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)

Protein kinase C was purified 6,900-fold from rabbit pancreas with a total yield of 15% by a procedure involving ammonium sulfate fractionation, diethyl aminoethyl ion exchange chromatography, hydroxylapatite chromatography, and finally protamine-agarose affinity chromatography. After these purification steps the protein kinase C preparation contained two major protein bands as judged by silver staining after SDS-polyacrylamide gel electrophoresis: 80 and 69-kDa bands. Monoclonal antibodies directed against bovine brain protein kinase C (alpha- and beta-subtype) recognized only the 80-kDa band. On the other hand, both the 80 and 69-kDa proteins were recognized by a polyclonal monospecific antibody directed against rat brain protein kinase C. Analysis of rabbit pancreas protein kinase C subtypes by means of hydroxylapatite chromatography showed the presence of the III (alpha) subtype as the major subtype. The enzyme depended absolutely on the presence of both phosphatidylserine and Ca2+ for its activity, with apparent Ka values of 3.1 micrograms/ml and 247 microM for phosphatidylserine and Ca2+, respectively. When dioctanoylglycerol or the phorbol ester 12-O-tetradecanoyl-phorbol 13 acetate (TPA) was present, the Ka value for Ca2+ decreased to 10 and 18 microM, respectively. In the presence of the phorbol ester, pancreatic protein kinase C could be activated without added Ca2+. The enzyme also required Mg2+ for its activity. The Ka value was 3.6 mM and maximal activity was reached at 10 mM Mg2+. Pancreatic protein kinase C activity showed a broad pH dependence, with optimal activity at pH 6.75. The Km value for ATP and for histone-H1 was 8.5 microM and 20.4 micrograms/ml, respectively. The present study shows that the kinetic properties of protein kinase C purified from rabbit pancreas closely resemble those found in other tissues.
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PMID:Purification and characterization of rabbit pancreas protein kinase C. 155 44

Previous results from our laboratory suggest that long-term treatment of primary cultured bovine adrenal medullary (BAM) chromaffin cells with nicotine or phorbol 12-myristate 13-acetate, either of which directly activates protein kinase C (PKC), increases the mRNA levels encoding catecholamine-synthesizing enzymes and proenkephalin. In the present study, we have examined the effects of nicotine on BAM cell PKC activity with special emphasis on long-term effects. Nicotine increased particulate PKC activity in a concentration-dependent manner when measured using in vitro enzyme assay with histone as the substrate. This effect is mediated through nicotinic cholinergic receptors, because 1,1-dimethylphenylpiperazinium, a nicotinic agonist, had a similar effect. In addition, chlorisondamine, a specific nicotine-receptor blocking drug, antagonized the effect of nicotine. Nicotine also increased specific [3H]phorbol 12,13-dibutyrate ([3H]PdBu) binding within 1 min, the effect of which was maximal between 3 and 12 min. This effect was reversed by chlorisondamine similarly after 12 min and after 18 h of nicotine treatment, indicating that continual nicotinic-receptor occupancy is required for persistent PKC activation. Compared to PKC activation, the onset of nicotine-stimulated diacylglycerol production was slow, and it was observed after 12 min of incubation with nicotine. The diacylglycerol levels, specific [3H]PdBu binding, and PKC activity remained significantly elevated for at least 18 h with continuous nicotine incubation. Furthermore, nicotine increased the PKC immunoreactivity of a particulate protein with a molecular mass of 82 kDa in the western blot. These results suggest that nicotinic-receptor activation increases PKC activity and immunoreactivity in BAM cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Long-term activation of protein kinase C by nicotine in bovine adrenal chromaffin cells. 156 Feb 24

The role of protein kinase C in migration of tumor cells from spheroid cultures was investigated using parental rat glioma cells and their TPA resistant counterparts. These two lines differed in their PKC content as judged by the histone phosphorylation method. Also 4 days of treatment with IRA led to PKC down-regulation. Cells having a drastically decreased PKC level migrated better than those having a normal PKC content.
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PMID:The role of protein kinase C in migration of rat glioma cells from spheroid cultures. 156 92

