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

Increases in the intracellular Ca2+ concentration of human platelets caused by receptor agonists, such as thrombin, 9,11-epithio-11,12-methanothromboxane A2 (STA2), platelet-activating factor (PAF) and arginine-vasopressin, were inhibited by prior addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) in time-dependent and concentration-dependent manners. The inhibitions were mostly reversed by staurosporine, and inhibitor of protein kinase C, added 1 min before TPA. Prior treatment of platelets with thrombin or STA2, the efficacious Ca2+ mobilizer, suppressed the increase in the intracellular Ca2+ concentration of the cells to other agonists, but treatment with less efficacious PAF or vasopressin did not. The heterologous receptor desensitizations were also reversed by staurosporine. The antibody, directed against the carboxy-terminal region of the alpha subunits 1 and 2 of the inhibitory guanine-nucleotide-binding proteins (Gi1 alpha and Gi2 alpha), was raised in rabbit and was used to immunoprecipitate Gi alpha in 32P-labeled platelets. The radioactivity was detected in Gi alpha after incubation of 32P-labeled platelets with TPA, thrombin or STA2, but not in the cells incubated with PAF or vasopressin. The time-dependency or concentration-dependency of TPA-induced phosphorylation of Gi alpha was similar to the dependency of its inhibitory action on agonist-induced Ca2+ mobilization. Thus, strong activation of Ca2+/phospholipid-dependent protein kinase C by phorbol ester or agonists of certain Ca(2+)-mobilizing receptors leads to phosphorylation of the alpha subunit of guanine-nucleotide-binding protein, thereby impairing the coupling of the G protein to receptors as a feedback regulatory component of the receptor-triggered intracellular Ca(2+)-mobilizing system.
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PMID:Phosphorylation of the inhibitory guanine-nucleotide-binding protein as a possible mechanism of inhibition by protein kinase C of agonist-induced Ca2+ mobilization in human platelet. 157 85

We investigated the intracellular processes of the shape change in the human megakaryoblastic leukemia cell, MEG-01, by platelet agonists. Thrombin induced the formation of many pseudopods. This shape change was also induced by TPA and A23187, but not by ADP, collagen, or epinephrine. Electron microscopy and FITC-labeled phalloidin staining revealed thick submembranous microfilament bundles in the pseudopods of the shape-changed cells induced by thrombin. Shape change was inhibited by cytochalasin B. Protein kinase C (RKC) inhibitor, H-7, markedly inhibited thrombin-induced shape change, while the myosin light chain kinase (MLCK) inhibitor, ML-9 did not. These results suggest that thrombin-induced reorganization of microfilaments and shape change of MEG-01 cells are mediated by PKC but not by MLCK.
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PMID:[Shape change in human megakaryoblastic leukemia cells, MEG-01]. 161 74

Several agonists of endothelial cell function (thrombin, histamine, dioctanoylglycerol, phorbol 12-myristate 13-acetate, interleukin-1) have previously been shown to enhance the level of phosphorylation of an undefined 29,000-M(r) protein (P29). Comparison of this protein with other phosphoproteins suggested that it may be related to the mammalian heat-shock protein HSP27. Immunoprecipitation and immunoblot analysis with antibodies specific for human HSP27 demonstrated that P29 was immunochemically identical with HSP27. Further characterization of agonist-induced phosphorylation of HSP27 indicated that phosphorylation occurred exclusively on serine residues, and phosphopeptide analysis of tryptic- and chymotryptic-cleavage products demonstrated that the phosphopeptides generated were identical for each agonist and okadaic acid. Down-regulation of protein kinase C-alpha by prolonged treatment with phorbol esters eliminated the ability of phorbol 12-myristate 13-acetate, dioctanoylglycerol, thrombin and histamine to phosphorylate HSP27 above background levels and deceased interleukin-1-stimulated HSP27 phosphorylation by 60%. These data suggest that the various agonists employed stimulate HSP27 phosphorylation through similar mechanisms and that protein kinase C is probably involved.
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PMID:Identification of a protein transiently phosphorylated by activators of endothelial cell function as the heat-shock protein HSP27. A possible role for protein kinase C. 162 89

