Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Avian myosin light chain kinase (MLCK) is inhibited by a range of plant-derived flavonoids. Maximal inhibition requires 2,3-unsaturation and polyhydroxylation of two of the three flavonoid rings. Phosphorylation of a synthetic myosin light chain-related peptide by wheat embryo Ca(2+)-dependent protein kinase (CDPK) is also inhibited by a range of flavonoids but phosphorylation of histone preparation III-S by wheat CDPK is not inhibited by flavonoids. The structural requirements for inhibition of wheat CDPK by flavonoids are more stringent than for inhibition of avian MLCK. Potent flavonoid inhibitors of wheat CDPK are unsaturated in 2,3 position, have hydroxyl groups in positions 3' and 4' and an additional hydroxyl in the chromone ring. Flavonoid glycosylation or methylation can abolish inhibition. A number of other naturally occurring plant phenolics including chalcones and gossypol also inhibit avian MLCK and wheat CDPK. Gossypol binds to calmodulin, abolishing Ca(2+)-dependent enhancement of dansyl-calmodulin fluorescence.
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PMID:Inhibition of wheat embryo calcium-dependent protein kinase and avian myosin light chain kinase by flavonoids and related compounds. 177 94

We studied the effects of various protein kinase inhibitors on the attachment of mouse lung carcinoma 3LL cells to the fibronectin (FN) substratum. Calmodulin antagonists (W-7 and W-13) and myosin light chain kinase inhibitors (ML-7 and ML-9) exhibited the inhibitory effect for the attachment, while inhibitors of protein kinases A and C were ineffective. Since Arg-Gly-Asp-containing hexapeptide blocked the attachment, cell surface FN receptor appeared to be involved in this mechanism. These results support the hypothesis that the cell attachment requires the rearrangement of the cytoskeleton in association with the phosphorylation of myosin light chain which would lead to the clustering of the cell surface FN receptors.
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PMID:Myosin light chain kinase inhibitors ML-7 and ML-9 inhibit mouse lung carcinoma cell attachment to the fibronectin substratum. 177 44

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

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

To determine the contribution of phosphate acceptor substrate to the pattern of activity of calcium-dependent, phospholipid-sensitive protein kinase (protein kinase C, PKC), we assayed cytosolic and particulate PKC activity for histone, troponin, myosin light chain (MLC), and endogenous cellular proteins in human neutrophils stimulated with phorbol myristate acetate (PMA), the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) and synergistic stimulation with both agonists. In general, phosphotransferase activity in neutrophil subfractions toward troponin and endogenous proteins paralleled that toward histone, but MLC was a poor substrate for PKC and the pattern of phosphotransferase activity differed from that seen with the other substrates. Furthermore, the phosphorylation of endogenous neutrophil cytosolic proteins increased significantly after stimulation with FMLP, suggesting an endogenous cytosolic substrate(s) which increased in concentration following stimulation. We conclude that histone is a useful phosphate acceptor for study of PKC activity in human neutrophils, but substrate variability occurs and may influence interpretation of results in assays of PKC activity.
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PMID:Substrate dependence of human neutrophil protein kinase C. 205 46

The central helical region of calmodulin (CaM) includes amino acids 65-92 and serves to separate the two pairs of Ca2(+)-binding sites. This region may impart conformational flexibility and also interact with target proteins. The functional effects of deleting two, three, five, or eight amino acids from the central helix were monitored by examining the activation of phosphodiesterase, smooth muscle myosin light chain (MLC) kinase, and Ca2+/CaM-dependent protein kinase II (CaM kinase II). CaMDM(-8), a calmodulin-deletion mutant with 8 amino acids deleted from the middle of the central helix, failed to activate MLC kinase, phosphodiesterase, or CaM kinase II at physiologically significant concentrations of activator but also had altered electrophoretic mobility and tyrosine fluorescence properties suggesting major changes in the structure of this mutant. Deletion of five amino acids (77-81) resulted in an increase in apparent Kact for phosphodiesterase (150-fold), CaM kinase II (25-fold), and MLC kinase (5-fold) relative to CaM. The maximal autophosphorylation activity of CaM kinase II was also diminished 70% with CaMDM(-5). For phosphodiesterase activation, CaMDM(-2) has a 15-fold increase in apparent Kact while CaMDM(-3) had an apparent Kact value only 3-fold higher than native CaM. In contrast, the activation of MLC kinase by the two (79-80)- and three (79-81)-amino acid deletion mutants were indistinguishable from each other or native CaM. CaMDM(-2) and CaMDM(-3) stimulated CaM kinase II autophosphorylation to 85 and 70%, respectively, of native CaM with less than a 2-fold increase in Kact. Therefore, all deletions in the central helix of CaM reduce the efficiency of phosphodiesterase activation as reflected by substantial alterations in Kact. MLC kinase activation, however, is relatively insensitive to small two or three amino acid deletions. CaM kinase II interacts with the central helix deletion mutants in a complex manner with alterations in both the Kact and the maximum activity. The data suggest the central helix of CaM may serve as a flexible tether for MLC kinase (and to a lesser extent CaM kinase II) but that an extended conformation of CaM, as predicted from the crystal structure, may be required for phosphodiesterase activation.
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PMID:Calmodulin activation of target enzymes. Consequences of deletions in the central helix. 215 85

