Phosphatidylinositol 3,4,5-trisphosphate-dependent stimulation of phospholipase C-gamma2 is an early key event in FcgammaRIIA-mediated activation of human platelets

Platelets express a single class of Fcgamma receptor (FcgammaRIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. FcgammaRIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-gamma2 (PLC-gamma2), and calcium signaling. Here, we show that in response to FcgammaRIIA cross-linking, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns(3,4,5)P3) is rapidly produced, whereas phosphatidylinositol (3,4)-bisphosphate accumulates more slowly, demonstrating a marked activation of phosphoinositide 3-kinase (PI 3-kinase). Inhibition of PI 3-kinase by wortmannin or LY294002 abolished platelet secretion and aggregation, as well as phospholipase C (PLC) activation, indicating a role of this lipid kinase in the early phase of platelet activation. Inhibition of PLCgamma2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin alphaIIb/beta3 engagement. In contrast, the stable association of PLCgamma2 to the membrane/cytoskeleton interface observed at early stage of platelet activation was fully abolished upon inhibition of PI 3-kinase. In addition, PLCgamma2 was able to preferentially interact in vitro with PtdIns(3,4,5)P3. Finally, exogenous PtdIns(3,4,5)P3 restored PLC activation in permeabilized platelets treated with wortmannin. We propose that PI 3-kinase and its product PtdIns(3,4,5)P3 play a key role in the activation and adequate location of PLCgamma2 induced by FcgammaRIIA cross-linking.     

Phosphoinositide 3-kinase regulates phospholipase Cgamma-mediated calcium signaling

It has been demonstrated that the lipid products of the phosphoinositide 3-kinase (PI3K) can associate with the Src homology 2 (SH2) domains of specific signaling molecules and modify their actions. In the current experiments, phosphatidylinositol 3,4, 5-trisphosphate (PtdIns-3,4,5-P3) was found to bind to the C-terminal SH2 domain of phospholipase Cgamma (PLCgamma) with an apparent Kd of 2.4 microM and to displace the C-terminal SH2 domain from the activated platelet-derived growth factor receptor (PDGFR). To investigate the in vivo relevance of this observation, intracellular inositol trisphosphate (IP3) generation and calcium release were examined in HepG2 cells expressing a series of PDGFR mutants that activate PLCgamma with or without receptor association with PI3K. Coactivation of PLCgamma and PI3K resulted in an approximately 40% increase in both intracellular IP3 generation and intracellular calcium release as compared with selective activation of PLCgamma. Similarly, the addition of wortmannin or LY294002 to cells expressing the wild-type PDGFR inhibited the release of intracellular calcium. Thus, generation of PtdIns-3,4,5-P3 by receptor-associated PI3K causes an increase in IP3 production and intracellular calcium release, potentially via enhanced PtdIns-4, 5-P2 substrate availability due to PtdIns-3,4,5-P3-mediated recruitment of PLCgamma to the lipid bilayer.     

A novel phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase associates with the interleukin-3 receptor

To gain insight into the intracellular signaling cascades that are activated by the binding of interleukin-3 (IL-3) to its target cells, we have embarked on the identification of proteins that are associated with the IL-3 receptor (IL-3R). In a previous study we reported that a 110-kDa serine/threonine protein kinase is constitutively associated with the IL-3R and activated following IL-3 stimulation. We now report that a phosphatidylinositol-3,4, 5-trisphosphate (PtdIns-3,4,5-P3) 5-phosphatase (5-ptase) is also constitutively associated with the IL-3R. This 5-ptase is magnesium-dependent and removes the 5-position phosphate from PtdIns-3,4,5-P3 but does not metabolize PtdIns-4,5-P2, inositol (Ins)-1,3,4,5-P4, or Ins-1,4,5-P3. This substrate specificity distinguishes it from any previously characterized 5-ptase. Interestingly, it may be bound indirectly via phosphatidylinositol 3-kinase (PI 3-kinase), another enzyme that is constitutively bound to the IL-3R. However, unlike PI 3-kinase which becomes activated following IL-3 stimulation, this receptor-associated 5-ptase activity does not increase following IL-3 stimulation, and its primary function may be to keep the principal in vivo product of PI 3-kinase, PtdIns-3,4,5-P3, at low levels in unstimulated cells, to terminate the PI 3-kinase signal following IL-3 stimulation or to metabolize PtdIns-3,4,5-P3 to a metabolically active second messenger, i.e. PtdIns-3,4-P2.     

