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)

Human platelet myosin forms 10S and 6S conformations, and its Ca(2+)- and Mg(2+)-ATPase activities are parallel with the transition between 10S and 6S conformation, as judged by the gel filtration, intrinsic fluorescence, and viscosity methods. The 20,000-dalton myosin light chain (LC20) is phosphorylated by both myosin light chain kinase (MLC kinase) and Ca2+, phospholipid-dependent protein kinase (protein kinase C [PKC]). The phosphorylation (1 mol of phosphate/mol of LC20) by MLC kinase shifts the equilibrium toward the 6S conformation, but that by PKC does not. The prephosphorylation of myosin by PKC prevents the effect of phosphorylation by MLC kinase on actin-activated Mg(2+)-ATPase activity, but not the effect on conformational change. Inhibition of actin-activated ATPase activity by PKC is due to a decreased affinity of myosin for actin, and no change in Vmax is observed. These results suggest that sequential phosphorylation of myosin by both kinases plays an important role in the ATPase activities of human platelet myosin.
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PMID:Effect of phosphorylation of myosin light chain by myosin light chain kinase and protein kinase C on conformational change and ATPase activities of human platelet myosin. 183 91

In the present study we tested the hypothesis that phosphorylation of the 20,000-dalton light chain subunit of smooth muscle myosin (LC20) by the calcium-activated and phospholipid-dependent protein kinase C regulates contraction of chemically-permeabilized (glycerinated) porcine carotid artery smooth muscle. Purified protein kinase C and oleic acid were used to phosphorylate LC20 in glycerinated muscles in the presence of a CaEGTA/EGTA buffer system (pCa 8) to prevent activation of myosin light chain kinase. Phosphorylation of the light chain to 1.3 mol of PO4/mol of LC20 did not stimulate contraction. Tryptic digests of glycerinated carotid artery LC20 contained two major phosphopeptides which contained phosphoserine but not phosphothreonine. Incubation of glycerinated muscles with calcium (20 microM) and calmodulin (10 microM) resulted in contraction and LC20 phosphorylation to 1.1 mol of PO4/mol of LC20; tryptic digests of LC20 from these muscles contained a single phosphopeptide which could be distinguished by phosphopeptide mapping from the two phosphopeptides derived from muscles phosphorylated with protein kinase C. Further phosphorylation of Ca2+/calmodulin-activated muscles to 2.0 mol of PO4/mol of LC20, by incubation with protein kinase C, had no effect on either the level of isometric force or the lightly-loaded shortening velocity (after-load = 0.1 peak active force); removal of Ca2+ and calmodulin, but not protein kinase C and oleic acid, resulted in normal relaxation in spite of maintained phosphorylation to 1.2 mol of PO4/mol of LC20. Comparison of LC20 phosphopeptide maps from glycerinated muscles incubated with protein kinase C plus Ca2+/calmodulin (2.0 mol of PO4/mol of LC20) to maps from intact muscles stimulated with 10(-6) M phorbol 12,13-dibutyrate (0.05 mol of PO4/mol of LC20) showed that the same three phosphopeptides were present in both the intact and glycerinated muscles. These findings show that phosphorylation of LC20 by protein kinase C in glycerinated muscles to levels at least 40 times higher than those present during contraction of intact, phorbol ester-stimulated muscles does not activate contraction nor does it significantly modify the contraction of smooth muscle which occurs in response to the Ca2+/calmodulin-dependent phosphorylation of Ser19 by myosin light chain kinase.
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PMID:Phosphorylation by protein kinase C of the 20,000-dalton light chain of myosin in intact and chemically skinned vascular smooth muscle. 230 24

We have examined the effect of protein kinase C phosphorylation on the actin-activated ATPase activity and filament stability of calf thymus myosin. Protein kinase C phosphorylated thymus myosin regulatory light chains, LC20, on two sites which are different from the site phosphorylated by myosin light chain kinase. The light meromyosin part of the thymus myosin heavy chain was also phosphorylated by protein kinase C, but at a rate about 10% that of LC20. Under conditions where both unphosphorylated thymus and myosin light chain kinase-phosphorylated thymus myosin were filamentous and under conditions where myosin light chain kinase phosphorylation induces myosin filament formation, protein kinase C phosphorylation had little effect on the actin-activated ATPase activity or filament stability of unphosphorylated or myosin light chain kinase-phosphorylated myosin. In contrast, protein kinase C phosphorylation has been reported to inhibit the actin-activated ATPase activity of gizzard myosin.
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PMID:Protein kinase C phosphorylation of thymus myosin. 253 57

