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)

Contractile performance of cardiac and skeletal muscles may be regulated by cyclic AMP or Ca2+, two second messengers that stimulate the phosphorylation of specific myofibrillar proteins. Cyclic AMP-dependent protein kinase catalyzed the rapid phosphorylation of a single site in the inhibitory subunit of cardiac troponin in vitro and in perfused hearts. Skeletal muscle troponin was not phosphorylated by this enzyme in vivo. Although there was a correlation between cardiac troponin phosphorylation and the positive inotropic response to catecholamines, a biochemical mechanism that could account for a functional relationship between the two processes has not been discovered. Phosphorylation of skeletal muscle myosin was catalyzed by myosin light chain kinase in the presence of Ca2+ and the ubiguitous, multifunctional Ca2+-dependent regulator protein (CDR). The activation of kinase activity appeared to proceed via a trimolecular reaction process in which Ca2+ bound to CDR and the Ca2+.CDR complex then interacted with the enzyme. In rat extensor digitorum longus muscle, a 1 sec tetanic contraction resulted in phosphorylation of myosin light chain with the maximal phosphate incorporated 20 sec after the contraction. The light chain phosphate content declined slowly and correlated to post-tetanic potentiation of isometric twitch tension. Phosphorylation of skeletal muscle myosin may be important in modulating contraction.
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PMID:Phosphorylation of contractile proteins in heart and skeletal muscle. 736 51

12(S)-HETE, a lipoxygenase metabolite of arachidonic acid, has been demonstrated to induce a reversible retraction of vascular endothelial cells (EC). 12(S)-HETE-induced microvascular EC retraction was blocked by a selective protein kinase C inhibitor, calphostin C, but not by the protein kinase A inhibitor, H8. EC exposed to 12(S)-HETE demonstrated a gradual dissolution of actin microfilaments and a decrease of vinculin-containing focal adhesions. The intermediate filaments, vimentin, also underwent extensive reorganization (i.e., filament bundling and enrichment to the cell filapodia) following 12(S)-HETE treatment. In vivo phosphorylation studies revealed that 12(S)-HETE induced a hyperphosphorylation of several major cytoskeletal proteins including myosin light chain, actin, and vimentin. The increased phosphorylation of these cytoskeletal proteins following 12(S)-HETE stimulation was abolished by calphostin C but not by H8. Confluent EC express alpha v beta 3 in focal adhesions at both the cell body and the cell-cell borders. 12(S)-HETE induced a sequential rearrangement of the alpha v beta 3-containing focal adhesions, resulting in a general decrease in alpha v beta 3 integrin receptors, especially in those retracted EC. 12(S)-HETE-induced rearrangement of alpha v beta 3 was inhibited by calphostin C but not by H8. In contrast to alpha v beta 3, confluent EC enrich alpha 5 beta 1 integrin receptors primarily at the cell-cell borders, colocalizing with extracellular fibronectin and cell cortical microfilaments. 12(S)-HETE treatment also disrupted the cell-border distribution pattern of alpha 5 beta 1 as EC retracted, but no distinct alterations (such as time-related redistribution and quantitative differences) in alpha 5 beta 1 were observed.
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PMID:12(S)-HETE-induced microvascular endothelial cell retraction results from PKC-dependent rearrangement of cytoskeletal elements and alpha V beta 3 integrins. 768 34

We report the discovery, semi-purification and characterization of a novel Ca2+/calmodulin-dependent protein kinase (peak I kinase) using syntide 2 as a substrate from the rabbit heart. In the study of dependence of peak I kinase on the concentration of calmodulin, half-maximal activation was obtained at approx. 2.0 x 10(-7) M calmodulin. Peak I kinase did not undergo autophosphorylation. This kinase phosphorylates the synthetic peptides such as syntide 2, autocamtide-2, site 3 in a Ca2+/CaM-dependent manner, but not myosin light chain-peptide, gamma-peptide, and cAMP Response Element Binding Protein (CREB) peptide. Elongation Factor-2, alpha-casein and histone-IIIs were not phosphorylated. These data indicate that this CaM kinase is different from other identified Ca2+/calmodulin-dependent protein kinases and therefore constitutes a novel protein kinase.
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PMID:A novel Ca2+/calmodulin-dependent protein kinase lacking autophosphorylation activity in the rabbit heart. 779 70

