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)

Platelets from stroke-prone spontaneously hypertensive rats (SHRSP) show severe hypofunctions accompanied by defective protein (P47) phosphorylation. To examine the mechanism of platelet hypofunctions, phospholipid metabolism in SHRSP was compared with that in Wistar Kyoto rats (WKY). Phosphatidylinositol (PI) content was 20% less in SHRSP than in WKY, but no changes were observed in other phospholipids. Incorporation of [3H]-arachidonic acid (AA) into PI and phosphatidylethanolamine (PE) was 12% and 11% lower, and that into phosphatidylcholine (PC) was 6% higher in SHRSP than in WKY. Thrombin-induced diacylglycerol and phosphatidic acid formation were similar in both groups of platelets. Thrombin-induced release of [14C]-AA from the labeled platelets and its metabolism to eicosanoids occurred at similar rates. These results suggest that reduced formation of diacylglycerol, an activator of protein kinase C (PKC), does not cause defective phosphorylation of P47, a substrate of PKC, in SHRSP. However it remains unclear how the lower PI content and the altered distribution of AA in PC and PE is related to SHRSP platelet hypofunctions.
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PMID:Phospholipid metabolism in platelets from stroke-prone spontaneously hypertensive rats and Wistar Kyoto rats. 132 96

We showed previously that direct platelet activation by collagen involves an increase in the platelet cytosolic free Ca2+ concentration ([Ca2+]i) but that this increase is not required for the adhesion of platelets to collagen. We now report that collagen-induced arachidonic acid liberation, myosin phosphorylation and 5-hydroxytryptamine secretion are dependent on increases in [Ca2+]i, as they were markedly inhibited in platelets loaded with the acetoxymethyl ester of the Ca2+ chelator BAPTA but not in cells loaded with the acetoxymethyl ester of the non-chelating diazo-3. BAPTA also partially inhibited the rate of collagen-induced phosphatidic acid (PtdA) formation but had little effect on increases in phosphorylation of pleckstrin (47 kDa protein; P47). From these results we infer that collagen-induced increases in [Ca2+]i are required for dense granule secretion and arachidonic acid liberation, but are not necessary for stimulation of the protein kinase C pathway.
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PMID:Cytosolic calcium as a second messenger for collagen-induced platelet responses. 147 5

A cytosolic factor of 47 kDa required for activation of the NADPH oxidase, and referred to as p47, has been purified in its functional form from the cytosol of resting bovine neutrophils. The purification was monitored by the determination of the activating potency of p47 in a cell-free system of oxidase activation. The recovery was around 10% and the purification factor greater than 1000. P47 was phosphorylated in vitro by protein kinase A and protein kinase C. [32P] labeled p47 was resolved by isoelectric focusing into two major labeled bands of pI 7.0 and 8.5. Polyclonal antibodies were used to demonstrate that p47 is localized specifically in the cytosol of resting neutrophils, and that, upon activation of neutrophils, p47 is translocated from the cytosol to the membrane.
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PMID:Purification and properties of a functional 47-kilodalton cytosolic factor required for NADPH-oxidase activation in bovine neutrophils. 149 61

The protein kinase C (PKC) activator phorbol 12,13-dibutyrate stimulated the phosphorylation of a 75 kDa protein (p75) in intact cultured A10 smooth-muscle cells and sonicated cell preparations; p75 was the only major substrate for endogenous PKC in sonicated A10 cells. The Ca(2+)-dependent phosphorylation of p75 in vitro was dramatically decreased in PKC-down-regulated A10 cells; however, p75 from identical sonicated cell preparations was still phosphorylated by an exogenous aortic PKC preparation. Calmodulin inhibited the phosphorylation of p75 by PKC, but not the phosphorylation of other PKC substrates (platelet P47 protein and histone). The addition of calmodulin after the phosphorylation reaction was started prevented further phosphorylation, but did not decrease the extent of phosphorylation of p75 that was reached before the addition of calmodulin. The inhibition of p75 phosphorylation was concentration-dependent, with IC50 values (concn. giving 50% inhibition) ranging from less than 0.5 to 10 micrograms of calmodulin/ml, and was Ca(2+)-dependent, requiring a free Ca2+ concentration of 10 microM or greater. These results suggest that the inhibition of the PKC-catalysed phosphorylation of p75 by calmodulin may be due to its interaction with the substrate, rather than a direct inhibitory effect on the enzyme, and that this inhibition could be regulated by intracellular Ca2+ concentration. Therefore, p75 may be a physiological link between the PKC and Ca2+/calmodulin pathways.
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PMID:Calmodulin inhibits the protein kinase C-catalysed phosphorylation of an endogenous protein in A10 smooth-muscle cells. 185 72

