular trafficking of the bradykinin B2 receptor: role of phosphoinositide 3-kinase and Rab5.  

PubMed Central

Wortmannin reportedly induces the formation of enlarged cytoplasmic endosomes. Such vesicles were observed in a definite time window after wortmannin treatment (250 nM) in HEK-293 cells stably expressing a B2R (B2 receptor)--green fluorescent protein conjugate and other cell types. The alternative PI3K (phosphoinositide 3-kinase) inhibitor LY 294002 (100 microM) and a dominant-negative form of the enzyme (p85alpha DeltaiSH2) induce a more modest vesicle enlargement. PI3K inhibition by drugs did not affect agonist-induced [3H]arachidonate release. The wortmannin-induced formation of giant endosomes also involves Rab5 activity, since a dominant-negative form of this GTPase (Rab5 S34N) partially inhibits the wortmannin effect and a constitutively active form of Rab5 (Rab5 Q79L) induces the formation of enlarged endosomes. Moreover, agonist stimulation targeted B2R-green fluorescent protein towards the periphery of the giant vesicles and led to partial receptor degradation only in wortmannin-treated cells. Receptor degradation was decreased by protease inhibitors and by bafilomycin A1, a drug that inhibits lysosome function. Accumulation of fluorescent material inside the enlarged endosomes was observed in cells treated with bafilomycin A1, wortmannin and an agonist. [3H]Bradykinin binding was decreased in HEK-293 cells treated with both wortmannin and the agonist, but not with either separately. Furthermore, a wortmannin-induced functional down-regulation of B2R was observed in rabbit jugular veins after repeated agonist stimulation (contractility assay). This is the first report of a G-protein-coupled receptor down-regulation induced by an alteration of its usual routing in the cell. These results suggest that both PI3K and Rab5 influence B2R intracellular trafficking.

Houle, Steeve; Marceau, Francois



Phosphorylation of the platelet p47 phosphoprotein is mediated by the lipid products of phosphoinositide 3-kinase.  


Platelet stimulation by thrombin or the thrombin receptor activating peptide (TRAP) results in the activation of phosphoinositide 3-kinase and the production of the novel polyphosphoinositides phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3). We have shown previously that these lipids activate calcium-independent protein kinase C (PKC) isoforms in vitro (Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M. and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367). Activation of platelet PKC in response to TRAP is detected by the phosphorylation of the major PKC substrate in platelets, the p47 phosphoprotein, also known as pleckstrin. Here we provide evidence for two phases of pleckstrin phosphorylation in response to TRAP. A rapid phase of pleckstrin phosphorylation (< 1 min) precedes the peak of PtdIns-3,4-P2 production and is unaffected by concentrations of wortmannin (10-100 nM) that block production of this lipid. However prolonged phosphorylation of pleckstrin (> 2 min) is inhibited by wortmannin concentrations that block PtdIns-3,4-P2 production. Phorbol ester-mediated pleckstrin phosphorylation was not affected by wortmannin and wortmannin had no effect on purified platelet PKC activity. Phosphorylation of pleckstrin could be induced using permeabilized platelets supplied with exogenous gamma-32P[ATP] and synthetic dipalmitoyl PtdIns-3,4,5-P3 and dipalmitoyl PtdIns-3,4-P2 micelles, but not with dipalmitoyl phosphatidylinositol 3-phosphate or phosphatidylinositol 4,5-bisphosphate. These results suggest two modes of stimulating pleckstrin phosphorylation: a rapid activation of PKC (via diacylglycerol and calcium) followed by a slower activation of calcium-independent PKCs via PtdIns-3,4-P2. PMID:7493994

Toker, A; Bachelot, C; Chen, C S; Falck, J R; Hartwig, J H; Cantley, L C; Kovacsovics, T J



Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase  

NASA Technical Reports Server (NTRS)

To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.

Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.; Brown, C. S. (Principal Investigator)



Phosphoinositide 3-Kinase ? Protects Against Catecholamine-Induced Ventricular Arrhythmia Through Protein Kinase A-Mediated Regulation of Distinct Phosphodiesterases  

PubMed Central

Background Phosphoinositide 3-kinase ? (PI3K?) signaling engaged by ?-adrenergic receptors is pivotal in the regulation of myocardial contractility and remodeling. However, the role of PI3K? in catecholamine-induced arrhythmia is currently unknown. Methods and Results Mice lacking PI3K? (PI3K??/?) showed runs of premature ventricular contractions on adrenergic stimulation that could be rescued by a selective ?2-adrenergic receptor blocker and developed sustained ventricular tachycardia after transverse aortic constriction. Consistently, fluorescence resonance energy transfer probes revealed abnormal cAMP accumulation after ?2-adrenergic receptor activation in PI3K??/? cardiomyocytes that depended on the loss of the scaffold but not of the catalytic activity of PI3K?. Downstream from ?-adrenergic receptors, PI3K? was found to participate in multiprotein complexes linking protein kinase A to the activation of phosphodiesterase (PDE) 3A, PDE4A, and PDE4B but not of PDE4D. These PI3K?-regulated PDEs lowered cAMP and limited protein kinase A–mediated phosphorylation of L-type calcium channel (Cav1.2) and phospholamban. In PI3K??/? cardiomyocytes, Cav1.2 and phospholamban were hyperphosphorylated, leading to increased Ca2+ spark occurrence and amplitude on adrenergic stimulation. Furthermore, PI3K??/? cardiomyocytes showed spontaneous Ca2+ release events and developed arrhythmic calcium transients. Conclusions PI3K? coordinates the coincident signaling of the major cardiac PDE3 and PDE4 isoforms, thus orchestrating a feedback loop that prevents calcium-dependent ventricular arrhythmia.

Ghigo, Alessandra; Perino, Alessia; Mehel, Hind; Zahradnikova, Alexandra; Morello, Fulvio; Leroy, Jerome; Nikolaev, Viacheslav O.; Damilano, Federico; Cimino, James; De Luca, Elisa; Richter, Wito; Westenbroek, Ruth; Catterall, William A.; Zhang, Jin; Yan, Chen; Conti, Marco; Gomez, Ana Maria; Vandecasteele, Gregoire; Hirsch, Emilio; Fischmeister, Rodolphe



How Cells Integrate Complex Stimuli: The Effect of Feedback from Phosphoinositides and Cell Shape on Cell Polarization and Motility  

PubMed Central

To regulate shape changes, motility and chemotaxis in eukaryotic cells, signal transduction pathways channel extracellular stimuli to the reorganization of the actin cytoskeleton. The complexity of such networks makes it difficult to understand the roles of individual components, let alone their interactions and multiple feedbacks within a given layer and between layers of signalling. Even more challenging is the question of if and how the shape of the cell affects and is affected by this internal spatiotemporal reorganization. Here we build on our previous 2D cell motility model where signalling from the Rho family GTPases (Cdc42, Rac, and Rho) was shown to organize the cell polarization, actin reorganization, shape change, and motility in simple gradients. We extend this work in two ways: First, we investigate the effects of the feedback between the phosphoinositides (PIs) , and Rho family GTPases. We show how that feedback increases heights and breadths of zones of Cdc42 activity, facilitating global communication between competing cell “fronts”. This hastens the commitment to a single lamellipodium initiated in response to multiple, complex, or rapidly changing stimuli. Second, we show how cell shape feeds back on internal distribution of GTPases. Constraints on chemical isocline curvature imposed by boundary conditions results in the fact that dynamic cell shape leads to faster biochemical redistribution when the cell is repolarized. Cells with frozen cytoskeleton, and static shapes, consequently respond more slowly to reorienting stimuli than cells with dynamic shape changes, the degree of the shape-induced effects being proportional to the extent of cell deformation. We explain these concepts in the context of several in silico experiments using our 2D computational cell model.

Edelstein-Keshet, Leah



Identification and chromosomal localisation by fluorescence in situ hybridisation of human gene of phosphoinositide-specific phospholipase C beta(1).  


Members of phosphoinositide-specific phospholipase C (PLC) families are central intermediary in signal transduction in response to the occupancy of receptors by many growth factors. Among PLC isoforms, the type beta(1) is of particular interest because of its reported nuclear localisation in addition to its presence at the plasma membrane. It has been previously shown that both the stimulation and the inhibition of the nuclear PLCbeta(1) under different stimuli implicate PLCbeta(1) as an important enzyme for mitogen-activated cell growth as well as for murine erythroleukaemia cell differentiation. The above findings hinting at a direct involvement of PLCbeta(1) in controlling the cell cycle in rodent cells, and the previously reported mapping of its gene in rat chromosome band 3q35-36, a region frequently rearranged in rat tumours induced by chemical carcinogenesis, prompted us to identify its human homologue. By screening a human foetal brain cDNA library with the rat PLCbeta(1) cDNA probe, we have identified a clone homologous to a sequence in gene bank called KIAA 0581, which encodes a large part of the human PLCbeta(1). By using this human cDNA in fluorescence in situ hybridisation on human metaphases, it has been possible to map human PLCbeta(1) on chromosome 20p12, confirming the synteny between rat chromosome 3 and human chromosome 20 and providing a novel locus of homology between bands q35-36 in rat and p12 in man. Since band 20p12 has been recently reported amplified and/or deleted in several solid tumours, the identification and chromosome mapping of human PLCbeta(1) could pave the way for further investigations on the role exerted both in normal human cells and in human tumours by PLCbeta(1), which has been shown to behave as a key signalling intermediate in the control of the cell cycle. PMID:10760467

Peruzzi, D; Calabrese, G; Faenza, I; Manzoli, L; Matteucci, A; Gianfrancesco, F; Billi, A M; Stuppia, L; Palka, G; Cocco, L



1,25(OH)2 vitamin D3 stimulates membrane phosphoinositide turnover, activates protein kinase C, and increases cytosolic calcium in rat colonic epithelium.  

PubMed Central

The hormonal form of vitamin D, 1,25(OH)2 vitamin D3 [1,25(OH)2D3], regulates colonic calcium absorption and colonocyte proliferation and differentiation. In this study, we have examined the effect of 1,25(OH)2D3 on membrane phosphoinositide turnover, protein kinase C activation, and regulation of intracellular calcium concentration [( Ca+2]i) in isolated rat colonic epithelium. In a concentration-dependent manner, 1,25(OH)2D3 stimulated breakdown of membrane phosphoinositides within 15 s, generating diacylglycerol and inositol 1,4,5-triphosphate (IP3). 1,25(OH)2D3 rapidly activated colonic protein kinase C, with maximal translocation of activity from the cytosol to the membrane occurring within 1 min of exposure to the secosteroid. Studies performed in isolated colonocytes with the fluorescent dye fura-2 demonstrated that 10(-8) M 1,25(OH)2D3 caused a rapid rise in [Ca+2]i which then transiently decreased before rising to a new plateau value. When these experiments were performed in a calcium-free buffer, an increase in [Ca+2]i was observed, but both the transient and secondary rise were diminished in magnitude, suggesting that 1,25(OH)2D3 may stimulate both release of intracellular calcium stores and calcium influx. 1,25(OH)2D3 stimulated [3H]thymidine uptake in rat colonocytes, 4 h after an in vivo injection. These studies indicate that 1,25(OH)2D3 exerts a rapid influence on membrane phosphoinositide metabolism which may mediate certain of the secosteroid's effects on colonocyte calcium transport and proliferation.

Wali, R K; Baum, C L; Sitrin, M D; Brasitus, T A



Piperine inhibits type II phosphatidylinositol 4-kinases: a key component in phosphoinositides turnover.  


Piperine has been shown to have anti-inflammatory properties. The molecular mechanisms by which it mediates anti-inflammatory activities remain elusive. Type II phosphatidylinositol 4-kinase(s) are key components in Fc?RI receptor-mediated signaling leading to inflammatory mediators release in RBL-2H3 cells. The effects of piperine on IgE-mediated signaling and mast cell degranulation were investigated. Pretreatment of RBL-2H3 cells with piperine inhibited IgE-induced activation of type II PtdIns 4-kinase(s). In vitro lipid kinase assays showed piperine-inhibited type II PtdIns 4-kinase activity in a dose-dependent fashion with no effect on PtdIns 3-kinase activity. Concomitantly, pretreatment of RBL-2H3 cells with piperine also inhibited IgE-induced ?-hexosaminidase release in RBL-2H3 cells. These results suggest that type II PtdIns 4-kinases are part of piperine-mediated anti-inflammatory signaling mechanisms. PMID:24671493

Bojjireddy, Naveen; Sinha, Ranjeet Kumar; Subrahmanyam, Gosukonda



Alterations in the MA and NC Domains Modulate Phosphoinositide-Dependent Plasma Membrane Localization of the Rous Sarcoma Virus Gag Protein  

PubMed Central

Retroviral Gag proteins direct virus particle assembly from the plasma membrane (PM). Phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] plays a role in PM targeting of several retroviral Gag proteins. Here we report that depletion of intracellular PI(4,5)P2 and phosphatidylinositol-(3,4,5)-triphosphate [PI(3,4,5)P3] levels impaired Rous sarcoma virus (RSV) Gag PM localization. Gag mutants deficient in nuclear trafficking were less sensitive to reduction of intracellular PI(4,5)P2 and PI(3,4,5)P3, suggesting a possible connection between Gag nuclear trafficking and phosphoinositide-dependent PM targeting.

Nadaraia-Hoke, Shorena; Bann, Darrin V.; Lochmann, Timothy L.; Gudleski-O'Regan, Nicole



Roles of mitogen-activated protein kinase and phosphoinositide 3?-kinase in ErbB2\\/ErbB3 coreceptor-mediated heregulin signaling  

Microsoft Academic Search

ErbB2\\/HER2 and ErbB3\\/HER3, two members of the ErbB\\/HER family, together constitute a heregulin coreceptor complex that elicits a potent mitogenic and transforming signal. Among known intracellular effectors of the ErbB2\\/ErbB3 heregulin coreceptor are mitogen-activated protein kinase (MAPK) and phosphoinositide (PI) 3-kinase. Activation of the distinct MAPK and PI 3-kinase signaling pathways by the ErbB2\\/ErbB3 coreceptor in response to heregulin and

Ulka Vijapurkar; Myong-Soo Kim; John G Koland



Light-induced phosphoinositide degradation and light-induced structural alterations in the rat retina are enhanced after chronic lithium treatment  

SciTech Connect

In the rat retina, light-induced break down of polyphosphoinositides and structural alterations of photoreceptor outer segment disk-membranes were enhanced by chronic lithium treatment at doses equivalent to therapeutic levels in humans. Furthermore, the recovery of phosphoinositide baseline levels after light exposure was delayed in lithium treated retinas. Light and lithium may thus affect phospholipid membranes in the retina. Initial processes in the pathogenesis of light damage in the retina are largely unknown to date. The hydrolysis of polyphosphoinositides may possibly constitute such an initial event.

Pfeilschifter, J.; Reme, C.; Dietrich, C.



Mass-analysed ion kinetic energy spectra and B1E-B2 triple sector mass spectrometric analysis of phosphoinositides by fast atom bombardment.  


Fast atom bombardment is shown to produce useful spectra of the three phosphoinositides and the metabolically related phospholipids, lysophosphatidylinositol and phosphatidic acid. Analysis of the [M-H]- ions for fatty ester composition by mass-analysed ion kinetic energy spectra (MIKES) is shown to be inadequate to resolve fatty acyl daughter ions when the parent ion contains isobaric species. However, analysis on a triple sector instrument with and without collisional activation does provide complete compositional information. Quantitative analysis of the fatty ester content of each lipid molecular species is complicated by dissimilar ion yields from fatty acyl-bearing fragments from compositionally different parent ions. PMID:2994769

Sherman, W R; Ackermann, K E; Bateman, R H; Green, B N; Lewis, I




PubMed Central

SUMMARY Polyphosphoinositides are lipid signaling molecules generated from phosphatidylinositol (PtdIns) with critical roles in vesicular trafficking and signaling. It is poorly understood where PtdIns is located within cells and how it moves around between membranes. Here we identify a hitherto unrecognized highly mobile membrane compartment as the site of PtdIns synthesis and a likely source of PtdIns of all membranes. We show that the PtdIns synthesizing enzyme, PIS associates with a rapidly moving compartment of ER origin that makes ample contacts with other membranes. In contrast, CDP-diacylglycerol synthases that provide PIS with its substrate reside in the tubular ER. Expression of a PtdIns-specific bacterial PLC generates diacylglycerol also in rapidly moving cytoplasmic objects. We propose a model in which PtdIns is synthesized in a highly mobile lipid distribution platform and is delivered to other membranes during multiple contacts by yet to be defined lipid transfer mechanisms.

