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)

Bovine platelet myosin is phosphorylated by protein kinase C at multiple sites. Most of the phosphate is incorporated in the 20,000-dalton light chain although some phosphate is incorporated in the heavy chain. Phosphorylation of the 20,000-dalton light chain of platelet myosin is 10 times faster than the phosphorylation of smooth muscle myosin. Platelet myosin light chain is first phosphorylated at a threonine residue followed by a serine residue. Dominant phosphorylation sites of the 20,000-dalton light chain are estimated as serine-1, serine-2, and threonine-9. Prolonged phosphorylation by protein kinase C resulted in an additional phosphorylation site which, on the basis of limited proteolysis, appears to be either serine-19 or threonine-18. Phosphorylation by protein kinase C causes an inhibition of actin-activated ATPase activity of platelet myosin prephosphorylated by myosin light chain kinase. Inhibition of ATPase activity is due to a decreased affinity of myosin for actin, and no change in Vmax is observed. It is shown that platelet myosin also exhibits the 6S to 10S conformation transition as judged by viscosity and gel filtration methods. Mg2(+)-ATPase activity of platelet myosin is paralleled with the 10S-6S transition. Phosphorylation by protein kinase C affects neither the 10S-6S transition nor the myosin filament formation. Therefore, the inhibition of actin-activated ATPase activity of platelet myosin is not due to the change in the myosin conformation.
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PMID:Phosphorylation of bovine platelet myosin by protein kinase C. 234 43

Monoclonal antibody P256, which is specific for glycoprotein IIb-IIIa complex, was found to induce aggregation of normal platelets in plasma. The mechanism of platelet activation induced by this monoclonal antibody was thoroughly studied. The divalent binding to the IIb-IIIa molecule was necessary for triggering aggregation since Fab' fragments did not induce aggregation as did IgG and F(ab')2 fragments; however, F(ab')2 did not induce the release as did the whole IgG. P256-induced aggregation was accompanied by release of all three granule constituents, namely dense granules, alpha-granules and lysosomes, with parallel kinetics showing half-maximum release 50 s after addition of P256. Thromboxane synthesis was initiated at the same time. Using 32P-prelabeled platelets, no variation in level of [32P]phosphatidylinositol 4,5-bisphosphate could be detected in the first minute after P256 addition, indicating no activation of the calcium-independent phospholipase C specific for polyphosphoinositol phospholipid. P256 induced a calcium mobilization as measured by Indo-1 fluorescence of about the third of that measured in the presence of a thrombin concentration giving the same intensity of aggregation. P256 induced phosphorylation of the myosin light chain p20 and of the main substrate of protein kinase C, p43. Addition of aspirin inhibited almost totally calcium mobilization and partially aggregation, release and protein phosphorylations. By contrast, in the absence of external calcium, although no aggregation could occur, the release reaction was only partially reduced. In this activation, the glycoprotein IIb-IIIa complex thus appears to play a role in modulating platelet response, not only via calcium fluxes but also in activating protein kinase C responsible for p43 phosphorylation.
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PMID:Activation of platelets induced by mAb P256 specific for glycoprotein IIb-IIIa. Possible evidence for a role for IIb-IIIa in membrane signal transduction. 236 45

The incorporation of [32P]phosphate into the 20 kDa myosin light chain of phorbol dibutyrate-contracted artery was slightly increased as compared to that of resting muscle. Addition of K+ to the 1-h phorbol dibutyrate-contracted artery immediately doubled the force and greatly increased the light chain phosphorylation. Two-dimensional phosphopeptide mapping of light chain from phorbol dibutyrate-contracted muscle showed distinct peptides phosphorylated on serine residues by myosin light chain kinase and protein kinase C. In addition, the peptide phosphorylated on threonine residue by protein kinase C was revealed for the first time in intact muscle. Upon addition of K+, the distribution of phosphopeptides shifted toward the myosin light chain kinase catalyzed pattern.
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PMID:Analysis of myosin light chain phosphopeptides in phorbol dibutyrate-contracted artery. 238 75

