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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



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:



Hydrogen peroxide activates focal adhesion kinase and c-Src by a phosphatidylinositol 3 kinase-dependent mechanism and promotes cell migration in Caco-2 cell monolayers  

PubMed Central

Recent studies showed that c-Src and phosphatidylinositol 3 (PI3) kinase mediate the oxidative stress-induced disruption of tight junctions in Caco-2 cell monolayers. The present study evaluated the roles of PI3 kinase and Src kinase in the oxidative stress-induced activation of focal adhesion kinase (FAK) and acceleration of cell migration. Oxidative stress, induced by xanthine and xanthine oxidase system, rapidly increased phosphorylation of FAK on Y397, Y925, and Y577 in the detergent-insoluble and soluble fractions and increased its tyrosine kinase activity. The PI3 kinase inhibitors, wortmannin and LY294002, and the Src kinase inhibitor, 4-amino-5[chlorophyll]-7-[t-butyl]pyrazolo[3–4-d]pyrimidine, attenuated tyrosine phosphorylation of FAK. Oxidative stress induced phosphorylation of c-Src on Y418 by a PI3 kinase-dependent mechanism, whereas oxidative stress-induced activation of PI3 kinase was independent of Src kinase activity. Hydrogen peroxide accelerated Caco-2 cell migration in a concentration-dependent manner. Promotion of cell migration by hydrogen peroxide was attenuated by LY294002 and PP2. Reduced expression of FAK by siRNA attenuated hydrogen peroxide-induced acceleration of cell migration. The expression of constitutively active c-SrcY527F enhanced cell migration, whereas the expression of dominant negative c-SrcK296R/Y528F attenuated hydrogen peroxide-induced stimulation of cell migration. Oxidative stress-induced activation of c-Src and FAK was associated with a rapid increase in the tyrosine phosphorylation and the levels of paxillin and p130CAS in actin-rich, detergent-insoluble fractions. This study shows that oxidative stress activates FAK and accelerates cell migration in an intestinal epithelium by a PI3 kinase- and Src kinase-dependent mechanism.

Basuroy, Shyamali; Dunagan, Mitzi; Sheth, Parimal; Seth, Ankur



Progesterone increases brain-derived neuroptrophic factor expression and protects against glutamate toxicity in a mitogen-activated protein kinase- and phosphoinositide-3 kinase-dependent manner in cerebral cortical explants.  


The higher prevalence and risk for Alzheimer's disease in women relative to men has been partially attributed to the precipitous decline in gonadal hormone levels that occurs in women following the menopause. Although considerable attention has been focused on the consequence of estrogen loss, and thus estrogen's neuroprotective potential, it is important to recognize that the menopause results in a precipitous decline in progesterone levels as well. In fact, progesterone is neuroprotective, although the precise mechanisms involved remain unclear. Based on our previous observation that progesterone elicits the phosphorylation of ERK and Akt, key effectors of the neuroprotective mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3-K) pathways, respectively, we determined whether activation of either of these pathways was necessary for progesterone-induced protection. With organotypic explants (slice culture) of the cerebral cortex, we found that progesterone protected against glutamate-induced toxicity. Furthermore, these protective effects were inhibited by either the MEK1/2 inhibitor UO126 or the PI3-K inhibitor LY294002, supporting the requirement for both the MAPK and PI3-K pathways in progesterone-induced protection. In addition, at a concentration and duration of treatment consistent with our neuroprotection data, progesterone also increased the expression of brain-derived neurotrophic factor (BDNF), at the level of both protein and mRNA. This induction of BDNF may be relevant to the protective effects of progesterone, in that inhibition of Trk signaling, with K252a, inhibited the protective effects of progesterone. Collectively, these data suggest that progesterone is protective via multiple and potentially related mechanisms. (c) 2007 Wiley-Liss, Inc. PMID:17549730

Kaur, Paramjit; Jodhka, Parmeet K; Underwood, Wendy A; Bowles, Courtney A; de Fiebre, Nancyellen C; de Fiebre, Christopher M; Singh, Meharvan



Role of PI3-kinase-dependent Bad phosphorylation and altered transcription in cytokine-mediated neutrophil survival  

Microsoft Academic Search

Phosphoinositide 3-kinase (PI3-kinase)- dependent phosphorylation of the pro- apoptotic Bcl-2 family member Bad has been proposed as an important regulator of apoptotic cell death. To understand the importance of this pathway in nontrans- formed hematopoietic cells, we have ex- amined the effect of survival cytokines on PI3-kinase activity and Bad expression and phosphorylation status in human neu- trophils. Granulocyte macrophage-colony-

Andrew S. Cowburn; Karen A. Cadwallader; Benjamin J. Reed; Neda Farahi; Edwin R. Chilvers



Transforming growth factor beta1 inhibits cystic fibrosis transmembrane conductance regulator-dependent cAMP-stimulated alveolar epithelial fluid transport via a phosphatidylinositol 3-kinase-dependent mechanism.  


Exogenous or endogenous beta(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 beta1 (TGF-beta1), a critical mediator of acute lung injury, inhibits beta(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 beta(2)-adrenergic receptors. Consistent with these in vitro results, inhibition of the PI3K pathway or pretreatment with soluble chimeric TGF-beta type II receptor restored beta(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-beta1 in impairing the beta- 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. PMID:19996317

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



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



Phosphoinositide 3-Kinase C2? Regulates Cytoskeletal Organization and Cell Migration via Rac-dependent Mechanisms  

PubMed Central

Receptor-linked class I phosphoinositide 3-kinases (PI3Ks) induce assembly of signal transduction complexes through protein–protein and protein–lipid interactions that mediate cell proliferation, survival, and migration. Although class II PI3Ks have the potential to make the same phosphoinositides as class I PI3Ks, their precise cellular role is currently unclear. In this report, we demonstrate that class II phosphoinositide 3-kinase C2? (PI3KC2?) associates with the Eps8/Abi1/Sos1 complex and is recruited to the EGF receptor as part of a multiprotein signaling complex also involving Shc and Grb2. Increased expression of PI3KC2? stimulated Rac activity in A-431 epidermoid carcinoma cells, resulting in enhanced membrane ruffling and migration speed of the cells. Conversely, expression of dominant negative PI3KC2? reduced Rac activity, membrane ruffling, and cell migration. Moreover, PI3KC2?-overexpressing cells were protected from anoikis and displayed enhanced proliferation, independently of Rac function. Taken together, these findings suggest that PI3KC2? regulates the migration and survival of human tumor cells by distinct molecular mechanisms.

Katso, Roy M.; Pardo, Olivier E.; Palamidessi, Andrea; Franz, Clemens M.; Marinov, Marin; De Laurentiis, Angela; Downward, Julian; Scita, Giorgio; Ridley, Anne J.; Waterfield, Michael D.



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



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



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.



Mechanism of Constitutive Phosphoinositide 3Kinase Activation by Oncogenic Mutants of the p85 Regulatory Subunit  

Microsoft Academic Search

p85\\/p110 phosphoinositide 3-kinases regulate multi- ple cell functions and are frequently mutated in human cancer. The p85 regulatory subunit stabilizes and inhib- its the p110 catalytic subunit. The minimal fragment of p85 capable of regulating p110 is the N-terminal SH2 domain linked to the coiled-coil iSH2 domain (referred to as p85ni). We have previously proposed that the con- formationally rigid

S. Chandra Shekar; Haiyan Wu; Zheng Fu; Shu-Chin Yip; Sean M. Cahill; Mark E. Girvin; Jonathan M. Backer



Vav1 Transduces T Cell Receptor Signals to the Activation of Phospholipase C- ? 1 via Phosphoinositide 3-Kinase-dependent and -independent Pathways  

Microsoft Academic Search

Vav1 is a signal transducing protein required for T cell receptor (TCR) signals that drive posi- tive and negative selection in the thymus. Furthermore, Vav1-deficient thymocytes show greatly reduced TCR-induced intracellular calcium flux. Using a novel genetic system which allows the study of signaling in highly enriched populations of CD4 ? CD8 ? double positive thymocytes, we have studied the

Lucinda F. Reynolds; Lesley A. Smyth; Trisha Norton; Norman Freshney; Julian Downward; Dimitris Kioussis


Phosphoinositide-dependent kinase 1 targets protein kinase A in a pathway that regulates interleukin 4  

PubMed Central

CD28 plays a critical role in T cell immune responses. Although the kinase Akt has been shown to act downstream of CD28 in T helper (Th)1 cytokine induction, it does not induce Th2 cytokines such as interleukin 4 (IL-4). We recently reported that phosphoinositide-dependent kinase 1 (PDK1) partially corrects the defect in IL-4 production present in CD28-deficient T cells, suggesting that PDK1 regulates IL-4 independently of Akt. We now describe a signaling pathway in which PDK1 targets IL-4 in the murine Th2 cell line D10. PDK1-mediated activation of this pathway is dependent on protein kinase A (PKA) and the nuclear factor of activated T cells (NFAT) P1 transcriptional element in the IL-4 promoter. PDK1 localizes to the immune synapse in a phosphatidylinositol 3-kinase–dependent manner, partially colocalizes with PKA at the synapse, and physically interacts with PKA. In RNA interference knockdown experiments, PDK1 is necessary for phosphorylation of PKA in T cells, as well as for activation of the IL-4 NFAT P1 element by the T cell receptor (TCR) and CD28. Phosphorylation of the critical PKA threonine residue is stimulated by engagement of TCR/CD28 via a PDK1-dependent mechanism. These findings together define a pathway linking the kinases PDK1 and PKA in the induction of the Th2 cytokine IL-4.

Nirula, Ajay; Ho, Mary; Phee, Hyewon; Roose, Jeroen; Weiss, Arthur



Phosphoinositides as Regulators of Protein-Chromatin Interactions  

NSDL National Science Digital Library

The molecular function of phospholipids in the nucleus has been only partially elucidated. The upsurge of epigenetic research has contributed to increased interest in nuclear phospholipids, such as phosphoinositides, and their involvement in gene transcription. However, the mechanisms by which phosphoinositides regulate transcription is still unknown at the molecular level. Certain phosphoinositide species can regulate protein-chromatin and protein–nucleic acid interactions, and specific nuclear target proteins link nuclear signaling lipids to gene expression. We propose that a phosphoinositide-mediated detachment of proteins from chromatin is a general biological mechanism that partly underlies the signaling effects of nuclear phosphoinositides.

Keijo Viiri (School of Medicine and Tampere University Hospital;University of Tampere REV); Markku Maki (School of Medicine and Tampere University Hospital;University of Tampere REV); Olli Lohi (School of Medicine and Tampere University Hospital;University of Tampere REV)



Mechanical stimuli regulate rapamycin-sensitive signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism.  

PubMed Central

In response to growth factors, mTOR (mammalian target of rapamycin) has been identified as a central component of the signalling pathways that control the translational machinery and cell growth. Signalling through mTOR has also been shown to be necessary for the mechanical load-induced growth of cardiac and skeletal muscles. Although the mechanisms involved for mechanically induced activation of mTOR are not known, it has been suggested that activation of PI3K (phosphoinositide 3-kinase) and protein kinase B (Akt), via the release of locally acting growth factors, underlies this process. In the present study, we show that mechanically stimulating (passive stretch) the skeletal muscle ex vivo results in the activation of mTOR-dependent signalling events. The activation of mTOR-dependent signalling events was necessary for an increase in translational efficiency, demonstrating the physiological significance of this pathway. Using pharmacological inhibitors, we show that activation of mTOR-dependent signalling occurs through a PI3K-independent pathway. Consistent with these results, mechanically induced signalling through mTOR was not disrupted in muscles from Akt1-/- mice. In addition, ex vivo co-incubation experiments, along with in vitro conditioned-media experiments, demonstrate that a mechanically induced release of locally acting autocrine/paracrine growth factors was not sufficient for the activation of the mTOR pathway. Taken together, our results demonstrate that mechanical stimuli can activate the mTOR pathway independent of PI3K/Akt1 and locally acting growth factors. Thus mechanical stimuli and growth factors provide distinct inputs through which mTOR co-ordinates an increase in the translational efficiency.

Hornberger, Troy A; Stuppard, Rudy; Conley, Kevin E; Fedele, Mark J; Fiorotto, Marta L; Chin, Eva R; Esser, Karyn A



Migration of Th1 lymphocytes is regulated by CD152 (CTLA-4)-mediated signaling via PI3 kinase-dependent Akt activation.  


Efficient adaptive immune responses require the localization of T lymphocytes in secondary lymphoid organs and inflamed tissues. To achieve correct localization of T lymphocytes, the migration of these cells is initiated and directed by adhesion molecules and chemokines. It has recently been shown that the inhibitory surface molecule CD152 (CTLA-4) initiates Th cell migration, but the molecular mechanism underlying this effect remains to be elucidated. Using CD4 T lymphocytes derived from OVA-specific TCR transgenic CD152-deficient and CD152-competent mice, we demonstrate that chemokine-triggered signal transduction is differentially regulated by CD152 via phosphoinositide 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt). In the presence of CD152 signaling, the chemoattractant CCL4 selectively induces the full activation of Akt via phosphorylation at threonine 308 and serine 473 in pro-inflammatory Th lymphocytes expressing the cognate chemokine receptor CCR5. Akt signals lead to cytoskeleton rearrangements, which are indispensable for migration. Therefore, this novel Akt-modulating function of CD152 signals affecting T cell migration demonstrates that boosting CD152 or its down-stream signal transduction could aid therapies aimed at sensitizing T lymphocytes for optimal migration, thus contributing to a precise and effective immune response. PMID:22412835

Knieke, Karin; Lingel, Holger; Chamaon, Kathrin; Brunner-Weinzierl, Monika C



Insulin Activation of Mitogen-Activated Protein (MAP) Kinase and Akt Is Phosphatidylinositol 3-Kinase-Dependent in Rat Adipocytes  

Microsoft Academic Search

To explore the mechanism of MAP kinase activation in adipocytes, we examined the possible involvement of several candidate signaling proteins. MAP kinase activity was markedly increased 2–4 min after treatment with insulin and declined to basal levels after 20 min. The insulin-dependent tyrosine phosphorylation of IRS-1 in the internal membrane and its association with phosphatidylinositol 3 (PI3) kinase preceded MAP

Hongzhi Liu; Bassil Kublaoui; Paul F. Pilch; Jongsoon Lee



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



Interferon-? activates transglutaminase 2 via a phosphatidylinositol-3-kinase-dependent pathway: implications for celiac sprue therapy.  


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. PMID:22228808

Diraimondo, Thomas R; Klöck, Cornelius; Khosla, Chaitan



Phosphoinositides and cell growth.  


The findings described above illustrate how the src kinase can influence several new pathways of inositol phosphate metabolism, both at the membrane level with the production of novel D-3 phosphoinositides and the activation of PI-3 kinase, and at the cytosolic level by altering the expression of certain inositol polyphosphates, in particular Ins(1,4,5,6)P4. At present, it is difficult to speculate on the role these phenomena play in cellular transformation by src, since the functions of D-3 phosphoinositides and most inositol polyphosphates are unclear. There is evidence, however, that these new pathways of phosphoinositide metabolism occur in response to other types of cellular stimulations besides src transformation. Novel D-3 phosphoinositides are expressed in a variety of nonneoplastic cells, including human platelets treated with thrombin, smooth muscle cells and stimulated neutrophils. In addition, unusual InsP4 isomers such as D/L-Ins(1,4,5,6)P4 are found in chicken erythrocytes, murine macrophages, AR4-2J rat pancreatoma cells and adrenal glomerulosa cells, to name only a few. Recently, associations have been reported between PI-3 kinases and cytoskeletal elements in thrombin- stimulated platelets, and between activated ras proteins in rat liver epithelial cells. The latter discovery is particularly intriguing since GTP-binding proteins such as ras are known to influence cell shape and serve as downstream effector proteins in the signal transduction pathways of numerous growth factor receptors. Thus, one function of novel phosphoinositides and their metabolites may lie at the level of cytoskeletal and cell shape regulation. Clearly, additional roles for phosphoinositides exist in cells besides their traditional use as precursors for the generation of Ins(1,4,5)P3 and diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1333166

Wasilenko, W J



TGF?-induced PI 3 kinase-dependent Mnk-1 activation is necessary for Ser-209 phosphorylation of eIF4E and mesangial cell hypertrophy.  


Transforming growth factor? (TGF?)-induced canonical signal transduction is involved in glomerular mesangial cell hypertrophy; however, the role played by the noncanonical TGF? signaling remains largely unexplored. TGF? time-dependently stimulated eIF4E phosphorylation at Ser-209 concomitant with enhanced phosphorylation of Erk1/2 (extracellular signal regulated kinase1/2) and MEK (mitogen-activated and extracellular signal-regulated kinase kinase) in mesangial cells. Inhibition of Erk1/2 by MEK inhibitor or by expression of dominant negative Erk2 blocked eIF4E phosphorylation, resulting in attenuation of TGF?-induced protein synthesis and mesangial cell hypertrophy. Expression of constitutively active (CA) MEK was sufficient to induce protein synthesis and hypertrophy similar to those induced by TGF?. Pharmacological or dominant negative inhibition of phosphatidylinositol (PI) 3 kinase decreased MEK/Erk1/2 phosphorylation leading to suppression of eIF4E phosphorylation. Inducible phosphorylation of eIF4E at Ser-209 is mediated by Mnk-1 (mitogen-activated protein kinase signal-integrating kinase-1). Both PI 3 kinase and Erk1/2 promoted phosphorylation of Mnk-1 in response to TGF?. Dominant negative Mnk-1 significantly inhibited TGF?-stimulated protein synthesis and hypertrophy. Interestingly, inhibition of mTORC1 activity, which blocks dissociation of eIF4E-4EBP-1 complex, decreased TGF?-stimulated phosphorylation of eIF4E without any effect on Mnk-1 phosphorylation. Furthermore, mutant eIF4E S209D, which mimics phosphorylated eIF4E, promoted protein synthesis and hypertrophy similar to TGF?. These results were confirmed using phosphorylation deficient mutant of eIF4E. Together our results highlight a significant role of dissociation of 4EBP-1-eIF4E complex for Mnk-1-mediated phosphorylation of eIF4E. Moreover, we conclude that TGF?-induced noncanonical signaling circuit involving PI 3 kinase-dependent Mnk-1-mediated phosphorylation of eIF4E at Ser-209 is required to facilitate mesangial cell hypertrophy. PMID:23359369

Das, Falguni; Ghosh-Choudhury, Nandini; Bera, Amit; Kasinath, Balakuntalam S; Choudhury, Goutam Ghosh



Early activation of mTORC1 signalling in response to mechanical overload is independent of phosphoinositide 3-kinase/Akt signalling  

PubMed Central

Abstract The mammalian target of rapamycin complex 1 (mTORC1) functions as a central integrator of a wide range of signals that modulate protein metabolism and cell growth. However, the contributions of individual pathways regulating mTORC1 activity in skeletal muscle are poorly defined. The purpose of this study was to determine the regulatory mechanisms that contribute to mTORC1 activation during mechanical overload-induced skeletal muscle hypertrophy. Consistent with previous studies, mechanical overload induced progressive hypertrophy of the plantaris muscle which was associated with significant increases in total RNA content and protein metabolism. mTORC1 was activated after a single day of overload as indicated by a significant increase in S6K1 phosphorylation at T389 and T421/S424. In contrast, Akt activity, as assessed by Akt phosphorylation status (T308 and S473), phosphorylation of direct downstream targets (glycogen synthase kinase 3 ?, proline-rich Akt substrate 40 kDa and tuberous sclerosis 2 (TSC2)) and a kinase assay, was not significantly increased until 2–3 days of overload. Inhibition of phosphoinositide 3-kinase (PI3K) activity by wortmannin was sufficient to block insulin-dependent signalling but did not prevent the early activation of mTORC1 in response to overload. We identified that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent pathway was activated at day 1 after overload. In addition, a target of MEK/ERK signalling, phosphorylation of TSC2 at S664, was also increased at this early time point. These observations demonstrate that in vivo, mTORC1 activation at the early phase of mechanical overload in skeletal muscle occurs independently of PI3K/Akt signalling and provide evidence that the MEK/ERK pathway may contribute to mTORC1 activation through phosphorylation of TSC2.

Miyazaki, Mitsunori; McCarthy, John J; Fedele, Mark J; Esser, Karyn A



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.



Insulin-like growth factor-1 stimulates rat prolactin gene expression by a Ras, ETS and phosphatidylinositol 3- kinase dependent mechanism  

Microsoft Academic Search

We have examined the influence of insulin-like growth factor I (IGF-1) on prolactin gene expression in rat pituitary GH4C1 cells. Incubation with IGF-1 increases prolactin mRNA levels and activates the prolactin promoter in transient transfection assays. A similar degree of activation is observed with constructs extending to ?3000 and ?176 base pairs of the prolactin 5? flanking region, indicating that

Ana I Castillo; Rosa M Tolon; Ana Aranda



Regulation of transient receptor potential (TRP) channels by phosphoinositides  

Microsoft Academic Search

This review summarizes the modulation of transient receptor potential (TRP) channels, by phosphoinositides. TRP channels are\\u000a characterized by polymodal activation and a surprising complexity of regulation mechanisms. Possibly, most if not all TRP\\u000a channels are modulated by phosphoinositides. Modulation by phosphatidylinositol 4,5-biphosphate (PIP2) has been shown in detail for TRP vanilloid (TRPV) 1, TRPV5, TRP melastatin (TRPM) 4, TRPM5, TRPM7,

Tibor Rohacs; Bernd Nilius



Molecular characteristics of phosphoinositide binding  

Microsoft Academic Search

Phosphoinositides in general and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) in particular have been recently found to function as important regulators of ion channels. Yet, while specific residues\\u000a have been identified that affect channel–PIP2 interactions, the precise binding site of PIP2 has not been determined in any case. In addition to binding ion channels, however, phosphoinositides interact with a plethora\\u000a of

Avia Rosenhouse-Dantsker; Diomedes E. Logothetis



Class III phosphoinositide 3-kinase--Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes.  

PubMed Central

Increased proteolysis contributes to muscle atrophy that prevails in many diseases. Elucidating the signalling pathways responsible for this activation is of obvious clinical importance. Autophagy is a ubiquitous degradation process, induced by amino acid starvation, that delivers cytoplasmic components to lysosomes. Starvation markedly stimulates autophagy in myotubes, and the present studies investigate the mechanisms of this regulation. In C(2)C(12) myotubes incubated with serum growth factors, amino acid starvation stimulated autophagic proteolysis independently of p38 and p42/p44 mitogen-activated protein kinases, but in a PI3K (phosphoinositide 3-kinase)-dependent manner. Starvation, however, did not alter activities of class I and class II PI3Ks, and was not sufficient to affect major signalling proteins downstream from class I PI3K (glycogen synthase kinase, Akt/protein kinase B and protein S6). In contrast, starvation increased class III PI3K activity in whole-myotube extracts. In fact, this increase was most pronounced for a population of class III PI3K that coimmunoprecipitated with Beclin1/Apg6 protein, a major determinant in the initiation of autophagy. Stimulation of proteolysis was reproduced by feeding myotubes with synthetic dipalmitoyl-PtdIns3 P, the class III PI3K product. Conversely, protein transfection of anti-class III PI3K inhibitory antibody into starved myotubes inverted the induction of proteolysis. Therefore, independently of class I PI3K/Akt, protein S6 and mitogen-activated protein kinase pathways, amino acid starvation stimulates proteolysis in myotubes by regulating class III PI3K-Beclin1 autophagic complexes.

Tassa, Amina; Roux, Marie Paule; Attaix, Didier; Bechet, Daniel M



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



The role of phosphoinositides in membrane transport  

Microsoft Academic Search

Phosphoinositides serve as intrinsic membrane signals that regulate intracellular membrane trafficking. Recently, phosphoinositides have been found to direct the localization and activity of effector proteins containing consensus sequence motifs such as FYVE, PH and ENTH domains. In addition, recent results show that regulated synthesis and turnover of phosphoinositides by membrane-associated phosphoinoside kinases and phosphatases spatially restrict the location of effectors

Anne Simonsen; Andrew E Wurmser; Scott D Emr; Harald Stenmark



The anti-apoptotic effect of IGF-1 on tissue resident stem cells is mediated via PI3-kinase dependent secreted frizzled related protein 2 (Sfrp2) release  

SciTech Connect

Previous studies suggest that IGF-1 may be used as an adjuvant to stem cell transfer in order to improve cell engraftment in ischemic tissue. In the current study, we investigated the effect of IGF-1 on serum deprivation and hypoxia induced stem cell apoptosis and the possible mechanisms involved. Exposure of adipose tissue derived stem cells (ASCs) to serum deprivation and hypoxia resulted in significant apoptosis in ASC which is partially prevented by IGF-1. IGF-1's anti-apoptotic effect was abolished in ASCs transfected with Sfrp2 siRNA but not by the control siRNA. Using Western blot analysis, we demonstrated that serum deprivation and hypoxia reduced the expression of nuclear {beta}-catenin, which is reversed by IGF-1. IGF-1's effect on {beta}-catenin expression was abolished by the presence of PI3-kinase inhibitor LY294002 or in ASCs transfected with Sfrp2 siRNA. These results suggest that IGF-1, through the release of the Sfrp2, contributes to cell survival by stabilizing {beta}-catenin.

Gehmert, Sebastian; Sadat, Sanga; Song Yaohua; Yan Yasheng [Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, SCRB2, Box 951, 7435 Fannin Street, Houston, TX 77030 (United States); Alt, Eckhard [Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, SCRB2, Box 951, 7435 Fannin Street, Houston, TX 77030 (United States)], E-mail:



Prostaglandin E2 induces interleukin-6 expression in human chondrocytes via cAMP/protein kinase A- and phosphatidylinositol 3-kinase-dependent NF-?B activation  

PubMed Central

Elevated levels of prostaglandin (PG)E2 and interleukin (IL)-6 have been reported in the cartilage and synovial fluid from patients with arthritic disorders. PGE2 regulates IL-6 production in numerous different cells including macrophages and synovial fibroblasts. Although PGE2 stimulates IL-6 expression in human chondrocytes, the underlying signaling pathway of this process has yet to be delineated. Here, we investigate the mechanism of IL-6 induction in human T/C-28a2 chondrocytes treated with exogenously added PGE2. PGE2 induces IL-6 mRNA and protein expression via a cAMP-dependent pathway, reaching maximal levels after 60 min of stimulation before declining to baseline levels at 6 h. Forskolin, an adenylyl cyclase activator, also stimulates IL-6 expression in human chondrocytes in a dose- and time-dependent fashion. Inhibition of downstream effectors of cAMP activity such as protein kinase A (PKA) or phosphatidylinositol 3 kinase (PI3K) blocks PGE2- and forskolin-induced IL-6 upregulation. Simultaneous inhibition of PKA and PI3K reduces IL-6 expression in stimulated chondrocytes well below the basal levels of untreated cells. Gel shift, supershift, and chromatin immunoprecipitation assays reveal the activation and binding of the nuclear factor (NF)-?B p65 subunit to the IL-6 promoter, which is markedly suppressed by selective PI3K or PKA pharmacological inhibitors. p65 knockdown completely abrogates IL-6 mRNA synthesis in PGE2- and forskolin-primed chondrocytes. Cumulatively, our data show that PGE2 and forskolin induce IL-6 expression in human chondrocytes via cAMP/PKA and PI3K-dependent pathways, which in turn regulate the activation and binding of p65 to the IL-6 promoter.

Wang, Pu; Zhu, Fei



Phosphoinositide Signaling: New Tools and Insights  

NSDL National Science Digital Library

Phosphoinositides constitute only a small fraction of cellular phospholipids, yet their importance in the regulation of cellular functions can hardly be overstated. The rapid metabolic response of phosphoinositides after stimulation of certain cell surface receptors was the first indication that these lipids could serve as regulatory molecules. These early observations opened research areas that ultimately clarified the plasma membrane role of phosphoinositides in Ca2+ signaling. However, research of the last 10 years has revealed a much broader range of processes dependent on phosphoinositides. These lipids control organelle biology by regulating vesicular trafficking, and they modulate lipid distribution and metabolism more generally via their close relationship with lipid transfer proteins. Phosphoinositides also regulate ion channels, pumps, and transporters as well as both endocytic and exocytic processes. The significance of phosphoinositides found within the nucleus is still poorly understood, and a whole new research concerns the highly phosphorylated inositols that also appear to control multiple nuclear processes. The expansion of research and interest in phosphoinositides naturally created a demand for new approaches to determine where, within the cell, these lipids exert their effects. Imaging of phosphoinositide dynamics within live cells has become a standard cell biological method. These new tools not only helped us localize phosphoinositides within the cell but also taught us how tightly phosphoinositide control can be linked with distinct effector protein complexes. The recent progress allows us to understand the underlying causes of certain human diseases and design new strategies for therapeutic interventions.



Melatonin agonists induce phosphoinositide hydrolysis in Xenopus laevis melanophores.  


Melatonin, the principal hormone of the vertebrate pineal gland, has been implicated in a variety of neurobiological processes such as circadian rhythmicity and reproductive function. One of the earliest described actions of melatonin was its ability to cause pigment translocation in the dermal melanophores of amphibians. Melatonin binding sites have been identified in the brain of many species and in pigmented tumour cell lines; however, the dermal melanophores of the frog Xenopus Laevis possess the highest known density of melatonin binding sites. These cells are the source from which a melatonin receptor has been cloned and provide an excellent model to study melatonin-mediated signal transduction in an isolated cell system. In Xenopus melanophores, melatonin induces a rapid perinuclear aggregation of intracellular pigment which is associated with a pertussis toxin-sensitive inhibition of cAMP. We have previously demonstrated that a subtype of melatonin binding sites found in selected regions of the pigeon brain and in Syrian Hamster RPMI 1846 melatonin cells are functionally coupled to phosphoinositide hydrolysis as a second messenger. Here we now present evidence to suggest that Xenopus Laevis melanophores also possess melatonin binding sites which are functionally linked to phosphoinositide hydrolysis. Melatonin agonists induced phosphoinositide hydrolysis in melanophores in a concentration-dependent manner with a rank order of potency of 2-iodomelatonin > 6-chloromelatonin > N-acetylserotonin > melatonin. Stimulatory response of 2-iodomelatonin was blocked by the melatonin antagonist N-acetyltryptamine and the alpha-adrenergic antagonist prazosin, which has been shown to have high affinity for melatonin binding sites. Phosphoinositide hydrolysis induced by melatonin agonists was not blocked by the serotonin antagonist ketanserin or by phentolamine, an alpha-adrenergic antagonist, indicating that the response observed was not due to stimulation of 5-HT2a/2c receptors or alpha-adrenergic receptors. Furthermore, incubation of melanophores with the non-hydrolyzable G-protein source GTP-gamma-S attenuated the phosphoinositide dose response induced by 2-iodomelatonin, and pre-incubation of the cells with pertussis toxin had no effect on 2-iodomelatonin-induced phosphoinositide hydrolysis. The present data suggest that Xenopus Laevis Melanophores possess G-protein linked pertussis toxin-insensitive melatonin binding sites which are functionally coupled to phosphoinositide hydrolysis as a signal transduction mechanism. PMID:9113416

Mullins, U L; Fernandes, P B; Eison, A S



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 metabolism and insulin secretion.  


Secretion of insulin from beta cells of the pancreatic islets is regulated by glucose, its anaerobic metabolism and its metabolites. The phospholipids of the cell membrane the phosphoinositides are broken down by the activation of the enzyme phospholipase C either through the occupation of the receptor by an agonist or through the metabolism of glucose in the anaerobic glycolytic pathway. The hydrolysis of the phosphotidyl inositide-bisphosphate yields to the generation of Inositol 1, 4, 5-trisphosphate and diacylglycerol. Ins-1, 4, 5-P3 increases the intracellular Ca2+ by releasing the sequestered Ca2+ in the endoplasmic reticulum and diacylglycerol activates the enzyme protein kinase C. PMID:7805952

Ilkova, H M


Ion Induced Changes in Phosphoinositide Monolayers at Phisiological Concentrations  

NASA Astrophysics Data System (ADS)

Phosphoinositides (PIPs) play a crucial role in many cellular process that occur at the plasma membrane such as calcium release, exocytosis or endocytosis. In order to specifically regulate these functions PIPs must segregate in pools at the plasma membrane. A possible mechanism that could induce and regulate such organization of phosphoinositides is their interaction with bivalent cations. 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 calcium and magnesium on the surface pressure-area/lipid isotherm of monolayer of phosphatidylinositol (PI), phosphatidylinositol bisphosphate (PIP2), dioleoylphosphatidylglycerol (DOPG) and palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). It is found that the decrease of area per lipid, i.e. the increase in aggregation, is mostly dependent on the lipid's head group charge but ion specific. In addition, we discuss changes in free energy and compressibility of these monolayer-ion systems.

Kazadi Badiambile, Adolphe; Forstner, Martin B.



The Phox homology (PX) domain, a new player in phosphoinositide signalling.  

PubMed Central

Phosphoinositides are key regulators of diverse cellular processes. The pleckstrin homology (PH) domain mediates the action of PtdIns(3,4)P(2), PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), while the FYVE domain relays the pulse of PtdIns3P. The recent establishment that the Phox homology (PX) domain interacts with PtdIns3P and other phosphoinositides suggests another mechanism by which phosphoinositides can regulate/integrate multiple cellular events via a spectrum of PX domain-containing proteins. Together with the recent discovery that the epsin N-terminal homologue (ENTH) domain interacts with PtdIns(4,5)P(2), it is becoming clear that phosphoinositides regulate diverse cellular events through interactions with several distinct structural motifs present in many different proteins.

Xu, Y; Seet, L F; Hanson, B; Hong, W



Crystal structure of a mammalian phosphoinositide-specific phospholipase Cdelta  

Microsoft Academic Search

Mammalian phosphoinositide-specific phospholipase C enzymes (PI-PLC) act as signal transducers that generate two second messengers, inositol-l,4,5-trisphosphate and diacylglycerol. The 2.4-Å structure of phospholipase Cdelta1 reveals a multidomain protein incorporating modules shared by many signalling proteins. The structure suggests a mechanism for membrane attachment and Ca2+ -dependent hydrolysis of second-messenger precursors. The regulation and reversible membrane association of PI-PLC may serve

Lars-Oliver Essen; Olga Perisic; Robert Cheung; Matilda Katan; Roger L. Williams



Aluminum interaction with phosphoinositide-associated signal transduction  

Microsoft Academic Search

Concerning molecular and cellular mechanisms of aluminum toxicity, recent studies support the hypothesis that interactions\\u000a of aluminum ions with elements of signal transduction pathways are apparently primary events in cells. In the case of the\\u000a phosphoinositide-associated signalling pathway of neuroblastoma cells, guanine nucleotide-binding proteins (G proteins) and\\u000a a phosphatidylinositol-4,5-diphosphate (PIP2)-specific phospholipase C are probable interaction sites for inhibitory actions of

Alfred Haug; Biao Shi; Victor Vitorello



Phosphoinositide signaling and the regulation of membrane trafficking in yeast  

Microsoft Academic Search

Phosphoinositides are key regulators of diverse cellular processes in eukaryotic cells. Genetic studies in yeast have advanced our understanding of how phosphoinositide-signaling pathways regulate membrane trafficking. Enzymes required for the synthesis (kinases) and turnover (phosphatases) of distinct phosphoinositides have been identified and several downstream effector molecules linked to phosphoinositide signaling have recently been characterized.

Greg Odorizzi; Markus Babst; Scott D Emr



Stress-ING Out: Phosphoinositides Mediate the Cellular Stress Response  

NSDL National Science Digital Library

Phosphoinositides regulate numerous cellular processes required for growth, proliferation, and motility. Whereas phosphoinositide signal transduction pathways within the cytosol have been well characterized, nuclear signaling pathways remain poorly understood. Accumulating experimental data have now started to uncover critical functions for nuclear phosphoinositides. In particular, phosphoinositides modulate the activity of the tumor suppressor protein ING2 in response to extracellular stress. These findings highlight a previously uncharacterized function for phosphoinositides and implicate their metabolism in signaling pathways critical for cell survival.

Matthew W. Bunce (University of Wisconsin;Department of Pharmacology REV); Michael L. Gonzales (University of Wisconsin;Department of Pharmacology REV); Richard A. Anderson (University of Wisconsin;Department of Pharmacology REV)



Membrane phosphoinositides and protein-membrane interactions.  


Proteins with polybasic clusters bind to negatively charged phosphoinositides at the cell membrane. In this review, I have briefly discussed the types of phosphoinositides naturally found on membrane surfaces and how they recruit protein complexes for carrying out the process of signal transduction. A large number of researchers from around the world are now focusing their attention on protein-membrane binding, as these interactions have started to offer us a much better insight into the process of cell signaling. The main areas discussed in this brief review article include the phosphoinositide binding specificities of proteins and the role of their lipid binding in signaling processes downstream of membrane recruitment. PMID:23824360

Gokhale, Nikhil A



Prostaglandins Activate Phosphoinositide Metabolism in Rat Aorta.  

National Technical Information Service (NTIS)

The ability of the five prostaglandins to activate phosphoinositide (PI) metabolism and to induce contraction was studied in rat aorta. All prostaglandins (PG) tested stimulated PI hydrolysis and elicited contractions. The activation of PI hydrolysis by t...

E. A. Suba B. L. Roth



Activation of Phosphoinositide Metabolism by Cholinergic Agents.  

National Technical Information Service (NTIS)

The primary acute, toxic effect of cholinergic agonists in the central nervous system is seizures. One system activated by cholinergic agonists is the hydrolysis of phosphoinositides (PI), a major site of action of lithium which potentiates convulsions as...

R. S. Jope



Pleckstrin homology (PH) domains and phosphoinositides.  


PH (pleckstrin homology) domains represent the 11th most common domain in the human proteome. They are best known for their ability to bind phosphoinositides with high affinity and specificity, although it is now clear that less than 10% of all PH domains share this property. Cases in which PH domains bind specific phosphoinositides with high affinity are restricted to those phosphoinositides that have a pair of adjacent phosphates in their inositol headgroup. Those that do not [PtdIns3P, PtdIns5P and PtdIns(3,5)P2] are instead recognized by distinct classes of domains including FYVE domains, PX (phox homology) domains, PHD (plant homeodomain) fingers and the recently identified PROPPINs (b-propellers that bind polyphosphoinositides). Of the 90% of PH domains that do not bind strongly and specifically to phosphoinositides, few are well understood. One group of PH domains appears to bind both phosphoinositides (with little specificity) and Arf (ADP-ribosylation factor) family small G-proteins, and are targeted to the Golgi apparatus where both phosphoinositides and the relevant Arfs are both present. Here, the PH domains may function as coincidence detectors. A central challenge in understanding the majority of PH domains is to establish whether the very low affinity phosphoinositide binding reported in many cases has any functional relevance. For PH domains from dynamin and from Dbl family proteins, this weak binding does appear to be functionally important, although its precise mechanistic role is unclear. In many other cases, it is quite likely that alternative binding partners are more relevant, and that the observed PH domain homology represents conservation of structural fold rather than function. PMID:17233582

Lemmon, Mark A



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


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. PMID:23492670

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



Acute Manipulation of Phosphoinositide Levels in Cells  

PubMed Central

Phosphoinositides are membrane-bound signaling phospholipids that function in a myriad of cellular processes, including membrane trafficking, cytoskeletal dynamics, ion channel and transporter function, and signal transduction. In order to better understand the role of phosphoinositides in cellular processes, different approaches to study the effects of the presence or absence of these lipids must be devised. Conventional approaches of manipulating phosphoinositide levels such as over-expression or genetic ablation of lipid enzymes cause prolonged exposure of the cells to changes in lipid levels that could result in compensatory actions by the cell or downstream alterations in cell physiology. In this chapter we present an approach used recently by various laboratories, including our own, to acutely manipulate phosphoinositide levels at target locations using chemically induced dimerization (CID) that can be spatially and temporally controlled. We discuss considerations when designing expression constructs for targeting specific cellular compartment membranes and present examples from the literature on different ways of perturbing phosphoinositide levels at particular organelle membranes using CID. In addition, we provide details on image acquisition, data collection and data interpretation. CID technology can be applied to many lipid enzymes to broaden the understanding of the role lipid signaling plays in cell physiology.

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



Phosphatidylserine binding is essential for plasma membrane recruitment and signaling function of 3-phosphoinositide-dependent kinase-1.  


3-Phosphoinositide-dependent kinase-1 (PDK1) is a ubiquitously expressed serine/threonine kinase that functions downstream of phosphoinositide 3-kinase. Although binding of 3'-phosphoinositides, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate, to the pleckstrin homology (PH) domain of PDK1 is known to be essential for its interaction with and activation of downstream kinases, the mechanism by which PDK1 is recruited to the plasma membrane remains controversial. Our surface plasmon resonance analysis of the PDK1 PH domain and selected mutants shows that the PH domain specifically binds phosphatidylserine using a site that is separate from the canonical phosphoinositide-binding site. Further cell studies show that this specific phosphatidylserine binding is important for the plasma membrane localization and signaling function of PDK1. PMID:21971045

Lucas, Nathan; Cho, Wonhwa



CDP-diacylglycerol synthetase-controlled phosphoinositide availability limits VEGFA signaling and vascular morphogenesis  

PubMed Central

Understanding the mechanisms that regulate angiogenesis and translating these into effective therapies are of enormous scientific and clinical interests. In this report, we demonstrate the central role of CDP-diacylglycerol synthetase (CDS) in the regulation of VEGFA signaling and angiogenesis. CDS activity maintains phosphoinositide 4,5 bisphosphate (PIP2) availability through resynthesis of phosphoinositides, whereas VEGFA, mainly through phospholipase C?1, consumes PIP2 for signal transduction. Loss of CDS2, 1 of 2 vertebrate CDS enzymes, results in vascular-specific defects in zebrafish in vivo and failure of VEGFA-induced angiogenesis in endothelial cells in vitro. Absence of CDS2 also results in reduced arterial differentiation and reduced angiogenic signaling. CDS2 deficit-caused phenotypes can be successfully rescued by artificial elevation of PIP2 levels, and excess PIP2 or increased CDS2 activity can promote excess angiogenesis. These results suggest that availability of CDS-controlled resynthesis of phosphoinositides is essential for angiogenesis.

Pan, Weijun; Pham, Van N.; Stratman, Amber N.; Castranova, Daniel; Kamei, Makoto; Kidd, Kameha R.; Lo, Brigid D.; Shaw, Kenna M.; Torres-Vazquez, Jesus; Mikelis, Constantinos M.; Gutkind, J. Silvio; Davis, George E.



Tandem regulation of phosphoinositide signaling and acute behavioral effects induced by antidepressant agents in rats  

Microsoft Academic Search

Rationale  Antidepressants increase synaptic monoamine concentrations, but the subsequent signaling events that produce the beneficial\\u000a clinical effects remain unclear. Diverse antidepressants increase CDP-diacylglycerol, a crucial step in phosphoinositide signaling.\\u000a Serotonin 5HT2 receptors, implicated in depression or the actions of some antidepressants, signal through phosphoinositide hydrolysis. Thus,\\u000a cross talk between antidepressant-induced CDP-diacylglycerol and 5HT2 signaling could contribute to the antidepressant mechanism.\\u000a \\u000a \\u000a \\u000a Objective  The

Kimberly R. Tyeryar; Ashiwel S. Undie



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.



Physical Foundations of PTEN/Phosphoinositide Interaction  

NASA Astrophysics Data System (ADS)

Phosphoinositides act as signaling molecules by recruiting critical effectors to specific subcellular membranes to regulate cell proliferation, apoptosis and cytoskeletal reorganization, which requires a tight regulation of phosphoinositide generation and turnover as well as a high degree of compartmentalization. PTEN is a phosphatase specific for the 3 position of the phosophoinositide ring that is deleted or mutated in many different disease states. PTEN association with membranes requires the interaction of its C2 domain with phosphatidylserine and the interaction of its N-terminal end with phosphatidylinositol-4,5-bisphophate (PI(4,5)P2). We have investigated PTEN/PI(4,5)P2 interaction and found that Lys13 is crucial for the observed binding. We also found that the presence of cholesterol enhances PTEN binding to mixed PI(4,5)P2/POPC vesicles. Fluorescence microscopy experiments utilizing GUVs yielded results consistent with enhanced phosphoinositide domain formation in the presence of cholesterol. These experiments were accompanied by zeta potential measurements and solid state MAS ^31P-NMR experiments aimed at investigating the ionization behavior of phosphoinositides.

Gericke, Arne; Jiang, Zhiping; Redfern, Roberta E.; Kooijman, Edgar E.; Ross, Alonzo H.



Maitotoxin: Effects on calcium channels, phosphoinositide breakdown, and arachidonate release in pheochromocytoma PC12 cells  

SciTech Connect

Maitotoxin (MTX) increases formation of (3H)inositol phosphates from phosphoinositides and release of (3H)arachidonic acid from phospholipids in pheochromocytoma PC12 cells. Formation of (3H)inositol phosphates is detected within 1 min of incubation even with concentrations as low as 0.3 ng/ml (90 pm) MTX, whereas release of (3H)arachidonic acid is not detected until 20 min even with concentrations as high as 1 ng/ml (300 pm) MTX. Stimulation of arachidonic acid release can be detected at 0.03 ng/ml (9 pm) MTX, whereas 0.1 ng/ml (30 pm) MTX is the threshold for detection of phosphoinositide breakdown. Organic and inorganic calcium channel blockers, except Cd2+ and a high concentration of Mn2+, have no effect on MTX-elicited phosphoinositide breakdown, whereas inorganic blockers (e.g., Co2+, Mn2+, Cd2+), but not organic blockers (nifedipine, verapamil, diltiazem), inhibit MTX-stimulated arachidonic acid release. All calcium channel blockers, however, inhibited MTX-elicited influx of 45Ca2+ and the MTX-elicited increase in internal Ca2+ measured with fura-2 was markedly reduced by nifedipine. MTX-elicited phosphoinositide breakdown and arachidonic acid release are abolished or reduced, respectively, in the absence of extracellular calcium plus chelating agent. The calcium ionophore A23187 has little or no effect alone but, in combination with MTX, A23187 inhibits MTX-elicited phosphoinositide breakdown and enhances arachidonic acid release, the latter even in the absence of extracellular calcium. The results suggest that different sites and/or mechanisms are involved in stimulation of calcium influx, breakdown of phosphoinositides, and release of arachidonic acid by MTX.

Choi, O.H.; Padgett, W.L.; Nishizawa, Y.; Gusovsky, F.; Yasumoto, T.; Daly, J.W. (National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD (USA))



Nuclear Phosphoinositides: A Signaling Enigma Wrapped in a Compartmental Conundrum  

PubMed Central

While the presence of phosphoinositides in the nuclei of eukaryotes and the identity of the enzymes responsible for their metabolism have been known for some time, their functions in the nucleus are only now emerging. This is illustrated by the recent identification of effectors for nuclear phosphoinositides. Like the cytosolic phosphoinositide signaling pathway, nuclear phosphatidylinositol 4,5 bisphosphate (PI4,5P2) is at the center of the pathway and acts both as a messenger and as a precursor for many additional messengers. Here, recent advances in the understanding of nuclear phosphoinositide signaling and its functions are reviewed with an emphasis on PI4,5P2 and its role in gene expression. The compartmentalization of nuclear phosphoinositide phosphates (PIPn) remains a mystery, but emerging evidence suggests that phosphoinositides occupy several functionally distinct compartments.

Barlow, Christy A.; Laishram, Rakesh S.; Anderson, Richard A.



TPIP: a novel phosphoinositide 3-phosphatase.  

PubMed Central

The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor is a phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] 3-phosphatase that plays a critical role in regulating many cellular processes by antagonizing the phosphoinositide 3-kinase signalling pathway. We have identified and characterized two human homologues of PTEN, which differ with respect to their subcellular localization and lipid phosphatase activities. The previously cloned, but uncharacterized, TPTE (transmembrane phosphatase with tensin homology) is localized to the plasma membrane, but lacks detectable phosphoinositide 3-phosphatase activity. TPIP (TPTE and PTEN homologous inositol lipid phosphatase) is a novel phosphatase that occurs in several differentially spliced forms of which two, TPIP alpha and TPIP beta, appear to be functionally distinct. TPIP alpha displays similar phosphoinositide 3-phosphatase activity compared with PTEN against PtdIns(3,4,5)P(3), PtdIns(3,5)P(2), PtdIns(3,4)P(2) and PtdIns(3)P, has N-terminal transmembrane domains and appears to be localized on the endoplasmic reticulum. This is unusual as most signalling-lipid-metabolizing enzymes are not integral membrane proteins. TPIP beta, however, lacks detectable phosphatase activity and is cytosolic. TPIP has a wider tissue distribution than the testis-specific TPTE, with specific splice variants being expressed in testis, brain and stomach. TPTE and TPIP do not appear to be functional orthologues of the Golgi-localized and more distantly related murine PTEN2. We suggest that TPIP alpha plays a role in regulating phosphoinositide signalling on the endoplasmic reticulum, and might also represent a tumour suppressor and functional homologue of PTEN in some tissues.

