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)

Dephosphorylation by the catalytic subunits of protein phosphatases 1 (CS1) and 2A (CS2) reveals that mature protein kinase C is phosphorylated at two distinct sites. Treatment of protein kinase C beta II with CS1 causes a significant increase in the protein's electrophoretic mobility (approximately 4 kDa) and a coincident loss in catalytic activity. The CS1-dephosphorylated enzyme cannot autophosphorylate or be phosphorylated by mature protein kinase C, indicating that a different kinase catalyzes the phosphorylation at this site. The loss of activity is consistent with dephosphorylation on protein kinase C's activation loop (Orr, J. W., and Newton, A. C., (1994) J. Biol. Chem. 269, 27715-27718). Treatment with CS2 results in a smaller shift in electrophoretic mobility (approximately 2 kDa) and no loss in catalytic activity. Furthermore, the CS2-dephosphorylated form can autophosphorylate and thus regain the electrophoretic mobility of mature enzyme, consistent with dephosphorylation at protein kinase C's carboxyl-terminal autophosphorylation site, which is modified in vivo (Flint, A. J., Paladini, R. D., and Koshland, D. E., Jr. (1990) Science 249, 408-411). In summary, two phosphorylations process protein kinase C to generate the mature form: a transphosphorylation that renders the kinase catalytically competent and an autophosphorylation that may be important for the subcellular localization of the enzyme.
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PMID:In vivo regulation of protein kinase C by trans-phosphorylation followed by autophosphorylation. 796 10

Translocation of protein kinase C (PKC) after PMA or LPS stimulation has been studied in thioglycolate (TGC)-elicited murine peritoneal macrophages. Among the PKC subtypes we examined (alpha, beta, gamma, delta, and epsilon) by indirect immunostaining and immunoblot analysis, conventional PKC-beta, as well as novel PKC-delta and PKC-epsilon were found to exist in TGC-elicited C3H/HeN mouse macrophages. Translocation of PKC-beta to the Triton-stable cytoskeleton could be seen in macrophages after stimulation by both PMA and LPS. On the other hand, novel PKCs redistributed only after PMA stimulation. Macrophages obtained from LPS-nonresponsive C3H/HeJ mice also exhibited PKC-beta, and the m.w., cellular distribution, and cellular content of this enzyme could not be distinguished from those of C3H/HeN macrophages. These macrophages exhibited PKC-delta and PKC-epsilon, as did the C3H/HeN macrophages. In these macrophages, however, LPS did not induce any remarkable change in the intracellular distribution of PKC-delta and PKC-epsilon or PKC-beta, whereas PMA was able to induce the translocation of PKC-beta to the cytoskeleton. These results suggest that LPS stimulation induces selective redistribution of PKC-beta in LPS-responsive macrophages, whereas a defect related to LPS unresponsiveness exists in C3H/HeJ mouse macrophages before the PKC activation. Translocation of PKC-beta can be understood to be an important event in LPS signaling in macrophages.
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PMID:LPS induces selective translocation of protein kinase C-beta in LPS-responsive mouse macrophages, but not in LPS-nonresponsive mouse macrophages. 798 73

In this paper we examine whether sodium orthovanadate activates diacylglycerol (DAG)/protein kinase C (PKC) signaling systems that are activated by insulin in BC3H-1 myocytes. Like insulin, sodium orthovanadate provoked increases in membrane DAG, PKC enzyme activity, and immunoreactive PKC-beta. Concomitantly, both PKC enzyme activity and immunoreactive PKC-beta decreased in the cytosol, suggesting that sodium orthovanadate, like insulin, stimulated the translocation of PKC-beta from the cytosol to the membrane fraction. Sodium orthovanadate was also found to activate phospholipid signaling pathways that were previously reported to be activated by insulin, viz., inositol-lipid synthesis/turnover; phosphatidylcholine hydrolysis; and de novo phospholipid synthesis by activation of glycerol-3-PO4 acyltransferase. Our findings suggest that vanadate mimics insulin in the activation of specific phospholipid/DAG/PKC signaling pathways.
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PMID:Insulin-like effects of sodium orthovanadate on diacylglycerol-protein kinase C signaling in BC3H-1 myocytes. 803 Nov 24

Earlier studies of a site-specific mutant of protein kinase C beta 1 (PKC beta 1) altered at Thr635 and Thr642 indicated that these phosphorylation sites are critical for enzymatic function (Zhang, J., Wang, L., Petrin, J., Bishop, W. R., and Bond, R. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,6130-6134). To determine the contribution of the individual threonines, we report here on two site-specific mutants in which either Thr635 or Thr642 was changed to alanine. When transiently overexpressed in Cos cells wild-type PKC beta 1 exists in two forms: a Triton-insoluble form with high electrophoretic mobility and a slower migrating Triton-soluble form. Mutation at Thr642 (but not Thr635) results in production of only the fast-migrating form. [35S]Methionine pulse-chase labeling indicates that wild-type PKC beta 1 is synthesized as the fast-migrating form and is subsequently converted to the slow-migrating form. 32P labeling shows that only the slow-migrating form is a phosphoprotein. Mutation of Thr642 abolishes this phosphorylation. Finally, the Thr642 mutant PKC beta 1 lacks enzymatic activity and, when expressed in NIH 3T3 cells, reduces phorbol ester-induced c-fos promoter activity. These results indicate that Thr642 phosphorylation is an early event in the processing of newly synthesized PKC beta 1 and is required for enzymatic function. These results support a role for a PKC kinase in PKC processing and activation.
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PMID:Phosphorylation of Thr642 is an early event in the processing of newly synthesized protein kinase C beta 1 and is essential for its activation. 803 26