Experiments were conducted to characterize the effects of amiloride on the regulatory mechanisms of vascular smooth muscle contraction. Intact, saponin-skinned and A23187-treated strips of rabbit aorta were used for these studies. Amiloride significantly (P less than 0.05) reduced the norepinephrine bitartrate (NE)-stimulated increase in intracellular Ca2+ in intact arteries. In saponin-skinned arteries, amiloride depressed both stress and concomitant levels of myosin light chain phosphorylation. This inhibition of stress appeared to be competitive with MgATP. In A23187-treated preparations, where the effects of amiloride were studied at physiological [MgATP] in the absence of functional membrane Ca2+-channels, amiloride caused a reduction in both stress and myosin light chain phosphorylation. In other experiments on intact arteries, the contractile response to phorbol 12,13-dibutyrate, an activator of protein kinase C, was reduced by amiloride. We conclude that the vasorelaxant effects of amiloride are mediated via inhibition of myosin light chain kinase and protein kinase C, in addition to the inhibition of Ca2+ influx.
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PMID:Effect of amiloride on regulatory mechanisms of vascular smooth muscle contraction. 244 93

Studies utilizing cultured muscle cells have shown that myoblast fusion requires extracellular Ca2+ and involves transient coordinated changes in cell membrane topography and cytoskeletal organization. However, neither the mechanisms by which Ca2+ influences these changes nor its cellular sites of action are known. We have investigated the effects of Ca2+ channel modulators and phorbol esters on fusion of embryonic chick myoblasts in culture. Myoblast fusion was inhibited by the Ca2+ channel blockers D600 and nitrendipine and stimulated by the Ca2+ channel activator Bay K 8644. We have obtained evidence that the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits fusion through activation of protein kinase C. Myoblasts prevented from fusing by Ca2+ channel blockers or TPA display a distinctive elongated morphology that is characteristic of cells prevented from fusion by Ca2+ deprivation. The inhibition of fusion by D600 and TPA is significantly diminished in the presence of the Ca2+ ionophore A23187. TPA arrest of myoblast fusion was found to be accompanied by an increase in phosphorylation of the 20-kDa light chain of cytoplasmic myosin in a dose- and time-dependent manner. The effects of TPA on myoblast fusion and phosphorylation of myosin light chain were mimicked by the cell permeant diacylglycerol sn-1,2-dioctanoylglycerol, a potent activator of protein kinase C. The present results suggest that activators of protein kinase C block fusion by interfering with a Ca2+ signal transduction pathway and that this interference may be associated with a protein kinase C catalyzed inhibitory phosphorylation of myosin light chain.
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PMID:Opposing effects of calcium entry and phorbol esters on fusion of chick muscle cells. 247 83

IgE-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells results in the secretion of histamine. Myosin immunoprecipitated from these cells shows an increase in the amount of radioactive phosphate incorporated into its heavy (200 kDa) and light (20 kDa) chains. In unstimulated cells two-dimensional mapping of tryptic peptides of the myosin light chain reveals one phosphopeptide containing the serine residue phosphorylated by myosin light chain kinase. Following stimulation a second phosphopeptide appears containing a serine residue phosphorylated by protein kinase C. Tryptic phosphopeptide maps derived from myosin heavy chains show that unstimulated cells contain three major phosphopeptides. Following stimulation a new tryptic phosphopeptide appears containing a serine site phosphorylated by protein kinase C. The stoichiometry of phosphorylation of the myosin light and heavy chains was determined before and after antigenic stimulation. Before stimulation, myosin light chains contained 0.4 mol of phosphate/mol of light chain all confined to a serine not phosphorylated by protein kinase C. Cells that secreted 44% of their total histamine in 10 min exhibited an increase in phosphate content at sites phosphorylated by protein kinase C from 0 mol of phosphate/mol of myosin subunit to 0.7 mol of phosphate/mol of light chain and to 1 mol of phosphate/mol of heavy chain. When RBL-2H3 cells were made permeable with streptolysin O they still showed a qualitatively similar pattern of secretion and phosphorylation. Our results show that the time course of histamine secretion from stimulated RBL-2H3 cells parallels that of myosin heavy and light chain phosphorylation by protein kinase C.
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PMID:Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells. 247 73

