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)

Desensitization or habituation to repeated or prolonged stimulation is a common property of secretory cells. Phosphorylation of receptors mediates some desensitization processes, but the relationship of phosphorylation to desensitization at postreceptor sites is not well understood. We have tested the effect of protein phosphorylation on desensitization in bovine chromaffin cells. To increase protein phosphorylation, we have used the protein phosphatase inhibitor okadaic acid at 12.5 nM, 100 microM 8-bromo-cyclic AMP to activate protein kinase A, and 10 nM phorbol 12,13-dibutyrate to activate protein kinase C. During repeated 6-s stimulation at 5-min intervals, catecholamine secretion from control cells decreases. Cells exposed to 8-bromo-cyclic AMP or okadaic acid alone show slightly decreased rates of desensitization. In cells pretreated with phorbol 12,13-dibutyrate, desensitization is blocked. Okadaic acid-treated cells stimulated in the presence of 8-bromo-cyclic AMP show potentiation of secretion with repeated stimulation. The protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) increases the desensitization rate. Because these phenomena are observed during secretion evoked with elevated K+ as well as by a nicotinic agonist, the effect of phosphorylation is at a postreceptor site. In contrast to desensitization to the repeated stimulations, desensitization to prolonged stimulation with high K+ is not altered by the above protocols in chromaffin cells.
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PMID:Protein phosphorylation at a postreceptor site can block desensitization and induce potentiation of secretion in chromaffin cells. 838 51

Autophagy, measured as the sequestration of electroinjected [3H]raffinose or endogenous lactate dehydrogenase, was inhibited in isolated rat hepatocytes by the protein phosphatase inhibitors okadaic acid, calyculin A and microcystin-LR. Okadaic acid, the most potent inhibitor, suppressed autophagy almost completely at 15 nM, suggesting inhibition of a protein phosphatase of type 2A. Okadaic acid had no effect on ATP levels, protein synthesis or cellular viability at this concentration, but caused a disruption of the hepatocytic cytoskeleton and a consequent reduction in organelle sedimentability, potentially interfering with the autophagy assay unless the necessary precautions are taken. Lysosomal (propylamine-sensitive) degradation of endogenous protein was inhibited by okadaic acid, whereas non-lysosomal (propylamine-resistant) degradation was unaffected. The autophagy-inhibitory effect of okadaic acid was not affected by inhibitors of cAMP-dependent protein kinase or protein kinase C (H-7, H-89, calphostin C) but eliminated by the non-specific inhibitor K-252a and its analogues (KT-5720, KT-5823, KT-5926) and by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. Protein phosphorylation by this kinase would thus seem to play a role in regulation of the autophagic-lysosomal degradation pathway.
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PMID:Inhibition of hepatocytic autophagy by okadaic acid and other protein phosphatase inhibitors. 839 87

Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, and teleocidin, an activator of protein kinase C, are both potent tumor promoters on mouse skin. The effects of simultaneous treatment of the two different types of tumor promoters on tumor promotion as well as on their biochemical activities were studied. Three independent experiments with different doses of tumor promoters revealed that simultaneous repeated applications of okadaic acid and teleocidin did not induce any synergistic or additive effects on tumor promotion in mouse skin initiated with 7,12-dimethylbenz(a)anthracene (DMBA). In Experiment 1, the group treated with a single application of DMBA, followed by repeated applications of 1.0 micrograms (1.2 nmol) okadaic acid and 2.5 micrograms (5.7 nmol) teleocidin, resulted in 64.3% tumor-bearing mice at week 20. But the groups treated with DMBA plus okadaic acid or DMBA plus teleocidin gave 73.3% and 71.4%, respectively. The biochemical activities were studied by means of induction of ornithine decarboxylase in mouse skin and protein phosphorylation in the cells. Simultaneous application of okadaic acid at three different doses with teleocidin did not induce ornithine decarboxylase activity synergistically or additively. Phosphorylation of proteins, cytokeratins, or heat shock protein 27 was not synergistically increased in human keratinocytes treated with okadaic acid and teleocidin, although the cotreatment in a cell-free system synergistically increased protein phosphorylation. Thus, the absence of synergistic effects on tumor promotion in mouse skin was also confirmed in two systems, induction of ornithine decarboxylase in mouse skin and protein phosphorylation in human keratinocytes. The effect of cotreatment of okadaic acid and teleocidin is discussed at the molecular level.
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PMID:Absence of synergistic effects on tumor promotion in CD-1 mouse skin by simultaneous applications of two different types of tumor promoters, okadaic acid and teleocidin. 843 47

