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)

To investigate the changes in the properties of cardiac contractile proteins due to neurohormonal stimulation, different agonists were applied to single cells isolated from rat ventricle. Cells were then rapidly skinned by Triton X-100, and force was recorded after gluing the cells to a strain gauge. The skinned cells had mechanical properties very similar to those described for thin trabeculas. Tension-pCa relations were highly reproducible from one cell to another, with sarcomere length fixed at 2.1 microns. The application of alpha 1-adrenergic and muscarinic agonists, which increase the turnover of phosphatidylinositol, for 5 minutes before skinning the cells increased the sensitivity of the myofilaments to calcium, as indicated by a leftward shift of the tension-pCa relation, whereas beta-adrenergic stimulation induced a rightward shift. The increase in calcium sensitivity was also evoked by protein kinase C activators such as 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate but not by protein kinase C itself or by purinergic agonists, although the latter also increased the turnover of phosphatidylinositol. Incubation of the skinned cells with phosphatase reversed the alterations in calcium sensitivity induced by previous agonist stimulation of the intact cells. In conclusion, this study demonstrates a potentially influential mechanism for the physiological regulation of cardiac muscle contractility.
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PMID:Neurohormonal control of calcium sensitivity of myofilaments in rat single heart cells. 211 24

In the Triton X-100-treated polymorphonuclear leukocytes (PMN), which were stimulated with formyl-Met-Leu-Phe (FMLP) for 1 min, a 64,000 molecular weight protein (p64) was preferentially phosphorylated by the incubation with [gamma-32P]ATP in the presence of Mg2+, but not in the presence of Ca2+. Phosphoamino acid analysis of pp64 revealed that the p64-kinase was a serine-specific protein kinase. The p64 was maximally phosphorylated in the first minute, suggesting that the rapid phosphorylation was related to the initial reaction for activation of the FMLP-stimulated PMN functions. The FMLP-stimulated phosphorylation of p64 was slightly inhibited by the addition of cGMP in the reaction mixture. However, addition of cAMP, the cyclic nucleotide-dependent kinase inhibitor (H-8), protein kinase C-inhibitor (H-7) or Ca/calmodulin-dependent kinase inhibitor (W-7), showed no effect on the phosphorylation. These data suggest that phosphorylation of p64 seems to be a novel protein kinase specific to p64.
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PMID:Formyl-Met-Leu-Phe-dependent serine kinase for a 64,000 molecular weight protein of polymorphonuclear leukocytes in a cell-lysate system. 214 31

Synaptic plasma membranes from rat brain cortex possess intrinsic ability to dephosphorylate the endogenous protein B-50. At low concentrations of [gamma-32P]ATP, B-50 phosphorylation in synaptic membranes is maximal at 30 seconds, followed by dephosphorylation for an additional 60 minutes. The dephosphorylation of 32P-labeled B-50 is not sensitive to the protease inhibitor leupeptin and not correlated with a loss of the B-50 content of synaptic membranes as measured with immunoblot analysis. Dephosphorylation of membrane-associated B-50 is stimulated to a small extent by Mg2+ but not by Ca2+. Heat-stable protein phosphatase inhibitors prevent dephosphorylation of 32P-labeled B-50. Dephosphorylation of B-50 in synaptic membranes is stimulated by ATP, ADP, or adenosine 5'-O-thiotriphosphate, but not by adenine, adenosine, other adenine or guanine nucleotides, nonhydrolyzable analogs of ATP or GTP, nor by adenosine 5'-O-(2-thiodiphosphate). B-50, phosphorylated by exogenous protein kinase C and purified to homogeneity, has been used as a substrate to follow the purification of B-50 phosphatase activity. B-50 phosphatase activity can be solubilized from synaptic membranes with 0.5% Triton X-100 and 75 mM KCl. Chromatography of the extract on DEAE-cellulose yields enhanced B-50 phosphatase activity.
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PMID:Dephosphorylation of B-50 in synaptic plasma membranes. 215 32

Investigations of protein kinase C (PKC) activity have focussed on protein phosphorylation using adenosine triphosphate (ATP), not guanosine triphosphate (GTP), as the phosphate donor. In a continuing study of the enzymology of the PKC of human neutrophils, we wanted to determine if there might be protein kinases that do use GTP as a phosphate donor. Soluble extracts or detergent-extracted fractions of human neutrophils were used as enzyme sources. Phosphorylation of histone using [gamma-32P]-GTP was 31% as effective as [gamma-32P]-ATP. Phosphorylation with GTP depended on Ca2+, Mg2+, and phospholipid, just as the ATP, and the Ca2+ requirements were similar. In all cases, H-7, an inhibitor of ATP-supported PKC activity, blocked GTP-utilizing activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that similar endogenous proteins were phosphorylated with ATP or GTP. The apparent Km and Vmax for the enzyme(s) for both phosphate donors were identical, although these were modified by treatment with Triton X-100. GTP competitively inhibited use of ATP by PKC; however, low concentrations of ATP enhanced GTP-utilizing kinase activity in some cases. Non-hydrolyzable forms of ATP and other nucleotide triphosphates were inhibitory. Detergent treatment also markedly altered the number of proteins phosphorylated by either nucleotide. The major protein phosphorylated in the soluble or detergent extract was a single polypeptide band in the 34 Kd range. These studies are the first to explicitly examine the possible phosphorylation by neutrophil PKC using GTP and point to a potential alternative mode of enzyme activity. Since high concentrations of GTP are available within neutrophils, the ability of PKC or a PKC-like enzyme to use this nucleotide may have important ramifications in signal transduction.
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PMID:Human neutrophils contain a protein kinase C-like enzyme that utilizes guanosine triphosphate as a phosphate donor. Cofactor requirements, kinetics, and endogenous acceptor proteins. 229 3

