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)

Myosin light chain kinase plays a central role in the regulation of smooth muscle contraction. The activity of this enzyme is controlled by protein-protein interaction (the Ca2+-dependent binding of calmodulin) and by phosphorylation catalyzed by cAMP-dependent protein kinase. The effects of these two regulatory mechanisms on the conformation of myosin light chain kinase and the locations of the phosphorylation sites, the calmodulin-binding site, and the active site have been probed by limited proteolysis. Phosphorylated and nonphosphorylated myosin light chain kinases were subjected to limited digestion by four proteases having different peptide bond specificities (trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and thrombin), both in the presence and in the absence of bound calmodulin. The digests were compared in terms of gel electrophoretic pattern, distribution of phosphorylation sites, and Ca2+ dependence of kinase activity. A 24 500-dalton chymotryptic peptide containing both sites of phosphorylation was purified and tentatively identified as the amino-terminal peptide. The following conclusions can be drawn: neither phosphorylation nor calmodulin binding induces dramatic changes in the conformation of the kinase; the kinase contains two regions that are particularly susceptible to proteolytic cleavage, one located approximately 25 000 daltons from the amino terminus and the other near the center of the molecule; the two phosphorylation sites are located within 24 500 (probably 17 500) daltons of the amino terminus; the active site is located close to the center of the molecule; the calmodulin-binding site is located in the amino-terminal half of the molecule, between the sites of phosphorylation and the active site, and this region is very susceptible to cleavage by trypsin.
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PMID:Limited proteolysis of smooth muscle myosin light chain kinase. 384 33

A major substrate, Mr 100,00 (100 kDa), for a Ca2+/calmodulin (CaM)-dependent protein kinase found in many mammalian tissues has been purified from rat pancreas. The purified substrate was used to identify and partially purify a CaM-dependent protein kinase (CaM kinase III) from rat pancreas. The physical properties and substrate specificity of CaM kinase III were distinct from those of all known CaM-dependent protein kinases. Only CaM kinase III was able to phosphorylate the 100-kDa protein; synapsin I, phosphorylase b, myosin light chain, and histone were poor substrates for this enzyme. Polyclonal antibodies, raised against the purified 100-kDa protein, recognized the protein in a variety of mammalian tissues and cell lines. Immunoassay revealed that the 100-kDa protein made up 0.3-1.7% of the total cytosolic protein in these samples. Analysis of CaM kinase III revealed that the enzyme had a similar widespread tissue distribution. These results demonstrate the existence of a fifth CaM-dependent protein phosphorylation system present in high levels in animal cells.
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PMID:Identification of calmodulin-dependent protein kinase III and its major Mr 100,000 substrate in mammalian tissues. 390 54

The substrate specificity of the multifunctional calmodulin-dependent protein kinase from skeletal muscle has been studied using a series of synthetic peptide analogs. The enzyme phosphorylated a synthetic peptide corresponding to the NH2-terminal 10 residues of glycogen synthase, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH2, stoichiometrically at Ser-7, the same residue phosphorylated in the parent protein. The synthetic peptide was phosphorylated with a Vmax of 12.5 mumol X min-1 X mg-1 and an apparent Km of 7.5 microM compared to values of 1.2 mumol X min-1 X mg-1 and 3.1 microM, respectively, for glycogen synthase. Similarly, a synthetic peptide corresponding to the NH2-terminal 23 residues of smooth muscle myosin light chain was readily phosphorylated on Ser-19 with a Km of 4 microM and a Vmax of 5.4 mumol X min-1 X mg-1. The importance of the arginine 3 residues NH2-terminal to the phosphorylated serine in each of these peptides was evident from experiments in which this arginine was substituted by either leucine or alanine, as well as from experiments in which its position in the myosin light chain sequence was varied. Positioning arginine 16 at residues 14 or 17 abolished phosphorylation, while location at residue 15 not only decreased Vmax 14-fold but switched the major site of phosphorylation from Ser-19 to Thr-18. It is concluded that the sequence Arg-X-Y-Ser(Thr) represents the minimum specificity determinant for the multifunctional calmodulin-dependent protein kinases. Studies with various synthetic peptide substrates and their analogs revealed that the specificity determinants of the multifunctional calmodulin-dependent protein kinase were distinct from several other "arginine-requiring" protein kinases.
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PMID:Substrate specificity of a multifunctional calmodulin-dependent protein kinase. 405 84