Protein kinase C is present in bovine epididymal sperm. The enzyme was partially purified by gel filtration on Sephacryl S-300. The Ca2+/phosphatidylserine-dependent histone phosphotransferase activity elutes from the gel filtration column in a manner corresponding to a Mr approximately 80 kDa. The activity peak also corresponds with [3H]phorbol 12,13-dibutyrate binding activity. Immunoblot analysis of the partially purified enzyme with isozyme-specific monoclonal antibodies revealed the presence of alpha-, beta-, and gamma-subspecies of protein kinase C. Indirect immunofluorescence showed that the antibodies against alpha-, beta-, and gamma-subspecies produced prominent staining of the postacrosomal region of the sperm head. In addition, beta-subspecies antibodies produced minor staining of the midpiece and gamma-subspecies antibodies produced a minor staining of the acrosomal region.
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PMID:Isotypes of protein kinase C in bovine sperm. 158 55

Of the recently identified protein kinase C (PKC) types of group B (delta, epsilon, zeta, eta, PKC-L), only PKC-epsilon has been characterized in great detail. In order to compare the regulatory and catalytic properties of these new kinases, we have expressed PKC-delta, -epsilon, -zeta and PKC-L as recombinant proteins from their cDNAs in insect cells via baculovirus vectors and in mammalian COS-1 cells. After expression in insect cells, phorbol ester binding and kinase activities of the group B enzymes were compared with the respective activities of a member of group A, PKC-gamma. Although PKC-delta and PKC-L(eta) bind phorbol ester to a similar or the same extent as PKC-gamma, they show a distinctively different behaviour towards conventional PKC substrates such as histone, myelin basic protein, protamine and protamine sulphate, suggesting either that phorbol esters are not able to fully activate these enzymes or that their substrate specificities are very different from those of the group A enzymes. PKC-zeta, a polypeptide of 80 kDa, does not bind phorbol ester and does not phosphorylate these substrates to a significant extent. Consistent with their ability to bind phorbol ester, recombinant PKC-delta and PKC-epsilon are down-regulated in COS cells by prolonged treatment with phorbol ester, whereas PKC-zeta protein levels remain unaltered.
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PMID:Protein kinase C group B members PKC-delta, -epsilon, -zeta and PKC-L(eta). Comparison of properties of recombinant proteins in vitro and in vivo. 159 Jul 67

1. The oligopeptide AAASFKAKK which contains recognition motifs similar to that found in the surrounding of the site of H1 histone phosphorylated by protein kinase C is unable to compete with H1 histone for the type II and type III isoenzymes, though it is a good substrate for protein kinase C and it is able to compete with a physiological substrate of the enzyme. 2. Among several oligopeptides tested as an alternative substrate a very basic peptide proved to be the most effective inhibitor of H1 histone phosphorylation. This oligopeptide substrate contains basic recognition motifs at both sides of the phosphorylated residue at variance with the sequence of H1 histone in the surrounding of the phosphorylated site.
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PMID:Synthetic oligopeptide substrates which fail to compete with H1 histone for type II and type III isoenzymes of protein kinase C. 159 53

A long-chain neutral phospholipid, dioleoylphosphatidylcholine, was found to support protein kinase C activation by cis-fatty acid and diacylglycerol (DAG). This effect of phosphatidylcholine (PC) is totally dependent on the presence of cis-fatty acid; PC greatly stimulates the cis-fatty acid-induced protein kinase C activity, but it does not activate protein kinase C at all, even in the presence of DAG, if cis-fatty acid is absent. DAG, however, plays a modulatory role in the presence of Ca2+; it further enhances the PC-potentiated cis-fatty acid activation of protein kinase C. Although the activities of all three protein kinase C subtypes tested (types I, II and III) are supported by this PC mechanism, type III is most sensitive to the DAG effect, and it is activated synergistically by cis-fatty acid and DAG. The potency of PC to support the synergistic activation of this subtype is equivalent to that of phosphatidylserine (PS). There are several differences, however, between PC- and PS-supported synergism observed in type III protein kinase C: (1) Ca(2+)-sensitivity is different; PC requires higher concentrations of Ca2+ (10-20 microM-Ca2+) than those required for PS (micromolar Ca2+); (2) PC/cis-fatty acid/DAG-induced autophosphorylation of protein kinase C subtypes (types I, II and III) is very weak, whereas PS/cis-fatty acid/DAG strongly stimulate autophosphorylation of these subtypes under the conditions at which both PC and PS systems fully activate the protein kinase C in terms of histone phosphorylation. These observations suggest that a neutral phospholipid such as PC may also participate in the activation and differential regulation of protein kinase C.
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PMID:Phosphatidylcholine-dependent protein kinase C activation. Effects of cis-fatty acid and diacylglycerol on synergism, autophosphorylation and Ca(2+)-dependency. 159 99