Kim, Yeun Ju; Guzman-Hernandez, Maria Luisa; Balla, Tamas



Light-dependent modulation of Shab channels via phosphoinositide depletion in Drosophila photoreceptors.  


The Drosophila phototransduction cascade transforms light into depolarizations that are further shaped by activation of voltage-dependent K+ (Kv) channels. In whole-cell recordings of isolated photoreceptors, we show that light selectively modulated the delayed rectifier (Shab) current. Shab currents were increased by light with similar kinetics to the light-induced current itself (latency approximately 20 ms), recovering to control values with a t(1/2) of approximately 60 s in darkness. Genetic disruption of PLCbeta4, responsible for light-induced PIP(2) hydrolysis, abolished this light-dependent modulation. In mutants of CDP-diaclyglycerol synthase (cds(1)), required for PIP(2) resynthesis, the modulation became irreversible, but exogenously applied PIP(2) restored reversibility. The modulation was accurately and reversibly mimicked by application of PIP(2) to heterologously expressed Shab channels in excised inside-out patches. The results indicate a functionally implemented mechanism of Kv channel modulation by PIP(2) in photoreceptors, which enables light-dependent regulation of signal processing by direct coupling to the phototransduction cascade. PMID:18760696

Krause, Yani; Krause, Stephan; Huang, Jiehong; Liu, Che-Hsiung; Hardie, Roger C; Weckström, Matti



SNX-BAR proteins in phosphoinositide-mediated, tubular-based endosomal sorting  

PubMed Central

The endocytic network is morphologically characterized by a wide variety of membrane bound compartments that are able to undergo dynamic re-modeling through tubular and vesicular structures. The precise molecular mechanisms governing such re-modeling, and the events that co-ordinated this with the major role of the endocytic network, cargo sorting, remain unclear. That said, recent work on a protein family of sorting nexins (SNX) – especially a subfamily of SNX that contain a BAR domain (SNX–BARs) – has begun to shed some much needed light on these issues and in particular the process of tubular–based endosomal sorting. SNX–BARs are evolutionary conserved in endosomal protein complexes such as retromer, where they co–ordinate membrane deformation with cargo selection. Furthermore a central theme emerges of SNX–BARs linking the forming membrane carrier to cytoskeletal elements for transport through motor proteins such as dynein. By studying these SNX–BARs, we are gaining an increasingly detailed appreciation of the mechanistic basis of endosomal sorting and how this highly dynamic process functions in health and disease.

van Weering, Jan R.T.; Verkade, Paul; Cullen, Peter J.



PTEN Regulation, a Novel Function for the p85 Subunit of Phosphoinositide 3-Kinase  

NSDL National Science Digital Library

Timely regulation of phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] abundance in cells is essential for the control of cellular homeostasis. The concentrations of these lipids are low in quiescent cells but rapidly and transiently increase following growth factor receptor (GFR) stimulation, which triggers cellular metabolic changes, proliferation, survival, and motility. Class IA phosphatidylinositol 3-kinase (PI3K), which is composed of a p85 (regulatory) and p110 (catalytic) subunits, is the enzyme generating PI(3,4)P2 and PI(3,4,5)P3 following GFR stimulation. Although the steps in GFR-induced activation of PI3K , are relatively well known, the mechanisms for subsequent 3-polyphospho-PI down-regulation are less understood. Examination of frequent genetic alterations in human cancer showed that PTEN (phosphatase with tensin homology on chromosome 10) is the major enzyme that decreases PI(3,4)P2 and PI(3,4,5)P3 cell content. Nonetheless, interpretation of the complexity of PTEN regulation remains a matter of debate. The recent description of diminished PTEN activity in liver-conditional knockout mice lacking the p85α PI3K regulatory subunit reveals a previously unknown p85α-dependent negative-feedback pathway that controls PI(3,4)P2 and PI(3,4,5)P3 half-life by regulating PTEN.

Domingo F. Barber (Universidad Autonoma de Madrid;Centro Nacional de Biotecnologia/Consejo Superiod de Investigaciones Cientificas REV); Maria Alvarado-Kristensson (Universidad Autonoma de Madrid;Centro Nacional de Biotecnologia/Consejo Superiod de Investigaciones Cientificas REV); Ana Gonzalez-Garcia (Universidad Autonoma de Madrid;Centro Nacional de Biotecnologia/Consejo Superiod de Investigaciones Cientificas REV); Rafael Pulido (Centro de Investigacion Principe Felipe; REV); Ana C. Carrera (Universidad Autonoma de Madrid;Centro Nacional de Biotecnologia/Consejo Superiod de Investigaciones Cientificas REV)



A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis.  


Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches. PMID:24403601

Ren, Jihui; Pei-Chen Lin, Coney; Pathak, Manish C; Temple, Brenda R S; Nile, Aaron H; Mousley, Carl J; Duncan, Mara C; Eckert, Debra M; Leiker, Thomas J; Ivanova, Pavlina T; Myers, David S; Murphy, Robert C; Brown, H Alex; Verdaasdonk, Jolien; Bloom, Kerry S; Ortlund, Eric A; Neiman, Aaron M; Bankaitis, Vytas A



A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis  

PubMed Central

Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches.

Ren, Jihui; Pei-Chen Lin, Coney; Pathak, Manish C.; Temple, Brenda R. S.; Nile, Aaron H.; Mousley, Carl J.; Duncan, Mara C.; Eckert, Debra M.; Leiker, Thomas J.; Ivanova, Pavlina T.; Myers, David S.; Murphy, Robert C.; Brown, H. Alex; Verdaasdonk, Jolien; Bloom, Kerry S.; Ortlund, Eric A.; Neiman, Aaron M.; Bankaitis, Vytas A.



Hepatocyte growth factor activates phosphoinositide 3-kinase C2 beta in renal brush-border plasma membranes.  

PubMed Central

Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH(3) and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2 beta activity, which is sensitive to wortmannin (10 nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices with HGF and could be mimicked by the Ca(2+) ionophore A23187 and blocked by the cell-penetrant Ca(2+) chelator BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. On Western blots PI3K-C2 beta revealed a single immunoreactive band of 180 kDa in BLM and BBM, while after stimulation with HGF a gel shift of 18 kDa was noticed only in BBM, suggesting that the observed enzyme activation is achieved by proteolysis. When BBM were subjected to short-term (15 min) exposure to mu-calpain, a similar gel shift together with an increase in PI3K-C2 beta activity was observed, when compared with the BBM harvested after HGF stimulation. The above-mentioned gel shift and increase in PI3K-C2 beta activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that in renal cells there is a spatial separation of the inositol lipid signalling system between BLM and BBM, and that HGF causes activation of PLC and PI3K primarily in BLM, which leads to calpain-mediated activation of PI3K-C2 beta in BBM with a concomitant increase in PtdIns3P.

Crljen, Vladiana; Volinia, Stefano; Banfic, Hrvoje



Inhibition of the phosphoinositide 3-kinase pathway decreases innate resistance to lipopolysaccharide toxicity in TLR4 deficient mice  

PubMed Central

Background Upon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model. Results The overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 ?g LPS from Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-?g LPS injection in Tlr4-/- mice. When the PI3K inhibitor LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4-/- mice was 30%. In the Tlr4-/- mice, the LPS injection induced no NF-?B activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 ?g LPS led to a significant induction in O2- detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4-/- mice. Addition of LY294002 only significantly increased the O2- level in the lung and liver of the Tlr4-/- mice but not in the C57BL/6 mice following 500-?g LPS injection. In addition, the serum IL-1? and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4-/- mice. Notably, IL-1? and IL-2 were significantly increased in Tlr4-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by LY294002 prior to LPS injection. Conclusions In this study, we demonstrate that innate resistance to LPS toxicity in Tlr4-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O2- and inflammatory cytokines.



Proinsulin C-peptide rapidly stimulates mitogen-activated protein kinases in Swiss 3T3 fibroblasts: requirement of protein kinase C, phosphoinositide 3-kinase and pertussis toxin-sensitive G-protein.  

PubMed Central

It has been demonstrated that proinsulin C-peptide possesses several biological activities and that its specific binding sites are present on the surface of cell membranes. However, the molecular and cellular mechanisms of C-peptide actions are poorly known. In the present study we examined the possible involvement of the mitogen-activated protein kinase (MAPK) pathway in C-peptide effects. C-peptide induced the phosphorylation of MAPK [p44 extracellular signal-regulated kinase 1 (ERK1) and p42 ERK2] in Swiss 3T3 and 3T3-F442A fibroblasts but not in 3T3-L1 fibroblasts and some other cell lines such as L(6)E(9) muscle cells. In Swiss 3T3 cells, C-peptide-induced phosphorylation of MAPK was dependent on time and concentration, being maximal at 1 min and at 1 nM C-peptide and was accompanied by an increase in MAPK activity and MAPK kinase (MEK) phosphorylation. The MAPK phosphorylation by C-peptide was abolished by treatment with pertussis toxin (PTX) and also with a MEK inhibitor, PD 98059. In addition, MAPK phosphorylation was attenuated by treatment with a phosphoinositide 3-kinase (PI-3K) inhibitor, wortmannin, and with a protein kinase C (PKC) inhibitor, GF109203X, and by down-regulation of PKC by prolonged treatment with PMA. Similar effects of the inhibitors and PTX were found on the MAPK phosphorylation induced by neuropeptide Y. These results suggest that C-peptide activates MAPK through a putative G(i)/G(o)-linked receptor for C-peptide and through PI-3K-dependent and PKC-dependent pathways.

Kitamura, T; Kimura, K; Jung, B D; Makondo, K; Okamoto, S; Canas, X; Sakane, N; Yoshida, T; Saito, M



EP1 Prostanoid Receptor Coupling to Gi/o Up-Regulates the Expression of Hypoxia-Inducible Factor-1? through Activation of a Phosphoinositide-3 Kinase Signaling Pathway  

PubMed Central

The EP1 prostanoid receptor is one of four subtypes whose cognate physiological ligand is prostaglandin-E2 (PGE2). It is in the family of G-protein-coupled receptors and is known to activate Ca2+ signaling, although relatively little is known about other aspects of E-type prostanoid receptor (EP) 1 receptor signaling. In human embryonic kidney (HEK) cells expressing human EP1 receptors, we now show that PGE2 stimulation of the EP1 receptor up-regulates the expression of hypoxia-inducible factor-1? (HIF-1?), which can be completely blocked by pertussis toxin, indicating coupling to Gi/o. This up-regulation of HIF-1? occurs under normoxic conditions and could be inhibited with wortmannin, Akt inhibitor, and rapamycin, consistent with the activation of a phosphoinositide-3 kinase/Akt/mammalian target of rapamycin (mTOR) signaling pathway, respectively. In contrast to the hypoxia-induced up-regulation of HIF-1?, which involves decreased protein degradation, the up-regulation of HIF-1? by the EP1 receptor was associated with the phosphorylation of ribosomal protein S6 (rpS6), suggesting activation of the ribosomal S6 kinases and increased translation. Stimulation of endogenous EP1 receptors in human HepG2 hepatocellular carcinoma cells recapitulated the normoxic up-regulation of HIF-1? observed in HEK cells, was sensitive to pertussis toxin, and involved the activation of mTOR signaling and phosphorylation of rpS6. In addition, treatment of HepG2 cells with sulprostone, an EP1-selective agonist, up-regulated the mRNA expression of vascular endothelial growth factor-C, a HIF-regulated gene. HIF-1? is known to promote tumor growth and metastasis and is often up-regulated in cancer. Our findings provide a potential mechanism by which increased PGE2 biosynthesis could up-regulate the expression of HIF-1? and promote tumorigenesis.

Ji, Ruyue; Chou, Chih-Ling; Xu, Wei; Chen, Xiao-Bo; Woodward, David F.



Protein Phosphatase 2A (PP2A)-specific Ubiquitin Ligase MID1 Is a Sequence-dependent Regulator of Translation Efficiency Controlling 3-Phosphoinositide-dependent Protein Kinase-1 (PDPK-1)*  

PubMed Central

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells.

Aranda-Orgilles, Beatriz; Rutschow, Desiree; Zeller, Raphael; Karagiannidis, Antonios I.; Kohler, Andrea; Chen, Changwei; Wilson, Timothy; Krause, Sven; Roepcke, Stefan; Lilley, David; Schneider, Rainer; Schweiger, Susann



Lower Phosphoinositide 3Kinase (PI 3-kinase) Activity and Differential Expression Levels of Selective Catalytic and Regulatory PI 3Kinase Subunit Isoforms in Prefrontal Cortex and Hippocampus of Suicide Subjects  

Microsoft Academic Search

Phosphoinositide 3 (PI 3)-kinase is one of the key signaling enzymes that participates in a myriad of physiological functions in brain and is utilized by neurotrophins to mediate neuronal plasticity, cell survival, and inhibition of apoptosis for several neuronal subtypes. Our recent demonstration that expression of neurotrophic factors and activation of the receptor tyrosine kinase B are significantly altered in

Yogesh Dwivedi; Hooriyah S Rizavi; Tara Teppen; Hui Zhang; Amal Mondal; Rosalinda C Roberts; Robert R Conley; Ghanshyam N Pandey



Contribution of phosphoinositide-dependent signalling to photomotility of Blepharisma ciliate 1 Paper presented at the 2nd Internet Photochemistry and Photobiology Conference, 16 July–7 September, 1999; http:\\/\\/\\/photobiology99\\/index.htm 1  

Microsoft Academic Search

The effect of experimental procedures designed to modify an intracellular phosphoinositide signalling pathway, which may be instrumental in the photophobic response of the protozoan ciliate Blepharisma japonicum, has been investigated. To assess this issue, the latency time of the photophobic response and the cell photoresponsiveness have been assayed employing newly developed computerized videorecording and standard macro-photographic [i1]methods. Cell incubation with

Hanna Fabczak



Evidence for attenuation of myo-inositol uptake, phosphoinositide turnover and inositol phosphate production in aortic vasculature of rats during pregnancy.  

PubMed Central

We postulated that vascular phosphoinositide metabolism is attenuated during pregnancy, and thereby could contribute to maternal vasodilation and reduced vascular reactivity. The basal rate of incorporation of [3H]myo-inositol and [3H]glycerol into phosphoinositides of aortae from pregnant rats in vitro was significantly reduced, when compared with vessels from virgin animals. After injection of [3H]myo-inositol intravenously into chronically instrumented conscious pregnant and virgin rats, the incorporation of the label by phosphatidylinositol was 66 +/- 4% less in aortae of gravid versus virgin animals (P less than 0.001), despite comparable plasma concentrations of radioactivity. Fold stimulation of total [3H]inositol phosphates by arginine vasopressin, norepinephrine, and angiotensin II over a 15-min period was not different between aortic segments from virgin and gravid rats, although both absolute basal and stimulated levels were significantly less in vessels from pregnant animals. After 45 s of incubation with 10(-7) M arginine vasopressin, however, the fold-stimulation of [3H]inositol trisplus tetrakisphosphate was reduced in aortae from gravid rats, when compared with vessels from virgin animals (P less than 0.005). By HPLC, greater than 90% of the radioactivity in the [3H]inositol trisplus tetrakisphosphate column fraction after 30 and 60 s of agonist stimulation was [3H]inositol-1,4,5-trisphosphate. We further observed that the rate of uptake of [3H]myo-inositol by aortic vasculature obtained from gravid rats was significantly (24%) less than uptake by vessels from virgin animals. Plasma myo-inositol concentrations were not significantly different, but presumably as a consequence of reduced uptake, aortic segments freshly isolated from pregnant rats contained 22 +/- 6% less myo-inositol than vessels from virgin controls as measured by gas chromatography-mass spectrometry (P less than 0.03). We conclude that myo-inositol uptake and content, phosphoinositide turnover, and inositol phosphate production are reduced in aortic vasculature of gravid rats.

