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)

Porcine carotid arterial muscles were 32P-labeled then contracted with 8 microM phorbol dibutyrate (PDBu) in normal physiological salt solution (PSS) and in Ca(2+)-free PSS containing 0.5 mM ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetate. Significant incorporation of [32P]phosphate into the 20-kDa myosin light chain, a 28-kDa protein, desmin and caldesmon was measured with no apparent difference between normal and Ca(2+)-depleted muscles. Ca-determination showed that the Ca(2+)-depleted muscle contained 15% of the total Ca of the normal muscle. However, determination of the actin-bound Ca revealed that all the Ca in the Ca(2+)-depleted muscle could be accounted for by its actin-bound Ca. Accordingly, protein phosphorylation during the slow PDBu-induced contraction may proceed in the virtual absence of free Ca2+. Phosphopeptide mapping of the myosin light chain isolated from muscles contracted with PDBu either in the presence or absence of Ca2+ showed that two-thirds of the incorporated [32P]phosphate was attributable to myosin light chain kinase catalyzed phosphorylation and one-third was due to phosphorylation by protein kinase C. PDBu increased the phosphorylation of the 28-kDa protein, desmin and caldesmon two- to threefold, as compared with that in muscles contracted by KCl depolarization or by the receptor mediated agonists norepinephrine and histamine. Muscles contracted by high concentration of PDBu in the presence or absence of Ca2+ could be fully relaxed and recontracted.
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PMID:Protein phosphorylation in arterial muscle contracted by high concentration of phorbol dibutyrate in the presence and absence of Ca2+. 155 47

Endothelin-1 (ET-1) is one of the most potent naturally occurring vasoconstrictors. The mechanism(s) by which ET-1 contracts vascular smooth muscle remains controversial. This study was designed to determine the effects of ET-1 on stress, stiffness, shortening velocity, and myosin light chain (MLC) phosphorylation in swine carotid media. The source of activator Ca2+ for the contractions and the role of protein kinase C (PKC) were determined. The results demonstrate that ET-1 contractions of the swine carotid media are supported primarily by the influx of extracellular Ca2+. The ET-1-stimulated contraction is characterized by rapid, transient increases in MLC phosphorylation levels and shortening velocity, whereas stiffness and stress increase monotonically. The PKC inhibitor staurosporine (80 nM) significantly decreased stiffness but had little effect on stress, MLC phosphorylation, or shortening velocity. Thus inhibition of PKC activity alters the stress-stiffness relationship during ET-1-induced contractions. This alteration could result from a change in the stress generated per cross bridge or by a cooperative mechanism for cross-bridge attachment that does not affect stress development.
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PMID:Staurosporine decreases stiffness but not stress in endothelin-1-stimulated arterial muscle. 156 14

We prepared anti-platelet 20-kDa myosin light chain (MLC-20) antibody and demonstrated diphosphorylation of MLC-20 in platelets ex vivo in the initial phase of activation by thrombin. Our results are as follows. (1) By Western blotting, using anti-MLC-20 antibody, both mono- and diphosphorylated myosin were seen in the initial phase of aggregation of platelets by thrombin. The peak of the diphosphorylation was later than that of monophosphorylation and the degree of both mono- and diphosphorylation reduced in the process of aggregation. (2) ML-7 (a synthetic inhibitor of MLCK) inhibited both mono- and diphosphorylation of myosin and also blocked aggregation of thrombin-activated platelets. However, H-7 (an inhibitor of protein kinase C) had little effect on either the (di)phosphorylation of myosin or the aggregation of thrombin-activated platelets. (3) Arg-Gly-Asp-Ser (RGDS) peptide, a synthetic anti-adhesive peptide, inhibited aggregation of thrombin-activated platelets in a dose-dependent manner (100-200 microM). However, it had little effect on either mono- or diphosphorylation of myosin in the process of the platelet aggregation stimulated by thrombin. From these results, we conclude that mono- and diphosphorylation of myosin by MLCK play a role in the initial phase of activation of thrombin-stimulated platelets in vivo and that mono- and diphosphorylation of myosin by MLCK precedes the secondary signal mediated by GPIIb/IIIa.
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PMID:Diphosphorylation of platelet myosin ex vivo in the initial phase of activation by thrombin. 164 15

