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)

The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.
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PMID:Myosin light chain phosphorylation in 32P-labeled rabbit aorta stimulated by phorbol 12,13-dibutyrate and phenylephrine. 268 75

We have examined regulation by protein kinase C (Ca2+/phospholipid-dependent enzyme) of thrombin-induced inositol polyphosphate accumulation in human platelets. When platelets are exposed to thrombin for 10 s, the protein kinase C inhibitor staurosporine causes inositol phosphate elevations over control values of 2.7-fold (inositol 1,4,5-trisphosphate (Ins(1,4,5)P3], 1.9-fold (inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4], and 1.2-fold (inositol 1,3,4-trisphosphate). In the same period, phosphatidic acid and diacylglycerol are unaffected. The myosin light chain kinase inhibitor ML-7 has no effect on inositol phosphate accumulations. Staurosporine does not inhibit Ins(1,4,5)P3 3-kinase and 5-phosphomonoesterase activities in saponin-permeabilized platelets incubated with exogenous Ins(1,4,5)P3 unless the platelets have been exposed to thrombin and protein kinase C is consequently activated. The protein kinase C agonist beta-phorbol 12,13-dibutyrate increases the Vmax of the 3-kinase 1.8-fold, with little effect on Km. Our results provide strong evidence for a role for protein kinase C in regulating inositol phosphate levels in thrombin-activated platelets. We propose that endogenously activated protein kinase C removes Ins(1,4,5)P3 by stimulating both 5-phosphomonoesterase and Ins(1,4,5)P3 3-kinase. Initial activation of phospholipase C does not appear to be affected by such protein kinase C. Inhibition of protein kinase C by staurosporine decreases 5-phosphomonoesterase activity. The resulting elevated Ins(1,4,5)P3, as substrate for Ins(1,4,5)P3 3-kinase, promotes production of Ins(1,3,4,5)P4, which also may accumulate through decreased 5-phosphomonoesterase activity and elevated Ca2+ levels. These factors apparently counteract the inhibitory effect on 3-kinase, yielding a net increase in Ins(1,3,4,5)P4.
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PMID:Inhibition of protein kinase C by staurosporine promotes elevated accumulations of inositol trisphosphates and tetrakisphosphate in human platelets exposed to thrombin. 270 80

Felodipine, a dihydropyridine Ca2+ channel blocker, appears to have intracellular sites of action in addition to its ability to attenuate voltage-dependent Ca2+ channels in smooth muscle cells. In vitro, felodipine inhibits several calmodulin-dependent enzymes such as myosin light chain kinase, cyclic nucleotide phosphodiesterase and caldesmon kinase [Walsh MP, Sutherland C and Scott-Woo GC, Biochem Pharmacol 37: 1569-1580, 1988]. Such effects may partially explain the relaxant effects of felodipine and related dihydropyridines on vascular smooth muscle. We have examined the effects of felodipine on the activity of another important enzyme which has been implicated in the regulation of the contractile state of smooth muscle, protein kinase C. We chose to use a physiologically relevant substrate of protein kinase C for these studies, viz. platelet P47 protein, rather than the more commonly used lysine-rich histone which is probably not a physiologically important substrate. Protein kinase C and P47 were purified from human platelets and their important structural and functional properties were characterized. Felodipine and the p-chloro analogue of felodipine enhanced both the rate and extent of P47 phosphorylation by protein kinase C. Half-maximal activation was observed at 9.5 microM felodipine and 8.5 microM p-chloro analogue. Activation by felodipine was dependent upon the presence of phospholipid but did not require diacylglycerol. These observations suggest that the pharmacological actions of felodipine and related dihydropyridines may involve activation of protein kinase C in addition to their known effects on voltage-dependent Ca2+ channels and calmodulin-dependent enzymes.
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PMID:Activation of protein kinase C by the dihydropyridine calcium channel blocker, felodipine. 270 18

We examined the extranuclear effects of thyroid hormones on human platelets. Pretreatment with DL-thyroxine or DL-triiodothyronine inhibited collagen-induced aggregation, in a dose-dependent manner, but other derivatives of thyroid hormone had no significant effects. In contrast to collagen, 12-O-tetradecanoylphorbol-13-acetate-induced aggregation was not affected by thyroid hormones at the same concentration range. Thyroxine also inhibited the release of [14C] serotonin from collagen-stimulated platelets, with a marked reduction in the phosphorylation of 20,000-dalton protein. Thyroxine and triiodothyronine had inhibitory effects on myosin light chain kinase purified from human platelets and inhibited more markedly the myosin light chain kinase than protein kinase C (Ca2+/phospholipid-dependent enzyme) and cAMP-dependent protein kinase. In addition, L-thyroxine behaved as a competitive inhibitor of myosin light chain kinase toward calmodulin, and the Ki value was calculated to be 2.6 microM. To determine whether or not thyroxine directly binds myosin light chain kinase, we prepared an affinity column, using L-thyroxine as the ligand. Myosin light chain kinase was selectively bound to the column while calmodulin passed through. We also designed a procedure for the purification of myosin light chain kinase from human platelets, using L-thyroxine-affinity chromatography. A markedly increased purification was thus achieved, and DEAE-cellulose and L-thyroxine-affinity chromatography were made feasible. These results suggest that thyroxine can serve as a pharmacological tool for elucidating the biological significance of myosin light chain kinase-mediated reactions and is a pertinent ligand which can be used to purify myosin light chain kinase from platelets as a substitute for calmodulin.
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PMID:Thyroid hormones inhibit platelet function and myosin light chain kinase. 272 89