In this report we identify the specific isozymes of protein kinase C (PKC) that are involved in c-fos and c-jun mRNA accumulation in the rat basophilic leukemia cell line RBL-2H3. These cells could be largely depleted of the endogenous PKC isozymes by chronic treatment with phorbol 12-myristate 13-acetate followed by permeabilization of the cells with streptolysin O. The reconstitution of these cells with defined concentrations of either PKC-beta or PKC-epsilon up to 10 nM and 20 nM, respectively, induced c-fos and c-jun in a dose-dependent manner. At high concentrations of PKC-beta and -epsilon the induction of c-fos and c-jun was independent of the aggregation of the high-affinity IgE receptors (Fc epsilon type I receptors). In contrast, at limiting concentrations of these two PKC isozymes, 1 nM, the increase in c-fos and c-jun mRNAs was dependent on the aggregation of the Fc epsilon type I receptors. Unlike PKC-beta and -epsilon, PKC-alpha and PKC-delta failed to reconstitute c-fos and c-jun induction at any dose over the range examined. We conclude that PKC-beta and PKC-epsilon serve as a link between the cell surface receptor and gene expression.
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PMID:Protein kinases C-beta and C-epsilon link the mast cell high-affinity receptor for IgE to the expression of c-fos and c-jun. 805 50

Human erythroleukemia (HEL) cells grow in suspension, but after treatment with nM PMA the cells adhere and spread on glass or fibronectin [Jarvinen et al., 1987: Eur. J. Cell Biol. 44:238-246]. We observed an early (20-30 min) stage of spreading in which F-actin was organized into peripheral arcs near the spreading margin and vinculin was localised to the cell's periphery at the ends of these arcs. By 1 h the cells were well spread with straight actin bundles many of which ended at more central sites terminating on patches containing vinculin and talin; thus the cells assemble typical stress fibers but do not appear to polarize. The cells also spread on RGD polymer. DiC8 (1,2-dioctanoyl-sn-glycerol, C8:0, Sigma Chemical Co., St. Louis, MO) induced spreading but only if DAG kinase inhibitor and A-23187 were also present; in their absence cells adhered but did not spread. Spreading was approximately 85% inhibited by 100 nM staurosporine. PKC-beta was shown to be present in the cells by immunoblotting. In cells spread for 1 h with PMA, F-actin increased to 180% of control levels as measured by RP binding and the actin sequestering complex of G-actin-thymosin beta 4 decreased significantly. To determine whether the F-actin increase required adhesion, we inhibited cell attachment to the substratum by adding RGDS, by coating glass surfaces with hemoglobin, or by a combined treatment. Under these conditions PMA-treated suspended cells still increased their F-actin to 126-137% of controls, a significant increase over control levels. Staurosporine inhibited F-actin increases under all the conditions studied. Permeabilized cell suspensions, incubated with rhodamine labelled G-actin, incorporated the labelled actin along cell membranes at a low level. A few minutes preincubation with either diC8 plus DAG kinase inhibitor or with PMA strongly increased the incorporation. This increased incorporation was reduced to below control levels by either staurosporine (100 nM) or cytochalasin D (1 microM). We conclude that both suspended and spreading HEL cells can be stimulated to polymerize actin by a mechanism dependent on PKC or a PKC-like molecule. In suspended cells, the polymerization occurs along the membrane. When cells spread, F-actin increased to a significantly greater extent. This second step could involve additional polymerization, perhaps at the observed adhesion sites, decreased turnover of the actin bundles, or a combined effect of both mechanisms.
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PMID:Two-step mechanism for actin polymerization in human erythroleukemia cells induced by phorbol ester. 806 40