We have isolated caldesmon (Mr = 145,000), by immunoprecipitation, from [32P]orthophosphate-loaded porcine carotid arteries. In resting muscles, caldesmon was phosphorylated to 0.45 mol of PO4/mol protein, while the 20,000-dalton myosin regulatory light chain (LC20) was phosphorylated to less than 0.05 mol/mol. After stimulation by KCl (110 mM) for 75 min and phorbol 12,13-dibutyrate (PDBu, 1 microM) for 60 min, caldesmon phosphorylation levels rose to 0.96 and 1.1 mol/mol, respectively. LC20 phosphorylation increased to 0.49 mol/mol at 1 min of stimulation by KCl and decreased to 0.17 mol/mol at 60 min. With PDBu, phosphate incorporation into LC20 rose only slightly, reaching 0.09 mol/mol after 90 min. Muscles contracted with histamine (10 microM) or ouabain (1 microM) also demonstrated elevated levels of phosphate incorporation into caldesmon. In these muscles, LC20 phosphorylation levels were less than 0.05 mol/mol. Three major phosphopeptides of indistinguishable mobility were identified on maps of caldesmon from resting, KCl-stimulated, and PDBu-stimulated muscles. There was, however, little similarity between the phosphopeptide maps of caldesmon phosphorylated in intact tissue and maps of purified caldesmon phosphorylated in vitro by protein kinase C (Ca2+/phospholipid-dependent enzyme) or Ca2+/calmodulin kinase II.
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PMID:Phosphorylation of caldesmon in arterial smooth muscle. 270 86

The relationship between phosphorylation of the 20-kDa myosin light chain, intracellular calcium levels ([Ca2+]i), and isometric force was studied during prolonged activation of arterial smooth muscle. Aequorin, preloaded into ferret aortic strips, was used as a [Ca2+]i indicator. Two dimensional polyacrylamide gel electrophoresis was used to determine the phosphorylation levels of the 20-kDa myosin light chain (LC20). During the 30-min depolarization of arterial smooth muscle by K+ (21 mM), both LC20 phosphorylation and [Ca2+]i increased significantly at all time points examined as did the steady state stress. A transient rise in LC20 phosphorylation and [Ca2+]i occurred within 30 s, followed by suprabasal levels through the 10-min period during a sustained alpha 1-mediated activation by 10(-5) M phenylephrine whereas a higher force was developed at a shorter time compared to K+. An active phorbol ester 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA, 10(-6) M) induced a slow contraction of similar magnitude to that induced by K+ without significantly changing either [Ca2+]i or LC20 phosphorylation over a 90-min period. These results demonstrate that the amount of LC20 phosphorylation correlates with the [Ca2+]i in all three types of activation. The initial levels of [Ca2+]i and LC20 phosphorylation correlate with the onset of force development but not the magnitude of steady state stress, suggesting a role for [Ca2+]i and LC20 phosphorylation in regulating the cross bridge cycling rate during tension development. The lack of a detectable increase in [Ca2+]i and LC20 phosphorylation during DPBA activation suggests that sites other than LC20, phosphorylated by protein kinase C, may be involved in regulating smooth muscle contraction.
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PMID:Agonist-specific myosin phosphorylation and intracellular calcium during isometric contractions of arterial smooth muscle. 272 26

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), which has been identified as a potent inhibitor of protein kinase C in vitro (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry, in press), enhanced serotonin release from human platelets that was induced by the 12-O-tetradecanoyl phorbol 13-acetate and correspondingly decreased incorporation of radioactive phosphate into a 20,000-dalton protein. H-7 did not affect the protein phosphorylation or the serotonin secretion in unstimulated platelets. A phosphopeptide with a molecular weight of 20,000 has previously been identified as a light chain (LC20) of platelet myosin and both protein kinase C and Ca2+-calmodulin-dependent myosin light-chain kinase have been shown to be involved in its phosphorylation. Two-dimensional peptide mapping following tryptic hydrolysis revealed that H-7 selectively inhibited the protein kinase C-catalyzed phosphorylation of myosin light chain. This pharmacological evidence suggests that Ca2+-activated, phospholipid-dependent myosin light-chain phosphorylation may play an inhibitory role in the release reaction.
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PMID:Serotonin secretion from human platelets may be modified by Ca2+-activated, phospholipid-dependent myosin phosphorylation. 623 60

