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)

The role of protein kinase C (PKC) in platelet activation by thrombin was assessed using a PKC inhibitor Ro 31-7549/001 (R2) which, in vitro, shows more selectivity for PKC than other kinase inhibitors. During early (1.5 s) thrombin-induced platelet activation, when phosphorylation of 47 kDa protein (pleckstrin) and myosin light chain by PKC and myosin light chain kinase, respectively, are most readily differentiated, R2 suppressed phosphorylation of pleckstrin more effectively than myosin light chain. R2-inhibited dense granule secretion (measured 0-10 s using quenched-flow techniques) with a dose dependency similar to that for inhibition of pleckstrin phosphorylation, supporting a role for PKC in this process. R2, at 0.5 microM inhibited 47 kDa protein phosphorylation by more than 60%, but had only minimal effects on the kinetics (0-3s) of ADP-induced primary aggregation. At this same concentration, R2 potentiated the thrombin-induced rise in cytosolic calcium during early (0-15 s) activation as measured in the presence or absence of external calcium. These data support the hypothesis that activation of PKC during early platelet function helps regulate cytosolic calcium levels by limiting calcium release into the cytosol.
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PMID:The role of protein kinase C in the initial events of platelet activation by thrombin assessed with a selective inhibitor. 846 70

This work was carried out to decide whether a non-specific perturbation of the platelet membrane with exogenous amphiphiles affects protein phosphorylation in platelets, especially phosphorylation mediated by PKC. Effects of amphiphiles per se on protein phosphorylation were also recorded. (i) Sublytic concentrations of the differently charged model surfactants cetyltrimethylammonium bromide (CTAB), Zwittergent 3-16, sodium tetradecyl sulphate, and octaethyleneglycol hexadecyl ether, as well as chlorpromazine, and Triton X-100, did not affect the thrombin-induced, PKC-mediated phosphorylation of pleckstrin, whereas sphingosine blocked this phosphorylation. (ii) The sphingosine-mediated phosphorylation blockade is not related to a non-specific perturbation of the membrane, but can instead be attributed to specific properties of sphingosine. (iii) The amphiphiles, per se, had differential effects on protein phosphorylation at sublytic concentrations: a treatment with CTAB, Zwittergent 3-16, and sodium tetradecyl sulphate for 1 min led to phosphorylation of a 49-kDa protein, while treatment with sphingosine for 1 min led to a transient phosphorylation of the myosin light chain as well as a weak phosphorylation of pleckstrin.
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PMID:Perturbation of the platelet plasma membrane is not sufficient for inhibition of thrombin-induced PKC-activity. 848 83

The bisindolylmaleimide, GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide ), a highly selective inhibitor of protein kinase C (PKC), was used to test the role of this enzyme in phorbol ester-induced megakaryocytic differentiation of HEL cells. Treatment of these cells with 10 nmol/L phorbol 12-myristate 13-acetate (PMA) for 3 days caused a complete inhibition of proliferation and a threefold increase in the surface expression of glycoprotein (GP) IIIa, a marker of megakaryocytic differentiation that forms part of the fibrinogen receptor complex, GPIIb/IIIa. A similar effect was observed with phorbol 12,13-dibutyrate, but not with the biologically inactive derivative PMA-4-O-methyl ether. The PMA-induced increase in GPIIIa expression was completely inhibited by GF109203X in a dose-dependent manner (IC50 = 0.5 mumol/L), with a maximal effect at 2.5 to 5.0 mumol/L. GF109203X also blocked the inhibitory effect of PMA on cell growth and inhibited PMA-stimulated phosphorylation of the 47-kD PKC substrate, pleckstrin. Incubation of HEL cells with 25 mumol/L hemin for 3 days caused a fourfold to fivefold increase in expression of the erythroid differentiation marker, glycophorin A. In contrast to the inhibitory effect of GF109203X on GPIIIa expression, hemin induction of glycophorin A was enhanced by this compound. Furthermore, GF109203X alone caused a dose-dependent increase in glycophorin A expression, and induced hemoglobinization. Consistent with these changes, Northern blot analysis revealed that GF109203X treatment reduced the steady-state level of GPIIb mRNA and increased those for glycophorin A and gamma-globin. These results suggest that PKC may act as a developmental switch controlling erythroid/megakaryocytic differentiation.
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PMID:Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells. 854 33