Conrad, K P; Barrera, S A; Friedman, P A; Schmidt, V M



Effects of guanosine 5'-[gamma-thio]triphosphate and thrombin on the phosphoinositide metabolism of electropermeabilized human platelets.  


Incubation of human platelets with myo-[3H]inositol in a low-glucose Tyrode's solution containing MnCl2 enhanced the labelling of phosphoinositides about sevenfold and greatly facilitated the measurement of [3H]inositol phosphates formed by the activation of phospholipase C. Labelled platelets were permeabilized by high-voltage electric discharges and equilibrated at 0 degree C with ATP, Ca2+ buffers and guanine nucleotides, before incubation in the absence or presence of thrombin. Incubation of these platelets with ATP in the presence or absence of Ca2+ ions led to the conversion of [3H]phosphatidylinositol to [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PtdInsP2). At a pCa of 6, addition of 100 microM GTP[gamma S] both prevented this accumulation of [3H]PtdInsP2 and stimulated its breakdown; the formation of [3H]inositol phosphates was increased ninefold. After 5 min these comprised 70% [3H]inositol monophosphate ([3H]InsP), 28% [3H]inositol bisphosphate ([3H]InsP2) and 2% [3H]inositol trisphosphate ([3H]InsP3). In shorter incubations higher percentages of [3H]InsP2 and [3H]InsP3 were found. In the absence of added Ca2+, the formation of [3H]inositol phosphates was decreased by over 90%. Incubation of permeabilized platelets with GTP[gamma S] in the presence of 10 mM Li+ decreased the accumulation of [3H]InsP and increased that of [3H]InsP2, without affecting [3H]InsP3 levels. Addition of unlabelled InsP3 decreased the intracellular hydrolysis of exogenous [32P]InsP3 but did not trap additional [3H]InsP3. These results and the time course of [3H]inositol phosphate formation suggest that GTP[gamma S] stimulated the action of phospholipase C on a pool of [3H]phosphatidylinositol 4-phosphate that was otherwise converted to [3H]PtdInsP2 and that much less hydrolysis of [3H]phosphatidylinositol to [3H]InsP or of [3H]PtdInsP2 to [3H]InsP3 occurred. At a pCa of 6, addition of thrombin (2 units/ml) to permeabilized platelets caused small increases in the formation of [3H]InsP and [3H]InsP2. This action of thrombin was enhanced twofold by 10-100 microM GTP and much more potently by 4-40 microM GTP[gamma S]. In the presence of the latter, thrombin also increased [3H]InsP3. The total formation of [3H]inositol phosphates by permeabilized platelets incubated with thrombin and GTP[gamma S] was comparable with that observed on addition of thrombin alone to intact platelets. However, HPLC of the [3H]inositol phosphates formed indicated that about 75% of the [3H]InsP accumulating in permeabilized platelets was the 4-phosphate, whereas in intact platelets stimulated by thrombin, up to 80% was the 1-phosphate.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2831052

Culty, M; Davidson, M M; Haslam, R J



Human adenosine A(3) receptor leads to intracellular Ca(2+) mobilization but is insufficient to activate the signaling pathway via phosphoinositide 3-kinase gamma in mice.  


Selective antagonists for the adenosine A(3) receptor (A3AR), a member of the G protein-coupled receptors, have been indicated as potential drugs for anti-asthma or anti-inflammation. However, potent antagonists for the rodent A3AR have not been identified. To evaluate the pharmacological effects of human A3AR antagonists in mice, we here generated A3AR-humanized mice, in which the mouse A3AR gene was replaced by its human counterpart. The expression levels of human A3AR in the A3AR-humanized mice were equivalent to those of mouse A3AR in wild-type mice. Elevation of the intracellular Ca(2+) concentration induced by an A3AR agonist was observed in bone marrow-derived mast cells from the A3AR-humanized mice and this Ca(2+) mobilization was completely antagonized by a human A3AR antagonist. However, antigen-dependent degranulation was not potentiated by the A3AR agonist in the mast cells from A3AR-humanized mice. The agonist-stimulated human A3AR did not lead to the phosphorylation of either extracellular signal-regulated kinase 1/2 or protein kinase B in A3AR-humanized mice. The rate of human A3AR internalization in the mast cells was also markedly decreased compared with that of mouse A3AR in the mast cells. These results demonstrate that the human A3AR is insufficient to activate phosphoinositide 3-kinase gamma-dependent signaling pathways in mice, probably due to the uncoupling of member(s) of the G proteins, which are capable of activating phosphoinositide 3-kinase gamma, to the human A3AR, despite the mouse G protein(s) responsible for the Ca(2+) elevation are coupled with the human A3AR. PMID:16157310

Yamano, Kazuya; Inoue, Miho; Masaki, Shigehiro; Saki, Mayumi; Ichimura, Michio; Satoh, Mitsuo



p70 S6 kinase is regulated by protein kinase Czeta and participates in a phosphoinositide 3-kinase-regulated signalling complex.  


p70 S6 kinase (p70S6K) is an important regulator of cell proliferation. Its activation by growth factor requires phosphorylation by various inputs on multiple sites. Data accumulated thus far support a model whereby p70S6K activation requires sequential phosphorylations at proline-directed residues in the putative autoinhibitory pseudosubstrate domain, as well as threonine 389. Threonine 229, a site in the catalytic loop is phosphorylated by phosphoinositide-dependent kinase 1 (PDK-1). Experimental evidence suggests that p70S6K activation requires a phosphoinositide 3-kinase (PI3-K)-dependent signal(s). However, the intermediates between PI3-K and p70S6K remain unclear. Here, we have identified PI3-K-regulated atypical protein kinase C (PKC) isoform PKCzeta as an upstream regulator of p70S6K. In coexpression experiments, we found that a kinase-inactive PKCzeta mutant antagonized activation of p70S6K by epidermal growth factor, PDK-1, and activated Cdc42 and PI3-K. While overexpression of a constitutively active PKCzeta mutant (myristoylated PKCzeta [myr-PKCzeta]) only modestly activated p70S6K, this mutant cooperated with PDK-1 activation of p70S6K. PDK-1-induced activation of a C-terminal truncation mutant of p70S6K was also enhanced by myr-PKCzeta. Moreover, we have found that p70S6K can associate with both PDK-1 and PKCzeta in vivo in a growth factor-independent manner, while PDK-1 and PKCzeta can also associate with each other, suggesting the existence of a multimeric PI3-K signalling complex. This work provides evidence for a link between a phorbol ester-insensitive PKC isoform and p70S6K. The existence of a PI3-K-dependent signalling complex may enable efficient activation of p70S6K in cells. PMID:10082559

Romanelli, A; Martin, K A; Toker, A; Blenis, J



Mutations of the lutropin/choriogonadotropin receptor that do not activate the phosphoinositide cascade allow hCG to induce aromatase expression in immature rat granulosa cells  

PubMed Central

Using primary cultures of immature rat granulosa cells and adenoviral infections we expressed two mutants of the human lutropin receptor (hLHR) that do not activate the phosphoinositide cascade. One mutant (hLFF) has the extracellular domain of the hLHR and the transmembrane and intracellular domains of the hFSHR. The other (hLHR-L457D) has a leucine to aspartate mutation in residue 457 of transmembrane helix 3. When expressed in immature rat granulosa cells the hLHR stimulates cAMP and inositol phosphate accumulation, transactivates the epidermal growth factor receptor (EGFR), elicits a transient increase in Akt phosphorylation, and a sustained increase in ERK1/2 phosphorylation but aromatase expression is not enhanced. When expressed at comparable densities, hLFF and hLHR-L457D support cAMP accumulation and transient Akt phosphorylation but do not support inositol phosphate accumulation, EGFR transactivation or a sustained phosphorylation of ERK1/2. Cells expressing either of these two mutants respond to hCG with increased aromatase expression. We also show that addition of hCG to cells expressing the hLHR antagonizes the effects of hFSH on aromatase expression whereas addition of hCG to cells expressing the hLHR-L457D mutant does not. These results show that activation of the phosphoinositide cascade is upstream of EGFR transactivation and ERK1/2 phosphorylation and that this pathway is a negative regulator of aromatase expression in granulosa cells.

Andric, Nebojsa; Ascoli, Mario



Autophosphorylation of serine 608 in the p85 regulatory subunit of wild type or cancer-associated mutants of phosphoinositide 3-kinase does not affect its lipid kinase activity  

PubMed Central

Background The ?-isoform of the Type 1A Phosphoinositide 3-kinases (PI3K?) has protein kinase activity as well as phosphoinositide lipid kinase activity. The best described substrate for its protein kinase activity is its regulatory subunit, p85?, which becomes phosphorylated on Serine 608. Phosphorylation of Serine 608 has been reported to down-regulate its lipid kinase activity. Results We have assessed whether oncogenic mutants of PI3K?, which have up-regulated lipid kinase activity, have altered levels of Serine 608 phosphorylation compared to wild type PI3K?, and whether differential phosphorylation of Serine 608 contributes to increased activity of oncogenic forms of PI3K? with point mutations in the helical or the kinase domains. Despite markedly increased lipid kinase activity, protein kinase activity was not altered in oncogenic compared to wild type forms of PI3K?. By manipulating levels of phosphorylation of Serine 608 in vitro, we found no evidence that the protein kinase activity of PI3K? affects its phosphoinositide lipid kinase activity in either wild-type or oncogenic mutants of PI3K?. Conclusions Phosphorylation of p85? S608 is not a significant regulator of wild-type or oncogenic PI3K? lipid kinase activity.



Temporal differences in the dependency on phosphoinositide-dependent kinase 1 distinguish the development of invariant Valpha14 NKT cells and conventional T cells.  


This study uses two independent genetic strategies to explore the requirement for phosphoinositide-dependent kinase-1 (PDK1) in the development of mature T cell populations from CD4/CD8 double-positive thymocytes. The data show that CD4/CD8 double-positive thymocytes that do not express PDK1 or express a catalytically inactive PDK1 mutant fail to produce mature invariant V?14 NKT cells but can differentiate to conventional CD4, CD8, or regulatory T cell subsets in the thymus. The PDK1 requirement for V?14 NKT cell development reflects that these cells require the PDK1 substrate protein kinase B to meet the metabolic demands for proliferative expansion in response to IL-15 or AgR stimulation. There is also constitutive PDK1 signaling in conventional ?/? T cells that is not required for lineage commitment of these cells but fine-tunes the expression of coreceptors and adhesion molecules. Also, although PDK1 is dispensable for thymic development of conventional ?/? T cells, peripheral cells are reduced substantially. This reflects a PDK1 requirement for lymphopenia-induced proliferation, a process necessary for initial population of the peripheral T cell niche in neonatal mice. PDK1 is thus indispensable for T cell developmental programs, but the timing of the PDK1 requirement is unique to different T cell subpopulations. PMID:20944007

Finlay, David K; Kelly, April P; Clarke, Rosemary; Sinclair, Linda V; Deak, Maria; Alessi, Dario R; Cantrell, Doreen A



Endothelium-independent hypoxic contraction of porcine coronary arteries may be mediated by activation of phosphoinositide 3-kinase/Akt pathway.  


Phosphoinositide 3-kinase (PI3K)/Akt signaling pathway plays an essential role in the regulation of vascular tone. The present study aimed to determine its role in hypoxic coronary vasoconstriction. Isometric tension of isolated porcine coronary arteries was measured with organ chamber technique; the protein levels of phosphorylated and total MLC were examined by Western blotting; the activities of PI3K and Rho kinase were determined by the phosphorylation of their respective target protein Akt and MTPT1. Acute hypoxia induced a rapid contraction followed by a short-term relaxation and then a sustained contraction in porcine coronary arteries. The rapid but not the sustained contraction was abolished by endothelium removal. The sustained contraction was attenuated by inhibitors of PI3K (LY294002) and Akt (Akt-I). The attenuation effect caused by LY294002 was not affected by nifedipine, but was abolished by Y27632, an inhibitor of Rho kinase. The sustained hypoxic contraction was associated with altered phosphorylation of MLC and Akt, which was inhibited by LY294002. The sustained hypoxic contraction was also accompanied with increased phosphorylation of MYPT1, which was inhibited by LY294002 and Y27632. This study demonstrates that sustained hypoxia causes porcine coronary artery to contract in an endothelium-independent manner. An increased PI3K/Akt/Rho kinase signaling may be involved. PMID:24685819

Liu, Huixia; Chen, Zhengju; Liu, Juan; Liu, Limei; Gao, Yuansheng; Dou, Dou



In vivo cooperation between Bcl-xL and the phosphoinositide 3-kinase-Akt signaling pathway for the protection of epidermal keratinocytes from apoptosis.  


To investigate the function of Bcl-xL in the skin, we established keratinocyte-specific Bcl-x gene-targeted mice under the keratin 5 promoter (K5). K5.Bcl-xL-/- mice were viable, devoid of alteration in the development of skin or appendages. However, they harbored spontaneous apoptotic keratinocytes in the epidermis. Bcl-xL-deficient keratinocytes cultured in vitro readily underwent apoptosis in the absence of growth factors, but the addition of HGF or EGF resulted in restoration of cell survival, which was reversed by treatment with wortmannin, an inhibitor of phosphoinositide-3 kinase (PI3K). Topical treatment of K5.Bcl-xL-/- mice with wortmannin sensitized the skin for apoptosis induced by UV (UV) B, although wild-type epidermis was only marginally affected by this treatment, suggesting that the resistance to UVB largely depended on PI3K-Akt signaling in Bcl-xL-deficient mice but not in wild-type mice. Furthermore, UVB irradiation resulted in redistribution of phosphorylated Akt from the basal layer to the suprabasal layer, indicating that Akt could spatially cooperate with Bcl-xL upon UVB exposure in the upper epidermis where Bcl-xL is normally localized. These results suggest that Bcl-xL and the PI3K-Akt pathway form a cooperative, intercompensatory axis for the protection of epidermal keratinocytes from apoptosis in vivo. PMID:12665473

Umeda, Jiro; Sano, Shigetoshi; Kogawa, Kazuhiko; Motoyama, Noboru; Yoshikawa, Kunihiko; Itami, Satoshi; Kondoh, Gen; Watanabe, Takeshi; Takeda, Junji



Loss of Cardiac Phosphoinositide 3-kinase p110? Results in Contractile Dysfunction: Lu et al PI3K p110? Regulates Myocyte Contractility  

PubMed Central

Background Phosphoinositide 3-kinase (PI3K) p110? plays a key role in insulin action and tumorigenesis. Myocyte contraction is initiated by an inward Ca2+ current (ICa,L) through the voltage-dependent L-type Ca2+ channel (LTCC). The aim of this study was to evaluate if p110? also controls cardiac contractility by regulating the LTCC. Methods and Results Genetic ablation of p110? (also known as Pik3ca), but not p110? (also known as Pik3cb), in cardiac myocytes of adult mice reduced ICa,L and blocked insulin signaling in the heart. p110?-null myocytes had a reduced number of LTCCs on the cell surface and a contractile defect that decreased cardiac function in vivo. Similarly, pharmacological inhibition of p110? decreased ICa,L and contractility in canine myocytes. Inhibition of p110? did not reduce ICa,L. Conclusions PI3K p110? but not p110? regulates the LTCC in cardiac myocytes. Decreased signaling to p110? reduces the number of LTCCs on the cell surface and thus attenuates ICa,L and contractility.

Lu, Zhongju; Jiang, Ya-Ping; Wang, Wei; Xu, Xin-Hua; Mathias, Richard T.; Entcheva, Emilia; Ballou, Lisa M.; Cohen, Ira S.; Lin, Richard Z.



