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)

Cyclosporine increases platelet aggregation as well as the risk of thromboembolism. To test the hypothesis that CsA stimulates platelets by activating cytosolic calcium [Ca2+]i and related mechanisms, we measured the effects of CsA on [Ca2+]i, protein kinase C (PKC), and sodium/proton (Na+/H+) exchange. [Ca2+]i was measured in human platelets with fura 2, PKC was determined by the phosphorylation of the endogenous 47 kDa protein, and Na+/H+ exchange was measured with BCECF after acidification of the platelets with propionic acid. CsA alone did not influence basal PKC activity in platelets. However, CsA augmented the thrombin-induced phosphorylation of the specific PKC substrate p47 in platelets in a dose-dependent fashion. CsA did not affect basal [Ca2+]i--however, it increased thrombin-induced calcium influx. The effect of CsA on PKC was not dependent on the CsA-induced calcium influx. CsA increased Na+/H+ exchange, which was blocked completely by a PKC inhibitor. Our results demonstrate that CsA directly augments PKC-dependent cellular mechanisms in platelets.
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PMID:The effect of cyclosporine on calcium, protein kinase C, and sodium-proton exchange in platelets. 819 17

Stimulation of polymorphonuclear neutrophils (PMN) by phorbol esters or formyl peptides (fMLP) generates large quantities of superoxide anion, the so-called respiratory burst (RB), a phenomenon associated with intense phosphorylation of a 47-kD protein (p47 phox). Staurosporine, a potent protein kinase C (PKC) antagonist, inhibits both responses when PMN are stimulated by phorbol myristate acetate (PMA), suggesting a positive role of PKC. In this study, we reassessed these PMN responses in fMLP-stimulated cells and found that staurosporine had opposite effects depending on the duration of PMN treatment with staurosporine. Short PMN incubation (0.5 to 3 minutes) with 25 to 100 nmol/L staurosporine inhibited the fMLP-induced RB, whereas longer treatment (15 to 20 minutes) enhanced it by up to about 200% relative to controls. In contrast, the PMA-mediated RB was depressed by staurosporine in a time-dependent manner. A primed fMLP-induced RB was also observed after long (15 minutes) PMN treatment with 5 to 100 mumol/L H-7, whereas shorter treatment (5 minutes) resulted in a small decrease in RB. By contrast, the tyrosine kinase inhibitor genistein (2 to 80 mumol/L) depressed fMLP-induced RB whatever the duration of PMN treatment. Analysis of 32P-phosphorylated proteins in fMLP-stimulated cells showed that short PMN treatment (< 8 minutes) with staurosporine abolished the phosphorylation of the 47-kD protein, which was identified as p47 phox, whereas long treatment partially restored p47 phox phosphorylation up to approximately 50% of the control value. In PMA-stimulated PMN, phosphorylation was reduced in a time-dependent manner. Furthermore, the staurosporine-primed RB and the staurosporine-induced recovery of phosphorylation were inhibited by sphingosine but not by genistein. Thus, in addition to its known depressive effect, staurosporine markedly potentiated fMLP-stimulated RB as a function of the duration of PMN treatment. The restoration of p47 phox phosphorylation suggests that staurosporine may alter the interactions between different protein kinases, producing marked time-dependent changes in signalling pathways. These data emphasize the care that should be taken in interpreting data obtained using this kinase inhibitor that may, however, be helpful analyzing in signalling pathways.
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PMID:Stimulation of the human neutrophil respiratory burst by formyl peptides is primed by a protein kinase inhibitor, staurosporine. 821 37

Phospholipase A2 (PLA2) inhibitors suppressed simultaneously, in a dose-dependent manner, the activation of NADPH oxidase and the release of 3H-labelled arachidonic acid ([3H]AA) stimulated by either phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OZ) in human neutrophils. In spite of total inhibition of superoxide production in the presence of the PLA2 inhibitors, 10 microM bromophenacyl bromide (BPB) or 20 microM quinacrine, a maximal phosphorylation of p47 and translocation of p47 and p67 to the neutrophil membranes induced by PMA or OZ was observed. Addition of 10 microM free AA, which by itself did not stimulate superoxide generation, restored oxidase activity in neutrophils treated with PLA2 inhibitors. These findings indicate that phosphorylation and translocation of the cytosolic factors to the membranes are not sufficient for generating superoxide; a functional PLA2 is also needed to stimulate the oxidase activity. The inhibition of PLA2 activity did not prevent the phosphorylation of p47, suggesting that the location of PLA2 is downstream of and does not activate protein kinase C.
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PMID:The requirement for phospholipase A2 for activation of the assembled NADPH oxidase in human neutrophils. 828 Jan 2