Walker, S M; Downes, C P; Leslie, N R



Role of phosphoinositides in STIM1 dynamics and store-operated calcium entry  

PubMed Central

Ca2+ entry through store-operated Ca2+ channels involves the interaction at ER–PM (endoplasmic reticulum–plasma membrane) junctions of STIM (stromal interaction molecule) and Orai. STIM proteins are sensors of the luminal ER Ca2+ concentration and, following depletion of ER Ca2+, they oligomerize and translocate to ER–PM junctions where they form STIM puncta. Direct binding to Orai proteins activates their Ca2+ channel function. It has been suggested that an additional interaction of the C-terminal polybasic domain of STIM1 with PM phosphoinositides could contribute to STIM1 puncta formation prior to binding to Orai. In the present study, we investigated the role of phosphoinositides in the formation of STIM1 puncta and SOCE (store-operated Ca2+ entry) in response to store depletion. Treatment of HeLa cells with inhibitors of PI3K (phosphatidylinositol 3-kinase) and PI4K (phosphatidylinositol 4-kinase) (wortmannin and LY294002) partially inhibited formation of STIM1 puncta. Additional rapid depletion of PtdIns(4,5)P2 resulted in more substantial inhibition of the translocation of STIM1–EYFP (enhanced yellow fluorescent protein) into puncta. The inhibition was extensive at a concentration of LY294002 (50 ?M) that should primarily inhibit PI3K, consistent with a major role for PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in puncta formation. Depletion of phosphoinositides also inhibited SOCE based on measurement of the rise in intracellular Ca2+ concentration after store depletion. Overexpression of Orai1 resulted in a recovery of translocation of STMI1 into puncta following phosphoinositide depletion and, under these conditions, SOCE was increased to above control levels. These observations support the idea that phosphoinositides are not essential but contribute to STIM1 accumulation at ER–PM junctions with a second translocation mechanism involving direct STIM1–Orai interactions.

Walsh, Ciara M.; Chvanov, Michael; Haynes, Lee P.; Petersen, Ole H.; Tepikin, Alexei V.; Burgoyne, Robert D.



FERM Domain Phosphoinositide Binding Targets Merlin to the Membrane and Is Essential for Its Growth-Suppressive Function ?  

PubMed Central

The neurofibromatosis type 2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of plasma membrane-actin cytoskeleton linkers. For ezrin, phosphatidylinositol 4,5-bisphosphate (PIP2) binding to the amino-terminal FERM domain is required for its conformational activation, proper subcellular localization, and function, but less is known about the role of phosphoinositide binding for merlin. Current evidence indicates that association with the membrane is important for merlin to function as a growth regulator; however, the mechanisms by which merlin localizes to the membrane are less clear. Here, we report that merlin binds phosphoinositides, including PIP2, via a conserved binding motif in its FERM domain. Abolition of FERM domain-mediated phosphoinositide binding of merlin displaces merlin from the membrane and releases it into the cytosol without altering the folding of merlin. Importantly, a merlin protein whose FERM domain cannot bind phosphoinositide is defective in growth suppression. Retargeting the mutant merlin into the membrane using a dual-acylated amino-terminal decapeptide from Fyn is sufficient to restore the growth-suppressive properties to the mutant merlin. Thus, FERM domain-mediated phosphoinositide binding and membrane association are critical for the growth-regulatory function of merlin.

Mani, Timmy; Hennigan, Robert F.; Foster, Lauren A.; Conrady, Deborah G.; Herr, Andrew B.; Ip, Wallace



Regulation of cytokine-independent survival kinase (CISK) by the Phox homology domain and phosphoinositides  

Microsoft Academic Search

KB\\/Akt and serum and glucocorticoid-regulated kinase (SGK) family kinases are important downstream tar- gets of phosphatidylinositol 3 (PI-3) kinase and have been shown to mediate a variety of cellular processes, in- cluding cell growth and survival. Although regulation of Akt can be achieved through several mechanisms, includ- ing its phosphoinositide-binding Pleckstrin homology (PH) domain, how SGK kinases are targeted and

Jun Xu; Dan Liu; Gordon Gill; Zhou Songyang



Cross-talk between phospholipase C and phosphoinositide 3-kinase signalling pathways.  


1321N1 astrocytoma cells have proved a valuable model system in which to study interactions between two major PtdIns (4,5) P2-utilizing signaling pathways, since they possess receptor populations which elicit independent activation of PI 3-kinase and a G-protein-dependent PLC respectively. Activation of PLC down-regulates PI 3-kinase by at least two mechanisms involving inhibition of IRS-1-associated PI 3-kinase and acute activation of a PtdIns (3,4,5) P3 5-phosphatase. PKB, which is an important early PI 3-kinase-dependent component of insulin signalling pathways, is also down-regulated by PLC-coupled agonists. The activation of PKB by insulin appears to involve a novel PtdIns (3,4,5) P3-dependent protein kinase, which we have named PDK1. The molecular mechanisms underlying PtdIns (3,4,5) P3-stimulated phosphorylation and activation of PKB by PDK1 are currently under investigation. PMID:9449962

Batty, I H; Hickinson, D M; Downes, C P



The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides  

SciTech Connect

Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.

Zhang, Yanfeng; Varnum, Susan M.



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))



Noradrenaline stimulation unbalances the phosphoinositide cycle in rat cerebral cortical slices.  


Muscarinic cholinergic and alpha 1-adrenoceptor-mediated stimulation of phosphoinositide hydrolysis in rat cerebral cortex were compared by measuring carbachol- and noradrenaline-induced accumulation of various intermediates of the phosphoinositide cycle. Unlike carbachol, noradrenaline in the presence of guanosine 5'-O-(3-thiotriphosphate) did not stimulate phospholipase C activity in brain cortical membranes. In cortical slices, the efficacy of noradrenaline to stimulate accumulation of 3H-inositol phosphates and [32P]phosphatidic acid was 2.5 to threefold that of carbachol. However, noradrenaline was less effective than carbachol in stimulating accumulation of [3H]CDP-diacylglycerol and resynthesis of phosphatidylinositol. This was not due to calcium inhibition of CTP:phosphatidate cytidyltransferase or to different lithium requirements for carbachol- and noradrenaline-stimulated accumulation of [3H]CDP-diacylglycerol. The noradrenaline-induced unbalance of the phosphoinositide cycle, which was most apparent at relatively high concentrations of calcium (2.5 mM) in the incubation buffer, was qualitatively reproduced with ionomycin. The use of the alpha 1a-subtype-selective adrenoceptor antagonists WB4101 and 5-methylurapidil revealed a single alpha 1a-like component mediating the effects of noradrenaline. Our results suggest that the primary mechanism for phospholipase C activation by brain alpha 1 adrenoceptors involves an increase in intracellular calcium concentration. PMID:8388033

Claro, E; Fain, J N; Picatoste, F



Cellular calcium mobilization in response to phosphoinositide delivery  

Microsoft Academic Search

Intracellular calcium [Ca2+]i is mobilized in many cell types in response to activation of phosphoinositide (PIPn) signaling pathways involving PtdIns(4,5)P2 or PtdIns(3,4,5)P3. To further explore the relationship between increases in intracellular PIPn concentrations and mobilization of [Ca2+]i, each of the seven phosphorylated phosphoinositides (PIPns) were delivered into cells and the metabolism and physiological effects of the exogenously administered PIPns were

Daryll B. DeWald; Shoichiro Ozaki; Swati Malaviya; Joseph C. Shope; Kelly Manabe; Lee Crosby; Paul Neilsen; Derrick Johnston; Sitaram Harihar; Glenn D. Prestwich



Regulation of Bin1 SH3 domain binding by phosphoinositides  

PubMed Central

Bin1/M-amphiphysin-II is an amphiphysin-II isoform highly expressed in transverse tubules of adult striated muscle and is implicated in their biogenesis. Bin1 contains a basic unique amino-acid sequence, Exon10, which interacts with certain phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), to localize to membranes. Here we found that Exon10 also binds to the src homology 3 (SH3) domain of Bin1 itself, and hence blocks the binding of the SH3 domain to its canonical PxxP ligands, including dynamin. This blockage was released by addition of PI(4,5)P2 in vitro or in cells overexpressing phosphatidylinositol 4-phosphate 5-kinase. The Exon10-binding interface of the Bin1 SH3 domain largely overlapped with its PxxP-binding interface. We also show that the PLC? pleckstrin homology domain, another PI(4,5)P2-binding module, cannot substitute for Exon10 in Bin1 function in transverse tubule formation, and suggest the importance of the dual biochemical properties of Exon10 in myogenesis. Our results exemplify a novel mechanism of SH3 domain regulation, and suggest that the SH3-mediated protein–protein interactions of Bin1 are regulated by Exon10 so that it may only occur when Bin1 localizes to certain submembrane areas.

Kojima, Chie; Hashimoto, Ari; Yabuta, Izumi; Hirose, Mayumi; Hashimoto, Shigeru; Kanaho, Yasunori; Sumimoto, Hideki; Ikegami, Takahisa; Sabe, Hisataka



Structure, function, and control of phosphoinositide-specific phospholipase C.  


Phosphoinositide-specific phospholipase C (PLC) subtypes beta, gamma, and delta comprise a related group of multidomain phosphodiesterases that cleave the polar head groups from inositol lipids. Activated by all classes of cell surface receptor, these enzymes generate the ubiquitous second messengers inositol 1,4, 5-trisphosphate and diacylglycerol. The last 5 years have seen remarkable advances in our understanding of the molecular and biological facets of PLCs. New insights into their multidomain arrangement and catalytic mechanism have been gained from crystallographic studies of PLC-delta(1), while new modes of controlling PLC activity have been uncovered in cellular studies. Most notable is the realization that PLC-beta, -gamma, and -delta isoforms act in concert, each contributing to a specific aspect of the cellular response. Clues to their true biological roles were also obtained. Long assumed to function broadly in calcium-regulated processes, genetic studies in yeast, slime molds, plants, flies, and mammals point to specific and conditional roles for each PLC isoform in cell signaling and development. In this review we consider each subtype of PLC in organisms ranging from yeast to mammals and discuss their molecular regulation and biological function. PMID:11015615

Rebecchi, M J; Pentyala, S N



Phosphoinositide metabolism and adrenergic receptors in astrocytes  

SciTech Connect

Agonist-induced phosphoinositide (PI) breakdown functions as a signal generating system. Diacylglycerol, one breakdown product of phosphotidylinositol-4,5-diphosphate hydrolysis, can stimulate protein kinase C, whereas inositol triphosphate, the other product, has been proposed to be a second messenger for Ca/sup + +/ mobilization. Using purified astrocyte cultures from neonatal rat brain, the effects of adrenergic agonists and antagonists at 10/sup -5/ M were measured on PI breakdown. Astrocytes grown in culture were prelabeled with (/sup 3/H)inositol, and basal (/sup 3/H) inositol phosphate (IP/sub 1/) accumulation was measured in the presence of Li/sup +/. Epinephrine > norepinephrine (NE) were the most active stimulants of IP/sub 1/ production. The ..cap alpha../sub 1/ adrenoreceptor blockers, phentolamine and phenoxybenzamine, added alone had no effect on IP/sub 1/ production was reduced below basal levels. Propranolol partially blocked the effects of NE. Clonidine and isoproterenol, separately added, reduced IP/sub 1/ below basal levels and when added together diminished IP/sub 1/ accumulation even further. The role of adrenergic stimulation in the production of c-AMP.

Noble, E.P.; Ritchie, T.; de Vellis, J.



The Phosphoinositide Kinase PIKfyve Is Vital in Early Embryonic Development  

PubMed Central

Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases. In mammals, PIKfyve synthesizes PtdIns(3,5)P2 and PtdIns5P lipids that regulate endosomal trafficking and responses to extracellular stimuli. The consequence of pikfyve gene ablation in mammals is unknown. To clarify the importance of PIKfyve and PIKfyve lipid products, in this study, we have characterized the first mouse model with global deletion of the pikfyve gene using the Cre-loxP approach. We report that nearly all PIKfyveKO/KO mutant embryos died before the 32–64-cell stage. Cultured fibroblasts derived from PIKfyveflox/flox embryos and rendered pikfyve-null by Cre recombinase expression displayed severely reduced DNA synthesis, consistent with impaired cell division causing early embryo lethality. The heterozygous PIKfyveWT/KO mice were born at the expected Mendelian ratio and developed into adulthood. PIKfyveWT/KO mice were ostensibly normal by several other in vivo, ex vivo, and in vitro criteria despite the fact that their levels of the PIKfyve protein and in vitro enzymatic activity in cells and tissues were 50–55% lower than those of wild-type mice. Consistently, steady-state levels of the PIKfyve products PtdIns(3,5)P2 and PtdIns5P selectively decreased, but this reduction (35–40%) was 10–15% less than that expected based on PIKfyve protein reduction. The nonlinear decrease of the PIKfyve protein versus PIKfyve lipid products, the potential mechanism(s) discussed herein, may explain how one functional allele in PIKfyveWT/KO mice is able to support the demands for PtdIns(3,5)P2/PtdIns5P synthesis during life. Our data also shed light on the known human disorder linked to PIKFYVE mutations.

Ikonomov, Ognian C.; Sbrissa, Diego; Delvecchio, Khortnal; Xie, Yufen; Jin, Jian-Ping; Rappolee, Daniel; Shisheva, Assia



Phosphoinositide 3-kinase inhibition enables retinoic acid-induced neurogenesis in monolayer culture of embryonic stem cells.  


Retinoic acid (RA) is able to induce the differentiation of embryonic stem cells into neuronal lineages. The mechanism of this effect is unknown but it has been evidenced to be dependent on the formation of floating spheroids called embryoid bodies. Results presented here show that the inhibition of phosphoinositide 3-kinase signaling pre-determines mouse embryonic stem cells to RA induced neurogenesis in monolayer culture with no need of embryoid bodies formation. PMID:21948563

Kotasová, Hana; Veselá, Iva; Ku?era, Jan; Houdek, Zbyn?k; Procházková, Ji?ina; Králi?ková, Milena; Pacherník, Ji?í



Dynamic Regulation of Phosphoinositide 3Kinase Activity and Adrenergic Receptor Trafficking in End-Stage Human Heart Failure  

Microsoft Academic Search

Background—Downregulation of -adrenergic receptors (ARs) under conditions of heart failure requires receptor targeting of phosphoinositide 3-kinase (PI3K)- and redistribution of ARs into endosomal compartments. Because support with a left ventricular assist device (LVAD) results in significant improvement of cardiac function in humans, we investigated the effects of mechanical unloading on regulation of PI3K activity and intracellular distribution of ARs. Additionally,

Cinzia Perrino; Jacob N. Schroder; Brian Lima; Nestor Villamizar; Jeffrey J. Nienaber; Carmelo A. Milano; Sathyamangla V. Naga Prasad



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


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. PMID:23359693

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



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



Structural basis of phosphoinositide binding to kindlin-2 protein pleckstrin homology domain in regulating integrin activation.  


Kindlins are a subclass of FERM-containing proteins that have recently emerged as key regulators of integrin receptor activation and signaling. As compared with the conventional FERM domain, the kindlin FERM domain contains an inserted pleckstrin homology (PH) domain that recognizes membrane phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). Using NMR spectroscopy, we show that PIP3 site-specifically binds to kindlin-2 PH with substantial chemical shift changes that are much larger than PIP2. This suggests an enhanced association of kindlin-2 with membrane as mediated by PIP3 upon its conversion from PIP2 by phosphoinositide-3 kinase, a known regulator of integrin activation. We determined the NMR structure of the kindlin-2 PH domain bound to the head group of PIP3, inositol 1,3,4,5-tetraphosphate (IP4). The structure reveals a canonical PH domain fold, yet with a distinct IP4 binding pocket that appears highly conserved for the kindlin family members. Functional experiments demonstrate that although wild type kindlin-2 is capable of cooperating with integrin activator talin to induce synergistic integrin ?(IIb)?(3) activation, this ability is significantly impaired for a phosphoinositide binding-defective kindlin-2 mutant. These results define a specific PIP3 recognition mode for the kindlin PH domain. Moreover, they shed light upon a mechanism as to how the PH domain mediates membrane engagement of kindlin-2 to promote its binding to integrin and cooperation with talin for regulation of integrin activation. PMID:22030399

Liu, Jianmin; Fukuda, Koichi; Xu, Zhen; Ma, Yan-Qing; Hirbawi, Jamila; Mao, Xian; Wu, Chuanyue; Plow, Edward F; Qin, Jun



Physiologic implications of phosphoinositides and phospholipase C in the regulation of insulin secretion.  


The secretion of insulin from the pancreatic beta-cell must be commensurate to satisfy the insulin requirements of the organism. This cell has a great flexibility to meet these requirements which are increased not only by the ingestion of nutrients (increase of plasma glucose) but also by the sensitivity of target tissues to insulin as well. The insulin secretion is a complex biochemical event regulated by a host of potential second messenger molecules acting alone or in concert. These events include the cation calcium, which gains access to the beta-cell via the opening of voltage-regulated channels, cAMP and phosphoinositide-derived second messenger molecules, generated as a consequence of phospholipase C (PLC) activation. In this review, we focused on phosphoinositides, PLC/Phosphokinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) cascade in the regulation of insulin secretion. We also described our studies on the mechanism of the beta-cell desensitization using perifused islets. It is suggested that a failure of the signaling events contribute to the pathogenesis of diabetes in which the beta-cell can no longer secrete the required amounts of insulin. It has been observed that chronic exposure to high glucose desensitizes the beta-cells to subsequent stimulation. We suggested that the failure of PLC activation can be attributed in the impairment of insulin secretion by chronic sustained glucose exposure. It may contribute to the vicious circle of impaired insulin secretion leading up to diabetes. PMID:20354339

Yamazaki, Hanae; Zawalich, Kathleen C; Zawalich, Walter S



Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis.  


Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH(2)PO(4) and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH(2)PO(4) concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2×10(5) in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10(-18)-10(-20)mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and robust separation method for SFL and S1PFL and various metabolites of phosphoinositide-related signal transduction is expected to enable improved enzymatic assays for biological and clinical applications. PMID:23000742

Wang, Kelong; Jiang, Dechen; Sims, Christopher E; Allbritton, Nancy L



Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis  

PubMed Central

Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH2PO4 and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH2PO4 concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2×105 in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10?18 to 10?20 mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and robust separation method for SFL and S1PFL and various metabolites of phosphoinositide-related signal transduction is expected to enable improved enzymatic assays for biological and clinical applications.

Wang, Kelong; Jiang, Dechen; Sims, Christopher E.; Allbritton, Nancy L.



Desensitization and antagonism of vasopressin-induced phosphoinositide metabolism and elevation of cytosolic free calcium concentration in human platelets.  

PubMed Central

The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration-dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM-vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1-receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane-A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization.

Pollock, W K; MacIntyre, D E



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.



Phosphoinositides Direct Equine Infectious Anemia Virus Gag Trafficking and Release  

PubMed Central

Phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), the predominant phosphoinositide on the plasma membrane, binds the matrix (MA) protein of Human Immunodeficiency Virus type 1 (HIV-1) and Equine Infectious Anemia Virus (EIAV) with similar affinities in vitro. Interaction with PI(4,5)P2 is critical for HIV-1 assembly on the plasma membrane. EIAV has been shown to localize in internal compartments hence the significance of its interaction with PI(4,5)P2 is unclear. We therefore investigated the binding in vitro of other phosphoinositides to EIAV MA and whether intracellular association with compartments bearing these phosphoinositides was important for assembly and release of virus-like particles (VLPs) formed by Gag. In vitro, EIAV MA bound PI(3)P with higher affinity than PI(4,5)P2 as revealed by NMR spectra upon lipid titration. Gag was detected on the plasma membrane and in compartments enriched in PI(3,5)P2. Treatment of cells with YM201636, a kinase inhibitor that blocks production of PI(3,5)P2 from PI(3)P, caused Gag to co-localize with aberrant compartments and inhibited VLP release. In contrast to HIV-1, release of EIAV VLPs was not significantly diminished by co-expression with 5-phosphatase IV, an enzyme that specifically depletes PI(4,5)P2 from the plasma membrane. However, co-expression with synaptojanin 2, a phosphatase with broader specificity, diminished VLP production. PI-binding pocket mutations caused striking budding defects, as revealed by electron microscopy. One of the mutations also modified Gag-Gag interaction, as suggested by altered bimolecular fluorescence complementation. We conclude that phosphoinositide-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release.

Fernandes, Fiona; Chen, Kang; Ehrlich, Lorna S.; Jin, Jing; Chen, Min H.; Medina, Gisselle N.; Symons, Marc; Montelaro, Ronald; Donaldson, Julie; Tjandra, Nico; Carter, Carol A.



Targeting the phosphoinositide 3-kinase pathway in cancer  

Microsoft Academic Search

The phosphoinositide 3-kinase (PI3K) pathway is a key signal transduction system that links oncogenes and multiple receptor classes to many essential cellular functions, and is perhaps the most commonly activated signalling pathway in human cancer. This pathway therefore presents both an opportunity and a challenge for cancer therapy. Even as inhibitors that target PI3K isoforms and other major nodes in

Pixu Liu; Hailing Cheng; Thomas M. Roberts; Jean J. Zhao



Phosphoinositide phosphatases: just as important as the kinases.  


Phosphoinositide phosphatases comprise several large enzyme families with over 35 mammalian enzymes identified to date that degrade many phosphoinositide signals. Growth factor or insulin stimulation activates the phosphoinositide 3-kinase that phosphorylates phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] to form phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)], which is rapidly dephosphorylated either by PTEN (phosphatase and tensin homologue deleted on chromosome 10) to PtdIns(4,5)P(2), or by the 5-phosphatases (inositol polyphosphate 5-phosphatases), generating PtdIns(3,4)P(2). 5-phosphatases also hydrolyze PtdIns(4,5)P(2) forming PtdIns(4)P. Ten mammalian 5-phosphatases have been identified, which regulate hematopoietic cell proliferation, synaptic vesicle recycling, insulin signaling, and embryonic development. Two 5-phosphatase genes, OCRL and INPP5E are mutated in Lowe and Joubert syndrome respectively. SHIP [SH2 (Src homology 2)-domain inositol phosphatase] 2, and SKIP (skeletal muscle- and kidney-enriched inositol phosphatase) negatively regulate insulin signaling and glucose homeostasis. SHIP2 polymorphisms are associated with a predisposition to insulin resistance. SHIP1 controls hematopoietic cell proliferation and is mutated in some leukemias. The inositol polyphosphate 4-phosphatases, INPP4A and INPP4B degrade PtdIns(3,4)P(2) to PtdIns(3)P and regulate neuroexcitatory cell death, or act as a tumor suppressor in breast cancer respectively. The Sac phosphatases degrade multiple phosphoinositides, such as PtdIns(3)P, PtdIns(4)P, PtdIns(5)P and PtdIns(3,5)P(2) to form PtdIns. Mutation in the Sac phosphatase gene, FIG4, leads to a degenerative neuropathy. Therefore the phosphatases, like the lipid kinases, play major roles in regulating cellular functions and their mutation or altered expression leads to many human diseases. PMID:22403078

Dyson, Jennifer M; Fedele, Clare G; Davies, Elizabeth M; Becanovic, Jelena; Mitchell, Christina A



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



Development of Phosphoinositide3 Kinase Pathway Inhibitors for Advanced Cancer  

Microsoft Academic Search

The phosphoinositide-3 kinase (PI3K) pathway plays a critical role in cancer cell growth and survival. PI3K is activated in\\u000a human cancers by elevated receptor tyrosine kinase activity, RAS mutation, as well as by mutation, amplification, and deletion\\u000a of genes encoding components of the pathway. Additionally, PI3K pathway activation plays an important role in acquired resistance\\u000a to both chemotherapy and targeted

James M. Cleary; Geoffrey I. Shapiro



Cloning and Characterization of a G Protein-Activated Human Phosphoinositide3 Kinase  

Microsoft Academic Search

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110gamma, was cloned and

Borislav Stoyanov; Stefano Volinia; Theodor Hanck; Ignacio Rubio; Michael Loubtchenkov; Daria Malek; Stefka Stoyanova; Bart Vanhaesebroeck; Ritu Dhand; Bernd Nurnberg; Peter Gierschik; Klaus Seedorf; J. Justin Hsuan; Michael D. Waterfield; Reinhard Wetzker



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


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 (PIP(n)), including phosphatidylinositol 3,4,5-triphosphate (PIP(3)) and phosphatidylinositol 4,5-bisphosphate (PIP(2)), 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 PIP(n) application. However, PIP(n) induced an ?2.5-fold increase in cAMP efficacy for A3 channels. The PIP(n)-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 PIP(n) 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 PIP(n) regulation of heteromeric A3+B3 channels. B3 subunits were not sufficient to confer PIP(n) sensitivity to heteromeric channels formed with PIP(n)-insensitive A subunits. Finally, channels formed by mixtures of PIP(n)-insensitive A3 subunits, having complementary mutations in N- and/or C-terminal regions, restored PIP(n) regulation, implying that intersubunit N-C interactions help control the phosphoinositide sensitivity of cone CNG channels. PMID:23530136

Dai, Gucan; Peng, Changhong; Liu, Chunming; Varnum, Michael D



Cloning and expression analysis of some genes involved in the phosphoinositide and phospholipid signaling pathways from maize ( Zea mays L.)  

Microsoft Academic Search

Previous studies have indicated the phosphoinositide and phospholipid signaling pathways play a key role in plant growth, development and responses to environmental stresses. However, little is known about the phosphoinositide and phospholipid signaling pathways in maize (Zea mays L.). To better understand the function of genes involved in the phosphoinositide and phospholipid signaling pathways in maize, the cDNA sequences of

Zhenhua Sui; Linyuan Niu; Guidong Yue; Aifang Yang; Juren Zhang



Structural Comparisons of Class I Phosphoinositide 3-kinases  

PubMed Central

Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that regulate cell growth. One of these kinases, PI3K?, is frequently mutated in diverse tumor types. The recently determined structure of PI3K? reveals features that distinguish this enzyme from related lipid kinases. In addition, wild-type PI3K? differs from PI3K? by a substitution identical to a PI3K? oncogenic mutant (H1047R), explaining differences in the enzymatic activities of the normal ? and ? enzymes. Comparison of the PI3K structures also identified structural features that could potentially be exploited for the design of isoform-specific inhibitors.

Amzel, L. Mario; Huang, Chuan-Hsiang; Mandelker, Diana; Lengauer, Christoph; Gabelli, Sandra B.; Vogelstein, Bert



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.



Phosphoinositide 3-kinase signalling regulates early development and developmental haemopoiesis.  


Phosphoinositide 3-kinase (PI3K)-dependent signalling regulates a wide variety of cellular functions including proliferation and differentiation. Disruption of class I(A) PI3K isoforms has implicated PI3K-mediated signalling in development of the early embryo and lymphohaemopoietic system. We have used embryonic stem (ES) cells as an in vitro model to study the involvement of PI3K-dependent signalling during early development and haemopoiesis. Both pharmacological inhibition and genetic manipulation of PI3K-dependent signalling demonstrate that PI3K-mediated signals, most likely via 3-phosphoinositide-dependent protein kinase 1 (PDK1), are required for proliferation of cells within developing embryoid bodies (EBs). Surprisingly, the haemopoietic potential of EB-derived cells was not blocked upon PI3K inhibition but rather enhanced, correlating with modest increases in expression of haemopoietic marker genes. By contrast, PDK1-deficient EB-derived progeny failed to generate terminally differentiated haemopoietic lineages. This deficiency appeared to be due to a requirement for PI3K signalling during the proliferative phase of blast-colony-forming cell (BL-CFC) expansion, rather than as a result of effects on differentiation per se. We also demonstrate that PI3K-dependent signalling is required for optimal generation of erythroid and myeloid progenitors and their differentiation into mature haemopoietic colony types. These data demonstrate that PI3K-dependent signals play important roles at different stages of haemopoietic development. PMID:17456549

Bone, Heather K; Welham, Melanie J



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.




PubMed Central

This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present only in tiny amounts compared to structural lipids but are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane-recruitment and activity of many protein signaling-complexes in specific membrane compartments and have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single cell level. The only available technique in live cell application is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules when fused to fluorescent proteins can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular resolution and rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. Here, we summarize the design and practical use of these constructs and also review important considerations for the interpretation of the data obtained by this technique.

Balla, Tamas; Varnai, Peter



Analysis of hormone-induced changes of phosphoinositide metabolism in rat liver  

SciTech Connect

The relationship between hormone-stimulated phosphoinositide turnover and Ca/sup 2 +/ flux can be investigated using radiolabelled hepatocytes and the subcellular fractions derived from them or from whole liver. Comparison of the results obtained using intact cells to those from subcellular fractions should ultimately lead to a reconstruction of the transmembrane signaling events through which hormone such as vasopressin, angiotensin, and catecholamines acutely activate liver glycogenolysis. The paper reviews hormone-stimulated phosphoinositide metabolism in intact hepatocytes as well as hepatic enzymes involved in phosphoinositide metabolism. Also discussed is the current status of studies on hormone action in broken cell preparations in liver.

Wallace, M.A.; Fain, J.N.



GTP and cytosol stimulate phosphoinositide hydrolysis in isolated platelet membranes  

SciTech Connect

Hydrolysis of polyphosphoinositides by phospholipase C was examined in isolated membranes prepared from (/sup 32/P)labelled platelets. In the presence of guanosine 5'-(3-O)-thiotriphosphate (GTP..gamma..S), thrombin increased the release of inositol triphosphate and inositol biphosphate approximately 500%. GTP..gamma..S alone stimulated release 2 fold. Maximal activation of thrombin-induced phosphoinositide hydrolysis was observed at 10 GTP. Although addition of calcium had no effect, 2 mM EGTA completely inhibited inositolphosphate release. Addition of high speed supernatant to (/sup 32/P)labelled membranes stimulated the release of inositolphosphates. This hydrolysis was further enhanced by the addition of GTP. These data demonstrate that the breakdown of polyphosphoinositides in isolated platelet membranes is dependent on GTP and stimulated by platelet cytosol.

Baldassare, J.J.; Fisher, G.J.



A Role for Phosphoinositides in Regulating Plant Nuclear Functions  

PubMed Central

Nuclear localized inositol phospholipids and inositol phosphates are important for regulating many essential processes in animal and yeast cells such as DNA replication, recombination, RNA processing, mRNA export and cell cycle progression. An overview of the current literature indicates the presence of a plant nuclear phosphoinositide (PI) pathway. Inositol phospholipids, inositol phosphates, and enzymes of the PI pathway have been identified in plant nuclei and are implicated in DNA replication, chromatin remodeling, stress responses and hormone signaling. In this review, the potential functions of the nuclear PI pathway in plants are discussed within the context of the animal and yeast literature. It is anticipated that future research will help shed light on the functional significance of the nuclear PI pathway in plants.

Dieck, Catherine B.; Boss, Wendy F.; Perera, Imara Y.



Activation of intracellular phosphoinositide signaling after a single 600 nanosecond electric pulse.  


Exposure to nanosecond pulsed electrical fields (nsPEFs) results in a myriad of observable effects in mammalian cells. While these effects are often attributed to the direct permeabilization of both the plasma and organelle membranes, the underlying mechanism(s) are not well understood. We hypothesize that nsPEF-induced membrane disturbance will initiate complex intracellular lipid signaling pathways, which ultimately lead to the observed multifarious effects. In this article, we show activation of one of these pathways - phosphoinositide signaling cascade. Here we demonstrate that nsPEF initiates phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis or depletion from the plasma membrane, accumulation of inositol-1,4,5-trisphosphate (IP3) in the cytoplasm and increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. All of these events are initiated by a single 16.2kV/cm, 600ns pulse exposure. To further this claim, we show that the nsPEF-induced activation mirrors the response of M1-acetylcholine Gq/11-coupled metabotropic receptor (hM1). This demonstration of PIP2 hydrolysis by nsPEF exposure is an important step toward understanding the mechanisms underlying this unique stimulus for activation of lipid signaling pathways and is critical for determining the potential for nsPEFs to modulate mammalian cell functions. PMID:23747521

Tolstykh, Gleb P; Beier, Hope T; Roth, Caleb C; Thompson, Gary L; Payne, Jason A; Kuipers, Marjorie A; Ibey, Bennett L



Phosphoinositide 3-OH kinase inhibition prevents ventilation-induced lung cell activation.  


In acute respiratory distress syndrome patients, protective ventilation strategies reduce mortality and proinflammatory mediator levels. It has been suggested that some of the side effects of mechanical ventilation are caused by the excessive release of mediators capable of causing pulmonary inflammation and tissue destruction (biotrauma). Selective inhibition of this process might be used to minimize the side effects of artificial mechanical ventilation. This study was designed to identify the cell types and specific signaling mechanisms that are activated by ventilation with increased pressure/volume (overventilation). In isolated perfused mouse lungs, overventilation caused nuclear translocation of nuclear factor-kappaB (NF-kappaB) and enhanced expression of interleukin-6 mRNA in alveolar macrophages and alveolar epithelial type II cells. The phosphoinositide 3-OH kinase inhibitor Ly294002 prevented nuclear translocation of NF-kappaB and the subsequent release of interleukin-6 and macrophage inflammatory protein-2alpha in overventilated but not in endotoxic lungs. Similar results were obtained in rats in vivo, where Ly294002 prevented NF-kappaB activation by overventilation but not by endotoxin. These findings show that alveolar macrophages and alveolar epithelial type II cells contribute to the ventilation-induced release of proinflammatory mediators and that selective inhibition of this process is possible without inhibiting the activation of NF-kappaB by endotoxin. PMID:14578214

Uhlig, Ulrike; Fehrenbach, Heinz; Lachmann, Robert A; Goldmann, Torsten; Lachmann, Burkhard; Vollmer, Ekkehard; Uhlig, Stefan



Effect of Repeated Organophosphate Administration on Carbachol-Stimulated Phosphoinositide Hydrolysis in the Rat Brain.  

National Technical Information Service (NTIS)

The effects of repeated exposure to two organophosphates on the turnover of phosphoinositides, the second messenger system coupled to the M1 and M3 subtypes of muscarinic receptors, were examined in the rat hippocampus. Repeated diisopropylfluorophosphate...

W. R. Mundy T. R. Ward V. F. Dulchinos H. A. Tilson



Inositol metabolism in WRK-1 cells. Relationship of hormone-sensitive to -insensitive pools of phosphoinositides  

SciTech Connect

Previous studies have indicated the existence of two separate pools of phosphoinositides in WRK-1 cells; one is labile and hormone-sensitive with respect to turnover, while the other is stable. Hormonal stimulation results in a rapid increase in /sup 32/Pi incorporation into the sensitive pool, while in the absence of hormone, incorporation of /sup 32/Pi into this pool is slow. Results are quite different when (/sup 3/H)inositol is the precursor utilized. Incorporation of (/sup 3/H)inositol into hormone-sensitive phosphoinositides is not stimulated in the presence of hormone, suggesting entry of this exogenous precursor into the cycle by a route other than the resynthetic phase of the cycle. Furthermore, failure of hormone to induce loss of (/sup 3/H)phosphoinositide in pulse-chase experiments in the absence of lithium suggests reutilization of the (/sup 3/H)inositol moiety generated by phosphodiesteratic cleavage of hormone-sensitive phosphoinositide. Time course studies indicate that the relative rates of incorporation of (/sup 3/H)inositol into sensitive and insensitive phosphoinositide remain constant from 2 to 24 h. Several factors are capable of increasing (/sup 3/H)inositol incorporation into hormone-insensitive phosphoinositide including vasopressin, calcium ionophores, and manganese. On the other hand, vasopressin treatment appears to decrease incorporation of (/sup 3/H)inositol into the hormone-sensitive pool, probably by shifting the equilibrium between phosphoinositides and inositol phosphates, since the decrease in radioactivity observed in the phosphoinositides is equaled by the increase observed in that in the inositol phosphates.

Monaco, M.E.



Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding.  

PubMed Central

Internalization of agonist-activated G protein-coupled receptors is mediated by non-visual arrestins, which also bind to clathrin and are therefore thought to act as adaptors in the endocytosis process. Phosphoinositides have been implicated in the regulation of intracellular receptor trafficking, and are known to bind to other coat components including AP-2, AP180 and COPI coatomer. Given these observations, we explored the possibility that phosphoinositides play a role in arrestin's function as an adaptor. High-affinity binding sites for phosphoinositides in beta-arrestin (arrestin2) and arrestin3 (beta-arrestin2) were identified, and dissimilar effects of phosphoinositide and inositol phosphate on arrestin interactions with clathrin and receptor were characterized. Alteration of three basic residues in arrestin3 abolished phosphoinositide binding with complete retention of clathrin and receptor binding. Unlike native protein, upon agonist activation, this mutant arrestin3 expressed in COS1 cells neither supported beta2-adrenergic receptor internalization nor did it concentrate in coated pits, although it was recruited to the plasma membrane. These findings indicate that phosphoinositide binding plays a critical regulatory role in delivery of the receptor-arrestin complex to coated pits, perhaps by providing, with activated receptor, a multi-point attachment of arrestin to the plasma membrane.

Gaidarov, I; Krupnick, J G; Falck, J R; Benovic, J L; Keen, J H



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

PubMed Central

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 PIP2 and the production of the secondary messenger IP3 in gametocytes. Both processes were selectively blocked by a PI-PLC inhibitor, which also reduced the early Ca2+ 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 PIP2/IP3 probe that translocates from the parasite plasmalemma to the cytosol upon cell activation. Our study revealed a complex interdependency of Ca2+ and PI-PLC activity, with PI-PLC being essential throughout gamete formation, possibly explaining the irreversibility of this process.

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



Specific function of phosphoinositide 3-kinase beta in the control of DNA replication  

PubMed Central

Class IA phosphoinositide 3-kinase (PI3K) are enzymes comprised of a p85 regulatory and a p110 catalytic subunit that induce formation of 3-polyphosphoinositides, which activate numerous downstream targets. PI3K controls cell division. Of the 2 ubiquitous PI3K isoforms, ? has selective action in cell growth and cell cycle entry, but no specific function in cell division has been described for ?. We report here a unique function for PI3K? in the control of DNA replication. PI3K? regulated DNA replication through kinase-dependent and kinase-independent mechanisms. PI3K? was found in the nucleus, where it associated PKB. Modulation of PI3K? activity altered the DNA replication rate by controlling proliferating cell nuclear antigen (PCNA) binding to chromatin and to DNA polymerase ?. PI3K? exerted this action by regulating the nuclear activation of PKB in S phase, and in turn phosphorylation of PCNA negative regulator p21Cip. Also, p110? associated with PCNA and controlled PCNA loading onto chromatin in a kinase-independent manner. These results show a selective function of PI3K? in the control of DNA replication.

Marques, Miriam; Kumar, Amit; Poveda, Ana M.; Zuluaga, Susana; Hernandez, Carmen; Jackson, Shaun; Pasero, Philippe; Carrera, Ana C.



Specific function of phosphoinositide 3-kinase beta in the control of DNA replication.  


Class I(A) phosphoinositide 3-kinase (PI3K) are enzymes comprised of a p85 regulatory and a p110 catalytic subunit that induce formation of 3-polyphosphoinositides, which activate numerous downstream targets. PI3K controls cell division. Of the 2 ubiquitous PI3K isoforms, alpha has selective action in cell growth and cell cycle entry, but no specific function in cell division has been described for beta. We report here a unique function for PI3Kbeta in the control of DNA replication. PI3Kbeta regulated DNA replication through kinase-dependent and kinase-independent mechanisms. PI3Kbeta was found in the nucleus, where it associated PKB. Modulation of PI3Kbeta activity altered the DNA replication rate by controlling proliferating cell nuclear antigen (PCNA) binding to chromatin and to DNA polymerase delta. PI3Kbeta exerted this action by regulating the nuclear activation of PKB in S phase, and in turn phosphorylation of PCNA negative regulator p21(Cip). Also, p110beta associated with PCNA and controlled PCNA loading onto chromatin in a kinase-independent manner. These results show a selective function of PI3Kbeta in the control of DNA replication. PMID:19416922

Marqués, Miriam; Kumar, Amit; Poveda, Ana M; Zuluaga, Susana; Hernández, Carmen; Jackson, Shaun; Pasero, Philippe; Carrera, Ana C



Phosphoinositide 3-kinase-gamma induces Xenopus oocyte maturation via lipid kinase activity.  

PubMed Central

Type-I phosphoinositide 3-kinases (PI3Ks) were characterized as a group of intracellular signalling proteins expressing both protein and lipid kinase activities. Recent studies implicate PI3Ks as mediators of oocyte maturation, but the molecular mechanisms are poorly defined. Here we used the Xenopus oocyte expression system as a model to investigate a possible contribution of the gamma-isoform of PI3K (PI3Kgamma) in the different pathways leading to cell-cycle progression by monitoring the time course of germinal vesicle breakdown (GVBD). Expression of a constitutive active PI3Kgamma (PI3Kgamma-CAAX) induced GVBD and increased the levels of phosphorylated Akt/protein kinase B and mitogen-activated protein kinase (MAPK). Furthermore, PI3Kgamma-CAAX accelerated progesterone-induced GVBD, but had no effect on GVBD induced by insulin. The effects of PI3Kgamma-CAAX could be suppressed by pre-incubation of the oocytes with LY294002, PD98059 or roscovitine, inhibitors of PI3K, MEK (MAPK/extracellular-signal-regulated protein kinase kinase) and cdc2/cyclin B kinase, respectively. Mutants of PI3Kgamma-CAAX, in which either lipid kinase or both lipid and protein kinase activities were altered or eliminated, did not induce significant GVBD. Our data demonstrate that expression of PI3Kgamma in Xenopus oocytes accelerates their progesterone-induced maturation and that lipid kinase activity is required to induce this effect.

Hehl, S; Stoyanov, B; Oehrl, W; Schonherr, R; Wetzker, R; Heinemann, S H



Phosphoinositide 3?kinase ?/? inhibition does not prevent concanavalin A?induced hepatitis.  


An increasing number of studies have suggested that phosphoinositide 3?kinase?? (PI3K?) and PI3K? are involved in the pathogenesis of autoimmune and inflammatory diseases, such as asthma and atherosclerosis. However, the underlying mechanism of acute hepatitis remains unknown. The present study aimed to determine the effect of PI3K?/? inhibition on hepatic injury in a murine model of hepatitis induced by concanavalin A (ConA). It was demonstrated that the pharmacological inhibition of PI3K?/? by TG100?115 did not prevent liver damage following ConA challenge. Furthermore, the PI3K?/? inhibition resulted in elevated transaminase activity in the serum, aggravated hepatic lesions characterized by hepatic necrosis, increased inflammatory cell infiltration and apoptosis of hepatocytes. Survival tests demonstrated that TG100?115 significantly increased the death rate of mice following ConA challenge. In addition, TG100?115 increased the serum levels of the proinflammatory cytokine IL?2 following ConA injection. These results may oppose the development of PI3K?/? inhibitors as therapeutic agents, particularly for the treatment of human hepatitis. PMID:23969545

Liu, Yuanyuan; Xiong, Li; Chang, Ying; Tang, Jianying; Ang, Wei; Yang, Tao; Pi, Weiyi; Yang, Xiaoyan; Ye, Weiwei; Luo, Youfu; Wang, Zhenling



The Novel Therapeutic Effect of Phosphoinositide 3-Kinase-? Inhibitor AS605240 in Autoimmune Diabetes  

PubMed Central

Type 1 diabetes (T1D) remains a major health problem worldwide, with a steadily rising incidence yet no cure. Phosphoinositide 3-kinase-? (PI3K?), a member of a family of lipid kinases expressed primarily in leukocytes, has been the subject of substantial research for its role in inflammatory diseases. However, the role of PI3K? inhibition in suppressing autoimmune T1D remains to be explored. We tested the role of the PI3K? inhibitor AS605240 in preventing and reversing diabetes in NOD mice and assessed the mechanisms by which this inhibition abrogates T1D. Our data indicate that the PI3K? pathway is highly activated in T1D. In NOD mice, we found upregulated expression of phosphorylated Akt (PAkt) in splenocytes. Notably, T regulatory cells (Tregs) showed significantly lower expression of PAkt compared with effector T cells. Inhibition of the PI3K? pathway by AS605240 efficiently suppressed effector T cells and induced Treg expansion through the cAMP response element-binding pathway. AS605240 effectively prevented and reversed autoimmune diabetes in NOD mice and suppressed T-cell activation and the production of inflammatory cytokines by autoreactive T cells in vitro and in vivo. These studies demonstrate the key role of the PI3K? pathway in determining the balance of Tregs and autoreactive cells regulating autoimmune diabetes.

Azzi, Jamil; Moore, Robert F.; Elyaman, Wassim; Mounayar, Marwan; El Haddad, Najib; Yang, Sunmi; Jurewicz, Mollie; Takakura, Ayumi; Petrelli, Alessandra; Fiorina, Paolo; Ruckle, Thomas; Abdi, Reza



Small-molecule agonists of SHIP1 inhibit the phosphoinositide 3-kinase pathway in hematopoietic cells.  


Because phosphoinositide 3-kinase (PI3K) plays a central role in cellular activation, proliferation, and survival, pharmacologic inhibitors targeting components of the PI3K pathway are actively being developed as therapeutics for the treatment of inflammatory disorders and cancer. These targeted drugs inhibit the activity of either PI3K itself or downstream protein kinases. However, a previously unexplored, alternate strategy is to activate the negative regulatory phosphatases in this pathway. The SH2-containing inositol-5'-phosphatase SHIP1 is a normal physiologic counter-regulator of PI3K in immune/hematopoietic cells that hydrolyzes the PI3K product phosphatidylinositiol-3,4,5-trisphosphate (PIP(3)). We now describe the identification and characterization of potent and specific small-molecule activators of SHIP1. These compounds represent the first small-molecule activators of a phosphatase, and are able to activate recombinant SHIP1 enzyme in vitro and stimulate SHIP1 activity in intact macrophage and mast cells. Mechanism of activation studies with these compounds suggest that they bind a previously undescribed, allosteric activation domain within SHIP1. Furthermore, in vivo administration of these compounds was protective in mouse models of endotoxemia and acute cutaneous anaphylaxis, suggesting that SHIP1 agonists could be used therapeutically to inhibit the PI3K pathway. PMID:17502453

Ong, Christopher J; Ming-Lum, Andrew; Nodwell, Matt; Ghanipour, Ali; Yang, Lu; Williams, David E; Kim, Joseph; Demirjian, Loutfig; Qasimi, Pooran; Ruschmann, Jens; Cao, Li-Ping; Ma, Kewei; Chung, Stephen W; Duronio, Vincent; Andersen, Raymond J; Krystal, Gerald; Mui, Alice L-F



Inositol Pentakisphosphate Isomers Bind PH Domains with Varying Specificity and Inhibit Phosphoinositide Interactions  

SciTech Connect

PH domains represent one of the most common domains in the human proteome. These domains are recognized as important mediators of protein-phosphoinositide and protein-protein interactions. Phosphoinositides are lipid components of the membrane that function as signaling molecules by targeting proteins to their sites of action. Phosphoinositide based signaling pathways govern a diverse range of important cellular processes including membrane remodeling, differentiation, proliferation and survival. Myo-Inositol phosphates are soluble signaling molecules that are structurally similar to the head groups of phosphoinositides. These molecules have been proposed to function, at least in part, by regulating PH domain-phosphoinositide interactions. Given the structural similarity of inositol phosphates we were interested in examining the specificity of PH domains towards the family of myo-inositol pentakisphosphate isomers. In work reported here we demonstrate that the C-terminal PH domain of pleckstrin possesses the specificity required to discriminate between different myo-inositol pentakisphosphate isomers. The structural basis for this specificity was determined using high-resolution crystal structures. Moreover, we show that while the PH domain of Grp1 does not possess this high degree of specificity, the PH domain of protein kinase B does. These results demonstrate that some PH domains possess enough specificity to discriminate between myo-inositol pentakisphosphate isomers allowing for these molecules to differentially regulate interactions with phosphoinositides. Furthermore, this work contributes to the growing body of evidence supporting myo-inositol phosphates as regulators of important PH domain-phosphoinositide interactions. Finally, in addition to expanding our knowledge of cellular signaling, these results provide a basis for developing tools to probe biological pathway.