1. The [Ca2+] sensitivity of myosin light chain phosphorylation in vascular smooth muscle is dependent on the form of stimulation. Contractile agonist stimulation, when compared to high-KCl depolarization, is associated with an increase in [Ca2+] sensitivity of phosphorylation. I evaluated potential mechanisms for this stimulus-specific response by measuring aequorin-estimated myoplasmic [Ca2+], myosin phosphorylation, and isometric stress in swine carotid media. 2. The relative [Ca2+] sensitivity of phosphorylation depended on the type of stimulus (ranked high to low sensitivity): contractile agonists (histamine, phenylephrine) = endothelin (sustained contraction) = combination of histamine and NaF greater than NaF alone = endothelin (initial contraction) = combination of histamine and depolarization = combination of NaF and depolarization greater than depolarization = Bay K 8644 = combination of depolarization and low-dose phorbol diester. 3. Activation of L-type Ca2+ channels with Bay K 8644 induced a [Ca2+] sensitivity of phosphorylation similar to depolarization, suggesting that any other effects of high KCl (such as cellular swelling) were not responsible for the low [Ca2+] sensitivity of phosphorylation. 4. The addition of either histamine or NaF (an activator of G proteins) to depolarized tissues produced similar increases in the [Ca2+] sensitivity of phosphorylation, suggesting that NaF (possibly by activation of a G protein) can mimic contractile agonist-induced increases in the [Ca2+] sensitivity of phosphorylation. 5. Phorbol dibutyrate enhanced the contractile effect of depolarization, and this enhancement was primarily caused by increases in [Ca2+] rather than an alteration in the [Ca2+] sensitivity of phosphorylation. 6. These data suggest that the [Ca2+] sensitivity of phosphorylation in smooth muscle may be regulated by agonists (possible by G protein activation); however, the role of protein kinase C activation or depolarization induced Ca2+ compartmentalization requires further study.
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PMID:Modulation of the [Ca2+] sensitivity of myosin phosphorylation in intact swine arterial smooth muscle. 170 75

Chronic exposure of differentiated avian skeletal muscle cells in culture to the phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (PMA), results in the selective disassembly of sarcomeric structures and loss of muscle-specific contractile proteins, leaving cytoskeletal structures and their associated proteins intact. We demonstrate here that these morphological and biochemical changes are accompanied by dramatic and selective decreases in the level of the mRNAs that encode the contractile proteins. We measured the effects of PMA on the transcriptional activity and mRNA stability of four contractile protein genes (alpha-cardiac and alpha-skeletal actin, cardiac troponin C [cTnC], and myosin light chain lf [MLClf]) and two nonmuscle genes (beta-cytoplasmic actin and the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). The transcriptional activity of the alpha-cardiac actin and cTnC genes dramatically decreased by 8 h after the addition of PMA, while other muscle and nonmuscle genes examined showed no change. Pulse-chase experiments of in vivo labeled RNA showed significant reductions in mRNA half-lifes for all the contractile protein mRNAs examined, while the half-lifes of beta-actin and GAPDH mRNA were unchanged. All of the above effects occurred under conditions in which cellular protein kinase C (PKC) levels had been reduced by greater than 90%. The fact that many of the contractile protein genes remained transcriptionally active despite the fact that the cells were unable to accumulate their mRNAs to any significant extent indicated that the treated cells were still committed to skeletal muscle differentiation. The selective changes in the stability of the contractile protein mRNAs suggest that the control of mRNA stability may be part of the normal regulatory program of skeletal muscle differentiation and that this control may be linked to the integrity of the contractile apparatus and mediated by second messenger pathways involving PKC activation.
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PMID:Phorbol esters selectively downregulate contractile protein gene expression in terminally differentiated myotubes through transcriptional repression and message destabilization. 171 91

We have found that a fungal strain, Talaromyces wortmannin KY12420, produces a potent inhibitor of smooth muscle myosin light chain kinase (MLCK). This active product, designated as MS-54, was isolated and purified from the culture broth of the fungus and identified as wortmannin. The inhibition of MLCK by wortmannin was prevented by a high concentration of ATP. The activity of the catalytic domain, which was disclosed by partial tryptic digestion, was also inhibited by wortmannin. These results suggest that wortmannin acts at or near to the catalytic site of the enzyme. It was shown clearly by kinetic analyses, preincubation studies, and dialysis experiments that the inhibitory action of wortmannin on MLCK was irreversible. Under the condition of preincubation for 3 min, 0.3 microM wortmannin inhibited the activity of MLCK, while 10 microM wortmannin had no effect on the activities of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and calmodulin-dependent protein kinase II, and had little effect on protein kinase C activity. These data expressed clearly the marked selectivity of the compound for MLCK. Furthermore, wortmannin also inhibited both the phosphorylation of myosin light chain and the contraction in rat thoracic aorta stimulated with KCl, which indicates the effectiveness of the compound in the cellular level as an MLCK inhibitor.
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PMID:Wortmannin, a microbial product inhibitor of myosin light chain kinase. 173 24