The biochemical mechanism(s) underlying the priming of the macrophage for an enhanced PMA-induced respiratory burst is not understood. Because the cellular receptor for PMA is thought to be protein kinase C (PKC), we have investigated the effects of priming agents on cellular PKC levels. Sonicates from unprimed bone marrow-derived macrophages (BMM) were found to contain PKC activity (309 +/- 51 pmol 32P-incorporated/mg/min; mean +/- SE, n = 17) as measured by the phospholipid-, diacylglycerol-, and calcium-dependent phosphorylation of histone. Exposure of BMM to priming agents such as TNF-alpha, LPS, and granulocyte/macrophage-CSF resulted in a significant increase in both histone-phosphorylating activity and levels of immunoreactive PKC protein in these cells. A minimum of 6-h exposure, with an increasing effect up to 48 h, was required for a detectable increase in PKC level. The activity from primed BMM, like that of the untreated cells, was predominantly cytosolic. The kinetics and concentration dependence of the priming agent-induced increase in the PKC content of BMM closely paralleled the enhancing effects of these agents on the PMA-stimulated respiratory burst. Furthermore, CSF-1, a cytokine that does not prime BMM, failed to increase PKC activity. We propose that the exposure of BMM to priming agents leads to an increase in the expression of a stimulatory isozyme(s) of PKC, resulting in an enhanced ability to mount a respiratory burst in response to stimulation with PMA.
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PMID:Priming of the respiratory burst of bone marrow-derived macrophages is associated with an increase in protein kinase C content. 140 16

Expression of rat protein kinase C-delta (PKC-delta) and PKC-zeta in insect cells using recombinant baculovirus resulted in the production of proteins with a molecular size of approximately 76 kD and 78 kD, respectively, as determined by immunoblotting with subtype-specific antisera. Although the PKC-zeta cDNA encoded for 592 amino acids, a 76 kD protein was also generated by in vitro transcription/translation. Extracts of cells expressing PKC-delta were able to bind phorbol ester to levels comparable to extracts of cells expressing PKC-alpha. No phorbol ester binding was, however, detected in insect cell extracts expressing PKC-zeta. However, similar levels of protein kinase activity were detected in lysates of cells expressing PKC-delta or PKC-zeta when protamine sulfate was used as exogenous substrate. Compared to protamine sulfate, both, myelin basic protein (MBP) or histone, were poor substrates for PKC-delta and PKC-zeta. In contrast to PKC-zeta, the PKC-delta enzyme activity phosphorylated MBP or histone in a phosphatidylserine-(PS)/diacylglycerol(DG)-dependent manner, albeit not to the same extent as PKC-alpha. Lack of stimulation of the enzyme activity of PKC-zeta by PS/DG, was confirmed by endogenous phosphorylation of insect cell proteins by PKC-zeta, whereas several insect cell proteins were phosphorylated by PKC-delta in a PS/DG-dependent manner, including a protein of 78 kD. Our data demonstrate that the 76 kD PKC-zeta, in contrast to PKC-delta, is unable to bind phorbol esters and displays a protein kinase activity that is independent of PS or PS/DG. In addition, staurosporine was about 2-4 order of magnitudes less effective in inhibiting the protein kinase activities of PKC-delta and PKC-zeta when compared to PKC-alpha.
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PMID:Expression and partial characterization of rat protein kinase C-delta and protein kinase C-zeta in insect cells using recombinant baculovirus. 164 61

To determine the mechanisms of cell signalling by asbestos in epithelial cells of the respiratory tract, the activity of protein kinase C (PKC) was examined in hamster tracheal epithelial (HTE) cells exposed to mitogenic concentrations of crocidolite asbestos. In the histone phosphorylation assay, asbestos significantly increased activity of PKC associated with the membrane fraction of HTE cells. However, in contrast to 12-O-tetradecanoylphorbol-13-acetate, which caused redistribution of almost all PKC activity from the cytosolic to the membrane fraction, the majority of the PKC activity was associated with the cytosolic fraction at all time periods examined. Asbestos did not inhibit binding of [3H]phorbol-12,13-dibutyrate to intact HTE cells, whereas binding was inhibited by the phorbol compounds phorbol dibutyrate and phorbol dibenzoate. Thus, crocidolite-induced activation of PKC does not appear to be mediated through the same mechanism as classical phorbol ester tumor promoters, compounds which activate PKC by structurally resembling diacylglycerol.
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PMID:Activation of protein kinase C by crocidolite asbestos in hamster tracheal epithelial cells. 165 Feb 93


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