Myosin light chain kinase from smooth muscle has been shown to be phosphorylated by cyclic AMP-dependent protein kinase, which leads to a decrease in the affinity of the kinase for Ca2+ . calmodulin and, hence, a decrease in enzymatic activity. This event has been proposed as a mechanism for the relaxation of smooth muscle in response to increased intracellular concentrations of cyclic AMP. The ratio of myosin light chain kinase activities measured in the presence of 4 microM or 100 microM Ca2+, at 1 microM calmodulin, permits evaluation of such a change in the calmodulin activation properties of myosin light chain kinase. This activity ratio was decreased by phosphorylation of either purified bovine tracheal smooth muscle myosin light chain kinase, or the endogenous myosin light chain kinase in a homogenate of tracheal smooth muscle, with the addition of the catalytic subunit of cyclic AMP-dependent protein kinase. The ratio was unchanged, however, by activation of the endogenous cyclic AMP-dependent protein kinase in homogenates of tracheal smooth muscle by the addition of cyclic AMP. Incubation of tracheal smooth muscle with isoproterenol, at a concentration sufficient to relax the muscle and to increase phosphorylase a formation, had no effect upon the activity ratio. Incubation of tracheal smooth muscle for 2 hr in the presence of carbachol resulted in a transient increase and then a decrease in myosin light chain phosphate content to control values with no decrease in isometric force. The addition of isoproterenol at 2 hr still resulted in relaxation. These findings are inconsistent with a role of myosin light chain kinase phosphorylation in mediating relaxation of tracheal smooth muscle by beta-adrenergic agonists. Cyclic AMP-dependent effects on cytoplasmic calcium concentrations may be more important in mediating relaxation.
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PMID:The role of myosin light chain kinase phosphorylation in beta-adrenergic relaxation of tracheal smooth muscle. 613 4

The mechanisms by which endothelium-dependent relaxants and nitrovasodilators cause relaxation of vascular smooth muscle has been reviewed. A model explaining these observations is summarized in Fig. 1. The endothelium-dependent vasodilators through interaction with their appropriate receptors are thought to activate phospholipase A2 and cause the release of an unsaturated fatty acid. The released unsaturated fatty acid or a metabolite is thought to be the "endothelial relaxant factor" that interacts with the smooth muscle component to cause relaxation. While the unsaturated fatty acid may be oxidized in either the endothelial cell or smooth muscle cell, the lability of the endothelial relaxant factor suggests that at least some of this processing occurs before its release from the endothelium. the model in Figure 1 suggests that an oxidized fatty acid or a derived free radical is responsible for activation of smooth muscle guanylate cyclase and increases in cyclic GMP levels. As pointed out above, the use of various inhibitors of fatty acid release and metabolism has not allowed us or others to predict the structure of the active material. To date the best evidence suggests that the unsaturated fatty acid is a product of either the lipoxygenase or P-450 pathways. Nitrovasodilators are thought to form nitric oxide free radical and directly activate guanylate cyclase as described above. Activated guanylate cyclase, whether by endothelium dependent agents or the nitrovasodilators, then increases the formation of cyclic GMP, which activates cyclic GMP-dependent protein kinase. The phosphorylation state of various proteins is then altered and, eventually, myosin light chain is dephosphorylated and relaxation occurs. Whether this mechanism involves cyclic GMP-dependent changes in activities of myosin light chain kinase and/or myosin light chain phosphatase remains to be determined. Although the altered phosphorylation state of myosin light chain that results from cyclic GMP accumulation may explain the mechanisms of action of cyclic GMP in smooth muscle relaxation, other mechanisms can not be excluded. For example, some additional studies which we have not summarized here indicate that the integrity of the membrane and Na+-K+ pump can modify both cyclic GMP synthesis and relaxation in rat aorta (38 and unpublished observations). Apparently complex interactions may exist in smooth muscle and other tissues which regulate cyclic GMP accumulation and/or its expression on some process. While several functions for cyclic GMP have been suggested, there is considerable evidence which suggests that one of its roles is relaxation of airway and vascular smooth muscle.
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PMID:Endothelium-dependent and nitrovasodilator-induced relaxation of vascular smooth muscle: role of cyclic GMP. 614 63