Smooth muscle myosin light chain (LC) can be phosphorylated by myosin light chain kinase (MLCK) at Ser19 and Thr18 and by protein kinase C (PKC) at Thr9 and Ser1 or Ser2 under the in vitro assay conditions. Conversion of PKC to the spontaneously active protein kinase M (PKM) by proteolysis resulted in a change in the substrate specificity of the kinase. PKM phosphorylated both sets of sites in LC recognized by MLCK and PKC as analyzed by peptide mapping analysis. The PKM-catalyzed phosphorylation of these sites was not greatly affected by a MLCK inhibitor, ML-9, nor by the activators of MLCK, Ca2+ and calmodulin.
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PMID:Catalytic fragment of protein kinase C exhibits altered substrate specificity toward smooth muscle myosin light chain. 174 84

The contractile state of smooth muscle is regulated primarily by the sarcoplasmic (cytosolic) free Ca2+ concentration. A variety of stimuli that induce smooth muscle contraction (e.g., membrane depolarization, alpha-adrenergic and muscarinic agonists) trigger an increase in sarcoplasmic free [Ca2+] from resting levels of 120-270 to 500-700 nM. At the elevated [Ca2+], Ca2+ binds to calmodulin, the ubiquitous and multifunctional Ca(2+)-binding protein. The interaction of Ca2+ with CaM induces a conformational change in the Ca(2+)-binding protein with exposure of a site(s) of interaction with target proteins, the most important of which in the context of smooth muscle contraction is the enzyme myosin light chain kinase. The interaction of calmodulin with myosin light chain kinase results in activation of the kinase that catalyzes phosphorylation of myosin at serine-19 of each of the two 20-kDa light chains (native myosin is a hexamer composed of two heavy chains (230 kDa each) and two pairs of light chains (one pair of 20 kDa each and the other pair of 17 kDa each)). This simple phosphorylation reaction triggers cycling of myosin cross-bridges along actin filaments and the development of force. Relaxation of the muscle follows removal of Ca2+ from the sarcoplasm, whereupon calmodulin dissociates from myosin light chain kinase regenerating the inactive kinase; myosin is dephosphorylated by myosin light chain phosphatase(s), whereupon it dissociates and remains detached from the actin filament and the muscle relaxes. A substantial body of evidence has been accumulated in support of this central role of myosin phosphorylation-dephosphorylation in the regulation of smooth muscle contraction. However, a wide range of physiological and biochemical studies supports the existence of additional, secondary Ca(2+)-dependent mechanisms that can modulate or fine-tune the contractile state of the smooth muscle cell. Three such mechanisms have emerged: (i) the actin-, tropomyosin-, and calmodulin-binding protein, calponin; (ii) the actin-, myosin-, tropomyosin-, and calmodulin-binding protein, caldesmon; and (iii) the Ca(2+)- and phospholipid-dependent protein kinase (protein kinase C).
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PMID:The Ayerst Award Lecture 1990. Calcium-dependent mechanisms of regulation of smooth muscle contraction. 181 84

The aim of the study was to determine the role of protein kinase C (PKC) in protein phosphorylation in hypertrophied C. myocytes, particularly the phosphorylation of the 19 kDa protein which corresponds to myosin light chains. In myocardial hypertrophy the PKC activity in the cytosolic fraction of tissue homogenate was increased up to 253% of control hearts, and in membrane fraction up to 140% of the control value. Phorbol ester (TPA), the specific activator of protein kinase C, stimulated phosphorylation of the 19 kDa protein obtained from isolated myocytes to 181 +/- 9% of control value in normal and to 248 +/- 66% in hypertrophic myocytes. Taken together, these data suggest that protein kinase C might be involved in the increased phosphorylation of cardiac myosin light chain protein in myocardial hypertrophy.
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PMID:Increased 19 kDa protein phosphorylation and protein kinase C activity in pressure-overload cardiac hypertrophy. 183 75

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


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