NMR Analyses of the Interaction between the FYVE Domain of Early Endosome Antigen 1 (EEA1) and Phosphoinositide Embedded in a Lipid Bilayer*  

PubMed Central

Phosphoinositides (PIs) are crucial lipid components of membranes and are involved in a number of cellular processes through interactions with their effector proteins. Recently, we have established a lipid-protein nanoscale bilayer (nanodisc) containing PIs, hereafter referred to as PI-nanodisc and demonstrated that it could be used for both qualitative and quantitative evaluations of protein-membrane interactions. Here, we report further NMR analyses for obtaining structural insights at the residue-specific level between PI-binding effector protein and PI-nanodisc, using the FYVE domain of early endosome antigen 1 (EEA1), denoted as EEA1 FYVE, and PI(3)P-nanodisc as a model system. We performed a combination of the NMR analyses including chemical shift perturbation, transferred cross-saturation, and paramagnetic relaxation enhancement experiments. These enabled an identification of the interaction surface, structural change, and relative orientation of EEA1 FYVE to the PI(3)P-incorporated lipid bilayer, substantiating that NMR analyses of protein-membrane interactions using nanodisc makes it possible to show the residue-specific interactions in the lipid bilayer environment.

Yokogawa, Mariko; Kobashigawa, Yoshihiro; Yoshida, Naoki; Ogura, Kenji; Harada, Kohsuke; Inagaki, Fuyuhiko



Phosphatidic acid and phosphoinositides facilitate liposome association of Yas3p and potentiate derepression of ARE1 (alkane-responsive element one)-mediated transcription control.  


In the n-alkane assimilating yeast Yarrowia lipolytica, the expression of ALK1, encoding a cytochrome P450 that catalyzes terminal mono-oxygenation of n-alkanes, is induced by n-alkanes. The transcription of ALK1 is regulated by a heterocomplex that comprises the basic helix-loop-helix transcription activators, Yas1p and Yas2p, and binds to alkane-responsive element 1 (ARE1) in the ALK1 promoter. An Opi1 family transcription repressor, Yas3p, represses transcription by binding to Yas2p. Yas3p localizes in the nucleus when Y. lipolytica is grown on glucose but localizes to the endoplasmic reticulum (ER) upon the addition of n-alkanes. In this study, we showed that recombinant Yas3p binds to the acidic phospholipids, phosphatidic acid (PA) and phosphoinositides (PIPs), in vitro. The ARE1-mediated transcription was enhanced in vivo in mutants defective in an ortholog of the Saccharomyces cerevisiae gene PAH1, encoding PA phosphatase, and in an ortholog of SAC1, encoding PIP phosphatase in the ER. Truncation mutation analyses for Yas3p revealed two regions that bound to PA and PIPs. These results suggest that the interaction with acidic phospholipids is important for the n-alkane-induced association of Yas3p with the ER membrane. PMID:24120453

Kobayashi, Satoshi; Hirakawa, Kiyoshi; Horiuchi, Hiroyuki; Fukuda, Ryouichi; Ohta, Akinori



Ca2+ influx and phosphoinositide signalling are essential for the establishment and maintenance of cell polarity in monospores from the red alga Porphyra yezoensis  

PubMed Central

The asymmetrical distribution of F-actin directed by cell polarity has been observed during the migration of monospores from the red alga Porphyra yezoensis. The significance of Ca2+ influx and phosphoinositide signalling during the formation of cell polarity in migrating monospores was analysed pharmacologically. The results indicate that the inhibition of the establishment of cell polarity, as judged by the ability of F-actin to localize asymmetrically, cell wall synthesis, and development into germlings, occurred when monospores were treated with inhibitors of the Ca2+ permeable channel, phospholipase C (PLC), diacylglycerol kinase, and inositol-1,4,5-trisphosphate receptor. Moreover, it was also found that light triggered the establishment of cell polarity via photosynthetic activity but not its direction, indicating that the Ca2+ influx and PLC activation required for the establishment of cell polarity are light dependent. By contrast, inhibition of phospholipase D (PLD) prevented the migration of monospores but not the asymmetrical localization of F-actin. Taken together, these findings suggest that there is functional diversity between the PLC and PLD signalling systems in terms of the formation of cell polarity; the former being critical for the light-dependent establishment of cell polarity and the latter playing a role in the maintenance of established cell polarity.

Saga, Naotsune; Mikami, Koji



Activity of the pan-class I phosphoinositide 3-kinase inhibitor NVP-BKM120 in T-cell acute lymphoblastic leukemia.  


Constitutively active phosphoinositide 3-kinase (PI3K) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival and drug resistance. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the pan-PI3K inhibitor NVP-BKM120 (BKM120), an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in G2/M phase cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T lymphoblasts, and promoting a dose- and time-dependent dephosphorylation of Akt and S6RP. BKM120 maintained its pro-apoptotic activity against Jurkat cells even when cocultured with MS-5 stromal cells, which mimic the bone marrow microenvironment. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. Moreover, in vivo administration of BKM120 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth, thus prolonging survival time. Taken together, our findings indicate that BKM120, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment for T-ALLs that have aberrant upregulation of the PI3K signaling pathway. PMID:24310736

Lonetti, A; Antunes, I L; Chiarini, F; Orsini, E; Buontempo, F; Ricci, F; Tazzari, P L; Pagliaro, P; Melchionda, F; Pession, A; Bertaina, A; Locatelli, F; McCubrey, J A; Barata, J T; Martelli, A M



cDNA sequence and gene locus of the human retinal phosphoinositide-specific phospholipase-C{beta}4 (PLCB4)  

SciTech Connect

Defects in the Drosophila norpA (no receptor potential A) gene encoding a phosphoinositide-specific phospholipase C (PLC) block invertebrate phototransduction and lead to retinal degeneration. The mammalian homolog, PLCB4, is expressed in rat brain, bovine cerebellum, and the bovine retina in several splice variants. To determine a possible role of PLCB4 gene defects in human disease, we isolated several overlapping cDNA clones from a human retina library. The composite cDNA sequence predicts a human PLC{beta}4 polypeptide of 1022 amino acid residues (MW 117,000). This PLC{beta}4 variant lacks a 165-amino-acid N-terminal domain characteristic for the rat brain isoforms, but has a distinct putative exon 1 unique for human and bovine retina isoforms. A PLC{beta}4 monospecific antibody detected a major (130 kDa) and a minor (160 kDa) isoform in retina homogenates. Somatic cell hybrids and deletion panels were used to localize the PCLB4 gene to the short arm of chromosome 20. The gene was further sublocalized to 20p12 by florescence in situ hybridization. 4 refs., 5 figs.

Alvarez, R.A.; Ghalayini, A.J.; Anderson, R.E. [Baylor College of Medicine, Houston, TX (United States)] [and others] [Baylor College of Medicine, Houston, TX (United States); and others



Nuclear Phosphoinositide-Specific Phospholipase C ?1 Controls Cytoplasmic CCL2 mRNA Levels in HIV-1 gp120-Stimulated Primary Human Macrophages  

PubMed Central

HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of CCL2 from macrophages. In turn, this chemokine acts as an autocrine factor enhancing viral replication. In this study, we show for the first time that phosphoinositide-specific phospholipase C (PI-PLC) is required for the production of CCL2 triggered by gp120 in macrophages. Using a combination of confocal laser-scanner microscopy, pharmacologic inhibition, western blotting and fluorescence-activated cell sorter analysis, we demonstrate that gp120 interaction with CCR5 leads to nuclear localization of the PI-PLC ?1 isozyme mediated by mitogen-activated protein kinase ERK-1/2. Notably, phosphatidylcholine-specific phospholipase C (PC-PLC), previously reported to be required for NF-kB-mediated CCL2 production induced by gp120 in macrophages, drives both ERK1/2 activation and PI-PLC ?1 nuclear localization induced by gp120. PI-PLC ?1 activation through CCR5 is also triggered by the natural chemokine ligand CCL4, but independently of ERK1/2. Finally, PI-PLC inhibition neither blocks gp120-mediated NF-kB activation nor overall accumulation of CCL2 mRNA, whereas it decreases CCL2 transcript level in the cytoplasm. These results identify nuclear PI-PLC ?1 as a new intermediate in the gp120-triggered PC-PLC-driven signal transduction pathway leading to CCL2 secretion in macrophages. The finding that a concerted gp120-mediated signaling involving both PC- and PI-specific PLCs is required for the expression of CCL2 in macrophages suggests that this signal transduction pathway may also be relevant for the modulation of viral replication in these cells. Thus, this study may contribute to identify novel targets for therapeutic intervention in HIV-1 infection.

Purificato, Cristina; Sabbatucci, Michela; Podo, Franca; Ramoni, Carlo; Gessani, Sandra; Fantuzzi, Laura



Phosphoinositide 3-Kinases Upregulate System xc(-) via Eukaryotic Initiation Factor 2? and Activating Transcription Factor 4?-?A Pathway Active in Glioblastomas and Epilepsy.  


Abstract Aims: Phosphoinositide 3-kinases (PI3Ks) relay growth factor signaling and mediate cytoprotection and cell growth. The cystine/glutamate antiporter system xc(-) imports cystine while exporting glutamate, thereby promoting glutathione synthesis while increasing extracellular cerebral glutamate. The aim of this study was to analyze the pathway through which growth factor and PI3K signaling induce the cystine/glutamate antiporter system xc(-) and to demonstrate its biological significance for neuroprotection, cell growth, and epilepsy. Results: PI3Ks induce system xc(-) through glycogen synthase kinase 3? (GSK-3?) inhibition, general control non-derepressible-2-mediated eukaryotic initiation factor 2? phosphorylation, and the subsequent translational up-regulation of activating transcription factor 4. This pathway is essential for PI3Ks to modulate oxidative stress resistance of nerve cells and insulin-induced growth in fibroblasts. Moreover, the pathway is active in human glioblastoma cells. In addition, it is induced in primary cortical neurons in response to robust neuronal activity and in hippocampi from patients with temporal lobe epilepsy. Innovation: Our findings further extend the concepts of how growth factors and PI3Ks induce neuroprotection and cell growth by adding a new branch to the signaling network downstream of GSK-3?, which, ultimately, leads to the induction of the cystine/glutamate antiporter system xc(-). Importantly, the induction of this pathway by neuronal activity and in epileptic hippocampi points to a potential role in epilepsy. Conclusion: PI3K-regulated system xc(-) activity is not only involved in the stress resistance of neuronal cells and in cell growth by increasing the cysteine supply and glutathione synthesis, but also plays a role in the pathophysiology of tumor- and non-tumor-associated epilepsy by up-regulating extracellular cerebral glutamate. Antioxid. Redox Signal. 20: 2907-2922. PMID:24219064

Lewerenz, Jan; Baxter, Paul; Kassubek, Rebecca; Albrecht, Philipp; Van Liefferinge, Joeri; Westhoff, Mike-Andrew; Halatsch, Marc-Eric; Karpel-Massler, Georg; Meakin, Paul J; Hayes, John D; Aronica, Eleonora; Smolders, Ilse; Ludolph, Albert C; Methner, Axel; Conrad, Marcus; Massie, Ann; Hardingham, Giles E; Maher, Pamela



Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells  

SciTech Connect

Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrations below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.

Okajima, F.; Sho, K.; Kondo, Y.



Genetic alterations in the phosphoinositide 3-kinase/Akt signaling pathway confer sensitivity of thyroid cancer cells to therapeutic targeting of Akt and mammalian target of rapamycin.  


We investigated the genotype-dependent therapeutic potential of targeting the phosphoinositide 3-kinase (PI3K)/Akt pathway for thyroid cancer. Proliferation of TPC1, Hth7, FTC133, OCUT1, K1, and BCPAP cells that harbored PI3K/Akt-activating genetic alterations was potently inhibited by the Akt inhibitor perifosine, whereas SW1736, Hth74, WRO, KAT18, and TAD2 cells that harbored no genetic alterations had no or only modest responses. Inhibition of Akt phosphorylation by perifosine was seen in these cells. Genetic-dependent apoptosis was induced by perifosine in cells selectively tested. Similarly, potent inhibition of cell proliferation by the mammalian target of rapamycin (mTOR) inhibitor temsirolimus occurred in virtually all the cells harboring genetic alterations, whereas modest inhibition was seen in some of the cells not harboring genetic alterations. Temsirolimus inhibited the phosphorylation of p70S6K, a substrate of mTOR. Knockdown of Akt1/2 or mTOR by shRNA approach inhibited the proliferation and colony formation of FTC133 and OCUT1 cells that harbored genetic alterations in the PI3K/Akt pathway but had no effect on SW1736 and KAT18 cells that did not. Transfection with PIK3CA mutants greatly sensitized SW1736 cells to perifosine and temsirolimus. Growth of xenograft tumors derived from FTC133 cells but not SW1736 cells in nude mice was dramatically inhibited by perifosine. Thus, this work for the first time shows that genetic alterations in the PI3K/Akt pathway confer thyroid cancer cells addiction to this pathway and their sensitivity to inhibition by targeting Akt and mTOR. This genotype-based targeting of the PI3K/Akt pathway using Akt and mTOR inhibitors may offer an effective therapeutic strategy for thyroid cancer and warrants further studies. PMID:19706758

Liu, Dingxie; Hou, Peng; Liu, Zhi; Wu, Guojun; Xing, Mingzhao



Regulation of Phosphoinositide Levels by the Phospholipid Transfer Protein Sec14p Controls Cdc42p/p21-Activated Kinase-Mediated Cell Cycle Progression at Cytokinesis?  

PubMed Central

Sec14p is an essential phosphatidylcholine/phosphatidylinositol transfer protein with a well-described role in the regulation of Golgi apparatus-derived vesicular transport in yeast. Inactivation of the CDP-choline pathway for phosphatidylcholine synthesis allows cells to survive in the absence of Sec14p function through restoration of Golgi vesicular transport capability. In this study, Saccharomyces cerevisiae cells containing a SEC14 temperature-sensitive allele along with an inactivated CDP-choline pathway were transformed with a high-copy-number yeast genomic library. Genes whose increased expression inhibited cell growth in the absence of Sec14p function were identified. Increasing levels of the Rho GTPase Cdc42p and its direct effector kinases Cla4p and Ste20p prevented the growth of cells lacking Sec14p and CDP-choline pathway function. Growth suppression was accompanied by an increase in large and multiply budded cells. This effect on polarized cell growth did not appear to be due to an inability to establish cell polarity, since both the actin cytoskeleton and localization of the septin Cdc12p were unaffected by increased expression of Cdc42p, Cla4p, or Ste20p. Nuclei were present in both the mother cell and the emerging bud, consistent with Sec14p regulation of the cell cycle subsequent to anaphase but prior to cytokinesis/septum breakdown. Increased expression of phosphatidylinositol 4-kinases and phosphatidylinositol 4-phosphate 5-kinase prevented growth arrest by CDC42, CLA4, or STE20 upon inactivation of Sec14p function. Sec14p regulation of phosphoinositide levels affects cytokinesis at the level of the Cdc42p/Cla4p/Ste20p signaling cascade.

Howe, Alicia G.; Fairn, Gregory D.; MacDonald, Kendra; Bankaitis, Vytas A.; McMaster, Christopher R.



Muscarinic-receptor-mediated inhibition of insulin-like growth factor-1 receptor-stimulated phosphoinositide 3-kinase signalling in 1321N1 astrocytoma cells.  

PubMed Central

In 1321N1 astrocytoma cells, stimulation of the IGF-1 (insulin-like growth factor-1) receptor increased the association of PI3K [phosphoinositide (PI) 3-kinase] activity with IRS-1 (insulin re-ceptor substrate 1), and increased the cellular concentration of PtdIns(3,4,5)P3. Carbachol, acting on M3 muscarinic receptors, inhibited insulin-, but not PDGF (platelet-derived growth factor)-, stimulated responses by approximately 50%. The inhibition of IRS-1-associated PI3K activity by carbachol (i) was rapid (<1 min), persistent (> or =60 min) and potent (half-maximal concentration approximately 1 microM); (ii) was reproduced by stimuli for several phospholipase-C-coupled receptors; (iii) was prevented by the inhibition of protein kinase C, but not by chelation of intracellular Ca2+; and (iv) was not blocked or reproduced by inhibitors or stimuli respectively of mitogen-activated protein kinase, PI3K, protein kinase B or the mammalian target of rapamycin. However, the effects of carbachol were prevented by sodium vanadate, a protein tyrosine phosphatase inhibitor, and were accompanied by reduced insulin-stimulated IRS-1 tyrosine phosphorylation and recruitment of the 85 kDa regulatory subunit of PI3K to IRS-1, but not by reduced IGF-1 receptor kinase activity. The inhibitory effect of carbachol was reproduced by okadaic acid, a protein serine/threonine phosphatase inhibitor, but not by PDGF, yet all three agents stimulated the serine phosphorylation of IRS-1 at residues Ser312, Ser616 and Ser636/639, albeit to different extents. Thus muscarinic receptors may inhibit insulin signalling by promoting IRS-1 tyrosine dephosphorylation and/or by uncoupling IRS-1 from the stimulated IGF-1 receptor by stimulating IRS-1 serine phosphorylation. However, the proportion of IRS-1 molecules phosphorylated at a particular site or the phosphorylation of additional IRS-1 serine residues other than those noted above must be important.