S Jackson; S Al-Saigh; C Schultz; M Junop



Attenuation of phosphoinositide 3-kinase ? signaling restrains autoimmune disease.  


Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by the production of autoantibodies against nuclear components. Lyn-deficient mice are an excellent animal model of SLE manifesting clinical, pathological and biochemical features seen in the human disease. They develop autoreactive antibodies, glomerulonephritis and show generalized inflammation, and their B cells have a hyperactive phenotype. Since loss of Lyn confers hyper-activation of phosphoinositide 3-kinase (PI3K) signaling, we studied the effect of down-modulating PI3K in Lyn-deficient mice. We found that heterozygous inactivation of the p110? isoform of PI3K was sufficient to restrain disease in Lyn-deficient mice, leading to significantly decreased autoantibody development and autoimmune-mediated kidney pathology, and improved survival. Intriguingly, haploinsufficiency of p110? did not dampen signaling in Lyn-deficient B cells. However, plasma cell numbers, serum immunoglobulin titers, inflammation and T cell signaling and activation were significantly moderated in Lyn(-/-)p110?(+/KD) mice. Importantly, we have shown that haploinsufficiency of p110? has minor effects on the B cell compartment per se but leads to significant defects in T cell activation and B cell class-switching. These studies suggest that agents targeting p110? PI3K need not achieve full blockade of the enzyme to be of great benefit in the treatment of SLE. PMID:22537464

Maxwell, Mhairi J; Tsantikos, Evelyn; Kong, Anne M; Vanhaesebroeck, Bart; Tarlinton, David M; Hibbs, Margaret L



Kappa opioid receptors stimulate phosphoinositide turnover in rat brain  

SciTech Connect

The effects of various subtype-selective opioid agonists and antagonists on the phosphoinositide (PI) turnover response were investigated in the rat brain. The {kappa}-agonists U-50,488H and ketocyclazocine produced a concentration-dependent increase in the accumulation of IP's in hippocampal slices. The other {kappa}-agonists Dynorphin-A (1-13) amide, and its protected analog D(Ala){sup 2}-dynorphin-A (1-13) amide also produced a significant increase in the formation of ({sup 3}H)-IP's, whereas the {mu}-selective agonists (D-Ala{sup 2}-N-Me-Phe{sup 4}-Gly{sup 5}-ol)-enkephalin and morphine and the {delta}-selective agonist (D-Pen{sup 2,5})-enkephalin were ineffective. The increase in IP's formation elicited by U-50,488H was partially antagonized by naloxone and more completely antagonized by the {kappa}-selective antagonists nor-binaltorphimine and MR 2266. The formation of IP's induced by U-50,488H varies with the regions of the brain used, being highest in hippocampus and amygdala, and lowest in striatum and pons-medullar. The results indicate that brain {kappa}- but neither {mu}- nor {delta}- receptors are coupled to the PI turnover response.

Periyasamy, S.; Hoss, W. (Univ. of Toledo, OH (USA))



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.



Glyburide priming of beta cells. Possible involvement of phosphoinositide hydrolysis.  


In the simultaneous presence of 5.5 mM glucose, exposure of isolated perifused islets to the sulfonylurea glyburide (500 nM) acutely stimulated insulin release and amplified the subsequent insulin secretory responses to 10 mM glucose or 10 mM arginine. This sensitizing effect of glyburide developed within 10 min, was maintained for at least 40 min after glyburide removal from the perifusion medium, and was attenuated by the calcium channel blocker nitrendipine. In islets whose inositol-containing lipids were prelabeled during a 2-hr incubation period with myo[2-3H]inositol, glyburide induced a concentration-dependent increase in labeled inositol phosphate accumulation. Nitrendipine abolished this stimulatory effect of glyburide. In perifused islets, the stimulatory effect of glyburide on phosphoinositide (PI) hydrolysis persisted after its removal from the medium and the duration of this effect paralleled the duration of sensitization. These findings suggest that glyburide-induced increases in PI hydrolysis account, at least in part, for its acute stimulatory effect on insulin output and its ability to sensitize islets to subsequent stimulation. PMID:1847815

Zawalich, W S



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.



Receptor-coupled phosphoinositide hydrolysis in human retinal pigment epithelium.  


Carbachol and histamine stimulated phosphoinositide (PPI) hydrolysis in cultured human retinal pigment epithelium (RPE), as reflected by an accumulation of 3H-inositol phosphates in the presence of 10 mM Li+. Carbachol increased PPI hydrolysis to greater than 600% of basal with an EC50 of 60 microM; stimulation was linear up to 60 min. This activation likely occurred via the M3 muscarinic cholinergic receptor based on the IC50 values for 4-diphenylacetoxy-N-methylpiperidine methiodide (0.47 nM), pirenzepine (280 nM), and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11- dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (1.4 microM). Carbachol-mediated PPI hydrolysis was decreased by 80% in the absence of extracellular Ca2+. Histamine stimulated PPI turnover in a linear manner by 180% with an EC50 of 20 microM by the H1 histaminergic receptor. Serotonin, glutamate, norepinephrine, and dopamine were inactive. In human RPE, the resting cytoplasmic Ca2+ concentration, as determined by fura-2 fluorescence, was 138 +/- 24 nM. On the addition of carbachol, there was a 180% increase in peak intracellular Ca2+; addition of histamine increased intracellular Ca2+ by 187%. These results suggest receptor-mediated, inositol lipid hydrolysis is coupled to intracellular Ca2+ flux in human RPE. PMID:1851211

Feldman, E L; Randolph, A E; Johnston, G C; DelMonte, M A; Greene, D A



Drosophila neuroligin 1 regulates synaptic growth and function in response to activity and phosphoinositide-3-kinase.  


Neuroligins are postsynaptic neural cell adhesion molecules that mediate synaptic maturation and function in vertebrates and invertebrates, but their mechanisms of action and regulation are not well understood. At the Drosophila larval neuromuscular junction (NMJ), previous analysis demonstrated a requirement for Drosophila neuroligin 1 (dnlg1) in synaptic growth and maturation. The goal of the present study was to better understand the effects and mechanisms of loss-of-function and overexpression of dnlg1 on synapse size and function, and to identify signaling pathways that control dnlg1 expression. Consistent with reduced synapse size, evoked excitatory junctional currents (EJCs) were diminished in dnlg1 mutants but displayed normal Ca(2+) sensitivity and short-term plasticity. However, postsynaptic function was also perturbed, in that glutamate receptor staining and the distribution of amplitudes of miniature excitatory junctional currents (mEJCs) were abnormal in mutants. All the above phenotypes were rescued by a genomic transgene. Overexpression of dnlg1 in muscle resulted in synaptic overgrowth, but reduced the amplitudes of EJCs and mEJCs. Overgrowth and reduced EJC amplitude required Drosophila neurexin 1 (dnrx1) function, suggesting that increased DNlg1/DNrx1 signaling attenuates synaptic transmission and regulates growth through a retrograde mechanism. In contrast, reduced mEJC amplitude was independent of dnrx1. Synaptic overgrowth, triggered by neuronal hyperactivity, absence of the E3 ubiquitin ligase highwire, and increased phosphoinositide-3-kinase (PI3K) signaling in motor neurons reduced synaptic DNlg1 levels. Likewise, postsynaptic attenuation of PI3K, which increases synaptic strength, was associated with reduced DNlg1 levels. These observations suggest that activity and PI3K signaling pathways modulate growth and synaptic transmission through dnlg1-dependent mechanisms. PMID:22954894

Mozer, Brian A; Sandstrom, David J



Class II Phosphoinositide 3-Kinases Contribute to Endothelial Cells Morphogenesis  

PubMed Central

The question of whether the distinct isoforms of the family of enzymes phosphoinositide 3-kinases (PI3Ks) play redundant roles within a cell or whether they control distinct cellular processes or distinct steps within the same cellular process has gained considerable importance in the recent years due to the development of inhibitors able to selectively target individual isoforms. It is important to understand whether inhibition of one PI3K can result in compensatory effect from other isoform(s) and therefore whether strategies aimed at simultaneously blocking more than one PI3K may be needed. In this study we investigated the relative contribution of distinct PI3K isoforms to endothelial cells (EC) functions specifically regulated by the sphingolipid sphingosine-1-phosphate (S1P) and by high density lipoproteins (HDL), the major carrier of S1P in human plasma. Here we show that a co-ordinated action of different PI3Ks is required to tightly regulate remodelling of EC on Matrigel, a process dependent on cell proliferation, apoptosis and migration. The contribution of each isoform to this process appears to be distinct, with the class II enzyme PI3K-C2? and the class IB isoform p110? mainly regulating the S1P- and HDL-dependent EC migration and PI3K-C2? primarily controlling EC survival. Data further indicate that PI3K-C2? and p110? control distinct steps involved in cell migration supporting the hypothesis that different PI3Ks regulate distinct cellular processes.

Chiozzotto, Daniela; Mavrommati, Ioanna; Wheeler, Ann P.; Norata, Giuseppe Danilo; Catapano, Alberico Luigi; Falasca, Marco



Multiple effects of increases in phosphoinositide hydrolysis on islets and their relationship to changing patterns of insulin secretion.  


Evidence continues to accumulate which suggests that increases in phosphoinositide hydrolysis play an integral role in beta cell insulin secretory response patterns. In the present report some of the methodological approaches utilized to monitor phosphoinositide-hydrolysis in islets are reviewed. The contribution of phosphoinositide-derived second messenger molecules to acute insulin release as well as the induction of time dependent potentiation are also discussed. Finally, the role of impaired phosphoinositide hydrolysis in the induction of time dependent suppression of insulin release is also explored. It is concluded that alterations in phosphoinositide hydrolysis contribute, at least in part, to both ordered and disordered patterns of insulin release noted under a variety of experimental conditions. PMID:1965429

Zawalich, W



Rapamycin regulates connective tissue growth factor expression of lung epithelial cells via phosphoinositide 3-kinase.  


The pathogenesis of idiopathic pulmonary fibrosis (IPF) remains largely unknown. It is believed that IPF is mainly driven by activated alveolar epithelial cells that have a compromised migration capacity, and that also produce substances (such as connective tissue growth factor, CTGF) that contribute to fibroblast activation and matrix protein accumulation. Because the mechanisms regulating these processes are unclear, the aim of this study was to determine the role of rapamycin in regulating epithelial cell migration and CTGF expression. Transformed epithelial cell line A549 and normal human pulmonary alveolar or bronchial epithelial cells were cultured in regular medium or medium containing rapamycin. Real time reverse transcriptase polymerase chain reaction was employed to determine CTGF mRNA expression. Western blotting and an enzyme-linked immunosorbent assay were used for detecting CTGF protein. Wound healing and migration assays were used to determine the cell migration potential. Transforming growth factor (TGF)-? type I receptor (T?RI) inhibitor, SB431542 and phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 were used to determine rapamycin's mechanism of action. It was found that treatment of A549 and normal human alveolar or bronchial epithelial cells with rapamycin significantly promoted basal or TGF-?1 induced CTGF expression. LY294002, not SB431542 attenuated the promotional effect of rapamycin on CTGF expression. Cell mobility was not affected by rapamycin in wound healing and migration assays. These data suggest rapamycin has a profibrotic effect in vitro and underscore the potential of combined therapeutic approach with PI3K and mammalian target of rapamycin inhibitors for the treatment of animal or human lung fibrosis. PMID:23986222

Xu, Xuefeng; Wan, Xuan; Geng, Jing; Li, Fei; Yang, Ting; Dai, Huaping



ARNO but not cytohesin-1 translocation is phosphatidylinositol 3-kinase-dependent in HL60 cells  

Microsoft Academic Search

Cytohesin-1 and ARNO are guanine nu- cleotide-exchange factors (GEFs) for ADP-ribosyla- tion factor (Arf). Here, we show that ARNO is ex- pressed in HL-60 cells and established that granulo- cytic differentiation induced with Me2SO stimulated cytohesin-1 but not ARNO expression. Cytohesin-1 levels in HL-60 granulocytes were similar to those in human neutrophils. Me2SO-differentiated HL-60 cells expressed ARNO and cytohesin-1 isoforms

Sylvain G. Bourgoin; Martin G. Houle; Indrapal N. Singh; Danielle Harbour; Steve Gagnon; Andrew J. Morris; David N. Brindley


ERBB-2 overexpression confers PI 3 ? kinase-dependent invasion capacity on human mammary epithelial cells  

PubMed Central

Amplification and overexpression of ERBB-2 in human breast cancer is thought to play a significant role in the progression of the disease; however, its precise role in the aetiology of altered phenotypes associated with human breast cancer is unknown. We have previously shown that exogenous overexpression of ERBB-2 conferred growth factor independence on human mammary epithelial cells. In this study, we show that ERBB-2 overexpression also causes the cells to acquire other characteristics exhibited by human breast cancer cells, such as anchorage-independent growth and invasion capabilities. ERBB-2-induced invasion is dependent on fibronectin and correlates with the down-regulation of cell surface ?4 integrin. In addition ERBB-2 co-immunoprecipitates with focal adhesion kinase (FAK) in these cells. We have also shown, by use of exogenously expressed PTEN and by treatment with the PI3?-kinase inhibitor LY294002, that ERBB-2-induced invasion is dependent on the PI3?-kinase pathway; however, PTEN does not dephosphorylate FAK in these cells. © 2000 Cancer Research Campaign

Ignatoski, K M Woods; Maehama, T; Markwart, S M; Dixon, J E; Livant, D L; Ethier, S P



IGF-1 Modulates N and L Calcium Channels in a PI 3-Kinase-Dependent Manner  

Microsoft Academic Search

Receptor tyrosine kinases (RTKs) have long been associated with proliferation in non-neural cells, although they are also expressed in postmitotic neurons. We demonstrate that insulin-like growth factor-1 (IGF-1) induces within seconds a large, tyrosine- kinase-dependent increase in calcium channel currents in cerebellar granule neurons. Separation of channel subtypes reveals that, while P, Q, and R channels are unaffected, N and

Lesley A. C Blair; John Marshall



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



Reduced Phosphoinositide 3-Kinase (p110?) Activation Increases the Susceptibility to Atrial Fibrillation  

PubMed Central

Atrial fibrillation (AF) is the most common sustained arrhythmia presenting at cardiology departments. A limited understanding of the molecular mechanisms responsible for the development of AF has hindered treatment strategies. The purpose of this study was to assess whether reduced activation of phosphoinositide 3-kinase (PI3K, p110?) makes the compromised heart susceptible to AF. Risk factors for AF, including aging, obesity, and diabetes, have been associated with insulin resistance that leads to depressed/defective PI3K signaling. However, to date, there has been no link between PI3K(p110?) and AF. To address this question, we crossed a cardiac-specific transgenic mouse model of dilated cardiomyopathy (DCM) with a cardiac-specific transgenic mouse expressing a dominant negative mutant of PI3K (dnPI3K; reduces PI3K activity). Adult (?4.5 months) double-transgenic (dnPI3K-DCM), single-transgenic (DCM-Tg, dnPI3K-Tg), and nontransgenic mice were subjected to morphological, functional/ECG, microarray, and biochemical analyses. dnPI3K-DCM mice developed AF and had depressed cardiac function as well as greater atrial enlargement and fibrosis than DCM-Tg mice. AF was not detected in other groups. Aged DCM-Tg mice (?15 months) with a similar phenotype to dnPI3K-DCM mice (4.5 months) did not develop AF, suggesting loss of PI3K activity directly contributed to the AF phenotype. Furthermore, increasing PI3K activity reduced atrial fibrosis and improved cardiac conduction in DCM-Tg mice. Finally, in atrial appendages from patients with AF, PI3K activation was lower compared with tissue from patients in sinus rhythm. These results suggest a link between PI3K(p110?) and AF.

Pretorius, Lynette; Du, Xiao-Jun; Woodcock, Elizabeth A.; Kiriazis, Helen; Lin, Ruby C.Y.; Marasco, Silvana; Medcalf, Robert L.; Ming, Ziqiu; Head, Geoffrey A.; Tan, Joon Win; Cemerlang, Nelly; Sadoshima, Junichi; Shioi, Tetsuo; Izumo, Seigo; Lukoshkova, Elena V.; Dart, Anthony M.; Jennings, Garry L.; McMullen, Julie R.



Phosphoinositide 3-Kinase ? Regulates Airway Smooth Muscle Contraction by Modulating Calcium Oscillations  

PubMed Central

Phosphoinositide 3-kinase ? (PI3K?) has been implicated in the pathogenesis of asthma, but its mechanism has been considered indirect, through release of inflammatory cell mediators. Because airway smooth muscle (ASM) contractile hyper-responsiveness plays a critical role in asthma, the aim of the present study was to determine whether PI3K? can directly regulate contractility of ASM. Immunohistochemistry staining indicated expression of PI3K? protein in ASM cells of mouse trachea and lung, which was confirmed by Western blot analysis in isolated mouse tracheal ASM cells. PI3K? inhibitor II inhibited acetylcholine (ACh)-stimulated airway contraction of cultured precision-cut mouse lung slices in a dose-dependent manner with 75% inhibition at 10 ?M. In contrast, inhibitors of PI3K?, PI3K?, or PI3K?, at concentrations 40-fold higher than their reported IC50 values for their primary targets, had no effect. It is noteworthy that airways in lung slices pretreated with PI3K? inhibitor II still exhibited an ACh-induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, the PI3K?-selective inhibitor had a small inhibitory effect on the ACh-stimulated initial Ca2+ transient in ASM cells of mouse lung slices or isolated mouse ASM cells but significantly attenuated the sustained Ca2+ oscillations that are critical for sustained airway contraction. This report is the first to show that PI3K? directly controls contractility of airways through regulation of Ca2+ oscillations in ASM cells. Thus, in addition to effects on airway inflammation, PI3K? inhibitors may also exert direct effects on the airway contraction that contribute to pathologic airway hyper-responsiveness.

Jiang, Haihong; Abel, Peter W.; Toews, Myron L.; Deng, Caishu; Casale, Thomas B.; Xie, Yan



Triggering Actin Comets Versus Membrane Ruffles: Distinctive Effects of Phosphoinositides on Actin Reorganization  

PubMed Central

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)P2]. First, we increased PI(4,5)P2 synthesis from phosphatidylinositol 4-phosphate [PI(4)P] using a membrane recruitable form of PI(4)P 5-kinase, and found that COS-7, HeLa, and HEK293 cells formed bundles of motile actin filaments known as actin comets. In contrast, a second technique that increased the concentration of PI(4,5)P2 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)P2 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.

Ueno, Tasuku; Falkenburger, Bjorn H.; Pohlmeyer, Christopher; Inoue, Takanari



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




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...



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...


Alternative splicing of caspase 9 is modulated by the phosphoinositide 3-kinase/Akt pathway via phosphorylation of SRp30a.  


Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology. Two splice variants are derived from the CASP9 gene via the inclusion (Casp9a) or exclusion (Casp9b) of a four-exon cassette. Here we show that alternative splicing of Casp9 is dysregulated in non-small cell lung cancers (NSCLC) regardless of their pathologic classification. Based on these findings we hypothesized that survival pathways activated by oncogenic mutation regulated this mechanism. In contrast to K-RasV12 expression, epidermal growth factor receptor (EGFR) overexpression or mutation dramatically lowered the Casp9a/9b splice isoform ratio. Moreover, Casp9b downregulation blocked the ability of EGFR mutations to induce anchorage-independent growth. Furthermore, Casp9b expression blocked inhibition of clonogenic colony formation by erlotinib. Interrogation of oncogenic signaling pathways showed that inhibition of phosphoinositide 3-kinase or Akt dramatically increased the Casp9a/9b ratio in NSCLC cells. Finally, Akt was found to mediate exclusion of the exon 3,4,5,6 cassette of Casp9 via the phosphorylation state of the RNA splicing factor SRp30a via serines 199, 201, 227, and 234. Taken together, our findings show that oncogenic factors activating the phosphoinositide 3-kinase/Akt pathway can regulate alternative splicing of Casp9 via a coordinated mechanism involving the phosphorylation of SRp30a. PMID:21045158

Shultz, Jacqueline C; Goehe, Rachel W; Wijesinghe, D Shanaka; Murudkar, Charuta; Hawkins, Amy J; Shay, Jerry W; Minna, John D; Chalfant, Charles E



Maintenance of Hormone-sensitive Phosphoinositide Pools in the Plasma Membrane Requires Phosphatidylinositol 4-Kinase III?  

PubMed Central

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca2+ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (? or ?) is responsible for supplying phosphoinositides during agonist-induced Ca2+ signaling. Using inhibitors that discriminate between the ?- and ?-isoforms of type III PI4Ks, PI4KIII? was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], and Ca2+ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P2 levels in 32P-labeled cells, but only PI4KIII? down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P2 resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca2+ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIII? enzyme in maintaining plasma membrane phosphoinositides.

Balla, Andras; Kim, Yeun Ju; Varnai, Peter; Szentpetery, Zsofia; Knight, Zachary; Shokat, Kevan M.



Normalization of immune response and phosphoinositide fatty acid composition of peripheral blood lymphocytes in an alcoholic patient after alcohol abstinence.  


After 10 months of alcohol abstinence a malnourished alcoholic patient improved his nutritional status. The analysis of peripheral blood lymphocyte response to mitogenic stimulation with the antibody anti-CD3 and of the fatty acid composition of the (poly)-phosphoinositide fraction derived from lymphocytes revealed: 1) a similar [3H]-thymidine uptake as in control (non-drinker) subjects; 2) a similar relative molar content of the main fatty acids in the (poly)-phosphoinositides as in control subjects. Alcohol abstinence can normalize both the parameters, which are greatly altered during alcohol abuse. This suggests a link between nutritional status and lymphocyte responsiveness via phosphoinositide fatty acid composition. PMID:8799464

Stefanini, G F; Castelli, E; Foschi, F G; Marsigli, L; Addolorato, G; Celadon, M; Biagi, P L; Bordoni, A; Gasbarrini, G; Hrelia, S



Subtype-specific regulation of P2X3 and P2X2/3 receptors by phosphoinositides in peripheral nociceptors  

PubMed Central

Background P2X3 and P2X2/3 purinergic receptor-channels, expressed in primary sensory neurons that mediate nociception, have been implicated in neuropathic and inflammatory pain responses. The phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) are involved in functional modulation of several types of ion channels. We report here evidence that these phospholipids are able to modulate the function of homomeric P2X3 and heteromeric P2X2/3 purinoceptors expressed in dorsal root ganglion (DRG) nociceptors and in heterologous expression systems. Results In dissociated rat DRG neurons, incubation with the PI3K/PI4K inhibitor wortmannin at 35 ?M induced a dramatic decrease in the amplitude of ATP- or ?,?-meATP-evoked P2X3 currents, while incubation with 100 nM wortmannin (selective PI3K inhibition) produced no significant effect. Intracellular application of PIP2 was able to fully reverse the inhibition of P2X3 currents induced by wortmannin. In Xenopus oocytes and in HEK293 cells expressing recombinant P2X3, 35 ?M wortmannin incubation induced a significant decrease in the rate of receptor recovery. Native and recombinant P2X2/3 receptor-mediated currents were inhibited by incubation with wortmannin both at 35 ?M and 100 nM. The decrease of P2X2/3 current amplitude induced by wortmannin could be partially reversed by application of PIP2 or PIP3, indicating a sensitivity to both phosphoinositides in DRG neurons and Xenopus oocytes. Using a lipid binding assay, we demonstrate that the C-terminus of the P2X2 subunit binds directly to PIP2, PIP3 and other phosphoinositides. In contrast, no direct binding was detected between the C-terminus of P2X3 subunit and phosphoinositides. Conclusion Our findings indicate a functional regulation of homomeric P2X3 and heteromeric P2X2/3 ATP receptors by phosphoinositides in the plasma membrane of DRG nociceptors, based on subtype-specific mechanisms of direct and indirect lipid sensing.

Mo, Gary; Bernier, Louis-Philippe; Zhao, Qi; Chabot-Dore, Anne-Julie; Ase, Ariel R; Logothetis, Diomedes; Cao, Chang-Qing; Seguela, Philippe



Dihydrotestosterone induces SREBP-1 expression and lipogenesis through the phosphoinositide 3-kinase/Akt pathway in HaCaT cells  

PubMed Central

Background The purpose of this study was to investigate the effects and mechanisms of dihydrotestosterone (DHT)-induced expression of sterol regulatory element binding protein-1 (SREBP-1), and the synthesis and secretion of lipids, in HaCaT cells. HaCaT cells were treated with DHT and either the phosphoinositide 3-kinase inhibitor LY294002 or the extracellular-signal-regulated kinase (ERK) inhibitor PD98059. Real time-PCR, Western blot, Oil Red staining and flow cytometry were employed to examine the mRNA and protein expressions of SREBP-1, the gene transcription of lipid synthesis, and lipid secretion in HaCaT cells. Findings We found that DHT upregulated mRNA and protein expressions of SREBP-1. DHT also significantly upregulated the transcription of lipid synthesis-related genes and increased lipid secretion, which can be inhibited by the addition of LY294002. Conclusions Collectively, these results indicate that DHT induces SREBP-1 expression and lipogenesis in HaCaT cells via activation of the phosphoinositide 3-kinase/Akt Pathway.



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

Microsoft Academic Search

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, b, g, d, 3 and z, have been identified in mammals. Plant PI-PLCs are structurally close to the mammalian PI-PLC-z isoform. The Arabidopsis genome contains nine

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


Involvement of non-muscarinic receptors in phosphoinositide signalling during soman-induced seizures.  


Previous investigations have indicated that soman-induced convulsions involve the inositol lipid signalling system. We previously reported that 10 min after the onset of seizures, inositol 1,4,5-triphosphate (IP3) build-up was coupled to activation of non-muscarinic receptor subtypes. In the present study, we demonstrate that (1) in addition to muscarinic receptors, histamine H1 subtypes and glutamate metabotropic receptors contribute to the first IP3 increase (first 10 min of seizures) and (2) the histamine H1 subtype and glutamate metabotropic receptors are also involved in the second step of inositol phosphate response (after 10 min of seizures). alpha 1-adrenoceptor and 5-HT2 receptors, known to be coupled to phosphoinositide turnover, did not participate in soman-induced IP3 response. Neurochemical interactions between cholinergic, histamine H1 and glutamate metabotropic systems, responsible of the phosphoinositide hydrolysis under soman are envisaged. PMID:7621903

Bodjarian, N; Carpentier, P; Baubichon, D; Blanchet, G; Lallement, G



Essential role for the p110delta phosphoinositide 3-kinase in the allergic response  

Microsoft Academic Search

Inflammatory substances released by mast cells induce and maintain the allergic response. Mast cell differentiation and activation are regulated, respectively, by stem cell factor (SCF; also known as Kit ligand) and by allergen in complex with allergen-specific immunoglobulin E (IgE). Activated SCF receptors and high-affinity receptors for IgE (Fc?RI) engage phosphoinositide 3-kinases (PI(3)Ks) to generate intracellular lipid second messenger signals.

Khaled Ali; Antonio Bilancio; Matthew Thomas; Wayne Pearce; Alasdair M. Gilfillan; Christine Tkaczyk; Nicolas Kuehn; Alexander Gray; June Giddings; Emma Peskett; Roy Fox; Ian Bruce; Christoph Walker; Carol Sawyer; Klaus Okkenhaug; Peter Finan; Bart Vanhaesebroeck



Phylogenomics of phosphoinositide lipid kinases: perspectives on the evolution of second messenger signaling and drug discovery  

Microsoft Academic Search

BACKGROUND: Phosphoinositide lipid kinases (PIKs) generate specific phosphorylated variants of phosatidylinositols (PtdIns) that are critical for second messenger signaling and cellular membrane remodeling. Mammals have 19 PIK isoforms spread across three major families: the PtIns 3-kinases (PI3Ks), PtdIns 4-kinases (PI4Ks), and PtdIns-P (PIP) kinases (PIPKs). Other eukaryotes have fewer yet varying PIK complements. PIKs are also an important, emerging class

James R Brown; Kurt R Auger



Class IA Phosphoinositide 3Kinase Regulates Heart Size and Physiological Cardiac Hypertrophy  

Microsoft Academic Search

Class IA phosphoinositide 3-kinases (PI3Ks) are activated by growth factor receptors, and they regulate, among other processes, cell growth and organ size. Studies using transgenic mice overexpressing constitutively active and dominant negative forms of the p110 catalytic subunit of class IA PI3K have implicated the role of this enzyme in regulating heart size and physiological cardiac hypertrophy. To further understand

Ji Luo; Julie R. McMullen; Cassandra L. Sobkiw; Li Zhang; Adam L. Dorfman; Megan C. Sherwood; M. Nicole Logsdon; James W. Horner; Ronald A. DePinho; Seigo Izumo; Lewis C. Cantley



Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells  

Microsoft Academic Search

The mitogen-activated protein kinase–extracellular signal–regulated kinase signaling element (MAPK-ERK) plays a critical role in natural killer (NK) cell lysis of tumor cells, but its upstream effectors were previously unknown. We show that inhibition of phosphoinositide-3 kinase (PI3K) in NK cells blocks p21-activated kinase 1 (PAK1), MAPK kinase (MEK) and ERK activation by target cell ligation, interferes with perforin and granzyme

Kun Jiang; Bin Zhong; Danielle L. Gilvary; Brian C. Corliss; Elizabeth Hong-Geller; Sheng Wei; Julie Y. Djeu



VEGF-Induced Activation of Phosphoinositide 3Kinase Is Dependent on Focal Adhesion Kinase  

Microsoft Academic Search

Vascular endothelial growth factor (VEGF)-A stimulates formation of new blood vessels (angiogenesis). This process includes migration of endothelial cells from the preexisting vessel toward the source of the growth factor. We show that VEGF-A-induced migration of porcine aortic endothelial cells expressing VEGF receptor-2 (VEGFR-2) is dependent on activation of phosphoinositide 3-kinase (PI3-kinase). There is no direct interaction between VEGF receptor-2

Jian Hua Qi; Lena Claesson-Welsh



Different Subcellular Localization and Phosphoinositides Binding of Insulin Receptor Substrate Protein Pleckstrin Homology Domains  

Microsoft Academic Search

Insulin evokes diverse biological effects through receptor-mediated tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins. Here, we show that, in vitro, the IRS-1, -2 and -3 pleckstrin homology (PH) domains bind with different speci- ficities to the 3-phosphorylated phosphoinositides. In fact, the IRS-1 PH domain binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdIns- 3,4,5-P3), the IRS-2 PH domain to phosphatidylino- sitol

Giorgia Razzini; Alessandra Ingrosso; Anna Brancaccio; Salvatore Sciacchitano; Diana L. Esposito; Marco Falasca



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



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 Nyåkern; G Tabellini; R Bortul; P L Tazzari; C Evangelisti; L Cocco



Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism  

Microsoft Academic Search

We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of (3H)choline and (3H)phosphorylcholine ((3H)Pchol) from cells containing (3H)choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of

E. A. Martinson; D. Goldstein; J. H. Brown



Activation of Phosphoinositide 3OH Kinase by the ?6?4 Integrin Promotes Carcinoma Invasion  

Microsoft Academic Search

We demonstrate that the ?6?4 integrin promotes carcinoma invasion through a preferential and localized targeting of phosphoinositide-3 OH kinase (PI3K) activity. Stable expression of ?6?4 increased carcinoma invasion in a PI3K-dependent manner, and transient expression of a constitutively active PI3K increased invasion in the absence of ?6?4. Ligation of ?6?4 stimulated significantly more PI3K activity than ligation of ?1 integrins,

Leslie M Shaw; Isaac Rabinovitz; Helen H.-F Wang; Alex Toker; Arthur M Mercurio



Calcium and a mitochondrial signal interact to stimulate phosphoinositide hydrolysis and insulin secretion in rat islets.  


Fuel metabolism generates multiple signals that interact to stimulate insulin secretion. These studies explored the mechanism by which fuels activate phosphoinositide (PI) hydrolysis and the role of this signal transduction pathway in fuel-stimulated insulin secretion. High potassium (30 mM), which depolarizes the membrane and increases Ca2+ influx, caused only a transient monophasic release of insulin. In contrast, glucose (20 mM) or monomethylsuccinate (MMSucc; 10 mM) markedly stimulated a sustained insulin secretory response, indicating that fuel metabolism generates a signal(s) in addition to Ca2+ influx that is required for a sustained secretory response. On the other hand, diazoxide, an ATP-sensitive K+ channel activator that prevents membrane depolarization and Ca2+ influx in response to fuel metabolism, reduced the secretory responses to glucose and MMSucc to baseline levels, demonstrating that Ca2+ influx was essential to fuel-stimulated insulin secretion. The further addition of high K+ bypassed the diazoxide block and restored insulin secretory rates. The insulin secretory response to glucose or MMSucc in the presence of diazoxide and K+ was inhibited by the Ca2+ channel antagonist nitrendipine and the protein kinase-C inhibitor staurosporine. Changes in PI hydrolysis paralleled those in insulin secretion. High potassium alone induced only a modest 2.5-fold increase in inositol phosphate accumulation. This response was significantly less than that to glucose or MMSucc, which increased inositol phosphate accumulation by 6.8- or 5.2-fold, respectively. Like its effect on secretion, diazoxide markedly reduced glucose- or MMSucc-stimulated PI hydrolysis, and this inhibition was reversed with high K+. In contrast, diazoxide had no effect on receptor-activated PI hydrolysis stimulated by 100 nM cholecystokinin (CCK), and the effects of CCK were not dependent on added fuel, indicating that fuel and CCK activate PI hydrolysis by distinct pathways. These findings demonstrate that mitochondrial metabolism of glucose or MMSucc generates a signal(s) that interacts with Ca2+ influx to stimulate PI hydrolysis and sustained insulin secretion. This pathway of fuel-activated PI hydrolysis is distinct from that of CCK receptor-activated PI hydrolysis. These studies suggest that fuel-activated PI hydrolysis plays an important role in fuel-stimulated insulin secretion. PMID:8137727

Kelley, G G; Zawalich, K C; Zawalich, W S



TRPV1 channels are intrinsically heat sensitive and negatively regulated by phosphoinositide lipids.  


The capsaicin receptor, TRPV1, is regulated by phosphatidylinositol-4,5-bisphosphate (PIP(2)), although the precise nature of this effect (i.e., positive or negative) remains controversial. Here, we reconstitute purified TRPV1 into artificial liposomes, where it is gated robustly by capsaicin, protons, spider toxins, and, notably, heat, demonstrating intrinsic sensitivity of the channel to both chemical and thermal stimuli. TRPV1 is fully functional in the absence of phosphoinositides, arguing against their proposed obligatory role in channel activation. Rather, introduction of various phosphoinositides, including PIP(2), PI4P, and phosphatidylinositol, inhibits TRPV1, supporting a model whereby phosphoinositide turnover contributes to thermal hyperalgesia by disinhibiting the channel. Using an orthogonal chemical strategy, we show that association of the TRPV1 C terminus with the bilayer modulates channel gating, consistent with phylogenetic data implicating this domain as a key regulatory site for tuning stimulus sensitivity. Beyond TRPV1, these findings are relevant to understanding how membrane lipids modulate other "receptor-operated" TRP channels. PMID:23439120

Cao, Erhu; Cordero-Morales, Julio F; Liu, Beiying; Qin, Feng; Julius, David



Phosphoinositide synthesis and Ca2+ gating in blowfly salivary glands exposed to 5-hydroxytryptamine.  

PubMed Central

Blowfly salivary glands, previously exposed to 10 microM-5-hydroxytryptamine for 30 min, demonstrated a rapid compensatory resynthesis of [3H]inositol-labelled phosphatidylinositol 4,5-bisphosphate when allowed to recover in medium containing 3-5 microM-inositol. Phosphatidylinositol 4,5-bisphosphate comprised 70% of the total [3H]-phosphoinositide, and there was a corresponding decrease in the formation of [3H]-phosphatidylinositol. Subsequent addition of 5-hydroxytryptamine produced an equivalent breakdown of the newly synthesized phosphoinositides but little 45Ca2+ gating. Increasing the inositol concentration in the medium to 300 microM produced a 14-fold stimulation of phosphatidylinositol synthesis but only a 5-fold increase in phosphatidylinositol 4,5-bisphosphate synthesis. Increasing the inositol concentration in the medium from 3 microM to 300 microM resulted in a progressively greater recovery of the 45Ca2+-gating response. At 300 microM-inositol there was an 85% recovery of 45Ca2+-gating response. These results indicate that conversion of phosphatidylinositol into phosphatidylinositol 4,5-bisphosphate occurs in blowfly salivary glands and is secondary to an initial breakdown of the phosphoinositides. Recovery of Ca2+ gating is dependent on the restoration of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate to appropriate concentrations. Images Fig. 1.

Sadler, K; Litosch, I; Fain, J N



Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a ?-propeller protein family.  


?-propellers that bind polyphosphoinositides (PROPPINs), a eukaryotic WD-40 motif-containing protein family, bind via their predicted ?-propeller fold the polyphosphoinositides PtdIns3P and PtdIns(3,5)P(2) using a conserved FRRG motif. PROPPINs play a key role in macroautophagy in addition to other functions. We present the 3.0-Å crystal structure of Kluyveromyces lactis Hsv2, which shares significant sequence homologies with its three Saccharomyces cerevisiae homologs Atg18, Atg21, and Hsv2. It adopts a seven-bladed ?-propeller fold with a rare nonvelcro propeller closure. Remarkably, in the crystal structure, the two arginines of the FRRG motif are part of two distinct basic pockets formed by a set of highly conserved residues. In comprehensive in vivo and in vitro studies of ScAtg18 and ScHsv2, we define within the two pockets a set of conserved residues essential for normal membrane association, phosphoinositide binding, and biological activities. Our experiments show that PROPPINs contain two individual phosphoinositide binding sites. Based on docking studies, we propose a model for phosphoinositide binding of PROPPINs. PMID:22753491

Krick, Roswitha; Busse, Ricarda A; Scacioc, Andreea; Stephan, Milena; Janshoff, Andreas; Thumm, Michael; Kühnel, Karin



Phosphoinositide fatty acid composition of peripheral blood lymphocytes from aging humans.  


The interaction of the T-cell receptor complex with the ligands is associated with early molecular events involved in the process of signal transduction implicating phosphoinositide breakdown. In elderly people, abnormalities in membrane signal transduction pathways are the basis of the immune deficiency associated with aging. Peripheral blood lymphocytes from aging humans and young subjects were stimulated with anti-CD3 monoclonal antibody and the phosphoinositide fractions were analyzed in order to determine the fatty acid composition in resting and stimulated conditions. In aging humans, in resting conditions, the all three phosphoinositide fractions appeared more saturated than the corresponding fractions in young subjects. Following anti-CD3 stimulation a decrease in arachidonic acid relative molar content was detected in both young and old subjects, but the arachidonic acid content in resting conditions greatly differed between the two groups, suggesting a different modulation of the microenvironment of the T-cell receptor complex in elderly people, so determining alterations in the early activation steps of lymphocytes. PMID:1336381

Hrelia, S; Celadon, M; Rossi, C A; Stefanini, G F; Bordoni, A; Biagi, P L



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



Phosphoinositide 3-kinase C2? regulates RhoA and the actin cytoskeleton through an interaction with Dbl.  


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. PMID:22984590

B?ajecka, Karolina; Marinov, Marin; Leitner, Laura; Uth, Kristin; Posern, Guido; Arcaro, Alexandre



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



Canonical autophagy dependent on the class III phosphoinositide-3 kinase Vps34 is required for naive T-cell homeostasis  

PubMed Central

The homeostasis of naive T cells is essential for protective immunity against infection, but the cell-intrinsic molecular mechanisms that control naïve T-cell homeostasis are poorly understood. Genetic ablation in lower organisms has revealed a critical role for Vps34, an evolutionary conserved class III phosphoinositide-3 kinase (PI3K), in regulating endocytosis and autophagy; however, the physiological function of Vps34 in the immune system, especially in T cells, is unclear. Here we report that Vps34 is required for the maintenance of naïve T cells, acting in a cell-intrinsic manner. T-cell–specific deletion of the gene encoding Vps34 resulted in reduced stability of Vps15 and Beclin-1, components of the class III PI3K complex, and impaired autophagy in T cells. Vps34 was dispensable for T-cell development but important for the survival of naïve T cells. Vps34-deficient T cells showed increased mitochondrial mass and accumulation of reactive oxygen species, consistent with deficient removal of damaged mitochondria. Thus, Vps34-dependent canonical autophagy plays a critical role in maintaining T-cell homeostasis by promoting T-cell survival through quality control of mitochondria.

Willinger, Tim; Flavell, Richard A.



Modulation of phosphoinositide metabolism in aortic smooth muscle cells by allylamine  

SciTech Connect

Aortic smooth muscle cells (SMC) modulate from a contractile to a proliferative phenotype upon subchronic exposure to allylamine. The present studies were designed to determine if this phenotypic modulation is associated with alterations in the metabolism of membrane phosphoinositides. 32P incorporation into phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA) was lower by 31, 35, and 22%, respectively, in SMC from allylamine-treated animals relative to controls. In contrast, incorporation of (3H)myoinositol into inositol phosphates did not differ in allylamine cells relative to control cells. Exposure to dibutyryl (db) cAMP (0.2 mM) and theophylline (0.1 mM) reduced 32P incorporation into PIP and PIP2 in SMC from both experimental groups. Under these conditions, a decrease in (3H)myoinositol incorporation into inositol 1-phosphate was only observed in allylamine cells. The effects of db cAMP and theophylline in allylamine and control SMC correlated with a marked decrease in cellular proliferation. These results suggest that alterations in phosphoinositide synthesis and/or degradation contribute to the enhanced proliferation of SMC induced by allylamine. To further examine this concept, the effects of agents which modulate protein kinase C (PKC) activity were evaluated. Sphingosine (125-500 ng/ml), a PKC inhibitor, decreased SMC proliferation in allylamine, but not control cells. 12-O-Tetradecanoylphorbol-13-acetate (1-100 ng/ml), a PKC agonist, stimulated proliferation in control cells, but inhibited proliferation in cells from allylamine-treated animals. We conclude that allylamine-induced phenotypic modulation of SMC is associated with alterations in phosphoinositide metabolism.

Cox, L.R.; Murphy, S.K.; Ramos, K. (Philadelphia College of Pharmacy and Science, PA (USA))



Role of phosphoinositide metabolism in induction of memory in isolated perifused rat islets.  


Prior exposure of isolated perifused rat islets to 20 mM glucose or 10 mM glyceraldehyde amplifies their subsequent insulin secretory response to 10 mM glucose. The involvement of phosphoinositide turnover in the induction of this "time-dependent potentiation" (TDP) was investigated. In islets in which inositol-containing phospholipids were prelabeled with myo-[2-3H]inositol, the addition of 20 mM glucose augments the efflux of 3H. This effect persists for approximately 50 min after the cessation of stimulation. Direct measurements of labeled inositol phosphate accumulation confirmed that this increase in 3H efflux is primarily the result of a persistent increase in phosphoinositide (PI) hydrolysis and not due to the slow efflux and/or degradation of performed [3H]inositol phosphates. The duration of the increase in 3H efflux parallels the duration of TDP. Mannoheptulose abolishes both the increase in 3H efflux evoked by 20 mM glucose and TDP. The omission of extracellular calcium plus 0.5 mM ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid also abolishes both of these effects of high glucose. D-Glyceraldehyde (10 mM) addition to 3H-inositol-prelabeled islets results in an acute efflux of 3H, a persistent efflux after removal of the D-glyceraldehyde from the perifusion medium, and the induction of TDP. Similar to the results obtained with high glucose, the return of 3H efflux rates to prestimulatory values is accompanied by the abolition of TDP. These results suggest that events associated with persistent stimulant-induced increases in phosphoinositide hydrolysis may participate in the induction and maintenance of TDP. PMID:2834960

Zawalich, W S; Diaz, V A; Zawalich, K C



Targeting the phosphoinositide 3-kinase (PI3K) pathway in cancer  

PubMed Central

The phosphoinositide 3-kinase (PI3K) pathway, a critical signal transduction system linking oncogenes and multiple receptor classes to many essential cellular functions, is perhaps the most commonly activated signaling pathway in human cancer. This pathway thus presents both an opportunity and a challenge for cancer therapy. Even as inhibitors that target PI3K isoforms and other major nodes in the pathway including AKT and mTOR reach clinical trials, major issues remain. Here we highlight recent progress made in our understanding of the PI3K pathway and discuss both the promises and challenges for the therapeutic development of agents targeting the PI3K pathway in cancer.

Liu, Pixu; Cheng, Hailing; Roberts, Thomas M.; Zhao, Jean J.



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



The phosphoinositide kinase PIKfyve is vital in early embryonic development: preimplantation lethality of PIKfyve-/- embryos but normality of PIKfyve+/- mice.  


Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases. In mammals, PIKfyve synthesizes PtdIns(3,5)P(2) and PtdIns5P lipids that regulate endosomal trafficking and responses to extracellular stimuli. The consequence of pikfyve gene ablation in mammals is unknown. To clarify the importance of PIKfyve and PIKfyve lipid products, in this study, we have characterized the first mouse model with global deletion of the pikfyve gene using the Cre-loxP approach. We report that nearly all PIKfyve(KO/KO) mutant embryos died before the 32-64-cell stage. Cultured fibroblasts derived from PIKfyve(flox/flox) embryos and rendered pikfyve-null by Cre recombinase expression displayed severely reduced DNA synthesis, consistent with impaired cell division causing early embryo lethality. The heterozygous PIKfyve(WT/KO) mice were born at the expected Mendelian ratio and developed into adulthood. PIKfyve(WT/KO) mice were ostensibly normal by several other in vivo, ex vivo, and in vitro criteria despite the fact that their levels of the PIKfyve protein and in vitro enzymatic activity in cells and tissues were 50-55% lower than those of wild-type mice. Consistently, steady-state levels of the PIKfyve products PtdIns(3,5)P(2) and PtdIns5P selectively decreased, but this reduction (35-40%) was 10-15% less than that expected based on PIKfyve protein reduction. The nonlinear decrease of the PIKfyve protein versus PIKfyve lipid products, the potential mechanism(s) discussed herein, may explain how one functional allele in PIKfyve(WT/KO) mice is able to support the demands for PtdIns(3,5)P(2)/PtdIns5P synthesis during life. Our data also shed light on the known human disorder linked to PIKFYVE mutations. PMID:21349843

Ikonomov, Ognian C; Sbrissa, Diego; Delvecchio, Khortnal; Xie, Yufen; Jin, Jian-Ping; Rappolee, Daniel; Shisheva, Assia



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.



Phosphoinositide 3-kinase and integrin signalling are involved in activation of Bruton tyrosine kinase in thrombin-stimulated platelets.  


Bruton tyrosine kinase (Btk) plays a crucial role in the differentiation of B lymphocytes and belongs to the group of Tec kinases, which are characterised by the presence of a pleckstrin homology domain. Here we show that Btk is activated and undergoes tyrosine phosphorylation upon challenge of platelet thrombin receptor, these responses requiring engagement of alphaIIb/beta3 integrin and phosphoinositide 3-kinase activity. These data unravel a novel signalling pathway involving Btk downstream of an adhesive receptor via a complex regulation implicating the products of phosphoinositide 3-kinase, which might act to anchor Btk at the membrane. PMID:9928954

Laffargue, M; Ragab-Thomas, J M; Ragab, A; Tuech, J; Missy, K; Monnereau, L; Blank, U; Plantavid, M; Payrastre, B; Raynal, P; Chap, H



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.



Phosphoinositide 3-kinase beta controls replication factor C assembly and function  

PubMed Central

Genomic integrity is preserved by the action of protein complexes that control DNA homeostasis. These include the sliding clamps, trimeric protein rings that are arranged around DNA by clamp loaders. Replication factor C (RFC) is the clamp loader for proliferating cell nuclear antigen, which acts on DNA replication. Other processes that require mobile contact of proteins with DNA use alternative RFC complexes that exchange RFC1 for CTF18 or RAD17. Phosphoinositide 3-kinases (PI3K) are lipid kinases that generate 3-poly-phosphorylated-phosphoinositides at the plasma membrane following receptor stimulation. The two ubiquitous isoforms, PI3Kalpha and PI3Kbeta, have been extensively studied due to their involvement in cancer and nuclear PI3Kbeta has been found to regulate DNA replication and repair, processes controlled by molecular clamps. We studied here whether PI3Kbeta directly controls the process of molecular clamps loading. We show that PI3Kbeta associated with RFC1 and RFC1-like subunits. Only when in complex with PI3Kbeta, RFC1 bound to Ran GTPase and localized to the nucleus, suggesting that PI3Kbeta regulates RFC1 nuclear import. PI3Kbeta controlled not only RFC1– and RFC–RAD17 complexes, but also RFC–CTF18, in turn affecting CTF18-mediated chromatid cohesion. PI3Kbeta thus has a general function in genomic stability by controlling the localization and function of RFC complexes.