Escherichia coli cytotoxic necrotizing factor 1 (CNF1), a toxin that activates the Rho GTPase

Cytotoxic necrotizing factor 1 (CNF1), a 110-kDa protein toxin from pathogenic Escherichia coli induces actin reorganization into stress fibers and retraction fibers in human epithelial cultured cells allowing them to spread. CNF1 is acting in the cytosol since microinjection of the toxin into HEp-2 cells mimics the effects of the externally applied CNF1. Incubation in vitro of CNF1 with recombinant small GTPases induces a modification of Rho (but not of Rac, Cdc42, Ras, or Rab6) as demonstrated by a discrete increase in the apparent molecular weight of the molecule. Preincubation of cells with CNF1 impairs the cytotoxic effects of Clostridium difficile toxin B, which inactivates Rho but not those of Clostridium sordellii LT toxin, which inhibits Ras and Rac. As shown for Rho-GTP, CNF1 activates, in a time- and dose-dependent manner, a cytoskeleton-associated phosphatidylinositol 4-phosphate 5-kinase. However, neither the phosphatidylinositol 4,5-bisphosphate (PIP2) nor the phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) or 3,4,5-trisphosphate (PIP3) cellular content were found increased in CNF1 treated HEp-2 cells. Cellular effects of CNF1 were not blocked by LY294002, a stable inhibitor of the phosphoinositide 3-kinase. Incubation of HEp-2 cells with CNF1 induces relocalization of myosin 2 in stress fibers but not in retraction fibers. Altogether, our data indicate that CNF1 is a toxin that selectively activates the Rho GTP-binding protein, thus inducing contractility and cell spreading.     

Integrin alpha IIb beta 3-mediated pp125FAK phosphorylation and platelet spreading on fibrinogen are regulated by PI 3-kinase

Activation of the focal adhesion kinase pp125FAK correlates with its phosphorylation on tyrosine residues and is mediated by multiple receptor-ligand pairs. In platelets, pp125FAK phosphorylation is triggered by alpha IIb beta 3 integrin or Fc gamma RII receptor interaction with immobilized fibrinogen and IgG, respectively. In this study we used platelets as a model system to explore the role of PI 3-kinase relative to pp125FAK phosphorylation. Treatment of the platelets with two PI 3-kinase inhibitors, wortmannin and LY294002, inhibited in a dose-dependent manner alpha IIb beta 3-mediated platelet spreading on fibrinogen having no effect on platelet spreading on IgG. Both inhibitors also completely abolished alpha IIb beta 3-mediated pp125FAK phosphorylation but not pp72syk phosphorylation. Furthermore, Fc gamma RII- and thrombin-induced pp125FAK phosphorylation were not affected by wortmannin and LY294002. Finally, the PI 3-kinase inhibitors' effect on alpha IIb beta 3-mediated spreading and pp125FAK phosphorylation was reversed by phorbol ester treatment. These results establish that the role of PI 3-kinase relative to pp125FAK phosphorylation in platelets is receptor type-specific yet essential for alpha IIb beta 3-mediated cell spreading and pp125FAK phosphorylation.     

Activation of phospholipase C-gamma by phosphatidylinositol 3,4,5-trisphosphate

Signal transduction across cell membranes often involves the activation of both phosphatidylinositol (PI)-specific phospholipase C (PLC) and phosphoinositide 3-kinase (PI 3-kinase). Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a substrate for both enzymes, is converted to phosphatidylinositol 3,4, 5-trisphosphate (PI(3,4,5)P3) by the action of PI 3-kinase. Here, we show that PI(3,4,5)P3 activates purified PLC-gamma isozymes by interacting with their Src homology 2 domains. Furthermore, the expression of an activated catalytic subunit of PI 3-kinase in COS-7 cells resulted in an increase in inositol phosphate formation, whereas platelet-derived growth factor-induced PLC activation in NIH 3T3 cells was markedly inhibited by the specific PI 3-kinase inhibitor LY294002. These results suggest that receptors coupled to PI 3-kinase may activate PLC-gamma isozymes indirectly, in the absence of PLC-gamma tyrosine phosphorylation, through the generation of PI(3,4,5)P3.     

Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.     