In previous reports, we have provided evidence indicating that newly formed histamine is an intracellular messenger in human platelets. The involvement of protein kinase C (PKC) and intracellular calcium (Ca2+i) in the synthesis of histamine was investigated. Human platelets were stimulated by phorbol 12-myristate 13-acetate (PMA), collagen and the Ca2+ ionophore A23187, with or without the PKC inhibitor staurosporine. Aggregation, histamine synthesis and phosphorylation of pleckstrin (47 kDa; P47) and myosin light chain (20 kDa; P20) proteins were monitored. Staurosporine inhibited PMA- and collagen-induced aggregation, histamine synthesis and phosphorylation of 47 kDa and 20 kDa proteins in a dose-dependent manner. For PMA, median inhibitory concentrations (IC50 values) for staurosporine inhibition of aggregation, histamine synthesis and phosphorylation were similar, suggesting that histamine synthesis induced by this agonist may be a consequence of PKC activation. Conversely, collagen-stimulated histamine synthesis was inhibited by staurosporine at concentrations significantly higher than those required to inhibit aggregation (P less than 0.005) or pleckstrin phosphorylation (P less than 0.01), indicating the possible involvement of non-PKC mechanism(s) in the synthesis of histamine induced by this agonist. A23187 failed to induce the synthesis of intracellular histamine in platelets, whereas staurosporine blocked A23187-induced aggregation and phosphorylation of the 20 kDa protein at significantly higher concentrations than those needed to inhibit PKC. When platelets were stimulated with a combination of A23187 and PMA, the increase in platelet histamine was less than that with PMA alone. The results provide evidence that the synthesis of intracellular histamine in platelets occurs as a consequence of PKC activation and may be down-regulated under conditions where there is a substantial rise in [Ca2+]i.
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PMID:Synthesis of intracellular histamine in platelets is associated with activation of protein kinase C, but not with mobilization of Ca2+. 189 34

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.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Changes in phosphoinositide turnover, Ca2+ mobilization, and protein phosphorylation in platelets from NIDDM patients. 217 10

Since protein kinase C (PKC) plays an important role in the control of platelet biological responses, we investigated whether it can be involved in the enhanced platelet reactivity to thrombin which is observed in spontaneously hypertensive rats (SHR) in comparison to that observed in controls (WKY). PKC activity was determined by measuring the phosphorylation of P47 protein (the endogenous substrate of PKC in the platelet). Mean effective concentration (EC50) values for phorbol ester and synthetic diacylglycerol (direct activators of PKC) were similar in SHR and WKY, thus revealing similar intrinsic activity of PKC in both rat strains. EC50 values for thrombin were approximately 30% lower in SHR v WKY. Enhanced PKC activity in SHR is likely the result of an increased diacylglycerol formation and release of Ca2+ from intracellular pools, consequent to an increased thrombin-induced phospholipase C activity.
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PMID:Protein kinase C and platelet reactivity in spontaneously hypertensive rats. 226 Nov 54

Aggregating agents including phorbol esters which activate protein kinase C induce the rapid phosphorylation of a Mr = 47,000 cytosolic protein in blood platelets (P47 or pleckstrin). This protein is well resolved by analytical 16-BAC----SDS two-dimensional PAGE and was purified from platelets by preparative 16-BAC----SDS PAGE. Polyclonal antibodies were raised to the protein in mice and rabbits. These antisera detected a single protein with the migration of P47 on Western blots of platelet extracts, and the rabbit antisera immunoprecipitated 32P-labelled P47 from platelet cytosol. The presence of P47 in other haematopoietic cells was determined by prelabelling them with 32P and observing increased 32P incorporation into the location of P47 on autoradiographs of 16-BAC----SDS analytical PAGE of cells exposed to phorbol ester. The identity of the phosphoprotein found in this location was further established by probing Western blots of SDS PAGE gels of cultured cell lines with the P47 antisera. P47 was detected in peripheral blood lymphocytes, monocytes and granulocytes (including the granulocytes of two unrelated patients with X-linked chronic granulomatous disease). P47 was also found in HL-60 promyelocytes (especially after differentiation with retinoic acid), U937 histiocytes, HEL leukaemia cells, and Raji 'B' lymphoblasts. It was not detected in normal erythrocytes, K562 leukaemic cells, MOLT-3 'T' lymphoblasts, or in wide range of non-haematopoietic cell lines. We conclude that P47 is a major target for the action of phorbol ester induced phosphorylation in platelets, normal leucocytes and some haematopoietic cell lines. These cells have as their common feature the ability when stimulated to develop adhesive functions on their plasma membranes.
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PMID:P47 phosphoprotein of blood platelets (pleckstrin) is a major target for phorbol ester-induced protein phosphorylation in intact platelets, granulocytes, lymphocytes, monocytes and cultured leukaemic cells: absence of P47 in non-haematopoietic cells. 231 54