Angiotensin II (ang II) induces c-fos gene expression in part via a protein kinase C-dependent mechanism in cultured vascular smooth muscle cells (VSMC). However, little is known about the mechanisms by which protein kinase C regulates nuclear functions. We examined the ability of ang II to phosphorylate nuclear lamina proteins in VSMC and the possibility that protein kinase C is involved in these putative phosphorylation events. Ang II stimulated the phosphorylation of Triton X-100- and high salt-insoluble nuclear envelope proteins with molecular weights of 70,000, 67,000, and 60,000. These proteins were identified as lamins A, B, and C, respectively, based on their mobilities on two-dimensional gel electrophoresis and interaction with antibodies to lamins as detected by immunoblot analyses. After a 2-min delay, phosphorylation levels of lamins increased, peaked at 20-30 min, and were sustained for at least 60 min after ang II stimulation. The threshold, half-maximal, and maximal concentrations of ang II which induced phosphorylation of lamins were 0.1, 0.5-1, and 100 nM, respectively. Phorbol 12-myristate 13-acetate also induced these reactions, whereas ionomycin did not. Down-regulation of protein kinase C by prolonged treatment with phorbol 12,13-dibutyrate attenuated ang II-induced phosphorylation of lamins. In vitro phosphorylation of nuclear envelope proteins by protein kinase C revealed that lamins served as substrates for this enzyme. These results indicate that ang II induces phosphorylation of lamins in cultured VSMC and suggest that protein kinase C is either directly or indirectly involved in these reactions. The results raise the possibility that phosphorylation of nuclear proteins is one of the important steps by which the protein kinase C signaling pathway regulates agonist-induced nuclear events.
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PMID:Angiotensin II stimulates phosphorylation of nuclear lamins via a protein kinase C-dependent mechanism in cultured vascular smooth muscle cells. 229 7

To determine whether or not PKC is present in the nuclei of nerve tissue we made use of biochemical and immunocytochemical techniques. A 219-fold purification of rabbit brain nuclear protein kinase C was achieved by sequential steps of Triton X-100 extraction of isolated nuclei, DEAE-cellulose, Butyl-toyopearl and hydroxylapatite chromatography. The major peak of protein kinase C activity was eluted from the hydroxylapatite column at the KPO4 concentration of 0.3 M. Both Ca2+ and Ptd Ser were required for stimulation of the enzyme. Immunoblot analysis revealed that the kinase fraction was immunoreactive with a polyclonal antibody, PC-zeta, that had been raised against a peptide synthesized according to the deduced sequence of rat zeta protein kinase C. Light-microscopy revealed strong immunoreactivity in the nuclei of Purkinje cells in cerebellum and pyramidal cells in the rat cerebral cortex. These observations suggest that a zeta-related protein kinase C is present in the nuclei of nerve cells.
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PMID:zeta-Related protein kinase C in nuclei of nerve cells. 232 95

A calcium-unresponsive, phorbol ester/phospholipid-activated protein kinase was purified to apparent homogeneity from a Triton X-100 extract of an EGTA/EDTA-preextracted particulate fraction of porcine spleen by chromatography on S-Sepharose Fast Flow, phenyl-Sepharose Fast Flow, protamine-agarose, and Superdex 200. The enzyme had a Mr of 76,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (p76-kinase). A similar value (78,000) was obtained by gel filtration. The purified p76-kinase proved to be much more stable than the enzyme in crude preparations. Storage in a buffer containing 50 mM mercaptoethanol and 20% glycerol at -20 degrees C for at least 4 months caused less than 20% loss in enzyme activity. The enzyme exhibited a pH optimum of 8.3. The affinity of the novel enzyme for substrates and cofactors differed to some extent from that of conventional alpha, beta, gamma protein kinase C (PKC). p76-kinase did not respond to calcium, had a lower requirement for magnesium, and a higher affinity for histone III-S than PKC. Both the p76-kinase-catalyzed phosphorylation of histone III-S and the autophosphorylation of the enzyme could be activated by the phorbol ester TPA (or diacylglycerol) plus phosphatidyl serine, but not by calcium plus phosphatidyl serine. The stoichiometry of autophosphorylation suggested that fully phosphorylated p76-kinase contained two phosphoserine residues and one phosphothreonine residue. Like PKC, p76-kinase bound TPA with high affinity (KD = 9.6 nM). In the absence of TPA, various unsaturated fatty acids, particularly arachidonic acid, were more potent as activators of the enzyme than phosphatidyl serine. The p76-kinase was recognized by an antiserum raised against a delta PKC-specific peptide, but not by an alpha, beta, gamma PKC-specific antiserum. The previously described p82-kinase of mouse epidermis and spleen exhibiting the same properties as the p76-kinase did also react with the p76-kinase-specific antiserum.
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PMID:Purification and characterization of a calcium-unresponsive, phorbol ester/phospholipid-activated protein kinase from porcine spleen. 239 47