Differential expression of PSA-NCAM and HNK-1 epitopes in the developing cardiac conduction system of the chick

Studies on a platelet-derived growth factor (PDGF) responsive osteosarcoma cell line, MG-63, were initiated to determine the effects of phosphatidylinositol (Ptdlns) 3-kinase inhibitors on serum-stimulated cell proliferation and PDGF-stimulated DNA replication, actin rearrangements, or Ptdlns 3-kinase activity. In a dose-dependent manner, the fungal metabolite wortmannin and a quercetin derivative, LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), inhibited serum-stimulated MG-63 cell proliferation. The mitogenic effects of PDGF on MG-63 cells, as determined by incorporation of [3H]-thymidine, were also substantially inhibited in the presence of 0.10 microM wortmannin or 10 microM Ly294002. Furthermore, MG-63 cells stimulated by PDGF form distinct actin-rich, finger-like membrane projections which are completely inhibited by either 0.10 microM wortmannin or 10 microM LY294002. At these same concentrations, wortmannin and LY294002 were also effective at reducing levels of phosphatidylinositol 3-phosphate in PDGF-stimulated MG-63 cells. Treatment of these cells with increasing concentrations of wortmannin reduced the level of PDGF stimulated tyrosine phosphorylation of the PDGF receptor but did not significantly affect the amount of the Ptdlns 3-kinase regulatory subunit, p85, associated with the receptor. Additionally, pretreatment of cells with 0.250 microM wortmannin followed by stimulation with PDGF resulted in a slightly reduced level of receptor autokinase activity; however, similar treatment with 50 microM LY294002 did not affect the level of autokinase activity. These results demonstrate the effects of two different Ptdlns 3-kinase inhibitors on serum- and PDGF-stimulated MG-63 cell proliferation and PDGF-stimulated morphological changes and suggest a greater role for Ptdlns 3-kinase in these processes.     

A new pathway for synthesis of phosphatidylinositol-4,5-bisphosphate

Phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2), a key molecule in the phosphoinositide signalling pathway, was thought to be synthesized exclusively by phosphorylation of PtdIns-4-P at the D-5 position of the inositol ring. The enzymes that produce PtdIns-4,5-P2 in vitro fall into two related subfamilies (type I and type II PtdInsP-5-OH kinases, or PIP(5)Ks) based on their enzymatic properties and sequence similarities'. Here we have reinvestigated the substrate specificities of these enzymes. As expected, the type I enzyme phosphorylates PtdIns-4-P at the D-5 position of the inositol ring. Surprisingly, the type II enzyme, which is abundant in some tissues, phosphorylates PtdIns-5-P at the D-4 position, and thus should be considered as a 4-OH kinase, or PIP(4)K. The earlier error in characterizing the activity of the type II enzyme is due to the presence of contaminating PtdIns-5-P in commercial preparations of PtdIns-4-P. Although PtdIns-5-P was previously thought not to exist in vivo, we find evidence for the presence of this lipid in mammalian fibroblasts, establishing a new pathway for PtdIns-4,5-P2 synthesis.     

Thrombospondin signaling of focal adhesion disassembly requires activation of phosphoinositide 3-kinase

Thrombospondin is an extracellular matrix protein involved in modulating cell adhesion. Thrombospondin stimulates a rapid loss of focal adhesion plaques and reorganization of the actin cytoskeleton in cultured bovine aortic endothelial cells. The focal adhesion labilizing activity of thrombospondin is localized to the amino-terminal domain, specifically amino acids 17-35. Use of a synthetic peptide (hep I), containing amino acids 17-35 of thrombospondin, enables us to examine the signaling mechanisms specifically involved in thrombospondin-induced disassembly of focal adhesions. We tested the hypothesis that activation of phosphoinositide 3-kinase is a necessary step in the thrombospondin-induced signaling pathway regulating focal adhesion disassembly. Both wortmannin and LY294002, membrane permeable inhibitors of phosphoinositide 3-kinase activity, blocked hep I-induced disassembly of focal adhesions. Similarly, wortmannin inhibited hep I-mediated actin microfilament reorganization and the hep I-induced translocation of alpha-actinin from focal adhesion plaques. Hep I also stimulated phosphoinositide 3-kinase activity approximately 2-3-fold as measured in anti-phosphoinositide 3-kinase and anti-phosphotyrosine immunoprecipitates. Increased immunoreactivity for the 85-kDa regulatory subunit in anti-phosphotyrosine immunoprecipitates suggests that the p85/p110 form of phosphoinositide 3-kinase is involved in this pathway. In 32Pi-labeled cells, hep I increased levels of phosphatidylinositol (3,4,5)-trisphosphate, the major product of phosphoinositide 3-kinase phosphorylation. These results suggest that thrombospondin signals the disassembly of focal adhesions and reorganization of the actin cytoskeleton by a pathway involving stimulation of phosphoinositide 3-kinase activity.     