We studied the extent of Ca2+ influx, myoplasmic free Ca2+ concentration changes, and phosphorylation of the regulatory 20-kDa myosin light chain (LC20) associated with the potentiation of stretch-induced myogenic tone in the rabbit facial vein. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), was used to augment Ca(2+)-dependent stretch-induced myogenic tone. Veins stretched to an optimal resting tension in physiological salt solution (PSS) containing 0.4 mM of Ca2+ developed stretch-induced myogenic tone. Tissues incubated in Ca(2+)-free PSS, either with or without PMA (0.1 microM) did not develop myogenic tone. Readmission of Ca2+ (0.4 mM) caused a three-fold increase in the contraction in PMA-treated segments (710 +/- 60 mg, n = 29 v control: 188 +/- 10 mg, n = 24). This increased contraction was not associated with additional increases in either Ca2+ influx (73.5 +/- 6.9 pmol/mg of tissue/min, n = 29 v control: 61.1 +/- 5.7 pmol/mg of tissue/min, n = 24), myoplasmic free Ca2+ concentration or LC20, (0.44 +/- 0.02 mol PO4/mol LC20, n = 9 v control: 0.43 +/- 0.03 mol PO4/mol LC20, n = 7). Our results suggest that PKC activation amplifies stretch-induced myogenic tone in the rabbit facial vein through target proteins that are not associated with regulation of Ca2+ influx, myoplasmic free Ca2+ concentration, or LC20 phosphorylation. We conclude that the PKC-mediated potentiation of stretch-induced myogenic tone is due to an increased sensitivity of the contractile apparatus to Ca2+.
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PMID:Phorbol ester-induced potentiation of myogenic tone is not associated with increases in Ca2+ influx, myoplasmic free Ca2+ concentration, or 20-kDa myosin light chain phosphorylation. 802 13

Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase inactivation due to slow dephosphorylation by SMP-I, whereas calponin and myosin are rapidly dephosphorylated by SMP-I and SMP-III/SMP-IV respectively. This may have important functional consequences in terms of the contractile properties of smooth muscle.
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PMID:Smooth-muscle caldesmon phosphatase is SMP-I, a type 2A protein phosphatase. 839 39

CPI-17 is a cytosolic protein of 17 kDa that becomes a potent inhibitor of certain type 1 protein serine/threonine phosphatases, including smooth muscle myosin light-chain phosphatase (MLCP), when phosphorylated at Thr38. Several protein kinases are capable of phosphorylating CPI-17 at this site in vitro; however, in intact tissue, compelling evidence only exists for phosphorylation by protein kinase C (PKC). Agonist-induced activation of heterotrimeric G proteins of the Gq/11 family via seven-transmembrane domain-containing, G protein-coupled receptors results in phospholipase Cbeta-mediated hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 triggers Ca2+ release from the sarcoplasmic reticulum. DAG and Ca2+ together activate classical isoforms of PKC, and DAG activates novel PKC isoforms without a requirement for Ca2+. Activated PKC phosphorylates CPI-17 at Thr38, enhancing its potency of inhibition of MLCP approx 1000-fold. The myosin light-chain kinase (MLCK):MLCP activity ratio is thereby increased at the prevailing cytosolic free-Ca2+ concentration ([Ca2+]i), resulting in an increase in phosphorylation of the 20-kDa light chains of myosin II (LC20) catalyzed by Ca2+- and calmodulin-dependent MLCK and contraction of the smooth muscle. Physiologically, this mechanism can account for some instances of Ca2+ sensitization of smooth muscle contraction (i.e., an increase in force in response to agonist stimulation without a change in [Ca2+]i).
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PMID:Phosphorylation of the protein phosphatase type 1 inhibitor protein CPI-17 by protein kinase C. 1720 May 64

Smooth muscle contractility is mainly regulated by phosphorylation of the 20 kDa myosin light chains (LC20), a process that is controlled by the opposing activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Recently, intensive research has revealed that various protein kinase networks including Rho-kinase, integrin-linked kinase, zipper-interacting protein kinase (ZIPK), and protein kinase C (PKC) are involved in the regulation of LC20 phosphorylation and have important roles in modulating smooth muscle contractile responses to Ca2+ (i.e., Ca2+ sensitization and Ca2+ desensitization). Here, we review the general background and structure of ZIPK and summarize our current understanding of its involvement in a number of cell processes including cell death (apoptosis), cell motility, and smooth muscle contraction. ZIPK has been found to induce the diphosphorylation of LC20 at Ser-19 and Thr-18 in a Ca2+-independent manner and to regulate MLCP activity directly through its phosphorylation of the myosin-targeting subunit of MLCP or indirectly through its phosphorylation of the PKC-potentiated inhibitory protein of MLCP. Future investigations of ZIPK function in smooth muscle will undoubtably focus on determining the mechanisms that regulate its cellular activity, including the identification of upstream signaling pathways, the characterization of autoinhibitory domains and regulatory phosphorylation sites, and the development of specific inhibitor compounds.
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PMID:The regulation of smooth muscle contractility by zipper-interacting protein kinase. 1748 47


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