Prompted by the reversal of skin hyperproliferation to normal by 13-hydroxyoctadecadienoic acid (13-HODE), a 15-lipoxygenase metabolite of linoleic acid, we investigated a possible mechanism for this antiproliferative action. To address this we first demonstrated that 13-HODE is incorporated into epidermal phosphatidyl 4,5-bisphosphate (PtdIns4,5-P2) and released as 13-HODE-containing diacylglycerol by epidermal phospholipase C. Secondly, we tested the possibility whether this putative 13-HODE-containing DAG (13HODE-DAG) could exert a modulatory effect on epidermal protein kinase C (PKC) activity which previously has been associated with skin hyperproliferation. Unlabeled 13HODE-DAG was generated from 13-HODE-containing phosphatidylcholine after phospholipase C hydrolytic cleavage. The effects of the 13HODE-DAG were determined on: i) total epidermal PKC activity; ii) diolein-activated PKC activity; and iii) the two identified epidermal PKC-isozymes (PKC-beta and PKC-alpha). Our data revealed over a twofold activation of total basal PKC activity by diolein. In contrast, replacement of diolein (1,2-dioleoylglycerol) with 13HODE-DAG (1-palmitoyl,2-13HODE-glycerol) in the incubation mixture exerted no effect on total basal PKC activity. In an another experiment, 13HODE-DAG inhibited diolein-activated PKC activity in a dose-dependent manner. To determine whether the effects of 13HODE-DAG are selective, we tested its effects on DEAE-Sephacel-purified and Western blot-confirmed PKC isozymes. Our data revealed that 13HODE-DAG selectively inhibited the activity of PKC-beta isozyme, while exerting negligible effect on the PKC-alpha isozyme. This selective inhibitory effect of 13HODE-DAG on a major epidermal PKC isozyme activity suggests that 13HODE-containing DAG seemingly can modulate epidermal PKC activity, which purportedly is associated with epidermal hyperproliferation.
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PMID:Expression of protein kinase C isozymes in guinea pig epidermis: selective inhibition of PKC-beta activity by 13-hydroxyoctadecadienoic acid-containing diacylglycerol. 807 13

In this study we explored the pattern of protein kinase C (PKC) isozyme selectivity of the bryostatins, a unique class of PKC activators that induce only a subset of the typical phorbol ester responses and antagonize those phorbol ester-mediated responses that they themselves fail to induce. The binding properties of individual recombinant PKC isozymes that had been expressed in insect cells, isolated, and reconstituted in Triton X-100/phosphatidylserine mixed micelles were determined. [3H]Bryostatin 1 showed lower affinity for PKC-beta 1 and -gamma, compared with PKC-alpha, -delta, -epsilon, and -eta. This pattern contrasts with that observed for other PKC ligands. These latter assays were conducted with isozymes reconstituted in phosphatidylserine, conditions that unfortunately do not permit quantitation of bryostatin 1 binding under equilibrium conditions. Using delta 19,20-bryostatin 10 and delta 19,20-isobryostatin 10, we could distinguish the respective roles of ligand and lipid in the pattern of selectivity. When isozymes were reconstituted in phosphatidylserine vesicles, delta 19,20-bryostatin 10 and delta 19,20-isobryostatin 10 showed similar affinities for PKC-alpha and -gamma, similarly to the phorbol esters. However, in the mixed micellar system, PKC-gamma showed a significantly lower binding affinity, as had been observed for bryostatin 1. These results suggest that the unique pattern of biological responses to the bryostatins does not represent a unique pattern of isotype recognition. Furthermore, the lipid environment of PKC plays an important role in determining the binding selectivity for individual isozymes.
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PMID:Binding of [26-3H]bryostatin 1 and analogs to calcium-dependent and calcium-independent protein kinase C isozymes. 807 99

The requirement for protein kinase C (PKC)-beta in phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation of human HL-60 promyelocytic leukemia cells was studied by using the variant HL-525, which is deficient in PKC-beta and is resistant to PMA-induced differentiation. Transfecting these resistant HL-525 cells with expression vectors containing either PKC-beta I or PKC-beta II cDNA resulted in clones that displayed PKC-beta transcript levels similar to or higher than those of the parental HL-60 cells or cells from a PMA-susceptible HL-60 clone, HL-205. These productive transfectants also exhibited PMA-induced cell attachment and spreading, inhibition of cell replication, reactivity to the OKM1 monoclonal antibody, and the ability to phagocytize opsonized beads, which are all characteristic macrophage markers. No PMA-induced differentiation markers were observed in any of the PKC-beta I or PKC-beta II transfectants that did not exhibit an increased PKC-beta RNA level or in cells transfected with control plasmids. These results indicate that restoration of the PKC-beta isozyme deficiency by productive gene transfection causes HL-525 cells to revert to a phenotype like that of the parental HL-60 cells, which is characterized by susceptibility to PMA-induced macrophage differentiation. Therefore, we can conclude that PKC-beta is one of the essential elements in the PMA-induced signal transduction pathway which leads to macrophage differentiation in HL-60 cells and perhaps in other related cell types.
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PMID:Protein kinase C-beta is required for macrophage differentiation of human HL-60 leukemia cells. 808 28

Protein kinase C is allosterically activated by diacylglycerol and phosphatidylserine. The enzyme is also activated by surprisingly dissimilar compounds such as short chained phosphatidylcholines and protamine sulfate. Here we show that conventional and nonconventional activators of protein kinase C beta II produce the same structural alteration. They expose Arg19 of the autoinhibitory pseudosubstrate domain to proteolysis. Molecular modeling of protein kinase C beta II's catalytic domain, based on the structure of the cAMP-dependent kinase's catalytic domain, indicates that Arg19 is shielded by a cluster of acidic residues when the pseudosubstrate occupies the substrate-binding site. Our biochemical data and structural modeling indicate a common mechanism of intrapeptide regulation of protein kinase C by all activators that involves release of the pseudosubstrate from the active site.
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PMID:Intrapeptide regulation of protein kinase C. 813 62


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