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)

Recently, one of the authors (K.I.) and other investigators reported that myosin light chain (MLC) of smooth muscle (gizzard, arterial and tracheal) was diphosphorylated by myosin light chain kinase (MLCK) and that diphosphorylated myosin showed a marked increase in the actin-activated myosin ATPase activity in vitro and ex vivo. In this study, we prepared myosin, actin, tropomyosin (human platelet), MLCK (chicken gizzard) and calmodulin (bovine brain) and demonstrated diphosphorylation of MLC of platelet by MLCK in vitro. Our results are as follows. (1) Platelet MLC was diphosphorylated by a relatively high concentration (greater than 20 micrograms/ml) of MLCK in vitro. As a result of diphosphorylation, the actin-activated myosin ATPase activity was increased 3 to 4-fold as compared to the monophosphorylation. (2) Both di- and monophosphorylation reactions showed similar Ca2+, KCl, MgCl2-dependence. Maximal reaction was seen at [Ca2+] greater than 10(-6) M, 60 mM KCl and 2 mM MgCl2. This condition was physiological in activated platelets. (3) Di- and monophosphorylated myosin showed similar Ca2+, KCl-dependence of ATPase activity but distinct MgCl2-dependence. Diphosphorylated myosin showed maximal ATPase activity at 2 mM MgCl2 and monophosphorylated myosin showed a maximum at 10 mM MgCl2. (4) The addition of tropomyosin stimulated actin-activated ATPase activity in both di- and monophosphorylated myosin to the same degree. (5) ML-9, a relatively specific inhibitor of MLCK, inhibited the aggregation of human platelets induced by thrombin ex vivo in a dose-dependent manner. Moreover, this drug also partially inhibited both di- and monophosphorylation reactions and actin-activated ATPase activity. On the other hand, H-7, a synthetic inhibitor of protein kinase C, had little effect on the aggregation of human platelets induced by thrombin ex vivo. From these results, we conclude that diphosphorylation of platelet myosin by MLCK may play an important role in activated platelets in vivo.
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PMID:Diphosphorylation of platelet myosin by myosin light chain kinase. 153 1

The role of protein kinase C (PKC) in regulating the contractile state of smooth muscle was investigated using the constitutively active catalytic fragment of PKC (PKM) with skinned (demembranated) chicken gizzard fibres. PKM attenuated a submaximal contraction in gizzard smooth muscle skinned fibres, but not in rabbit cardiac skinned fibres. PKM-mediated relaxation of submaximal contractions of smooth muscle was accompanied by a reduction in the rate of ATP hydrolysis in the fibre and by phosphorylation of the 20 kDa light chain of gizzard myosin at the PKC sites (serine-1, serine-2 and threonine-9). In addition, several other endogenous proteins were phosphorylated by PKM. However, the inhibitory effects on tension and ATPase are consistent with the biochemical effects of PKC-catalysed phosphorylation of myosin, i.e. reduction of the actin-activated MgATPase activity of myosin prephosphorylated at serine-19 by myosin light chain kinase. Pretreatment of skinned fibres with PKM and ATP gamma S in the absence of Ca2+ had no inhibitory effect on the subsequent submaximal Ca(2+)-activation of force. Consistent with this observation, PKC was not able to utilize ATP gamma S as a substrate, confirming that the observed effects were the result of PKM-catalysed protein phosphorylation. We suggest that PKC may have two distinct effects on smooth muscle contraction: translocation of PKC to the sarcolemma on stimulation results in phosphorylation of a protein(s) other than myosin and a slow, sustained contraction; in some circumstances PKC may undergo proteolysis to PKM resulting in myosin phosphorylation at PKC-specific sites, a reduction in ATPase activity and relaxation of the muscle.
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PMID:Effects of the constitutively active proteolytic fragment of protein kinase C on the contractile properties of demembranated smooth muscle fibres. 153 85

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

1. Circular strips from ferret aorta were used to investigate the mechanism of the intrinsic basal tone. 2. Determinations of stiffness using small sinusoidal length changes showed an abolition of both stiffness and force with cooling, but the temperature dependence of the change in active stiffness did not parallel that of force. At temperatures below 22 degrees C there appeared to be a relatively large population of attached, non-force-generating cross-bridges, indicating that separate mechanisms are involved in regulating cross-bridge attachment and the force per cross-bridge. 3. Active intrinsic tone was not affected by removal of extracellular Ca2+ or removal of endothelium. 4. Intracellular ionized Ca2+ concentrations ([Ca2+]i) as measured with the photoprotein aequorin, did not significantly change when intrinsic tone was abolished by cooling. 5. Myosin light chain phosphorylation, as measured by 2-dimensional polyacrylamide gel electrophoresis, significantly decreased on cooling, but the temperature dependence of phosphorylation did not parallel that of force. The change in phosphorylation in the absence of a change in [Ca2+]i suggests the presence of a constitutively active Ca(2+)-independent form of myosin light chain kinase. 6. Maximal concentrations of staurosporine inhibited but did not eliminate intrinsic tone. 7. Changes in myosin light chain kinase and protein kinase C activities may explain part but not all of the intrinsic tone.
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PMID:Mechanisms of intrinsic tone in ferret vascular smooth muscle. 159 66