Redondo-Munoz, Javier; Josefa Rodriguez, Maria; Silio, Virginia; Perez-Garcia, Vicente; Maria Valpuesta, Jose; Carrera, Ana C.



Phosphoinositide 3-kinase signaling pathway mediated by p110? regulates invadopodia formation  

PubMed Central

Invadopodia are extracellular matrix–degrading protrusions formed by invasive cancer cells that are thought to function in cancer invasion. Although many invadopodia components have been identified, signaling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. We investigate the role of phosphoinositide 3-kinase (PI3K) signaling during invadopodia formation. We find that in human breast cancer cells, both invadopodia formation and degradation of a gelatin matrix were blocked by treatment with PI3K inhibitors or sequestration of D-3 phosphoinositides. Functional analyses revealed that among the PI3K family proteins, the class I PI3K catalytic subunit p110?, a frequently mutated gene product in human cancers, was selectively involved in invadopodia formation. The expression of p110? with cancerous mutations promoted invadopodia-mediated invasive activity. Furthermore, knockdown or inhibition of PDK1 and Akt, downstream effectors of PI3K signaling, suppressed invadopodia formation induced by p110? mutants. These data suggest that PI3K signaling via p110? regulates invadopodia-mediated invasion of breast cancer cells.

Yoshida, Shuhei; Muroi, Emi; Yoshida, Nachi; Kawamura, Masahiro; Kouchi, Zen; Nakamura, Yoshikazu; Sakai, Ryuichi; Fukami, Kiyoko



Targeting phosphoinositide 3-kinase pathways in pancreatic cancer--from molecular signalling to clinical trials.  


Pancreatic cancer has one of the poorest prognoses among all cancers partly because of its silent nature and tendency for late discovery but also because of its persistent resistance to chemotherapy. At present there are very limited treatment alternatives for pancreatic cancer, hence the need to develop novel and more efficient drugs. It is well known that mutations in K-Ras oncogene accumulate early in the disease progression and occur in almost all of pancreatic ductal adenocarcinomas (PDAC). A key downstream target of the Ras family is phosphoinositide 3-kinase (PI3K), the enzyme responsible for generation of 3-phosphorylated phosphoinositides and activation of Akt (Protein Kinase B/Akt). The PI3K/Akt pathway is involved in inhibition of apoptosis and stimulation of cell proliferation and it is has been estimated that at least 50% of all cancer types are related to deregulation of this signalling pathway. In this review we will discuss how the PI3K/Akt/mTOR signaling network is altered in pancreatic cancer and further give an overview of preclinical and clinical studies where this pathway has been targeted. PMID:21521159

Falasca, Marco; Selvaggi, Federico; Buus, Richard; Sulpizio, Sara; Edling, Charlotte E



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.



Stimulation of phosphoinositide hydrolysis by a novel substance partially purified from rat and bovine brain  

SciTech Connect

This study demonstrates the partial purification of a potentially novel substance from rat and bovine brain. Whole brains were homogenized in distilled water, then heated at 100/sup 0/C for 30 min. The water extract was dialyzed and the < 2000 dalton dialysate was fractionated on a Bio-Gel P-4 gel filtration column. Fractions were assayed for stimulation of /sup 3/H-inositol monophosphate (/sup 3/H-IP) using lithium-treated slices of rat cerebral cortex prelabelled with /sup 3/H-myo-inositol. A major peak of activity was observed in fractions from the molecular weight range of 800-1300 daltons. Stimulation of phosphoinositide hydrolysis by this material was time-dependent and dose-related. Maximal stimulation of /sup 3/H-IP (323% of control) required 10mg/ml of bovine material and was observed at 30 minutes. These effects could not be mimicked by a number of substances of similar molecular weight (e.g. substance P, neurotensin, angiotensin II, bradykinin). Furthermore, the effects of this material were not blocked by antagonist drugs which act at the alpha-adrenoceptor, muscarinic cholinoceptor, 5-HT2 receptor, substance P receptor, or neurotensin receptor. These results indicate that the substance isolated may be a novel neuroactive molecule which has receptors coupled to phosphoinositide hydrolysis in brain.

Schoepp, D.; Wilson, T.; Elliott, C.; Wright, G.; McCumbee, W.



Interleukin 1 is a potent stimulator of islet insulin secretion and phosphoinositide hydrolysis.  


The insulin stimulatory effect of 7 mM glucose on isolated perifused rat islets is dramatically potentiated by the monokine interleukin 1 (IL-1). At levels (10(-10) -10(-8) M) noted in vivo during sepsis, it reversibly amplifies peak second phase insulin release to the hexose. At 2.75 mM glucose, however, IL-1 has no effect on insulin secretion. IL-1 also potentiates glyceraldehyde (2 mM)- and alpha-ketoisocaproate (5 mM)-induced insulin secretion. In islets whose phosphoinositides were prelabeled with myo-[2-3H]inositol, 2.0-5.0 nM IL-1 increases the efflux of [3H]inositol from subsequently perifused islets, the parallel accumulation of labeled inositol phosphates, and insulin secretion in the simultaneous presence of 7 mM glucose but not 2.75 mM glucose. In support of these in vitro observations, the in vivo infusion of IL-1 (40 micrograms/kg body wt) elevated circulating plasma insulin levels two-to fourfold. These results establish IL-1 as a potent, readily reversible, glucose-dependent modulator of stimulated insulin secretion and further suggest that its positive impact on insulin release is mediated, at least in part, by phosphoinositide-derived second messenger molecules. IL-1-induced insulin secretion may participate in the multiple metabolic and immunologic adaptations occurring during sepsis. PMID:2536231

Zawalich, W S; Zawalich, K C



Cholecystokinin-induced alterations in beta-cell sensitivity. Duration, specificity, and involvement of phosphoinositide metabolism.  


Prior exposure of isolated perifused rat islets to the sulfated gut hormone cholecystokinin-8 (CCK-8S) dramatically increased their insulin secretory response to 7.5 mM glucose, 10 mM arginine, and 10 mM alpha-ketoisocaproate. In the case of glucose, the heightened secretory response was still apparent 60-80 min after CCK-8S removal from the perifusion medium. Prior exposure of perifused islets to arginine (10 mM), tolbutamide (25 microM), or forskolin (1.0 microM) did not sensitize them to 7.5 mM glucose. CCK-8S exposure increased 3H efflux from islets prelabeled with [3H]inositol, and the increase in 3H efflux was sustained after CCK-8S removal from the perifusion medium. The duration of this increase in 3H efflux paralleled the temporal characteristics of this sensitization process and was significantly attenuated by 25 microM asperlicin, a competitive antagonist of CCK binding to its membrane receptor. Arginine, tolbutamide, or forskolin treatment of islets did not increase 3H efflux from [3H]inositol-prelabeled islets. The results suggest that the turnover of membrane phosphoinositides induced by CCK-8S is largely responsible for this heightened state of secretory responsiveness to various stimulants. Second-messenger molecules generated during phosphoinositide turnover may be responsible for the phenomenon of sensitization displayed by islet tissue to CCK-8S addition. PMID:2824261

Zawalich, W S; Diaz, V A; Zawalich, K C



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)



Microscopic modes and free energies of 3-phosphoinositide-dependent kinase-1 (PDK1) binding with celecoxib and other inhibitors.  


Celecoxib, also known as Celebrex (approved by FDA in 1998) and remembered as the fastest-selling drug in history, was used as a cyclooxygenase-2 (COX-2) selective inhibitor having both anti-inflammatory and anticancer activities. Most recent studies have revealed that the apoptotic activity of celecoxib (and its derivatives) is actually independent of the COX-2 inhibitory activity and that celecoxib also inhibits the kinase activity of 3-phosphoinositide-dependent protein kinase-1 (PDK1), suggesting that the well-known anticancer activity of celecoxib is not due to the inhibition of COX-2, but possibly is due to the inhibition of PDK1. It is highly desirable to develop new celecoxib derivatives as PDK1-specifc inhibitors to avoid the side effects of COX-2 inhibitors. To understand how PDK1 binds with celecoxib and its derivatives, we have performed extensive molecular docking and combined molecular dynamics (MD) simulations and molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations on eight representative PDK1 inhibitors, leading to the finding of a new, more favorable binding mode which is remarkably different from the previously proposed binding mode. Based on the determined most stable binding structures, the calculated binding free energies are all in good agreement with the corresponding experimental data, and the biological activity data available for celecoxib and its derivatives can be better interpreted. The obtained new insights, concerning both the binding mode and computational protocol, will be valuable not only for future rational design of novel, more potent PDK1-specific inhibitors as promising anticancer therapeutics, but also for rational design of drugs targeting other proteins. PMID:17181296

Abdulhameed, Mohamed Diwan M; Hamza, Adel; Zhan, Chang-Guo



A stimulatory TSH receptor antibody enhances adipogenesis via phosphoinositide 3-kinase activation in orbital preadipocytes from patients with Graves' ophthalmopathy  

PubMed Central

Graves' ophthalmopathy (GO) is characterized by expanded volume of the orbital tissues associated with elevated serum levels of TSH receptor (TSHR) autoantibodies. Because previous studies have demonstrated evidence of adipogenesis within the GO orbit, we sought to determine whether M22, a human monoclonal antibody directed against TSHR, enhances adipogenesis in orbital fibroblasts from patients with GO and, if so, to identify signaling mechanisms involved. GO orbital fibroblast cultures (n=10) were treated for 10 days with bovine TSH (1 or 10·0?U/l) or M22 (1 or 10?ng/ml) in serum-free adipocyte differentiation medium. Some cultures also received a phosphoinositide 3-kinase (PI3K) inhibitor or an inhibitor of cAMP production. In other experiments, confluent cultures (n=8) were treated for between 1 and 30?min with TSH (0·1–10·0?U/l) or M22 (0·1–100?ng/ml) with measurement of cAMP production or levels of phosphorylated AKT (pAKT). We found levels of adiponectin, leptin, and TSHR mRNA to be increased in GO cultures treated for 10 days with either M22 (2·6 mean fold ±0·7; P=0·03) or TSH (13·2±5·8-fold, P=0·048). In other studies, M22 and TSH stimulated cAMP production and pAKT levels in GO cells. Inhibition of PI3K activity during 10 days in culture decreased the levels of M22-stimulated mRNA encoding adiponectin (67±12%; P=0·021), as well as adiponectin and CCAAT/enhancer-binding protein ? protein levels. In conclusion, M22 is a pro-adipogenic factor in GO orbital preadipocytes. This antibody appears to act via the PI3K signaling cascade, suggesting that inhibition of PI3K signaling may represent a potential novel therapeutic approach in GO.

Kumar, Seema; Nadeem, Sarah; Stan, Marius N; Coenen, Michael; Bahn, Rebecca S



Phosphoinositide-3-kinase inhibition induces sodium/iodide symporter expression in rat thyroid cells and human papillary thyroid cancer cells.  


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 lines and human papillary thyroid cancer cells. A PI3K inhibitor, LY294002, significantly enhanced iodide uptake in two rat thyroid cell lines, FRTL-5 and PCCL3. The induction of Nis mRNA by LY294002 occurred 6 h after treatment, and was abolished by a translation inhibitor, cycloheximide. Expression of the transcription factor, Pax8, which stimulates NIS expression, was significantly increased in PCCL3 cells after LY294002 treatment. Removal of insulin abrogated the stimulatory effects of LY294002 on NIS mRNA and protein expression, but not on iodide uptake. These findings suggest that PI3K pathway inhibition results in post-translational stimulation of NIS. Inhibition of the PI3K pathway also significantly increased iodide uptake ( approximately 3.5-fold) in BHP 2-7 papillary thyroid cancer cells (Ret/PTC1 positive), engineered to constitutively express NIS. Pharmacological inhibition of Akt, a factor stimulated by the PI3K pathway, increased exogenous NIS expression in BHP 2-7 as was seen with LY294002, but not increase the endogenous NIS expression in FRTL-5 cells. PI3K pathway inhibition increases functional NIS expression in rat thyroid cells and some papillary thyroid cancer cells by several mechanisms. PI3K inhibitors have the potential to increase radioiodide accumulation in some differentiated thyroid cancer. PMID:18762555

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



Serum withdrawal kills U937 cells by inducing a positive mutual interaction between reactive oxygen species and phosphoinositide 3-kinase  

Microsoft Academic Search

Reactive oxygen species (ROS) can be generated following cell stimulation and function as intracellular signaling molecules. To determine signaling components involved in ROS induction, human U937 blood cells grown in 10% serum were exposed to serum-free media. It was previously reported that serum withdrawal (SW) killed cells by elevating cellular ROS levels. This study showed that SW activates phosphoinositide 3-kinase

Seung Bum Lee; Eun Sook Cho; Hyun Sook Yang; Hoguen Kim; Hong-Duck Um



Phosphoinositide metabolism in the developing conceptus. Effects of hyperglycemia and scyllo-inositol in rat embryo culture.  


Culture of the postimplantation rat conceptus in hyperglycemic medium causes developmental abnormalities and is associated with a diminished water-soluble myo-inositol content. We investigated the effect myo-inositol depletion has on lipid-soluble phosphoinositides, precursors, and water-soluble inositol phosphates. Rat conceptuses were cultured from gestational day 9.5 (presomite, early head fold) to day 10.5 (7-15 somites) in 6.7-73.3 mM D-glucose. Significant decreases in the phosphoinositides of the embryo were observed with increased culture D-glucose concentrations. PI was reduced 15-34%, PIP 18-46%, and PIP2 26-46%. Yolk sac phosphoinositides also were reduced but to a lesser degree. Culture in hyperglycemic media also mediated significant reductions of conceptus inositol phosphates. To investigate whether effects similar to those induced by D-glucose could be mediated by another agent capable of decreasing myo-inositol content, we used scyllo-inositol, a transported but nonmetabolized isomer of myo-inositol. Conceptuses cultured in medium containing scyllo-inositol (0.06-16.7 mM) had dose-dependent decreases of myo-[3H]inositol in water-soluble and lipid-soluble fractions. Incorporation of myo-[3H]inositol into phosphoinositides and inositol phosphates was decreased concomitantly. Developmental effects of D-glucose and scyllo-inositol were assessed in rat conceptuses cultured from day 9.5 (presomite, early head fold) to day 11.5 (22-28 somites). Culture in 40.0-73.3 mM glucose and 0.06-33.3 mM scyllo-inositol impaired growth while increasing dysmorphogenesis in a dose-dependent manner. The results suggest that decreases in conceptus myo-inositol and associated diminution of phosphoinositides, which are the inositol/lipid cycle precursors, are dysmorphogenic and may contribute to the etiology of diabetic embryopathy. PMID:1321063

Strieleman, P J; Connors, M A; Metzger, B E



Contribution of phosphoinositide-dependent signalling to photomotility of Blepharisma ciliate.  


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 methods. Cell incubation with neomycin, heparin and Li+, drugs known to greatly impede phosphoinositide turnover, causes evident dose-dependent changes in cell photomotile behaviour. The strongest effect on photoresponses is exerted by neomycin, a potent inhibitor of polyphosphoinositide hydrolysis. The presence of micromolar concentrations of neomycin in the cell medium causes both prolongation of response latency and decrease of cell photoresponsiveness. Neomycin at higher concentrations (> 10 microM) abolishes the cell response to light at the highest applied intensity. A slightly lower inhibition of cell responsiveness to light stimulation and prolongation of response latency are observed in cells incubated in the presence of heparin, an inositol trisphosphate receptor antagonist. Lithium ions, widely known to deplete the intracellular phosphoinositide pathway intermediate, inositol trisphosphate, added to the cell medium at millimolar level, also cause a slowly developing inhibitory effect on cell photoresponses. Mastoparan, a specific G-protein activator, efficiently mimics the effect of light stimulation. In dark-adapted ciliates, it elicits ciliary reversal with the response latency typical for ciliary reversal during the photophobic response. Sustained treatment of Blepharisma cells with mastoparan also suppresses the photoresponsiveness, as in the case of cell adaptation to light during prolonged illumination. The mastoparan-induced responses can be eliminated by pretreatment of the cells with neomycin. Moreover, using antibodies raised against bovine transducin, a cross-reacting protein with an apparent molecular mass of about 55 kDa in the Blepharisma cortex fraction is detected on immunoblots. The obtained results indirectly suggest that the changes in internal inositol trisphosphate level, possibly elicited by G-protein-coupled phospholipase C, might play a role in the photophobic response of Blepharisma. However, further experiments are necessary to clarify the possible coupling between the G-protein and the putative phospholipase C. PMID:10942076

Fabczak, H


Glucose and scyllo-inositol impair phosphoinositide hydrolysis in the 10.5-day cultured rat conceptus: a role in dysmorphogenesis?  


Culture of the postimplantation rat conceptus from gestational day 9.5-10.5 in media supplemented with d-glucose or scyllo-inositol decreases tissue myo-inositol and phosphoinositides with a concomitant increase in dysmorphogenesis. A number of mitogenic agents initiate cellular proliferation and differentiation through receptors coupled to phosphoinositide hydrolysis. To test whether the decrease in conceptus phosphoinositides is associated with a reduced phosphoinositide hydrolytic response, we developed a protocol to stimulate phosphoinositide hydrolysis. Phosphoinositide hydrolysis was monitored by measurement of [3H]inositol phosphates after preincubation in serum free media. We examined the ability of serum, platelet-derived growth factor (PDGF), epidermal-derived growth factor (EGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), endothelin-1 (ET-1), and endothelin-2 (ET-2), to stimulate phosphoinositide hydrolysis. As measured by [3H]inositol monophosphate ([3H]InsP1) accumulation, normal rat seru, ET-1, and ET-2 stimulated phosphoinositide hydrolysis 47%, 420%, and 154% above the basal rate observed in serum free controls. EGF stimulated a statistically insignificant 15% increase while PDGF, IGF-1, or IGF-2 were without effect. We further characterized ET-1 stimulated phosphoinositide hydrolysis. Dose-response studies disclosed that incremental increases in [3H]InsP1 (129-420%) are observed over a concentration range of 10-1,000 nM. Maximal stimulation was not reached even at 1,000 nM. Temporally [3H]InsP1 and [3H]InsP3 levels increased linearly during incubation periods of 15-60 min. We further analyzed ET-1 stimulated phosphoinositide hydrolysis in 10.5-day conceptuses cultured for 24 hr in media containing high concentrations of glucose (23.3-56.6 mM) or scyllo-inositol (0.55, 5.5 mM). Under these dysmorphogenic conditions that concomitantly decrease the phosphoinositide precursor pool the response to ET-1 was blunted 28-76% for glucose and 29-65% for scyllo-inositol. This suggests that the effect of glucose and scyllo-inositol on lowering phosphoinositide precursor pools also results in a decrease in the response to agonists using the inositol/lipid intracellular pathway. This impaired signaling response may contribute to initiating dysmorphogenic events in diabetic embryopathy. PMID:8248864

Strieleman, P J; Metzger, B E



Novel Phosphoinositides in Barley Aleurone Cells (Additional Evidence for the Presence of Phosphatidyl-scyllo-Inositol).  


A novel isomer of phosphatidylinositol that differs in the structure of the head group was detected in barley (Hordeum vulgare cv Himalaya) seeds. In this paper we describe our efforts to elucidate the structure of the novel isomer. Evidence from a variety of techniques, including chemical modification of in vivo 32Pi- and myo-[3H]inositol-labeled compounds, gas chromatography-mass spectrometry analysis, in vivo incorporation of scyllo-[3H]inositol, and enzymatic studies that suggest that the structure is phosphatidylscyllo-inositol (scyllo-PI), is presented. The use of microwave energy to significantly enhance the slow rate of hydrolysis of phosphoinositides is described. The presence of scyllo-PI can be easily overlooked by the methods commonly employed; therefore, experimental considerations important for the detection of scyllo-PI are discussed. PMID:12223679

Narasimhan, B.; Pliska-Matyshak, G.; Kinnard, R.; Carstensen, S.; Ritter, M. A.; Von Weymarn, L.; Murthy, PPN.



Continuous spectrophotometric assay of mammalian phosphoinositide-specific phospholipase Cdelta1 with a thiophosphate substrate analog.  


1,2-Dimyristoyloxypropane-3-thiophospho(1D-1-myo-inositol) (D-thio-DMPI) was used as a substrate for the continuous assay of phosphoinositide-specific phospholipase C (PI-PLC). Its activity with a Delta(1-132) deletion mutant of mammalian PI-PLCdelta1 is about one-fourth that with PI under similar conditions. Optimal conditions for the assay include 0.2 mM substrate, 0.2 mM Ca2+, and a mole ratio of hexadecylphosphocholine detergent to substrate of 2.0. A minimum of about 60 ng of pure enzyme can be detected. The apparent bulk Km for PI-PLC with D-thio-DMPI under these conditions is about 6 microM. Enzyme activity as a function of surface concentration of substrate shows no sign of saturation up to the maximum mole fraction. PMID:9593806

Hendrickson, H S



Actin and phosphoinositide recruitment to fully-formed Candida albicans phagosomes in mouse macrophages  

PubMed Central

Candida albicans is a dimorphic yeast that enters macrophages via the ?-glucan receptor Dectin-1. Phagocytosis of C. albicans is characterized by actin polymerization, Syk kinase activation and rapid acquisition of phagolysosomal markers. In mice, C. albicans are able to resist the harsh environment of the phagosome and form pseudohyphae inside the phagolysosomal compartment, eventually extending from the macrophage. In this study, we investigated these unique C. albicans phagosomes and found that actin dynamically localized around the phagosomes, before disintegrating. Membrane phosphoinositides, PI(4,5)P2, PI(3,4,5)P3, PI(3,4)P2, and PI(3)P also localized to the phagosomes. This localization was not related to actin polymerization and inhibitor studies showed that polymerization of actin on the C. albicans phagosome was independent of PI3K. The ability of mature C. albicans phagosomes to stimulate actin polymerization could facilitate the escape of the growing yeast from the macrophage.

Heinsbroek, Sigrid E.M.; Kamen, Lynn A.; Taylor, Philip R.; Brown, Gordon D.; Swanson, Joel; Gordon, Siamon



Platelet alpha-2 adrenergic receptor-mediated phosphoinositide responses in endogenous depression  

SciTech Connect

We have previously indicated that epinephrine stimulates phosphoinositide (PI) hydrolysis by activating alpha-2 adrenergic receptors in human platelets. This method involves the measurement of the accumulation of ({sup 3}H)-inositol-1-phosphate (IP-1) as an index of Pl hydrolysis; lithium is added to inhibit the metabolism of IP-1, thus giving an enhanced signal. In the present study, we assessed the platelet alpha-2 adrenergic receptor-mediated PI responses in samples from 15 unmedicated patients with endogenous depression and 15 age- and sex-matched control subjects. The responses to epinephrine in the depressed patients were significantly higher than those of the controls, whereas the basal values did not differ significantly. These results support the hypothesis that platelet alpha-2 adrenergic receptors may be supersensitive in patients with endogenous depression.

Mori, Hideki (Municipal Second Hospital Otaru (Japan)); Koyama, Tsukasa; Yamashita, Itaru (Hokkaido Univ. School of Medicine, Sapporo (Japan))



Intranuclear 3'-phosphoinositide metabolism and apoptosis protection in PC12 cells.  


Lipid second messengers, particularly those derived from the polyphosphoinositide metabolism, play a pivotal role in multiple cell signaling networks. Phosphoinositide 3-kinase (PI3K) generates specific 3'-phosphorylated inositol lipids that have been implicated in a multitude of cell functions. One of the best characterized targets of PI3K lipid products is the serine/threonine protein kinase Akt (protein kinase B). Recent findings have implicated the PI3K/Akt pathway in cancer progression because it stimulates cell proliferation and suppresses apoptosis. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid cycle, and strongly suggests that lipid molecules are important components of signaling networks operating within the nucleus. PI3K, its lipid products, and Akt have also been identified at the nuclear level. In this review, we shall summarize the most updated findings about these molecules in relationship with suppression of apoptotic stimuli in PC12 cells. PMID:17465329

Martelli, Alberto M; Evangelist, Camilla; Billi, Anna Maria; Manzoli, Lucia; Papa, Veronica; Cocco, Lucio



Involvement of the phosphoinositide 3-kinase/Akt pathway in apoptosis induced by capsaicin in the human pancreatic cancer cell line PANC-1  

PubMed Central

Capsaicin, one of the major pungent ingredients found in red peppers, has been recently demonstrated to induce apoptosis in various malignant cell lines through an unclear mechanism. In this study, the effect of capsaicin on proliferation and apoptosis in the human pancreatic cancer cell line PANC-1 and its possible mechanism(s) of action were investigated. The results of a Cell Counting Kit-8 (CCK-8) assay revealed that capsaicin significantly decreased the viability of PANC-1 cells in a dose-dependent manner. Capsaicin induced G0/G1 phase cell cycle arrest and apoptosis in PANC-1 cells as demonstrated by a flow cytometric assessment. Caspase-3 expression at both the protein and mRNA level was promoted following capsaicin treatment. Furthermore, we revealed that phospho-PI3 Kinase p85 (Tyr458) and phospho-Akt (Ser473) in PANC-1 cells were downregulated in response to capsaicin. Moreover, capsaicin gavage significantly inhibited the growth of pancreatic cancer PANC-1 cell xenografts in athymic nude mice. An increased number of TUNEL-positive cells and cleaved caspase-3 were observed in capsaicin-treated mice. In vivo, capsaicin downregulated the expression of phospho-PI3 Kinase p85 (Tyr458) and phospho-Akt (Ser473). In conclusion, we have demonstrated that capsaicin is an inhibitor of growth of PANC-1 cells, and downregulation of the phosphoinositide 3-kinase/Akt pathway may be involved in capsaicin-induced apoptosis in vitro and in vivo.




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.



Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism  

SciTech Connect

We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of (3H)choline and (3H)phosphorylcholine ((3H)Pchol) from cells containing (3H)choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of (3H)phosphatidic acid ((3H)PA) in cells containing (3H)myristate-labeled PC. (3H)Diacylglycerol ((3H)DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with (3H)myristate and (14C)arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol.

Martinson, E.A.; Goldstein, D.; Brown, J.H. (Univ. of California, San Diego, La Jolla (USA))



Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes activated after stimulation with lipopolysaccharide.  


Signal transduction pathways, involved in cell cycle and activities, depend on various components including lipid signalling molecules, such as phosphoinositides and related enzymes. Many evidences support the hypothesis that inositol lipid cycle is involved in astrocytes activation during neurodegeneration. Previous studies investigated the pattern of expression of phosphoinositide-specific phospholipase C (PI-PLC) family isoforms in astrocytes, individuating in cultured neonatal rat astrocytes, supposed to be quiescent cells, the absence of some isoforms, accordingly to their well known tissue specificity. The same study was conducted in cultured rat astrocytoma C6 cells and designed a different pattern of expression of PI-PLCs in the neoplastic counterpart, accordingly to literature suggesting a PI signalling involvement in tumour progression. It is not clear the role of PI-PLC isoforms in inflammation; recent data demonstrate they are involved in cytokines production, with special regard to IL-6. PI-PLCs expression in LPS treated neonatal rat astrocytes performed by using RT-PCR, observed at 3, 6, 18 and 24 h intervals, expressed: PI-PLC beta1, beta4 and gamma1 in all intervals analysed; PI-PLC delta1 at 6, 18 and 24 h; PI-PLC delta3 at 6 h after treatment. PI-PLC beta3, delta4 and epsilon, present in untreated astrocytes, were not detected after LPS treatment. Immunocytochemical analysis, performed to visualize the sub-cellular distribution of the expressed isoforms, demonstrated different patterns of localisation at different times of exposure. These observations suggest that PI-PLCs expression and distribution may play a role in ongoing inflammation process of CNS. PMID:20082315

Lo Vasco, Vincenza Rita; Fabrizi, Cinzia; Fumagalli, Lorenzo; Cocco, L



Chronic in vivo hyperglycemia impairs phosphoinositide hydrolysis and insulin release in isolated perifused rat islets.  


We examined the effect of chronic hyperglycemia on phosphoinositide hydrolysis and insulin secretion in isolated perifused rat islets. Rats were infused for 44 h with 40% dextrose in order to raise and maintain the plasma glucose concentration at 350 mg/dl. Control animals were infused with equiosmolar amounts of mannitol. In vivo insulin secretion and rats of glucose disposal were monitored throughout the study. At the end of the infusion, islets were collagenase isolated, and phosphoinositide (PI) hydrolysis (assessed by measuring the increment in [3H]inositol efflux as well as labeled inositol phosphates) and insulin output in response to a 20-mM glucose challenge were quantitated. Plasma insulin concentration and in vivo glucose disposal rates decreased significantly, by 47% and 35% respectively, after 6-8 h of hyperglycemia. In islets perifused immediately after isolation, prior in vivo hyperglycemia markedly altered the pattern of insulin output in response to 20-mM glucose challenge. Compared to mannitol infusion, 20 mM glucose stimulation resulted in an exaggerated first phase insulin secretory response (1121 +/- 88 vs. 467 +/- 75 pg/islets.min) and a blunted second phase insulin secretory response (392 +/- 90 vs. 1249 +/- 205 pg/islet.min). In islets prelabeled with myo-[2-3H]inositol for 2 h, PI hydrolysis, particularly [3H]inositol efflux in response to glucose stimulation was also reduced (0.28 +/- 0.03%/min) compared to that in mannitol-infused animals (0.53 +/- 0.08%/min). Two hours of preincubation in a low glucose medium (2.75 mM) were able to completely reverse the islet defect in both PI hydrolysis and insulin secretion. Our results demonstrate that chronic in vivo hyperglycemia impairs PI hydrolysis in perifused rat islets and suggest that this defect accounts in part for the abnormal pattern of glucose-induced insulin secretion. PMID:2152864

Zawalich, W S; Zawalich, K C; Shulman, G I; Rossetti, L



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.



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




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



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


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. PMID:24124465

Juvin, Veronique; Malek, Mouhannad; Anderson, Karen E; Dion, Carine; Chessa, Tamara; Lecureuil, Charlotte; Ferguson, G John; Cosulich, Sabina; Hawkins, Phillip T; Stephens, Len R



Requirement for phosphoinositide 3-OH kinase in growth hormone signalling to the mitogen-activated protein kinase and p70s6k pathways.  

PubMed Central

Pituitary growth hormone (GH) co-ordinately stimulates three distinct signalling pathways in 3T3-F442A preadipocytes, the STAT (signal transducer and activator of transcription) pathway, the mitogen-activated protein (MAP) kinase cascade and p70s6k. The mechanisms linking the GH receptor to these signals have not been fully identified. In this study we have examined the role of phosphoinositide 3-OH kinase (PI 3-kinase). Pretreatment of cells with wortmannin, a specific inhibitor of PI 3-kinase, prevented the activation of p70s6k and partially inhibited the activation of p42 and p44 MAP kinases by GH. In contrast, wortmannin failed to appreciably affect the GH-stimulated tyrosyl phosphorylation of JAK-2 or STAT-1. GH transiently increased the activity of PI 3-kinase recovered in antiphosphotyrosine immunoprecipitates. In addition, several tyrosyl-phosphorylated proteins were specifically adsorbed from lysates of cells exposed to GH by a glutathione S-transferase fusion protein containing the 85 kDa regulatory subunit of PI 3-kinase. GH also induced an increase in the PI 3-kinase activity associated with both JAK-2 and insulin receptor substrate-1 (IRS-1) immunoprecipitates. These results establish PI 3-kinase as an important mediator of GH signalling to the MAP kinase and p70s6k pathways and suggest that PI 3-kinase is activated by a mechanism involving JAK-2 and IRS-1.

Kilgour, E; Gout, I; Anderson, N G



Distinct Cell Cycle Timing Requirements for Extracellular Signal-Regulated Kinase and Phosphoinositide 3-Kinase Signaling Pathways in Somatic Cell Mitosis  

PubMed Central

Mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase (PI3K) pathways are necessary for cell cycle progression into S phase; however the importance of these pathways after the restriction point is poorly understood. In this study, we examined the regulation and function of extracellular signal-regulated kinase (ERK) and PI3K during G2/M in synchronized HeLa and NIH 3T3 cells. Phosphorylation and activation of both the MAP kinase kinase/ERK and PI3K/Akt pathways occur in late S and persist until the end of mitosis. Signaling was rapidly reversed by cell-permeable inhibitors, indicating that both pathways are continuously activated and rapidly cycle between active and inactive states during G2/M. The serum-dependent behavior of PI3K/Akt versus ERK pathway activation indicates that their mechanisms of regulation differ during G2/M. Effects of cell-permeable inhibitors and dominant-negative mutants show that both pathways are needed for mitotic progression. However, inhibiting the PI3K pathway interferes with cdc2 activation, cyclin B1 expression, and mitotic entry, whereas inhibiting the ERK pathway interferes with mitotic entry but has little effect on cdc2 activation and cyclin B1 and retards progression from metaphase to anaphase. Thus, our study provides novel evidence that ERK and PI3K pathways both promote cell cycle progression during G2/M but have different regulatory mechanisms and function at distinct times.

Roberts, Elisabeth C.; Shapiro, Paul S.; Nahreini, Theresa Stines; Pages, Gilles; Pouyssegur, Jacques; Ahn, Natalie G.



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



The phosphoinositide 3'-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia.  


In lymphocytes, the phosphoinositide 3'-kinase (PI3K) isoform p110? (PI3K?) transmits signals from surface receptors, including the B-cell receptor (BCR). CAL-101, a selective inhibitor of PI3K?, displays clinical activity in CLL, causing rapid lymph node shrinkage and a transient lymphocytosis. Inhibition of pro-survival pathways, the presumed mechanism of CAL-101, does not explain this characteristic pattern of activity. Therefore, we tested CAL-101 in assays that model CLL-microenvironment interactions in vitro. We found that CAL-101 inhibits CLL cell chemotaxis toward CXCL12 and CXCL13 and migration beneath stromal cells (pseudoemperipolesis). CAL-101 also down-regulates secretion of chemokines in stromal cocultures and after BCR triggering. CAL-101 reduces survival signals derived from the BCR or from nurse-like cells, and inhibits BCR- and chemokine-receptor-induced AKT and MAP kinase (ERK) activation. In stromal cocultures, CAL-101 sensitizes CLL cells toward bendamustine, fludarabine, and dexamethasone. These results are corroborated by clinical data showing marked reductions in circulating CCL3, CCL4, and CXCL13 levels, and a surge in lymphocytosis during CAL-101 treatment. Thus, CAL-101 displays a dual mechanism of action, directly decreasing cell survival while reducing interactions that retain CLL cells in protective tissue microenvironments. These data provide an explanation for the clinical activity of CAL-101, and a roadmap for future therapeutic development. PMID:21803855

Hoellenriegel, Julia; Meadows, Sarah A; Sivina, Mariela; Wierda, William G; Kantarjian, Hagop; Keating, Michael J; Giese, Neill; O'Brien, Susan; Yu, Albert; Miller, Langdon L; Lannutti, Brian J; Burger, Jan A



Phosphoinositide-specific phospholipase C is involved in cytokinin and gravity responses in the moss Physcomitrella patens.  


The phosphoinositide signalling pathway is important in plant responses to extracellular and intracellular signals. To elucidate the physiological functions of phosphoinositide-specific phopspholipase C, PI-PLC, targeted knockout mutants of PpPLC1, a gene encoding a PI-PLC from the moss Physcomitrella patens, were generated via homologous recombination. Protonemal filaments of the plc1 lines show a dramatic reduction in gametophore formation relative to wild type: this was accompanied by a loss of sensitivity to cytokinin. Moreover, plc1 appeared paler than the wild type, the result of an altered differentiation of chloroplasts and reduced chlorophyll levels compared with wild type filaments. In addition, the protonemal filaments of plc1 have a strongly reduced ability to grow negatively gravitropically in the dark. These effects imply a significant role for PpPLC1 in cytokinin signalling and gravitropism. PMID:15447651

Repp, Alexander; Mikami, Koji; Mittmann, Franz; Hartmann, Elmar



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


Effect of aging on alpha-1 adrenergic stimulation of phosphoinositide hydrolysis in various regions of rat brain  

Microsoft Academic Search

The effects of aging were examined on the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis in three brain regions. Tissue minces of thalamus, cerebral cortex and hippocampus from 3-, 18- and 28-month-old male Fischer 344 rats were prelabeled with (³H)myoinositol. Exposure of these prelabeled minces to phenylephrine and (-)-norepinephrine revealed that accumulation of (³H)inositol phosphates was selectively

D. M. Burnett; J. F. Bowyer; J. M. Masserano; N. R. Zahniser



Blockade of Inflammation and Airway Hyperresponsiveness in Immune-sensitized Mice by Dominant-Negative Phosphoinositide 3-Kinase-TAT  

Microsoft Academic Search

Phosphoinositide 3-kinase (PI3K) is thought to contribute to the pathogenesis of asthma by effecting the recruitment, activation, and apoptosis of inflammatory cells. We examined the role of class IA PI3K in antigen-induced airway inflammation and hyperresponsiveness by i.p. administration into mice ofp85 protein, a dominant negative form of the class IA PI3K regu- latory subunit, p85 ? , which was

Shigeharu Myou; Alan R. Leff; Saori Myo; Evan Boetticher; Jiankun Tong; Angelo Y. Meliton; Jie Liu; Nilda M. Munoz; Xiangdong Zhu



Amyloid Beta Peptide Impaired Carbachol but not Glutamate-Mediated Phosphoinositide Pathways in Cultured Rat Cortical Neurons  

Microsoft Academic Search

Signal transduction systems, including cholinergic pathways, which are likely to be of pathophysiological significance are altered in Alzheimer's disease (AD). Muscarinic cholinergic receptors are linked to the hydrolysis of phosphoinositide, involving the production of inositol 1,4,5-trisphosphate [Ins (1,4,5)P3] and the mobilization of cytosolic free calcium concentrations ([Ca2+]i). Effects of amyloid peptide (Aß) on these signals prior to neuronal degeneration were

Hsueh-Meei Huang; Hsio-Chung Ou; Shon-Jean Hsieh



5-Hydroxytryptamine-Induced Phosphoinositide Hydrolysis and Ca 2+ Mobilisation in Canine Cultured Aorta Smooth Muscle Cells  

Microsoft Academic Search

The effect of 5-hydroxytryptamine (5-HT) on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and intracellular Ca2+ ([Ca2+]i) changes was investigated in canine cultured aorta smooth muscle cells (ASMCs). 5-HT-stimulated inositol phosphate (IP) accumulation was time and concentration dependent with a half-maximal response (pEC50) and a maximal response at 6.4 and 10 ?M, n = 6, respectively. Stimulation of ASMCs by 5-HT

Chi-Tso Chiu; Hui-Liang Tsao; Lir-Wan Fan; Chuan-Chwan Wang; Chin-Sung Chien; Chuen-Mao Yang



Phosphoinositide 3-kinase regulates myogenin expression at both the transcriptional and post-transcriptional level during myogenesis  

Microsoft Academic Search

It is well-established that phosphoinositide 3-kinase (PI3-kinase) regulates myogenesis by inducing transcription of myogenin, a key muscle regulatory factor, at the initiation of myoblast differentiation. In this study, we investigated the role of PI3-kinase in cells that have committed to differentiation. PI3-kinase activity increases during myogenesis, and this increase is sustained during the myogenic process; however, its function after the

Joo Hong Woo; Min Jeong Kim; Hye Sun Kim



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



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



Direct metabolic regulation of ?-catenin activity by the p85? regulatory subunit of phosphoinositide 3OH kinase  

Microsoft Academic Search

Class IA phosphoinositide 3-OH kinases (PI3K) are lipid kinases composed of catalytic and regulatory subunits. These lipid kinases can regulate the metabolic stability and signaling activity of ?-catenin, a central component of the E-cadherin\\/catenin cell–cell adhesion complex, and of the Wnt signaling pathway. This regulation occurs at the level of glycogen synthase kinase 3 (GSK3), a serine\\/threonine kinase that marks

Jesús Espada; Héctor Peinado; Manel Esteller; Amparo Cano



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



Abscisic acid induces oscillations in guard-cell cytosolic free calcium that involve phosphoinositide-specific phospholipase C  

PubMed Central

Oscillations in cytosolic free Ca2+ concentration ([Ca2+]cyt) are an important component of Ca2+-based signal transduction pathways. This fact has led us to investigate whether oscillations in [Ca2+]cyt are involved in the response of stomatal guard cells to the plant hormone abscisic acid (ABA). We show that ABA induces oscillations in guard-cell [Ca2+]cyt. The pattern of the oscillations depended on the ABA concentration and correlated with the final stomatal aperture. We examined the mechanism by which ABA generates oscillations in guard-cell [Ca2+]cyt by using 1-(6-{[17?-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122), an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC)-dependent processes in animals. U-73122 inhibited the hydrolysis of phosphatidylinositol 4,5-bisphosphate by a recombinant PI-PLC, isolated from a guard-cell-enriched cDNA library, in a dose-dependent manner. This result confirms that U-73122 is an inhibitor of plant PI-PLC activity. U-73122 inhibited both ABA-induced oscillations in [Ca2+]cyt and stomatal closure. In contrast, U-73122 did not inhibit external Ca2+-induced oscillations in guard-cell [Ca2+]cyt and stomatal closure. Furthermore, there was no effect of the inactive analogue 1-(6-{[17?-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-2,5-pyrrolidinedione on recombinant PI-PLC activity or ABA-induced and external Ca2+-induced oscillations in [Ca2+]cyt and stomatal closure. This lack of effect suggests that the effects of U-73122 in guard cells are the result of inhibition of PI-PLC and not a consequence of nonspecific effects. Taken together, our data suggest a role for PI-PLC in the generation of ABA-induced oscillations in [Ca2+]cyt and point toward the involvement of oscillations in [Ca2+]cyt in the maintenance of stomatal aperture by ABA.

Staxen, Irina; Pical, Christophe; Montgomery, Lucy T.; Gray, Julie E.; Hetherington, Alistair M.; McAinsh, Martin R.



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


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

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



Evolution of the voltage sensor domain of the voltage-sensitive phosphoinositide phosphatase VSP/TPTE suggests a role as a proton channel in eutherian mammals.  


The voltage-sensitive phosphoinositide phosphatases provide a mechanism to couple changes in the transmembrane electrical potential to intracellular signal transduction pathways. These proteins share a domain architecture that is conserved in deuterostomes. However, gene duplication events in primates, including humans, give rise to the paralogs TPTE and TPTE2 that retain protein domain organization but, in the case of TPTE, have lost catalytic activity. Here, we present evidence that these human proteins contain a functional voltage sensor, similar to that in nonmammalian orthologs. However, domains of these human proteins can also generate a noninactivating outward current that is not observed in zebra fish or tunicate orthologs. This outward current has the anticipated characteristics of a voltage-sensitive proton current and is due to the appearance of a single histidine residue in the S4 transmembrane segment of the voltage sensor. Histidine is observed at this position only during the eutherian radiation. Domains from both human paralogs generate proton currents. This apparent gain of proton channel function during the evolution of the TPTE protein family may account for the conservation of voltage sensor domains despite the loss of phosphatase activity in some human paralogs. PMID:22396523

Sutton, Keith A; Jungnickel, Melissa K; Jovine, Luca; Florman, Harvey M



Identification of myo-inositol 1,2-cyclic monophosphate by electrospray tandem mass spectrometry, a major constituent of EGF-stimulated phosphoinositide turnover in MDA 468 cells.  


Epidermal growth factor (EGF) caused an increase in phosphoinositide (PI) turnover in MDA 468 cells. This EGF-stimulated effect was inhibited by the protein tyrosine kinase inhibitor lavendustin A (LA). MDA 468 cells generated an atypical PI turnover profile. Examination and quantitation of the PI metabolite profile showed that even control cells produced a metabolite which was acid-labile and which formed about 60% of the total PI metabolites. By using the technique of electrospray ionization tandem mass spectrometry, we were able to confirm the identity of this acid-labile metabolite through the specific fragmentation as compared with the standard. The precursor molecule fragmented into two distinct productions with molar masses identical to that of the standard myo-inositol 1,2-cyclic monophosphate (cInsP). Changes in the PI turnover profile could be accounted for by the alterations in myo-inositol 1,2-cyclic monophosphate generated in these cells. We thus conclude that, by some as-yet-unidentified mechanism, cyclic inositol monophosphate forms a major constituent of EGF-stimulated PI turnover in MDA 468 cells. PMID:12083795

Mandal, Soma; Richardson, Vernon J; Banoub, Joseph; Church, Jon G



TULP3 bridges the IFT-A complex and membrane phosphoinositides to promote trafficking of G protein-coupled receptors into primary cilia  

PubMed Central

Primary cilia function as a sensory signaling compartment in processes ranging from mammalian Hedgehog signaling to neuronal control of obesity. Intraflagellar transport (IFT) is an ancient, conserved mechanism required to assemble cilia and for trafficking within cilia. The link between IFT, sensory signaling, and obesity is not clearly defined, but some novel monogenic obesity disorders may be linked to ciliary defects. The tubby mouse, which presents with adult-onset obesity, arises from mutation in the Tub gene. The tubby-like proteins comprise a related family of poorly understood proteins with roles in neural development and function. We find that specific Tubby family proteins, notably Tubby-like protein 3 (TULP3), bind to the IFT-A complex. IFT-A is linked to retrograde ciliary transport, but, surprisingly, we find that the IFT-A complex has a second role directing ciliary entry of TULP3. TULP3 and IFT-A, in turn, promote trafficking of a subset of G protein-coupled receptors (GPCRs), but not Smoothened, to cilia. Both IFT-A and membrane phosphoinositide-binding properties of TULP3 are required for ciliary GPCR localization. TULP3 and IFT-A proteins both negatively regulate Hedgehog signaling in the mouse embryo, and the TULP3–IFT-A interaction suggests how these proteins cooperate during neural tube patterning.

Mukhopadhyay, Saikat; Wen, Xiaohui; Chih, Ben; Nelson, Christopher D.; Lane, William S.; Scales, Suzie J.; Jackson, Peter K.



The basal level of intracellular calcium gates the activation of phosphoinositide 3-kinase - Akt signaling by brain-derived neurotrophic factor in cortical neurons  

PubMed Central

Brain derived neurotrophic factor (BDNF) mediates survival and neuroplasticity through the activation of phosphoinositide 3-kinase (PI3K)-Akt pathway. Although previous studies suggested the roles of MAPK, PLC-?-mediated intra-cellular calcium ([Ca2+]i) increase, and extra-cellular calcium influx in regulating Akt activation, the cellular mechanisms are largely unknown. We demonstrated that sub-nanomolar BDNF significantly induced Akt activation in developing cortical neurons. The TrkB-dependent Akt phosphorylation at S473 and T308 required only PI3K, but not PLC and MAPK activity. Blocking NMDA receptors, L-type voltage-gated calcium channels, and chelating extra-cellular calcium by EGTA failed to block BDNF-induced Akt phosphorylation. In contrast, chelating [Ca2+]i by BAPTA-AM abolished Akt phosphorylation. Interestingly, sub-nanomolar BDNF did not stimulate [Ca2+]i increase under our culture conditions. Together with that NMDA- and membrane depolarization-induced [Ca2+]i increase did not activate Akt, we conclude that the basal level of [Ca2+]i gates BDNF function. Furthermore, inhibiting calmodulin by W13 suppressed Akt phosphorylation. On the other hand, inhibition of protein phosphatase 1 by okadaic acid and tautomycin rescued Akt phosphorylation in BAPTA- and W13-treated neurons. We further demonstrated that the phosphorylation of PDK1 did not correlate with Akt phosphorylation at T308. Our results suggested novel roles of basal [Ca2+]i, rather than activity-induced calcium elevation, in BDNF-Akt signaling.

Zheng, Fei; Soellner, Deborah; Nunez, Joseph; Wang, Hongbing



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.