A soluble protein kinase purified from winged bean (Psophocarpus tetragonolobus) shoots, has been assessed as a monomeric enzyme with an approximate M(r) of 60,000 in spite of the presence of two polypeptides of 61 and 58 kDa determined by SDS/PAGE. Immunoblot analyses using either of the two antisera raised individually against the polypeptides, detect both of them in purified preparations and a single larger polypeptide (62 kDa) in freshly prepared tissue homogenates, clearly indicating the likelihood of the doublet being formed from the larger one by proteolysis. Histone H1, syntide 2 and a synthetic myosin light chain-related peptide (MLC-peptide) have been identified as exogenous substrates of the enzyme. Complete Ca(2+)-dependence for substrate phosphorylation, a drastic inhibition of the reaction by a calmodulin (CaM) antagonist which can be partially reversed by a heterologous CaM and direct 45Ca(2+)-binding on blot, form compelling evidence in favour of a CaM-like domain of the enzyme. Both the polypeptides of the purified enzyme undergo intramolecular autophosphorylation on serine residue(s). Unlike the substrate phosphorylation reaction, autophosphorylation is Ca(2+)-independent and is not inhibited by the CaM antagonist. Down-regulation of substrate phosphorylation by auto-phosphorylation, and stimulation of the autophosphorylation by histone H1 and MLC-peptide, are novel regulatory features of the enzyme.
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PMID:Characterization of a winged bean (Psophocarpus tetragonolobus) protein kinase with calmodulin-like domain: regulation by autophosphorylation. 782 30

The sites of action of many chemical agents that modify the contraction of smooth muscle are in the smooth muscle membrane. However, a few agents, such as calmodulin inhibitors and protein kinase inhibitors, interact directly with contractile elements of the actomyosin system so as to modify smooth muscle contraction. Here, we describe experimental procedures that are applicable for the screening of smooth muscle relaxants with this mode of action. Myosin B was extracted from chicken gizzard smooth muscle. Because myosin B was a crude preparation of smooth muscle actomyosin, it consisted of regulatory proteins of calmodulin, myosin light chain kinase and protein phosphatase in addition to the contractile proteins of actin and myosin. Interaction of chemical agents with these proteins could be detected by measuring the Mg-ATPase activity of the myosin B preparation. Then we examined whether the agents that altered the ATPase activity was associated with changes in phosphorylation of myosin light chain. If the levels are altered, the agents may interact with the regulatory protein(s). If not, the site of their action was in the contractile proteins. The analysis with these respective proteins will be also described.
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PMID:[Studies on agonists and antagonists of smooth muscle contraction by the use of an actomyosin preparation]. 782 22

The mechanisms by which guanosine 3',5'-cyclic monophosphate (cGMP) modulates the contraction induced by ATP were investigated in small mesenteric resistance arteries of the rat. The nitric oxide donors 3-morpholinosydnonimine (SIN-1, 10 microM) and sodium nitroprusside (SNP, 10 microM) increased cGMP but not adenosine 3',5'-cyclic monophosphate (cAMP) content of the tissue. SIN-1, SNP, and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 100 microM) inhibited the myosin light chain phosphorylation and the contractile response to ATP. Both effects were completely reversed by the selective inhibitor of cGMP protein kinase, Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate (30 microM). The sensitivity to Ca2+ of arteries permeabilized with Staphylococcus aureus alpha-toxin (4,000 hemolytic units/ml) was not affected by 8-BrcGMP. The two nitric oxide donors and 8-BrcGMP decreased the rise in intracellular Ca2+ induced by ATP. The vasodilator agents abolished the contractile response to the exogenous calcium in vessels that were exposed to 3 mM ATP after depletion of intracellular Ca2+ stores. Thapsigargin (1 microM), an inhibitor of the sarcoplasmic reticulum Ca(2+)-adenosinetriphosphatase, reversed the inhibitory effect of the vasodilator agents when the contraction induced by ATP was elicited in the presence of the Ca2+ entry blocker nitrendipine (1 microM) or in Ca(2+)-free medium. These results show that cGMP inhibits ATP-induced contraction by decreasing intracellular Ca2+ concentration in small resistance arteries. They indicate that this effect results from decreased Ca2+ influx and enhanced Ca2+ sequestration through a thapsigargin-sensitive pump via activation of a cGMP protein kinase.
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PMID:Effects of cGMP on calcium handling in ATP-stimulated rat resistance arteries. 790 Aug 76

