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)

In order to further evaluate the role of protein kinase C activation in glucose-induced insulin secretion, the extent of phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) was examined in freshly isolated rat pancreatic islets prelabeled with [32P]orthophosphate. The islets were incubated with either 2.75 mM glucose alone, 2.75 mM glucose + 1 microM phorbol myristate acetate, 20 mM glucose, or 20 mM glucose + 50 nM staurosporine. After stimulation, the homogenized islets were processed by immunoprecipitation with a specific polyclonal anti-MARCKS antibody, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Densitometric analysis of autoradiograms revealed that phorbol myristate acetate caused a 3.78 +/- 0.97-fold increase in MARCKS phosphorylation over control. In the islets exposed to 20 mM glucose, an increase of 3.43 +/- 0.46-fold over control was observed. In islets exposed to G20 + 50 nM staurosporine, MARCKS phosphorylation was inhibited by 90 +/- 4% compared with control islets exposed to 20 mM glucose alone. Islets similarly treated (but incubated without 32P) were examined by immunocytochemistry using an alpha-PKC-specific monoclonal antibody and visualized by confocal immunofluorescence microscopy. The alpha-PKC redistributed from the cytosol to the plasma membrane in the beta-cells of islets exposed to 20 mM glucose. In separate experiments, unlabeled but similarly treated islets were shown to respond with a 5-7-fold increase in insulin secretion in static incubation. Thus, when freshly isolated rat pancreatic islets are exposed to stimulatory glucose concentrations, they exhibit both a translocation of alpha-PKC and a significant increase in the extent of phosphorylation of MARCKS protein. These data suggest that alpha-PKC is activated during glucose-induced insulin secretion.
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PMID:Glucose-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) in isolated rat pancreatic islets. 152 3

Ras has been thought to be involved in neuronal differentiation of rat pheochromocytoma PC12 cells. PC12 cells are immature adrenal chromaffin-like cells which undergo differentiation to sympathetic neuron-like cells in response to nerve growth factor (NGF). Fibroblast growth factor (FGF) and interleukin (IL)-6 can also induce differentiation of PC12 cells. In this paper, we report that NGF, FGF, and IL-6 induce an accumulation of an active Ras.GTP complex. In the serum-starved culture of PC12 cells, 6% of the Ras protein was complexed with GTP. Upon stimulation with NGF, the percentage of Ras.GTP increased to 24% after 2 min, and the high level of Ras.GTP was maintained for at least 16 h. On the other hand, the activation of Ras by FGF and IL-6 showed distinct kinetics; about 3-fold increase of Ras.GTP was detected at 10 min, and afterward, the level returned to the basal level within 60 min. These observations provide direct evidence that activation of Ras is involved in signal transduction from these differentiation factors. In addition, it was found that growth factors, including epidermal growth factor, insulin, and insulin-like growth factor-I, and a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), can also activate Ras under the same conditions. A tyrosine kinase-specific inhibitor, genistein, inhibited the increase of Ras.GTP induced by NGF and other factors. On the other hand, down-regulation of protein kinase C (PKC) by prolonged treatment with TPA, which sufficiently blocked TPA-induced Ras activation, did not abolish the formation of Ras.GTP by NGF. These results suggest that tyrosine kinases rather than PKC play a major role in the NGF-induced activation of Ras in PC12 cells.
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PMID:Differentiation factors, including nerve growth factor, fibroblast growth factor, and interleukin-6, induce an accumulation of an active Ras.GTP complex in rat pheochromocytoma PC12 cells. 152 65

A cytosolic insulin-sensitive serine kinase has been purified to apparent homogeneity in parallel from livers of control or acutely insulin-treated rats. The kinase is labile and requires rapid purification for stability. The kinase migrates as a band of apparent Mr = 90,000 on denaturing gels and elutes as a monomer on Superose 12 gel filtration. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation, the 90-kDa band presumed to be the kinase shows kinase activity toward myelin basic protein in situ. Substrates of the kinase include Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), ribosomal protein S6, S6 peptide, a proline-rich peptide substrate, microtubule-associated protein 2, and myelin basic protein. The kinase also phosphorylates histones H1 and H2B, but does not autophosphorylate to a significant stoichiometry. The activity of the kinase is inhibited by fluoride, glycerophosphate, p-nitrophenyl phosphate, p-nitrophenol, heparin, quercetin, poly-L-lysine, and potassium phosphate, but is unaffected by calcium, cAMP, spermine, protein kinase inhibitor peptide, phorbol myristate acetate, calcium plus phosphatidylserine, or vanadate. The kinase will utilize magnesium (10 mM) as well as manganese (1 mM) as a cofactor for maximal phosphotransferase activity. The kinase is not detected by immunoblotting with antibodies directed against protein kinase C or type II S6 kinase. Taken together, these properties distinguish this kinase from other insulin-sensitive kinases that have been described previously. The purified kinase from livers of insulin-treated rats shows a 5-20-fold higher specific activity compared to enzyme prepared from control rats, suggesting a covalent modification as the mechanism of activation. Incubation of purified, insulin-stimulated kinase with purified phosphatase 2A leads to deactivation of the kinase activity, and the phosphatase inhibitor nitrophenyl phosphate blocks this deactivation. The insulin-activated kinase fails to immunoblot with anti-tyrosine phosphate antibodies. Taken together, these results indicate that insulin activates this novel cytosolic protein kinase by a mechanism that causes its phosphorylation on serine or threonine residues.
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PMID:Purification and characterization of a cytosolic insulin-stimulated serine kinase from rat liver. 153 38