We have partially purified a protein kinase from rat pancreas that phosphorylates two light-chain subunits of pancreatic myosin, a doublet with components of 18 and 20 kDa. This protein kinase was purified approx. 1000-fold by sequential (NH4)2SO4 fractionation, gel filtration, ion-exchange and affinity chromatography on calmodulin-Sepharose 4B. The resultant enzyme preparation is free of cyclic AMP-dependent protein kinase, protein kinase C and calmodulin-dependent type I or II kinase activities. The purified protein kinase is completely dependent on Ca2+ and calmodulin, and phosphorylates a 20 kDa light-chain subunit of intact gizzard myosin, suggesting that it belongs to a class of enzymes known as myosin light-chain kinase (MLCK). The apparent Km values of the putative pancreatic MLCK for ATP (73 microM), gizzard myosin light chains (18 microM) and calmodulin (2 nM) are similar to those reported for MLCKs isolated from smooth muscle, platelet and other sources. The enzyme is half-maximally activated at a free Ca2+ concentration of 2.5 microM. A single component of the affinity-purified kinase reacts with antibodies to turkey gizzard MLCK. The apparent molecular mass of this component is 138 kDa. Immunoprecipitation of a pancreatic homogenate with these antibodies decreases calmodulin-dependent kinase activity for pancreatic myosin by over 85%. The immunoprecipitate contains a single electrophoretic band of 138 kDa. Tryptic phosphopeptide analyses of pancreatic myosin, phosphorylated by either gizzard or pancreatic MLCK, are identical. Thus the enzyme that we have purified from rat pancreas is a MLCK, as judged by (1) absolute dependence on Ca2+ and calmodulin, (2) high affinity for calmodulin, (3) narrow substrate specificity for the light-chain subunit of myosin, and (4) reactivity with antibodies to turkey gizzard MLCK. These studies establish the existence of a pancreatic MLCK which may be responsible for regulating myosin phosphorylation and enzyme secretion in situ.
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PMID:Purification and characterization of myosin light-chain kinase from the rat pancreas. 273 May 65

The transport of cholesterol to the inner mitochondrial membrane, a key step in steroidogenesis, is subject to hormonal modulation that, at least in part, could be mediated by protein phosphorylation. This step is stimulated by sterol carrier protein 2 (SCP2) and Ca2+. To explore whether SCP2 itself is a potential control point for regulation by Ca2+-dependent phosphorylation we investigated whether highly purified SCP2 could serve as a substrate for major type Ca2+ and non-Ca2+-dependent protein kinases. Phosphorylation by calmodulin protein kinase II (CaM-PK II), myosin light chain kinase (MLCK), cAMP-dependent kinase (PKA) and protein kinase C (PKC) was monitored under optimal conditions for each enzyme. PKA, CaM-PK II and MLCK catalyzed the radiolabeling of histone 2A, synapsin I and myosin light chain (MLC), known substrates for these kinases, respectively, yet no phosphate transfer to SCP2 was observed. In contrast, PKC from two different sources (rat and calf brain) effectively catalyzed the phosphorylation of the highly purified SCP2. The phosphorylation of SCP2 depended on the addition of Ca2+ and phospholipids and was completely blocked by Polymyxin B, a PKC inhibitor. PKC catalyzed phosphorylation of SCP2 displayed a similar dependence on the concentration of ATP. Lineweaver Burk plots of the data indicate Km values for ATP of approximately 6 microM for the phosphorylation of SCP2. Our results, which have revealed for the first time that SCP2 is a substrate for PKC, are consistent with the possibilities that the control of steroidogenesis by tropic hormones and by PKC activation are mediated, at least in part, by the phosphorylation/dephosphorylation of SCP2.
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PMID:Protein kinase C catalyzed phosphorylation of sterol carrier protein 2. 273 66