Yeast calmodulin binds only three calcium ions in the presence of millimolar concentrations of magnesium due to a defective calcium-binding sequence in its carboxyl terminal domain. Yeast calmodulin's diminished calcium-binding activity can be restored to that of other calmodulins by the use of site-directed mutagenesis to substitute its fourth calcium-binding domain with that of a vertebrate calmodulin sequence. However, the repair of yeast calmodulin's calcium-binding activity is not sufficient to repair quantitatively yeast calmodulin's defective protein kinase activator activity. Yeast calmodulin's activator activity with smooth muscle and skeletal muscle myosin light chain kinases and brain calmodulin-dependent protein kinase II can be progressively repaired by additional substitutions of vertebrate calmodulin sequences, provided that the four calcium-binding sites remain intact. An unexpected result obtained during the course of these studies was the observation that myosin light chain kinases from smooth and skeletal muscle tissues can respond differently to mutations in calmodulin. These and previous results indicate that the binding of four calcium ions by calmodulin is necessary but not sufficient to bring about quantitative activation of protein kinases, and are consistent with the conformational selection/restriction model of the dynamic equilibrium among calcium, calmodulin and each calmodulin regulated enzyme.
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PMID:Gain of function mutations for yeast calmodulin and calcium dependent regulation of protein kinase activity. 791 68

In this article we review the various amino acids present in vertebrate nonmuscle and smooth muscle myosin that can undergo phosphorylation. The sites for phosphorylation in the 20 kD myosin light chain include serine-19 and threonine-18 which are substrates for myosin light chain kinase and serine-1 and/or -2 and threonine-9 which are substrates for protein kinase C. The sites in vertebrate smooth muscle and nonmuscle myosin heavy chains that can be phosphorylated by protein kinase C and casein kinase II are also summarized. Original data indicating that treatment of human T-lymphocytes (Jurkat cell line) with phorbol 12-myristate 13-acetate results in phosphorylation of both the 20 kD myosin light chain as well as the 200 kD myosin heavy chain is presented. We identified the amino acids phosphorylated in the human T-lymphocytes myosin light chains as serine-1 or serine-2 and in the myosin heavy chains as serine-1917 by 1-dimensional isoelectric focusing of tryptic phosphopeptides. Untreated T-lymphocytes contain phosphate in the serine-19 residue of the myosin light chain, and in a residue tentatively identified as serine-1944 in the myosin heavy chain.
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PMID:Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains. 793 53

Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans. It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases. We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains. The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation. The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism. Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase. By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core. Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated.
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PMID:Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region. 806 27

The changes in protein phosphorylation and cytoskeletal structure preceding the dramatic morphological changes in staurosporine-treated rat astrocytes were examined, and the dependence of these effects on protein kinase C (PKC) was studied. Fluorescence and photoelectron microscopy revealed that a 20-min exposure to the kinase inhibitor staurosporine at 100 nM substantially decreased the thickness and linear appearance of actin microfilament bundles (stress fibers) prior to major changes in cell shape, while 60 min of staurosporine depleted virtually all microfilament bundles and caused arborization and contraction of the cell body. The distribution of myosin light chain (MLC) labeling within the cytoplasm was also dramatically altered by staurosporine, progressing from a linear punctate pattern coincident with the linear pattern of filamentous actin to a diffuse pattern in cells in which microfilament dissolution was taking place. Two-dimensional gel analysis of astrocyte phosphoproteins demonstrated 50-80% reduction of 32P incorporation into four 20-kDa spots, one of which was recognized by an antibody to MLC, following a 15-min treatment with 100 nM staurosporine. Depletion of functinal PKC from astrocytes by a 24-h exposure to phorbol myristate acetate prior to staurosporine exposure did not reduce the extent of the cytoskeletal alterations or alter the decrease in protein phosphorylation. Two other protein kinase inhibitors which affect astrocyte morphology, H-7 and the MLC kinase inhibitor ML-9, were also observed to disrupt microfilament bundles with accompanying decreases in 32P incorporation into these same phosphoproteins, whereas the more selective PKC inhibitor Ro 31-8220 did not do either. The early onset of decreased phosphorylation of the 20-kDa proteins supports a direct relationship between the rapid dissociation of myosin light chain from actin microfilament bundles, the disruption of actin patterns, and the subsequent morphological alterations. These data also suggest that staurosporine and H-7 may exert their effects via a pathway involving inhibition of MLC kinase.
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PMID:Decreased phosphorylation of four 20-kDa proteins precedes staurosporine-induced disruption of the actin/myosin cytoskeleton in rat astrocytes. 808 48


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