Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human platelets undergo a rapid, major reorganization of the cytoskeletal matrix upon exposure to thrombin, and accumulate 3-phosphorylated phosphoinositides in a protein kinase C (PKC)-dependent manner. These phosphoinositides have been suggested to be involved in actin polymerization/depolymerization. We reasoned that, if newly generated 3-phosphorylated phosphoinositide modulates cytoskeletal reorganization, a prerequisite for such action would be generation near cytoskeletal proteins. We have found that, after platelet activation, phosphatidylinositol 3-kinase and phosphatidylinositol(4)P 3-kinase activities, antibody-detectable phosphoinositide 3-kinase, and PKC become markedly and specifically enriched in a Triton X-100-insoluble cytoskeletal fraction that contains GPIIb/IIIa (integrin) and pp60c-src. The cytoskeletal fraction then accounts for up to 70% of total phosphoinositide 3-kinase activity, a function of recruited activated enzyme. These proteins are not occluded or directly associated with newly polymerized actin, since blockage by cytochalasin D of actin polymerization, and consequent inhibition of accumulation of about 40% of incremental protein and actin in this fraction, has no effect on its content of phosphoinositide 3-kinase, GPIIb/IIIa, pp60c-src, or PKC. Depolymerization of actin with DNase I, or inhibition of ligand binding to GPIIb/IIIa by RGDS, however, in combination with cytochalasin D, further depletes actin and significantly decreases sedimentability of GPIIb/IIIa as well as phosphoinositide 3-kinase, pp60c-src, and PKC, without inhibiting total 3-kinase activity. Our results suggest that, as a function of platelet activation, enzymes that regulate the synthesis of 3-phosphorylated phosphoinositides rapidly associate with the membrane skeleton and that skeletally associated phosphoinositide 3-kinase is more active than the Triton-soluble form.
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PMID:Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets. 131 17

Platelet stimulation by thrombin or the thrombin receptor activating peptide (TRAP) results in the activation of phosphoinositide 3-kinase and the production of the novel polyphosphoinositides phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3). We have shown previously that these lipids activate calcium-independent protein kinase C (PKC) isoforms in vitro (Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M. and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367). Activation of platelet PKC in response to TRAP is detected by the phosphorylation of the major PKC substrate in platelets, the p47 phosphoprotein, also known as pleckstrin. Here we provide evidence for two phases of pleckstrin phosphorylation in response to TRAP. A rapid phase of pleckstrin phosphorylation (< 1 min) precedes the peak of PtdIns-3,4-P2 production and is unaffected by concentrations of wortmannin (10-100 nM) that block production of this lipid. However prolonged phosphorylation of pleckstrin (> 2 min) is inhibited by wortmannin concentrations that block PtdIns-3,4-P2 production. Phorbol ester-mediated pleckstrin phosphorylation was not affected by wortmannin and wortmannin had no effect on purified platelet PKC activity. Phosphorylation of pleckstrin could be induced using permeabilized platelets supplied with exogenous gamma-32P[ATP] and synthetic dipalmitoyl PtdIns-3,4,5-P3 and dipalmitoyl PtdIns-3,4-P2 micelles, but not with dipalmitoyl phosphatidylinositol 3-phosphate or phosphatidylinositol 4,5-bisphosphate. These results suggest two modes of stimulating pleckstrin phosphorylation: a rapid activation of PKC (via diacylglycerol and calcium) followed by a slower activation of calcium-independent PKCs via PtdIns-3,4-P2.
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PMID:Phosphorylation of the platelet p47 phosphoprotein is mediated by the lipid products of phosphoinositide 3-kinase. 749 94

The platelet membrane is lined by a membrane skeleton, which in turn appears to be associated with underlying cytoplasmic actin filaments. Glycoprotein IIb-IIIa appears to associate with the membrane skeleton in unstimulated platelets. Upon platelet activation, unidentified intracellular signals cause GP IIb-IIIa to become competent to bind adhesive ligand. We suggest that the membrane skeleton may play a role in allowing this inside-out signaling. Signaling molecules that appear to associate with the membrane skeleton in unstimulated platelets include pp60c-src, pp62c-yes, and GAP. Preliminary evidence suggests that components of the membrane skeleton may become phosphorylated on tyrosine residues prior to GP IIb-IIIa-ligand interactions. Once GP IIb-IIIa binds adhesive ligand in a platelet aggregate, there is signaling in the opposite direction. One consequence of the outside-in transmembrane signaling is that the membrane skeleton becomes more tightly associated with the underlying actin filaments as focal contact-like structures form. Proteins that accumulate in these focal contact-like structures with a time course identical to that of GP IIb-IIIa and in a GP IIb-IIIa-dependent manner include talin, vinculin, and spectrin. Signaling molecules that accumulate in the focal contact-like structures include pp60c-src, pp62c-yes, phosphoinositide 3-kinase, and protein kinase C. These are potential candidates for the enzymes that mediate the ligand-induced transmembrane signaling. Another enzyme involved in the ligand-induced signaling is calpain. This enzyme is activated as a consequence of ligand-GP IIb-IIIa interactions and cleaves components of the membrane skeleton. Future experiments will be needed to identify other signaling enzymes activated as a consequence of GP IIb-IIIa interactions and to determine which ones are responsible for inducing the cytoskeletal reorganizations that occur in platelets and other cells when integrins bind their adhesive ligands.
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PMID:Transmembrane signaling across the platelet integrin glycoprotein IIb-IIIa. 801 93