A newly synthesized isoquinolinesulfonamide, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), was shown to have a potent and selective inhibitory action against cyclic AMP-dependent protein kinase (protein kinase A), with an inhibition constant of 0.048 +/- 0.008 microM. H-89 exhibited weak inhibitory action against other kinases and Ki values of the compound for these kinases, including cGMP-dependent protein kinase (protein kinase G), Ca2+/phospholipid-dependent protein kinase (protein kinase C), casein kinase I and II, myosin light chain kinase, and Ca2+/calmodulin-dependent protein kinase II were 0.48 +/- 0.13, 31.7 +/- 15.9, 38.3 +/- 6.0, 136.7 +/- 17.0, 28.3 +/- 17.5, and 29.7 +/- 8.1 microM, respectively. Kinetic analysis indicated that H-89 inhibits protein kinase A, in competitive fashion against ATP. To examine the role of protein kinase A in neurite outgrowth of PC12 cells, H-89 was applied along with nerve growth factor (NGF), forskolin, or dibutyryl cAMP. Pretreatment with H-89 led to a dose-dependent inhibition of the forskolin-induced protein phosphorylation, with no decrease in intracellular cyclic AMP levels in PC12D cells, and the NGF-induced protein phosphorylation was not not inhibited. H-89 also significantly inhibited the forskolin-induced neurite outgrowth from PC12D cells. This inhibition also occurred when H-89 was added before the addition of dibutyryl cAMP. Pretreatment of PC12D cells with H-89 (30 microM) inhibited significantly cAMP-dependent histone IIb phosphorylation activity in cell lysates but did not affect other protein phosphorylation activity such as cGMP-dependent histone IIb phosphorylation activity, Ca2+/phospholipid-dependent histone IIIs phosphorylation activity, Ca2+/calmodulin-dependent myosin light chain phosphorylation activity, and alpha-casein phosphorylation activity. However, this protein kinase A inhibitor did not inhibit the NGF-induced neurite outgrowth from PC12D cells. Thus, the forskolin- and dibutyryl cAMP-induced neurite outgrowth is apparently mediated by protein kinase A while the NGF-induced neurite outgrowth is mediated by a protein kinase A-independent pathway.
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PMID:Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. 215 66

Dynamic reorganization of the actin microfilament networks is dependent on the reversible phosphorylation of myosin light chain. To assess the potential role of protein phosphatases in this process in living nonmuscle cells, we have microinjected the purified type-1 and type-2A phosphatases into the cytoplasm of mammalian fibroblasts. Our studies reveal that elevating type-1 phosphatase levels led to the rapid (within 30 min) and fully reversible disassembly of the actin microfilament network as determined by immunofluorescence analysis. In contrast, microinjection of equivalent amounts of the purified type-2A phosphatase had no effect on actin microfilament organization. Metabolic labeling of cells after injection of purified phosphatases was used to analyze changes in protein phosphorylation. Concomitant with the disassembly of the actin microfilaments induced by type-1 phosphatase, there was an extensive dephosphorylation of myosin light chain. No such change was observed when cells were injected with type-2A phosphatase. In addition, after extraction of fibroblasts with Triton X-100, the type-1 phosphatase could be specifically localized by immunofluorescence to a fibrillar network of microfilaments. Furthermore, neutralizing type-1 phosphatase activity in vivo by microinjection of an affinity-purified antibody, prevented the reorganization of actin microfilaments that we had previously described following injection of cAMP-dependent protein kinase. These data support the notion that type 1 and type-2 phosphatases have distinct substrate specificity in living cells, and that type-1 phosphatase plays a predominant role in the dephosphorylation of myosin light chain and thus in the modulation of actin microfilament organization in vivo in intact nonmuscle cells.
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PMID:Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells. 216 27

Previously, it was reported that smooth muscle caldesmon is a protein kinase and is autophosphorylated [Scott-Woo, G.C., & Walsh, M.P. (1988) Biochem. J. 252, 463-472]. We separated a Ca2+/calmodulin-dependent protein kinase from caldesmon in the presence of 15 mM MgCl2. The Ca2+/calmodulin-dependent caldesmon kinase was purified by using a series of liquid chromatography steps and was characterized. The subunit molecular weight (MW) of the kinase was 56K by SDS gel electrophoresis and was autophosphorylated. After the autophosphorylation, the kinase became active even in the absence of Ca2+/calmodulin. The substrate specificity of caldesmon kinase was similar to the rat brain calmodulin-dependent multifunctional protein kinase II (CaM PK-II) and phosphorylated brain synapsin and smooth muscle 20-kDa myosin light chain. The purified kinase bound to caldesmon, and the binding was abolished in the presence of high MgCl2. Enzymological parameters were measured for smooth muscle caldesmon kinase, and these were KCaM = 32 nM, KATP = 12 microM, Kcaldesmon = 4.9 microM, and KMg2+ = 1.1 mM. Optimum pH was 7.5-9.5. The observed properties were similar to brain CaM PK-II, and, therefore, it was concluded that smooth muscle caldesmon kinase is the isozyme of CaM PK-II in smooth muscle.
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PMID:Purification and characterization of calmodulin-dependent multifunctional protein kinase from smooth muscle: isolation of caldesmon kinase. 217 96


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