Batty, Ian H; Fleming, Ian N; Downes, C Peter



Endogenous expression of histamine H1 receptors functionally coupled to phosphoinositide hydrolysis in C6 glioma cells: regulation by cyclic AMP.  

PubMed Central

1. The effects of histamine receptor agonists and antagonists on phospholipid hydrolysis in rat-derived C6 glioma cells have been investigated. 2. Histamine H1 receptor-stimulation caused a concentration-dependent increase in the accumulation of total [3H]-inositol phosphates in cells prelabelled with [3H]-myo-inositol. The rank order of agonist potencies was histamine (EC50 = 24 microM) > N alpha-methylhistamine (EC50 = 31 microM) > 2-thiazolylethylamine (EC50 = 91 microM). 3. The response to 0.1 mM histamine was antagonized in a concentration-dependent manner by the H1-antagonists, mepyramine (apparent Kd = 1 nM) and (+)-chlorpheniramine (apparent Kd = 4 nM). In addition, (-)-chlorpheniramine was more than two orders of magnitude less potent than its (+)-stereoisomer. 4. Elevation of intracellular cyclic AMP accumulation with forskolin (10 microM, EC50 = 0.3 microM), isoprenaline (1 microM, EC50 = 4 nM) or rolipram (0.5 mM), significantly reduced the histamine-mediated (0.1 mM) inositol phosphate response by 37%, 43% and 26% respectively. In contrast, 1,9-dideoxyforskolin did not increase cyclic AMP accumulation and had no effect on the phosphoinositide response to histamine. 5. These data indicate the presence of functionally coupled, endogenous histamine H1 receptors in C6 glioma cells. Furthermore, the results also indicate that H1 receptor-mediated phospholipid hydrolysis is inhibited by the elevation of cyclic AMP levels in these cells.

Peakman, M C; Hill, S J



Dual Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235 Has a Therapeutic Potential and Sensitizes Cisplatin in Nasopharyngeal Carcinoma  

PubMed Central

Phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells and thus has been considered as a promising drug target. To ascertain a therapeutical approach of nasopharyngeal carcinoma (NPC), we hypothesized NVP-BEZ235, a novel and potent imidazo[4,5-c] quinolone derivative, that dually inhibits both PI3K and mTOR kinases activities, had antitumor activity in NPC. Expectedly, we found that NVP-BEZ235 selectively inhibited proliferation of NPC cells rather than normal nasopharyngeal cells using MTT assay. In NPC cell lines, with the extended exposure, NVP-BEZ235 selectively inhibited proliferation of NPC cells harboring PIK3CA mutation, compared to cells with wild-type PIK3CA. Furthermore, exposure of NPC cells to NVP-BEZ235 resulted in G1 growth arrest by Propidium iodide uptake assay, reduction of cyclin D1and CDK4, and increased levels of P27 and P21 by Western blotting, but negligible apoptosis. Moreover, we found that cisplatin (CDDP) activated PI3K/AKT and mTORC1 pathways and NVP-BEZ235 alleviated the activation by CDDP through dually targeting PI3K and mTOR kinases. Also, NVP-BEZ235 combining with CDDP synergistically inhibited proliferation and induced apoptosis in NPC cells. In CNE2 and HONE1 nude mice xenograft models, orally NVP-BEZ235 efficiently attenuated tumor growth with no obvious toxicity. In combination with NVP-BEZ235 and CDDP, there was dramatic synergy in shrinking tumor volumes and inducing apoptosis through increasing Noxa, Bax and decreasing Mcl-1, Bcl-2. Based on the above results, NVP-BEZ235, which has entered phase I/II clinical trials in patients with advanced solid tumors, has a potential as a monotherapy or in combination with CDDP for NPC treatment.

Deng, Rong; Wu, Xiao-Qi; Qin, Juan; Feng, Gong-Kan; Zhu, Xiao-Feng



Hydrogen sulfide regulates Na+/H+ exchanger activity via stimulation of phosphoinositide 3-kinase/Akt and protein kinase G pathways.  


Intracellular pH (pH(i)) is an important endogenous modulator of cardiac function. Inhibition of Na(+)/H(+) exchanger-1 (NHE-1) protects the heart by preventing Ca(2+) overload during ischemia/reperfusion. Hydrogen sulfide (H(2)S) has been reported to produce cardioprotection. The present study was designed to investigate the pH regulatory effect of H(2)S in rat cardiac myocytes and evaluate its contribution to cardioprotection. It was found that sodium hydrosulfide (NaHS), at a concentration range of 10 to 1000 ?M, produced sustained decreases in pH(i) in the rat myocytes in a concentration-dependent manner. NaHS also abolished the intracellular alkalinization caused by trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate (U50,488H), which activates NHEs. Moreover, when measured with an NHCl(4) prepulse method, NaHS was found to significantly suppress NHE-1 activity. Both NaHS and cariporide or [5-(2-methyl-5-fluorophenyl)furan-2-ylcarbonyl]guanidine (KR-32568), two NHE inhibitors, protected the myocytes against ischemia/reperfusion injury. However, coadministration of NaHS with KR-32568 did not produce any synergistic effect. Functional study showed that perfusion with NaHS significantly improved postischemic contractile function in isolated rat hearts subjected to ischemia/reperfusion. Blockade of phosphoinositide 3-kinase (PI3K) with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), Akt with Akt VIII, or protein kinase G (PKG) with (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6

Hu, Li-Fang; Li, Yu; Neo, Kay Li; Yong, Qian Chen; Lee, Shiau Wei; Tan, Benny Kwong Huat; Bian, Jin-Song



Selective inhibitors of phosphoinositide 3-kinase delta: modulators of B-cell function with potential for treating autoimmune inflammatory diseases and B-cell malignancies  

PubMed Central

The delta isoform of the p110 catalytic subunit (p110?) of phosphoinositide 3-kinase is expressed primarily in hematopoietic cells and plays an essential role in B-cell development and function. Studies employing mice lacking a functional p110? protein, as well as the use of highly-selective chemical inhibitors of p110?, have revealed that signaling via p110?-containing PI3K complexes (PI3K?) is critical for B-cell survival, migration, and activation, functioning downstream of key receptors on B cells including the B-cell antigen receptor, chemokine receptors, pro-survival receptors such as BAFF-R and the IL-4 receptor, and co-stimulatory receptors such as CD40 and Toll-like receptors (TLRs). Similarly, this PI3K isoform plays a key role in the survival, proliferation, and dissemination of B-cell lymphomas. Herein we summarize studies showing that these processes can be inhibited in vitro and in vivo by small molecule inhibitors of p110? enzymatic activity, and that these p110? inhibitors have shown efficacy in clinical trials for the treatment of several types of B-cell malignancies including chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). PI3K? also plays a critical role in the activation, proliferation, and tissue homing of self-reactive B cells that contribute to autoimmune diseases, in particular innate-like B-cell populations such as marginal zone (MZ) B cells and B-1 cells that have been strongly linked to autoimmunity. We discuss the potential utility of p110? inhibitors, either alone or in combination with B-cell depletion, for treating autoimmune diseases such as lupus, rheumatoid arthritis, and type 1 diabetes. Because PI3K? plays a major role in both B-cell-mediated autoimmune inflammation and B-cell malignancies, PI3K? inhibitors may represent a promising therapeutic approach for treating these diseases.

Puri, Kamal D.; Gold, Michael R.



Inhibitory Role of ?6?4-Associated Erbb-2 and Phosphoinositide 3-Kinase in Keratinocyte Haptotactic Migration Dependent on ?3?1 Integrin  

PubMed Central

Keratinocytes and other epithelial cells express two receptors for the basement membrane (BM) extracellular matrix component laminin-5 (Ln-5), integrins ?3?1 and ?6?4. While ?3?1 mediates adhesion, spreading, and migration (Kreidberg, J.A. 2000. Curr. Opin. Cell Biol. 12:548–553), ?6?4 is involved in BM anchorage via hemidesmosomes (Borradori, L., and A. Sonnenberg. 1999. J. Invest. Dermatol. 112:411–418). We investigated a possible regulatory interplay between ?3?1 and ?6?4 in cell motility using HaCaT keratinocytes as a model. We found that ?6?4 antibodies inhibit ?3?1-mediated migration on Ln-5, but only when migration is haptotactic (i.e., spontaneous or stimulated by ?3?1 activation), and not when chemotactic (i.e., triggered by epidermal growth factor receptor). Inhibition of migration by ?6?4 depends upon phosphoinositide 3-kinase (PI3-K) since it is abolished by PI3-K blockers and by dominant-negative PI3-K, and constitutively active PI3-K prevents haptotaxis. In HaCaT cells incubated with anti–?6?4 antibodies, activation of PI3-K is mediated by ?6?4-associated erbB-2, as indicated by erbB-2 autophosphorylation and erbB-2/p85 PI3-K coprecipitation. Furthermore, dominant-negative erbB-2 abolishes inhibition of haptotaxis by anti–?6?4 antibodies. These results support a model whereby (a) haptotactic cell migration on Ln-5 is regulated by concerted action of ?3?1 and ?6?4 integrins, (b) ?6?4-associated erbB-2 and PI3-K negatively affect haptotaxis, and (c) chemotaxis on Ln-5 is not affected by ?6?4 antibodies and may require PI3-K activity. This model could be of general relevance to motility of epithelial cells in contact with BM.

Hintermann, Edith; Bilban, Martin; Sharabi, Andrew; Quaranta, Vito



Platelet activation by bacterial phospholipase C involves phosphoinositide turnover and phosphorylation of 47,000 dalton but not 20,000 dalton protein  

SciTech Connect

This study was conducted to examine the role of phosphoinositides (PIns) and phosphorylation of 47,000 dalton (P47) and 20,000 dalton (P20) proteins in platelet activation by bacterial phospholipase C (PLC). PLC induced serotonin secretion (SS) and platelet aggregation (PA) in a concentration dependent manner. PLC (0.02 U/ml) caused phosphorylation of P47 in a time dependent manner (27% at 0.5 min to 378% at 7 min). PLC did not induce more than 15% phosphorylation of P20 by 7 min. Aspirin (500 blocked phosphorylation of P20 but did not inhibit SS, PA or phosphorylation of P47. PLC (0.04 U/ml) decreased radioactivity (cpm) in /sup 32/P labeled phosphatidylinositol (PI), PI-4,5-bis-PO4 (PIP2) and PI-4-PO4 (PIP) by 20%, 12% and 7.5% respectively at 15 sec. The level of PI but not that of PIP2 returned to base line in 3 min. PIP level increased above control values within one min. PLC increased phosphatidic acid level (75% at 0.5 min. to 1545% at 3 min). In other experiments PLC produced diacylglycerol (DAG) in a time and concentration dependent manner. However, no DAG was detectable in the first 60 sec. These data suggest that: (a) PIns turnover and phosphorylation of P47 but not that of P20 is involved in platelet activation by PLC; and (b) DAG production from outer membrane phospholipids is not a prerequisite for platelet activation by PLC.

Huzoor-Akbar; Anwer, K.



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

PubMed Central

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 Gq-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.

Purvis, Jeremy E.; Chatterjee, Manash S.; Brass, Lawrence F.



Phosphoinositide [PI(3,5)P2] lipid-dependent regulation of the general transcriptional regulator Tup1.  


Transcriptional activity of a gene is governed by transcriptional regulatory complexes that assemble/disassemble on the gene and control the chromatin architecture. How cytoplasmic components influence the assembly/disassembly of transcriptional regulatory complexes is poorly understood. Here we report that the budding yeast Saccharomyces cerevisiae has a chromatin architecture-modulating mechanism that is dependent on the endosomal lipid PI(3,5)P(2). We identified Tup1 and Cti6 as new, highly specific PI(3,5)P(2) interactors. Tup1--which associates with multiple transcriptional regulators, including the HDAC (histone deacetylase) and SAGA complexes--plays a crucial role in determining an activated or repressed chromatin state of numerous genes, including GAL1. We show that, in the context that the Gal4 activation pathway is compromised, PI(3,5)P(2) plays an essential role in converting the Tup1-driven repressed chromatin structure into a SAGA-containing activated chromatin structure at the GAL1 promoter. Biochemical and cell biological experiments suggest that PI(3,5)P(2) recruits Cti6 and the Cyc8-Tup1 corepressor complex to the late endosomal/vacuolar membrane and mediates the assembly of a Cti6-Cyc8-Tup1 coactivator complex that functions to recruit the SAGA complex to the GAL1 promoter. Our findings provide important insights toward understanding how the chromatin architecture and epigenetic status of a gene are regulated by cytoplasmic components. PMID:21536737

Han, Bong-Kwan; Emr, Scott D



Alpha 1-adrenergic receptor-mediated phosphoinositide hydrolysis and prostaglandin E2 formation in Madin-Darby canine kidney cells. Possible parallel activation of phospholipase C and phospholipase A2  

SciTech Connect

alpha 1-Adrenergic receptors mediate two effects on phospholipid metabolism in Madin-Darby canine kidney (MDCK-D1) cells: hydrolysis of phosphoinositides and arachidonic acid release with generation of prostaglandin E2 (PGE2). The similarity in concentration dependence for the agonist (-)-epinephrine in eliciting these two responses implies that they are mediated by a single population of alpha 1-adrenergic receptors. However, we find that the kinetics of the two responses are quite different, PGE2 production occurring more rapidly and transiently than the hydrolysis of phosphoinositides. The antibiotic neomycin selectively decreases alpha 1-receptor-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis without decreasing alpha 1-receptor-mediated arachidonic acid release and PGE2 generation. In addition, receptor-mediated inositol trisphosphate formation is independent of extracellular calcium, whereas release of labeled arachidonic acid is largely calcium-dependent. Moreover, based on studies obtained with labeled arachidonic acid, receptor-mediated generation of arachidonic acid cannot be accounted for by breakdown of phosphatidylinositol monophosphate, phosphatidylinositol bisphosphate, or phosphatidic acid. Further studies indicate that epinephrine produces changes in formation or turnover of several classes of membrane phospholipids in MDCK cells. We conclude that alpha 1-adrenergic receptors in MDCK cells appear to regulate phospholipid metabolism by the parallel activation of phospholipase C and phospholipase A2. This parallel activation of phospholipases contrasts with models described in other systems which imply sequential activation of phospholipase C and diacylglycerol lipase or phospholipase A2.

Slivka, S.R.; Insel, P.A.



Normal insulin-dependent activation of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 diabetes  

PubMed Central

To determine whether the serine/threonine kinase Akt (also known as protein kinase B) is activated in vivo by insulin administration in humans, and whether impaired activation of Akt could play a role in insulin resistance, we measured the activity and phosphorylation of Akt isoforms in skeletal muscle from 3 groups of subjects: lean, obese nondiabetic, and obese type 2 diabetic. Vastus lateralis biopsies were taken in the basal (overnight fast) and insulin-stimulated (euglycemic clamp) states. Insulin-stimulated glucose disposal was reduced 31% in obese subjects and 63% in diabetic subjects, compared with lean subjects. Glycogen synthase (GS) activity in the basal state was reduced 28% in obese subjects and 49% in diabetic subjects, compared with lean subjects. Insulin-stimulated GS activity was reduced 30% in diabetic subjects. Insulin treatment activated the insulin receptor substrate-1–associated (IRS-1–associated) phosphoinositide 3-kinase (PI 3-kinase) 6.1-fold in lean, 3.7-fold in obese, and 2.4-fold in diabetic subjects. Insulin also stimulated IRS-2–associated PI 3-kinase activity 2.2-fold in lean subjects, but only 1.4-fold in diabetic subjects. Basal activity of Akt1/Akt2 (Akt1/2) and Akt3 was similar in all groups. Insulin increased Akt1/2 activity 1.7- to 2.0-fold, and tended to activate Akt3, in all groups. Insulin-stimulated phosphorylation of Akt1/2 was normal in obese and diabetic subjects. In lean subjects only, insulin-stimulated Akt1/2 activity correlated with glucose disposal rate. Thus, insulin activation of Akt isoforms is normal in muscle of obese nondiabetic and obese diabetic subjects, despite decreases of approximately 50% and 39% in IRS-1– and IRS-2–associated PI 3-kinase activity, respectively, in obese diabetic subjects. It is therefore unlikely that Akt plays a major role in the resistance to insulin action on glucose disposal or GS activation that is observed in muscle of obese type 2 diabetic subjects.