Ligation of the membrane immunoglobulin M receptor as well as stimulation with the protein kinase C agonist phorbol 12-myristate 13-acetate leads to a B-lymphocyte proliferation and differentiation. Both stimuli activate p42 mitogen-activated protein (MAP) kinase in human B-lymphocytes [Casillas, Hanekom, Williams, Katz and Nel (1991) J. Biol. Chem. 266, 19088-19094]. MAP kinase activation is dependent on tyrosine as well as threonine phosphorylation of the kinase and its activity is inhibited by tyrosine as well as threonine/serine phosphatases. Okadaic acid, a specific inhibitor of type 1 and 2A serine/threonine phosphatases, induced MAP kinase activity in a potent and dose-dependent fashion, but failed to induce [3H]thymidine incorporation into normal human tonsil B-cells. Moreover, in combination with membrane immunoglobulin M ligation, okadaic acid decreased rather than increased [3H]thymidine incorporation. The kinetics of MAP kinase activation by okadaic acid differed from phorbol 12-myristate 13-acetate and anti-membrane immunoglobulin M stimulation. Okadaic acid induced tyrosine phosphorylation of 42 kDa and 44 kDa proteins which co-electrophoresed and co-chromatographed with ERK-2 and ERK-1 respectively. Ramos cells also contained a constitutively active 46 kDa MAP kinase which appeared as a separate peak in chromatography and could be immunoprecipitated by an antiserum against a rat ERK-1 fusion protein.
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PMID:Okadaic acid activates p42 mitogen-activated protein kinase (MAP kinase; ERK-2) in B-lymphocytes but inhibits rather than augments cellular proliferation: contrast with phorbol 12-myristate 13-acetate. 845 45

Vanadate, an inhibitor of protein tyrosine phosphatases (PTPases), elicited time-and-dose-dependent increases in glucose transport in rat muscle L6 cells in culture: the rate was increased by 150-175% over control in 24 h at 75-100 microM. In contrast, molybdate, another inhibitor of PTPases, failed to stimulate glucose transport. The effect of vanadate was not blocked by tyrosine kinase inhibitors, genistein or tyrphostin RG 50864, implying that tyrosine kinase activation may not mediate the action of vanadate. The ability of vanadate to stimulate glucose transport was preserved in cells whose protein kinase C (PKC) activity was down-regulated by prior exposure to phorbol esters (TPA), suggesting that the vanadate effect was unrelated to the TPA-sensitive PKC isoform(s). Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, was a potent activator of glucose transport increasing the rate 7-fold in 24 h at a concentration of 50 nM. The increases in GLUT-1 mRNA level in response to vanadate and TPA were paralleled bh much smaller increases in immunoreactive GLUT-1 protein level, whereas okadaic acid treatment markedly elevated GLUT-1 protein without a concomitant change in GLUT-1 mRNA levels.
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PMID:Differential regulation of glucose transport and glucose transporter (GLUT-1) gene expression by vanadate, phorbol ester and okadaic acid in L6 skeletal muscle cells. 858 51

Interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) induce tissue factor in endothelium, which results in activation of the coagulation cascade. Despite extensive investigation, in which various stimuli that induce tissue factor have been defined, the intracellular processes that control tissue factor expression are not well understood. It has been proposed that protein kinase C regulates tissue factor expression primarily because phorbol myristate acetate, the protein kinase C activator, induces tissue factor expression. In this study we examined whether IL-1 alpha- or TNF-alpha-stimulated tissue factor production is regulated through a protein kinase C-dependent mechanism. Northern blot analysis showed that cytokine-induced tissue factor mRNA was significantly reduced in human umbilical vein endothelial cells treated with calphostin C, a specific protein kinase C inhibitor. Tissue factor functional activity was decreased in the presence of calphostin C as well. Calphostin C also inhibited phorbol myristate acetate-induced tissue factor expression. In contrast, calphostin C did not alter cytokine induction of E-selectin or prostacyclin release. Because calcium stimulates protein kinase C binding to the membrane and its resulting catalytic activity, human umbilical vein endothelial cells were exposed to IL-1 alpha or TNF-alpha in the presence of calcium ionophore A23187. A23187 had little effect alone but significantly augmented cytokine stimulation of tissue factor mRNA. Okadaic acid, a phosphatase inhibitor, increased cytokine-induced tissue factor mRNA compared with cytokine alone, which suggests that a phosphorylation event is important in tissue factor expression. These results indicate that protein kinase C is involved in cytokine activation of endothelial cell tissue factor expression.
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PMID:Protein kinase C regulates cytokine-induced tissue factor transcription and procoagulant activity in human endothelial cells. 859

Washed intact human platelets were prelabelled with [3H]arachidonic acid ([3H]AA) and stimulated with thrombin or with AlF4-, a known unspecific activator of G-proteins. Both stimuli induced the liberation of [3H]AA, the release of beta-thromboglobulin (beta-TG) and platelet aggregation. PMA did not induce liberation of [3H]AA although it induced beta-TG release and aggregation; preincubation with PMA did not modify significantly the amounts of [3H]AA and beta-TG released by thrombin or AlF4-. Different inhibitors of PKC (staurosporine, H-7 and calphostin C) increased the release of [3H]AA and inhibited beta-TG release and aggregation induced by AlF4- but they had no effect when platelets were stimulated with thrombin (0.5 U/ml). Calphostin C was able to release [3H]AA by itself without inducing aggregation of beta-TG release. Okadaic acid (a serine/threonine phosphoprotein phosphatase inhibitor) greatly inhibited the release of [3H]AA, beta-TG and aggregation in AlF4--stimulated platelets. These results indicate the presence of a G-protein mediated mechanism for the activation of a platelet phospholipase A2 which is negatively affected by a protein kinase, sensible to putative inhibitors of protein kinase C, and it is activated by a protein phosphatase, sensible to okadaic acid.
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PMID:Protein kinase C inhibitors enhance G-protein induced phospholipase A2 activation in intact human platelets. 860 64

The intracellular signalling systems involved in the chronic insulin-antagonistic, anti-lipogenic effects and also the lipolytic effect of GH have been investigated in sheep adipose tissue in an in vitro tissue culture system. During culture, chronic exposure to GH decreased the rate of lipogenesis and prevented the increase in lipogenesis induced by insulin. GH also increased glycerol release into the culture medium. GH had no acute, insulin-like effect on lipogenesis in sheep adipose tissue. Pretreatment with phorbol ester to down-regulate isoforms of protein kinase C or addition of the protein serine kinase inhibitor staurosporine decreased the anti-lipogenic effect of GH while the protein serine kinase inhibitor H7 eliminated it completely. Pretreatment with phorbol ester or addition of H7 also decreased the insulin-antagonistic effect of GH on lipogenesis. Addition of the protein serine phosphatase inhibitor okadaic acid or the phosphatidyl choline phospholipase C inhibitor D609 both diminished the anti-lipogenic and insulin-antagonistic effects of GH. Chronic exposure of adipose tissue to GH had no effect on the total activity of acetyl CoA carboxylase or its activation status but it did diminish the increase in activation status induced by insulin. H7 and okadaic acid also diminished the increase in activation status of acetyl CoA carboxylase induced by insulin but did not alter the effect of GH on this variable. Okadaic acid decreased total acetyl CoA carboxylase activity. Pretreatment with phorbol ester or the addition of H7, staurosporine or okadaic acid increased glycerol release into the culture medium to the same extent as GH itself; the effects of GH and these various agents were not additive. These studies suggest that the anti-lipogenic, insulin-antagonistic effects of GH involve both protein serine kinases and phosphatases, possibly including one or more isoforms of protein kinase C, and a phosphatidyl choline-specific phospholipase C. Comparison with studies by others on the GH enhancement of preadipocyte differentiation and prolactin stimulation of lipogenesis in mammary tissue suggests involvement of protein kinase C at an early stage in all three systems. In contrast, effects of okadaic acid vary with the system, suggesting the involvement of protein serine phosphatase activity in a late stage of the action of GH. The effects of GH on lipogenesis and lipolysis do not occur via identical mechanisms.
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PMID:GH inhibition of lipogenesis and stimulation of lipolysis in sheep adipose tissue: involvement of protein serine phosphorylation and dephosphorylation and phospholipase C. 870 54