Structural basis for substrate recognition by a unique Legionella phosphoinositide phosphatase  

PubMed Central

Legionella pneumophila is an opportunistic intracellular pathogen that causes sporadic and epidemic cases of Legionnaires’ disease. Emerging data suggest that Legionella infection involves the subversion of host phosphoinositide (PI) metabolism. However, how this bacterium actively manipulates PI lipids to benefit its infection is still an enigma. Here, we report that the L. pneumophila virulence factor SidF is a phosphatidylinositol polyphosphate 3-phosphatase that specifically hydrolyzes the D3 phosphate of PI(3,4)P2 and PI(3,4,5)P3. This activity is necessary for anchoring of PI(4)P-binding effectors to bacterial phagosomes. Crystal structures of SidF and its complex with its substrate PI(3,4)P2 reveal striking conformational rearrangement of residues at the catalytic site to form a cationic pocket that specifically accommodates the D4 phosphate group of the substrate. Thus, our findings unveil a unique Legionella PI phosphatase essential for the establishment of lipid identity of bacterial phagosomes.

Hsu, FoSheng; Zhu, Wenhan; Brennan, Lucy; Tao, Lili; Luo, Zhao-Qing; Mao, Yuxin



The stereoselective recognition of substrates by phosphoinositide kinases. Studies using synthetic stereoisomers of dipalmitoyl phosphatidylinositol.  


Soluble phosphatidylinositol (PtdIns) 4- and 3-kinase activities were partially purified and characterized from human placental extracts. The placental PtdIns 4-kinase (type 3) has a Km for ATP of 460 microM and is kinetically different to a partially purified human erythrocyte, membrane-bound, PtdIns 4-kinase (type 2). These three inositol lipid kinases were then used to compare their substrate specificities against the four synthetic stereoisomers of dipalmitoyl PtdIns. Only the placental 4-kinase was influenced by the chirality of the glycerol moiety of PtdIns. However, neither of the 4-kinases was able to phosphorylate L-PtdIns and, therefore, these kinases have an absolute requirement for the inositol ring to be linked to the glyceryl backbone of the lipid through the D-1 position. Phosphoinositide 3-kinase, on the other hand, was found to phosphorylate both D- and L-PtdIns. While the 3-kinase phosphorylated exclusively the D-3 position of D-PtdIns, further analyses demonstrated that the same enzyme phosphorylated two sites on L-PtdIns, namely the D-6 and D-5 positions of the inositol ring. Some implications of these findings are discussed. PMID:1317855

Macphee, C H; Carter, A N; Ruiz-Larrea, F; Ward, J G; Young, R C; Downes, C P



Voltage sensitive phosphoinositide phosphatases of Xenopus: their tissue distribution and voltage dependence.  


Voltage-sensitive phosphatases (VSPs) are unique proteins in which membrane potential controls enzyme activity. They are comprised of the voltage sensor domain of an ion channel coupled to a lipid phosphatase specific for phosphoinositides, and for ascidian and zebrafish VSPs, the phosphatase activity has been found to be activated by membrane depolarization. The physiological functions of these proteins are unknown, but their expression in testis and embryos suggests a role in fertilization or development. Here we investigate the expression pattern and voltage dependence of VSPs in two frog species, Xenopus laevis and Xenopus tropicalis, that are well suited for experimental studies of these possible functions. X. laevis has two VSP genes (Xl-VSP1 and Xl-VSP2), whereas X. tropicalis has only one gene (Xt-VSP). The highest expression of these genes was observed in testis, ovary, liver, and kidney. Our results show that while Xl-VSP2 activates only at positive membrane potentials outside of the physiological range, Xl-VSP1 and Xt-VSP phosphatase activity is regulated in the voltage range that regulates sperm-egg fusion at fertilization. PMID:21618529

Ratzan, William J; Evsikov, Alexei V; Okamura, Yasushi; Jaffe, Laurinda A



Prostaglandin F/sub 2. cap alpha. activates phosphoinositide hydrolysis in rat aorta  

SciTech Connect

The authors have previously demonstrated that norepinephrine (NE) and serotonin (5HT) activate a phosphoinositide-(PI) specific phospholipase C in rat aorta by interaction with ..cap alpha../sub 1/-adrenergic receptors and 5HT/sub 2/ receptor, respectively. They have subsequently noted that angiotensin II and vasopressin as well activate PI hydrolysis in the tissue. The most active agent they have thus far investigated is prostaglandin F/sub 2..cap alpha../ (PGF/sub 2..cap alpha../). Rat aortic rings were pre-labelled with (/sup 3/H)-inositol and then, in the presence of 10 mM LiCl, exposed to various doses of PGF/sub 2..cap alpha../. (/sup 3/H)-inositol monophosphate was the quantified by anion-exchange chromatography. After a 60 min incubation, PGF/sub 2..cap alpha../ caused a 10-15 fold increase over basal at maximal concentrations (0.1-1.0 mM). An EC/sub 50/ for PI hydrolysis was between 0.1-1.0 PGF/sub 2..cap alpha../ caused maximal aortic contraction at 10 PGF/sub 2..cap alpha../-induced PI hydrolysis, was inhibited by phorbol esters. These results suggest that PGF/sub 2..cap alpha../, similar to 5HT, NE, vasopressin and angiotensin II, causes vasoconstriction by activation of PI hydrolysis.

Not Available



Chronic ethanol inhibits receptor-stimulated phosphoinositide hydrolysis in rat liver slices  

SciTech Connect

The effects of chronic ethanol feeding on norepinephrine (NE)- and arginine-vasopressin (AVP)-stimulated phosphoinositide (PI) hydrolysis in rat liver slices was determined. The maximum NE-stimulated PI response was significantly reduced by 40% in liver slices from 8-month-old rats which had been treated for 5 months with a liquid diet containing ethanol compared to pair-fed controls. The maximum AVP-stimulated PI response was decreased by 39% in liver slices from the ethanol-fed rats compared to control. EC50 values for NE- and AVP-stimulated PI hydrolysis in liver slices were not affected by the chronic ethanol treatment. Similar reductions in the maximal NE- and AVP-stimulated PI hydrolysis (28% and 27%, respectively) were found in 22-month-old rats which had been maintained on an ethanol containing diet for 5 months compared to pair-fed controls. The binding of (3H)prazosin and (3H)AVP to liver plasma membranes from 8-month-old ethanol-fed rats was not significantly different from binding to liver membranes from sucrose-fed controls. Our data suggest that chronic ethanol ingestion may lead to a reduction in PI-linked signal transduction in liver.

Gonzales, R.A.; Crews, F.T. (Department of Pharmacology, University of Texas, Austin (USA))



Alpha-1 adrenergic receptor: Binding and phosphoinositide breakdown in human myometrium  

SciTech Connect

Alpha-1 adrenergic receptors were examined in both inner and outer layers of human pregnant myometrium using radioligand binding of (3H)prazosin. (3H)prazosin bound rapidly and reversibly to a single class of high affinity binding sites in myometrial membrane preparations. Scatchard analysis gave similar values of equilibrium dissociation constants in both myometrial layers. In contrast, more alpha-1 adrenergic receptors were detected in the outer layer than in the inner layer. Antagonist inhibited (3H)prazosin binding with an order of potency of prazosin greater than phentolamine greater than idazoxan. Competition experiments have also revealed that a stable guanine nucleotide decreases the apparent affinity of norepinephrine for myometrial (3H)prazosin binding sites. The functional status of these alpha-1 adrenergic receptors was also assessed by measuring the norepinephrine-induced accumulation of inositol phosphates in myometrial tissue. Norepinephrine produced a concentration-dependent accumulation of inositol phosphates in both myometrial layers. However, norepinephrine-induced increases in inositol 1,4,5-triphosphate were only observed in the outer layer. These results indicate that alpha-1 adrenergic receptors in human myometrium at the end of pregnancy are linked to phosphoinositide hydrolysis and that this response occurs mainly in the outer layer.

Breuiller-Fouche, M.; Doualla-Bell Kotto Maka, F.; Geny, B.; Ferre, F. (INSERM U.166 Groupe de recherches sur l'Endocrinologie de la Reproduction, Maternite Baudelocque, Paris (France))



Phosphoinositide-3 kinase ? required for LPS-induced transepithelial neutrophil trafficking in the lung  

PubMed Central

Phosphoinositide 3-kinase ? (PI3K?) is a critical mediator of directional cell movement. Here, we sought to characterize the role of PI3K? in mediating the different steps of PMN trafficking in the lung. In a murine model of LPS-induced lung injury, PMN migration into the different lung compartments was determined in PI3K? gene-deficient (PI3K??/?) and wildtype mice. Bone marrow chimeras were created to characterize the role of PI3K? on hematopoietic vs. non-hematopoietic cells. A small molecule PI3K? inhibitor was tested in vitro and in vivo. PMN adhesion to the pulmonary endothelium and transendothelial migration into the lung interstitium was enhanced in PI3K??/? mice. However, transepithelial migration into the alveolar space was reduced in these mice. When irradiated PI3K??/? mice were reconstituted with bone marrow from wildtype mice, migratory activity into the alveolar space was restored partially. A small molecule PI3K? inhibitor reduced chemokine-induced PMN migration in vitro when PMNs or epithelial cells but not when endothelial cells were treated. The inhibitor also reduced LPS-induced PMN migration in vivo. We conclude that PI3K? is required for transepithelial but not for transendothelial migration in LPS-induced lung injury. Inhibition of PI3K? activity may be effective at curbing excessive PMN infiltration in lung injury.

Reutershan, Jorg; Saprito, Mary S.; Wu, Dan; Ruckle, Thomas; Ley, Klaus



Voltage sensitive phosphoinositide phosphatases of Xenopus: their tissue distribution and voltage dependence  

PubMed Central

Voltage sensitive phosphatases (VSPs) are unique proteins in which membrane potential controls enzyme activity. They are comprised of the voltage sensor domain of an ion channel coupled to a lipid phosphatase specific for phosphoinositides, and for ascidian and zebrafish VSPs, the phosphatase activity has been found to be activated by membrane depolarization. The physiological functions of these proteins are unknown, but their expression in testis and embryos suggests a role in fertilization or development. Here we investigate the expression pattern and voltage dependence of VSPs in two frog species, Xenopus laevis and Xenopus tropicalis, that are well suited for experimental studies of these possible functions. X. laevis has two VSP genes (Xl-VSP1 and Xl-VSP2), whereas X. tropicalis has only one gene (Xt-VSP). The highest expression of these genes was observed in testis, ovary, liver, and kidney. Our results show that while Xl-VSP2 activates only at positive membrane potentials outside of the physiological range, Xl-VSP1 and Xt-VSP phosphatase activity is regulated in the voltage range that regulates sperm-egg fusion at fertilization.

Ratzan, William J.; Evsikov, Alexei V.; Okamura, Yasushi; Jaffe, Laurinda A.



Blockade of class IB phosphoinositide-3 kinase ameliorates obesity-induced inflammation and insulin resistance  

PubMed Central

Obesity and insulin resistance, the key features of metabolic syndrome, are closely associated with a state of chronic, low-grade inflammation characterized by abnormal macrophage infiltration into adipose tissues. Although it has been reported that chemokines promote leukocyte migration by activating class IB phosphoinositide-3 kinase (PI3K?) in inflammatory states, little is known about the role of PI3K? in obesity-induced macrophage infiltration into tissues, systemic inflammation, and the development of insulin resistance. In the present study, we used murine models of both diet-induced and genetically induced obesity to examine the role of PI3K? in the accumulation of tissue macrophages and the development of obesity-induced insulin resistance. Mice lacking p110? (Pik3cg?/?), the catalytic subunit of PI3K?, exhibited improved systemic insulin sensitivity with enhanced insulin signaling in the tissues of obese animals. In adipose tissues and livers of obese Pik3cg?/? mice, the numbers of infiltrated proinflammatory macrophages were markedly reduced, leading to suppression of inflammatory reactions in these tissues. Furthermore, bone marrow-specific deletion and pharmacological blockade of PI3K? also ameliorated obesity-induced macrophage infiltration and insulin resistance. These data suggest that PI3K? plays a crucial role in the development of both obesity-induced inflammation and systemic insulin resistance and that PI3K? can be a therapeutic target for type 2 diabetes.

Kobayashi, Naoki; Ueki, Kohjiro; Okazaki, Yukiko; Iwane, Aya; Kubota, Naoto; Ohsugi, Mitsuru; Awazawa, Motoharu; Kobayashi, Masatoshi; Sasako, Takayoshi; Kaneko, Kazuma; Suzuki, Miho; Nishikawa, Yoshitaka; Hara, Kazuo; Yoshimura, Kotaro; Koshima, Isao; Goyama, Susumu; Murakami, Koji; Sasaki, Junko; Nagai, Ryozo; Kurokawa, Mineo; Sasaki, Takehiko; Kadowaki, Takashi



Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes.  


Signal transduction from plasma membrane to cell nucleus is a complex process depending on various components including lipid signaling molecules, in particular phosphoinositides and their related enzymes, which act at cell periphery and/or plasma membrane as well as at nuclear level. As far as the nervous system may concern the inositol lipid cycle has been hypothesized to be involved in numerous neural as well as glial functions. In this context, however, a precise panel of glial PLC isoforms has not been determined yet. In the present experiments we investigated astrocytic PLC isoforms in astrocytes obtained from foetal primary cultures of rat brain and from an established cultured (C6) rat astrocytoma cell line, two well known cell models for experimental studies on glia. Identification of PLC isoforms was achieved by using a combination of RT-PCR and immunocytochemistry experiments. While in both cell models the most represented PI-PLC isoforms were beta4, gamma1, delta4, and epsilon, isoforms PI-PLC beta2 and delta3 were not detected. Moreover, in primary astrocyte cultures PI-PLC delta3 resulted well expressed in C6 cells but was absent in astrocytes. Immunocytochemistry performed with antibodies against specific PLC isoforms substantially confirmed this pattern of expression both in astrocytes and C6 glioma cells. In particular while some isoenzymes (namely isoforms beta3 and beta4) resulted mainly nuclear, others (isoforms delta4 and epsilon) were preferentially localized at cytoplasmic and plasma membrane level. PMID:17063484

Lo Vasco, Vincenza Rita; Fabrizi, Cinzia; Artico, Marco; Cocco, Lucio; Billi, Anna Maria; Fumagalli, Lorenzo; Manzoli, Francesco Antonio



C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C.  


Phosphoinositide-specific phospholipase C (PLC) is involved in Ca²? mediated signalling events that lead to altered cellular status. Using various sequence-analysis methods, we identified two conserved motifs in known PLC sequences. The identified motifs are located in the C2 domain of plant PLCs and are not found in any other protein. These motifs are specifically found in the Ca²? binding loops and form adjoining beta strands. Further, we identified certain conserved residues that are highly distinct from corresponding residues of animal PLCs. The motifs reported here could be used to annotate plant-specific phospholipase C sequences. Furthermore, we demonstrated that the C2 domain alone is capable of targeting PLC to the membrane in response to a Ca²? signal. We also showed that the binding event results from a change in the hydrophobicity of the C2 domain upon Ca²? binding. Bioinformatic analyses revealed that all PLCs from Arabidopsis and rice lack a transmembrane domain, myristoylation and GPI-anchor protein modifications. Our bioinformatic study indicates that plant PLCs are located in the cytoplasm, the nucleus and the mitochondria. Our results suggest that there are no distinct isoforms of plant PLCs, as have been proposed to exist in the soluble and membrane associated fractions. The same isoform could potentially be present in both subcellular fractions, depending on the calcium level of the cytosol. Overall, these data suggest that the C2 domain of PLC plays a vital role in calcium signalling. PMID:22124893

Rupwate, Sunny D; Rajasekharan, Ram



RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms.  


RAS proteins are important direct activators of p110?, p110?, and p110? type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110? isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110?, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110? via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110?. Cells from mice carrying mutations in the p110? RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110? downstream of GPCRs. PMID:23706742

Fritsch, Ralph; de Krijger, Inge; Fritsch, Kornelia; George, Roger; Reason, Beth; Kumar, Madhu S; Diefenbacher, Markus; Stamp, Gordon; Downward, Julian



A specific role for phosphoinositide 3-kinase and AKT in osteoblasts?  

PubMed Central

The phosphoinositide 3-kinase and AKT (protein kinase B) signaling pathway (PI3K/AKT) plays a central role in the control of cell survival, growth, and proliferation throughout the body. With regard to bone, and particularly in osteoblasts, there is an increasing amount of evidence that the many signaling molecules exert some of their bone-specific effects in part via selectively activating some of the generic effects of the PI3K/AKT pathway in osteoblasts. There is further data demonstrating that PI3K/AKT has the capacity to specifically cross-talk with other signaling pathways and transcriptional networks controlling bone cells’ development in order to fine-tune the osteoblast phenotype. There is also evidence that perturbations in the PI3K/AKT pathway may well be responsible for certain bone pathologies. In this review, we discuss some of these findings and suggest that the PI3K/AKT pathway is a central nexus in the extensive network of extracellular signaling pathways that control the osteoblast.

McGonnell, Imelda M.; Grigoriadis, Agamemnon E.; Lam, Eric W.-F.; Price, Joanna S.; Sunters, Andrew



Synergistic interaction of glucose and neurohumoral agonists to stimulate islet phosphoinositide hydrolysis.  


The interaction between neurohumoral agonists and glucose to stimulate phosphoinositide (PI)-specific phospholipase C (PLC) and insulin release was examined. In freshly isolated rat islets, maximal glucose (40 mM), cholecystokinin (CCK; 300 nM), or carbachol (CCh; 1 mM) stimulated PI hydrolysis 6.5-, 9.8-, and 5.7-fold, respectively, above basal. The combination of glucose and CCK or of glucose and CCh, but not of CCK and CCh, synergistically increased PI hydrolysis 23.2- and 21.6-fold, respectively, indicating that these secretagogues activate PLC by distinct pathways and that there is an interaction between them. This synergy was maximal at physiological concentrations of stimulatory glucose (8-10 mM) and was paralleled by a marked synergistic stimulation of insulin secretion. The enhanced PI response was partially Ca2+ dependent and may involve the activation of distinct isozymes of PLC, which we identify in islets. These studies demonstrate for the first time a unique and highly sensitive synergistic interaction between glucose and neurohumoral agonists to stimulate PI hydrolysis, and they suggest that enhanced PI hydrolysis is important in the potentiation of glucose- and neurohumoral-stimulated insulin secretion. PMID:7573436

Kelley, G G; Zawalich, K C; Zawalich, W S



Modulation of insulin secretion from beta-cells by phosphoinositide-derived second-messenger molecules.  


In isolated islets, the hydrolysis of membrane phosphoinositides (PI) participates in the transduction of both extracellular and intracellular signals into an effective insulin secretory response. A wide variety of potential second-messenger molecules are generated during the phospholipase C-mediated cleavage of these strategically situated membrane phospholipids. Several distinct but interrelated issues are addressed in this perspective. These include 1) methodological approaches utilized to assess PI turnover, 2) the synergistic relationship between PI-derived second messengers and cAMP, 3) the contribution of changing PI turnover rates to the biphasic pattern of insulin output induced by 20 mM glucose, and 4) the role played by PI turnover in the phenomenon of "memory" displayed by islets after prior stimulation with various agonists. The concept that events unique to PI turnover contribute to beta-cell activation is well founded. Because of uncertainty regarding the exact nature of all PI-derived messengers, however, it is not yet possible to mold the available information into a comprehensive theory of beta-cell activation. Future studies will have to address various important unresolved issues. PMID:2839385

Zawalich, W S



Islet phosphoinositide hydrolysis and insulin secretory responses from prediabetic fa/fa ZDF rats.  


The sequence of events that culminate in the development of diabetes in the fa/fa Zucker diabetic fatty (ZDF) rat is unclear. In the present series of experiments islets from 5 week old prediabetic fa/fa male rats were isolated and their phosphoinositide (PI) hydrolysis and insulin secretory responses compared to those obtained from lean nondiabetic age- and weight-matched control rats. Peak first and second phase insulin secretory responses to 20mM glucose averaged 77 +/- 10 (mean +/- SE, n = 7) and 491 +/- 47 pg/islet/min from lean, nondiabetic control islets. The comparable responses from fa/fa prediabetic rat islets were significantly greater, 264 +/- 51 and 810 +/- 78 pg/islet/min. In a parallel fashion 3H-inositol efflux and inositol phosphate responses from prediabetic rat islets were also greater than comparable control responses. These findings demonstrate that significant increases in the phospholipase C-mediated hydrolysis of islet PI pools and insulin release in response to hyperglycemic stimulation can be detected prior to the emergence of diabetes in the fa/fa ZDF rat. These early changes in beta cell responsiveness to glucose may contribute to the hyperinsulinemia and subsequent insulin resistance characteristic of this animal model of non-insulin dependent diabetes mellitus. PMID:7733992

Zawalich, W S; Zawalich, K C; Kelley, G G; Shulman, G I



Interleukin-1 induces time-dependent potentiation in isolated rat islets: possible involvement of phosphoinositide hydrolysis.  


Prior exposure of isolated perfused rat islets to the monokine interleukin-1 (IL-1) amplifies their subsequent insulin secretory response to 10 mM glucose. This potentiating effect of the monokine is dose dependent, lasts for at least 45 min after IL-1 removal from the medium, and is not confined to glucose; IL-1 also potentiates the insulin secretory responses to tolbutamide and glyceraldehyde. IL-1 exposure of islets incubated with myo-[2-3H]inositol to label their phosphoinositides (PI) results in an increase in [3H]inositol efflux, an event that persists long after removal of IL-1 from the medium. Direct measurements of labeled inositol phosphate accumulation substantiate the concept that this sustained [3H]inositol efflux response is the direct result of a sustained increase in PI hydrolysis. These results expand the list of compounds that induce time-dependent potentiation in islets to include IL-1. This action of the monokine, mediated at least in part by PI-derived second messenger molecules, may contribute to its postulated effects on insulin and glucose homeostasis. PMID:2536318

Zawalich, W S; Dierolf, B; Zawalich, K C



Influence of monooleoylglycerol on islet cell phosphoinositide hydrolysis and insulin secretion.  


The diacylglycerol kinase inhibitor monooleoylglycerol (MOG) produced a dose-dependent increase in both phosphoinositide (PI) hydrolysis and insulin secretion in the presence of a substimulatory glucose level (2.75 mM). This effect could not be reproduced by the combination of oleic acid plus glycerol, potential metabolic products derived from MOG catabolism. At a level (25 microM) which has no significant effect on beta cell insulin secretion or PI hydrolysis in the presence of 2.75 mM glucose, MOG significantly potentiated the insulin stimulatory effect of the sulfonylurea tolbutamide (200 microM) in the presence of 7 mM glucose. This heightened insulin secretory response and PI hydrolysis were effectively attenuated by the calcium channel blocker nitrendipine (0.5 microM). These findings indicate that MOG has complex effects on beta cell performance. It promises, however, to be a useful probe in assessing how events associated with increases in PI hydrolysis influence insulin secretion. PMID:2155836

Zawalich, W S; Zawalich, K 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



Regulation of glucose metabolism in T cells: new insight into the role of Phosphoinositide 3-kinases  

PubMed Central

Naïve T cells are relatively quiescent cells that only require energy to prevent atrophy and for survival and migration. However, in response to developmental or extrinsic cues T cells can engage in rapid growth and robust proliferation, produce of a range of effector molecules and migrate through peripheral tissues. To meet the significantly increased metabolic demands of these activities, T cells switch from primarily metabolizing glucose to carbon dioxide through oxidative phosphorylation to utilizing glycolysis to convert glucose to lactate (termed aerobic glycolysis). This metabolic switch allows glucose to be used as a source of carbon to generate biosynthetic precursors for the production of protein, DNA, and phospholipids, and is crucial for T cells to meet metabolic demands. Phosphoinositide 3-kinases (PI3K) are a family of inositol lipid kinases linked with a broad range of cellular functions in T lymphocytes that include cell growth, proliferation, metabolism, differentiation, survival, and migration. Initial research described a critical role for PI3K signaling through Akt (also called protein kinase B) for the increased glucose uptake and glycolysis that accompanies T cell activation. This review article relates this original research with more recent data and discusses the evidence for and against a role for PI3K in regulating the metabolic switch to aerobic glycolysis in T cells.

Finlay, David K.



A PH domain within OCRL bridges clathrin-mediated membrane trafficking to phosphoinositide metabolism  

PubMed Central

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5-phosphatase domain followed by an ASH and a RhoGAP-like domain. Their divergent NH2-terminal portions remain uncharacterized. We show that the NH2-terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin-coated pits, was earlier shown to contain another binding site for clathrin in its COOH-terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2-terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin-binding site in OCRL maps to an unusual clathrin-box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin-dependent membrane trafficking.

Mao, Yuxin; Balkin, Daniel M; Zoncu, Roberto; Erdmann, Kai S; Tomasini, Livia; Hu, Fenghua; Jin, Moonsoo M; Hodsdon, Michael E; De Camilli, Pietro



The Phosphoinositide 3-Kinase Pathway in Human Cancer: Genetic Alterations and Therapeutic Implications  

PubMed Central

The phosphoinositide 3-kinase (PI3K) pathway is frequently activated in human cancer and represents an attractive target for therapies based on small molecule inhibitors. PI3K isoforms play an essential role in the signal transduction events activated by cell surface receptors including receptor tyrosine kinases (RTKs) and G-protein-coupled receptors (GPCRs). There are eight known PI3K isoforms in humans, which have been subdivided into three classes (I-III). Therefore PI3Ks show considerable diversity and it remains unclear which kinases in this family should be targeted in cancer. The class IA of PI3K comprises the p110?, p110? and p110? isoforms, which associate with activated RTKs. In human cancer, recent reports have described activating mutations in the PIK3CA gene encoding p110?, and inactivating mutations in the phosphatase and tensin homologue (PTEN) gene, a tumour suppressor and antagonist of the PI3K pathway. The PIK3CA mutations described in cancer constitutively activate p110? and, when expressed in cells drive oncogenic transformation. Moreover, these mutations cause the constitutive activation of downstream signaling molecules such as Akt/protein kinase B (PKB), mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K) that is commonly observed in cancer cells. In addition to p110?, the other isoforms of the PI3K family may also play a role in human cancer, although their individual functions remain to be precisely identified. In this review we will discuss the evidence implicating individual PI3K isoforms in human cancer and their potential as drug targets in this context.

Arcaro, Alexandre; Guerreiro, Ana S



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



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))



Phosphoinositides Play Differential Roles in Regulating Phototropin1- and Phototropin2-Mediated Chloroplast Movements in Arabidopsis  

PubMed Central

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 Ca2+(c) signaling. Using the transgenic aequorin system, we show that the application of these inhibitors suppresses the blue-light-induced transient Ca2+(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 Ca2+ signaling during movements.

Aggarwal, Chhavi; Labuz, Justyna; Gabrys, Halina



Changes in phosphoinositide turnover, Ca sup 2+ mobilization, and protein phosphorylation in platelets from NIDDM patients  

SciTech Connect

Enhanced platelet functions have been demonstrated in patients with non-insulin-dependent diabetes mellitus (NIDDM). This study evaluated abnormalities in platelet signal transduction in diabetic patients, including turnover of phosphoinositides, mobilization of intracellular Ca2+, and phosphorylation of 20,000- and 47,000-Mr proteins (P20 and P47). Washed platelets were obtained from 6 patients with NIDDM whose platelet aggregation rates were abnormally elevated (DM-A group), 11 NIDDM patients with normal platelet aggregation rates (DM-B group), and 8 age-matched healthy control subjects. The mass and specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), and phosphatidic acid (PA) in 32P-labeled platelets were not different among the three groups. Hydrolysis of PIP2, PIP, and PI; accumulation of PA; and phosphorylation of P20 in platelets stimulated by 0.05 U/ml thrombin were significantly increased in the DM-A group compared with the control or DM-B group. There was no difference in P47 phosphorylation among the three groups. On the contrary, P20 and P47 phosphorylation induced by 50 nM of 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, was significantly decreased in the DM-A group. Additionally, the intracellular free Ca2+ concentration (( Ca2+)i) was measured with the fluorescent Ca2+ indicator fura 2. Although the basal (Ca2+)i value was similar in the three groups, the rise in (Ca2+)i induced by 0.05 U/ml thrombin in the presence and the absence of extracellular Ca2+ was significantly higher in the DM-A group than the other groups.

Ishii, H.; Umeda, F.; Hashimoto, T.; Nawata, H. (Kyushu Univ., Fukuoka (Japan))



Emerging roles of phosphoinositide-specific phospholipases C in the ciliates Tetrahymena and Paramecium  

PubMed Central

Phospholipases C (PLCs) that hydrolyze inositol phospholipids regulate vital cellular functions in both eukaryotic and prokaryotic organisms. The PLC superfamily consists of eukaryotic phosphoinositide-specific PLCs (PI-PLCs), bacterial PLCs and trypanosomal PLCs.1 PI-PLCs hydrolyze phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2) to produce inositol-1,4,5-trisphosphate (Ins1,4,5P3) and constitute a hallmark feature of eukaryotic cells. In metazoa, this reaction is coupled to receptor signaling via specific PI-PLC isoforms and results in acute increase of cytosolic Ca2+ levels by Ins1,4,5P3-sensitive Ca2+ channels (IP3-receptors, IP3Rs).2 A striking result of many studies so far has been the presence of a single PI-PLC gene in all unicellular eukaryotes investigated, as opposed to expansion of PI-PLC isoforms in metazoa;3 this has suggested that a single housekeeping PI-PLC represents an archetypal and simplified form of PI-PLC signaling.3 Several studies however have noted a unique expansion of PI-PLC/IP3R pathway components in ciliates.4,5 In a recent paper we showed the presence of multiple functional PI-PLC genes in Tetrahymena thermophila and biochemical characterization, pharmacological studies and study of their expression patterns suggested that they are likely to serve distinct non-redundant roles.4 In this report we discuss these studies and how they advance our understanding of PI-PLC functions in ciliates.

Leondaritis, George



Prenatal ethanol exposure reduces phosphoinositide hydrolysis stimulated by quisqualate in rat cerebellar granule cell cultures.  


Prenatal ethanol exposure-induced alteration in poly-phosphoinositide (PPI) hydrolysis stimulated by excitatory amino acids (EAA) was studied in rat cerebellar granule cells previously labeled with [3H]myoinositol. The prenatal exposure to ethanol was achieved via maternal consumption of a Sustacal (chocolate flavored) liquid diet containing either 5% ethanol (w/v, 35% of calories) or isocaloric sucrose (pair-fed) substituted for ethanol from gestation d 11 until the day of parturition. The ionotropic glutamate receptor agonists, N-methyl-D-aspartate, kainate or (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) (100 microM each) induced a two- to four-fold increase in PPI hydrolysis over the basal level, regardless of the liquid dietary treatment. Stimulation with quisqualate (QA), an agonist activating both metabotropic and ionotropic glutamate receptors, resulted in a much stronger and dose-dependent response in PPI hydrolysis and exposure in utero to ethanol significantly reduced this response. Tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or (+/-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) had no effect on QA-stimulated PPI hydrolysis nor on the suppression of this hydrolysis by ethanol. Exposure in utero to ethanol did not affect PPI hydrolysis stimulated by a selective metabotropic glutamate receptor agonist, trans-(+/-)-l-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD). Although the PPI hydrolysis stimulated by t-ACPD could be blocked by (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG), an antagonist of the metabotropic glutamate receptor, MCPG was incapable of affecting QA-induced PPI hydrolysis and the suppressive effects of prenatal ethanol exposure on this hydrolysis. Taken together, the data suggest that the long-lasting suppressive effects of prenatal ethanol exposure on QA-stimulated PPI hydrolysis in cerebellar granule cell cultures is through a metabotropic QA receptor pathway that may be different from the one activated by t-ACPD. PMID:7893331

Rhodes, P G; Cai, Z; Zhu, N



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



Regulation of Interleukin-5-Induced ?2-Integrin Adhesion of Human Eosinophils by Phosphoinositide 3-Kinase  

PubMed Central

We examined the role of phosphoinositide 3-kinase (PI3K) in integrin-mediated eosinophil adhesion. ?p85, a dominant-negative form of the class IA PI3K adaptor subunit, was fused to an HIV-TAT protein transduction domain (TAT-?p85). Recombinant TAT-?p85 inhibited interleukin (IL)-5–stimulated phosphorylation of protein kinase B, a downstream target of PI3K. ?2-Integrin–dependent adhesion caused by IL-5 to the plated intracellular adhesion molecule-1 surrogate, bovine serum albumin, was inhibited by TAT-?p85 in a concentration-dependent manner. Similarly, two PI3K inhibitors, wortmannin and LY294002, blocked eosinophil adhesion to plated bovine serum albumin. By contrast, ?1-integrin–mediated eosinophil adhesion to vascular cell adhesion moelcule-1 was not blocked by TAT-?p85, wortmannin, or LY294002. Rottlerin, a protein kinase C (PKC)-? inhibitor, also blocked ?2-integrin adhesion of eosinophils caused by IL-5, whereas ?1 adhesion to vascular cell adhesion molecule-1 was not affected. IL-5 caused translocation of PKC? from the cytosol to cell membrane; inhibition of PI3K by wortmannin blocked translocation of PKC?. Western blot analysis demonstrated that extracellular signal–regulated kinase phosphorylation, a critical intermediary in adhesion elicited by IL-5, was blocked by inhibition of either PI3K or PKC-?. These data suggest that extracellular signal–regulated kinase–mediated adhesion of ?2-integrin caused by IL-5 is mediated in human eosinophils by a class IA PI3K through activation of a PKC? pathway.

Sano, Masaaki; Leff, Alan R.; Myou, Shigeharu; Boetticher, Evan; Meliton, Angelo Y.; Learoyd, Jonathan; Lambertino, Anissa T.; Munoz, Nilda M.; Zhu, Xiangdong



Phosphoinositide hydrolysis and insulin release from isolated perifused rat islets. Studies with glucose.  


The ability of glucose to promote the hydrolysis of prelabeled [2-3H]inositol-containing phosphoinositides (PI) was assessed by measuring the efflux of 3H in response to glucose and the accumulation of labeled inositol phosphates. The inclusion of nonradioactive inositol (1 mM) in the perifusion medium dramatically improved our ability to monitor glucose-induced increases in 3H efflux. Efflux studies with this method revealed the following. 1) 3H efflux is significantly greater at 7 than at 2.75 mM glucose, and this parallels a small but significant increase in insulin secretion. 2) D-manno-Heptulose reduces 3H efflux with 7 mM glucose to a level approximating that seen in the presence of 2.75 mM glucose and has no effect on 3H efflux with 2.75 mM glucose. 3) In the presence of 20 mM glucose plus 1 mM inositol, 3H efflux is rapid and biphasic, a response that parallels the timing and amplitude of the biphasic pattern of insulin secretion. Direct measurements of labeled inositol and inositol phosphate levels in islets revealed the following. 4) After 50 min of perifusion with 2.75 or 7 mM glucose, labeled inositol phosphates were significantly greater with 7 mM glucose. 5) In response to 20 mM glucose alone, islet levels of free inositol, inositol monophosphate (IP1), and inositol bisphosphate (IP2) increased. 6) In response to 20 mM glucose plus 1 mM cold inositol, islet levels of free inositol increased, whereas islet levels of IP1, IP2, and inositol trisphosphate (IP3) were reduced compared with values obtained with 20 mM glucose alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2842212

Zawalich, W S; Zawalich, K C



Influence of staurosporine, nitrendipine and monooleoylglycerol on interleukin-1-induced insulin secretion and phosphoinositide hydrolysis.  


The monokine interleukin-1 alpha (IL-1) induces a glucose-dependent increase in insulin secretion, an effect tentatively attributed to its ability to increase beta cell phosphoinositide (PI) hydrolysis. In the present experiments, the effects of the protein kinase C inhibitor staurosporine (20 nM), the calcium channel antagonist nitrendipine (5 microM), and the diacylglycerol kinase inhibitor monooleoylglycerol (MOG, 25 microM) on 40 nM IL-1-induced increments in insulin release from perifused islets and inositol phosphate levels in [3H]inositol prelabeled islets were assessed. In perifused islets, insulin secretion in response to IL-1 in the presence of 7 mM glucose averaged 313 +/- 43 pg/islet/min 35-40 min after the onset of stimulation. Release from control islets perifused in the presence of 7 mM glucose alone averaged 56 +/- 6 pg/islet/min at this time point. The addition of staurosporine together with IL-1 reduced insulin secretion at this time point to 88 +/- 21 pg/islet/min. This level of IL-1 caused significant increases in inositol phosphate accumulation in the presence of 7 mM glucose but not 2.75 mM glucose. Staurosporine was without a significant effect on inositol phosphate accumulation in response to the monokine. In contrast, nitrendipine (5 microM) inhibited insulin release and inositol phosphate accumulation in a parallel fashion. Finally, MOG significantly amplified release to the monokine without significantly affecting its impact on inositol phosphate accumulation. Nitrendipine or staurosporine blocked this amplifying effect of MOG on secretion. These results emphasize the role of PI hydrolysis in IL-1-induced insulin secretion and suggest further that calcium influx is essential for IL-1 to fully activate both PI hydrolysis and insulin secretion. PMID:1665456

Zawalich, W S; Zawalich, K C



PPAR? Activation Acts Cooperatively with 3-Phosphoinositide-Dependent Protein Kinase-1 to Enhance Mammary Tumorigenesis  

PubMed Central

Peroxisome proliferator-activated receptor? (PPAR?) is a transcription factor that is associated with metabolic gene regulation and inflammation. It has been implicated in tumor promotion and in the regulation of 3-phosphoinositide-dependent kinase-1 (PDK1). PDK1 is a key regulator of the AGC protein kinase family, which includes the proto-oncogene AKT/PKB implicated in several malignancies, including breast cancer. To assess the role of PDK1 in mammary tumorigenesis and its interaction with PPAR?, transgenic mice were generated in which PDK1 was expressed in mammary epithelium under the control of the MMTV enhancer/promoter region. Transgene expression increased pT308AKT and pS9GSK3?, but did not alter phosphorylation of mTOR, 4EBP1, ribosomal protein S6 and PKC?. The transgenic mammary gland also expressed higher levels of PPAR? and a gene expression profile resembling wild-type mice maintained on a diet containing the PPAR? agonist, GW501516. Both wild-type and transgenic mice treated with GW501516 exhibited accelerated rates of tumor formation that were more pronounced in transgenic animals. GW501516 treatment was accompanied by a distinct metabolic gene expression and metabolomic signature that was not present in untreated animals. GW501516-treated transgenic mice expressed higher levels of fatty acid and phospholipid metabolites than treated wild-type mice, suggesting the involvement of PDK1 in enhancing PPAR?-driven energy metabolism. These results reveal that PPAR? activation elicits a distinct metabolic and metabolomic profile in tumors that is in part related to PDK1 and AKT signaling.

Pollock, Claire B.; Yin, Yuzhi; Yuan, Hongyan; Zeng, Xiao; King, Sruthi; Li, Xin; Kopelovich, Levy; Albanese, Chris; Glazer, Robert I.



Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival  

PubMed Central

The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110? by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.

Green, Benjamin D.; Barry, Emma F.; Ma, Yuefang; Woodcock, Joanna; Fitter, Stephen; Zannettino, Andrew C. W.; Pitson, Stuart M.; Hughes, Timothy P.; Lopez, Angel F.; Shepherd, Peter R.; Wei, Andrew H.; Ekert, Paul G.; Guthridge, Mark A.



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

PubMed Central

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.



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)



Pharmacological characteristics and regulation of 5HT receptor-stimulated phosphoinositide hydrolysis in the rat spinal cord  

Microsoft Academic Search

In slices from immature rat spinal cord, both 5-hydroxytryptamine (5-HT) and the 5-HT2A\\/C receptor agonists (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and ?-methyl-5-HT (?-Me-5-HT) stimulate phosphoinositide (PI) hydrolysis. PI breakdown is also increased by the 5-HT3 receptor agonist 2-Me-5-HT but not by phenylbiguanide. The effect of either 5-HT or DOI is blocked by selective 5-HT2A receptor antagonists such as spiperone and ketanserin and more

E. Toscano; G. Romero; C. Oset; J. Del R??o



Substrates of semicarbazide-sensitive amine oxidase co-operate with vanadate to stimulate tyrosine phosphorylation of insulin-receptor-substrate proteins, phosphoinositide 3-kinase activity and GLUT4 translocation in adipose cells.  

PubMed Central

It has been shown that the combination of benzylamine or tyramine and low concentrations of vanadate markedly stimulates glucose transport in rat adipocytes by a mechanism that requires semicarbazide-sensitive amine oxidase (SSAO) activity and H(2)O(2) formation. Here we have further analysed the insulin-like effects of the combination of SSAO substrates and vanadate and we have studied the signal-transduction pathway activated in rat adipocytes. We found that several SSAO substrates (benzylamine, tyramine, methylamine, n-decylamine, histamine, tryptamine or beta-phenylethylamine), in combination with low concentrations of vanadate, stimulate glucose transport in isolated rat adipocytes. Furthermore, SSAO substrates together with vanadate stimulated the recruitment of GLUT4 to the cell surface in isolated rat adipocytes. Benzylamine plus vanadate also stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes. Benzylamine or tyramine in combination with vanadate potently stimulated the tyrosine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-3. In contrast, benzylamine and vanadate caused only a weak stimulation of insulin receptor kinase. Benzylamine or tyramine in combination with vanadate also stimulated phosphoinositide 3-kinase activity; wortmannin abolished the stimulatory effect of benzylamine and vanadate on glucose transport in adipose cells. Furthermore, the administration of benzylamine and vanadate in vivo caused a rapid lowering of plasma glucose levels, which took place in the absence of alterations in plasma insulin. On the basis of these results we propose that SSAO activity regulates glucose transport in adipocytes. SSAO oxidative activity stimulates glucose transport via the translocation of GLUT4 carriers to the cell surface, resulting from a potent tyrosine phosphorylation of IRS-1 and IRS-3 and phosphoinositide 3-kinase activation. Our results also indicate that substrates of SSAO might regulate glucose disposal in vivo.

Enrique-Tarancon, G; Castan, I; Morin, N; Marti, L; Abella, A; Camps, M; Casamitjana, R; Palacin, M; Testar, X; Degerman, E; Carpene, C; Zorzano, A



Effect of aging on alpha-1 adrenergic stimulation of phosphoinositide hydrolysis in various regions of rat brain  

SciTech Connect

The effects of aging were examined on the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis in three brain regions. Tissue minces of thalamus, cerebral cortex and hippocampus from 3-, 18- and 28-month-old male Fischer 344 rats were prelabeled with ({sup 3}H)myoinositol. Exposure of these prelabeled minces to phenylephrine and (-)-norepinephrine revealed that accumulation of ({sup 3}H)inositol phosphates was selectively reduced by 20 to 30% in the thalamus and cerebral cortex of the oldest age group. Analysis of concentration-response and competition binding curves indicated that this decrease was due to diminished agonist efficacy rather than diminished receptor affinity. The reduction in responsiveness to phenylephrine and (-)-norepinephrine in the cerebral cortex and the lack of any changes in the hippocampus parallel previously reported changes in the density of alpha-1 adrenergic receptors with aging. These data indicate that the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis is reduced in some, but not all, brain regions of aged Fischer 344 rats.

Burnett, D.M.; Bowyer, J.F.; Masserano, J.M.; Zahniser, N.R. (Univ. of Colorado Health Sciences Center, Denver (USA))



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

PubMed Central

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 p85? constitutively led to stress fiber disruption, cortical actin recruitment, extensive ruffling, and macropinosome formation, as measured by a selective acceleration of fluid-phase endocytosis. These alterations closely correlated with activation of PI3K and phosphatidylinositol-specific phospholipase C (PI-PLC), as assayed by 3-phosphoinositide synthesis in situ and in vitro and inositol 1,4,5 trisphosphate steady-state levels, respectively; they were abolished by stable transfection of v-Src–transformed cells for dominant-negative truncated p85? expression and by pharmacological inhibitors of PI3K and PI-PLC, indicating a requirement for both enzymes. Whereas PI3K activation resisted PI-PLC inhibition, PI-PLC activation was abolished by a PI3K inhibitor and dominant-negative transfection, thus placing PI-PLC downstream of PI3K. Together, these data suggest that permanent sequential activation of both PI3K and PI-PLC is necessary for the dramatic reorganization of the actin cytoskeleton in oncogene-transformed fibroblasts, resulting in constitutive ruffling and macropinocytosis.

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



Phylogenomics of phosphoinositide lipid kinases: perspectives on the evolution of second messenger signaling and drug discovery  

PubMed Central

Background Phosphoinositide lipid kinases (PIKs) generate specific phosphorylated variants of phosatidylinositols (PtdIns) that are critical for second messenger signaling and cellular membrane remodeling. Mammals have 19 PIK isoforms spread across three major families: the PtIns 3-kinases (PI3Ks), PtdIns 4-kinases (PI4Ks), and PtdIns-P (PIP) kinases (PIPKs). Other eukaryotes have fewer yet varying PIK complements. PIKs are also an important, emerging class of drug targets for many therapeutic areas including cancer, inflammatory and metabolic diseases and host-pathogen interactions. Here, we report the genomic occurrences and evolutionary relationships or phylogenomics of all three PIK families across major eukaryotic groups and suggest potential ramifications for drug discovery. Results Our analyses reveal four core eukaryotic PIKs which are type III PIK4A and PIK4B, and at least one homolog each from PI3K (possibly PIK3C3 as the ancestor) and PIP5K families. We also applied evolutionary analyses to PIK disease ontology and drug discovery. Mutated PIK3CA are known to be oncogenic and several inhibitors are in anti-cancer clinical trials. We found conservation of activating mutations of PIK3CA in paralogous isoforms suggesting specific functional constraints on these residues. By mapping published compound inhibition data (IC50s) onto a phylogeny of PI3Ks, type II PI4Ks and distantly related, MTOR, ATM, ATR and PRKDC kinases, we also show that compound polypharmacology corresponds to kinase evolutionary relationships. Finally, we extended the rationale for drugs targeting PIKs of malarial Plasmodium falciparum, and the parasites, Leishmania sp. and Trypanosoma sp. by identifying those PIKs highly divergent from human homologs. Conclusion Our phylogenomic analysis of PIKs provides new insights into the evolution of second messenger signaling. We postulate two waves of PIK diversification, the first in metazoans with a subsequent expansion in cold-blooded vertebrates that was post-emergence of Deutrostomia\\Chordata but prior to the appearance of mammals. Reconstruction of the evolutionary relationships among these lipid kinases also adds to our understanding of their roles in various diseases and assists in their development as potential drug targets.




Technology Transfer Automated Retrieval System (TEKTRAN)

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 organization, and vesicle trafficking. Nine genes encoding SAC domain-contai...


Activation and Membrane Binding of Retinal Protein Kinase B?/Akt1 is Regulated through Light-Dependent Generation of Phosphoinositides  

PubMed Central

Akt is a phospholipid-binding protein and the downstream effector of the phosphoinositide 3-kinase (PI3K) pathway. Akt has three isoforms: Akt1, Akt2, and Akt3. All of these isoforms are expressed in rod photoreceptor cells, but the individual functions of each isoform are not known. In this study we found that light induces the activation of Akt1. The membrane binding of Akt1 to rod outer segments (ROS) is insulin receptor (IR)/PI3K-dependent as demonstrated by reduced binding of Akt1 to ROS membranes of photoreceptor-specific IR knockout mice. Membrane binding of Akt1 is mediated through its Pleckstrin homology (PH) domain. To determine whether binding of the PH domain of Akt1 to photoreceptor membranes is regulated by light, various green fluorescent protein (GFP)/Akt1-PH domain fusion proteins were expressed in rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The R25C mutant PH domain of Akt1, which does not bind phosphoinositides, failed to associate with plasma membranes in a light-dependent manner. This study suggests that light-dependent generation of phosphoinositides regulates the activation and membrane binding of Akt1 in vivo. Our results also suggest that actin cytoskeletal organization may be regulated through light-dependent generation of phosphoinositides.

Li, Guiyuan; Rajala, Ammaji; Wiechmann, Allan F.; Anderson, Robert E.; Rajala, Raju V.S.



Attenuation of PTEN increases p21 stability and cytosolic localization in kidney cancer cells: a potential mechanism of apoptosis resistance  

Microsoft Academic Search

BACKGROUND: The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on

Pei-Yin Lin; Susan P Fosmire; See-Hyoung Park; Jin-Young Park; Shairaz Baksh; Jaime F Modiano; Robert H Weiss



Stimulatory effects of the putative metabotropic glutamate receptor antagonist L-AP3 on phosphoinositide turnover in neonatal rat cerebral cortex.  