Kim, Young-Bum; Nikoulina, Svetlana E.; Ciaraldi, Theodore P.; Henry, Robert R.; Kahn, Barbara B.



A Novel HPLC-Based Approach Makes Possible the Spatial Characterization of Cellular PtdIns5P and Other Phosphoinositides  

PubMed Central

SYNOPSIS Phosphatidylinositol-5-phosphate (PtdIns5P) was discovered in 1997 (Rameh, L.E.; Tolias, K.F.; Duckworth, B.C. and Cantley, L.C. 1997, Nature 390, 192–196) but very little is known about its regulation and function. Hitherto, studies of PtdIns5P regulation have been hindered by the inability to measure cellular PtdIns5P using conventional HPLC, owing to poor separation from PtdIns4P. Here we present a new HPLC method for resolving PtdIns5P from PtdIns4P, which makes possible accurate measurements of basal and inducible levels of cellular PtdIns5P in the context of other phosphoinositides. Using this new method, we found that PtdIns5P is constitutively present in all cells examined (epithelial cells, fibroblasts and myoblasts, among others) at levels typically 1% to 2 % of PtdIns4P levels. In the ?-pancreatic cell line BTC6, which is specialized in insulin secretion, PtdIns5P levels were higher than in most cells (2.5% to 4% of PtdIns4P). Using subcellular fractionation, we found that the majority of the basal PtdIns5P is present in the plasma membrane, but it is also enriched in intracellular membrane compartments, especially in smooth endoplasmic reticulum (SER) and/or Golgi, where high levels of PtdIns3P were also detected. Unlike PtdIns3P, PtdIns5P was also found in fractions containing very low-density vesicles. Knockdown of PtdIns5P 4-kinase (PIP4k) leads to accumulation of PtdIns5P in light fractions and fractions enriched in SER/Golgi, while treatment with Brefeldin A results in a subtle, but reproducible, change in PtdIns5P distribution. These results indicate that basal PtdIns5P and the PtdIns5P pathway for PtdIns(4,5)P2 synthesis may play a role in Golgi-mediated vesicle trafficking.

Sarkes, Deborah; Rameh, Lucia E.



Ca21Permeable AMPA Receptors Induce Phosphorylation of cAMP Response Element-Binding Protein through a Phosphatidylinositol 3-Kinase-Dependent Stimulation of the Mitogen-Activated Protein Kinase Signaling Cascade in Neurons  

Microsoft Academic Search

Ca 21-permeable AMPA receptors may play a key role during developmental neuroplasticity, learning and memory, and neu- ronal loss in a number of neuropathologies. However, the intra- cellular signaling pathways used by AMPA receptors during such processes are not fully understood. The mitogen- activated protein kinase (MAPK) cascade is an attractive target because it has been shown to be involved

Michael S. Perkinton; Talvinder S. Sihra; Robert J. Williams


Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism  

NASA Technical Reports Server (NTRS)

Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at Ser(1179) in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including Ser(116), Thr(497), and Ser(635). Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser(635), which was consistently slower than that at Ser(1179). Thr(497) was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated Ser(116). Whereas shear-stimulated Ser(635) phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and 2) PKA-dependent but PI3K-independent pathways. Phosphorylation of Ser(635) may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli.

Boo, Yong Chool; Hwang, Jinah; Sykes, Michelle; Michell, Belinda J.; Kemp, Bruce E.; Lum, Hazel; Jo, Hanjoong



Insulin-Like Growth Factor-1 Regulates Glutathione Peroxidase Expression and Activity in Vascular Endothelial Cells: Implications for Atheroprotective Actions of Insulin-Like Growth Factor-1  

PubMed Central

Oxidative stress promotes endothelial cell senescence and endothelial dysfunction, important early steps in atherogenesis. To investigate potential antioxidant effects of IGF-1 we treated human aortic endothelial cells (hAECs) with 0–100 ng/mL IGF-1 prior to exposure to native or oxidized low-density lipoprotein (oxLDL). IGF-1 dose- and time- dependently reduced basal- and oxLDL-induced ROS generation. IGF-1 did not alter superoxide dismutase or catalase activity but markedly increased activity of glutathione peroxidase (GPX), a crucial antioxidant enzyme, via a phosphoinositide-3 kinase dependent pathway. IGF-1 did not increase GPX1 mRNA levels but increased GPX1 protein levels by 2.6-fold at 24 hr, and altered selenocysteine-incorporation complex formation on GPX1 mRNA. Furthermore, IGF-1 blocked hydrogen peroxide induced premature cell senescence in hAECs. In conclusion, IGF-1 upregulates GPX1 expression in hAECs via a translational mechanism, which may play an important role in the ability of IGF-1 to reduce endothelial cell oxidative stress and premature senescence. Our findings have major implications for understanding vasculoprotective effects of IGF-1.

Higashi, Yusuke; Pandey, Arvind; Goodwin, Brett; Delafontaine, Patrice



Retinoic acid signaling in axonal regeneration.  


Following an acute central nervous system (CNS) injury, axonal regeneration and functional recovery are extremely limited. This is due to an extrinsic inhibitory growth environment and the lack of intrinsic growth competence. Retinoic acid (RA) signaling, essential in developmental dorsoventral patterning and specification of spinal motor neurons, has been shown through its receptor, the transcription factor RA receptor ?2 (RAR?2), to induce axonal regeneration following spinal cord injury (SCI). Recently, it has been shown that in dorsal root ganglion neurons (DRGs), cAMP levels were greatly increased by lentiviral RAR?2 expression and contributed to neurite outgrowth. Moreover, RAR?agonists, in cerebellar granule neurons (CGN) and in the brain in vivo, induced phosphoinositide 3-kinase dependent phosphorylation of AKT that was involved in RAR?-dependent neurite outgrowth. More recently, RA-RAR?pathways were shown to directly transcriptionally repress a member of the inhibitory Nogo receptor (NgR) complex, Lingo-1, under an axonal growth inhibitory environment in vitro as well as following spinal injury in vivo. This perspective focuses on these newly discovered molecular mechanisms and future directions in the field. PMID:22287943

Puttagunta, Radhika; Di Giovanni, Simone



Puzzling Mechanisms  

ERIC Educational Resources Information Center

The basis of a good mechanical puzzle is often a puzzling mechanism. This article will introduce some new puzzling mechanisms, like two knots that engage like gears, a chain whose links can be interchanged, and flat gears that do not come apart. It illustrates how puzzling mechanisms can be transformed into real mechanical puzzles, e.g., by…

van Deventer, M. Oskar



Yeast 3-Phosphoinositide-dependent Protein Kinase-1 (PDK1) Orthologs Pkh1-3 Differentially Regulate Phosphorylation of Protein Kinase A (PKA) and the Protein Kinase B (PKB)/S6K Ortholog Sch9*  

PubMed Central

Pkh1, -2, and -3 are the yeast orthologs of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1). Although essential for viability, their functioning remains poorly understood. Sch9, the yeast protein kinase B and/or S6K ortholog, has been identified as one of their targets. We now have shown that in vitro interaction of Pkh1 and Sch9 depends on the hydrophobic PDK1-interacting fragment pocket in Pkh1 and requires the complementary hydrophobic motif in Sch9. We demonstrated that Pkh1 phosphorylates Sch9 both in vitro and in vivo on its PDK1 site and that this phosphorylation is essential for a wild type cell size. In vivo phosphorylation on this site disappeared during nitrogen deprivation and rapidly increased again upon nitrogen resupplementation. In addition, we have shown here for the first time that the PDK1 site in protein kinase A is phosphorylated by Pkh1 in vitro, that this phosphorylation is Pkh-dependent in vivo and occurs during or shortly after synthesis of the protein kinase A catalytic subunits. Mutagenesis of the PDK1 site in Tpk1 abolished binding of the regulatory subunit and cAMP dependence. As opposed to PDK1 site phosphorylation of Sch9, phosphorylation of the PDK1 site in Tpk1 was not regulated by nitrogen availability. These results bring new insight into the control and prevalence of PDK1 site phosphorylation in yeast by Pkh protein kinases.

Voordeckers, Karin; Kimpe, Marlies; Haesendonckx, Steven; Louwet, Wendy; Versele, Matthias; Thevelein, Johan M.



Mechanical Systems  

NASA Technical Reports Server (NTRS)

The presentation provides an overview of requirement and interpretation letters, mechanical systems safety interpretation letter, design and verification provisions, and mechanical systems verification plan.

Davis, Robert E.



Osteopontin is a myosphere-derived secretory molecule that promotes angiogenic progenitor cell proliferation through the phosphoinositide 3-kinase\\/Akt pathway  

Microsoft Academic Search

We have reported that skeletal myosphere-derived progenitor cells (MDPCs) can differentiate into vascular cells, and that MDPC transplantation into cardiomyopathic hearts improves cardiac function. However, the autocrine\\/paracrine molecules and underlying mechanisms responsible for MDPC growth have not yet been determined. To explore the molecules enhancing the proliferation of MDPCs, we performed serial analysis of gene expression and signal sequence trap

Takehiro Ogata; Tomomi. Ueyama; Tetsuya Nomura; Satoshi Asada; Masashi Tagawa; Tomoyuki Nakamura; Tomosaburo Takahashi; Hiroaki Matsubara; Hidemasa. Oh



Moving Forward: Mechanisms of Chemoattractant Gradient Sensing  

NSDL National Science Digital Library

Cells use an internal compass to sense the direction of chemoattractant gradients. This is used to bias pseudopod extension at the front of the cell and to orient cell polarization. Recent studies have highlighted the important roles played by phosphoinositide-3,4,5-triphosphate and small G proteins, but many questions remain.

PhD Jonathan Franca-Koh (Johns Hopkins University School of Medicine Department of Cell Biology); PhD Peter N. Devreotes (Johns Hopkins University School of Medicine Department of Cell Biology)



Phosphoinositide 3-Kinase Pathway Activation in Phosphate and Tensin Homolog (PTEN)-deficient Prostate Cancer Cells Is Independent of Receptor Tyrosine Kinases and Mediated by the p110? and p110? Catalytic Subunits*  

PubMed Central

Class IA phosphoinositide 3-kinase (PI3K) p110 catalytic subunits are activated upon Src homology 2 domain-mediated binding of their p85 regulatory subunits to tyrosine-phosphorylated pYXXM motifs in receptor tyrosine kinases (RTKs) or adaptor proteins. The PI3K pathway is activated by phosphate and tensin homolog (PTEN) loss in most prostate cancers (PCa), but the contribution of upstream RTKs that may be targeted therapeutically has not been assessed. Immunoblotting of p85-associated proteins in serum-starved PTEN-deficient LNCaP and C4-2 PCa cells showed a small set of discrete tyrosine-phosphorylated proteins, but these proteins were not recognized by an anti-pYXXM motif antibody and were not found in PTEN-deficient PC3 PCa cells. LC/MS/MS using label-free proteomics and immunoblotting showed that p85 was associated primarily with p110? and p110?. An interaction with ErbB3 was also detected but was independent of ErbB3 tyrosine phosphorylation and was not required for basal PI3K activity. Basal tyrosine phosphorylation of p110? and p110? could be blocked by c-Src inhibitors, but this did not suppress PI3K activity, which was similarly independent of Ras. Basal PI3K activity was mediated by p110? in PC3 cells and by both p110? and p110? in LNCaP cells, whereas p110? was required for PI3K activation in response to RTK stimulation by heregulin-?1. These findings show that basal PI3K activity in PTEN-deficient PCa cells is RTK-independent and can be mediated by p110? and p110?. Increased p110? expression in PCa may be required for RTK-independent PI3K pathway activation in adult prostate epithelium with genetic or epigenetic PTEN down-regulation.

Jiang, Xinnong; Chen, Sen; Asara, John M.; Balk, Steven P.



Invasion of HeLa cells by group B streptococcus requires the phosphoinositide-3-kinase signalling pathway and modulates phosphorylation of host-cell Akt and glycogen synthase kinase-3.  


The group B streptococcus (GBS) is an opportunistic bacterial pathogen with the ability to cause invasive disease. While the ability of GBS to invade a number of host-cell types has been clearly demonstrated, the invasion process is not well understood at the molecular level. What has been well established is that modulation of host-cell actin microfilaments is essential for GBS invasion to occur. Phosphoinositide-3 kinase (PI3K) is a key regulator of the cytoskeleton in eukaryotic cells. Our goal in this investigation was to explore the role of the PI3K/Akt signalling pathway in epithelial cell invasion by GBS. The epithelial cell invasion process was mimicked using the HeLa 229 cell-culture model. Treating HeLa cells with chemical inhibitors of PI3K, Akt or Ras prior to bacterial infection inhibited GBS invasion but not attachment; treatment with 30 microM LY294002 (PI3K inhibitor) reduced GBS invasion by 75%, 20 microM L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (ICIO) (Akt inhibitor) reduced GBS invasion by 50%, and 10 microM manumycin A (Ras inhibitor) inhibited GBS invasion by 90%. Genetic inactivation of the p85alpha or p110alpha PI3K subunits in HeLa cells also reduced GBS invasion by 55 and 30%, respectively. Western blot analysis revealed that phosphorylation of host-cell Akt and glycogen synthase kinase-3 (GSK-3) occurs in response to GBS infection, and that this is mediated upstream by PI3K. Infection of HeLa cells with GBS triggers pro-survival signalling and protects the HeLa cells from camptothecin-induced caspase-3 cleavage. The results from this investigation show that GBS both requires and activates the PI3K/Akt host-cell signalling pathway during invasion of epithelial cells. PMID:18048937

Burnham, Carey-Ann D; Shokoples, Sandra E; Tyrrell, Gregory J



Fluorodeoxyglucose uptake in laryngeal carcinoma is associated with the expression of glucose transporter-1 and hypoxia-inducible-factor-1? and the phosphoinositide 3-kinase/protein kinase B pathway  

PubMed Central

High fluorodeoxyglucose (FDG) uptake by human carcinomas, including head and neck cancers, is associated with a poor prognosis. Glucose transporter-1 (Glut-1) is believed to be an intrinsic marker of hypoxia in malignant tumors. The expression of hypoxia-inducible factor-1? (HIF-1?) and correlated target genes, including Glut-1, is regulated by the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway. However, it remains unclear whether the PI3K/Akt signaling pathway is involved in regulating FDG uptake directly. In the present study, 24 consecutive patients with laryngeal carcinoma were examined pre-operatively and the standardized uptake values (SUV) of the laryngeal carcinomas were determined. Glut-1, HIF-1?, PI3K and phosphorylated-Akt (p-Akt) expression was detected by immunohistochemical staining of paraffin sections from the tumor specimens. Associations among SUVmax, Glut-1, HIF-1?, PI3K and p-Akt protein expression and the other clinical parameters were analyzed. The univariate analyses revealed a significantly shorter survival time along with higher HIF-1? (P=0.018) and PI3K (P=0.008) expression, but the survival time was not significantly correlated with Glut-1 or p-Akt expression. The multivariate analysis demonstrated that higher SUVmax (P=0.043) and PI3K expression (P=0.012) were significantly correlated with a poor survival time. Spearman’s rank analysis showed significant correlations between SUVmax and HIF-1? (r=0.577; P=0.003), PI3K (r=1.0; P<0.0001) and p-Akt (r=0.577; P=0.003) expression. PI3K was associated with poorly- and moderately-differentiated laryngeal carcinoma (P=0.012). In conclusion, a high SUVmax indicates a poor prognosis for laryngeal carcinoma. Also, a high SUVmax may be associated with the increased expression of Glut-1, HIF-1?, PI3K and p-Akt.