The identification of three highly conserved phosphorylation sites in the cytoplasmic domain of each of the monomeric subunits of the macrophage scavenger receptor suggests that protein phosphorylation may regulate this receptor pathway. To investigate this, mouse peritoneal macrophages were pretreated with either the protein phosphatase inhibitor okadaic acid or the protein kinase inhibitor staurosporine to modulate cellular protein phosphorylation and their effects on the metabolism of acetyl-LDL were measured. Both okadaic acid and staurosporine inhibited the degradation of acetyl-low density lipoprotein (LDL) without affecting cellular lactic dehydrogenase (LDH) levels. The inhibition by okadaic acid was due to a 70% decrease in acetyl-LDL binding whereas post-receptor processing was minimally affected. Calyculin A, another serine/threonine phosphatase inhibitor, also reduced acetyl-LDL binding, whereas lithium chloride, an inositol phosphatase inhibitor, did not. Okadaic acid did not decrease steady state receptor mRNA levels nor decrease the number of total cellular receptors, consistent with a posttranslational mechanism of action. Interestingly, protease sensitivity studies showed that the receptors were still located on the cell surface. These studies suggest that okadaic acid inhibits acetyl-LDL binding by causing the redistribution of surface receptors into a sequestered compartment or inactivating the receptors. In contrast, staurosporine produced a paradoxical increase in receptor expression (30%) but slowed post-receptor processing (2.3-fold decrease). The latter was due to an inhibition of ligand internalization (2.9-fold decrease) via a protein kinase C-independent mechanism. Macrophage pinocytosis was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also investigated and it was established that the receptor can be phosphorylated; however, changes in receptor function did not correlate with changes in the degree of receptor phosphorylation. Together these studies demonstrate that changes in cellular protein phosphorylation affect the expression, surface transport, and internalization of the macrophage scavenger receptor and suggest that the regulated phosphorylation/dephosphorylation of cellular proteins may be an important biochemical mechanism that controls normal processing of ligands by this receptor pathway.
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PMID:Modulation of macrophage scavenger receptor transport by protein phosphorylation. 872 20

C6 glioma cells were used as a model system to study the regulation of EAAC1-mediated Na(+)-dependent L-[3H]glutamate transport. Although a 30-min preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12-myristate 13-acetate (PMA) increased transport activity two- to threefold. PMA caused a time-dependent and concentration-dependent increase in EAAC1-mediated L-[3H]glutamate transport activity. A 2-min preincubation with PMA was sufficient to cause more than a twofold increase in transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase. These data suggest that this increase is independent of protein synthesis. The EC50 value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5-fold increase in Vmax with no change in Km. PMA also increased the transport of the nonmetabolizable analogue, D-[3H] aspartate to the same extent. In parallel assays, PMA did not, however, increase Na(+)-dependent glycine transport activity in C6 glioma. The inactive phorbol ester alpha-phorbol 12,13- didecanoate, did not stimulate L-[3H]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca2+ ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride-sensitive Na(+)/H+ transport activity in C6 glioma. In the present study, pre- and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transport activity. This rapid increase in Na(+)-dependent L-[3H]-glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS.
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PMID:Rapid stimulation of EAAC1-mediated Na+-dependent L-glutamate transport activity in C6 glioma cells by phorbol ester. 876 74


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