PubMed Central

1. The effects of the metabotropic glutamate receptor (mGluR) antagonist, L-2-amino-3-phosphonopropionate (L-AP3) on phosphoinositide turnover in neonatal rat cerebral cortex slices has been investigated. 2. At concentrations of < or = 300 microM, L-AP3 inhibited total [3H]-inositol phosphate ([3H]-InsPx) and Ins(1,4,5)P3 mass responses stimulated by the selective mGluR agonist, 1-amino-cyclopentane-1S, 3R-dicarboxylic acid (1S, 3R-ACPD). Comparison with the competitive mGluR antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine ((+/-)-MCPG) clearly demonstrated that L-AP3 caused inhibition by a mechanism that was not competitive, as L-AP3 decreased the maximal response to 1S, 3R-ACPD (by approximately 40% at 300 microM L-AP3) without significantly affecting the concentration of 1S, 3R-ACPD required to cause half-maximal stimulation of the [3H]-InsPx response. 3. In contrast, at a higher concentration L-AP3 (1 mM) caused a large increase in [3H]-InsPx accumulation which was similar in magnitude in both the absence and presence of 1S, 3R-ACPD (300 microM). D-AP3 (1 mM) had no stimulatory effect alone and did not affect the response evoked by 1S, 3R-ACPD. L-AP3 (1 mM) also caused a large increase in Ins(1,4,5)P3 accumulation. The magnitude of the response (4-5 fold increase over basal) approached that evoked by a maximally effective concentration of 1S, 3R-ACPD, but differed substantially in the time-course of the response. The stimulatory effects of 1S, 3R-ACPD and L-AP3 on Ins(1,4,5)P3 accumulation were also similarly affected by decreases in extracellular calcium concentration. 4. Detailed analysis of the inositol phospholipid labelling pattern and the inositol (poly)phosphate isomeric species generated following addition of L-AP3 was also performed. In the continued presence of myo-[3H]-inositol, L-AP3 (1 mM) stimulated a significant increase in phosphatidylinositol labelling, but not that of the polyphosphoinositides, and the inositol (poly)phosphate profile suggested that substantial Ins(1,4,5)P3 metabolism occurs via both 5-phosphatase and 3-kinase routes. 5. A significant stimulatory effect of L-AP3 (1 mM) on [3H]-InsPx accumulation was also observed in neonatal rat hippocampus, and cerebral cortex and hippocampus slices prepared from adult rat brain. 6. These data demonstrate that whilst L-AP3 antagonizes mGluR-mediated phosphoinositide responses at concentrations of < or = 300 microM, higher concentrations substantially stimulate this response. The ability of (+/-)-MCPG (1 mM) to attenuate significantly L-AP3-stimulated [3H]-InsPx accumulation, suggests that both the inhibitory and stimulatory effects of L-AP3 may be mediated by mGluRs.

Mistry, R.; Prabhu, G.; Godwin, M.; Challiss, R. A.



Structure-based design of potent and selective 3-phosphoinositide-dependent kinase-1 (PDK1) inhibitors.  


Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor. PMID:21341675

Medina, Jesús R; Becker, Christopher J; Blackledge, Charles W; Duquenne, Celine; Feng, Yanhong; Grant, Seth W; Heerding, Dirk; Li, William H; Miller, William H; Romeril, Stuart P; Scherzer, Daryl; Shu, Arthur; Bobko, Mark A; Chadderton, Antony R; Dumble, Melissa; Gardiner, Christine M; Gilbert, Seth; Liu, Qi; Rabindran, Sridhar K; Sudakin, Valery; Xiang, Hong; Brady, Pat G; Campobasso, Nino; Ward, Paris; Axten, Jeffrey M



Regioselective synthesis of 5- and 6-methoxybenzimidazole-1,3,5-triazines as inhibitors of phosphoinositide 3-kinase.  


Phosphoinositide 3-kinases (PI3K) hold significant therapeutic potential as novel targets for the treatment of cancer. ZSTK474 (4a) is a potent, pan-PI3K inhibitor currently under clinical evaluation for the treatment of cancer. Structural studies have shown that derivatisation at the 5- or 6-position of the benzimidazole ring may influence potency and isoform selectivity. However, synthesis of these derivatives by the traditional route results in a mixture of the two regioisomers. We have developed a straightforward regioselective synthesis that gave convenient access to 5- and 6-methoxysubstituted benzimidazole derivatives of ZSTK474. While 5-methoxy substitution abolished activity at all isoforms, the 6-methoxy substitution is consistently 10-fold more potent. This synthesis will allow convenient access to further 6-position derivatives, thus allowing the full scope of the structure-activity relationships of ZSTK474 to be probed. PMID:23265896

Miller, Michelle S; Pinson, Jo-Anne; Zheng, Zhaohua; Jennings, Ian G; Thompson, Philip E



Increase in cytosolic calcium and phosphoinositide metabolism induced by angiotensin II and [Arg]vasopressin in vascular smooth muscle cells.  


Effects of angiotensin II and [Arg]vasopressin on cytosolic free Ca2+ concentration ([Ca2+]i) and phosphoinositide metabolism were studied in cultured aortic smooth muscle cells obtained from Wistar-Kyoto rats and their spontaneously hypertensive substrain. [Ca2+]i was measured using the fluorescent Ca2+ indicator quin2. No clear differences in basal [Ca2+]i were detected between cells derived from the two strains. High concentrations of angiotensin II (greater than or equal to 10 nM) and [Arg]vasopressin (greater than or equal to 100 nM) elicited large and rapid increases in [Ca2+]i, followed by a rapid return to control values. Low concentrations of these peptides (less than or equal to 1.0 nM) elicited small and slow increases in [Ca2+]i that persisted for minutes. These responses were blocked by specific antagonists for each of these peptides. Only high concentrations of angiotensin II caused [Ca2+]i increases in "Ca2+-free" medium, which suggested that high concentrations of angiotensin II could release Ca2+ from intracellular pools. A high concentration of angiotensin II and [Arg]vasopressin elicited progressive accumulations of inositol phosphates. Only high concentrations of angiotensin II caused inositol phosphate accumulation in Ca2+-free medium. Maximal accumulation of inositol phosphate elicited by angiotensin II and [Arg]vasopressin was found to be additive. A desensitization to the effects of both peptides on Ca2+ mobilization occurred despite the continued accumulation of inositol phosphates. These observations indicated that angiotensin II and [Arg]vasopressin interacted with independent receptors, both of which are linked to phosphoinositide breakdown and Ca2+ mobilization. PMID:2985562

Nabika, T; Velletri, P A; Lovenberg, W; Beaven, M A



Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca2+ mobilization and degranulation in mast cells  

PubMed Central

Protein kinase C ? (PKC?) participates in antigen-stimulated mast cell degranulation mediated by the high-affinity receptor for immunoglobulin E, Fc?RI, but the molecular basis is unclear. We investigated the hypothesis that the polybasic effector domain (ED) of the abundant intracellular substrate for protein kinase C known as myristoylated alanine-rich protein kinase C substrate (MARCKS) sequesters phosphoinositides at the inner leaflet of the plasma membrane until MARCKS dissociates after phosphorylation by activated PKC. Real-time fluorescence imaging confirms synchronization between stimulated oscillations of intracellular Ca2+ concentrations and oscillatory association of PKC?–enhanced green fluorescent protein with the plasma membrane. Similarly, MARCKS-ED tagged with monomeric red fluorescent protein undergoes antigen-stimulated oscillatory dissociation and rebinding to the plasma membrane with a time course that is synchronized with reversible plasma membrane association of PKC?. We find that MARCKS-ED dissociation is prevented by mutation of four serine residues that are potential sites of phosphorylation by PKC. Cells expressing this mutated MARCKS-ED SA4 show delayed onset of antigen-stimulated Ca2+ mobilization and substantial inhibition of granule exocytosis. Stimulation of degranulation by thapsigargin, which bypasses inositol 1,4,5-trisphosphate production, is also substantially reduced in the presence of MARCKS-ED SA4, but store-operated Ca2+ entry is not inhibited. These results show the capacity of MARCKS-ED to regulate granule exocytosis in a PKC-dependent manner, consistent with regulated sequestration of phosphoinositides that mediate granule fusion at the plasma membrane.

Gadi, Deepti; Wagenknecht-Wiesner, Alice; Holowka, David; Baird, Barbara



Serotonergic, 5-HT2, receptor-mediated phosphoinositide turnover and mobilization of calcium in cultured rat retinal pigment epithelium cells.  


Cultured rat retinal pigment epithelium cells are shown to contain serotonergic, 5-HT2, receptors associated with phosphoinositide turnover and mobilization of intracellular calcium. Serotonin at a concentration of 10 microM induced a 2.5-fold increase in [3H]-inositol phosphates (more than 75% is in the form of [3H]-inositol-1-phosphate) accumulation within 30 min in cells preincubated in [3H]-myo-inositol and exposed to 5 mM lithium chloride. The EC50 value of serotonin was approx. 0.9 microM and the saturation concentration was 100 microM. Serotonin analogues like tryptamine, 5-methoxytryptamine, alpha-methyl-serotonin and the 5-HT2 agonists quipazine and DOI (1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane) all stimulated InsPs accumulation to some degree. Carbachol, noradrenaline, isoproterenol, dopamine, tryptophan, 5-hydroxytryptophan, 8-hydroxy-2(di-n-propyl-amino) tetralin, 2-methyl-serotonin and NECA (5'-[N-ethyl]-carboxamidoadenosine) were inactive. The serotonin-induced response was blocked most effectively by ketanserin and methysergide but not by 5-HT3 or 5-HT1 antagonists. The serotonin response was attenuated by the active phorbol ester, 4 beta-phorbol 12-myristate 13-acetate and this was attenuated by the non-selective protein kinase C inhibitor, staurosporine. Pertussis toxin failed to influence the serotonin-mediated phosphoinositide turnover. Addition of serotonin to cultures loaded with Fura-2 showed a transient increase in calcium concentrations in most of the cells. This change in calcium was independent of external calcium and the serotonin response was attenuated by ketanserin but not by the 5-HT3 antagonist granisetron.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8273284

Osborne, N N; Fitzgibbon, F; Nash, M; Liu, N P; Leslie, R; Cholewinski, A



5-HT1A and histamine H1 receptors in HeLa cells stimulate phosphoinositide hydrolysis and phosphate uptake via distinct G protein pools.  


Regulation of phosphate uptake was studied in a HeLa cell line after transfection with DNA encoding the human 5-HT1A receptor. In these cells, 5-HT stimulates sodium-dependent phosphate uptake via protein kinase C activation. Endogenous histamine H1 receptors (739 +/- 20 fmol/mg protein) were identified with [3H]pyrilamine. Histamine (i) stimulated phosphoinositide hydrolysis (EC50 = 8.6 +/- 4.1 microM), (ii) activated protein kinase C (2.4-fold increase in activity), and (iii) increased phosphate uptake (EC50 = 3.2 +/- 1.8 microM) by increasing maximal transport (Vmax(basal) = 6.2 +/- 0.3 versus Vmax(histamine) = 9.1 +/- 0.4) without changing the affinity of the transport process for phosphate. Prolonged treatment with 16 microM phorbol 12-myristate 13-acetate completely blocked protein kinase C activation and markedly attenuated the stimulation of phosphate uptake induced by histamine, establishing that 5-HT and histamine stimulate phosphate uptake through the common pathway of protein kinase C activation. The linkages of the histamine H1 and 5-HT1A receptors to G protein pools were assessed in two ways. (i) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake associated with histamine were insensitive to pertussis toxin, whereas those associated with 5-HT were very sensitive to pertussis toxin. (ii) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake induced by histamine and 5-HT were additive. These findings suggest that distinct receptor types can stimulate phosphoinositide hydrolysis, protein kinase C, and phosphate uptake in an additive fashion through distinct pools of G proteins in a single cell type. PMID:1845968

Raymond, J R; Albers, F J; Middleton, J P; Lefkowitz, R J; Caron, M G; Obeid, L M; Dennis, V W



Opposing effects of a ras oncogene on growth factor-stimulated phosphoinositide hydrolysis: desensitization to platelet-derived growth factor and enhanced sensitivity to bradykinin  

SciTech Connect

Expression of a transforming Harvey or Kirsten ras gene caused opposing effects in the ability of platelet-derived growth factor (PDGF) and bradyknin to activate phospholipase C-mediated phosphoinositide hydrolysis. In (/sup 3/H)inositol-labeled rat-1 fibroblasts, PDGF resulted in a 2-fold increase in the level of (/sup 3/H)inositol trisphosphate (InsP/sub 3/) after 2 min and, in the presence of LiCl, a 3- to 8-fold increase in the level of (/sup 3/H)inositol monophosphate (InsP/sub 1/) after 30 min. However, in EJ-ras-transfected rat-1 cells, which exhibit near normal levels of PDGF receptors, PDGF resulted in little or no accumulation of either (/sup 3/H)InsP/sub 3/ or (/sup 3/H)InsP/sub 1/. Similarly, marked stimulations by PDGF were observed in NIH 3T3 cells, as well as in v-src-transformed 3T3 cells, but not in 3T3 cells transformed by Kirsten sarcoma virus or by transfection with v-Ha-ras DNA. This diminished phosphoinositide response in ras-transformed cells was associated with a markedly attenuated mitogenic response to PDGF. On the other hand, both phosphoinositide metabolism and DNA synthesis in ras-transformed fibroblasts were stimulated several-fold by serum. In NIH 3T3 cells carrying a glucocorticoid-inducible v-Ha-ras gene, a close correlation was found between the expression of p21/sup ras/ and the loss of PDGF-stimulated (/sup 3/H)InsP/sub 1/ accumulation. The authors propose that a ras gene product (p21) can, directly or indirectly, influence growth factor-stimulated phosphoinositide hydrolysis, as well as DNA synthesis, via alterations in the properties of specific growth factor receptors.

Parries, G.; Hoebel, R.; Racker, E.



A Direct Linkage between the Phosphoinositide 3-Kinase-AKT Signaling Pathway and the Mammalian Target of Rapamycin in Mitogen-stimulated and Transformed Cells1  

Microsoft Academic Search

The microbially derived antiproliferative agent rapamycin inhibits cell growth by interfering with the signaling functions of the mammalian target of rapamycin (mTOR). In this study, we demonstrate that inter- leukin-3 stimulation induces a wortmannin-sensitive increase in mTOR kinase activity in a myeloid progenitor cell line. The involvement of phosphoinositide 3*-kinase (PI3K) in the regulation of mTOR activity was further suggested

Aleksandar Sekulic; Christine C. Hudson; James L. Homme; Peng Yin; Diane M. Otterness; Larry M. Karnitz; Robert T. Abraham



Phosphoinositides and Rho proteins spatially regulate actin polymerization to initiate and maintain directed movement in a 1D model of a motile cell  

Microsoft Academic Search

Gradient sensing, polarization, and chemotaxis of motile cells involves the actin cytoskeleton, and regulatory modules, including the phosphoinositides (PIs), their ki- nases\\/phosphatases, and small GTPases (Rho proteins). Here we model their indi- vidual components (PIP, PIP2, PIP3; PTEN, PI3K, PI5K; Cdc42, Rac, Rho; Arp2\\/3, and actin), their interconversions, interactions, and modular functions in the context of a 1D dynamic model

Adriana T. Dawes; Leah Edelstein-Keshet


Phosphoinositides and Rho Proteins Spatially Regulate Actin Polymerization to Initiate and Maintain Directed Movement in a One-Dimensional Model of a Motile Cell  

Microsoft Academic Search

Gradient sensing, polarization, and chemotaxis of motile cells involve the actin cytoskeleton, and regulatory modules, including the phosphoinositides (PIs), their kinases\\/phosphatases, and small GTPases (Rho proteins). Here we model their individual components (PIP1, PIP2, PIP3; PTEN, PI3K, PI5K; Cdc42, Rac, Rho; Arp2\\/3, and actin), their interconversions, interactions, and modular functions in the context of a one-dimensional dynamic model for protrusive

Adriana T. Dawes; Leah Edelstein-Keshet



v-Src accelerates spontaneous motility via phosphoinositide 3-kinase, phospholipase C and phospholipase D, but abrogates chemotaxis in Rat1 and MDCK cells  

Microsoft Academic Search

In Rat-1 fibroblasts, v-Src causes a profound remodelling of cortical actin cytoskeleton. This transformation includes membrane ruffling, a hallmark of the leading edge in migrating cells, and results from activation of phosphoinositide 3-kinase (PI 3-kinase), phospholipase C (PLC) and phospholipase D (PLD). We therefore re- examined whether motility is constitutively triggered by v- Src and studied whether this response is

Anna Platek; Marcel Mettlen; Isabelle Camby; Robert Kiss; Mustapha Amyere; Pierre J. Courtoy



Limits for the detection of (poly-)phosphoinositides by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS)  

Microsoft Academic Search

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently established as a powerful tool for the analysis of biomolecules. Here, MALDI-TOF MS was used for the detection of (poly-)phosphoinositides (PPI). PPI possess higher molecular weights than other phospholipids and a high phosphorylation-dependent negative charge. Both features affect the MALDI detection limits expressed as the minimum of

Matthias Müller; Jürgen Schiller; Marijana Petkovi?; Wolf Oehrl; Regina Heinze; Reinhard Wetzker; Klaus Arnold; Jürgen Arnhold



Effects of UV-C irradiation on phosphoinositide turnover in plant cells: similarities with those occurring via the formation of reactive oxygen intermediates in animal cells  

Microsoft Academic Search

With the aim of examining the response of plant cells to UV-C irradiation, we investigated the behaviour of the phosphatidylinositol 4,5 bisphosphate (PtdIns 4,5-P2) molecule (the precursor of the phosphoinositide signal transduction cascade) by exposing callus cells from Peucedanum verticillare to UV-C (130 J m?2) and by examining the level and the fatty acid composition of PtdIns 4,5-P2 at different

Maria Piera Piacentini; Donata Ricci; Daniele Fraternale; Elena Piatti; Antonio Manunta; Augusto Accorsi



2-(3-Oxo-1,3-diphenylpropyl)malonic acids as potent allosteric ligands of the PIF pocket of phosphoinositide-dependent kinase-1: development and prodrug concept.  


The protein kinase C-related kinase 2 (PRK2)-interacting fragment (PIF) pocket of phosphoinositide-dependent kinase-1 (PDK1) was proposed as a novel target site for allosteric modulators. In the present work, we describe the design, synthesis, and structure-activity relationship of a series of 2-(3-oxo-1,3-diphenylpropyl)malonic acids as potent allosteric activators binding to the PIF pocket. Some congeners displayed AC(50) values for PDK1 activation in the submicromolar range. The potency of the best compounds to stabilize PDK1 in a thermal stability shift assay was in the same order of magnitude as that of the PIF pocket binding peptide PIFtide, suggesting comparable binding affinities to the PIF pocket. The crystal structure of PDK1 in complex with compound 4h revealed that additional ionic interactions are mainly responsible for the increased potency compared to the monocarboxylate analogues. Notably, several compounds displayed high selectivity for PDK1. Employing a prodrug strategy, we were able to corroborate the novel mechanism of action in cells. PMID:23106316

Wilhelm, Adriana; Lopez-Garcia, Laura A; Busschots, Katrien; Fröhner, Wolfgang; Maurer, Frauke; Boettcher, Stefan; Zhang, Hua; Schulze, Jörg O; Biondi, Ricardo M; Engel, Matthias



MicroRNA-375 targets the 3-phosphoinositide-dependent protein kinase-1 gene in pancreatic carcinoma  

PubMed Central

Pancreatic carcinoma (PC) is an aggressive malignancy with one of the poorest mortality rates. It is the sixth leading cause of mortality from malignant disease in China and the fourth leading cause of cancer-related mortality in the United States. The poor outcome reflects the requirement for an improved understanding of the transcriptional control of oncogenic signaling pathways. 3-phosphoinositide-dependent protein kinase-1 (PDK1) is a potent oncogenic driver of PC. The present study aimed to elucidate the transcriptional regulation of microRNA (miR)-375-targeted PDK1. miR-375 is a putative target and, in the present study, was observed to be significantly downregulated in the tumor compared with non-tumor tissues from patients with PC (n=44). As determined by a luciferase reporter assay, the ectopic expression of miR-375 was identified to diminish the transcriptional activity of PDK1. Furthermore, immunoblotting revealed that miR-375 suppressed endogenous PDK1 protein levels. Functional assays showed that miR-375 was able to inhibit proliferation and promote apoptosis of the PC cells. miR-375 is a significant regulator of the PDK1 oncogene, suggesting that it may have a potential therapeutic role in the treatment of PC.




Guanosine 5'-thiotriphosphate may stimulate phosphoinositide messenger production in sea urchin eggs by a different route than the fertilizing sperm.  

PubMed Central

We show that microinjecting guanosine-5'-thiotriphosphate (GTP gamma S) into unfertilized sea urchin eggs generates an intracellular free calcium concentration [( Ca]i) transient apparently identical in magnitude and duration to the calcium transient that activates the egg at fertilization. The GTP gamma S-induced transient is blocked by prior microinjection of the inositol trisphosphate (InsP3) antagonist heparin. GTP gamma S injection also causes stimulation of the egg's Na+/H+ antiporter via protein kinase C, even in the absence of a [Ca]i increase. These data suggest that GTP gamma S acts by stimulating the calcium-independent production of the phosphoinositide messengers InsP3 and diacylglycerol (DAG). However, the fertilization [Ca]i transient is not affected by heparin, nor can the sperm cause calcium-independent stimulation of protein kinase C. It seems that the bulk of InsP3 and DAG production at fertilization is triggered by the [Ca]i transient, not by the sperm itself. GDP beta S, a G-protein antagonist, does not affect the fertilization [Ca]i transient. Our findings do not support the idea that signal transduction at fertilization operates via a G-protein linked directly to a plasma membrane sperm receptor.

Crossley, I; Whalley, T; Whitaker, M



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.



A Novel Function of eIF2? Kinases as Inducers of the Phosphoinositide-3 Kinase Signaling Pathway  

PubMed Central

Phosphoinositide-3 kinase (PI3K) plays an important role in signal transduction in response to a wide range of cellular stimuli involved in cellular processes that promote cell proliferation and survival. Phosphorylation of the ? subunit of the eukaryotic translation initiation factor eIF2 at Ser51 takes place in response to various types of environmental stress and is essential for regulation of translation initiation. Herein, we show that a conditionally active form of the eIF2? kinase PKR acts upstream of PI3K and turns on the Akt/PKB-FRAP/mTOR pathway leading to S6 and 4E-BP1 phosphorylation. Also, induction of PI3K signaling antagonizes the apoptotic and protein synthesis inhibitory effects of the conditionally active PKR. Furthermore, induction of the PI3K pathway is impaired in PKR?/? or PERK?/? mouse embryonic fibroblasts (MEFs) in response to various stimuli that activate each eIF2? kinase. Mechanistically, PI3K signaling activation is indirect and requires the inhibition of protein synthesis by eIF2? phosphorylation as demonstrated by the inactivation of endogenous eIF2? by small interfering RNA or utilization of MEFs bearing the eIF2? Ser51Ala mutation. Our data reveal a novel property of eIF2? kinases as activators of PI3K signaling and cell survival.

Kazemi, Shirin; Mounir, Zineb; Baltzis, Dionissios; Raven, Jennifer F.; Wang, Shuo; Krishnamoorthy, Jothi-Latha; Pluquet, Olivier; Pelletier, Jerry



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



Phosphoinositide 3-kinase(p110?) plays a critical role for the induction of physiological, but not pathological, cardiac hypertrophy  

PubMed Central

An unresolved question in cardiac biology is whether distinct signaling pathways are responsible for the development of pathological and physiological cardiac hypertrophy in the adult. Physiological hypertrophy is characterized by a normal organization of cardiac structure and normal or enhanced cardiac function, whereas pathological hypertrophy is associated with an altered pattern of cardiac gene expression, fibrosis, cardiac dysfunction, and increased morbidity and mortality. The elucidation of signaling cascades that play distinct roles in these two forms of hypertrophy will be critical for the development of more effective strategies to treat heart failure. We examined the role of the p110? isoform of phosphoinositide 3-kinase (PI3K) for the induction of pathological hypertrophy (pressure overload-induced) and physiological hypertrophy (exercise-induced) by using transgenic mice expressing a dominant negative (dn) PI3K(p110?) mutant specifically in the heart. dnPI3K transgenic mice displayed significant hypertrophy in response to pressure overload but not exercise training. dnPI3K transgenic mice also showed significant dilation and cardiac dysfunction in response to pressure overload. Thus, PI3K(p110?) appears to play a critical role for the induction of physiological cardiac growth but not pathological growth. PI3K(p110?) also appears essential for maintaining contractile function in response to pathological stimuli.

McMullen, Julie R.; Shioi, Tetsuo; Zhang, Li; Tarnavski, Oleg; Sherwood, Megan C.; Kang, Peter M.; Izumo, Seigo



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



Requirement of Phosphatidylinositol 3-Kinase-Dependent Pathway and Src for Gas6-Axl Mitogenic and Survival Activities in NIH 3T3 Fibroblasts  

Microsoft Academic Search

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




Centaurin-alpha1 is a phosphatidylinositol 3-kinase-dependent activator of ERK1/2 mitogen-activated protein kinases.  


Centaurin-alpha1 is known to be a phosphatidylinositol 3,4,5-triphosphate (PIP3)-binding protein that has two pleckstrin homology domains and a putative ADP ribosylation factor GTPase-activating protein domain. However, the physiological function of centaurin-alpha1 is still not understood. Here we have shown that transient expression of centaurin-alpha1 in COS-7 cells results in specific activation of ERK, and the activation is inhibited by co-expression of a dominant negative form of Ras. We have also found that a mutant form of centaurin-alpha1 that is unable to bind PIP3 fails to induce ERK activation and that a phosphatidylinositol 3-kinase inhibitor LY294002 inhibits centaurin-alpha1-dependent ERK activation. Furthermore, transient knockdown of centaurin-alpha1 by small interfering RNAs results in reduced ERK activation after epidermal growth factor stimulation in T-REx 293 cells. These results suggest that centaurin-alpha1 contributes to ERK activation in growth factor signaling, linking the PI3K pathway to the ERK mitogen-activated protein kinase pathway through its ability to interact with PIP3. PMID:16287813

Hayashi, Hideko; Matsuzaki, Osamu; Muramatsu, Shuji; Tsuchiya, Yoshiki; Harada, Takeshi; Suzuki, Yutaka; Sugano, Sumio; Matsuda, Akio; Nishida, Eisuke



The Role of Phosphoinositide 3-Kinase-Akt Signaling in Virus Infection  

Microsoft Academic Search

Successful virus infection of host cells requires efficient viral replication, production of virus progeny and spread of newly\\u000a synthesized virus particles. This success, however also depends on the evasion of a multitude of antiviral signaling mechanisms.\\u000a Many viruses are capable of averting antiviral signals through modulation of host cell signaling pathways. Apoptotic inhibition,\\u000a for example, is a universal intracellular antiviral

Samantha Cooray


Spatial and Temporal Regulation of 3-Phosphoinositides by PI 3Kinase and PTEN Mediates Chemotaxis  

Microsoft Academic Search

We have investigated the mechanisms of leading edge formation in chemotaxing Dictyostelium cells. We demonstrate that while phosphatidylinositol 3-kinase (PI3K) transiently translocates to the plasma membrane in response to chemoattractant stimulation and to the leading edge in chemotaxing cells, PTEN, a negative regulator of PI3K pathways, exhibits a reciprocal pattern of localization. By uniformly localizing PI3K along the plasma membrane,

Satoru Funamoto; Ruedi Meili; Susan Lee; Lisa Parry; Richard A. Firtel



Par3/Bazooka and phosphoinositides regulate actin protrusion formation during Drosophila dorsal closure and wound healing.  


Effective wound closure mechanisms are essential for maintenance of epithelial structure and function. The repair of wounded epithelia is primarily driven by the cells bordering the wound, which become motile after wounding, forming dynamic actin protrusions along the wound edge. The molecular mechanisms that trigger wound edge cells to become motile following tissue damage are not well understood. Using wound healing and dorsal closure in Drosophila, we identify a direct molecular link between changes in cell-cell adhesion at epithelial edges and induction of actin protrusion formation. We find that the scaffolding protein Par3/Bazooka and the lipid phosphatase Pten are specifically lost from cell-cell junctions at epithelial edges. This results in a localized accumulation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), which promotes the formation of actin protrusions along the epithelial edge. Depleting PIP3 results in defective epithelial closure during both dorsal closure and wound healing. These data reveal a novel mechanism that directly couples loss of epithelial integrity to activation of epithelial closure. PMID:23318638

Pickering, Karen; Alves-Silva, Juliana; Goberdhan, Deborah; Millard, Tom H



Leptin inhibits insulin secretion by activation of phosphodiesterase 3B.  

PubMed Central

The molecular signaling events by which leptin exerts its functions in vivo are not well delineated. Here, we show a novel leptin signaling mechanism that requires phosphoinositide 3-kinase (PI 3-kinase)-dependent activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and subsequent suppression of cAMP levels. In pancreatic beta cells, leptin causes the activation of PDE3B, which leads to marked inhibition of glucagon-like peptide-1-stimulated insulin secretion. The effect of leptin is abolished when insulin secretion is induced with cAMP analogues that cannot be hydrolyzed by PDE3B. Selective inhibitors of PDE3B and PI 3-kinase completely prevent the leptin effect on insulin secretion and cAMP accumulation. The results demonstrate that one of the physiological effects of leptin, suppression of insulin secretion, is mediated through activation of PDE3B and suggest PDE3B as a mediator of leptin action in other tissues.

Zhao, A Z; Bornfeldt, K E; Beavo, J A



How cells integrate complex stimuli: the effect of feedback from phosphoinositides and cell shape on cell polarization and motility.  


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) PIP?, PIP? 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. PMID:22396633

Marée, Athanasius F M; Grieneisen, Verônica A; Edelstein-Keshet, Leah



Modulation of NMDA effects on agonist-stimulated phosphoinositide turnover by memantine in neonatal rat cerebral cortex.  

PubMed Central

1. The ability of memantine (1-amino-3,5-dimethyladamantane) to antagonize the modulatory effects of N-methyl-D-aspartate (NMDA) on phosphoinositide turnover stimulated by muscarinic cholinoceptor- and metabotropic glutamate receptor-agonists has been examined in neonatal rat cerebral cortex slices. 2. Memantine antagonized the inhibitory effect of NMDA (100 microM) on both total [3H]-inositol phosphate ([3H]-InsPx) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulations stimulated by carbachol (1 mM) with EC50 values of 21 and 16 microM respectively. 3. Memantine concentration-dependently antagonized (IC50 24 microM) the ability of NMDA (10 microM) to potentiate [3H]-InsPx accumulation in response to a sub-maximal concentration of the metabotropic glutamate receptor agonist, 1S,3R-ACPD (10 microM). 4. The small (approx. 3 fold), concentration-dependent increase in [3H]-InsPx accumulation stimulated by NMDA was completely antagonized by the prototypic NDMA receptor-channel blocker, MK-801 (1 microM) at all concentrations of NDMA studied (1-1000 microM). In contrast, antagonism by memantine (100 microM) was observed only at low concentrations of NMDA (1-10 microM), whilst [3H]-InsPx accumulation stimulated by high concentrations of NMDA (300-1000 microM) was markedly enhanced by memantine. 5. Assessment of the incorporation of [3H]-inositol into inositol phospholipids revealed that memantine (100 microM) caused an approximate 2 fold increase in the labelling of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Mistry, R; Wilke, R; Challiss, R A



Genetic association of the Phosphoinositide-3 kinase in schizophrenia and bipolar disorder and interaction with a BDNF gene polymorphism  

PubMed Central

Phosphoinositide-3-kinase, class III (PIK3C3) is a member of the phosphoinosite-3-kinases family, involved in cell signaling, membrane trafficking, and neurodevelopment. Previous studies have indeed shown an association between PIK3C3 gene variants and both bipolar disorder (BD) and schizophrenia (SZ). Brain-derived neurotrophic factor (BDNF) is a neurodevelopmental factor, which can regulate the PI3K signaling pathway. Associations have been reported between BDNF gene polymorphisms and affective and psychotic disorders. The aim of the present study was to replicate an association between PIK3C3 and BDNF gene variants in SZ and BD and a putative epistasis between the two genes. Patients meeting the DSM-IV criteria of BD and SZ were included in this study (98 BD and 79 SZ) as well as 158 healthy controls. Blood DNA was extracted and genotyping was performed either by the polymerase chain reaction (PCR) technique followed by enzymatic digestion or by the high-resolution melt (HRM) method. Genotype and haplotype association was assessed with the UNPHASED statistical program.The results showed one nominal association with BD (P < 0.02) and two risk haplotypes in both SZ (P < 0.001) and BP (P < 0.0005), which survived multiple testing correction. A modest interaction between a BDNF variant and PI3KC3 polymorphism was observed (P < 0.04).These preliminary results confirm the genetic association of PI3K gene variants with both SZ and BD, and support the hypothesis that SZ and BD share a genetic background.

Carrard, Anthony; Salzmann, Annick; Perroud, Nader; Gafner, Jeremie; Malafosse, Alain; Karege, Felicien



Characterization of muscarinic receptors on intact human neuroblastoma cells: coupling to phosphoinositide hydrolysis and phosphorylation by phorbol esters  

SciTech Connect

Cloned human neuroblastoma cells (SH-SY5Y) were grown. High affinity binding of (/sup 3/H)(-)quinuclidinyl benzilate ((/sup 3/H)(-)QNB) and its quaternary derivative (/sup 3/H)(-)methyl QNB to muscarinic receptors (MR) on intact SH-SY5Y cells was studied. A 30 min rinse time gave a ratio of specific/total binding of 90% for both ligands. Association rates of (/sup 3/H)(-)QNB and (/sup 3/H)(-)methyl QNB were determined. Both ligands reached steady state by 60 min at 37/sup 0/C. Rates of dissociation for both radioligands were biphasic, although (/sup 3/H)(-)methyl QNB was faster. Saturation studies yielded K/sub d/ (dissociation constant) values of 16 and 260 pM and B/sub max/ (maximal MR density) values of 172 and 134 fmoles/mg prot for (/sup 3/H)(-)QNB and (/sup 3/H)(-)methyl QNB, respectively. Activation of protein kinase C by phorbol esters produced increased phosphorylation of cellular proteins. Pretreatment with 100 nM of 4..beta..-phorbol 12..beta..-myristate 13..cap alpha..-acetate (PMA) induced a decrease in agonist affinity for MR, suggesting a PMA-promoted phosphorylation of the MR protein. Phosphoinositide (PhI) turnover was measured by MR agonist-induced accumulation of inositol-1-phosphate in the presence of Li/sup + +/ (10 mM). Only carbachol and acetylcholine elicited potent responses with oxotremorine (16%) pilocarpine (17%) and McN-A-343 (8%) appearing to be weak partial agonist of low efficacy.

Serra, M.; Watson, M.; Roeske, W.R.; Yamamura, H.I.



Cloning of a human phosphoinositide 3-kinase with a C2 domain that displays reduced sensitivity to the inhibitor wortmannin.  

PubMed Central

The generation of phosphatidylinositide 3-phosphates has been observed in a variety of cellular responses. The enzymes that mediate synthesis are the phosphoinositide 3-kinases (PI3-Ks) that form a family of structurally diverse enzymes with distinct substrate specificities. In this paper, we describe the cloning of a novel human PI3-K, namely PI3-K-C2 alpha, which contains a C-terminal C2 domain. This enzyme can be assigned to the class II PI3-Ks, which was defined by characterization of the Drosophila 68D enzyme and includes the recently described murine enzymes m-cpk and p170. Despite the overall similarity in the amino acid sequence of the murine and human enzymes, which suggests that they are encoded by closely related genes, these molecules show marked sequence heterogeneity at their N-termini. Biochemical analysis of recombinant PI3-K-C2 alpha demonstrates a restricted lipid substrate specificity. As reported for other members of this class, the enzyme only phosphorylates PtdIns and PtdIns4P when the lipids are presented alone. However, when lipids were presented together with phosphatidylserine acting as a carrier, phosphorylation of PtdIns(4,5)P2 was also observed. The catalytic activity of PI3-K-C2 alpha is refractory to concentrations of wortmannin and LY294002 which inhibit the PI3-K activity of other family members. The comparative insensitivity of PI3-K-C2 alpha to these inhibitors suggests that their use should be reevaluated in the study of PI3-Ks.

Domin, J; Pages, F; Volinia, S; Rittenhouse, S E; Zvelebil, M J; Stein, R C; Waterfield, M D



Induction of memory in rat pancreatic islets by tolbutamide. Dependence on ambient glucose level, calcium, and phosphoinositide hydrolysis.  


The ability of the sulfonylurea tolbutamide to induce insulin output, increase phosphoinositide (PI) hydrolysis, and modulate the insulin response to other agonists was assessed. At 200 microM, tolbutamide increased both insulin release and the efflux of 3H from [3H]inositol-prelabeled islets only in the presence of 5.5 or 7 mM glucose. When the glucose level was maintained at 2.75 mM, tolbutamide (200 microM) had no positive impact on either parameter. The calcium-influx inhibitor nitrendipine (200 nM) blocked the effects of 200 microM tolbutamide (with 7 mM glucose) on 3H efflux and insulin output. Prior exposure of islets to tolbutamide (200 microM) in the presence of 7 mM glucose amplified their subsequent insulin response to 10 mM glucose and 5 mM glyceraldehyde. The effect of 200 microM tolbutamide (with 7 mM glucose) was blocked by nitrendipine. Furthermore, the effect of 200 microM tolbutamide was not observed with 2.75 mM glucose; however, if the level of tolbutamide was increased to 1 mM, both PI hydrolysis and potentiated release to subsequent stimulation with 10 mM glucose were observed. Tolbutamide (200 microM with 7 mM glucose) stimulation for 20 min resulted in an increase in 3H efflux from [3H]inositol-prelabeled islets. Despite the rapid fall in insulin secretion, elevated rates of 3H efflux persisted long after the removal of the sulfonylurea from the medium. The duration of the 3H-efflux response paralleled the duration of potentiation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2838355

Zawalich, W S; Zawalich, K C



Effects of short-term culturing on islet phosphoinositide and insulin secretory responses to glucose and carbachol.  


The ability of glucose and carbachol, alone or in combination, to stimulate islet cell phosphoinositide (PI) hydrolysis and insulin secretory responses in freshly isolated or in 20-24 h cultured rat islets was assessed. In freshly isolated, 3H-inositol-prelabeled islets, 20 mM glucose alone or 1 mM carbachol alone stimulated significant increments in 3H-inositol efflux and inositol phosphate (IP) accumulation. When stimulated with both agonists, a dramatic and synergistic effect on IP accumulation was noted. Carbachol (1 mM) alone had no sustained stimulatory effect on insulin secretion. Glucose (20 mM) alone induced a biphasic insulin secretory response. When compared to prestimulatory secretory rates of 18 +/- 4 pg/islet/min, peak first and second phase responses now averaged 422 +/- 61 and 1016 +/- 156 pg/islet/min, respectively. In contrast to freshly studied islets, culturing islets for 20-24 h in CMRL-1066 medium attenuated all measured responses. The increases in 3H-inositol efflux rates in response to glucose, carbachol, or their combination were significantly less than those observed with fresh islets. The IP responses were also attenuated. Second phase insulin secretory responses to 20 mM glucose alone 68 +/- 9 pg/islet/min) or the combination of 20 mM glucose plus 1 mM carbachol (358 +/- 85 pg/islet/min) were also significantly decreased when compared with fresh islets. We conclude from these studies that the process of culturing islets for one day in CMRL-1066 significantly decreases islet cell PI hydrolysis and insulin secretory responsiveness. These observations may help to explain the discordant conclusions reached concerning the involvement of PI hydrolysis and protein kinase C activation in the regulation of insulin release from freshly isolated versus cultured islets. PMID:8590784

Zawalich, W S; Zawalich, K C; Kelley, G G



Suppression of the {alpha}-isoform of class II phosphoinositide 3-kinase gene expression leads to apoptotic cell death  

SciTech Connect

Phosphoinositide 3-kinases (PI3Ks) have known to be key enzymes activating intracellular signaling molecules when a number of growth factors bind to their cell surface receptors. PI3Ks are divided into three classes (I, II, and III) and enzymes of each class have different tissue-specificities and physiological functions. Class II PI3Ks consist of three isoforms ({alpha}, {beta}, {gamma}). Although the {alpha}-isoform (PI3K-C2{alpha}) is considered ubiquitous and preferentially activated by insulin rather than the {beta}-isoform, the physiological significance of PI3K-C2{alpha} is poorly understood. The present study aimed to determine whether PI3K-C2{alpha} is associated with the suppression of apoptotic cell death. Different sense- and antisense oligonucleotides (ODNs) were synthesized based on the sequence of C2 domain of PI3K-C2{alpha} gene. Transfection of CHO-IR cells with two different antisense ODNs clearly reduced the protein content as well as mRNA levels of PI3K-C2{alpha} whereas neither the nonspecific mock- nor sense ODNs affected. The decrease of PI3K-C2{alpha} gene expression was paralleled by cellular changes indicating apoptotic cell death such as nuclear condensation, formation of apoptotic bodies, and DNA fragmentation. PI3K-C2{alpha} mRNA levels were also reduced when cells were incubated in growth factor-deficient medium. Supplementing growth factors (serum or insulin) into medium lead to an increase of PI3K-C2{alpha} mRNA levels. This finding strongly suggests that PI3K-C2{alpha} is a crucial survival factor.

Kang, Shinhae [Technology Innovation Center, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Song, Jihoon [Department of Life Science, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Kang, Jihoon [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Kang, Heekyoung [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Lee, Daeho [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Lee, Youngki [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Park, Deokbae [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of)]. E-mail:



The CIL-1 phosphoinositide 5-phosphatase regulates ciliary localization of the TRP polycystins and sperm function in C. elegans  

PubMed Central

Summary Background C. elegans male sexual behaviors include chemotaxis and response to hermaphrodites, backing/turning, vulva location, spicule insertion and sperm transfer, culminating in cross fertilization of hermaphrodite oocytes with male sperm. The LOV-1 and PKD-2 transient receptor potential polycystin (TRPP) complex localizes to ciliated endings of C. elegans male-specific sensory neurons and mediates several aspects of male mating behavior. TRPP complex ciliary localization and sensory function is evolutionarily conserved. A genetic screen for C. elegans mutants with PKD-2 ciliary localization (Cil) defects led to the isolation of a mutation in the cil-1 gene. Results Here, we report that a phosphoinositide (PI) 5-phosphatase CIL-1 regulates TRPP complex ciliary receptor localization and sperm activation. cil-1 does not regulate the localization of other ciliary proteins, including intraflagellar transport (IFT) components, sensory receptors, or other TRP channels in different cell types. Rather, cil-1 specifically controls TRPP complex trafficking in male-specific sensory neurons and does so in a cell autonomous fashion. In these cells, cil-1 is required for normal PI(3)P distribution, indicating that a balance between PI(3,5)P2 and PI(3)P is important for TRPP localization. cil-1 mutants are infertile due to sperm activation and motility defects. In sperm, the CIL-1 5-phosphatase and a wortmannin sensitive PI 3-kinase act antagonistically to regulate the conversion of sessile spermatids into motile spermatozoa, implicating PI(3,4,5)P3 signaling in nematode sperm activation. Conclusion Our studies identify the CIL-1 5-phosphatase as key regulator of PI metabolism in cell types that are important in several aspects of male reproductive biology.

Bae, Young-Kyung; Kim, Eunsoo; L'Hernault, Steven W.; Barr, Maureen M.



Activation of Myocardial Phosphoinositide-3-Kinase p110? Ameliorates Cardiac Dysfunction and Improves Survival in Polymicrobial Sepsis  

PubMed Central

Phosphoinositide-3-kinase (PI3K)/Akt dependent signaling has been shown to improve outcome in sepsis/septic shock. There is also ample evidence that PI3K/Akt dependent signaling plays a crucial role in maintaining normal cardiac function. We hypothesized that PI3K/Akt signaling may ameliorate septic shock by attenuating sepsis-induced cardiac dysfunction. Cardiac function and survival were evaluated in transgenic mice with cardiac myocyte specific expression of constitutively active PI3K isoform, p110? (caPI3K Tg). caPI3K Tg and wild type (WT) mice were subjected to cecal ligation/puncture (CLP) induced sepsis. Wild type CLP mice showed dramatic cardiac dysfunction at 6 hrs. Septic cardiomyopathy was significantly attenuated in caPI3K CLP mice. The time to 100% mortality was 46 hrs in WT CLP mice. In contrast, 80% of the caPI3K mice survived at 46 hrs after CLP (p<0.01) and 50% survived >30 days (p<0.01). Cardiac caPI3K expression prevented expression of an inflammatory phenotype in CLP sepsis. Organ neutrophil infiltration and lung apoptosis were also effectively inhibited by cardiac PI3k p110? expression. Cardiac high mobility group box–1 (HMGB-1) translocation was also inhibited by caPI3K p110? expression. We conclude that cardiac specific activation of PI3k/Akt dependent signaling can significantly modify the morbidity and mortality associated with sepsis. Our data also indicate that myocardial function/dysfunction plays a prominent role in the pathogenesis of sepsis and that maintenance of cardiac function during sepsis is essential. Finally, these data suggest that modulation of the PI3K/p110? signaling pathway may be beneficial in the prevention and/or management of septic cardiomyopathy and septic shock.

Li, Chuanfu; Hua, Fang; Ha, Tuanzhu; Singh, Krishna; Lu, Chen; Kalbfleisch, John; Breuel, Kevin F.; Ford, Tiffany; Kao, Race L.; Gao, Ming; Ozment, Tammy R.; Williams, David L.



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

PubMed Central

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 isogenic cell culture for the analysis of cell signalling pathways in pharmacological research. The model also supports an increasingly important ethical view of research, involving the reduction, replacement and refinement of animals in biomedical research. This review outlines the use of Dictyostelium in understanding the pharmacological action of two commonly used bipolar disorder treatments (valproic acid and lithium). Both of these compounds regulate mitogen activated protein (MAP) kinase and inositol phospholipid-based signalling by unknown means. Analysis of the molecular pathways targeted by these drugs in Dictyostelium and translation of discoveries to animal systems has helped to further understand the molecular mechanisms of these bipolar disorder treatments.

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



Phosphoinositide-specific inositol polyphosphate 5-phosphatase IV inhibits Akt/protein kinase B phosphorylation and leads to apoptotic cell death.  


Phosphoinositide-specific inositol polyphosphate 5- phosphatase IV has the affinity for PI(3,4,5)P(3) (K(m) = 0.65 microM) that is approximately 10-fold greater than the other inositol polyphosphate 5-phosphatases, which use this substrate including SHIP, OCRL, and 5ptase II, suggesting that it may be important in controlling intracellular levels of this metabolite. We created cell lines stably expressing the enzyme to study its effect on cell function. We found that overexpression of 5ptase IV in 293 cells caused the rapid depletion of both PI(4,5)P(2) and PI(3,4,5)P(3) in cells with corresponding increases in the products, PI(4)P and PI(3,4)P(2), changing the balance of two phosphoinositol products of phosphoinositide 3-kinase, PI(3,4)P(2) and PI(3,4,5)P(3), in the cell. One of the targets of these phosphoinositides is the serine/threonine kinase Akt, which plays an important role in the control of apoptosis. We were able to address the relative roles of PI(3,4)P(2) and PI(3,4,5)P(3) in the activation of Akt by selective depletion of these phosphoinositides in cells stably transfected with 5ptase IV and inositol polyphosphate 4-phosphatase (4ptase I). In cells transfected with 4ptase I, the level of PI(3,4)P(2) was reduced, and PI(3,4,5)P(3) was increased. Expression of the two enzymes had the opposite effect on the phosphorylation of Akt in response to stimulation with growth factors or heat shock. Akt phosphorylation was inhibited in cells expressing 5ptase IV but increased in 4ptase I cells and correlated with the intracellular level of PI(3,4,5)P(3) and not that of PI(3,4)P(2). The inhibition of Akt phosphorylation in cells expressing 5ptase IV makes them highly susceptible to FAS-induced apoptosis, whereas overexpressing of the 4ptase I protects cells from apoptosis. Our results place 5ptase IV as a relevant biological regulator of PI3K/Akt pathway in cells. PMID:11706019

Kisseleva, Marina V; Cao, Li; Majerus, Philip W



Ca2+-induced changes in the secondary structure of a 60 kDa phosphoinositide-specific phospholipase C from bovine brain cytosol.  

PubMed Central

The purification to homogeneity of a 60 kDa phosphoinositide-specific phospholipase C from bovine brain cytosol is reported here. This enzyme exhibits the same properties, in terms of response to Ca2+, as does the cytosolic activity in a variety of cell types. We show here that Ca2+ does not appear to modulate the binding of the enzyme to the substrate, but induces dramatic changes in its secondary structure. Therefore we suggest that a decrease in the alpha-helix content of this enzyme correlates with its ability to be activated by Ca2+. Images Fig. 1.