Insulin is known to rapidly stimulate and/or translocate Ca2+/phospholipid-dependent protein kinase (conventional PKC; cPKC) in rat adipocytes. Presently we examined whether insulin also stimulates/translocates Ca(2+)-independent, phospholipid-dependent protein kinase (novel PKC; nPKC). Total Mono Q column-elutable nPKC (like cPKC) activities were decreased in cytosolic and increased in membrane fractions with insulin treatment. Immunoblot study of novel PKC epsilon also showed insulin-induced translocation of immunoreactive PKC from cytosol to membrane, similar to the translocation of cPKC, PKC beta. These results suggest that nPKC has an important role in insulin-induced signal transduction.
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PMID:Insulin stimulates novel protein kinase C in rat adipocytes. 155 May 87

The regulation of 3-O-methyl-D-glucose (OMG) uptake by insulin and phorbol esters was studied in cultured human skin fibroblasts. Insulin rapidly stimulated OMG uptake through a mechanism independent of new protein synthesis. Maximal insulin effect was reached in 30 min and remained constant up to 12 h. The protein kinase C activators 12-O-tetradecanoyl phorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PdBU) promoted an initial rapid stimulation followed by a secondary long-term rise of OMG influx. This latter effect of phorbol esters on OMG influx began after 1 h, reached a maximum in 12-15 h, and was prevented by the simultaneous addition of protein synthesis inhibitors, suggesting that phorbol esters increased the synthesis of new glucose transporters. In accord with this interpretation, phorbol esters, but not insulin, increased mRNA levels for two distinct glucose transporters (GLUT1 and GLUT3) in human fibroblasts. Both the rapid and the long-term effects of phorbol esters on OMG influx were dose-dependent and half-maximal stimulations occurred at 15 nM for both PdBU and TPA. Kinetic analysis of OMG uptake indicated that both effects of phorbol esters were associated with an increase in the Vmax of the transport process, with no significant changes of the Km (4-6 mM). These results suggest that, in human fibroblasts, phorbol esters, unlike insulin, produce a long-term stimulation of OMG uptake, which is dependent upon protein synthesis and is associated with increased levels of GLUT1 and GLUT3 mRNA.
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PMID:Glucose transport by cultured human fibroblasts: regulation by phorbol esters and insulin. 155 Aug 50

The late phase of the time-dependent epidermal growth factor (EGF)-induced biphasic activation of the p70s6k is selectively attenuated by the specific PKC inhibitor, CGP 41,251, a staurosporine derivative. At a 40-fold lower concentration than CGP 41,251, staurosporine inhibits both phases of S6 kinase activation to the same extent, whereas the inactive staurosporine derivative CGP 42,700 shows no effect on either phase. Platelet-derived growth factor (PDGF) and insulin also induce biphasic S6 kinase activation, but in neither case is either phase of activation affected by the presence of CGP 41,251. This finding was unexpected in the case of PDGF, which is a potent activator of PKC and whose receptor directly interacts with phospholipase C gamma 1. However, similar results were obtained following down-regulation of PKC by prolonged 12-O-tetradecanoylphorbol-13-acetate treatment. Therefore, even though EGF and PDGF induce PKC activation, PDGF, unlike EGF, does not appear to use this signaling pathway for late phase p70s6k activation.
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PMID:Inhibition or down-regulation of protein kinase C attenuates late phase p70s6k activation induced by epidermal growth factor but not by platelet-derived growth factor or insulin. 155 99

