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)

This study investigates the role of protein kinase C and of myosin light chain kinase in mediating platelet hyperresponsiveness in spontaneously hypertensive rats (SHR). For this purpose, 32P-labeled washed platelets of both SHR and normotensive controls Wistar-Kyoto (WKY) were challenged either with a receptor-mediated agonist (thrombin) or with direct activators of myosin light chain kinase and protein kinase C. Such enzymatic activities were assessed by measuring changes in 32P-labeling of their respective target proteins, namely myosin light chain (20 KDa) and the 47 KDa protein. In resting platelets, the patterns of protein phosphorylation were similar between SHR and WKY, suggesting that the two cell types were in a comparable quiescent status. By contrast, in both dose-response and time-course studies, thrombin promoted a significantly greater phosphorylation of the 20- and 47 KDa proteins in platelets of SHR compared with that for WKY. Sensitivity of myosin light chain kinase to the calcium ionophore A23187 and of protein kinase C to both phorbol ester and dioctanoylglycerol was apparently not different between the two cell types. The data indicate that the exaggerated thrombin-induced protein phosphorylation observed for platelets of SHR is not linked to alterations in protein kinase C and/or myosin light chain kinase per se. These results therefore suggest that platelet hyperresponsiveness in SHR is likely to be related, at least in part, to abnormalities in receptor-mediated transmembrane signalling.
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PMID:Receptor-dependent and -independent protein phosphorylation in platelets of spontaneously hypertensive rats. 217 66

Sustained smooth muscle contraction has been proposed to be regulated by either 1) sustained increases in intracellular Ca2+ concentration [(Ca2+]i)-dependent myosin phosphorylation or 2) diacylglycerol-dependent protein kinase C activation. We measured diacylglycerol mass with the diacylglycerol kinase assay and myoplasmic [Ca2+] with aequorin in swine carotid medial smooth muscle. Sustained and significant increases in [Ca2+], myosin light chain phosphorylation, and isometric stress were observed with histamine or endothelin stimulation. Neither stimuli, however, induced significant increases in diacylglycerol mass. Relaxation of histamine-stimulated tissues was induced by removal of histamine or removal of extracellular CaCl2 in the continued presence of histamine. The rate of decline of both [Ca2+] and force was similar in both protocols, suggesting that removal of Ca2+ (without removing the stimulus) was equivalent to removal of the stimulus. These data suggest that [Ca2+]i is the primary regulator of sustained swine arterial smooth muscle contraction, whereas diacylglycerol has, at most, only a minor role.
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PMID:[Ca2+], not diacylglycerol, is the primary regulator of sustained swine arterial smooth muscle contraction. 219 Sep 21

Experiments using 32P-labeled strips of swine carotid artery medial smooth muscle were performed to define the relative contribution of myosin light chain (MLC) phosphorylation as an activation mechanism mediating contractile responses stimulated by phorbol dibutyrate (PDB). Tryptic phosphopeptide mapping of phosphorylated MLC indicated that near-maximal force responses were associated with increases in functional MLC phosphorylation of less than 10% of the total MLC content following tonic (45 min) stimulation by PDB. Significant phosphorylation of MLC residues, consistent with the specificity of protein kinase C, occurred in response to high concentrations of PDB (greater than 0.1 microM). Histamine (10 microM)-induced MLC phosphorylation after 2 min (72.5% of total MLC) or 45 min (61.7%) was restricted to serine residues on peptides thought to contain serine19. Although agonist (histamine)-induced responses were eliminated under conditions of Ca2+ depletion, near-maximal force in response to 10 microM PDB (89.4% of a standard KCl response) was associated with monophosphorylation of less than 9% of the total MLC on peptides interpreted as containing serine19. A substantial fraction of this was localized to threonine residues. The quantitative analysis of the relation between PDB-stimulated force and the residues in MLC phosphorylated supports the concept that PDB stimulation results in activation of arterial smooth muscle cross bridges by MLC-phosphorylation-independent mechanisms.
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PMID:Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle. 222 Oct 41