Inhibition of phosphatidylinositol 3-kinase activity blocks cellular differentiation mediated by glial cell line-derived neurotrophic factor in dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for midbrain dopaminergic neurons. To begin to understand the intracellular signaling pathways used by GDNF, we investigated the role of phosphatidylinositol 3-kinase activity in GDNF-stimulated cellular function and differentiation of dopaminergic neurons. We found that treatment of dopaminergic neuron cultures with 10 ng/ml GDNF induced maximal levels of Ret phosphorylation and produced a profound increase in phosphatidylinositol 3-kinase activity, as measured by western blot analysis and lipid kinase assays. Treatment with 1 microM 2-(4-morpholinyl)-8-phenylchromone (LY294002) or 100 nM wortmannin, two distinct and potent inhibitors of phosphatidylinositol 3-kinase activity, completely inhibited GDNF-induced phosphatidylinositol 3-kinase activation, but did not affect Ret phosphorylation. Furthermore, we examined specific biological functions of dopaminergic neurons: dopamine uptake activity and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. GDNF significantly increased dopamine uptake activity and promoted robust morphological differentiation. Treatment with LY294002 completely abolished the GDNF-induced increases of dopamine uptake and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. Our findings show that GDNF-induced differentiation of dopaminergic neurons requires phosphatidylinositol 3-kinase activation.     

Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons

Recent studies have suggested a role for phosphatidylinositol (PI) 3-kinase in cell survival, including the survival of neurons. We used rat sympathetic neurons maintained in vitro to characterize the potential survival signals mediated by PI 3-kinase and to test whether the Akt protein kinase, a putative effector of PI 3-kinase, functions during nerve growth factor (NGF)-mediated survival. Two PI 3-kinase inhibitors, LY294002 and wortmannin, block NGF-mediated survival of sympathetic neurons. Cell death caused by LY294002 resembles death caused by NGF deprivation in that it is blocked by a caspase inhibitor or a cAMP analog and that it is accompanied by the induction of c-jun, c-fos, and cyclin D1 mRNAs. Treatment of neurons with NGF activates endogenous Akt protein kinase, and LY294002 or wortmannin blocks this activation. Expression of constitutively active Akt or PI 3-kinase in neurons efficiently prevents death after NGF withdrawal. Conversely, expression of dominant negative forms of PI 3-kinase or Akt induces apoptosis in the presence of NGF. These results demonstrate that PI 3-kinase and Akt are both necessary and sufficient for the survival of NGF-dependent sympathetic neurons.     

Protein kinase C-zeta as a downstream effector of phosphatidylinositol 3-kinase during insulin stimulation in rat adipocytes. Potential role in glucose transport

Insulin provoked rapid increases in enzyme activity of immunoprecipitable protein kinase C-zeta (PKC-zeta) in rat adipocytes. Concomitantly, insulin provoked increases in 32P labeling of PKC-zeta both in intact adipocytes and during in vitro assay of immunoprecipitated PKC-zeta; the latter probably reflected autophosphorylation, as it was inhibited by the PKC-zeta pseudosubstrate. Insulin-induced activation of immunoprecipitable PKC-zeta was inhibited by LY294002 and wortmannin; this suggested dependence upon phosphatidylinositol (PI) 3-kinase. Accordingly, activation of PI 3-kinase by a pYXXM-containing peptide in vitro resulted in a wortmannin-inhibitable increase in immunoprecipitable PKC-zeta enzyme activity. Also, PI-3,4-(PO4)2, PI-3,4,5-(PO4)3, and PI-4,5-(PO4)2 directly stimulated enzyme activity and autophosphoralytion in control PKC-zeta immunoprecipitates to levels observed in insulin-treated PKC-zeta immunoprecipitates. In studies of glucose transport, inhibition of immunoprecipitated PKC-zeta enzyme activity in vitro by both the PKC-zeta pseudosubstrate and RO 31-8220 correlated well with inhibition of insulin-stimulated glucose transport in intact adipocytes. Also, in adipocytes transiently expressing hemagglutinin antigen-tagged GLUT4, co-transfection of wild-type or constitutive PKC-zeta stimulated hemagglutinin antigen-GLUT4 translocation, whereas dominant-negative PKC-zeta partially inhibited it. Our findings suggest that insulin activates PKC-zeta through PI 3-kinase, and PKC-zeta may act as a downstream effector of PI 3-kinase and contribute to the activation of GLUT4 translocation.     