Aspirin-pretreated, 32P-prelabeled, washed human platelets resuspended in a buffer containing apyrase and 2% plasma were exposed to epinephrine and the Ca2+ ionophore A23187. Epinephrine potentiated platelet aggregation (not secretion), the production of [32P]phosphatidic acid and myosin light chain phosphorylation induced by A23187. No phosphorylation of the 40 kDa protein, the substrate of protein kinase C, was observed. We conclude that G1-protein activation evoked by epinephrine and Ca2+ mobilization caused by A23187 represents a novel synergism for platelet aggregation and that protein kinase C activation, under these conditions is not needed for platelet aggregation.
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PMID:Epinephrine and the Ca2+ ionophore A23187 synergistically induce platelet aggregation without protein kinase C activation. 249 53

The effect of ethanol on signal generation in collagen-stimulated human platelets was evaluated. Incubation of washed human platelets with physiologically relevant concentrations of ethanol (25-150 mM) resulted in a dose-dependent inhibition of aggregation and secretion in response to collagen (0.5-10 micrograms/ml), but did not inhibit shape change. In platelets labeled with [3H]arachidonic acid, ethanol significantly inhibited the release of arachidonic acid from phospholipids, in both the presence and the absence of indomethacin. Thromboxane B2 formation was also inhibited in proportion to the reduction in free arachidonic acid. There was a close correlation between the extent of inhibition of arachidonic acid release and secretion. The inhibition of platelet aggregation and secretion by ethanol was partially overcome by the addition of exogenous arachidonic acid. In the presence of indomethacin, ethanol had no effect on the activation of phospholipase C by collagen as determined by the formation of inositol phosphates and phosphatidic acid. Moreover, ethanol had no effect on the mobilization of intracellular calcium by collagen and only minimally inhibited the early phases of the phosphorylation of myosin light chain (20 kDa) and a 47-kDa protein, a known substrate for protein kinase C. Arachidonic acid formation was also inhibited by ethanol in response to ionomycin under conditions where phospholipase C activation was prevented. The results suggest that the functional effects of ethanol on collagen-stimulated platelets are due, at least in part, to an inhibition of phospholipase A2.
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PMID:Ethanol interferes with collagen-induced platelet activation by inhibition of arachidonic acid mobilization. 249 46

Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.
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PMID:Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases. 249 92

Two types of myosin light chain phosphatase from aortic smooth muscle extract were separated by chromatography on heparin-agarose. The phosphatase which appeared in the flow-through fractions had low activity on actomyosin, its apparent molecular mass was 260 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 36-39 kDa as determined by gel filtration. This phosphatase preferentially dephosphorylated the alpha-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. The phosphatase retained by heparin-agarose had high activity on actomyosin, its apparent molecular mass was 150 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 39-42 kDa. It preferentially dephosphorylated the beta-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. Myosin light chain was phosphorylated by myosin light chain kinase in peptides AB (Ser-P) and CD (Thr-P), and/or by protein kinase C in peptides E (Ser-P) and F (Thr-P) as determined by one-dimensional phosphopeptide mapping. The catalytic subunit of heparin-agarose flow-through phosphatase preferentially dephosphorylated peptide F over peptides AB, CD and E in both isolated light chain and actomyosin. The catalytic subunit of heparin-agarose bound phosphatase could effectively dephosphorylate all sites in isolated light chain, whereas it was less effective on dephosphorylation of peptide E in actomyosin.
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PMID:Dephosphorylation of distinct sites in myosin light chain by two types of phosphatase in aortic smooth muscle. 253 49


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