We investigated the role of protein kinase C in the mechanical responses evoked by high K or by acetylcholine (ACh) in intact vascular smooth muscle tissues, and by Ca in skinned vascular smooth muscle tissues. To activate protein kinase C, the phorbol ester 12-o-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, or 1,2-diolein, plus phosphatidylserine (PS) was used. TPA enhanced or reduced the amplitude of the contraction evoked by increased concentrations of K below 39 mmol/L or over 90 mmol/L, respectively, but consistently enhanced the resting tension at any given concentration of high K. Similar effects of TPA were observed on the Ca-induced contraction in saponin skinned muscle tissues. The enhancing action of TPA on the K-induced contraction was not related to activation of either the voltage-dependent Ca channel or the sarcoplasmic reticulum, and did not occur in the case of Ca-independent contraction in skinned muscle tissues. During the enhancement of the contraction induced by TPA, the phosphorylation of myosin light chain and the shortening velocity of contraction as measured using the slack test, were enhanced with no remarkable change in the free Ca concentration in the cytosol. TPA consistently inhibited the ACH-induced contraction accompanied by a marked reduction in free Ca due to inhibition of the hydrolysis of phosphatidyl inositol 4,5-bisphosphate. Under the assumption that TPA possesses the same action as DG, activation of protein kinase C increased the Ca sensitivity of contractile proteins in vascular smooth muscles.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Roles of protein kinase C on the mechanical activity of vascular smooth muscles. 222 71

Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol [32P]phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the [32P]phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in [32P]phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.
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PMID:Stretch activates myosin light chain kinase in arterial smooth muscle. 225 11

Prostaglandin (PG) F2 alpha (30 microM) stimulated both monophosphorylation and diphosphorylation of myosin light chain (MLC) in a smooth muscle cell line (SM-3). The diphosphorylation was significantly decreased by treatment with the protein kinase C inhibitor staurosporine (30 nM, 30 min) from 20.1% of total MLC to 4.5%. The protein kinase C down-regulation treatment of SM-3 cells with phorbol dibutyrate suppressed to 8.7% the MLC diphosphorylation activity in the SM-3 cells. This down-regulation treatment had little effect on the monophosphorylation. We propose that the MLC diphosphorylation in PGF2 alpha-stimulated SM-3 cells in culture may be regulated through mechanisms sensitive to protein kinase C.
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PMID:Diphosphorylation of myosin light chain in smooth muscle cells in culture. Possible involvement of protein kinase C. 226 95

Endothelin-1 contracts porcine carotid arterial smooth muscle with an ED50 of 10 nM. Contraction is associated with phosphorylation of the 20,000 dalton-regulatory light chain subunits of vascular myosin. Phosphopeptide mapping of light chains isolated from 32PO4-loaded muscle strips stimulated by endothelin-1 (5 x 10(-8) M) and comparison with maps generated from light chains phosphorylated in vitro or muscles stimulated with KCl (110 mM) or angiotensin-II (5 x 10(-8) M) indicates that Ca2(+)-calmodulin activation of myosin light chain kinase is a biochemical pathway stimulated by all three agonists. However, a small amount of phosphate (17%) was detected in a light chain peptide phosphorylated by protein kinase C. Endothelin-1 also stimulated phosphorylation of the thin filament protein, caldesmon, (from 0.35 mol PO4/mol caldesmon to 0.52 mol PO4/mol). Collectively, these results provide evidence that the effects of endothelin-1 on force generation and maintenance in vascular muscle may be dependent upon myosin light chain phosphorylation by Ca2+ calmodulin--requiring myosin light chain kinase and upon a thin filament mechanism that is modulated by phosphorylation of caldesmon.
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PMID:Myosin light chain and caldesmon phosphorylation in arterial muscle stimulated with endothelin-1. 228 Apr 12

The CD9 molecule is a 24 kDa surface-membrane glycoprotein present on platelets and a variety of haematopoetic and non-haematopoetic tissues. In the present study we utilized specific inhibitors of thromboxane A2 (TxA2) formation (aspirin), protein kinase C [H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine]] and autocrine stimulation by secreted ADP (apyrase) to modify platelet activation by a monoclonal antibody ALB-6 to the CD9 antigen. This activation is only partially inhibited by aspirin alone but, in combination with either H-7 or apyrase, more than 50% inhibition of platelet aggregation and secretion was observed. This combination of inhibitors was also required to inhibit effectively the phosphorylation of myosin light chain and the 47 kDa substrate of protein kinase C. Intracellular Ca2+ flux monitored by the fluorescent dye fura-2 showed that this was almost completely mediated by the aspirin-sensitive TxA2 pathway. We suggest that the aspirin-insensitive pathway is primarily mediated by phospholipase C formation of diacylglycerol to activate protein kinase C. The inhibition by apyrase suggests a strong dependency on autocrine stimulation by secreted ADP to fully activate both phospholipase C and express fibrinogen-binding sites mediating platelet aggregation. This alternate pathway of phospholipase C activation by ALB-6 may be mediated by cytoplasmic alkalinization [monitored by SNARF-1 (5'(6')-carboxy-10-bismethylamino-3-hydroxy-spiro-[7H- benzo[c]xanthine-1',7(3H)-isobenzofuran]-3'-one) fluorescence of the dye]. Both activation pathways are dependent on intact antibodies, since F(ab')2 fragments of SYB-1, a monoclonal antibody against the CD9 antigen with activation characteristics identical with those of ALB-6, do not elicit activation. Besides thrombin, collagen is another physiological agonist shown to induce aspirin-insensitive activation. Similarities to ALB-6 in collagen sensitivity to apyrase in combination with aspirin inhibitors were noted with respect to aggregation and secretion, as well as a complete block of Ca2+ flux by aspirin. However, it is unlikely that collagen activation is mediated by the CD9 antigen, since SYB-1 F(ab')2 fragments had no effect on collagen activation and aspirin also completely blocked the alkalinization response to collagen, in contrast with ALB-6.
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PMID:Stimulus-response coupling in human platelets activated by monoclonal antibodies to the CD9 antigen, a 24 kDa surface-membrane glycoprotein. 231 2