Cathepsin G, an enzyme released by stimulated polymorphonuclear neutrophils, and thrombin are two human proteinases which potently trigger platelet activation. Unlike thrombin, the mechanisms by which cathepsin G initiates platelet activation have yet to be elucidated. The involvement of the phospholipase C (PLC)/protein kinase C (PKC) pathway in cathepsin G-induced activation was investigated and compared with stimulation by thrombin. Exposure of 5-[14C]hydroxytryptamine-labelled platelets to cathepsin G, in the presence of acetylsalicylic acid and phosphocreatine/creatine kinase, induced platelet aggregation and degranulation in a concentration-dependent manner (0.1-3.0 microM). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 microM) and thrombin (0.5 unit/ml) resulted in very similar transient hydrolysis of phosphatidylinositol 4,5-bisphosphate and steady accumulation of phosphatidic acid. In addition cathepsin G, like thrombin, initiated the production of inositol phosphates. The neutrophil-derived proteinase also induced phosphorylation of both the myosin light chain and pleckstrin, a substrate for PKC, to levels similar to those observed in platelets challenged with thrombin. Inhibition of PKC by GF 109203X, a specific inhibitor, suppressed platelet aggregation and degranulation to the same extent for both proteinases. Using fura 2-loaded platelets, the rise in the cytosolic free Ca2+ concentration induced by cathepsin G was shown to result, as for thrombin, from both mobilization of internal stores and Ca2+ entry across the plasma membrane. These findings provide evidence that cathepsin G stimulates the PLC/PKC pathway as potently as does thrombin, independently of thromboxane A2 formation and ADP release, and that this pathway is required for platelet functional responses.
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PMID:The phospholipase C/protein kinase C pathway is involved in cathepsin G-induced human platelet activation: comparison with thrombin. 857 71

The intrinsic signal(s) responsible for the onset of human keratinocyte terminal differentiation is not yet fully understood. Evidence has been recently accumulated linking the phospholipase-mediated activation of protein kinase C to the coordinate changes in gene expression occurring during keratinocyte terminal differentiation. Here we report the purification of a keratinocyte-derived protein enhancing protein kinase C enzymatic activity. The stimulator eluted as a peak with estimated molecular mass of approximately 70 kDa, while analysis by SDS-PAGE showed a 30 kDa protein migrating as a distinct doublet, suggesting the formation of a 30 kDa homodimer. The amino acid sequence analysis allowed the unambigous identification of the protein kinase C stimulator as a mixture of the highly homologous sigma (stratifin) and zeta isoforms of 14-3-3 proteins, which are homodimers of identical 30 kDa subunits. Mono Q anion exchange chromatography and immunoblot analysis further confirmed that stratifin enhances protein kinase C activity. Stratifin was originally sequenced from a human keratinocyte protein database, but its function was unknown. The pleckstrin homology domain has been recently related to protein translocation to the cell membrane as well as to functional interactions of intracellular proteins involved in signal transduction. We show here that stratifin (and 14-3-3 zeta) harbors a pleckstrin homology domain, and the consequent functional implications will be discussed.
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PMID:Stratifin, a keratinocyte specific 14-3-3 protein, harbors a pleckstrin homology (PH) domain and enhances protein kinase C activity. 858 68

Platelet-agonist interaction results in activation of glycoprotein (GP) IIb-IIIa complex and fibrinogen binding, a prerequisite for platelet aggregation. Fibrinogen binding exposes new antibody binding sites on GPIIb-IIIa (ligand-induced binding sites: LIBS). Signal transduction events, including pleckstrin phosphorylation by protein kinase C (PKC), are considered to regulate GPIIb-IIIa activation. We studied a 16-year-old white male with lifelong mucocutaneous bleeding manifestations and abnormal platelet aggregation and secretion in response to multiple agonists. Pleckstrin phosphorylation was diminished in response to platelet-activating factor (PAF; 4 and 400 nmol/L) and thrombin (0.05 U/mL). Binding of monoclonal antibodies (MoAbs) 10E5 and A2A9, which bind to both resting and activated GPIIb-IIIa, was normal. Binding of MoAb PAC1, which binds to only activated GPIIb-IIIa, was diminished upon activation with PAF, adenosine diphosphate (ADP), thrombin receptor agonist peptide (SFLLRN), A23187, and 1,2-dioctonylglycerol (DiC8). Signal transduction-dependent LIBS expression (studied using MoAb 62) induced by ADP, SFLLRN, and DiC8 and signal transduction-independent LIBS expression induced by RGDS peptide or disintegrin albolabrin were normal or minimally decreased, indicating the presence of intact ligand binding sites. We conclude that the patient's platelets have a defect in inside-out signal transduction-dependent GPIIb-IIIa activation due to an upstream defect in the signal transduction mechanisms rather than in the GPIIb-IIIa complex itself. Our findings extend the spectrum of congenital mechanisms leading to impaired aggregation from defects in GPIIb-IIIa per se to aberrations in signaling mechanisms.
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PMID:Abnormal inside-out signal transduction-dependent activation of glycoprotein IIb-IIIa in a patient with impaired pleckstrin phosphorylation. 860 26