The phosphorylation of lipocortin (a substrate of EGF-receptor kinase, and a putative phospholipase A2 inhibitor) was examined in T51B cells. By using Western blot procedures and antisera specific to lipocortin I, we found that most immunoreactive lipocortin I was located in the cytosol (lipocortin(cvt] of cells extracted in Ca2+-free buffers These cells however had another pool of immunoreactive lipocortin I located in the particulate fraction that was Triton X-100 extractable (lipocortin(mem]. Increasing Ca2+ concentrations in the extraction buffer resulted in more lipocortin(mem) recovered. In vitro phosphorylation of endogenous proteins demonstrated that lipocortin I became phosphorylated in a Ca2+ and phosphatidylserine-dependent manner, suggesting an involvement of protein kinase C. Treatment of cells with 100 ng/ml 12-0-tetradecanoylphorbol-13-acetate (TPA) but not with 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) resulted in the in vitro phosphorylation of lipocortin(mem) by protein kinase C. TPA also increased the phosphorylation of lipocortin(mem) in [32P]phosphate-labeled cells.
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PMID:Tumor promoter-dependent phosphorylation of a Triton X-100 extractable form of lipocortin I in T51B rat liver cells. 253 98

Using Triton X-100/lipid mixed micellar methods, we observed that the adriamycin-iron(III) complex was a potent inhibitor of protein kinase C while uncomplexed adriamycin itself was a poor inhibitor in the absence of heavy metal contaminants. The 3:1 adriamycin-iron complex was more potent than 2:1, 1:1, and 1:0 complexes. Inhibition of protein kinase C was reversible, and 50% inhibition occurred at 13 microM (adriamycin)3Fe3+. Both the catalytic and the regulatory domain of protein kinase C were affected by adriamycin-iron(III). Adriamycin-iron(III) was a competitive inhibitor of the catalytic domain of protein kinase C with respect to MgATP but not with respect to magnesium (IC50 350 microM). The predominant interaction of adriamycin-iron(III) with native protein kinase C was as a competitive inhibitor with respect to diacylglycerol. Inhibition was not competitive with respect to phosphatidylserine, calcium, magnesium, MgATP, or histone. Interaction with the regulatory domain was demonstrated by the ability of adriamycin-iron(III) to inhibit phorbol dibutyrate binding. Other adriamycin transitional metal complexes showed little inhibition of protein kinase C activity. Acetylation of the amine on the daunosamine moeity of adriamycin did not preclude the formation of a ferric complex but resulted in total loss of inhibitory activity. These results suggest that the presence of free amines in a highly structured adriamycin-iron complex is necessary for inhibition. The implications of inhibition of protein kinase C by adriamycin-iron(III) are discussed.
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PMID:The adriamycin-iron(III) complex is a potent inhibitor of protein kinase C. 254 37

The Triton X-100 extract of the particulate fraction of porcine spleen contains a protein kinase which can be activated by phospholipid and the phorbol ester TPA but does not respond to phospholipid and calcium. The partially purified kinase has a molecular weight of 76 kDa (p76-kinase) and hence is somewhat smaller than the similarly behaving p82-kinase from mouse epidermis and spleen. The p76-kinase shows strong autophosphorylation. The protein kinase inhibitor K252a clearly differentiates between the Ca2+-unresponsive p76-kinase and Ca2+-responsive PKC. At concentrations of up to 5 x 10(-7)M it fails to suppress p76-kinase-catalyzed autophosphorylation and histone phosphorylation, but it inhibits PKC-catalyzed phosphorylation up to 50%. The IC50 values of K252a regarding PKC and the p76-kinase differ by two orders of magnitude. At low concentrations, K252a appears to slightly activate further TPA-activated p76-kinase. It is not able, however, to replace TPA and to stimulate the p76-kinase in the presence of phospholipid alone.
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PMID:Differentiative action of K252a on protein kinase C and a calcium-unresponsive, phorbol ester/phospholipid-activated protein kinase. 259 Feb 10


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