The mechanisms by which activators of protein kinase C (PKC) stimulate contractile responses in arterial smooth muscle is not known. In this study, we assessed the relative contribution of CA(++)-dependent and independent pathways in mediating phorbol ester-induced 20 kdalton myosin light chain (MLC)-phosphorylation and force in medial smooth muscle strips from swine carotid artery. Phorbol 12,13-dibutyrate (PDB; 10(-7)M)-stimulated stress development was associated with a significant increase in the fraction of phosphorylated MLC, from 0.08 +/- 0.02 to 0.24 +/- 0.02 after 30 min of stimulation. Under conditions of Ca++ depletion, which normally do not support Ca++/calmodulin-dependent activation of myosin light chain kinase (MLCK) by physiological stimuli, PDB-induced contractile responses were reduced significantly. However, after Ca2++ depletion, PDB (10(-6) M; 30 min) still caused an increase in MLC-phosphorylation from 0.10 +/- 0.02 at rest to 0.19 +/- 0.03. Preincubation with nifedipine (10(-7) M) had no significant effect on contractile responses to PDB, indicating that Ca++ influx through nifedipine-sensitive voltage channels did not contribute significantly to the observed Ca++ dependency of the PDB responses. Staurosporine (0.1-0.3 microM), a putative PKC inhibitor, significantly inhibited PDB-induced contractile and MLC phosphorylation responses. Tonic histamine (3 microM)- and KCl-induced contractile and MLC-phosphorylation responses were inhibited by the same concentrations of staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phorbol ester-induced stress and myosin light chain phosphorylation in swine carotid medial smooth muscle. 231 59

KT5926, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3 ,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde] trinden-1-one, was found to be a potent and selective inhibitor of myosin light chain kinase. The compound inhibited both Ca2+/calmodulin-dependent and -independent smooth muscle myosin light chain kinases to a similar extent. The inhibition was not affected by the concentration of calmodulin. Kinetic analyses showed that the mode of inhibition was of the competitive type with respect to ATP (Ki, 18 nM) and of the noncompetitive type with respect to myosin light chain (Ki, 12 nM). These results indicated that KT5926 directly interacted with the enzyme at the catalytic site. KT5926 also inhibited other protein kinases, but with relatively high Ki values; the values for protein kinase C, cAMP-dependent protein kinase, and cGMP-dependent protein kinase were 723, 1200, and 158 nM, respectively. Ca2(+)-ATPase, Na+/K(+)-ATPase, hexokinase, and 5'-nucleotidase were not inhibited by KT5926 at less than 10 microM. The effect of KT5926 on serotonin secretion and protein phosphorylation induced by platelet-activating factor or phorbol ester was examined in rabbit platelets. KT5926 inhibited the phosphorylation of a 20-kDa protein but had no effect on the phosphorylation of a 40-kDa protein, thereby indicating that the compound exerts its selective inhibition of myosin light chain kinase in intact cells. The compound inhibited serotonin secretion induced by platelet-activating factor, but its potency was significantly less than that of K-252a, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9, 10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b, 11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, which inhibited the phosphorylation of both the 20-kDa protein and the 40-kDa protein. Phorbol ester-induced secretion was not suppressed by KT5926. These results provide the evidence that both the 20-kDa protein phosphorylation by myosin light chain kinase and the 40-kDa protein phosphorylation by protein kinase C substantially contribute to the secretion response in platelets.
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PMID:KT5926, a potent and selective inhibitor of myosin light chain kinase. 232 35


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