We investigated the intracellular processes of the shape change in the human megakaryoblastic leukemia cell, MEG-01, by platelet agonists. Thrombin induced the formation of many pseudopods. This shape change was also induced by TPA and A23187, but not by ADP, collagen, or epinephrine. Electron microscopy and FITC-labeled phalloidin staining revealed thick submembranous microfilament bundles in the pseudopods of the shape-changed cells induced by thrombin. Shape change was inhibited by cytochalasin B. Protein kinase C (RKC) inhibitor, H-7, markedly inhibited thrombin-induced shape change, while the myosin light chain kinase (MLCK) inhibitor, ML-9 did not. These results suggest that thrombin-induced reorganization of microfilaments and shape change of MEG-01 cells are mediated by PKC but not by MLCK.
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PMID:[Shape change in human megakaryoblastic leukemia cells, MEG-01]. 161 74

Addition of ionophore A23187 to washed human platelets caused a time- and dose-dependent increase in the phosphotyrosyl content of 135, 124 and 76 kDa proteins. Platelets loaded with intracellular Ca2+ chelator 5,5'-demethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid before addition of A23187 exhibited no protein-tyrosine phosphorylation. Replenishment of such platelets with extracellular CaCl2 restored A23187-induced protein-tyrosine phosphorylation. Upon stimulation with A23187, both aspirin and ADP scavengers-treated platelets exhibited protein-tyrosine phosphorylation without phosphoinositide hydrolysis or protein kinase C activation. Its protein-tyrosine phosphorylation was not inhibited by ML-9, a selective inhibitor of myosin light chain kinase. Genistein, a selective inhibitor of protein-tyrosine kinase, inhibited A23187-induced platelet aggregation but not secretion. These data show (a) that A23187 stimulates protein-tyrosine phosphorylation by elevation of intracellular Ca2+, (b) that A23187-induced protein-tyrosine phosphorylation is independent of formation of endoperoxides/thromboxane A2, released ADP, phosphoinositide hydrolysis, protein kinase C activation, fibrinogen binding and myosin light chain kinase, and (c) that A23187-induced protein-tyrosine phosphorylation may be involved in platelet aggregation but not in secretion. Furthermore, a synergistic effect of A23187 and protein kinase C activators in stimulating protein-tyrosine phosphorylation is suggested.
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PMID:[Protein-tyrosine phosphorylation of human platelets and its function]. 161 80

Effects of a series of novel inhibitors of calmodulin or protein kinases on amylase release were studied in rat parotid slices. Amylase release induced by a cholinergic agonist, carbamylcholine, was inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, 1-(5-chloronaphthalen-1-sulfonyl)-1H-hexahydro-1, 4-diazepine (ML-9), a myosin light chain kinase (MLCK) inhibitor, and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of Ca(2+)-activated, phospholipid-dependent protein kinase (protein kinase C), while N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), an inhibitor of cyclic AMP-dependent protein kinase (protein kinase A), did not inhibit the release. On the other hand, amylase release induced by a beta-adrenergic agonist, isoproterenol, was inhibited only by H-8, but not by W-7, ML-9 or H-7. These results suggest that cholinergic stimulation evokes amylase release via the Ca(2+)-dependent system which involves calmodulin, MLCK and protein kinase C, while beta-adrenergic stimulation via the cyclic AMP-dependent system involves protein kinase A.
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PMID:Effects of inhibitors of intracellular messenger systems on amylase release from rat parotid gland. 171 7

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

NPC 15437 inhibited protein kinase C (PKC) activity and [3H]phorbol 12,13-dibutyrate (PDBu) binding to the enzyme in a concentration-dependent manner (IC50 values, 19 +/- 2 microM and 23 +/- 4 microM, respectively). No inhibition of cAMP-dependent protein kinase A (PKA) or calcium/calmodulin-dependent myosin light chain kinase (MLCK) was observed. A detailed kinetic analysis of the interaction of NPC 15437 and a homogeneous preparation of PKC-alpha revealed a competitive type of inhibition with respect to activation of the enzyme by both phorbol 12-myristate 13-acetate (PMA) (Ki = 5 +/- 3 microM) and phosphatidylserine (PS) (Ki = 12 +/- 4 microM). Mixed inhibition (predominantly of the non-competitive type), with respect to activation of the enzyme by calcium, was also observed. These studies indicate that NPC 15437 is a selective inhibitor of PKC, interacting at the regulatory region of the molecule. NPC 15437 inhibited phorbol ester-induced ear edema in mouse (IC50 = 175 micrograms/ear) demonstrating the ability of NPC 15437 to inhibit PKC-mediated activity in intact cells.
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PMID:2,6-Diamino-N-([1-oxotridecyl)-2-piperidinyl]methyl)hexanamide (NPC 15437): a selective inhibitor of protein kinase C. 179 19


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