Use of the immunosuppressant drug cyclosporine A (CSA) has resulted in improved renal graft survival. However, an increased incidence of arterial and venous thrombotic diseases, hemolytic-uremic type syndrome, and findings resembling vasculitis in the kidneys of patients with CSA nephrotoxicity and accelerated atherogenesis have been reported. These disorders may be related to CSA-induced abnormalities in platelet function. We report here that CSA causes increased ADP-stimulated aggregation in isolated platelet suspensions indicating that CSA has a direct effect on platelet function, independent of CSA interactions with plasma factors. Maximal hyperaggregability of ADP-stimulated platelets occurred following a 1 h preincubation period with CSA. Hyperaggregability of platelets due to the presence of CSA was dose-dependent and approached plateau between 200-500 ng/ml CSA. We determined that CSA exerted its effects through a signal transduction pathway involving the phosphorylation of two intracellular proteins, a 40 kD substrate of PKC (p47) and the 20 kD light chain of myosin (p20), a substrate of calcium/calmodulin dependent kinase. Preincubation with CSA resulted in a 200% increase in the phosphorylation of these proteins in platelets stimulated with ADP. We conclude that CSA enhances ADP-induced platelet aggregation and secretion, in part, by potentiating the phosphorylative response of specific intracellular proteins to stimulation by agonists. This process may be responsible for the increased thrombosis and atherogenesis observed in CSA-treated patients.
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PMID:Cyclosporine A enhances agonist-induced aggregation of human platelets by stimulating protein phosphorylation. 829 40

High levels of fluid shear stress at the blood vessel wall directly stimulate von Willebrand factor (vWF)-mediated platelet adhesion and aggregation and thereby contribute to the pathogenesis of arterial thrombosis. We have found that a pathological level of arterial wall shear stress (90 dynes/cm2) induces platelet aggregation that is associated with the phosphorylation of pleckstrin, a M(r) 47,000 protein kinase C substrate (p47). Shear-induced p47 phosphorylation depends entirely on vWF binding to platelet glycoprotein (Gp) Ib and GpIIb-IIIa, and the specific inhibition of protein kinase C with the staurosporine analogue Ro 31-7549 inhibits the full aggregation response to shear. Shear stress-induced platelet p47 phosphorylation occurs independent of any measurable change in diacylglycerol mass or hydrolysis of phosphatidylinositol 4,5-bisphosphate. These results indicate that mechanical shear stress induces vWF to bind to platelet GpIb and GpIIb-IIIa, stimulating a diacylglycerol-independent pathway of protein kinase C activation that contributes to platelet aggregation in response to shear.
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PMID:Protein kinase C is activated in platelets subjected to pathological shear stress. 842 27

Previously employed non-selective protein kinase inhibitors yielded inconclusive results regarding involvement of protein kinase C (PKC) in phosphorylation of 47 kDa protein (p47 phox) in intact neutrophils stimulated with physiologic agonists of superoxide generation. In the present study, phosphorylation of p47 phox in formylMet-Leu-Phe (fMLP) stimulated neutrophils was potently inhibited in the presence of 0.3 microM RO 31-8220, a selective inhibitor of PKC. These results provide experimental evidence in support of the currently considered essential involvement of PKC in p47 phox phosphorylation in response to physiologic stimulation of neutrophil surface receptors. The fMLP-induced phosphorylation of p47 phox was enhanced and prolonged by calyculin A, a specific inhibitor of protein phosphatases of types 1 and 2A, and such enhanced phosphorylation was also effectively inhibited by RO 31-8220. Our results suggest that the extent and duration of p47 phox phosphorylation in intact fMLP-stimulated neutrophils is probably controlled by a balance between the activities of PKC, on the one hand, and of protein phosphatase(s) of type(s) 1 and/or 2A, on the other. Effects of RO 31-8220 and of calyculin A on the fMLP-induced p47 phox phosphorylation were paralleled by similar effects on superoxide release. Calyculin A and RO 31-8220 were also used to study signal transduction by a post-receptor agonist of superoxide generation, a calcium ionophore A23187. The results of the latter study indicated that PKC was activated in A23187-stimulated neutrophils and was essentially involved in superoxide generation and p47 phox phosphorylation. Further, these results suggested that protein phosphatase(s) of type(s) 1 and/or 2A were also activated in A23187-signalling pathway, and limited the extent of superoxide release and p47 phox phosphorylation.
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PMID:Involvement of protein kinase C and of protein phosphatases 1 and/or 2A in p47 phox phosphorylation in formylmet-Leu-Phe stimulated neutrophils: studies with selective inhibitors RO 31-8220 and calyculin A. 851 1