During activation of platelets by agonists, a number of proteins become phosphorylated at tyrosine residues. Using immunoblotting with a monoclonal anti-phosphotyrosine antibody, we have compared the different phosphotyrosine-protein (PTP) profiles of platelets stimulated with thrombin, collagen, ADP, arachidonic acid, phorbol myristate acetate and P256, an anti-glycoprotein-IIb-IIIa (GPIIb-IIIa) monoclonal antibody (mAb). Only a few PTPs were observed in resting platelets, of molecular masses 130, 64, 56-60 and 36 kDa. After stimulation by different agonists these proteins were more intensely phosphorylated and additional PTPs appeared with molecular masses of 170, 150, 140, 120, 105/97 (doublet), 85, 80, 75 and 45 kDa. The kinetics of phosphorylation differed from one agonist to another, but no significant differences in the overall patterns were detected, except in presence of ADP and P256-F(ab')2, which induced only the additional tyrosine phosphorylation of the 64 kDa protein and to a lesser extent that of a 75 kDa protein. The use of various agonists and the inhibitors (staurosporine, ajoene and RGDS) permitted a better characterization of the relationship between the different steps of activation and phosphorylation on tyrosine residues. The studies suggest the following conclusions: (i) stimulation of tyrosine phosphorylation occurs after activation of protein kinase C; (ii) there is a relationship between ligand binding to GPIIb-IIIa and the tyrosine phosphorylation of the 64 kDa protein; and (iii) there is a close relationship between PTP formation and the intensity of platelet activation and aggregation.
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PMID:Functional implications of tyrosine protein phosphorylation in platelets. Simultaneous studies with different agonists and inhibitors. 162 7

1. The effect of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A), on human platelets has been investigated. 2. Okadaic acid exerts a general increase in phosphorylation of platelet proteins but did not induce aggregation or secretion of 5-hydroxytryptamine (5-HT). Okadaic acid, however, did inhibit thrombin-induced functional responses. 3. Maximally effective concentrations of prostacyclin, to elevate adenosine 3'-5'-cyclic monophosphate (cyclic AMP), or phorbol dibutyrate, to activate protein kinase C, inhibited the formation of inositol phosphates by thrombin by approximately 60%. When used in combination, prostacyclin and phorbol dibutyrate reduced the levels of inositol phosphates induced by thrombin to 11%. 4. Okadaic acid (1 microM) decreased thrombin-induced formation of inositol phosphates by approximately 55% and increased the inhibitory action of prostacyclin or phorbol dibutyrate. Okadaic acid had no further effect when prostacyclin and phorbol dibutyrate were used in combination. 5. These results suggest that protein kinases A and C act to inhibit phospholipase C by distinct mechanisms and that their action is reversed by PP1 and/or PP2A.
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PMID:Okadaic acid inhibits activation of phospholipase C in human platelets by mimicking the actions of protein kinases A and C. 162 49

Human platelets were found by immunoblot analysis to express protein kinase C (PKC) isozymes alpha, beta, delta, and zeta, but not gamma, epsilon, or eta. Exposure of platelets to thrombin, in the presence of 1 mM calcium, induced increased membrane association of PKC-alpha, -beta, and -zeta, while the subcellular distribution of PKC-delta remained unaltered. Maximal membrane association (2-fold) of PKC-alpha, -beta, and -zeta occurred within 1 min and was sustained for at least 10 min after the addition of thrombin. Similar results were obtained in the presence of the RGDS peptide, which blocks thrombin-induced binding of fibrinogen to its receptor, which indicates that PKC translocation was independent of fibrinogen binding. In the absence of added extracellular calcium, thrombin-induced translocation of PKC-alpha, -beta, and -zeta was transient, reaching a maximum at 1 min and returning to base line by 10 min. In the presence of calcium, thrombin induced a rapid (within 15 s) 8-fold rise in inositol 1,4,5-trisphosphate, which returned to baseline levels within 1 min, and a biphasic increase in sn-1,2-diacylglycerol (DAG), with peaks at 15 s and 2 min, which remained elevated for at least 5 min. Chelation of external calcium abolished the second phase of DAG formation but had no effect on the kinetics or magnitude of the increase in inositol 1,4,5-trisphosphate or the first phase of DAG formation. Two early PKC-dependent functions, serotonin release and 40-kDa protein phosphorylation, were independent of extracellular calcium and sustained DAG. These data demonstrate that in thrombin-stimulated human platelets the duration of the increased PKC membrane association closely parallels that of increased DAG content, and sustained elevations in DAG content and PKC translocation are dependent on extracellular calcium.
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PMID:Translocation of protein kinase C isozymes in thrombin-stimulated human platelets. Correlation with 1,2-diacylglycerol levels. 163 99

We prepared anti-platelet 20-kDa myosin light chain (MLC-20) antibody and demonstrated diphosphorylation of MLC-20 in platelets ex vivo in the initial phase of activation by thrombin. Our results are as follows. (1) By Western blotting, using anti-MLC-20 antibody, both mono- and diphosphorylated myosin were seen in the initial phase of aggregation of platelets by thrombin. The peak of the diphosphorylation was later than that of monophosphorylation and the degree of both mono- and diphosphorylation reduced in the process of aggregation. (2) ML-7 (a synthetic inhibitor of MLCK) inhibited both mono- and diphosphorylation of myosin and also blocked aggregation of thrombin-activated platelets. However, H-7 (an inhibitor of protein kinase C) had little effect on either the (di)phosphorylation of myosin or the aggregation of thrombin-activated platelets. (3) Arg-Gly-Asp-Ser (RGDS) peptide, a synthetic anti-adhesive peptide, inhibited aggregation of thrombin-activated platelets in a dose-dependent manner (100-200 microM). However, it had little effect on either mono- or diphosphorylation of myosin in the process of the platelet aggregation stimulated by thrombin. From these results, we conclude that mono- and diphosphorylation of myosin by MLCK play a role in the initial phase of activation of thrombin-stimulated platelets in vivo and that mono- and diphosphorylation of myosin by MLCK precedes the secondary signal mediated by GPIIb/IIIa.
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PMID:Diphosphorylation of platelet myosin ex vivo in the initial phase of activation by thrombin. 164 15