A requirement for phosphatidylinositol 3-kinase in pseudopod extension

Phagocytosis requires actin assembly and pseudopod extension, two cellular events that coincide spatially and temporally. The signal transduction events underlying both processes may be distinct. We tested whether phagocytic signaling resembles that of growth factor receptors, which induce actin polymerization via activation of phosphatidylinositol 3-kinase (PI 3-kinase). Fcgamma receptor-mediated phagocytosis was accompanied by a rapid increase in the accumulation of phosphatidylinositol 3,4,5-trisphosphate in vivo, and addition of wortmannin (WM) or LY294002, two inhibitors of PI 3-kinase(s), inhibited phagocytosis but not Fcgamma receptor-directed actin polymerization. However, both compounds prevented maximal pseudopod extension, suggesting that PI 3-kinase inhibition produced a limitation in membrane required for pseudopod extension. Availability of plasma membrane was not limiting for phagocytosis, because blockade of ingestion in the presence of WM was not overcome by reducing the number of particles adhering to macrophages. However, decreasing bead size, and hence the magnitude of pseudopod extension required for particle engulfment, relieved the inhibition of phagocytosis in the presence of WM or LY294002 by up to 80%. The block in phagocytosis of large particles occurred before phagosomal closure, because both compounds inhibited spreading of macrophages on substrate-bound IgG. Macrophage spreading on IgG was accompanied by exocytic insertion of membrane from an intracellular source, as measured by the dye FM1-43. These results indicate that one or more isoforms of PI 3 kinase are required for maximal pseudopod extension but not phagocytosis per se. We suggest that PI 3-kinase is required for coordinating exocytic membrane insertion and pseudopod extension.     

Investigation of neutrophil signal transduction using a specific inhibitor of phosphatidylinositol 3-kinase

Neutrophils contain a multicomponent NADPH oxidase system that is involved in the production of microbicidal oxidants. Stimulation of human neutrophils with the peptide FMLP activates this respiratory burst enzyme to produce superoxide and also has been shown to result in activation of phosphatidylinositol (Ptdlns) 3-kinase. Treatment of human neutrophils with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a potent and specific inhibitor of Ptdlns 3-kinase, resulted in complete inhibition of Ptdlns 3-kinase activity as well as in inhibition of superoxide production in FMLP-treated neutrophils in suspension; FMLP-stimulated oxidant production in adherent cells was also abolished. Treatment of human neutrophils with PMA resulted in production of superoxide without activation of Ptdlns 3-kinase; LY294002 did not block superoxide production in neutrophils exposed to PMA. In addition, LY294002 did not inhibit cellfree NADPH oxidase activation, CD11b-dependent adhesion, actin polymerization in response to FMLP, or FMLP-induced calcium flux. These results suggest that the signal transduction pathway of the FMLP-receptor involves activation of Ptdlns 3-kinase, which is required for subsequent superoxide production induced by the chemotactic peptide. Furthermore, Ptdlns 3-kinase may be located directly upstream of protein kinase C or other protein kinases, which in turn activate the NADPH oxidase system.     

The CC chemokine monocyte chemotactic peptide-1 activates both the class I p85/p110 phosphatidylinositol 3-kinase and the class II PI3K-C2alpha