Uterine cervical and corpus cancers have been reported to synthesize and secrete a putative peptide mitogen, which elicits a potent proliferative response in fibroblasts by a mechanism independent of phosphoinositide turnover. The extract from human uterine cervical cancer stimulated [3H]thymidine incorporation into human endometrial fibroblasts in a dose-dependent manner. Concomitant exposure of the fibroblasts to thrombin or fibroblast growth factor (FGF) led to synergistic enhancement of the extract-stimulated [3H]thymidine incorporation into fibroblasts. An apparent maximal activity of the extract in the presence of thrombin or FGF was 2-fold higher than that in the absence of them, implying that thrombin or FGF acted at a stage after the interaction of the mitogen in the extract with its specific receptor. Insulin or epidermal growth factor failed to augment the growth-promoting activity in the extract. The stimulatory action of thrombin or FGF was mimicked by protein kinase C activators, phorbol-12-myristate-13-acetate (PMA) or 1-oleoyl-2-acetyl glycerol, but not by Ca2+ ionophore A23187. When the fibroblasts were first exposed to the extract for 4 h and then to PMA, PMA succeeded to augment the mitogenic activity in the same manner. The identical effects of protein kinase C activators with thrombin or FGF suggested that both types of ligand share a similar signaling cascade of action, activation of protein kinase C. These results demonstrated that the growth-promoting activity in uterine cancer extract could be enhanced by the agents which promote phosphoinositide metabolism through activation of protein kinase C. These findings could give a new insight into pathophysiology of the interaction between malignant cells and their stromal cells, fibroblasts.
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PMID:Enhancement of growth-promoting activity in extract from uterine cancers by protein kinase C in human endometrial fibroblasts. 155 22

Insulin and the phorbol ester, phorbol 12-myristate 13-acetate, induce beta-actin gene transcription in H4 cells. This occurred rapidly, with a maximum 15-fold stimulation following 15 min of insulin (5 x 10(-9) M) or phorbol ester (1 microgram/ml) exposure. The increase in beta-actin transcription was transitory, returning to baseline within 120 min. Pretreatment of cells with phorbol ester for 24 h, reducing functional protein kinase C activity, abolished the ability of phorbol esters to increase beta-actin transcription. When insulin was added to phorbol ester-pretreated cells the insulin-induced increase in beta-actin transcription was reduced by 40-60%. These findings support our hypothesis that a common regulatory signal is utilized by both insulin and phorbol esters for the complete induction of specific genes.
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PMID:Regulation of beta-actin gene transcription by insulin and phorbol esters. 157 14

One of insulin's actions is the induction of DNA synthesis and cell division, but little is known about the molecular mechanisms involved. Previous studies indicate that insulin stimulates cell division and regulates the expression of several genes in rat H4IIE (H4) hepatoma cells. One of these genes is the proto-oncogene c-fos, a cellular gene whose deregulation has been implicated in the process of cellular differentiation and division. We have shown that insulin induces transcription of the c-fos gene in H4 cells. In the present study, the phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated c-fos transcription in a rapid and dose-dependent manner with an 800% increase in transcription following 15-30 min of addition. This increase in c-fos transcription was transitory, returning towards baseline transcription rates within 120 min. PMA stimulated the translocation of protein kinase C (PKC) from the cytoplasm to the membrane in H4 hepatoma cells, as evidenced by a 77% decrease in cytosolic PKC and a 29% increase in membrane PKC activity following 10 min of treatment. Insulin addition to H4 cells for 10 min also resulted in a 31% decrease in cytosolic PKC activity, suggesting a translocation response. When H4 cells were pretreated with PMA for 24 h, there was a decrease of 20-45% in both cytosolic and membrane PKC activity and a complete loss of PMA's induction of c-fos transcription. Thus, the cells were functionally desensitized to further PMA addition. When cells were pretreated with PMA for 24 h, the insulin-induced increase in transcription of c-fos was reduced by 50%. Western blot analysis indicated that the PKC-beta isozyme followed a translocation pattern almost identical with that of total PKC activity. These results suggest that a PMA-sensitive form of PKC is preferentially lost upon PMA pretreatment and that this PKC subtype may be necessary for insulin to fully induce c-fos gene expression.
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PMID:Role of protein kinase C in insulin's regulation of c-fos transcription. 157 56

Both 86Rb+ efflux experiments and electrophysiological studies have shown that arachidonic acid and other nonesterified fatty acids activate ATP-sensitive K+ channels in insulinoma cells (HIT-T15). Activation was observed with arachidonic, oleic, linoleic, and docosahexaenoic acid but not with myristic, stearic, and elaidic acids. Fatty acid activation of ATP-sensitive K+ channels was blocked by antidiabetic sulfonylureas such as glibenclamide. The activating effect of arachidonic acid was unaltered by indomethacin and by nordihydroguaiaretic acid, indicating that it is not due to metabolites of arachidonic acid via cyclooxygenase or lipoxygenase pathways. Moreover, the nonmetabolizable analogue of arachidonic acid, eicosatetraynoic acid, was an equally potent activator. Activation of ATP-sensitive K+ channels by fatty acids was potentiated by diacylglycerol and was inhibited by calphostin C, an inhibitor of protein kinase C. These findings indicate that fatty acid activation of ATP-sensitive K+ channels is most likely due to the participation of arachidonic acid (and other fatty acid)-activated protein kinase C isoenzymes. Activation of ATP-sensitive K+ channels by nonesterified fatty acids is not involved in the control of insulin secretion since arachidonic acid stimulates insulin secretion from insulinoma cells instead of inhibiting it.
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PMID:ATP-sensitive K+ channels in insulinoma cells are activated by nonesterified fatty acids. 158 15


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