The platelet cytoskeleton contains two actin filament-based components. One is the cytoplasmic actin filaments that fill the cytoplasm and mediate contractile events. The other is the membrane skeleton that coats the plasma membrane and regulates properties of the membrane such as its contours and stability and the lateral distribution of membrane glycoproteins. Recent work reviewed in this article indicates that the GP IIb-IIIa complex can associate with the membrane skeleton. Upon platelet activation, GP IIb-IIIa becomes competent to bind its adhesive ligand, fibrinogen. This induces a reorganization of the cytoskeleton such that the membrane skeletal proteins with which GP IIb-IIIa is associated become associated with underlying cytoplasmic filaments. As in focal contacts of cultured cells, this ligand-induced association of GP IIb-IIIa with cytoplasmic actin filaments regulates the ability of GP IIb-IIIa to bind adhesive ligand. Intracellular enzymes that are activated as a consequence of ligand binding to the GP IIb-IIIa complex include tyrosine kinase(s) and calpain, making these potential candidates for enzymes inducing the two-way signaling across the membrane. Additional candidates include phosphoinositide 3-kinase and protein kinase C, other enzymes that have been detected in focal contacts of aggregating platelets. Future studies identifying interactions between the GP IIb-IIIa complex and membrane skeletal proteins should help to further elucidate the significance of the GP IIb-IIIa in cytoskeleton interaction in regulating integrin-mediated transmembrane signaling in platelets.
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PMID:Regulation of platelet function by the cytoskeleton. 820 86

Activation of tyrosine kinase-linked receptors has been shown to stimulate Ca2+-independent protein kinase C isoforms in nonneuronal cells. We have examined this signaling pathway in the nervous system. Incubating bag cell neurons from the marine mollusk Aplysia californica with concentrations of insulin known to stimulate a tyrosine kinase-linked receptor in these cells persistently activated and down-regulated the Ca2+-independent protein kinase C (Apl II), whereas insulin only transiently activated and did not down-regulate the Ca2+-activated protein kinase C (Apl I). The effects of insulin may be mediated by activation of phosphoinositide 3-kinase because (a) diC16phosphatidylinositol 3,4,5-trisphosphate, a synthetic phosphoinositide 3-kinase product, stimulated autophosphorylation of baculovirus-expressed Apl II, but not of Apl I, and (b) wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the activation and down-regulation of Apl II by insulin but not the transient activation of Apl I. These results suggest that activators of tyrosine kinase-linked receptors may mediate some of their effects in neurons through activation of Ca2+-independent protein kinase C isoforms.
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PMID:Stimulation of an insulin receptor activates and down-regulates the Ca2+-independent protein kinase C, Apl II, through a Wortmannin-sensitive signaling pathway in Aplysia. 866 95

Synaptic vesicle recycling is a neuronal specialization of endocytosis that requires the GTPase activity of dynamin I and is triggered by membrane depolarization and Ca2+ entry. To establish the relationship between dynamin I GTPase activity and Ca2+, we used purified dynamin I and analyzed its interaction with Ca2+ in vitro. We report that Ca2+ bound to dynamin I and this was abolished by deletion of dynamin's C-terminal tail. Phosphorylation of dynamin I by protein kinase C promoted formation of a dynamin I tetramer and increased Ca2+ binding to the protein. Moreover, Ca2+ inhibited dynamin I GTPase activity after stimulation by phosphorylation or by phospholipids but not after stimulation with a GST-SH3 fusion protein containing the SH3 domain of phosphoinositide 3-kinase. These results suggest that in resting nerve terminals, phosphorylation of dynamin I by protein kinase C converts it to a tetramer that functions as a Ca(2+)-sensing protein. By binding to Ca2+, dynamin I GTPase activity is specifically decreased, possibly to regulate synaptic vesicle recycling.
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PMID:Calcium binds dynamin I and inhibits its GTPase activity. 878 38

