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)

Myosin binding protein C (MyBP-C) is one of the major sarcomeric proteins involved in the pathophysiology of familial hypertrophic cardiomyopathy (FHC). The cardiac isoform is tris-phosphorylated by cAMP-dependent protein kinase (cAPK) on beta-adrenergic stimulation at a conserved N-terminal domain (MyBP-C motif), suggesting a role in regulating positive inotropy mediated by cAPK. Recent data show that the MyBP-C motif binds to a conserved segment of sarcomeric myosin S2 in a phosphorylation-regulated way. Given that most MyBP-C mutations that cause FHC are predicted to result in N-terminal fragments of the protein, we investigated the specific effects of the MyBP-C motif on contractility and its modulation by cAPK phosphorylation. The diffusion of proteins into skinned fibers allows the investigation of effects of defined molecular regions of MyBP-C, because the endogenous MyBP-C is associated with few myosin heads. Furthermore, the effect of phosphorylation of cardiac MyBP-C can be studied in a defined unphosphorylated background in skeletal muscle fibers only. Triton skinned fibers were tested for maximal isometric force, Ca(2+)/force relation, rigor force, and stiffness in the absence and presence of the recombinant cardiac MyBP-C motif. The presence of unphosphorylated MyBP-C motif resulted in a significant (1) depression of Ca(2+)-activated maximal force with no effect on dynamic stiffness, (2) increase of the Ca(2+) sensitivity of active force (leftward shift of the Ca(2+)/force relation), (3) increase of maximal rigor force, and (4) an acceleration of rigor force and rigor stiffness development. Tris-phosphorylation of the MyBP-C motif by cAPK abolished these effects. This is the first demonstration that the S2 binding domain of MyBP-C is a modulator of contractility. The anchorage of the MyBP-C motif to the myosin filament is not needed for the observed effects, arguing that the mechanism of MyBP-C regulation is at least partly independent of a "tether," in agreement with a modulation of the head-tail mobility. Soluble fragments occurring in FHC, lacking the spatial specificity, might therefore lead to altered contraction regulation without affecting sarcomere structure directly.
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PMID:Myosin binding protein C, a phosphorylation-dependent force regulator in muscle that controls the attachment of myosin heads by its interaction with myosin S2. 1062 98

The potent vasodilator action of cyclic GMP-dependent protein kinase (cGK) involves decreasing the Ca(2+) sensitivity of contraction of smooth muscle via stimulation of myosin light chain phosphatase through unknown mechanisms (Wu, X., Somlyo, A. V., and Somlyo, A. P. (1996) Biochem. Biophys. Res. Commun. 220, 658-663). Myosin light chain phosphatase activity is controlled by the small GTPase RhoA and its target Rho kinase. Here we demonstrate cGMP effects mediated by cGK that inhibit RhoA-dependent Ca(2+) sensitization of contraction of blood vessels and actin cytoskeleton organization in cultured vascular myocytes. Ca(2+) sensitization and actin organization were inhibited by both 8-bromo-cGMP and sodium nitroprusside (SNP). SNP also caused translocation of activated RhoA from the membrane to the cytosol. SNP-induced actin disassembly was lost in vascular myocytes in culture after successive passages but was restored by transfection of cells with cGK I. Furthermore, cGK phosphorylated RhoA in vitro, and addition of cGK I inhibited RhoA-induced Ca(2+) sensitization in permeabilized smooth muscle. 8-Bromo-cGMP-induced actin disassembly was inhibited in vascular myocytes expressing RhoA(Ala-188), a mutant that could not be phosphorylated. Collectively, these results indicate that cGK phosphorylates and inhibits RhoA and suggest that the consequent inhibition of RhoA-induced Ca(2+) sensitization and actin cytoskeleton organization contributes to the vasodilator action of nitric oxide.
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PMID:Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle. 1078 86

1. The present study was undertaken to determine whether Ca2+-calmodulin-dependent protein kinase II (CaMKII) participates in the regulation of vascular smooth muscle contraction, and if so, to investigate the nature of the downstream effectors. 2. The contractility of isolated ferret aorta was measured while inhibiting CaMKII either with antisense oligodeoxynucleotides against CaMKII or with the CaMKII inhibitor KN93. 3. Treatment with antisense oligodeoxynucleotides against CaMKII resulted in, on average, a decrease in protein levels of CaMKII to 56 % of control levels and significantly decreased the magnitude of the contraction in response to 51 mM potassium physiological saline solution (KCl). Contraction in response to the phorbol ester DPBA was not significantly affected. 4. The CaMKII blocker KN93 also resulted in a significant decrease in the force induced by 51 mM KCl but caused no significant change in the contraction in response to DPBA or the alpha-adrenoceptor agonist phenylephrine. 5. During contraction with 51 mM KCl, both CaMKII and mitogen-activated protein kinase (MAPK) activity increased, as determined by phospho-specific antibodies. The MAPK phosphorylation level was inhibited by KN93, PD098059 (a MAPK kinase (MEK) inhibitor) and calcium depletion. 6. Myosin light chain (LC20) phosphorylation also increased during contraction with KCl and the increase was significantly blocked by PD098059 as well as by both KN93 and antisense oligodeoxynucleotides to CaMKII. 7. The data indicate that CaMKII plays a significant role in the regulation of smooth muscle contraction and suggest that CaMKII activates a pathway by which MAPK activation leads to phosphorylation of LC20 via activation of myosin light chain kinase.
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PMID:Ca2+-calmodulin-dependent protein kinase II-dependent activation of contractility in ferret aorta. 1089 25