Platelet function is inhibited by agents such as prostaglandin E1 (PGE1) that elevate the cytoplasmic concentration of cyclic AMP. Inhibition presumably results from the cyclic AMP-stimulated phosphorylation of intracellular proteins. Polypeptides that become phosphorylated are actin-binding protein, P51 (Mr = 51,000), P36 (Mr = 36,000), P24 (Mr = 24,000), and P22 (Mr = 22,000). Recently, we identified P24 as the beta-chain of glycoprotein (GP) Ib, a component of the plasma membrane GP Ib.IX complex. The existence of Bernard-Soulier syndrome, a hereditary disorder in which platelets selectively lack the GP Ib.IX complex, enabled us to examine whether the phosphorylation of GP Ib beta (P24) is responsible for any of the inhibitory effects of elevated cyclic AMP on platelet function. Exposure of control platelets to PGE1 increased phosphorylation of actin-binding protein, P51, P36, GP Ib beta, and P22. Prostaglandin E1 induced the same phosphorylation reactions in Bernard-Soulier platelets, except that of GP Ib beta, which is absent. In control platelets, PGE1 inhibited collagen-induced phosphorylation of myosin light chain, phosphorylation of P47 (an unidentified Mr 47,000 cytoplasmic protein that is phosphorylated by protein kinase C in stimulated platelets), aggregation, and the secretion of granule contents. Despite the absence of GP Ib beta, PGE1 also inhibited these collagen-induced responses in Bernard-Soulier platelets. However, while PGE1 inhibited collagen-induced polymerization of actin in control platelets, it did not inhibit actin polymerization in Bernard-Soulier platelets. These results suggest that cyclic AMP-induced phosphorylation of GP Ib inhibits collagen-induced actin polymerization in platelets. Because actin polymerization is required for at least some of the functional responses of platelets to an agonist, phosphorylation of Gp Ib beta may be one way in which cyclic AMP inhibits platelet function.
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PMID:Cyclic AMP-dependent phosphorylation of glycoprotein Ib inhibits collagen-induced polymerization of actin in platelets. 254 12

The protein kinase C activators phorbol myristate acetate (PMA), mezerein, oleoylacetylglycerol, and (-)-indolactam V, although without direct effect on arachidonic acid release, greatly enhance the release of platelet arachidonic acid caused by the Ca2+ ionophores A23187 and ionomycin. In contrast, 4 alpha-phorbol 12,13-didecanoate and (+)-indolactam V, which lack the ability to activate kinase C, do not potentiate arachidonate release. Release of arachidonic acid occurs without activation of phospholipase C and is therefore mediated by phospholipase A2. Synergism between PMA and A23187 is not affected by inactivation of the Na+/H+ exchanger with dimethylamiloride. The time course and dose-response for the effect of PMA at 23 degrees C closely correlate with the phosphorylation of a set of relatively "slowly" phosphorylated proteins (P20, P35, P41, P60), but not the rapidly phosphorylated P47 protein. P20 is myosin light chain, and P41 is probably Gi alpha, but the other proteins have not been positively identified. Depletion of metabolic ATP stores by antimycin A plus 2-deoxyglucose abolishes both protein phorphorylation and the potentiation of arachidonate release by PMA, but does not prevent fatty acid release by the ionophores. Similarly, the kinase C inhibitors H-7 and staurosporine produce, respectively, partial and complete inhibition of PMA-potentiated arachidonic acid release and protein phosphorylation, without affecting the direct response to ionophores. These results indicate that protein phosphorylation, mediated by kinase C, promotes the phospholipase A2 dependent release of arachidonic acid in platelets when intracellular Ca2+ is elevated by Ca2+ ionophores.
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PMID:Synergistic release of arachidonic acid from platelets by activators of protein kinase C and Ca2+ ionophores. Evidence for the role of protein phosphorylation in the activation of phospholipase A2 and independence from the Na+/H+ exchanger. 255 68


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