Herrero, C; Cornet, M E; Lopez, C; Barreno, P G; Municio, A M; Moscat, J



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



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


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 endosomes, 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. PMID:19914387

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



PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42.  


Formation of the apical surface and lumen is a fundamental, yet poorly understood, step in epithelial organ development. We show that PTEN localizes to the apical plasma membrane during epithelial morphogenesis to mediate the enrichment of PtdIns(4,5)P2 at this domain during cyst development in three-dimensional culture. Ectopic PtdIns(4,5)P2 at the basolateral surface causes apical proteins to relocalize to the basolateral surface. Annexin 2 (Anx2) binds PtdIns(4,5)P2 and is recruited to the apical surface. Anx2 binds Cdc42, recruiting it to the apical surface. Cdc42 recruits aPKC to the apical surface. Loss of function of PTEN, Anx2, Cdc42, or aPKC prevents normal development of the apical surface and lumen. We conclude that the mechanism of PTEN, PtdIns(4,5)P2, Anx2, Cdc42, and aPKC controls apical plasma membrane and lumen formation. PMID:17254974

Martin-Belmonte, Fernando; Gassama, Ama; Datta, Anirban; Yu, Wei; Rescher, Ursula; Gerke, Volker; Mostov, Keith



PTEN-mediated segregation of phosphoinositides at the apical membrane controls epithelial morphogenesis through Cdc42  

PubMed Central

Summary Formation of the apical surface and lumen is a fundamental, yet poorly understood, step in epithelial organ development. We show that PTEN localizes to the apical plasma membrane during epithelial morphogenesis to mediate the enrichment of PtdIns(4,5)P2 at this domain during cyst development in three dimensional culture. Ectopic PtdIns(4,5)P2 at the basolateral surface causes apical proteins to relocalize to the basolateral surface. Annexin 2 (Ax2) binds PtdIns(4,5)P2 and is recruited to the apical surface. Ax2 binds Cdc42, recruiting it to the apical surface. Cdc42 recruits aPKC to the apical surface. Loss of function of PTEN, Ax2, Cdc42 or aPKC prevents normal development of the apical surface and lumen. We conclude that the mechanism of PTEN, PtdIns(4,5)P2, Ax2, Cdc42 and aPKC controls apical plasma membrane and lumen formation.

Martin-Belmonte, Fernando; Gassama, Ama; Datta, Anirban; Yu, Wei; Rescher?, Ursula; Gerke?, Volker; Mostov, Keith



Bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal epithelial cells  

PubMed Central

Experiments were designed to differentiate the mechanisms and subtype of kinin receptors mediating the changes in intracellular Ca2+ concentration ([Ca2+]i) induced by bradykinin (BK) in canine cultured tracheal epithelial cells (TECs). BK and Lys-BK caused an initial transient peak of [Ca2+]i in a concentration-dependent manner, with half-maximal stimulation (pEC50) obtained at 7.70 and 7.23, respectively. Kinin B2 antagonists Hoe 140 (10?nM) and [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK (1??M) had high affinity in antagonizing BK-induced Ca2+ response with pKB values of 8.90 and 6.99, respectively. Pretreatment of TECs with pertussis toxin (100?ng?ml?1) or cholera toxin (10??g?ml?1) for 24?h did not affect the BK-induced IP accumulation and [Ca2+]i changes in TECs. Removal of Ca2+ by the addition of EGTA or application of Ca2+-channel blockers, verapamil, diltiazem, and Ni2+, inhibited the BK-induced IP accumulation and Ca2+ mobilization, indicating that Ca2+ influx was required for the BK-induced responses. Addition of thapsigargin (TG), which is known to deplete intracellular Ca2+ stores, transiently increased [Ca2+]i in Ca2+-free buffer and subsequently induced Ca2+ influx when Ca2+ was re-added to this buffer. Pretreatment of TECs with TG completely abolished BK-induced initial transient [Ca2+]i, but had slight effect on BK-induced Ca2+ influx. Pretreatment of TECs with SKF96365 and U73122 inhibited the BK-induced Ca2+ influx and Ca2+ release, consistent with the inhibition of receptor-gated Ca2+ -channels and phospholipase C in TECs, respectively. These results demonstrate that BK directly stimulates kinin B2 receptors and subsequently phospholipase C-mediated IP accumulation and Ca2+ mobilization via a pertussis toxin-insensitive G protein in canine TECs. These results also suggest that BK-induced Ca2+ influx into the cells is not due to depletion of these Ca2+ stores, as prior depletion of these pools by TG has no effect on the BK-induced Ca2+ influx that is dependent on extracellular Ca2+ in TECs.

Luo, Shue-Fen; Pan, Shiow-Lin; Wu, Wen-Bin; Wang, Chuan-Chwan; Chiu, Chi-Tso; Tsai, Yih-Jeng; Yang, Chuen-Mao



Interaction between phosphoinositide turnover system and cyclic AMP pathway for the secretion of pancreastatin and somatostatin from QGP-1N cells.  


It is found that secretion of pancreastatin and somatostatin from QGP-1N cells is regulated through muscarinic receptor-mediated activation of phosphatidylinositide hydrolysis system. In this report, whether the cAMP pathway interacts with the phosphoinositide turnover system for the secretion of pancreastatin and somatostatin from QGP-1N cells through muscarinic receptors was studied. Stimulation of QGP-1N cells with carbachol increased intracellular cAMP levels. The carbachol-induced increase in cAMP levels was inhibited by atropine. Calcium ionophore (A23187) and phorbol 12-myristate 13-acetate increased cAMP synthesis. Dibutyryl cAMP, forskolin and theophylline stimulated secretion of pancreastatin and somatostatin. When either dibutyryl cAMP, forskolin or theophylline was added in culture medium with A23187, phorbol ester or carbachol, a synergistic effect was found on pancreastatin and somatostatin secretion. These results suggest that interaction between the phosphoinositide turnover system and the cAMP pathway occurs in QGP-1N cells through muscarinic receptor stimulation for the secretion of pancreastatin and somatostatin. PMID:1352680

Tateishi, K; Funakoshi, A; Kitayama, N; Matsuoka, Y



D1-like dopaminergic activation of phosphoinositide hydrolysis is independent of D1A dopamine receptors: evidence from D1A knockout mice.  


Accumulated evidence suggests that dopamine and dopamine D1 agonists can activate phospholipase C in both brain and peripheral tissue. The receptor that mediates the hydrolysis of phosphoinositides has not been identified. The cloned dopamine D1A receptor that is generally thought to be linked to adenylyl cyclase, has also been proposed to couple to phospholipase C. However, a number of studies have suggested that this signaling pathway is mediated via a distinct D1-like dopamine receptor. We tested whether the D1A site plays a role in stimulating phosphoinositide hydrolysis by using the dopamine D1A-deficient mutant mice as a test model. Results show that although D1 dopamine receptor-mediated product on of cAMP is completely absent in membranes of D1A-deficient mice, D1 receptor-mediated accumulation of inositol phosphate is identical in tissues of mutant and wild-type animals. Furthermore, the coupling of [3H]SCH23390 binding sites in striatal or frontal cortex membranes to G alpha s is markedly reduced, although coupling of [3H]SCH23390 binding sites to G alpha q was unaltered in tissue taken from D1A mutant mice compared with control animals. These results clearly demonstrate that dopaminergic stimulation of inositol phosphate formation is mediated by a D1 dopamine receptor subtype that is distinct from the D1A receptor that activates adenylyl cyclase. PMID:9016340

Friedman, E; Jin, L Q; Cai, G P; Hollon, T R; Drago, J; Sibley, D R; Wang, H Y



Emergence of the Phosphoinositide 3-Kinase-Akt-Mammalian Target of Rapamycin Axis in Transforming Growth Factor-?-Induced Epithelial-Mesenchymal Transition  

PubMed Central

During development and in pathological contexts such as fibrosis and cancer progression, epithelial cells can initiate a complex transcriptional reprogramming, accompanied by dramatic morphological changes, in a process named ‘epithelial-mesenchymal transition’ (EMT). In this transition, epithelial cells lose their epithelial characteristics to acquire mesenchymal properties and increased motile and invasive behavior. Transforming growth factor-? (TGF-?) has emerged as a major inducer of EMT through activation of downstream signaling pathways, including Smad and non-Smad signaling pathways. Among the non-Smad pathways, increasing evidence is emerging that the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin axis plays a major role in TGF-?-induced EMT, notably through the regulation of translation and cell invasion. Pharmacological inhibitors of the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin pathway may therefore represent an opportunity to selectively target essential aspects of TGF-?-induced EMT and provide an approach to prevent cancer cell dissemination toward metastasis, without the need to fully inactivate TGF-? signaling.

Lamouille, Samy; Derynck, Rik



Influence of a ras oncogene on platelet-derived growth factor (PDGF)-stimulated phosphoinositide hydrolysis in murine fibroblasts  

SciTech Connect

The authors have examined the effects of transfection of rat-1 fibroblasts with the ras oncogene on the metabolism of phosphatidylinositol (PI). Incubation of (/sup 3/H)inositol-labeled rat-1 cells with PDGF resulted in a 2- to 3-fold increase in (/sup 3/H)IP3 levels within 90 s. In the presence of 25 mM Li+, (/sup 3/H)IP1 levels were increased 8-fold after 30 min. In contrast, incubation of ras-transfected fibroblasts (EJ-2 line) with PDGF had little or no effect on the level of either (/sup 3/H)IP3 or (/sup 3/H)IP1. Similar stimulations by PDGF were observed in NIH 3T3 cells, but not in Kirsten virus-transformed or Harvey ras-transfected cell lines. On the other hand, NIH 3T3 cells transfected with v-src responded to PDGF by stimulation of PI turnover similar to the parent cell line. In NIH 3T3 cells transfected with an expression vector containing the v-Ha-ras gene under transcriptional control of the glucocorticoid-inducible mouse mammary tumor virus promoter, the PDGF stimulation of (/sup 3/H)inositol incorporation into PI was reduced from 10-fold in the absence of dexamethasone to 1.8-fold when the cells were pretreated for 26 h with 2 dexamethasone. In the parental 3T3 cells PDGF stimulation was reduced by about 40% in the presence of dexamethasone. In the absence of PDGF the rate of PI turnover (i.e., the kinetics of (/sup 3/H)IP1 accumulation in the presence of Li+) in EJ-2 cells was similar to that in rat-1 cells. Thus, in the presence of PDGF, the rate of PI turnover in rat-1 cells was several fold higher than in the transfected cells. These results suggest that the ras gene product (p21) may exert an inhibitory effect on PDGF-stimulated phosphoinositide metabolism.

Parries, G.; Racker, E.



Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction.  

PubMed Central

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 wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.

Wymann, M P; Bulgarelli-Leva, G; Zvelebil, M J; Pirola, L; Vanhaesebroeck, B; Waterfield, M D; Panayotou, G



Uncoupling of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells  

PubMed Central

Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca2+]i peak in a concentration-dependent manner.Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1??M) for 30?min attenuated the BK-induced IPs formation and Ca2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2?h.The concentrations of PMA that gave half-maximal (pEC50) inhibition of BK-induced IPs accumulation and an increase in [Ca2+]i were 7.07?M and 7.11?M, respectively. Inactive phorbol ester, 4?-phorbol 12,13-didecanoate at 1??M, did not inhibit these responses. Prior treatment of TECs with staurosporine (1??M), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC.In parallel with the effect of PMA on the BK-induced IPs formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-?, ?I, ?II, ?, ?, ?, ? and ?. With PMA treatment of the cells for various times, translocation of PKC-?, ?I, ?II, ?, ?, ? and ? from cytosol to the membrane was seen after 5?min, 30?min, 2?h, and 4?h treatment. However, 6?h treatment caused a partial down-regulation of these PKC isozymes. PKC-? was not significantly translocated and down-regulated at any of the times tested.Treatment of TECs with 1??M PMA for either 30?min or 6?h did not significantly change the KD and Bmax receptor for BK binding (control: KD=1.7±0.3?nM; Bmax=50.5±4.9?fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses.In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit independently both responses to BK. The translocation of pKC-?, ?I, ?II, ?, ?, ?, and ? induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilization in TECs.

Yang, Chuen-Mao; Luo, Shue-Fen; Wu, Wen-Bin; Pan, Shiow-Lin; Tsai, Yih-Jeng; Chiu, Chi-Tso; Wang, Chuan-Chwan



Glucosamine-induced desensitization of beta-cell responses: possible involvement of impaired information flow in the phosphoinositide cycle.  


The influence of glucosamine on beta-cell response characteristics of collagenase-isolated rat islets was determined. Groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide (PI) pools. Also included in some experiments was glucosamine (0.1-10 mM). Subsequently, these islets were perifused, and their responses to 10 mM glucose, 10 mM alpha-ketoisocaproate (KIC), and 1 microM of the phorbol ester phorbol 12-myristate 13-acetate were assessed. Increases in PI hydrolysis were monitored during the perfusion by measuring fractional efflux rates of [3H]inositol. The accumulation of inositol phosphates after the perifusion was also determined. In other experiments, the use of 10 mM glucose was measured after a 2-h exposure to 5 or 10 mM glucosamine. Finally, the ability of glucosamine itself to augment release and activate PI hydrolysis was assessed. The following observations were made. 1) A prior 2-h exposure to 5-10 mM glucosamine resulted in parallel dose-dependent impairments in 10 mM glucose-induced insulin release and PI hydrolysis. 2) Glucosamine (5-10 mM) also impaired the subsequent response to alpha-ketoisocaproate (KIC). Parallel deficits in KIC-induced PI hydrolysis were noted under conditions where insulin secretion was impaired. 3) Under several conditions where glucosamine impaired glucose-induced secretion, it had no adverse effect on phorbol 12-myristate 13-acetate-induced release. 4) The desensitizing effect of 10 mM glucosamine on 10 mM glucose-induced release and PI hydrolysis developed within 30 min of exposure to it. 5) Glucosamine (5-10 mM) preexposure had no adverse effect on the use of 10 mM glucose by desensitized islets. 6) Short term (5-min) exposure to glucosamine (10 mM) alone stimulated PI hydrolysis, while a 30-min exposure to the same level of the hexosamine depressed it. 7) In the presence of 0.25 microM forskolin, 10 mM glucosamine also had a transient stimulatory effect on insulin release. These findings support the concept that the acute and chronic effects of glucosamine on the beta-cell result at least in part from its ability to influence PI hydrolysis in islets. PMID:1317776

Zawalich, W S; Zawalich, K C



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



Muscarinic stimulation of SK-N-BE(2) human neuroblastoma cells elicits phosphoinositide and phosphatidylcholine hydrolysis: relationship to diacylglycerol and phosphatidic acid accumulation.  

PubMed Central

Muscarinic stimulation of the human neuroblastoma cell line SK-N-BE(2) elicits hydrolysis of phosphoinositides and phosphatidylcholine (PtdCho) and produces a rapid and sustained elevation of diacylglycerol (DG) mass. PtdIns(4,5)P2 cleavage by phospholipase C (PLC) occurred immediately after carbachol (CCh) addition, and phosphoinositide hydrolysis was then sustained for at least 5 min. Cell stimulation, after extensive PtdCho labelling by long-term [3H]choline administration, resulted in an enhanced release of [3H]phosphocholine (PCho) into the external medium; enhanced [3H]PCho release, which occurred with a 15 s delay with respect to CCh addition, was particularly pronounced within the first minute of stimulation and proved to be caused by PtdCho-specific PLC activation. In fact, when cells were exposed to [3H]choline for a short period, to extensively label the intracellular PCho pool but not PtdCho, stimulation did not result in an enhanced release of [3H]PCho into the medium. PtdCho-specific phospholipase D (PLD) activation was documented by the accumulation of [3H]phosphatidylethanol in cells prelabelled with [3H]myristic acid and stimulated in the presence of 1% (v/v) ethanol; this metabolic pathway, however, proved to be a minor one leading to generation of phosphatidic acid (PtdOH) during cell stimulation, whereas DG production by the sequential action of PtdCho-specific PLD and PtdOH phosphohydrolase was not observed. Studies on cells which were double-labelled with [3H]myristic acid and [14C]arachidonic acid indicated that within 15 s of stimulation DG is uniquely derived from PtdIns(4,5)P2, whereas PtdCho is the major source at later times. Evidence is provided that rapid and selective conversion of phosphoinositide-derived DG into PtdOH may play an important role in determining the temporal accumulation profile of DG from the above-mentioned sources.

Pacini, L; Limatola, C; Frati, L; Luly, P; Spinedi, A



Influence of pyruvic acid methyl ester on rat pancreatic islets. Effects on insulin secretion, phosphoinositide hydrolysis, and sensitization of the beta cell.  


The methyl ester of pyruvic acid (methyl pyruvate) stimulated a dose-dependent increase in insulin secretion from isolated perifused rat islets. The threshold level for release was about 10 mM, and at 20 mM the addition of MP to perifused islets resulted in a large first phase of secretion followed by an insulin-secretory response that was sustained for at least 40 min. When compared to the effects of 20 mM glucose, peak first-phase release rates in response to 20 mM methyl pyruvate were comparable, but the second phase of release was only about 10-15% of that observed with an equimolar level of the hexose. The stimulatory effects of 20 mM methyl pyruvate on secretion were abolished by the K1+-ATP channel blocker diazoxide (200 microM) and by the calcium channel antagonist nitrendipine (500 nM). The glucokinase inhibitor mannoheptulose (20 mM) had no adverse effect on the secretory response to 20 mM methyl pyruvate, whereas 10 microM forskolin amplified the insulinotropic action of MP. Sodium pyruvate alone or in combination with 10 microM forskolin had no insulinotropic effect. In additional experiments islet phosphoinositide pools were labeled with myo-2-[3H]inositol, and the subsequent accumulation of labeled inositol phosphates was used to monitor the activation of phospholipase C. Methyl pyruvate stimulated a dose-dependent increase in inositol phosphate levels when measured after a 30-min incubation period with a maximal increase of about 300% at 20 mM methyl pyruvate. The increase in phosphoinositide hydrolysis caused by methyl pyruvate (20 mM) was, like insulin secretion, reduced by both diazoxide and nitrendipine but was immune to inhibition by mannoheptulose. Pyruvate (20 mM) had no effect on inositol phosphate accumulation. Prior short-term exposure to methyl pyruvate sensitized islets to subsequent stimulation with 15 mM glucose. Sodium pyruvate did not sensitize islets. These findings support the concept that the mitochondrial metabolism of nutrient molecules is an event sufficient to acutely augment insulin release from the beta cell, to increase phospholipase C-mediated phosphoinositide hydrolysis, and to induce time-dependent potentiation of insulin secretion. PMID:9013600

Zawalich, W S; Zawalich, K C



Interleukin-1 alpha exerts glucose-dependent stimulatory and inhibitory effects on islet cell phosphoinositide hydrolysis and insulin secretion.  


Isolated rat islets were incubated with myo-[2-3H]inositol to label their phosphoinositides (PI). Labeling was carried out in the presence of various glucose levels (2.75-10 mM) with or without human recombinant interleukin-1 alpha (IL-1). After the labeling period, insulin release, [3H]inositol efflux, and the accumulation of labeled inositol phosphates in perfused islets were assessed under various conditions. The following major observations were made. 1) In islets labeled for 2 h with [3H]inositol in the presence of 2.75 mM glucose, subsequent perifusion with 5.0 nM IL-1 increased insulin output, [3H]inositol efflux, and [3H]inositol phosphate accumulation in the simultaneous presence of 7 mM, but not 2.75 mM, glucose. 2) Mannoheptulose, a competitive inhibitor of islet glucokinase, blocked the stimulatory effects of IL-1 noted in the presence of 7 mM glucose. In other experiments, the conditions used during the 2-h labeling period with myo-[2-3H]inositol were varied. The following major observations were made in islets subsequently stimulated during the perifusion with 20 mM glucose. 3) Islets labeled with [3H]inositol in the presence of 2.75 mM glucose with or without 5.0 nM IL-1 responded with similar increases in PI hydrolysis and insulin output. 4) Compared to that with 2.75 mM glucose alone, labeling in the presence of 7 mM glucose alone was without any adverse effect on the subsequent PI and insulin responses of perifused islets to 20 mM glucose. 5) Labeling in the presence of 7 mM glucose plus 5.0 nM IL-1 resulted in a significant reduction in the subsequent PI and insulin responses. 6) These inhibitory effects of IL-1 were abolished if mannoheptulose was included during the 2-h incubation with 7 mM glucose plus 5.0 nM IL-1. 7) The diacylglycerol kinase inhibitor 1-monooleoylglycerol (100 microM) significantly restored insulin output after IL-1 exposure (with 7 mM glucose). 8) Similar to the results obtained with 7 mM glucose plus IL-1, incubation of islets with 8-10 mM glucose alone produced dose-dependent impairments of [3H]inositol efflux patterns and inositol phosphate accumulation. Insulin secretion was also impaired. These results demonstrate that IL-1 has glucose-dependent stimulatory and inhibitory effects on beta-cell function. Both effects appear to involve alterations in islet PI hydrolysis. PMID:2539976

Zawalich, W S; Zawalich, K C; Rasmussen, H



PDGF-induced receptor phosphorylation and phosphoinositide hydrolysis are unaffected by protein kinase C activation in mouse swiss 3T3 and human skin fibroblasts  

SciTech Connect

Short (1-10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 myristate, 13-acetate, PMA) affects the activity of a variety of surface receptors (for growth factors, hormones and neurotransmitters), with inhibition of transmembrane signal generation. In two types of fibroblasts it is demonstrated that the PDGF receptor is unaffected by PMA. Exposure to PMA at concentrations up to 100 nM for 10 min failed to inhibit either one of the agonist-induced, receptor-coupled responses of PDGF: the autophosphorylation of receptor molecules at tyrosine residues, and the hydrolysis of membrane polyphosphoinositides. In contrast, the EGF receptor autophosphorylation (in A 431 cells) and the bombesin-induced phosphoinositide hydrolysis were readily inhibited by PMA.

Sturani, E.; Vicentini, L.M.; Zippel, R.; Toschi, L.; Pandiella-Alonso, A.; Comoglio, P.M.; Meldolesi, J.



Phosphoinositide signalling enzymes in rat liver nuclei: phosphoinositidase C isoform beta 1 is specifically, but not predominantly, located in the nucleus.  

PubMed Central

The presence of phosphoinositide-mobilizing enzymes has been investigated in purified rat liver nuclei by radiolabelling and by probing with antibodies. A Ca(2+)-activated phosphoinositidase C (PIC) is present and was shown immunologically to be the beta 1 isoform. No gamma- or delta-PIC was found. However, only 5% of the total beta 1-PIC isoform is nuclear, with the majority being cytosolic. G alpha q and G alpha 11, the suggested physiological activators of beta 1-PIC, were not present. A PtdIns4P 5-kinase is also present, which immunologically is shown to be the C isoform. All of these nuclear inositide enzymes still remained after the removal of the nuclear envelope with Triton X-100, consistent with the concept of an intranuclear inositide cycle [Divecha, Banfic and Irvine (1991) EMBO. J. 10, 3207-3214]. Images Figure 2 Figure 3

Divecha, N; Rhee, S G; Letcher, A J; Irvine, R F



Time-dependent potentiation of insulin release induced by alpha-ketoisocaproate and leucine in rats: possible involvement of phosphoinositide hydrolysis.  


The ability of the amino acid leucine and its keto acid, alpha-ketoisocaproate, to induce insulin release, to initiate phosphoinositide hydrolysis, and to amplify the subsequent insulin secretory response to glucose was assessed. In islets whose inositol-containing lipids were prelabelled with myo[2-3H]inositol, the addition of either compound resulted in an increase in insulin output, an increase in 3H efflux, rapid and significant increases in labelled inositol phosphate accumulation and a sustained increase in 3H efflux after removal of the stimulant. Direct measurements of labelled inositol phosphate accumulation in islets previously stimulated with alpha-ketoisocaproate demonstrate that this sustained increase in 3H efflux was the result of a persistent increase in phosphoinositide hydrolysis and was not simply a consequence of the hydrolysis of preformed inositol phosphates into more membrane permeable species. Prior exposure of islets to alpha-ketoisocaproate or leucine also resulted in an amplified secretory response to a subsequent glucose (10 mmol/l) stimulus. While peak first phase insulin release averaged 66 +/- 4 (mean +/- SEM, n = 18) pg.islet-1. min-1 from control islets, this value increased to 204 +/- 14 and 246 +/- 11 pg.islet-1.min-1 in the leucine or alpha-keto-isocaproate pretreated islets respectively. The duration of this amplified response paralleled the duration of the persistent increase in 3H efflux. Prior alpha-ketoisocaproate exposure also amplified the subsequent insulin secretory response to tolbutamide and glyceraldehyde.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2851470

Zawalich, W S



A novel regulatory mechanism links PLC?1 to PDK1.  


3-Phosphoinositide-dependent protein kinase-1 (PDK1) and phospholipase C (PLC)?1 are two key enzymes in signal transduction that control several intracellular processes. Despite the fact that PLC?1 has been investigated for several years, the mechanisms of activation of this enzyme are still not completely clear. Similarly, although PDK1 has been mostly investigated for its role in activation of Akt, a crucial enzyme in regulation of several cellular processes, it has become evident recently that the role of PDK1 in physiological and pathological conditions is not limited to Akt activation. Here we demonstrate that PDK1 regulates PLC?1 activation in a mechanism involving association of the two enzymes and modulation of PLC?1 tyrosine phosphorylation. We further show that this novel PDK1-PLC?1 pathway is important for cancer cell invasion. The identification of a PDK1-PLC?1 pathway reveals the existence of a previously undetected link between two of the most important enzymes in signal transduction. This is likely to have profound consequences for our understanding of several cellular functions that are dependent on phosphoinositides and controlled by PDK1 and PLC?1. PMID:22454520

Raimondi, Claudio; Chikh, Anissa; Wheeler, Ann P; Maffucci, Tania; Falasca, Marco



Epigallocatechin gallate protects nerve growth factor differentiated PC12 cells from oxidative-radical-stress-induced apoptosis through its effect on phosphoinositide 3-kinase\\/Akt and glycogen synthase kinase-3  

Microsoft Academic Search

The effects of epigallocatechin gallate (EGCG) on the phosphoinositide 3-kinase (PI3K)\\/Akt and glycogen synthase kinase-3 (GSK-3) pathway during oxidative-stress-induced injury were studied using H2O2-treated PC12 cells, which were differentiated by nerve growth factor (NGF). Following 100 ?M H2O2 exposure, the viability of differentiated PC12 cells (EGCG or z-VAD-fmk pretreated vs. not pretreated) was evaluated the number of viable cell with

Seong-Ho Koh; Seung H. Kim; Hyugsung Kwon; Ki Sok Kim; Chi Won Song; Juhan Kim; Myung-Ho Kim; Hyun-Jeung Yu; Jenny S. Henkel; Hai Kwan Jung



The phosphoinositide 3-kinase\\/Akt pathway regulates cell cycle progression of HL60 human leukemia cells through cytoplasmic relocalization of the cyclin-dependent kinase inhibitor p27Kip1 and control of cyclin D1 expression  

Microsoft Academic Search

The serine\\/threonine protein kinase Akt, a downstream effector of phosphoinositide 3-kinase (PI3K), plays a pivotal role in tumorigenesis because it affects the growth and survival of cancer cells. Several laboratories have demonstrated that Akt inhibits transcriptional activation of a number of related forkhead transcription factors now referred to as FoxO1, FoxO3, and FoxO4. Akt-regulated forkhead transcription factors are involved in

A Cappellini; G Tabellini; M Zweyer; R Bortul; P L Tazzari; A M Billi; F Falà; L Cocco; A M Martelli



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  

Microsoft Academic Search

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

S. R. Slivka; P. A. Insel



EP1 prostanoid receptor coupling to G i/o up-regulates the expression of hypoxia-inducible factor-1 alpha through activation of a phosphoinositide-3 kinase signaling pathway.  


The EP1 prostanoid receptor is one of four subtypes whose cognate physiological ligand is prostaglandin-E2 (PGE(2)). It is in the family of G-protein-coupled receptors and is known to activate Ca(2+) 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 PGE(2) stimulation of the EP1 receptor up-regulates the expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha), which can be completely blocked by pertussis toxin, indicating coupling to G(i/o). This up-regulation of HIF-1 alpha 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 alpha, which involves decreased protein degradation, the up-regulation of HIF-1 alpha 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 alpha 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 alpha is known to promote tumor growth and metastasis and is often up-regulated in cancer. Our findings provide a potential mechanism by which increased PGE(2) biosynthesis could up-regulate the expression of HIF-1 alpha and promote tumorigenesis. PMID:20335389

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



Tyrosine 766 in the fibroblast growth factor receptor-1 is required for FGF-stimulation of phospholipase C, phospholipase D, phospholipase A(2), phosphoinositide 3-kinase and cytoskeletal reorganisation in porcine aortic endothelial cells.  


Fibroblast growth factor-mediated signalling was studied in porcine aortic endothelial cells expressing either wild-type fibroblast growth factor receptor-1 or a mutant receptor (Y766F) unable to bind phospholipase C-(&ggr;). Stimulation of cells expressing the wild-type receptor resulted in activation of phospholipases C, D and A(2) and increased phosphoinositide 3-kinase activity. Stimulation of the wild-type receptor also resulted in stress fibre formation and a cellular shape change. Cells expressing the Y766F mutant receptor failed to stimulate phospholipase C, D and A(2) as well as phosphoinositide 3-kinase. Furthermore, no stress fibre formation or shape change was observed. Both the wild-type and Y766F receptor mutant activated MAP kinase and elicited proliferative responses in the porcine aortic endothelial cells. Thus, fibroblast growth factor receptor-1 mediated activation of phospholipases C, D and A(2) and phosphoinositide 3-kinase was dependent on tyrosine 766. Furthermore, whilst tyrosine 766 was not required for a proliferative response, it was required for fibroblast growth factor receptor-1 mediated cytoskeletal reorganisation. PMID:10652257

Cross, M J; Hodgkin, M N; Roberts, S; Landgren, E; Wakelam, M J; Claesson-Welsh, L



A regulatory subunit of phosphoinositide 3-kinase increases the nuclear accumulation of X-box-binding protein-1 to modulate the unfolded protein response.  


Class Ia phosphoinositide 3-kinase (PI3K), an essential mediator of the metabolic actions of insulin, is composed of a catalytic (p110alpha or p110beta) and regulatory (p85alphaalpha, p85betaalpha or p55alpha) subunit. Here we show that p85alphaalpha interacts with X-box-binding protein-1 (XBP-1), a transcriptional mediator of the unfolded protein response (UPR), in an endoplasmic reticulum (ER) stress-dependent manner. Cell lines with knockout or knockdown of p85alphaalpha show marked alterations in the UPR, including reduced ER stress-dependent accumulation of nuclear XBP-1, decreased induction of UPR target genes and increased rates of apoptosis. This is associated with a decreased activation of inositol-requiring protein-1alpha (IRE1alpha) and activating transcription factor-6alphaalpha (ATF6alpha). Mice with deletion of p85alpha in liver (L-Pik3r1(-/-)) show a similar attenuated UPR after tunicamycin administration, leading to an increased inflammatory response. Thus, p85alphaalpha forms a previously unrecognized link between the PI3K pathway, which is central to insulin action, and the regulation of the cellular response to ER stress, a state that when unresolved leads to insulin resistance. PMID:20348923

Winnay, Jonathon N; Boucher, Jeremie; Mori, Marcelo A; Ueki, Kohjiro; Kahn, C Ronald



Phosphorylation-independent dual-site binding of the FHA domain of KIF13 mediates phosphoinositide transport via centaurin [alpha]1  

SciTech Connect

Phosphatidylinositol 3,4,5-triphosphate (PIP3) plays a key role in neuronal polarization and axon formation. PIP3-containing vesicles are transported to axon tips by the kinesin KIF13B via an adaptor protein, centaurin {alpha}1 (CENTA1). KIF13B interacts with CENTA1 through its forkhead-associated (FHA) domain. We solved the crystal structures of CENTA1 in ligand-free, KIF13B-FHA domain-bound, and PIP3 head group (IP4)-bound conformations, and the CENTA1/KIF13B-FHA/IP4 ternary complex. The first pleckstrin homology (PH) domain of CENTA1 specifically binds to PIP3, while the second binds to both PIP3 and phosphatidylinositol 3,4-biphosphate (PI(3,4)P2). The FHA domain of KIF13B interacts with the PH1 domain of one CENTA1 molecule and the ArfGAP domain of a second CENTA1 molecule in a threonine phosphorylation-independent fashion. We propose that full-length KIF13B and CENTA1 form heterotetramers that can bind four phosphoinositide molecules in the vesicle and transport it along the microtubule.

Tong, Yufeng; Tempel, Wolfram; Wang, Hui; Yamada, Kaori; Shen, Limin; Senisterra, Guillermo A.; MacKenzie, Farrell; Chishti, Athar H.; Park, Hee-Won (Toronto); (UICM)



Deletion of the p110beta isoform of phosphoinositide 3-kinase in platelets reveals its central role in Akt activation and thrombus formation in vitro and in vivo.  


During platelet activation, phosphoinositide 3-kinases (PI3Ks) produce lipid second messengers participating in the regulation of functional responses. Here, we generated a megakaryocyte-restricted p110beta null mouse model and demonstrated a critical role of PI3Kbeta in platelet activation via an immunoreceptor tyrosine-based activation motif, the glyco-protein VI-Fc receptor gamma-chain complex, and its contribution in response to G-protein-coupled receptors. Interestingly, the production of phosphatidylinositol 3,4,5-trisphosphate and the activation of protein kinase B/Akt were strongly inhibited in p110beta null platelets stimulated either via immunoreceptor tyrosine-based activation motif or G-protein-coupled receptors. Functional studies showed an important delay in fibrin clot retraction and an almost complete inability of these platelets to adhere onto fibrinogen under flow condition, suggesting that PI3Kbeta is also acting downstream of alpha(IIb)beta(3). In vivo studies showed that these mice have a normal bleeding time and are not protected from acute pulmonary thromboembolism but are resistant to thrombosis after FeCl(3) injury of the carotid, suggesting that PI3Kbeta is a potential target for antithrombotic drugs. PMID:20065293

Martin, Valérie; Guillermet-Guibert, Julie; Chicanne, Gaétan; Cabou, Cendrine; Jandrot-Perrus, Martine; Plantavid, Monique; Vanhaesebroeck, Bart; Payrastre, Bernard; Gratacap, Marie-Pierre



Insulin/phosphoinositide 3-kinase pathway accelerates the glucose-induced first-phase insulin secretion through TrpV2 recruitment in pancreatic ?-cells.  


Functional insulin receptor and its downstream effector PI3K (phosphoinositide 3-kinase) have been identified in pancreatic ?-cells, but their involvement in the regulation of insulin secretion from ?-cells remains unclear. In the present study, we investigated the physiological role of insulin and PI3K in glucose-induced biphasic insulin exocytosis in primary cultured ?-cells and insulinoma Min6 cells using total internal reflection fluorescent microscopy. The pretreatment of ?-cells with insulin induced the rapid increase in intracellular Ca2+ levels and accelerated the exocytotic response without affecting the second-phase insulin secretion. The inhibition of PI3K not only abolished the insulin-induced rapid development of the exocytotic response, but also potentiated the second-phase insulin secretion. The rapid development of Ca2+ and accelerated exocytotic response induced by insulin were accompanied by the translocation of the Ca2+-permeable channel TrpV2 (transient receptor potential V2) in a PI3K-dependent manner. Inhibition of TrpV2 by the selective blocker tranilast, or the expression of shRNA (short-hairpin RNA) against TrpV2 suppressed the effect of insulin in the first phase, but the second phase was not affected. Thus our results demonstrate that insulin treatment induced the acceleration of the exocytotic response during the glucose-induced first-phase response by the insertion of TrpV2 into the plasma membrane in a PI3K-dependent manner. PMID:20854263

Aoyagi, Kyota; Ohara-Imaizumi, Mica; Nishiwaki, Chiyono; Nakamichi, Yoko; Nagamatsu, Shinya



Recruitment of OCRL and Inpp5B to phagosomes by Rab5 and APPL1 depletes phosphoinositides and attenuates Akt signaling  

PubMed Central

Sealing of phagosomes is accompanied by the disappearance of phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2) from their cytoplasmic leaflet. Elimination of PtdIns(4,5)P2, which is required for actin remodeling during phagosome formation, has been attributed to hydrolysis by phospholipase C and phosphorylation by phosphatidylinositol 3-kinase. We found that two inositol 5-phosphatases, OCRL and Inpp5B, become associated with nascent phagosomes. Both phosphatases, which are Rab5 effectors, associate with the adaptor protein APPL1, which is recruited to the phagosomes by active Rab5. Knockdown of APPL1 or inhibition of Rab5 impairs association of OCRL and Inpp5B with phagosomes and prolongs the presence of PtdIns(4,5)P2 and actin on their membranes. Even though APPL1 can serve as an anchor for Akt, its depletion accentuated the activation of the kinase, likely by increasing the amount of PtdIns(4,5)P2 available to generate phosphatidylinositol (3,4,5)-trisphosphate. Thus, inositol 5-phosphatases are important contributors to the phosphoinositide remodeling and signaling that are pivotal for phagocytosis.

Bohdanowicz, Michal; Balkin, Daniel M.; De Camilli, Pietro; Grinstein, Sergio



Platelet alpha IIb-beta 3 integrin engagement induces the tyrosine phosphorylation of Cbl and its association with phosphoinositide 3-kinase and Syk.  

PubMed Central

Agonist-induced platelet activation triggers 'inside-out' signalling which activates alpha IIb-beta 3, the most abundant integrin in platelet membranes. The engagement of activated alpha IIb-beta 3 integrin by linking fibrinogen is necessary for platelet aggregation, and this induces subsequent outside-in signalling, which enhances platelet activation. Here we studied the involvement of Cbl during alpha IIb-beta 3-integrin-mediated signal transduction. During thrombin-induced platelet activation, Cbl was tyrosine phosphorylated, and phosphoinositide 3-kinase (PI 3-kinase) activity measured in Cbl immunoprecipitates was increased. Both Cbl phosphorylation and its association with PI 3-kinase were dependent on alpha IIb-beta 3 engagement by linking fibrinogen. The P256 and anti-LIBS6 (ligand-induced binding site 6) antibodies, which activate platelets directly through alpha IIb-beta 3, induced Cbl phosphorylation and increased the PI 3-kinase activity associated with Cbl. Both thrombin and antibodies to alpha IIb-beta 3 induced association of Cbl with the tyrosine kinase, Syk. Experiments performed with inhibitors of tyrosine kinases indicated that both Src-family kinases and Syk contribute to phosphorylation of Cbl and its consequent association with PI 3-kinase. The results show that, following integrin alpha IIb-beta 3 engagement, Cbl is tyrosine phosphorylated, recruits PI 3-kinase to this integrin signalling pathway and possibly enhances PI 3-kinase activity, downstream of Src-family tyrosine kinases and Syk activation.

Saci, A; Rendu, F; Bachelot-Loza, C



Expression pattern and sub-cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U-73122 in rat astrocytoma cells.  


Phosphoinositide specific phospholipase C (PI-PLC) enzymes interfere with the metabolism of inositol phospholipids (PI), molecules involved in signal transduction, a complex process depending on various components. Many evidences support the hypothesis that, in the glia, isoforms of PI-PLC family display different expression and/or sub cellular distribution under non-physiological conditions such as the rat astrocytes activation during neurodegeneration, the tumoural progression of some neoplasms and the inflammatory cascade activation after lipopolysaccharide administration, even if their role remains not completely elucidated. Treatment of a cultured established glioma cell line (C6 rat astrocytoma cell line) induces a modification in the pattern of expression and of sub cellular distribution of PI-PLCs compared to untreated cells. Special attention require PI-PLC beta3 and PI-PLC gamma2 isoforms, whose expression and sub cellular localization significantly differ after U-73122 treatment. The meaning of these modifications is unclear, also because the use of this N-aminosteroid compound remains controversial, inasmuch it has further actions which might contribute to the global effect recorded on the treated cells. PMID:20564200

Lo Vasco, Vincenza Rita; Fabrizi, Cinzia; Panetta, Barbara; Fumagalli, Lorenzo; Cocco, Lucio



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



An essential function of a phosphoinositide-specific phospholipase C is relieved by inhibition of a cyclin-dependent protein kinase in the yeast Saccharomyces cerevisiae.  

PubMed Central

The PLC1 gene product of Saccharomyces cerevisiae is a homolog of the delta isoform of mammalian phosphoinositide-specific phospholipase C (PI-PLC). We found that two genes (SPL1 and SPL2), when overexpressed, can bypass the temperature-sensitive growth defect of a plc1delta cell. SPL1 is identical to the PHO81 gene, which encodes an inhibitor of a cyclin (Pho80p)-dependent protein kinase (Pho85p) complex (Cdk). In addition to overproduction of Pho81p, two other conditions that inactivate this Cdk, a cyclin (pho80delta) mutation and growth on low-phosphate medium, also permitted growth of plc1delta cells at the restrictive temperature. Suppression of the temperature sensitivity of plc1delta cells by pho80delta does not depend upon the Pho4p transcriptional regulator, the only known substrate of the Pho80p/Pho85p Cdk. The second suppressor, SPL2, encodes a small (17-kD) protein that bears similarity to the ankyrin repeat regions present in Pho81p and in other known Cdk inhibitors. Both pho81delta and spl2delta show a synthetic phenotype in combination with plc1delta. Unlike single mutants, plc1delta pho81delta and plc1delta spl2delta double mutants were unable to grow on synthetic complete medium, but were able to grow on rich medium.

Flick, J S; Thorner, J



VEGF Induces Tie2 Shedding via a Phosphoinositide 3Kinase\\/Akt-Dependent Pathway to Modulate Tie2 Signaling  

Microsoft Academic Search

Objective—Tie2 and its ligands, the angiopoietins (Ang), are required for embryonic and postnatal angiogenesis. Previous studies have demonstrated that Tie2 is proteolytically cleaved, resulting in the production of a 75-kDa soluble receptor fragment (sTie2). We investigated mechanisms responsible for Tie2 shedding and its effects on Tie2 signaling and endothelial cellular responses. Methods and Results—sTie2 bound both Ang1 and Ang2 and

Clarence M. Findley; Melissa J. Cudmore; Asif Ahmed; Christopher D. Kontos



The inhibition of phosphoinositide synthesis and muscarinic-receptor-mediated phospholipase C activity by Li+ as secondary, selective, consequences of inositol depletion in 1321N1 cells.  

PubMed Central

Conditions are described for culture of 1321N1 cells under which cellular inositol is decreased from approximately 20 mM to < 0.5 mM but phosphoinositide concentrations are unaffected. The effects of the muscarinic-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinositide turnover in these or in inositol-replete cells was examined after steady-state [3H]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar initial (0-15 min) rates of phospholipase C (PLC) activity, in the presence of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [3H]PtdIns concentrations declined dramatically only in depleted cells. In inositol-depleted cells, carbachol alone evoked increased concentrations of [3H]inositol, [3H]InsP1, [3H]InsP2, [3H]InsP3 and [3H]InsP4, which were largely sustained over 90 min, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased only to approximately 82, 84 and 93% of control respectively. In the presence of Li+ in these cells, the stimulated rise in [3H]inositol was prevented and, although accumulation of [3H]InsP1, [3H]InsP2 and [3H]InsP3 was initially (0-30 min) potentiated, rates of accumulation of [3H]InsP1 and concentrations of [3H]polyphosphates later (> 30-60 min) declined, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased respectively to approximately 39, 48 and 81% of control. After 60 min in the presence of both carbachol and Li+, stimulated PLC activity was decreased by approximately 70% compared with the initial rate in depleted cells. This decreased PLC activity was reflected by changes in the stimulated concentrations of [3H]Ins(1,3,4)P3 but not of [3H]Ins(1,4,5)P3, but effects of Li+ on the latter may have been obscured by the demonstrated, concomitant and equal stimulated accumulation of [3H]inositol 1:2cyclic,4,5-trisphosphate. These data suggest that receptor-mediated PLC activity is selectively impaired by Li+ as a secondary consequence of inositol monophosphatase inhibition in cells which are highly dependent on inositol re-cycling, but imply that, although Li+ attenuation of PLC activity correlates closely with parameters indicative of limiting inositol supply, it is not readily attributed to decreased PtdInsP2 availability. The potential for complex regulation of PLC and PtdIns synthase is discussed.

Batty, I H; Downes, C P



Phosphoinositide-dependent kinase-1 and protein kinase C? contribute to endothelin-1 constriction and elevated blood pressure in intermittent hypoxia.  


Obstructive sleep apnea (OSA) is associated with cardiovascular complications including hypertension. Previous findings from our laboratory indicate that exposure to intermittent hypoxia (IH), to mimic sleep apnea, increases blood pressure in rats. IH also increases endothelin-1 (ET-1) constrictor sensitivity in a protein kinase C (PKC) ?-dependent manner in mesenteric arteries. Because phosphoinositide-dependent kinase-1 (PDK-1) regulates PKC? activity, we hypothesized that PDK-1 contributes to the augmented ET-1 constrictor sensitivity and elevated blood pressure following IH. Male Sprague-Dawley rats were exposed to either sham or IH (cycles between 21% O(2)/0% CO(2) and 5% O(2)/5% CO(2)) conditions for 7 h/day for 14 or 21 days. The contribution of PKC? and PDK-1 to ET-1-mediated vasoconstriction was assessed in mesenteric arteries using pharmacological inhibitors. Constrictor sensitivity to ET-1 was enhanced in arteries from IH-exposed rats. Inhibition of PKC? or PDK-1 blunted ET-1 constriction in arteries from IH but not sham group rats. Western analysis revealed similar levels of total and phosphorylated PDK-1 in arteries from sham and IH group rats but decreased protein-protein interaction between PKC? and PDK-1 in arteries from IH- compared with sham-exposed rats. Blood pressure was increased in rats exposed to IH, and treatment with the PDK-1 inhibitor OSU-03012 [2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}-acetamide] (33 mg/day) lowered blood pressure in IH but not sham group rats. Our results suggest that exposure to IH unmasks a role for PDK-1 in regulating ET-1 constrictor sensitivity and blood pressure that is not present under normal conditions. These novel findings suggest that PDK-1 may be a uniquely effective antihypertensive therapy for OSA patients. PMID:23093023

Webster, Bradley R; Osmond, Jessica M; Paredes, Daniel A; DeLeon, Xavier A; Jackson-Weaver, Olan; Walker, Benjimen R; Kanagy, Nancy L



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



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.



Differential regulation of class IA phosphoinositide 3-kinase catalytic subunits p110? and ? by protease-activated receptor 2 and ?-arrestins  

PubMed Central

PAR-2 (protease-activated receptor 2) is a GPCR (G-protein-coupled receptor) that can elicit both G-protein-dependent and -independent signals. We have shown previously that PAR-2 simultaneously promotes G?q/Ca2+-dependent activation and ?-arrestin-1-dependent inhibition of class IA PI3K (phosphoinositide 3-kinase), and we sought to characterize further the role of ?-arrestins in the regulation of PI3K activity. Whereas the ability of ?-arrestin-1 to inhibit p110? (PI3K catalytic subunit ?) has been demonstrated, the role of ?-arrestin-2 in PI3K regulation and possible differences in the regulation of the two catalytic subunits (p110? and p110?) associated with p85? (PI3K regulatory subunit) have not been examined. In the present study we have demonstrated that: (i) PAR-2 increases p110?- and p110?-associated lipid kinase activities, and both p110? and p110? are inhibited by over-expression of either ?-arrestin-1 or -2; (ii) both ?-arrestin-1 and -2 directly inhibit the p110? catalytic subunit in vitro, whereas only ?-arrestin-2 directly inhibited p110?; (iii) examination of upstream pathways revealed that PAR-2-induced PI3K activity required the small GTPase Cdc (cell-division cycle)42, but not tyrosine phosphorylation of p85; and (iv) ?-arrestins inhibit PAR-2-induced Cdc42 activation. Taken together, these results indicated that ?-arrestins could inhibit PAR-2-stimulated PI3K activity, both directly and through interference with upstream pathways, and that the two ?-arrestins differ in their ability to inhibit the p110? and p110? catalytic subunits. These results are particularly important in light of the growing interest in PAR-2 as a pharmacological target, as commonly used biochemical assays that monitor G-protein coupling would not screen for ?-arrestin-dependent signalling events.

Wang, Ping; Kumar, Puneet; Wang, Chang; DeFea, Kathryn A.