Roles of MKK1/2-ERK1/2 and phosphoinositide 3-kinase-AKT signaling pathways in erlotinib-induced Rad51 suppression and cytotoxicity in human non-small cell lung cancer cells.  


Erlotinib (Tarceva) is a selective epidermal growth factor receptor tyrosine kinase inhibitor in the treatment of human non-small cell lung cancer (NSCLC). In this study, we investigated the roles of ERK1/2 and AKT signaling pathways in regulating Rad51 expression and cytotoxic effects in different NSCLC cell lines treated with erlotinib. Erlotinib decreased cellular levels of phosphorylated ERK1/2, phosphorylated AKT, Rad51 protein, and mRNA in erlotinib-sensitive H1650, A549, and H1869 cells, leading to cell death via apoptosis, but these results were not seen in erlotinib-resistant H520 and H1703 cells. Erlotinib decreased Rad51 protein levels by enhancing Rad51 mRNA and protein instability. Enforced expression of constitutively active MKK1 or AKT vectors could restore Rad51 protein levels, which were inhibited by erlotinib, and decrease erlotinib-induced cytotoxicity. Knocking down endogenous Rad51 expression by si-Rad51 RNA transfection significantly enhanced erlotinib-induced cytotoxicity. In contrast, overexpression of Rad51 by transfection with Rad51 vector could protect the cells from cytotoxic effects induced by erlotinib. Blocking the activations of ERK1/2 and AKT by MKK1/2 inhibitor (U0126) and phosphoinositide 3-kinase inhibitor (wortmannin) suppressed the expression of Rad51 and enhanced the erlotinib-induced cell death in erlotinib-resistant cells. In conclusion, suppression of Rad51 may be a novel therapeutic modality in overcoming drug resistance of erlotinib in NSCLC. PMID:19671683

Ko, Jen-Chung; Ciou, Shih-Ci; Jhan, Jhih-Yuan; Cheng, Chao-Min; Su, Ying-Jhen; Chuang, Show-Mei; Lin, Szu-Ting; Chang, Chia-Che; Lin, Yun-Wei



Discovery of 2-{3-[2-(1-isopropyl-3-methyl-1H-1,2-4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl]-1H-pyrazol-1-yl}-2-methylpropanamide (GDC-0032): a ?-sparing phosphoinositide 3-kinase inhibitor with high unbound exposure and robust in vivo antitumor activity.  


Dysfunctional signaling through the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway leads to uncontrolled tumor proliferation. In the course of the discovery of novel benzoxepin PI3K inhibitors, we observed a strong dependency of in vivo antitumor activity on the free-drug exposure. By lowering the intrinsic clearance, we derived a set of imidazobenzoxazepin compounds that showed improved unbound drug exposure and effectively suppressed growth of tumors in a mouse xenograft model at low drug dose levels. One of these compounds, GDC-0032 (11l), was progressed to clinical trials and is currently under phase I evaluation as a potential treatment for human malignancies. PMID:23662903

Ndubaku, Chudi O; Heffron, Timothy P; Staben, Steven T; Baumgardner, Matthew; Blaquiere, Nicole; Bradley, Erin; Bull, Richard; Do, Steven; Dotson, Jennafer; Dudley, Danette; Edgar, Kyle A; Friedman, Lori S; Goldsmith, Richard; Heald, Robert A; Kolesnikov, Aleksandr; Lee, Leslie; Lewis, Cristina; Nannini, Michelle; Nonomiya, Jim; Pang, Jodie; Price, Steve; Prior, Wei Wei; Salphati, Laurent; Sideris, Steve; Wallin, Jeffery J; Wang, Lan; Wei, BinQing; Sampath, Deepak; Olivero, Alan G



The Vr-PLC3 gene encodes a putative plasma membrane-localized phosphoinositide-specific phospholipase C whose expression is induced by abiotic stress in mung bean ( Vigna radiata L.) 1 1 EMBL accession numbers: AY394079 ( Vr-PLC1), AY461431 ( Vr-PLC2) and AY394078 ( Vr-PLC3)  

Microsoft Academic Search

Phosphoinositide-specific phospholipase C (PI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate and diacylglycerol, both of which act as secondary messengers in animal cells. In this report, we identified in Vigna radiata L. (mung bean) three distinct partial cDNAs (pVr-PLC1, pVr-PLC2, and pVr-PLC3), which encode forms of putative PI-PLC. All three Vr-PLC genes were transcriptionally active and displayed

Yun Ju Kim; Jee Eun Kim; Jae-Hoon Lee; Myoung Hui Lee; Ho Won Jung; Young Yil Bahk; Byung Kook Hwang; Inhwan Hwang; Woo Taek Kim



CD28 Costimulation: A Source of Vav-1 for TCR Signaling with the Help of SLP-76?  

NSDL National Science Digital Library

T cells require dual stimulation to become activated. When T cells encounter antigen-presenting cells, both the T cell receptor (TCR) and the CD28 coreceptor are ligated and activated. Michel and Acuto discuss how the adaptor SLP-76, which is recruited to the activated TCR complex, and the Rho family guanosine triphosphatase exchanger Vav-1, which is recruited by the CD28 receptor and TCR, may form a macromolecular complex that results in T cells activation. Vav-1 may serve as a central integrator between CD28 signaling and TCR signaling through its indirect effects on phosphoinositide 3-kinase-dependent signaling.

Frederique Michel (Institut Pasteur;Molecular Immunology Unit, Department of Immunology REV); Oreste Acuto (Institut Pasteur;Molecular Immunology Unit, Department of Immunology REV)



Physical Mechanisms of Signal Integration by WASP Family Proteins  

PubMed Central

The proteins of the Wiskott-Aldrich Syndrome protein (WASP) family are activators of the ubiquitous actin nucleation factor, Arp2/3 complex. WASP family proteins contain a C-terminal VCA domain that binds and activates Arp2/3 complex in response to numerous inputs, including Rho family GTPases, phosphoinositide lipids, SH3 domain containing proteins, kinases and phosphatases. In the archetypal members of the family, WASP and N-WASP, these signals are integrated through two levels of regulation, an allosteric autoinhibitory interaction, in which the VCA is sequestered from Arp2/3 complex, and dimerization/oligomerization, in which multi-VCA complexes are better activators of Arp2/3 complex than monomers. Here we review the structural, biochemical and biophysical details of these mechanisms and illustrate how they work together to control WASP protein activity in response to multiple inputs. These regulatory principles, derived from studies of WASP and N-WASP, are likely to apply broadly across the family.

Padrick, Shae B.; Rosen, Michael K.



Computational mechanics  

Microsoft Academic Search

The Computational Mechanics thrust area is a vital and growing facet of the Mechanical Engineering Department at Lawrence Livermore National Laboratory (LLNL). This work supports the development of computational analysis tools in the areas of structural mechanics and heat transfer. Over 75 analysts depend on thrust area-supported software running on a variety of computing platforms to meet the demands of




Lactate Engages Receptor Tyrosine Kinases Axl, Tie2, and Vascular Endothelial Growth Factor Receptor 2 to Activate Phosphoinositide 3-Kinase/Akt and Promote Angiogenesis*  

PubMed Central

Although a high level of lactate is quintessential to both tumors and wound healing, the manner by which lactate impacts endothelial cells to promote angiogenesis and thereby create or restore vascular perfusion to growing tissues has not been fully elucidated. Here we report that lactate activated the PI3K/Akt pathway in primary human endothelial cells. Furthermore, activating this signaling pathway was required for lactate-stimulated organization of endothelial cells into tubes and for sprouting of vessels from mouse aortic explants. Lactate engaged the PI3K/Akt pathway via ligand-mediated activation of the three receptor tyrosine kinases Axl, Tie2, and VEGF receptor 2. Neutralizing the ligands for these receptor tyrosine kinases, pharmacologically inhibiting their kinase activity or suppressing their expression largely eliminated the ability of cells and explants to respond to lactate. Elucidating the mechanism by which lactate communicates with endothelial cells presents a previously unappreciated opportunity to improve our understanding of the angiogenic program and to govern it.

Ruan, Guo-Xiang; Kazlauskas, Andrius



Lactate engages receptor tyrosine kinases Axl, Tie2, and vascular endothelial growth factor receptor 2 to activate phosphoinositide 3-kinase/Akt and promote angiogenesis.  


Although a high level of lactate is quintessential to both tumors and wound healing, the manner by which lactate impacts endothelial cells to promote angiogenesis and thereby create or restore vascular perfusion to growing tissues has not been fully elucidated. Here we report that lactate activated the PI3K/Akt pathway in primary human endothelial cells. Furthermore, activating this signaling pathway was required for lactate-stimulated organization of endothelial cells into tubes and for sprouting of vessels from mouse aortic explants. Lactate engaged the PI3K/Akt pathway via ligand-mediated activation of the three receptor tyrosine kinases Axl, Tie2, and VEGF receptor 2. Neutralizing the ligands for these receptor tyrosine kinases, pharmacologically inhibiting their kinase activity or suppressing their expression largely eliminated the ability of cells and explants to respond to lactate. Elucidating the mechanism by which lactate communicates with endothelial cells presents a previously unappreciated opportunity to improve our understanding of the angiogenic program and to govern it. PMID:23754286

Ruan, Guo-Xiang; Kazlauskas, Andrius



The role of phosphoinositide 3-kinase in the sorting and transport of newly synthesized tyrosinase-related protein-1 (TRP-1).  


Tyrosinase-related protein-1 (TRP-1) is a 75 kDa type-1 transmembrane glycoprotein localized to the melanosome. The mechanism by which newly synthesized TRP-1 reaches its ultimate destination is currently unknown, but has been speculated to occur via the endosomal pathway. Recently, it has been shown that phosphatidylinositide (PI) 3-kinase is involved in various cellular functions, including regulating the constitutive movement of proteins from one intracellular compartment to another; however, whether PI 3-kinase participates in the trafficking of proteins such as TRP-1 to the melanosome is unknown. In this study we investigate the role of PI 3-kinase on the trafficking of TRP-1 in human melanoma MeWo cells using wortmannin, a potent inhibitor of PI 3-kinase. Our investigations demonstrate that wortmannin interferes with the membrane trafficking of TRP-1 in MeWo cells, and that it specifically results in the redistribution of the protein within a novel vesicular compartment with characteristics of the endosomal and lysosomal compartments [positive for LAMP-1, and partially positive for CD63 and cation-independent mannose 6-phosphate receptors (CI-M6PR)], and is accessible to internalized proteins such as immunoglobulins. Movement within this novel compartment is microtubule and GTPase dependent. These findings have led us to postulate that TRP-1 is sorted from the trans-Golgi network to a compartment in the vicinity of late endosomes, trafficking from which to the melanosome appears to be dependent on PI 3-kinase as it is blocked by wortmannin. PMID:11764278

Chen, H; Salopek, T G; Jimbow, K



Threonine-120 Phosphorylation Regulated by Phosphoinositide-3-Kinase/Akt and Mammalian Target of Rapamycin Pathway Signaling Limits the Antitumor Activity of Mammalian Sterile 20-Like Kinase 1*  

PubMed Central

Mst1/Stk4, a hippo-like serine-threonine kinase, is implicated in many cancers, including prostate cancer. However, the mechanisms regulating Mst1 remain obscure. Here, we characterized the effects of phospho-Thr-120 on Mst1 in prostate cancer cells. We demonstrated that phospho-Thr-120 did not alter the nuclear localization or cleavage of Mst1 in a LNCaP or castration-resistant C4-2 prostate tumor cell model, as revealed by a mutagenesis approach. Phospho-Thr-120 appeared to be specific to cancer cells and predominantly localized in the nucleus. In contrast, phospho-Thr-183, a critical regulator of Mst1 cell death, was exclusively found in the cytoplasm. As assessed by immunohistochemistry, a similar distribution of phospho-Mst1-Thr-120/Thr-183 was also observed in a prostate cancer specimen. In addition, the blockade of PI3K signaling by a small molecule inhibitor, LY294002, increased cytoplasmic phospho-Mst1-Thr-183 without having a significant effect on nuclear phospho-Mst1-Thr-120. However, the attenuation of mammalian target of rapamycin (mTOR) activity by a selective pharmacologic inhibitor, Ku0063794 or CCI-779, caused the up-regulation of nuclear phospho-Mst1-Thr-120 without affecting cytoplasmic phospho-Mst1-Thr-183. This suggests that PI3K and mTOR pathway signaling differentially regulate phospho-Mst1-Thr-120/Thr-183. Moreover, mutagenesis and RNAi data revealed that phospho-Thr-120 resulted in C4-2 cell resistance to mTOR inhibition and reduced the Mst1 suppression of cell growth and androgen receptor-driven gene expression. Collectively, these findings indicate that phospho-Thr-120 leads to the loss of Mst1 functions, supporting cancer cell growth and survival.

Collak, Filiz Kisaayak; Yagiz, Kader; Luthringer, Daniel J.; Erkaya, Bahriye; Cinar, Bekir



Neuregulin-1 Regulates Cell Adhesion via an ErbB2/Phosphoinositide-3 Kinase/Akt-Dependent Pathway: Potential Implications for Schizophrenia and Cancer  

PubMed Central

Background Neuregulin-1 (NRG1) is a putative schizophrenia susceptibility gene involved extensively in central nervous system development as well as cancer invasion and metastasis. Using a B lymphoblast cell model, we previously demonstrated impairment in NRG1?-mediated migration in cells derived from patients with schizophrenia as well as effects of risk alleles in NRG1 and catechol-O-methyltransferase (COMT), a second gene implicated both in schizophrenia susceptibility and in cancer. Methodology/Principal Findings Here, we examine cell adhesion, an essential component process of cell motility, using an integrin-mediated cell adhesion assay based on an interaction between ICAM-1 and the CD11a/CD18 integrin heterodimer expressed on lymphoblasts. In our assay, NRG1? induces lymphoblasts to assume varying levels of adhesion characterized by time-dependent fluctuations in the firmness of attachment. The maximum range of variation in adhesion over sixty minutes correlates strongly with NRG1?-induced migration (r2?=?0.61). NRG1?-induced adhesion variation is blocked by erbB2, PI3K, and Akt inhibitors, but not by PLC, ROCK, MLCK, or MEK inhibitors, implicating the erbB2/PI3K/Akt1 signaling pathway in NRG1-stimulated, integrin-mediated cell adhesion. In cell lines from 20 patients with schizophrenia and 20 normal controls, cells from patients show a significant deficiency in the range of NRG1?-induced adhesion (p?=?0.0002). In contrast, the response of patient-derived cells to phorbol myristate acetate is unimpaired. The COMT Val108/158Met genotype demonstrates a strong trend towards predicting the range of the NRG1?-induced adhesion response with risk homozygotes having decreased variation in cell adhesion even in normal subjects (p?=?0.063). Conclusion/Significance Our findings suggest that a mechanism of the NRG1 genetic association with schizophrenia may involve the molecular biology of cell adhesion.

Kanakry, Christopher G.; Li, Zhen; Nakai, Yoko; Sei, Yoshitatsu; Weinberger, Daniel R.



Modern mechanics  

NSDL National Science Digital Library

We consider the goals of the introductory course in classical mechanics taken by physics majors and argue both that these goals are not well met in actual courses and that the goals themselves should be rethought. We propose alternative goals and describe an introductory "modern mechanics" course that addresses these alternative goals. Included in the description are several genres of homework problems that are nearly absent from traditional mechanics courses at both the introductory and intermediate levels. The intermediate mechanics course could be restructured to exploit a broader foundation laid by the introductory course.