During platelet activation, receptor-coupled phospholipid hydrolysis stimulates protein kinase C (PKC) and results in the phosphorylation of several proteins, the most prominent being pleckstrin. Pleckstrin is composed of two repeated domains, now called pleckstrin homology (PH) domains, separated by a spacer region that contains several consensus PKC phosphorylation sites. To determine the role of PKC-dependent phosphorylation in pleckstrin function, we mapped the phosphorylation sites in vivo of wild-type and site-directed mutants of pleckstrin expressed in COS cells. Phosphorylation was found to occur almost exclusively on Ser-113 and Ser-117 within the sequence 108-KFARKS*TRRS*IRL-120. Phosphorylation of these sites was confirmed by phosphorylation of the corresponding wild-type and mutant synthetic peptides in vitro.
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PMID:Phosphorylation of human pleckstrin on Ser-113 and Ser-117 by protein kinase C. 861 92

The major substrate of protein kinase C(PKC) in platelets is the 40 kDa protein, pleckstrin. Addition of the homobifunctional reagent, bis(sulphosuccinimidyl)suberate (BS3), to platelet lysate, cytosol fraction or to electropermeabilized platelets resulted in cross-linking of pleckstrin to give higher-molecular-mass complexes of 68 kDa, 90 kDa and 100-120 kDa respectively, which were visualized by immunoblotting with an anti-pleckstrin antibody. Higher levels of cross-linking were observed in permeabilized platelets than in platelet lysates. The yields of the cross-linked complexes were much reduced after dilution of platelet lysate or lysis of electropermeabilized platelets and, in the case of the 90 kDa and 100-120 kDa species, after activation of PKC by phorbol 12-myristate 13-acetate. Similar experiments with purified pleckstrin indicated that the 90 kDa and 100-120 kDa species consist, at least in part, of pleckstrin dimers and higher oligomers. After incubation of purified pleckstrin (0.45 mg/ml) for 1 h with 2 mM BS3, about 25% of the protein was present in cross-linked species. The results indicate that pleckstrin undergoes a reversible self-association that can be prevented by phosphorylation of the protein, and also interacts with an unidentified platelet protein of about 28 kDa.
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PMID:Chemical cross-linking of pleckstrin in human platelets: evidence for oligomerization of the protein and its dissociation by protein kinase C. 869 52

Vav, a 95 kDa proto-oncogene product expressed specifically in hematopoietic cells, was originally isolated as a transforming human oncogene. Vav contains an array of functional domains that are involved in interactions with other proteins and, possibly, with lipids. These include, among others, a putative guanine nucleotide exchange domain, a cysteine-rich region similar to the phorbol ester/diacylglycerol-binding domain of protein kinase C, a pleckstrin-homology domain, and Src-homology 2 and 3 (SH2 and SH3, respectively) domains. The presence of these domains, the transforming activity of the vav oncogene, and the rapid increase in tyrosine phosphorylation of Vav induced by triggering of diverse receptors indicate that it plays an important role in hematopoietic cell signaling pathways. Such a role is supported by recent studies using "knockout" mice and transiently transfected T cells, in which Vav deletion or overexpression, respectively, had marked effects on lymphocyte development or activation. The presence of a putative guanine nucleotide exchange domain, the prototype of which is found in the dbl oncogene product, implies that Vav functions as a guanine nucleotide exchange factor (GEF) for one (or more) members of the Ras-like family of small GTP-binding proteins. In support of such a role, Vav preparations were found in some (but not other) studies to mediate in vitro-specific GEF activity for Ras. Additional studies are required to identify the physiological regulators and targets of Vav, and its exact role in hematopoietic cell development and signaling.
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PMID:Vav: function and regulation in hematopoietic cell signaling. 872 92

The biochemical mechanisms of platelet activation by lysophosphatidic acid were investigated. Lysophosphatidic acid interacts with a membrane receptor coupled to the inhibitory GTP-binding protein Gi and produces a rapid decrease of the intracellular concentration of cAMP. Aggregation of gel-filtered platelets by lysophosphatidic acid requires the presence of extracellular CaCl2, as this phospholipid does not induce secretion of platelet dense granules. Platelet activation by lysophosphatidic acid in the absence of extracellular CaCl2 does not involve phospholipase C activation, as evaluated by measuring mobilization of Ca2+ from internal stores and pleckstrin phosphorylation, but causes the rapid tyrosine phosphorylation of several intracellular proteins. Our results indicate that activation of intracellular tyrosine kinases is not secondary to Ca2+ mobilization and protein kinase C activation in lysophosphatidic acid-stimulated platelets.
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PMID:Activation of human platelets by lysophosphatidic acid. 873 32


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