Synergistic interactions between imatinib mesylate and the novel phosphoinositide-dependent kinase-1 inhibitor OSU-03012 in overcoming imatinib mesylate resistance.  


Resistance to the Ableson protein tyrosine (Abl) kinase inhibitor imatinib mesylate has become a critical issue for patients in advanced phases of chronic myelogenous leukemia. Imatinib-resistant tumor cells develop, in part, as a result of point mutations within the Abl kinase domain. As protein kinase B (Akt) plays a pivotal role in Abl oncogene-mediated cell survival, we hypothesize that concurrent inhibition of Akt will sensitize resistant cells to the residual apoptotic activity of imatinib mesylate, thereby overcoming the resistance. Here, we examined the effect of OSU-03012, a celecoxib-derived phosphoinositide-dependent kinase-1 (PDK-1) inhibitor, on imatinib mesylate-induced apoptosis in 2 clinically relevant breakpoint cluster region (Bcr)-Abl mutant cell lines, Ba/F3p210(E255K) and Ba/F3p210(T315I). The 50% inhibitory concentration (IC50) values of imatinib mesylate to inhibit the proliferation of Ba/F3p210(E255K) and Ba/F3p210(T315I) were 14 +/- 4 and 30 +/- 2 microM, respectively. There was no cross-resistance to OSU-03012 in these mutant cells with an IC50 of 5 microM irrespective of mutations. Nevertheless, in the presence of OSU-03012 the susceptibility of these mutant cells to imatinib-induced apoptosis was significantly enhanced. This synergistic action was, at least in part, mediated through the concerted effect on phospho-Akt. Together these data provide a novel therapeutic strategy to overcome imatinib mesylate resistance, especially with the Abl mutant T315I. PMID:15665113

Tseng, Ping-Hui; Lin, Ho-Pi; Zhu, Jiuxiang; Chen, Kuen-Feng; Hade, Erinn M; Young, Donn C; Byrd, John C; Grever, Michael; Johnson, Kara; Druker, Brian J; Chen, Ching-Shih



Synergistic interactions between imatinib mesylate and the novel phosphoinositide-dependent kinase-1 inhibitor OSU-03012 in overcoming imatinib mesylate resistance  

PubMed Central

Resistance to the Ableson protein tyrosine (Abl) kinase inhibitor imatinib mesylate has become a critical issue for patients in advanced phases of chronic myelogenous leukemia. Imatinib-resistant tumor cells develop, in part, as a result of point mutations within the Abl kinase domain. As protein kinase B (Akt) plays a pivotal role in Abl oncogene-mediated cell survival, we hypothesize that concurrent inhibition of Akt will sensitize resistant cells to the residual apoptotic activity of imatinib mesylate, thereby overcoming the resistance. Here, we examined the effect of OSU-03012, a celecoxib-derived phosphoinositide-dependent kinase-1 (PDK-1) inhibitor, on imatinib mesylate-induced apoptosis in 2 clinically relevant breakpoint cluster region (Bcr)-Abl mutant cell lines, Ba/F3p210E255K and Ba/F3p210T315I. The 50% inhibitory concentration (IC50) values of imatinib mesylate to inhibit the proliferation of Ba/F3p210E255K and Ba/F3p210T315I were 14 ± 4 and 30 ± 2 ?M, respectively. There was no cross-resistance to OSU-03012 in these mutant cells with an IC50 of 5 ?M irrespective of mutations. Nevertheless, in the presence of OSU-03012 the susceptibility of these mutant cells to imatinib-induced apoptosis was significantly enhanced. This synergistic action was, at least in part, mediated through the concerted effect on phospho-Akt. Together these data provide a novel therapeutic strategy to overcome imatinib mesylate resistance, especially with the Abl mutant T315I.

Tseng, Ping-Hui; Lin, Ho-Pi; Zhu, Jiuxiang; Chen, Kuen-Feng; Hade, Erinn M.; Young, Donn C.; Byrd, John C.; Grever, Michael; Johnson, Kara; Druker, Brian J.; Chen, Ching-Shih



Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca2+]i,ICa, and Ca2+ transients in HL-1 cardiomyocytes  

PubMed Central

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca2+]i, Ca2+ transients and membrane Ca2+ current, ICa, in cultured murine HL-1 cardiomyocytes. LY294002 (1–20??M), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca2+]i, Ca2+ transients and ICa. We also examined the effect of PI3K isoform specific inhibitors, i.e. ? (PI3-kinase ? inhibitor 2; 2–8?nM); ? (TGX-221; 100?nM) and ? (AS-252424; 100?nM), to determine the contribution of specific isoforms to HL-1 [Ca2+]i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca2+]i, and inhibited Ca2+ transients. Triciribine (1–20??M), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca2+]i, and Ca2+ transients and ICa. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca2+]i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca2+]i required for excitation-contraction coupling in cardiomyoctyes.



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



Ellagic acid prevents rat colon carcinogenesis induced by 1, 2 dimethyl hydrazine through inhibition of AKT-phosphoinositide-3 kinase pathway.  


Colon cancer is the third most malignant neoplasm in the world and chemoprevention through dietary intervention is an emerging option to reduce its mortality. Ellagic acid (EA) a major component of berries possesses attractive biological deeds. This study is aimed to investigate the effect of ellagic acid in fostering apoptosis in 1,2-dimethyl hydrazine (DMH) mediated experimental colon carcinogenesis model. Wistar male rats were segregated into four groups: group I-control rats, group II-rats received ellagic acid (60 mg/kg body weight p.o. every day), rats in group III-induced with DMH (20 mg/kg body weight, s.c.) for 15 weeks, DMH-induced group IV rats were initiated with ellagic acid treatment. The present study is designed to explore the significance of phosphoinositide-3-kinase (PI3K)/Akt molecular pathway as well as ellagic acid's chemopreventive effect in colon cancer. DMH-induced rats exhibited elevated expressions of PI3K and Akt as confirmed by immunofluorescence, immunoblot and confocal microscopic analysis. Mechanistically, ellagic acid was found to prevent PI3K/Akt activation that in turn, results in modulation of its downstream Bcl-2 family proteins. Bax expression and caspase-3 activation was noted after ellagic acid supplementation leading to elevation of cytochrome c (cyt c) levels and finally cell death. These observations were supported by the DNA fragmentation results, which showed the occurrence of apoptosis. This study reveals the involvement of PI3K-Akt signaling through which ellagic acid induces apoptosis and subsequently suppresses colon cancer during DMH-induced rat colon carcinogenesis. In conclusion, our findings demonstrate that ellagic acid begets apoptosis in DMH-induced colon carcinoma. PMID:21463623

Umesalma, Syed; Sudhandiran, Ganapasam



Discovery of PDK1 Kinase Inhibitors with a Novel Mechanism of Action by Ultrahigh Throughput Screening  

PubMed Central

The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen® screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, Km values were determined as 5.6 ?m for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1× and 10× Km concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.

Bobkova, Ekaterina V.; Weber, Michael J.; Xu, Zangwei; Zhang, Yan-Ling; Jung, Joon; Blume-Jensen, Peter; Northrup, Alan; Kunapuli, Priya; Andersen, Jannik N.; Kariv, Ilona



Phosphatidylinositol 3-kinase, phosphoinositide-specific phospholipase-C? and protein kinase-C signal myelin phagocytosis mediated by complement receptor-3 alone and combined with scavenger receptor-AI\\/II in macrophages  

Microsoft Academic Search

Complement-receptor-3 (CR3\\/MAC-1), scavenger-receptor-AI\\/II (SRAI\\/II) and Fc?-receptor (Fc?R) can mediate phagocytosis of degenerated myelin in macrophages and microglia. However, CR3\\/MAC-1 and SRAI\\/II, but not Fc?R, mediate phagocytosis after axonal injury. We tested for phosphatidylinositol 3-kinase (PI3K), phosphoinositide-specific phospholipase-C? (PLC?) and protein kinase-C (PKC) signaling in myelin phagocytosis mediated by CR3\\/MAC-1 alone and by CR3\\/MAC-1 combined with SRAI\\/II. Phagocytosis was inhibited by

Chen Makranz; Goni Cohen; Ayellet Baron; Lital Levidor; Tatsuhiko Kodama; Fanny Reichert; Shlomo Rotshenker



Rapid light-induced changes in phosphoinositide kinases and H(+)-ATPase in plasma membrane of sunflower hypocotyls.  


Irradiation of sunflower (Helianthus annuus L. cv. Russian Mammoth) hypocotyls with white light resulted in a 51% decrease in plasma membrane phosphatidylinositol monophosphate (PIP) kinase activity. As little as 10 s of white light irradiation was sufficient to lower the phosphatidylinositol bisphosphate (PIP2) produced in the in vitro phosphorylation assay. This decrease was not caused by an increase in phospholipase C activity since analysis of the water-soluble products indicated no increase in inositol bisphosphate or inositol trisphosphate. Treatment of the plasma membrane with 200 microM vanadate prior to phosphorylation enhanced the PIP kinase and appeared to overcome the light inhibition. In addition to decreasing the PIP kinase activity, light irradiation resulted in a corresponding decrease in the H(+)-ATPase activity to 53% of the dark control values. The plasma membrane ATPase activity increased approximately 2-fold when PIP or PIP2 was added to the isolated membranes. Thus, effects of external stimuli on the level of plasma membrane PIP or PIP2 could affect plasma membrane ATPase activity directly and thereby provide an alternative mechanism for control of cell growth. PMID:2168392

Memon, A R; Boss, W F



Phosphoinositide [PI(3,5)P2] lipid-dependent regulation of the general transcriptional regulator Tup1  

PubMed Central

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)P2. We identified Tup1 and Cti6 as new, highly specific PI(3,5)P2 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)P2 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)P2 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.

Han, Bong-Kwan; Emr, Scott D.



Cross-talk of phosphoinositide- and cyclic nucleotide-dependent signaling pathways in differentiating avian nasal gland cells.  


In many bird species, the nasal glands secrete excess salt ingested with drinking water or food. In ducks ( Anas platyrhynchos), osmotic stress results in adaptive cell proliferation and differentiation in the gland. Using 'naive' nasal gland cells isolated from animals that had never ingested excess salt or 'differentiated' cells from animals fed with a 1% NaCl solution for 48 h, we investigated the allocation of metabolic energy to salt excretory processes and to other cellular activities. Activation of muscarinic acetylcholine receptors (carbachol) or beta-adrenergic receptors (isoproterenol) in nasal gland cells resulted in a transient peak in metabolic rate followed by an elevated plateau level that was maintained throughout the activation period. Activation of cells using vasoactive intestinal peptide, however, had only marginal effects on metabolic rate. In differentiated cells, sequential stimulation with carbachol and isoproterenol resulted in additive changes in metabolic rate during the plateau phase. Naive cells, however, developed supra-additive plateau levels in metabolic rates indicating cross-talk of both signaling pathways. Using bumetanide, TEA or barium ions to block different components of the ion transport machinery necessary for salt secretion, the relative proportion of energy needed for processes related to ion transport or other cellular processes was determined. While differentiated cells in the activated state allocated virtually all metabolic energy to processes related to salt secretion, naive cells reserved a significant amount of energy for other processes, possibly sustaining cellular signaling and regulating biosynthetic mechanisms related to adaptive growth and differentiation. PMID:15185116

Krohn, M; Hildebrandt, J-P



Studies of inositol analogues as inhibitors of the phosphoinositide pathway, and incorporation of 2-deoxy-2-fluoro-myo-inositol to give analogues of phosphatidylinositol intermediates.  


The incorporation of [3H]Ins into PtdIns by exchange of free and lipid-bound inositol moieties occurs via the action of at least two types of Mg2+/Mn(2+)-dependent enzymes in turkey erythrocytes. One is a nucleotide-independent PtdIns/Ins exchange enzyme and its function is, as yet, unknown, whereas the other is CMP-dependent and appears to be an exchange reaction catalysed by PtdIns synthase. The effects of analogues with modifications of the substituent at the 1-, 2-, 3-, 4- and 5-positions on the incorporation of [3H]Ins into PtdIns under both synthase and exchange reaction conditions were investigated in turkey erythrocytes. Analogues causing substantial inhibition of [3H]Ins incorporation were then used in kinetic experiments to determine the type of inhibition involved. The analogues 1-deoxy-1-fluoro-scyllo-inositol and 5-O-methyl-myo-inositol exhibited the greatest effects on the incorporation of [3H]Ins via both the synthase and exchange reactions, and the kinetic analysis indicated that they were competitive inhibitors of Ins. Ki values of 0.37 mM and 2.87 mM were observed for 1-deoxy-1-fluoro-scyllo-inositol under exchange and synthase reaction conditions respectively; similar Ki values of 0.26 mM and 2.80 mM were observed for 5-O-methyl-myo-inositol in the exchange and synthase reactions respectively. The ability of 1-deoxy-1-fluoro-scyllo-inositol and its diastereoisomer, 2-deoxy-2-fluoro-myo-inositol, to act as substrates for the synthase and exchange reactions in turkey erythrocytes was investigated. The radiolabelled derivative of the former analogue was not incorporated into phospholipids, whereas the radiolabelled derivative of the latter analogue was a poor substrate for the synthase and exchange enzymes. In the presence of ATP, the labelled analogue of PtdIns, derived from 2-deoxy-2-fluoro-myo-[2-3H]inositol, appeared to be converted into phosphorylated PtdIns analogues, presumably by the enzymes of the phosphoinositide pathway. PMID:1650182

McPhee, F; Downes, C P; Lowe, G



Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of plcdelta1.  


Structural studies of phospholipase C delta1 (PLCdelta1) in complexes with the inositol-lipid headgroup and calcium identified residues within the catalytic domain that could be involved in substrate recognition, calcium binding, and catalysis. In addition, the structure of the PLCdelta1 catalytic domain revealed a cluster of hydrophobic residues at the rim of the active site opening (hydrophobic ridge). To assess a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His311, Asn312, Glu341, Asp343, His356, Glu390, Lys438, Lys440, Ser522, Arg549, and Tyr551) and residues from the hydrophobic ridge (Leu320, Phe360, and Trp555). The replacements of most active site residues by alanine resulted in a great reduction (1,000-200,000-fold) of PLC activity analyzed in an inositol lipid/sodium cholate mixed micelle assay. Measurements of the enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4, 5-bis-phosphate (PIP2) identified Ser522, Lys438, and Arg549 as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys440 selectively affected only hydrolysis of PIP2. When PLC activity was analyzed at different calcium concentrations, substitutions of Asn312, Glu390, Glu341, and Asp343 resulted in a shift toward higher calcium concentrations required for PIP2 hydrolysis, suggesting that all these residues contribute toward Ca2+ binding. Mutational analysis also confirmed the importance of His311 ( approximately 20,000-fold reduction) and His356 ( approximately 6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP2 hydrolysis in the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mutational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca2+ ion, catalysis, and surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs. PMID:9565585

Ellis, M V; James, S R; Perisic, O; Downes, C P; Williams, R L; Katan, M



Lysophosphatidic acid-mediated Ca2+ mobilization in human SH-SY5Y neuroblastoma cells is independent of phosphoinositide signalling, but dependent on sphingosine kinase activation.  

PubMed Central

Extracellular application of lysophosphatidic acid (LPA) elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) in human SH-SY5Y neuroblastoma cells. The maximal response to LPA occurred between 0. 1 and 1 microM, at which point [Ca(2+)](i) was increased by approx. 500 nM. This increase was of similar magnitude to that caused by the muscarinic acetylcholine receptor agonist methacholine (MCh), although the initial rate of release by LPA was slower. Both LPA and MCh released Ca(2+) from intracellular stores, as assessed by inhibition of their effects by thapsigargin, a blocker of endoplasmic reticular Ca(2+) uptake, and by the persistence of their action in nominally Ca(2+)-free extracellular medium. Similarly, both agonists appeared to stimulate store-refilling Ca(2+) entry. MCh produced a marked elevation in cellular Ins(1,4,5)P(3) and stimulated [(3)H]InsP accumulation in the presence of Li(+). In contrast, LPA failed to stimulate detectable phosphoinositide turnover. Chronic down-regulation of Ins(1,4,5)P(3) receptor (InsP(3)R) proteins with MCh did not affect Ca(2+) responses to LPA. In addition, heparin, a competitive antagonist of InsP(3)Rs, blocked Ca(2+)-mobilization in permeabilized SH-SY5Y cells in response to MCh or exogenously added Ins(1,4,5)P(3), but failed to inhibit Ca(2+)-release induced by LPA. Elevation of [Ca(2+)](i) elicited by LPA was blocked by guanosine 5'-[beta-thio]-diphosphate, indicating that this agonist acts via a G-protein-coupled receptor. However, pertussis toxin was without effect on LPA-evoked [Ca(2+)](i) responses, suggesting that G(i/o)-proteins were not involved. In the absence of extracellular Ca(2+), N,N-dimethylsphingosine (DMS, 30 microM), a competitive inhibitor of sphingosine kinase, blocked LPA-induced Ca(2+) responses by almost 90%. In addition, MCh-induced Ca(2+) responses were also diminished by the addition of DMS, although to a lesser extent than with LPA. We conclude that LPA mobilizes intracellular Ca(2+)-stores in SH-SY5Y cells independently of the generation and action of Ins(1,4,5)P(3). Furthermore, the Ca(2+)-response to LPA appears to be dependent on sphingosine kinase activation and the potential generation of the putative second messenger sphingosine 1-phosphate.

Young, K W; Challiss, R A; Nahorski, S R; MacKrill, J J



The essential phosphoinositide kinase MSS-4 is required for polar hyphal morphogenesis, localizing to sites of growth and cell fusion in Neurospora crassa.  


Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P(2). In N. crassa hyphae, PtdIns(4,5)P(2) localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P(2) that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms. PMID:23272106

Mähs, Anette; Ischebeck, Till; Heilig, Yvonne; Stenzel, Irene; Hempel, Franziska; Seiler, Stephan; Heilmann, Ingo



Glucagon-like peptide-1 stimulates insulin secretion but not phosphoinositide hydrolysis from islets desensitized by prior exposure to high glucose or the muscarinic agonist carbachol.  


In the present series of experiments, the ability of the postulated incretin factor, glucagon-like peptide-1 (GLP-1), to stimulate insulin release from desensitized islets was determined. Compared with responses observed from control islets incubated for 3.5 hours with 5.6 mmol/L glucose alone, prior exposure to 10 mmol/L glucose, 20 mmol/L glucose, or 10 micromol/L carbachol reduced peak second-phase insulin release rates to a subsequent 20-mmol/L glucose stimulus by 63%, 81%, or 70%, respectively. Efflux of 3H-inositol from prior high-glucose- or carbachol-exposed islets was abolished and accumulation of inositol phosphates (IPs) in response to 20 mmol/L glucose was reduced. Further addition of 10 nmol/L GLP-1 together with 20 mmol/L glucose significantly increased insulin output from desensitized islets. Carbachol (10 micromol/L) preexposure also abolished the subsequent insulin secretory and 3H-inositol efflux responses to 8 mmol/L glucose plus 10 micromol/L carbachol. Inclusion of 10 nmol/L GLP-1 together with 8 mmol/L glucose plus 10 micromol/L carbachol improved but did not normalize secretion from these islets. These improvements in secretory responsiveness from high-glucose- or carbachol- desensitized islets occurred despite the lack of any apparent restorative effect of GLP-1 on agonist-induced increases in phosphoinositide (PI) hydrolysis. Finally, unlike the situation observed with carbachol or high-glucose preexposure, chronic exposure of islets to GLP-1 (100 nmol/L) did not desensitized islets to a subsequent 20 mmol/L glucose stimulus. We conclude from these studies that the incretin factor GLP-1 may play an important role in maintaining insulin output from islets in which phospholipase C (PLC)-mediated hydrolysis of islet PI pools in impaired. GLP-1 may prevent a further decline in beta-cell function and the associated deterioration in glucose tolerance that accompanies chronic exposure of islets to one of several agonists, including high glucose. PMID:8596502

Zawalich, W S; Zawalich, K C



Biochemical mechanisms involved in monomethyl succinate-induced insulin secretion.  


Esters of succinic acid stimulate insulin secretion from pancreatic beta-cells. Using collagenase-isolated rat islets, the transduction mechanisms involved were investigated. In freshly isolated perifused islets, monomethyl succinate (MMSucc), in the presence of basal (2.75 mM) glucose, stimulated insulin release in a biphasic pattern. This secretory response was dependent on extracellular calcium movement into the beta-cell, since the calcium channel blocker nitrendipine (5 microM) abolished it. The glucokinase inhibitor mannoheptulose (20 mM) had no effect on its secretory action, while the protein kinase-C inhibitor staurosporine (20 nM) reduced secretion to MMSucc. In addition, while ineffective alone, the diacylglycerol kinase inhibitor monooleoylglycerol (25 microM) potentiated MMSucc-induced insulin release. A similarly amplified response occurred in the presence of forskolin (0.25 microM), a compound that elevates islet cAMP levels. The sodium salt of succinic acid (20 mM) had no effect on insulin release in the presence or absence of forskolin. Prior treatment with MMSucc in the presence of 2.75 mM glucose sensitized islets to the usually weak insulin secretory effect of 7.5 mM glucose. Other groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide pools. These islets were subsequently stimulated, and the kinetics of [3H]inositol efflux and insulin secretion were measured. MMSucc induced a rapid and sustained dose-dependent increase in [3H]inositol efflux rates. In batch-incubated islets, MMSucc increased inositol phosphate levels. Finally, MMSucc (20 mM), in the presence of 8 mM glucose, did not influence the detritiation of [5-3H]glucose, but reduced the oxidation of [U-14C] glucose. These results support the following conclusions. First, MMSucc is a potent activator of islet phosphoinositide hydrolysis. Second, the activation of protein kinase-C appears to contribute to the acute insulin secretory effect of MMSucc. Third, MMSucc-induced increases in phosphoinositide hydrolysis contribute at least in part to its ability to acutely stimulate insulin release and prime the beta-cell to subsequent stimulation. Finally, mitochondrial events associated with the oxidative metabolism of MMSucc may underlie its insulinotropic action. PMID:1322278

Zawalich, W S; Zawalich, K C



Metabolic activation of Ca(2+)-independent phosphoinositide hydrolysis in beta-cells and its role in the control of insulin secretion.  


Recent studies have led to the proposal that the oxidative metabolism of glucose leads to the generation of messengers, in addition to ATP, that are important in the ability of changes in extracellular glucose concentration to stimulate insulin secretion from pancreatic beta-cells. In particular, there is now evidence that glucose induces both a Ca(2+)-dependent and Ca(2+)-independent increase in phosphoinositide (PI) hydrolysis. To explore the relationship between oxidative metabolism and PI hydrolysis, we examined the effect of low concentrations (2.5 mM) of alpha-ketoisocaproate (KIC) and monomethylsuccinate (MMSucc) either alone or in combination on insulin secretion and PI hydrolysis in isolated rat pancreatic islets incubated with either no glucose, 5 mM glucose, or 20 mM glucose. A combination of KIC and MMSucc leads to a marked increase in largely (80%) Ca(2+)-independent PI hydrolysis in either the absence or presence of 5 mM glucose. When glucose is absent, this combination of substrates induces a very small and transient first phase of insulin secretion but no significant second phase of secretion. In the presence of 5 mM glucose, either KIC or MMSucc alone induces a first phase of insulin secretion with a peak secretory rate 10-fold greater than the basal rate but only a small second phase of secretion approximately 5-fold above control. However, in the presence of 5 mM glucose, the combination of KIC plus MMSucc induces a large biphasic increase in insulin secretion: peak first-phase secretion is increased 30-fold, and second-phase 40-fold. These response are comparable to those induced by 20 mM glucose and are completely inhibited by 0.5 microM nitrendipine. In contrast, KIC plus MMSucc do not enhance the insulin secretory response induced by 20 mM glucose. Previous data showed that when 20 mM glucose acts, the resulting increase in PI hydrolysis is only partially Ca2+ dependent. A reanalysis of these data shows that raising the glucose concentration from 5 to 7 mM causes a 2-fold increase in Ca(2+)-independent PI hydrolysis, and a further increase to 20 mM leads to a further 2-fold increase in Ca(2+)-dependent PI hydrolysis. These data show that these two pathways are regulated by different ranges of glucose concentration. They raise the interesting possibility that these distinct pathways have different signaling functions. In particular, raising the glucose concentration from 5 to 7 mM is known to alter the responsiveness of beta-cells to a variety of neurohumoral agonists and to tolbutamide.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7835291

Rasmussen, H; Zawalich, K C; Zawalich, W S



Phosphoinositide 3-kinase targeting by the ? galactoside binding protein cytokine negates akt gene expression and leads aggressive breast cancer cells to apoptotic death  

PubMed Central

Introduction Phosphoinositide 3-kinase (PI3K)-activated signalling has a critical role in the evolution of aggressive tumourigenesis and is therefore a prime target for anticancer therapy. Previously we have shown that the ? galactoside binding protein (?GBP) cytokine, an antiproliferative molecule, induces functional inhibition of class 1A and class 1B PI3K. Here, we have investigated whether, by targeting PI3K, ?GBP has therapeutic efficacy in aggressive breast cancer cells where strong mitogenic input is fuelled by overexpression of the ErbB2 (also known as HER/neu, for human epidermal growth factor receptor 2) oncoprotein receptor and have used immortalised ductal cells and non-aggressive mammary cancer cells, which express ErbB2 at low levels, as controls. Methods Aggressive BT474 and SKBR3 cancer cells where ErbB2 is overexpressed, MCF10A immortalised ductal cells and non-invasive MCF-7 cancer cells which express low levels of ErbB2, both in their naive state and when forced to mimic aggressive behaviour, were used. Class IA PI3K was immunoprecipitated and the conversion of phosphatidylinositol (4,5)-biphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3) assessed by ELISA. The consequences of PI3K inhibition by ?GBP were analysed at proliferation level, by extracellular signal-regulated kinase (ERK) activation, by akt gene expression and by apoptosis. Apoptosis was documented by changes in mitochondrial membrane potential, alteration of the plasma membrane, caspase 3 activation and DNA fragmentation. Phosphorylated and total ERK were measured by Western blot analysis and akt mRNA levels by Northern blot analysis. The results obtained with the BT474 and SKBR3 cells were validated in the MCF10A ductal cells and in non-invasive MCF-7 breast cancer cells forced into mimicking the in vitro behaviour of the BT474 and SKBR3 cells. Results In aggressive breast cancer cells, where mitogenic signalling is enforced by the ErbB2 oncoprotein receptor, functional inhibition of the catalytic activity of PI3K by the ?GBP cytokine and loss of akt mRNA results in apoptotic death. A functional correlation between ERK and the kt gene was also found. The relationship between ERK, akt mRNA, PI3K and cell vulnerability to ?GBP challenge was sustained both in mammary ductal cells forced to mimic an aggressive behaviour and in non-aggressive breast cancer cells undergoing an enforced shift into an aggressive phenotype. Conclusions ?GBP, a newly discovered physiological inhibitor of PI3K, is a selective and potent inducer of apoptosis in aggressive breast cancer cells. Due to its physiological nature, which carries no chemotherapeutic disadvantages, ?GBP has the potential to be safely tested in clinical trials.

Wells, Valerie; Mallucci, Livio



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.  


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 0-100ng/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 24h, 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. PMID:23261989

Higashi, Yusuke; Pandey, Arvind; Goodwin, Brett; Delafontaine, Patrice



Oleanolic acid induces relaxation and calcium-independent release of endothelium-derived nitric oxide  

PubMed Central

Background and purpose: The present study investigated the mechanisms by which oleanolic acid, a component of olive oil, increases release of nitric oxide (NO). Experimental approach: Measurements of isometric tension, NO concentration, or endothelial cell calcium were made in rat isolated mesenteric arteries. Immunoblotting for endothelial NOS (eNOS) and Akt kinase were performed in primary cultures of human umbilical vein endothelial cells (HUVECs). Key results: Oleanolic acid (3–30??M) evoked endothelium-dependent relaxations in noradrenaline-contracted rat superior and small mesenteric arteries. In rat superior mesenteric arteries, oleanolic acid induced simultaneous increases in NO concentration and relaxation, and these responses were inhibited by an inhibitor of NOS, asymmetric dimethyl-L-arginine (300??M) and by the NO scavenger, oxyhaemoglobin (10??M). Oleanolic acid-evoked NO increases were not reduced in Ca2+-free solution and in the presence of an inhibitor of endoplasmic reticulum calcium-ATPase, thapsigargin (1??M). Oleanolic acid evoked relaxation without changes in endothelial cell calcium, but decreased smooth muscle calcium in arterial segments. Oleanolic acid failed to increase calcium in HUVECs, but increased time-dependently phosphorylation of Akt kinase at Serine473 (Akt-Ser473) and eNOS at Serine1177 (eNOS-Ser1177), which was attenuated by inhibitors of phosphoinositide-3-kinase. Conclusions and implications: This study provides direct evidence that a component of olive oil, oleanolic acid, activated endothelium-dependent release of NO and decreased smooth muscle cell calcium followed by relaxation. The oleanolic acid-evoked endothelium-derived NO release was independent of endothelial cell calcium and involved phosphoinositide-3-kinase-dependent phosphorylation of Akt-Ser473 followed by phosphorylation of eNOS-Ser1177.

Rodriguez-Rodriguez, R; Stankevicius, E; Herrera, M D; ?stergaard, L; Andersen, M R; Ruiz-Gutierrez, V; Simonsen, U



Quantitative elucidation of a distinct spatial gradient-sensing mechanism in fibroblasts  

PubMed Central

Migration of eukaryotic cells toward a chemoattractant often relies on their ability to distinguish receptor-mediated signaling at different subcellular locations, a phenomenon known as spatial sensing. A prominent example that is seen during wound healing is fibroblast migration in platelet-derived growth factor (PDGF) gradients. As in the well-characterized chemotactic cells Dictyostelium discoideum and neutrophils, signaling to the cytoskeleton via the phosphoinositide 3-kinase pathway in fibroblasts is spatially polarized by a PDGF gradient; however, the sensitivity of this process and how it is regulated are unknown. Through a quantitative analysis of mathematical models and live cell total internal reflection fluorescence microscopy experiments, we demonstrate that PDGF detection is governed by mechanisms that are fundamentally different from those in D. discoideum and neutrophils. Robust PDGF sensing requires steeper gradients and a much narrower range of absolute chemoattractant concentration, which is consistent with a simpler system lacking the feedback loops that yield signal amplification and adaptation in amoeboid cells.

Schneider, Ian C.; Haugh, Jason M.



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



Molecular mechanism of membrane targeting by the GRP1 PH domain*  

PubMed Central

The general receptor for phosphoinositides isoform 1 (GRP1) is recruited to the plasma membrane in response to activation of phosphoinositide 3-kinases and accumulation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]. GRP1's pleckstrin homology (PH) domain recognizes PtdIns(3,4,5)P3 with high specificity and affinity, however, the precise mechanism of its association with membranes remains unclear. Here, we detail the molecular basis of membrane anchoring by the GRP1 PH domain. Our data reveal a multivalent membrane docking involving PtdIns(3,4,5)P3 binding, regulated by pH and facilitated by electrostatic interactions with other anionic lipids. The specific recognition of PtdIns(3,4,5)P3 triggers insertion of the GRP1 PH domain into membranes. An acidic environment enhances PtdIns(3,4,5)P3 binding and increases membrane penetration as demonstrated by NMR and monolayer surface tension and surface plasmon resonance experiments. The GRP1 PH domain displays a 28 nM affinity for POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/PtdIns(3,4,5)P3 vesicles at pH 6.0, but binds 22-fold weaker at pH 8.0. The pH sensitivity is attributed in part to the His355 residue, protonation of which is required for the robust interaction with PtdIns(3,4,5)P3 and significant membrane penetration, as illustrated by mutagenesis data. The binding affinity of the GRP1 PH domain for PtdIns(3,4,5)P3-containing vesicles is further amplified (by ?6-fold) by nonspecific electrostatic interactions with phosphatidylserine/phosphatidylinositol. Together, our results provide new insight into the multivalent mechanism of the membrane targeting and regulation of the GRP1 PH domain.

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



Seizure Mechanisms  


Seizure Mechanisms You are here: Home About Epilepsy What is Epilepsy The Brain Seizure Mechanisms Email Print Twitter Facebook MySpace Delicous Stumble Digg More... [ Seizure Mechanisms | Seizure Threshold | Brain Structure | Brain Hemispheres | Brain ...


Gallium: mechanisms  

SciTech Connect

The mechanisms of /sup 67/Ga localization in the body are discussed. Included in the discussion are: general biological considerations; specific mechanisms of uptake; and mechanisms of localization in tumors. (ERB)

Hoffer, P.



Stem cell factor/c-kit up-regulates cyclin D3 and promotes cell cycle progression via the phosphoinositide 3-kinase/p70 S6 kinase pathway in spermatogonia.  


Stem cell factor (SCF)/c-kit plays an important role in the regulation of hematopoiesis, melanogenesis, and spermatogenesis. In the testis, the SCF/c-kit system is believed to regulate germ cell proliferation, meiosis, and apoptosis. Studies with type A spermatogonia in vivo and in vitro have indicated that SCF induces DNA synthesis and proliferation. However, the signaling pathway for this function of SCF/c-kit has not been elucidated. We now demonstrate that SCF activates phosphoinositide 3-kinase (PI3-K) and p70 S6 kinase (p70S6K) and that rapamycin, a FRAP/mammalian target of rapamycin-dependent inhibitor of p70S6K, completely inhibited bromodeoxyuridine incorporation induced by SCF in primary cultures of spermatogonia. SCF induced cyclin D3 expression and phosphorylation of the retinoblastoma protein through a pathway that is sensitive to both wortmannin and rapamycin. Furthermore, AKT, but not protein kinase C-zeta, is used by SCF/c-kit/PI3-K to activate p70S6K. Dominant negative AKT-K179M completely abolished p70S6K phosphorylation induced by the constitutively active PI3-K catalytic subunit p110. Constitutively active v-AKT highly phosphorylated p70S6K, which was totally inhibited by rapamycin. Thus, SCF/c-kit uses a rapamycin-sensitive PI3-K/AKT/p70S6K/cyclin D3 pathway to promote spermatogonial cell proliferation. PMID:10849422

Feng, L X; Ravindranath, N; Dym, M



MicroRNA-7-regulated TLR9 signaling-enhanced growth and metastatic potential of human lung cancer cells by altering the phosphoinositide-3-kinase, regulatory subunit 3/Akt pathway  

PubMed Central

Recent evidence shows that microRNAs (miRNAs) contribute to the biological effects of Toll-like receptor (TLR) signaling on various cells. Our previous data showed that TLR9 signaling could enhance the growth and metastatic potential of human lung cancer cells. However, the potential role of miRNAs in the effects of TLR9 signaling on tumor biology remains unknown. In this paper, we first report that TLR9 signaling could reduce intrinsic miR-7 expression in human lung cancer cells. Furthermore, overexpression of miR-7 can significantly inhibit TLR9 signaling–enhanced growth and metastatic potential of lung cancer cells in vitro and in vivo. Notably, we identify phosphoinositide-3-kinase, regulatory subunit 3 (PIK3R3) as a novel target molecule of miR-7 in lung cancer cells by Western blotting and luciferase report assay. Further study shows that miR-7 inhibits the effects of TLR9 signaling on lung cancer cells through regulation of the PIK3R3/Akt pathway. These data suggest that miR-7 could act as a fine-tuner in regulating the biological effects of TLR9 signaling on human lung cancer cells, which might be helpful to the understanding of the potential role of miRNAs in TLR signaling effects on tumor biology.

Xu, Lin; Wen, Zhenke; Zhou, Ya; Liu, Zhongmin; Li, Qinchuan; Fei, Guangru; Luo, Junmin; Ren, Tao



MicroRNA-7-regulated TLR9 signaling-enhanced growth and metastatic potential of human lung cancer cells by altering the phosphoinositide-3-kinase, regulatory subunit 3/Akt pathway.  


Recent evidence shows that microRNAs (miRNAs) contribute to the biological effects of Toll-like receptor (TLR) signaling on various cells. Our previous data showed that TLR9 signaling could enhance the growth and metastatic potential of human lung cancer cells. However, the potential role of miRNAs in the effects of TLR9 signaling on tumor biology remains unknown. In this paper, we first report that TLR9 signaling could reduce intrinsic miR-7 expression in human lung cancer cells. Furthermore, overexpression of miR-7 can significantly inhibit TLR9 signaling-enhanced growth and metastatic potential of lung cancer cells in vitro and in vivo. Notably, we identify phosphoinositide-3-kinase, regulatory subunit 3 (PIK3R3) as a novel target molecule of miR-7 in lung cancer cells by Western blotting and luciferase report assay. Further study shows that miR-7 inhibits the effects of TLR9 signaling on lung cancer cells through regulation of the PIK3R3/Akt pathway. These data suggest that miR-7 could act as a fine-tuner in regulating the biological effects of TLR9 signaling on human lung cancer cells, which might be helpful to the understanding of the potential role of miRNAs in TLR signaling effects on tumor biology. PMID:23135998

Xu, Lin; Wen, Zhenke; Zhou, Ya; Liu, Zhongmin; Li, Qinchuan; Fei, Guangru; Luo, Junmin; Ren, Tao



Cocoa polyphenols suppress TNF-?-induced vascular endothelial growth factor expression by inhibiting phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase kinase-1 (MEK1) activities in mouse epidermal cells.  


Cocoa polyphenols have antioxidant and anti-inflammatory effects. TNF-? is a pro-inflammatory cytokine that has a vital role in the pathogenesis of inflammatory diseases such as cancer and psoriasis. Vascular endothelial growth factor (VEGF) expression is associated with tumorigenesis, CVD, rheumatoid arthritis and psoriasis. We tested whether cocoa polyphenol extract (CPE) inhibited TNF-?-induced VEGF expression in promotion-sensitive JB6 mouse epidermal cells. CPE significantly inhibited TNF-?-induced up-regulation of VEGF via reducing TNF-?-induced activation of the nuclear transcription factors activator protein-1 (AP-1) and NF-?B, which are key regulators of VEGF expression. CPE also inhibited TNF-?-induced phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinase. CPE blocked activation of their downstream kinases, p70 kDa ribosomal protein S6 kinase and p90 kDa ribosomal protein S6 kinase. CPE suppressed phosphoinositide 3-kinase (PI3K) activity via binding PI3K directly. CPE did not affect TNF-?-induced phosphorylation of mitogen-activated protein kinase kinase-1 (MEK1) but suppressed TNF-?-induced MEK1 activity. Collectively, these results indicate that CPE reduced TNF-?-induced up-regulation of VEGF by directly inhibiting PI3K and MEK1 activities, which may contribute to its chemopreventive potential. PMID:20550744

Kim, Jong-Eun; Son, Joe Eun; Jung, Sung Keun; Kang, Nam Joo; Lee, Chang Yong; Lee, Ki Won; Lee, Hyong Joo



Wnt1 Inducible Signaling Pathway Protein 1 (WISP1) Blocks Neurodegeneration through Phosphoinositide 3 Kinase/Akt1 and Apoptotic Mitochondrial Signaling Involving Bad, Bax, Bim, and Bcl-xL  

PubMed Central

Wnt1 inducible signaling pathway protein 1 (WISP1) is a member of the CCN family of proteins that determine cell growth, cell differentiation, immune system activation, and cell survival in tissues ranging from the cardiovascular-pulmonary system to the reproductive system. Yet, little is known of the role of WISP1 as a neuroprotective entity in the nervous system. Here we demonstrate that WISP1 is present in primary hippocampal neurons during oxidant stress with oxygen-glucose deprivation (OGD). WISP1 expression is significantly enhanced during OGD exposure by the cysteine-rich glycosylated protein Wnt1. Similar to the neuroprotective capabilities known for Wnt1 and its signaling pathways, WISP1 averts neuronal cell injury and apoptotic degeneration during oxidative stress exposure. WISP1 requires activation of phosphoinositide 3-kinase (PI 3-K) and Akt1 pathways to promote neuronal cell survival, since blockade of these pathways abrogates cellular protection. Furthermore, WISP1 through PI 3-K and Akt1 phosphorylates Bad and GSK-3?, minimizes expression of the Bim/Bax complex while increasing the expression of Bcl-xL/Bax complex, and prevents mitochondrial membrane permeability, cytochrome c release, and caspase 3 activation in the presence of oxidant stress. These studies provide novel considerations for the development of WISP1 as an effective and robust therapeutic target not only for neurodegenerative disorders, but also for disease entities throughout the body.

Wang, Shaohui; Chong, Zhao Zhong; Shang, Yan Chen; Maiese, Kenneth



Insulin stimulation of glycogen synthesis and glycogen synthase activity is blocked by wortmannin and rapamycin in 3T3-L1 adipocytes: evidence for the involvement of phosphoinositide 3-kinase and p70 ribosomal protein-S6 kinase.  

PubMed Central

We have investigated the involvement of phosphoinositide (PI) 3-kinase and p70 ribosomal protein-S6 kinase (p70s6k) in mediating insulin stimulation of glycogen synthesis in 3T3-L1 adipocytes using specific inhibitors. Wortmannin inhibited PI 3-kinase activity (IC50 approximately 10 nM), inhibition being complete at 100 nm. Wortmannin (100 nM) completely blocked the ability of insulin to activate glycogen synthase in 3T3-L1 adipocytes and the ability of insulin to stimulate glucose incorporation into glycogen in 3T3-L1 fibroblasts. Rapamycin, which blocks insulin-stimulated activation of p70s6k, decreased insulin activation of glycogen synthase in a dose-dependent manner (IC50 approximately 0.8 ng/ml), with a maximum approx. 75% inhibition of insulin's stimulatory effect. Rapamycin inhibited insulin-stimulated glucose incorporation into glycogen to a similar extent and with similar dose-dependency, while having no effect on insulin-stimulated glucose transport. We conclude that PI 3-kinase and p70s6k are involved in the signalling pathways by which insulin stimulates glycogen synthase in 3T3-L1 adipocytes.

Shepherd, P R; Nave, B T; Siddle, K



Chemokine receptor 7 activates phosphoinositide-3 kinase-mediated invasive and prosurvival pathways in head and neck cancer cells independent of EGFR  

Microsoft Academic Search

Chemokine receptor 7 (CCR7) upregulation, which mediates immune cell survival and migration to lymph nodes, has recently been associated with nodal metastasis of squamous cell carcinoma of the head and neck (SCCHN). However, the mechanism of CCR7 in tumor progression, its downstream signaling mediators, and interactions with other pathways contributing to metastasis of SCCHN have not been determined. We hypothesized

Jun Wang; Xin Zhang; Sufi M Thomas; Jennifer R Grandis; Alan Wells; Robert L Ferris



Computational mechanics.  

National Technical Information Service (NTIS)

The Computational Mechanics thrust area sponsors research into the underlying solid, structural and fluid mechanics and heat transfer necessary for the development of state-of-the-art general purpose computational software. The scale of computational capa...

G. L. Goudreau



Classical Mechanics  

NSDL National Science Digital Library

This textbook, a standard in advanced classical mechanics, covers the breadth of the subject. This latest edition contains sections in contemporary classical mechanics, applications, and computer and mathematical techniques for solving systems.

Goldstein, Herbert; Poole Jr., Charles P.; Safko, John L.



Role of X-linked inhibitor of apoptosis protein in chemoresistance in ovarian cancer: possible involvement of the phosphoinositide-3 kinase\\/Akt pathway  

Microsoft Academic Search

Although cisplatin derivatives are first-line chemotherapeutic agents for the treatment of epithelial ovarian cancer, chemoresistance remains a major hurdle to successful therapy and the molecular mechanisms involved are poorly understood. Apoptosis is the cellular underpinning of cisplatin-induced cell death, which is associated with expression of specific “death” genes and down-regulation of “survival” counterparts. The X-linked inhibitor of apoptosis proteins (Xiap),

Jin Q Cheng; Xiuxian Jiang; Michael Fraser; Ming Li; Han C Dan; Mei Sun; Benjamin K Tsang



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)



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



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



Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110alpha and p110beta catalytic subunits of class-Ia phosphoinositide 3-kinases.  

PubMed Central

Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110alpha and p110beta isoforms. The lipid-kinase activity did not display Michaelis-Menten kinetics but modelling the kinetic data demonstrated that p110alpha has a higher V(max) and a 25-fold higher K(m) for PtdIns than p110beta. A similar situation occurs with PtdIns(4,5)P(2), because at low concentration of PtdIns(4,5)P(2) p110beta is a better PtdIns(4,5)P(2) kinase than p110alpha, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110beta functions better in areas of membranes containing low levels of substrate whereas p110alpha would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110beta phosphorylated p85 to a lower degree than did p110alpha. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110alpha and p110beta. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110alpha has a higher K(m) (550 microM) than p110beta (K(m) 8 microgM). Similarly, the relative V(max) towards peptide substrate of p110alpha was three times higher than that of p110beta. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110alpha and p110beta in vivo.

Beeton, C A; Chance, E M; Foukas, L C; Shepherd, P R



Endothelin-1 Promotes Myofibroblast Induction through the ETA Receptor via a rac/Phosphoinositide 3-Kinase/Akt-dependent Pathway and Is Essential for the Enhanced Contractile Phenotype of Fibrotic Fibroblasts  

PubMed Central

The endothelins are a family of endothelium-derived peptides that possess a variety of functions, including vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and promotes myofibroblast contraction and migration, hence contributing to matrix remodeling during tissue repair. Here, we show that addition of ET-1 to normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including ?-smooth muscle actin (?-SMA), ezrin, moesin, and paxillin. We confirm that ET-1 enhances the ability of lung fibroblasts to contract extracellular matrix, a function essential for tissue repair, through induction of de novo protein synthesis. Blockade of the Akt/phosphoinositide 3-kinase (PI3-kinase) pathway with LY294002 and wortmannin prevents the ability of ET-1 to induce ?-SMA, ezrin, paxillin, and moesin and to promote matrix contraction. Dominant negative rac and Akt blocked the ability of ET-1 to promote formation of ?-SMA stress fibers. Using specific ET-1 receptor inhibitors, we show that ET-1 induces collagen matrix contraction through the ETA, but not the ETB, receptor. Relative to normal pulmonary fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease systemic sclerosis (scleroderma) show enhanced ET-1 expression and binding. Systemic sclerosis lung fibroblasts show increased ability to contract a collagen matrix and elevated expression of the procontractile proteins ?-SMA, ezrin, paxillin, and moesin, which are greatly reduced by antagonizing endogenous ET-1 signaling. Thus, blocking ET-1 or the PI3-kinase/Akt cascades might be beneficial in reducing scar formation in pulmonary fibrosis.

Shi-Wen, Xu; Chen, Yunliang; Denton, Christopher P.; Eastwood, Mark; Renzoni, Elisabetta A.; Bou-Gharios, George; Pearson, Jeremy D.; Dashwood, Michael; du Bois, Roland M.; Black, Carol M.; Leask, Andrew; Abraham, David J



Phosphoinositide-3 Kinase-Rac1-c-Jun NH2-terminal Kinase Signaling Mediates Collagen I-induced Cell Scattering and Up-Regulation of N-Cadherin Expression in Mouse Mammary Epithelial Cells  

PubMed Central

During epithelial-to-mesenchymal transitions (EMTs), cells must change their interactions with one another and with their extracellular matrix in a synchronized manner. To characterize signaling pathways cells use to coordinate these changes, we used NMuMG mammary epithelial cells. We showed that these cells become fibroblastic and scattered, with increased N-cadherin expression when cultured on collagen I. Rac1 and c-Jun NH2-terminal kinase (JNK) were activated when cells were plated on collagen I, and dominant inhibitory Rac1 (RacN17) or inhibition of JNK signaling prevented collagen I–induced morphological changes and N-cadherin up-regulation. Furthermore, inhibiting phosphoinositide-3 kinase (PI3K) activity prevented Rac1 and JNK activation as well as collagen I–induced N-cadherin up-regulation. These data implicate PI3K–Rac1–JNK signaling in collagen I–induced changes in NMuMG cells. To establish a role for N-cadherin in collagen I–induced cell scattering, we generated N-cadherin overexpressing and knockdown NMuMG cells and showed that knocking down N-cadherin expression prevented collagen I–induced morphological changes. Motility assays showed that cells overexpressing N-cadherin were significantly more motile than mock-transfected cells and that N-cadherin-mediated motility was collagen I dependent. In addition, we showed that cord formation and branching in three-dimensional culture (EMT-dependent events) required N-cadherin expression and PI3K–Rac1–JNK signaling.

Shintani, Yasushi; Johnson, Keith R.