Human renal glomerular epithelial cells possess membrane urokinase receptors. Addition of purified active urokinase to these cells in serum free minimum medium induced a dose-dependent increase in 3H-thymidine incorporation and a doubling of cell number after 48 hours of incubation. Both receptor occupancy and enzymatic activity of u-PA were required to stimulate cell proliferation. This effect was inhibited by down regulation of protein kinase C (PKC) or by H7, an inhibitor of PKC. It involved a pertussis toxin-sensitive pathway. This effect of urokinase was additive with EGF but not with thrombin growth factor activity and was not inhibited by aprotinin, an inhibitor of plasmin.
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PMID:Growth factor-like effect of urokinase type plasminogen activator in human renal cells. 164 44

We have found that thrombin-induced activation of protein kinase C (PKC) in platelets, measured by phosphorylation of the 47 kDa protein, is synergistically enhanced by the amiloride analogue ethylisopropylamiloride (EIA), a specific inhibitor of Na+/H+ exchange. This EIA effect was further synergistically enhanced by lowering intracellular pH (pHi) with either nigericin or sodium propionate, and reversed by raising pHi with monensin or ammonium chloride. The synergistic enhancement of thrombin-activated PKC by EIA plus nigericin was not observed when PKC was directly activated by phorbol esters. EIA and EIA plus nigericin caused a 3- to 6-fold increase in thrombin-induced diacylglycerol (DAG), but not phosphatidic acid (PA), production. EIA and nigericin also caused a marked increase in thrombin-induced breakdown and inhibition of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). In summary, we have presented evidence that inhibition of Na+/H+ exchange causes primarily a H(+)-mediated interruption of the phosphoinositide cycle in activated platelets, including the accumulation of DAG associated with the enhancement of PKC activation, the inhibition of conversion of DAG to PA, and increased PIP2 breakdown. These data suggest a model in which Na+/H+ and pHi play an important regulatory role in permitting the phosphoinositide cycle to proceed in thrombin-activated platelets.
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PMID:Regulation of the phosphoinositide cycle by Na+/H+ exchange and intracellular pH in human platelets. 164 5

Human platelets were loaded with the fluorescent Na(+)-sensitive dye sodium-binding benzofuran isophtalate (SBFI), and changes in the fluorescence excited at 345 and 385 nm were analyzed after manipulations that evoked predictable changes in the cytosolic Na+ concentration ([Na+]i). Raising [Na+]i by either gramicidin D or monensin specifically increased the fluorescence excited at 345 nm and decreased that excited at 385 nm. Hence, calculation of changes in the 345/385 nm excitation ratio yields an estimate of actual changes in [Na+]i. A transient activation of Na+/H+ exchange evoked by addition of acidified platelets to buffer, pH 7.4, evoked a transient rise in [Na+]i. The re-establishment of basal [Na+]i could be prevented by ouabain, indicating an involvement of the Na+,K(+)-ATPase. Upon stimulation by 0.5 unit/ml of thrombin, [Na+]i immediately increased by 16 +/- 4 mM and this rise continued for at least 60 min after addition of agonist, albeit at a lower rate. This latter sustained rise could not be curtailed by scavenging thrombin by means of hirudin. Addition of ouabain or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate induced a comparable slow rise in the 345/385 excitation ratio. This may indicate a protein kinase C-mediated inhibition by thrombin of the Na+,K(+)-ATPase. In the absence of extracellular Ca2+ (Ca2+o), the [Na+]i gain was augmented to 38 +/- 9 mM. This additional uptake of Na+ was prevented by (i) Mn2+ ions, (ii) La3+ ions, (iii) the blocker of receptor-mediated Ca2+ entry (1-[beta[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-im ida zole hydrochloride), and (iv) by hirudin which reversed receptor occupancy by thrombin. These findings suggest that the additional thrombin-induced [Na+]i gain in the absence of Ca2+o is due to Na+ influx through a Ca2+ entry pathway. The increase in [Na+]i in the presence of Ca2+o results from Na+ influx via Na+/H+ exchange.
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PMID:Further characterization of the mechanisms mediating the rise in cytosolic free Na+ in thrombin-stimulated platelets. Evidence for inhibition of the Na+,K(+)-ATPase and for Na+ entry via a Ca2+ influx pathway. 164 80


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