The effects of cell-permeable C2 and C6-ceramides on human platelet responses were investigated. In thrombin-activated platelets, C6(5-30 microM) potentiated Ca2+ mobilization and Ca2+ influx, and decreased the rate of removal of Ca2+ from cytosol. The effect of C2 was not significant. Phorbol ester or calyculin A inhibition of thrombin-induced rises in platelet [Ca2+]i was attenuated by C6. Assays show that C6 either prolonged the generation, or retarded the metabolism of inositol trisphosphates. Previous studies indicate that protein kinase C (PKC) acts in a negative feedback manner by inhibiting phosphatidylinositol breakdown, accelerating inositol trisphosphate metabolism, and increasing Ca2+ pump activity. C6 may counter these PKC effects indirectly. The synthetic ceramides inhibited platelet aggregation weakly and had no effect on pleckstrin (p47) phosphorylation. Recently we reported that C2 but not C6 inhibits superoxide generation and store-regulated Ca2+ influx in neutrophils at similar concentrations. Cellular differences in ceramide metabolism or ceramide-sensitive enzymes and their substrates may account for the disparate results.
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PMID:C6-ceramide maintains elevated cytosolic calcium levels in activated platelets. 882 37

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A(PKA) is considered to be a physiologic modulator of superoxide generation by stimulated neutrophils. Mechanisms of the inhibitory action of PKA are poorly understood. In this study, we investigated effects of cAMP-elevating agents on the phosphorylation of p47 phox in human neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe (fMLP). We observed that the fMLP-induced phosphorylation of p47 phox, an essential component of neutrophil NADPH oxidase, was significantly attenuated in the presence of dibutyryl-cAMP or of receptor agonists of adenylate cyclase. This attenuation was reversed in the presence of 0.4 microM KT 5720, a selective inhibitor of PKA. The effects of cAMP agonists and of KT 5720 on the phosphorylation of p47 phox were paralleled by similar effects on superoxide generation. In neutrophils stimulated with phorbol myristate acetate (PMA), which directly activates protein kinase C (PKC), neither cAMP agonists nor dibutyryl cAMP exerted any effects on p47 phox phosphorylation or superoxide generation. These results indicated that the PKA-dependent downregulation of fMLP-induced p47 phox phosphorylation apparently involves step(s) in the fMLP-signaling pathway that are upstream of PKC. The inhibition demonstrated here of p47 phox phosphorylation by cAMP agonists may underlie a physiologically significant mechanism whereby cAMP modulates the receptor-mediated respiratory burst in neutrophils.
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PMID:Protein kinase A downregulates the phosphorylation of p47 phox in human neutrophils: a possible pathway for inhibition of the respiratory burst. 884 30

1. Hypofunction of stroke-prone spontaneously hypertensive rat (SHRSP) platelets at developmental ages of hypertension is due to the defective protein (p47) phosphorylation which is mediated by protein kinase C. This study was undertaken to examine the genetic association of platelet functions and vascular reactivity with hypertension using male WKY, SHRSP, F1 and backcross generations at 16-18 weeks of age. 2. The distribution of blood pressure was continuous in each generation. 3. Contraction of mesenteric vascular bed with norepinephrine was positively correlated with blood pressure in the five generations (r = 0.77, n = 128). 4. Thrombin-induced platelet aggregation was inversely correlated with blood pressure (r = -0.87, n = 127). 5. The distribution of platelet aggregation and contraction was continuous in each generation, and backcross generations were not likely to have 1:1 segregation. 6. These results suggest the possibility that platelet hypoaggregability and peripheral vascular resistance are due to the pleiotropic effect of hypertensive genes, or that genes controlling these three characters are closely linked each other.
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PMID:Correlation analysis of blood pressure and platelet aggregation/vascular reactivity in SHRSP, WKY, F1 and backcross generations. 907 15

The leukocyte NADPH oxidase catalyzes the 1-electron reduction of oxygen to O2- at the expense of NADPH: 2 O2 + NADPH --> 2 O2- + NADP+ + H+. The oxidase is dormant in resting cells but acquires activity when the cells are stimulated with a suitable agent. Activation in whole cells is accompanied by extensive phosphorylation of p47(PHOX), an oxidase subunit located in the cytosol of resting cells that during oxidase activation migrates to the plasma membrane to complex with cytochrome b558, an oxidase-specific flavohemoprotein. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile as activating agent. We now report a cell-free system in which the oxidase can be activated in two stages using phosphorylated p47(PHOX). The first stage, which effects a change in the membrane, requires ATP and GTP and is blocked by the protein kinase inhibitor GF-109203X, suggesting a protein kinase requirement. The second stage requires phosphorylated p47(PHOX) and GTP, but no ATP, and is unaffected by GF-109203X; assembly of the oxidase may take place during this stage. Activation is accomplished by p47(PHOX) phosphorylated by protein kinase C but not protein kinase A or mitogen-activated protein kinase. We believe that activation by phosphorylated p47(PHOX) is more physiological than activation by amphiphiles, because the mutant p47(PHOX) S379A, which is inactive in whole cells, is also inactive in this system but works in systems activated by amphiphiles.
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PMID:Kinase-dependent activation of the leukocyte NADPH oxidase in a cell-free system. Phosphorylation of membranes and p47(PHOX) during oxidase activation. 911 Sep 96


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