Chabay, Ruth; Sherwood, Bruce



Inhibition of phosphoinositide 3-kinase/Akt pathway decreases hypoxia inducible factor-1? expression and increases therapeutic efficacy of paclitaxel in human hypoxic gastric cancer cells  

PubMed Central

The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway plays an important role in cell proliferation, transformation, apoptosis, tumor growth and angiogenesis. Paclitaxel is commonly used to treat multiple human malignancies; however, the underlying mechanisms of paclitaxel in gastric cancer (GC) have not been fully investigated. In the present study, specimens from 45 GC and 36 chronic gastritis patients were collected, and the correlations of PI3K, phosphorylated-Akt (p-Akt) and hypoxia-inducible factor-1? (HIF-1?) expression with the clinicopathological characteristics of GC were analyzed by immunohistochemistry. The human SGC-7901 GC cells under hypoxic conditions were pretreated with the PI3K inhibitor, LY294002 (40 ?M), and paclitaxel (0.1 ?M). The expression levels of PI3K, p-Akt and HIF-1? were detected by quantitative polymerase chain reaction and western blotting. Cell proliferative activity and apoptosis were evaluated by the Cell Counting Kit-8 assay and flow cytometry. As a result, the rates of positive expression of PI3K, p-Akt and HIF-1? were significantly higher in GC compared with chronic gastritis patients (each P<0.01), and were positively associated with the tumor-node-metastasis (TNM) staging, lymph node metastases, lymphatic infiltration and vascular infiltration (each P<0.01), but inversely correlated with tumor differentiation (P<0.01) in patients with GC. Under hypoxic conditions, the combined inhibition of the PI3K/Akt pathway with paclitaxel markedly reduced the proliferative activity and induced cell apoptosis in GC cells compared with the single treatment of PI3K inhibitor or paclitaxel (each P<0.01), and was accompanied by a decreased expression of HIF-1?. Overall, our findings indicate that the increased expression of the PI3K/Akt/HIF-1? pathway was closely correlated with tumor differentiation, TNM staging, lymph node metastases and lymphatic and vascular infiltration. The inhibition of the PI3K/Akt pathway enhanced the therapeutic efficacy of paclitaxel in GC cells under hypoxic conditions, suggesting that the PI3K/Akt/HIF-1? pathway may act as an important therapeutic target for paclitaxel treatment of GC.




YC-1-induced cyclooxygenase-2 expression is mediated by cGMP-dependent activations of Ras, phosphoinositide-3-OH-kinase, Akt, and nuclear factor-kappaB in human pulmonary epithelial cells.  


We demonstrated previously that 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), an activator of soluble guanylate cyclase (sGC), induces cyclooxygenase-2 (COX-2) expression via cGMP- and p44/42 mitogen-activated protein kinase-dependent pathways in human pulmonary epithelial A549 cells. In this study, we explore the role of Ras, phosphoinositide-3-OH-kinase (PI3K), Akt, and transcription factor nuclear factor-kappaB (NF-kappaB) in YC-1-induced COX-2 expression in A549 cells. A Ras inhibitor (manumycin A), a PI3K inhibitor (wortmannin), an Akt inhibitor (1l-6-Hydroxymethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate]), and an NF-kappaB inhibitor [pyrrolidine dithiocarbamate (PDTC)] all reduced YC-1-induced COX-2 expression. The YC-1-induced increase in COX activity was also blocked by manumycin A, wortmannin, PDTC, and the dominant-negative mutants for Ras (RasN17), Akt (Akt DN), and IkappaBalpha (IkappaBalphaM). The YC-1-induced increase in Ras activity was inhibited by an sGC inhibitor [1H-(1,2,4)oxadiazolo[4,3-a]quinozalin-1-one (ODQ)], a protein kinase G (PKG) inhibitor [1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT-5823)], and manumycin A. YC-1-induced Akt activation was also inhibited by ODQ, KT-5823, manumycin A, and wortmannin. YC-1 caused the formation of an NF-kappaB-specific DNA-protein complex and an increase in kappaB-luciferase activity. YC-1-induced kappaB-luciferase activity was inhibited by ODQ, KT-5823, manumycin A, wortmannin, an Akt inhibitor, PDTC, RasN17, Akt DN, and IkappaBalphaM. Likewise, YC-1-induced IKKalpha/beta activation was inhibited by ODQ, KT-5823, manumycin A, wortmannin, and an Akt inhibitor. Furthermore, YC-1-induced COX-2 promoter activity was inhibited by manumycin A, RasN17, Akt DN, PDTC, and IkappaBalphaM. Taken together, these results indicate that YC-1 might activate the sGC/cGMP/PKG pathway to induce Ras and PI3K/Akt activation, which in turn initiates IKKalpha/beta and NF-kappaB activation and finally induces COX-2 expression in A549 cells. PMID:15322248

Chang, Ming-Shyan; Lee, Wen-Sen; Chen, Bing-Chang; Sheu, Joen-Rong; Lin, Chien-Huang



Galanin-Like Peptide (GALP) Neurone-Specific Phosphoinositide 3-Kinase Signalling Regulates GALP mRNA Levels in the Hypothalamus of Males and Luteinising Hormone Levels in Both Sexes.  


Galanin-like peptide (GALP) neurones participate in the metabolic control of reproduction and are targets of insulin and leptin regulation. Phosphoinositide 3-kinase (PI3K) is common to the signalling pathways utilised by both insulin and leptin. Therefore, we investigated whether PI3K signalling in neurones expressing GALP plays a role in the transcriptional regulation of the GALP gene and in the metabolic control of luteinising hormone (LH) release. Accordingly, we deleted PI3K catalytic subunits p110? and p110? via conditional gene targeting (cKO) in mice (GALP-p110?/? cKO). To monitor PI3K signalling in GALP neurones, these animals were also crossed with Cre-dependent FoxO1GFP reporter mice. Compared to insulin-infused control animals, the PI3K-Akt-dependent FoxO1GFP nuclear exclusion in GALP neurones was abolished in GALP-p110?/? cKO mice. We next used food deprivation to investigate whether the GALP-neurone specific ablation of PI3K activity affected the susceptibility of the gonadotrophic axis to negative energy balance. Treatment did not affect LH levels in either sex. However, a significant genotype effect on LH levels was observed in females. By contrast, no genotype effect on LH levels was observed in males. A sex-specific genotype effect on hypothalamic GALP mRNA was observed, with fed and fasted GALP-p110?/? cKO males having lower GALP mRNA expression compared to wild-type fed males. Finally, the effects of gonadectomy and steroid hormone replacement on GALP mRNA levels were investigated. Compared to vehicle-treated mice, steroid hormone replacement reduced mediobasal hypothalamus GALP expression in wild-type and GALP-p110?/? cKO animals. In addition, within the castrated and vehicle-treated group and compared to wild-type mice, LH levels were lower in GALP-p110?/? cKO males. Double immunofluorescence using GALP-Cre/R26-YFP mice showed androgen and oestrogen receptor co-localisation within GALP neurones. Our data demonstrate that GALP neurones are direct targets of steroid hormones and that PI3K signalling regulates hypothalamic GALP mRNA expression and LH levels in a sex-specific fashion. PMID:24796383

Aziz, R; Beymer, M; Negrón, A L; Newshan, A; Yu, G; Rosati, B; McKinnon, D; Fukuda, M; Lin, R Z; Mayer, C; Boehm, U; Acosta-Martínez, M



Grasping Mechanism  

NASA Technical Reports Server (NTRS)

Grasping mechanism captures and locks onto rodlike handle without being precisely aligned with it initially. Mechanism includes two faceplates and three rotatable fingers meshing odd finger between two members of opposite finger pair. Power for fingers supplied by motor powered via harmonic drive reduces speed and increases torque by factor of 160.

Myers, W. Neil; Forbes, John C.



Fractofusion Mechanism  

Microsoft Academic Search

With respect to cold fusion, a fractofusion mechanism is investigated. Deuterons are accelerated by the electric field generated between crack surfaces in a crystal. By assuming the possible magnitude of the potential difference, we calculate the fusion rate and energy multiplication factor. The results are consistent with those of the cold fusion experiments, and the fractofusion mechanism can explain the

Tatsuoki Takeda; Tomonori Takizuka



Geometric Mechanics  

NASA Astrophysics Data System (ADS)

Mechanics for the nonmathematician-a modern approach For physicists, mechanics is quite obviously geometric, yet the classical approach typically emphasizes abstract, mathematical formalism. Setting out to make mechanics both accessible and interesting for nonmathematicians, Richard Talman uses geometric methods to reveal qualitative aspects of the theory. He introduces concepts from differential geometry, differential forms, and tensor analysis, then applies them to areas of classical mechanics as well as other areas of physics, including optics, crystal diffraction, electromagnetism, relativity, and quantum mechanics. For easy reference, Dr. Talman treats separately Lagrangian, Hamiltonian, and Newtonian mechanics-exploring their geometric structure through vector fields, symplectic geometry, and gauge invariance respectively. Practical perturbative methods of approximation are also developed. Geometric Mechanics features illustrative examples and assumes only basic knowledge of Lagrangian mechanics. Of related interest . . . APPLIED DYNAMICS With Applications to Multibody and Mechatronic Systems Francis C. Moon A contemporary look at dynamics at an intermediate level, including nonlinear and chaotic dynamics. 1998 (0-471-13828-2) 504 pp. MATHEMATICAL PHYSICS Applied Mathematics for Scientists and Engineers Bruce Kusse and Erik Westwig A comprehensive treatment of the mathematical methods used to solve practical problems in physics and engineering. 1998 (0-471-15431-8) 680 pp.

Talman, Richard



Cytoskeletal Mechanics  

NASA Astrophysics Data System (ADS)

1. Introduction and the biological basis for cell mechanics Mohammad R. K. Mofrad and Roger Kamm; 2. Experimental measurements of intracellular mechanics Paul Janmey and Christoph Schmidt; 3. The cytoskeleton as a soft glassy material Jeffrey Fredberg and Ben Fabry; 4. Continuum elastic or viscoelastic models for the cell Mohammad R. K. Mofrad, Helene Karcher and Roger Kamm; 5. Multiphasic models of cell mechanics Farshid Guuilak, Mansoor A. Haider, Lori A. Setton, Tod A. Laursen and Frank P. T. Baaijens; 6. Models of cytoskeletal mechanics based on tensegrity Dimitrije Stamenovic; 7. Cells, gels and mechanics Gerald H. Pollack; 8. Polymer-based models of cytoskeletal networks F. C. MacKintosh; 9. Cell dynamics and the actin cytoskeleton James L. McGrath and C. Forbes Dewey, Jr; 10. Active cellular motion: continuum theories and models Marc Herant and Micah Dembo; 11. Summary Mohammad R. K. Mofrad and Roger Kamm.

Mofrad, Mohammad R. K.; Kamm, Roger D.



Cytoskeletal Mechanics  

NASA Astrophysics Data System (ADS)

1. Introduction and the biological basis for cell mechanics Mohammad R. K. Mofrad and Roger Kamm; 2. Experimental measurements of intracellular mechanics Paul Janmey and Christoph Schmidt; 3. The cytoskeleton as a soft glassy material Jeffrey Fredberg and Ben Fabry; 4. Continuum elastic or viscoelastic models for the cell Mohammad R. K. Mofrad, Helene Karcher and Roger Kamm; 5. Multiphasic models of cell mechanics Farshid Guuilak, Mansoor A. Haider, Lori A. Setton, Tod A. Laursen and Frank P. T. Baaijens; 6. Models of cytoskeletal mechanics based on tensegrity Dimitrije Stamenovic; 7. Cells, gels and mechanics Gerald H. Pollack; 8. Polymer-based models of cytoskeletal networks F. C. MacKintosh; 9. Cell dynamics and the actin cytoskeleton James L. McGrath and C. Forbes Dewey, Jr; 10. Active cellular motion: continuum theories and models Marc Herant and Micah Dembo; 11. Summary Mohammad R. K. Mofrad and Roger Kamm.

Mofrad, Mohammad R. K.; Kamm, Roger D.



Signal transduction at point-blank range: analysis of a spatial coupling mechanism for pathway crosstalk.  


The plasma membrane provides a physical platform for the orchestration of molecular interactions and biochemical conversions involved in the early stages of receptor-mediated signal transduction in living cells. In that context, we introduce here the concept of spatial coupling, wherein simultaneous recruitment of different enzymes to the same receptor scaffold facilitates crosstalk between different signaling pathways through the local release and capture of activated signaling molecules. To study the spatiotemporal dynamics of this mechanism, we have developed a Brownian dynamics modeling approach and applied it to the receptor-mediated activation of Ras and the cooperative recruitment of phosphoinositide 3-kinase (PI3K) by activated receptors and Ras. Various analyses of the model simulations show that cooperative assembly of multimolecular complexes nucleated by activated receptors is facilitated by the local release and capture of membrane-anchored signaling molecules (such as active Ras) from/by receptor-bound signaling proteins. In the case of Ras/PI3K crosstalk, the model predicts that PI3K is more likely to be recruited by activated receptors bound or recently visited by the enzyme that activates Ras. By this mechanism, receptor-bound PI3K is stabilized through short-range, diffusion-controlled capture of active Ras and Ras/PI3K complexes released from the receptor complex. We contend that this mechanism is a means by which signaling pathways are propagated and spatially coordinated for efficient crosstalk between them. PMID:18502802

Monine, Michael I; Haugh, Jason M



Signal Transduction at Point-Blank Range: Analysis of a Spatial Coupling Mechanism for Pathway Crosstalk  

PubMed Central

The plasma membrane provides a physical platform for the orchestration of molecular interactions and biochemical conversions involved in the early stages of receptor-mediated signal transduction in living cells. In that context, we introduce here the concept of spatial coupling, wherein simultaneous recruitment of different enzymes to the same receptor scaffold facilitates crosstalk between different signaling pathways through the local release and capture of activated signaling molecules. To study the spatiotemporal dynamics of this mechanism, we have developed a Brownian dynamics modeling approach and applied it to the receptor-mediated activation of Ras and the cooperative recruitment of phosphoinositide 3-kinase (PI3K) by activated receptors and Ras. Various analyses of the model simulations show that cooperative assembly of multimolecular complexes nucleated by activated receptors is facilitated by the local release and capture of membrane-anchored signaling molecules (such as active Ras) from/by receptor-bound signaling proteins. In the case of Ras/PI3K crosstalk, the model predicts that PI3K is more likely to be recruited by activated receptors bound or recently visited by the enzyme that activates Ras. By this mechanism, receptor-bound PI3K is stabilized through short-range, diffusion-controlled capture of active Ras and Ras/PI3K complexes released from the receptor complex. We contend that this mechanism is a means by which signaling pathways are propagated and spatially coordinated for efficient crosstalk between them.

Monine, Michael I.; Haugh, Jason M.



Mechanical memory  


A first-in-first-out (FIFO) microelectromechanical memory apparatus (also termed a mechanical memory) is disclosed. The mechanical memory utilizes a plurality of memory cells, with each memory cell having a beam which can be bowed in either of two directions of curvature to indicate two different logic states for that memory cell. The memory cells can be arranged around a wheel which operates as a clocking actuator to serially shift data from one memory cell to the next. The mechanical memory can be formed using conventional surface micromachining, and can be formed as either a nonvolatile memory or as a volatile memory.

Gilkey, Jeffrey C. (Albuquerque, NM); Duesterhaus, Michelle A. (Albuquerque, NM); Peter, Frank J. (Albuquerque, NM); Renn, Rosemarie A. (Alburquerque, NM); Baker, Michael S. (Albuquerque, NM)



Mechanical memory  


A first-in-first-out (FIFO) microelectromechanical memory apparatus (also termed a mechanical memory) is disclosed. The mechanical memory utilizes a plurality of memory cells, with each memory cell having a beam which can be bowed in either of two directions of curvature to indicate two different logic states for that memory cell. The memory cells can be arranged around a wheel which operates as a clocking actuator to serially shift data from one memory cell to the next. The mechanical memory can be formed using conventional surface micromachining, and can be formed as either a nonvolatile memory or as a volatile memory.

Gilkey, Jeffrey C. (Albuquerque, NM); Duesterhaus, Michelle A. (Albuquerque, NM); Peter, Frank J. (Albuquerque, NM); Renn, Rosemarie A. (Albuquerque, NM); Baker, Michael S. (Albuquerque, NM)