Kaempferol, 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, was found to inhibit bovine aorta myosin light chain kinase with a Ki of 0.3-0.5 microM. It was found to be competitive with ATP and non-competitive with isolated myosin light chains. The specificity of this inhibitor was studied relative to protein kinase C and cAMP dependent protein kinase (IC50 = 15 microM and 150 microM, respectively). It appears not to interact strongly with calmodulin binding proteins, such as Ca2+-calmodulin dependent phosphodiesterase (IC50 = 45 microM), and had little effect on actin-activated myosin subfragment-1 ATPase activity (IC50 greater than 100 microM) or smooth muscle phosphatase activities (IC50 greater than 100 microM).
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PMID:Kaempferol inhibits myosin light chain kinase. 280 9

We have shown that the phosphorylation of smooth muscle regulatory myosin light chain (L20) with myosin light chain kinase (MLCK) produces faster moving bands (GMP1: heterodimer myosin with 1 unphosphorylated L20 and 1 mono-phosphorylated L20, GMP2: homodimer myosin with 2 mono-phosphorylated L20S) on native pyrophosphate polyacrylamide gel electrophoresis (PP1 PAGE) (J. Biochem. 100, 259-268, 1986; J. Biochem. 100, 1681-1684, 1986). However, the mobility of the myosin phosphorylated, at its L20, with protein kinase C (PK-C) was the same that of the unphosphorylated myosin (GM) on PPi PAGE. When the myosin prephosphorylated with MLCK was further phosphorylated with PK-C, PPi PAGE analysis showed only one band comigrating with GM, i.e., GMP1 and GMP2 migrated to the same position as GM. Conversely, when the myosin prephosphorylated with PK-C was further phosphorylated with MLCK, GMP1 and GMP2 were not produced. Thus the effect of L20 phosphorylated with PK-C is quite the opposite of that with MLCK, and the former predominated over the latter. We speculate that phosphorylation of L20 with PK-C "freezes" myosin in the inactive state.
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PMID:Native pyrophosphate gel analysis of smooth muscle myosin phosphorylated with protein kinase C. 283 Feb 55

Myosin light chain phosphorylation in aortic smooth muscle homogenate reached a maximal level of 0.75 mol phosphate/mol light chain, and then declined. Addition of okadaic acid led to a sustained phosphorylation level of 1.7 mol/mol. In the absence of okadaic acid, phosphorylation was predominantly due to myosin light chain kinase, whereas in the presence of okadaic acid both myosin light chain kinase and protein kinase C were involved in phosphorylation. Okadaic acid inhibited dephosphorylation of the distinct sites in LC phosphorylated by either myosin light chain kinase or protein kinase C, suggesting that it exerts its effect through inhibition of myosin light chain phosphatases present in aortic homogenate.
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PMID:Effect of okadaic acid on phosphorylation-dephosphorylation of myosin light chain in aortic smooth muscle homogenate. 283 97

Ca2+-dependent myosin phosphorylation by Ca2+/calmodulin-dependent myosin light chain kinase (MLC-kinase) and protein kinase C were studied using selective inhibitors, isoquinolinesulfonamide derivatives. Both protein kinases were potently inhibited by 1-(8-chloro-5-isoquinolinesulfonyl)piperazine (HA-156) and its derivatives. Kinetic analysis indicated that HA-156 inhibited both enzymes competitively with respect to ATP, and Ki values of HA-156 for MLC-kinase and protein kinase C were 7.3 and 7.2 microM, respectively. To clarify molecular mechanisms of the isoquinolinesulfonamides to inhibit the Ca2+-dependent protein kinases, we examined the structure-activity relationships of HA-156 and its derivatives. The dechlorinated analogues, HA-100 and HA-142, markedly decreased the affinity for MLC-kinase, suggesting that the inhibitory effect of isoquinolinesulfonamide derivatives depends upon hydrophobicity of the compounds. There is a good correlation between MLC-kinase inhibition and hydrophobicity determined by reverse phase chromatography. In contrast, HA-140 and HA-142 showed weak inhibition of protein kinase C, suggesting that the electron density of the nitrogen in the isoquinoline ring of the compounds correlates with the potency to inhibit protein kinase C activity. These pairs of isoquinolinesulfonamides will aid in elucidating the biological roles of Ca2+-dependent myosin phosphorylation in intact cells. HA-156 and HA-140 inhibited myosin light chain phosphorylation in platelets exposed to collagen, whereas HA-142 and HA-100 did not, significantly. These isoquinolinesulfonamide derivatives should prove to be useful tools for distinguishing between the biological functions of Ca2+-activated, phospholipid-dependent, and Ca2+/calmodulin-dependent myosin light chain phosphorylation, in vivo.
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PMID:Selective modulation of calcium-dependent myosin phosphorylation by novel protein kinase inhibitors, isoquinolinesulfonamide derivatives. 295 13


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