Pleckstrin, the prototypic protein containing two copies of the pleckstrin homology domain, is a prominent substrate of protein kinase C in platelets and neutrophils. Both cell types have p85 subunit-containing phosphoinositide 3-kinase (p85/PI3K) and non-p85-containing PI3K (PI3Kgamma) that is activated by betagamma subunits of heterotrimeric GTP-binding proteins. We have shown that a PI3K product, phosphatidylinositol (PI) 3,4,5-trisphosphate, promotes pleckstrin phosphorylation in platelets. Since pleckstrin homology domains are thought to interact with Gbetagamma heterodimers and/or PI(4,5)P2, we have examined the effects of recombinant pleckstrins on platelet PI3Kgamma and p85/PI3K activities. Depending upon its phosphorylation/charged state, pleckstrin inhibits PI3Kgamma, but not p85/PI3K. Pleckstrin-mediated inhibition of PI3Kgamma is overcome by excess Gbetagamma and is restricted to PI(4,5)P2 as substrate, i.e. pleckstrin does not inhibit phosphorylation of PI()P or PI. Consistent with this, activation of protein kinase C by exposure of platelets to beta-phorbol diester (to increase endogenous pleckstrin phosphorylation) prior to platelet lysis causes inhibition of Gbetagamma-stimulatable PI3K activity only with respect to PI(4,5)P2 substrate. This phosphopleckstrin-mediated inhibition is overcome by increasing concentrations of Gbetagamma. We propose that phosphorylation of pleckstrin may constitute an important inhibitory mechanism for PI3Kgamma-mediated cell signaling.
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PMID:Phosphopleckstrin inhibits gbetagamma-activable platelet phosphatidylinositol-4,5-bisphosphate 3-kinase. 881 Feb 77

Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by >/=5 microM thrombin receptor-activating peptide (TRAP), which causes rapid and massive wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (<1 microM) of TRAP. Phosphatidylinositol 3,4-bisphosphate inhibits actin filament severing and capping by human gelsolin in vitro. The findings implicate D3 polyphosphoinositides and integrin signaling in PMA-mediated platelet stimulation and implicate D3 containing phosphoinositides generated in response to protein kinase C activation and GPIIb-IIIa signaling as late-acting intermediates leading to filopodial actin assembly.
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PMID:D3 phosphoinositides and outside-in integrin signaling by glycoprotein IIb-IIIa mediate platelet actin assembly and filopodial extension induced by phorbol 12-myristate 13-acetate. 895 43

The present study takes a novel approach to explore the mode of action of phosphoinositide 3-kinase lipid products by identifying a synthetic peptide W-NG(28-43) (WAAKIQASFRGHMARKK) that displays discriminative affinity with phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). This PtdIns(3,4,5)P3-binding peptide was discovered by a gel filtration-based binding assay and exhibits a high degree of stereochemical selectivity in phosphoinositide recognition. It forms a 1:1 complex with PtdIns(3,4,5)P3 with Kd of 2 microM, but binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) with substantially lower affinity (5- and 40-fold, respectively) despite the largely shared structural motifs with PtdIns(3,4,5)P3. Other phospholipids examined, including phosphatidylserine, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, show low or negligible affinity with the peptide. Several lines of evidence indicate that this phosphoinositide-peptide interaction is not due to nonspecific electrostatic interactions or phospholipid aggregation, and requires a cooperative action among the hydrophobic and basic residues to exert the selective recognition. CD data suggest that the peptide acquires an ordered structure upon binding to PtdIns(3,4,5)P3. Further, we demonstrate that PtdIns(3,4,5)P3 enhances the phosphorylation rate of this binding peptide by protein kinase C (PKC)-alpha in a dose-dependent manner. In view of the findings that this stimulatory effect is not noted with other PKC peptide substrates lacking affinity with PtdIns(3,4,5)P3 and that PKC-alpha is not susceptible to PtdIns(3,4,5)P3 activation, the activity enhancement is thought to result from the substrate-concentrating effect of the D-3 phosphoinositide, i.e. the presence of PtdIns(3,4,5)P3 allows the peptide to bind to the same vesicles/micelles to which PKC is bound. Moreover, it is noteworthy that neurogranin, the full-length protein of W-NG(28-43) and a relevant PKC substrate in the forebrain, binds PtdIns(3,4,5)P3 with high affinity. Taken together, it is plausible that, in addition to PKC activation, PtdIns(3,4,5)P3 provides an alternative mechanism to regulate PKC activity in vivo by recruiting and concentrating its target proteins at the interface to facilitate the subsequent PKC phosphorylation.
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PMID:Selective recognition of phosphatidylinositol 3,4,5-trisphosphate by a synthetic peptide. 899 84

Phosphoinositide 3-kinase has been implicated as an activator of cell motility in a variety of recent studies, yet the role of its lipid product, phosphatidylinositol 1,4,5-trisphosphate (PtdIns-3,4,5-P3), has yet to be elucidated. In this study, three independent preparations of PtdIns-3,4,5-P3 were found to increase the motility of NIH 3T3 cells when examined utilizing a microchemotaxis chamber. Dipalmitoyl L-alpha-phosphatidyl-D-myo-inositol 3,4,5-triphosphate (Di-C16-PtdIns-3,4,5-P3) also produced actin reorganization and membrane ruffling. Cells pretreated with 12-O-tetradecanoylphorbol-13-acetate to cause down-regulation of protein kinase C (PKC) exhibited complete inhibition of cell motility induced by Di-C16-PtdIns-3,4,5-P3. These results are consistent with previous observations that PtdIns-3,4,5-P3 activates Ca2+-independent PKC isoforms in vitro and in vivo and provide the first demonstration of an in vivo role for the lipid products of the phosphoinositide 3-kinase. PtdIns-3,4,5-P3 appears to directly initiate cellular motility via activation of a PKC family member.
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PMID:The lipid products of phosphoinositide 3-kinase increase cell motility through protein kinase C. 904 71


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