The cellular effects of MCP-1 are mediated primarily by binding to CC chemokine receptor-2. We report here that MCP-1 stimulates the formation of the lipid products of phosphatidylinositol (PI) 3-kinase, namely phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P3) in THP-1 cells that can be inhibited by pertussis toxin but not wortmannin. MCP-1 also stimulates an increase in the in vitro lipid kinase activity present in immunoprecipitates of the class 1A p85/p110 heterodimeric PI 3-kinase, although the kinetics of activation were much slower than observed for the accumulation of PI 3,4,5-P3. In addition, this in vitro lipid kinase activity was inhibited by wortmannin (IC50 = 4.47 +/- 1.88 nM, n = 4), and comparable concentrations of wortmannin also inhibited MCP-stimulated chemotaxis of THP-1 cells (IC50 = 11.8 +/- 4.2 nM, n = 4), indicating that p85/p110 PI 3-kinase activity is functionally relevant. MCP-1 also induced tyrosine phosphorylation of three proteins in these cells, and a fourth tyrosine-phosphorylated protein co-precipitates with the p85 subunit upon MCP-1 stimulation. In addition, MCP-1 stimulated lipid kinase activity present in immunoprecipitates of a class II PI 3-kinase (PI3K-C2alpha) with kinetics that closely resembled the accumulation of PI 3,4,5-P3. Moreover, this MCP-1-induced increase in PI3K-C2alpha activity was insensitive to wortmannin but was inhibited by pertussis toxin pretreatment. Since this mirrored the effects of these inhibitors on MCP-1-stimulated increases in D-3 phosphatidylinositol lipid accumulation in vivo, these results suggest that activation of PI3K-C2alpha rather than the p85/p110 heterodimer is responsible for mediating the in vivo formation of D-3 phosphatidylinositol lipids. These data demonstrate that MCP-1 stimulates protein tyrosine kinases as well as at least two separate PI 3-kinase isoforms, namely the p85/p110 PI 3-kinase and PI3K-C2alpha. This is the first demonstration that MCP-1 can stimulate PI 3-kinase activation and is also the first indication of an agonist-induced activation of the PI3K-C2alpha enzyme. These two events may play important roles in MCP-1-stimulated signal transduction and biological consequences.     

Differential regulation of muscarinic acetylcholine receptor-sensitive polyphosphoinositide pools and consequences for signaling in human neuroblastoma cells

In this study we have quantitatively assessed the basal turnover of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and M3-muscarinic receptor-mediated changes in phosphoinositides in the human neuroblastoma cell line, SH-SY5Y. We demonstrate that the polyphosphoinositides represent a minor fraction of the total cellular phosphoinositide pool and that in addition to rapid, sustained increases in [3H]inositol phosphates dependent upon the extent of receptor activation by carbachol, there are equally rapid and sustained reductions in the levels of polyphosphoinositides. Compared with phosphatidylinositol 4-phosphate (PtdIns(4)P), PtdIns(4,5)P2 was reduced with less potency by carbachol and recovered faster following agonist removal suggesting protection of PtdIns(4,5)P2 at the expense of PtdIns(4)P and indicating specific regulatory mechanism(s). This does not involve a pertussis toxin-sensitive G-protein regulation of PtdIns(4)P 5-kinase. Using wortmannin to inhibit PtdIns 4-kinase activity, we demonstrate that the immediate consequence of blocking the supply of PtdIns(4)P (and therefore PtdIns(4,5)P2) is a failure of agonist-mediated phosphoinositide and Ca2+ signaling. The use of wortmannin also indicated that PtdIns is not a substrate for receptor-activated phospholipase C and that 15% of the basal level of PtdIns(4,5)P2 is in an agonist-insensitive pool. We estimate that the agonist-sensitive pool of PtdIns(4,5)P2 turns over every 5 s (0.23 fmol/cell/min) during sustained receptor activation by a maximally effective concentration of carbachol. Immediately following agonist addition, PtdIns(4,5)P2 is consumed >3 times faster (0.76 fmol/cell/min) than during sustained receptor activation which represents, therefore, utilization by a partially desensitized receptor. These data indicate that resynthesis of PtdIns(4,5)P2 is required to allow full early and sustained phases of receptor signaling. Despite the critical dependence of phosphoinositide and Ca2+ signaling on PtdIns(4,5)P2 resynthesis, we find no evidence that this rate resynthesis is limiting for agonist-mediated responses.     