The purified Ca2+- and calmodulin-dependent protein kinase from rat brain, which has a M.W. of 120,000 by gel filtration analysis, showed a broad substrate specificity. In addition to myosin light chain from chicken gizzard, the enzyme phosphorylated myelin basic protein, casein and two endogenous substrates in a Ca2+- and calmodulin-dependent manner. In contrast, chicken gizzard myosin light chain kinase exclusively phosphorylated myosin light chain.
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PMID:Multiple specificities of brain Ca2+- and calmodulin-dependent protein kinase for substrate. 618 Feb 75

9, 11-Epithio-11, 12-methano-thromboxane A2 (STA2), a stable analogue of thromboxane A2, caused a rapid rise in cytoplasmic free Ca2+ concentration ([Ca2+]i) in human platelets as measured with the fluorescent Ca2+ indicator quin2. Concomitantly, this compound induced phosphorylation of myosin light chain which is catalyzed by Ca2+, calmodulin-dependent protein kinase. These reactions were fast enough to trigger serotonin release. 13-Azaprostanoic acid, a receptor level antagonist of thromboxane A2 inhibited STA2-induced elevation of [Ca2+]i, phosphorylation of myosin light chain and serotonin release. These results provide evidence that STA2 interacts with a thromboxane A2 receptor which leads to elevation of [Ca2+]i.
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PMID:Elevation of cytoplasmic free calcium concentration by stable thromboxane A2 analogue in human platelets. 623 78

Cd2+ was found to mimic effectively, potentiate and antagonize the stimulatory action of Ca2+ on myosin light chain kinase (MLCK) and phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK, or protein kinase C). PL-Ca-PK, however, was slightly less sensitive to Cd2+ regulation than was MLCK. Cd2+ also biphasically regulates (i.e., stimulation followed by inhibition) phosphorylation, in the homogenates of the rat caudal artery, of myosin light chain and other endogenous proteins catalyzed by MLCK and PL-Ca-PK. The activation by Cd2+ of MLCK was inhibited by anticalmodulins (e.g., R-24571), whereas the inhibition by a higher Cd2+ concentration of MLCK and PL-Ca-PK was reversed by thiol agents (e.g., cysteine). The present findings may provide one mechanism underlying the vascular toxicity of Cd2+, a major environmental pollutant.
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PMID:Environmental pollutant Cd2+ biphasically and differentially regulates myosin light chain kinase and phospholipid/Ca2+-dependent protein kinase. 623 26

Smooth muscle myosin light chain kinase, purified to homogeneity, has a molecular weight of 130,000 +/- 5,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme has a specific activity under maximal conditions of 30 mumol Pi transferred to myosin light chain/mg kinase/min at 24 C and is totally dependent on calmodulin and calcium for activity. Incubation of myosin kinase with the catalytic subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase results in the covalent incorporation of up to one mol of phosphate per mol of myosin kinase in the absence of bound calmodulin. Limited tryptic digestion of the radioactively labeled kinase indicates that all of the label has been incorporated into a single tryptic peptide (mol wt approximately 22,000), suggesting that a single site is being phosphorylated. Phosphorylation of myosin kinase lowers the rate at which the kinase phosphorylates myosin light chain. The lower rate of light chain phosphorylation is due to a weaker binding of calmodulin to the phosphorylated kinase than to the unphosphorylated kinase. Cyclic adenosine 3':5'-monophosphate-dependent phosphorylation of the kinase actin-myosin interaction represents a possible link between hormonal binding to smooth muscle receptors and muscle relaxation. A scheme for this sequence of events is presented.
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PMID:Phosphorylation by cyclic adenosine 3':5'-monophosphate-dependent protein kinase regulates myosin light chain kinase. 624 81

A cAMP-independent glycogen synthase kinase has been purified from rabbit liver. This kinase is completely dependent on the presence of calmodulin and Ca2+ for activity. Half-maximal activation required about 0.1 microM calmodulin. Complete inhibition was obtained in the presence of ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid or trifluoperazine. This calmodulin-dependent synthase kinase does not phosphorylate phosphorylase, myosin light chain, casein, or histone. It rapidly incorporates 0.4 to 0.5 mol of 32P/mol of synthase subunit into the NH2-terminal domain, resulting in partial inactivation of glycogen synthase. These results indicate the existence of a calmodulin-dependent kinase which may be specific for glycogen synthase.
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PMID:Calmodulin-dependent glycogen synthase kinase. 625 Oct 41


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