Type I phosphatidylinositol-4-phosphate 5-kinases synthesize the novel lipids phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 5-phosphate

Inositol phospholipids regulate a variety of cellular processes including proliferation, survival, vesicular trafficking, and cytoskeletal organization. Recently, two novel phosphoinositides, phosphatidylinositol-3,5-bisphosphate (PtdIns-3,5-P2) and phosphatidylinositol- 5-phosphate (PtdIns-5-P), have been shown to exist in cells. PtdIns-3,5-P2, which is regulated by osmotic stress, appears to be synthesized by phosphorylation of PtdIns-3-P at the D-5 position. No evidence yet exists for how PtdIns-5-P is produced in cells. Understanding the regulation of synthesis of these molecules will be important for identifying their function in cellular signaling. To determine the pathway by which PtdIns-3,5-P2 and Ptd-Ins-5-P might be synthesized, we tested the ability of the recently cloned type I PtdIns-4-P 5-kinases (PIP5Ks) alpha and beta to phosphorylate PtdIns-3-P and PtdIns at the D-5 position of the inositol ring. We found that the type I PIP5Ks phosphorylate PtdIns-3-P to form PtdIns-3,5-P2. The identity of the PtdIns-3,5-P2 product was determined by anion exchange high performance liquid chromatography analysis and periodate treatment. PtdIns-3,4-P2 and PtdIns-3,4,5-P3 were also produced from PtdIns-3-P phosphorylation by both isoforms. When expressed in mammalian cells, PIP5K Ialpha and PIP5K Ibeta differed in their ability to synthesize PtdIns-3,5-P2 relative to PtdIns-3,4-P2. We also found that the type I PIP5Ks phosphorylate PtdIns to produce PtdIns-5-P and phosphorylate PtdIns-3,4-P2 to produce PtdIns-3,4,5-P3. Our findings suggest that type I PIP5Ks synthesize the novel phospholipids PtdIns-3,5-P2 and PtdIns-5-P. The ability of PIP5Ks to produce multiple signaling molecules indicates that they may participate in a variety of cellular processes.     

Chronic exposure to kappa-opioids enhances the susceptibility of immortalized neurons (F-11kappa 7) to apoptosis-inducing drugs by a mechanism that may involve ceramide

Chronic exposure of embryonic brain to opioids leads to microcephaly and developmental abnormalities. An immortalized mouse neuroblastoma x dorsal root ganglion hybrid cell line stably transfected to overexpress kappa-opioid receptors (F-11kappa7) showed complete loss of kappa-receptor binding to [3H]U69,593 after exposure to the kappa-agonist U69,593 for 24 h. U69,593 had no measurable effect on cell viability as determined by either cell viability or DNA fragmentation assays. However, when cell death (apoptosis) was induced by either staurosporine or the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002, cells pretreated with U69,593 for 24 h showed increased apoptosis compared with untreated cells. Thus, staurosporine (50 nM), wortmannin (4 microM), and LY294002 (30 microM) treatment for 24 h induced a 50% loss of cell viability and DNA fragmentation in 24 h. U69,593 pretreatment produced the same killing at lower concentrations, namely, 20 nM staurosporine, 2 microM wortmannin, and 14 microM LY294002, respectively. The effects of U69,593 were time-, dose-, and naloxone-reversible, suggesting that they are receptor-mediated. However, coaddition of U69,593 at the same time as staurosporine, wortmannin, or LY294002 did not enhance apoptosis. All three drugs that induced apoptosis were found to increase the level of ceramide, and pretreatment with U69,593 further increased the rate of formation of ceramide, a lipid that induces apoptosis in cells. We propose that chronic exposure to kappa-receptor agonists promotes increased vulnerability of neurons to apoptosis.     

Protein kinase C delta specifically associates with phosphatidylinositol 3-kinase following cytokine stimulation

Phosphatidylinositol (PI) 3-kinase is activated as a result of cytokine-induced association of the enzyme with specific tyrosine-phosphorylated proteins. PI 3-kinase lipid products, PI 3, 4-P2 and PI 3,4,5-P3, have been shown, in vitro, to directly activate novel and atypical protein kinase C (PKC) isozymes. However, the mechanism by which PI 3-kinase may be involved in regulation of PKC isoforms in vivo is presently unknown. We investigated a possible relationship by looking for associations between these enzymes. We found that in a human erythroleukemia cell line, as well as in rabbit platelets, PI 3-kinase and PKCdelta associate in a specific manner that is modulated by cell activation. Granulocyte-macrophage colony-stimulating factor treatment of cells caused increased association of PKCdelta and PI 3-kinase as did treatment of platelets with platelet-activating factor. Results using two PI 3-kinase inhibitors, wortmannin and LY-294002, showed that the former inhibited this association, while the latter did not, suggesting that PI 3-kinase lipid products may not be a prerequisite for the PI 3-kinase/PKCdelta association. Our results also suggest that tyrosine phosphorylation of PKCdelta is not involved in its association with PI 3-kinase.