Myosin-binding protein-C (MyBP-C) is a component of all striated-muscle sarcomeres, with a well established structural role and a possible function for force regulation. Multiple mutations within the gene for cardiac MyBP-C, one of three known isoforms, have been linked to familial hypertrophic cardiomyopathy. Here we generated a knock-in mouse model that carries N-terminal-shortened cardiac MyBP-C. The mutant protein was designed to have a similar size as the skeletal MyBP-C isoforms, whereas known myosin and titin binding sites as well as the phosphorylatable MyBP-C motif were not altered. We have shown that mutant cardiac MyBP-C is readily incorporated into the sarcomeres of both heterozygous and homozygous animals and can still be phosphorylated by cAMP-dependent protein kinase. Although histological characterization of wild-type and mutant hearts did not reveal obvious differences in phenotype, left ventricular fibers from homozygous mutant mice exhibited an increased Ca(2+) sensitivity of force development, particularly at lower Ca(2+) concentrations, whereas maximal active force levels remained unchanged. The results allow us to propose a model of how cMyBP-C may affect myosin-head mobility and to rationalize why N-terminal mutations of the protein in some cases of familial hypertrophic cardiomyopathy could lead to a hypercontractile state.
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PMID:Hypercontractile properties of cardiac muscle fibers in a knock-in mouse model of cardiac myosin-binding protein-C. 1109 95

Dictyostelium expresses 12 different myosins, including seven single-headed myosins I and one conventional two-headed myosin II. In this review we focus on the signaling pathways that regulate Dictyostelium myosin I and myosin II. Activation of myosin I is catalyzed by a Cdc42/Rac-stimulated myosin I heavy chain kinase that is a member of the p21-activated kinase (PAK) family. Evidence that myosin I is linked to the Arp2/3 complex suggests that pathways that regulate myosin I may also influence actin filament assembly. Myosin II activity is stimulated by a cGMP-activated myosin light chain kinase and inhibited by myosin heavy chain kinases (MHCKs) that block bipolar filament assembly. Known MHCKs include MHCK A and MHCK B, which have a novel type of kinase catalytic domain joined to a WD repeat domain, and MHC-protein kinase C (PKC), which contains both diacylglycerol kinase and PKC-related protein kinase catalytic domains. A Dictyostelium PAK (PAKa) acts indirectly to promote myosin II filament formation, suggesting that the MHCKs may be indirectly regulated by Rac GTPases.
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PMID:Regulation of Dictyostelium myosin I and II. 1125 38

Raf-1 serine/threonine protein kinase plays an important role in cell survival, proliferation, and migration; however, the specific targets of Raf-1 in diverse cellular processes are not clearly defined. Myosin phosphatase activity is critical to the regulation of cytoskeletal reorganization, cytokinesis, and cell motility. Here, we describe the association of Raf-1 with myosin phosphatase and phosphorylation of the regulatory myosin-binding subunit (MBS) of myosin phosphatase by Raf-1. Treatment of cells with phorbol 12-myristate 13-acetate has been shown to stimulate Raf-1 protein kinase. To determine the effect of enzymatic activation of Raf-1 on MBS phosphorylation, COS-1 cells were transiently transfected with FLAG-tagged full-length Raf-1. A significantly higher phosphorylation of purified glutathione S-transferase-tagged truncated MBS protein (amino acids 654-880) occurred in the presence of FLAG-Raf-1 immunoprecipitated from phorbol 12-myristate 13-acetate-treated cells compared with untreated cells ( approximately 3.0-fold). Using a sequential kinase-phosphatase assay and phosphorylated myosin light chain as substrate in the phosphatase reaction, we showed that Raf-1-associated protein phosphatase-specific activity was inhibited (relative phosphatase activity without and with adenosine 5'-O-(3-thiotriphosphate): 100 and approximately 30%, respectively). Previously, ionizing radiation has been shown to activate Raf-1 (Kasid, U., Suy, S., Dent, P., Ray, S., Whiteside, T. L., and Sturgill, T. W. (1996) Nature 382, 813-816). Exposure of cells to ionizing radiation resulted in the increased association of Raf-1 with MBS (3-6-fold versus unirradiated control) and inhibition of Raf-1-associated protein phosphatase-specific activity (relative phosphatase activity without and with ionizing radiation: 100 and approximately 54%, respectively). Our studies identify MBS as a new substrate of Raf-1 and implicate a role for Raf-1 in the regulation of pathways involving myosin phosphatase activity.
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PMID:Phosphorylation of the myosin-binding subunit of myosin phosphatase by Raf-1 and inhibition of phosphatase activity. 1171 7

The class III myosin is the most divergent member of the myosin superfamily, having a domain with homology to a protein kinase. However, the function of class III myosin at a molecular level is not known at all, and it has been questioned whether it is actually an actin-based motor molecule. Here, we showed that human myosin III has an ATPase activity that is significantly activated by actin (20-fold) with Kactin of 112 microm and Vmax of 0.34 s-1, indicating the mechanoenzymatic activity of myosin III. Furthermore, we found that human myosin III has actin translocating activity (0.11 +/- 0.05 microm/s) using an in vitro actin gliding assay, and it moves toward the plus end of actin filaments. Myosin III containing calmodulin as the light chain subunit showed a protein kinase activity and underwent autophosphorylation. The autophosphorylation was the intramolecular process, and the sites were at the C-terminal end of the motor domain. Autophosphorylation significantly activated the kinase activity, although it did not affect the ATPase activity. The present study is the first report that clearly demonstrates that the class III myosin is an actin-based motor protein having a protein kinase activity.
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PMID:Determination of human myosin III as a motor protein having a protein kinase activity. 1267 20

Regulation of vascular smooth muscle cell contractile state is critical for the maintenance of blood vessel tone. Abnormal vascular smooth muscle cell contractility plays an important role in the pathogenesis of hypertension, blood vessel spasm, and atherosclerosis. Myosin phosphatase, the key enzyme controlling myosin light chain dephosphorylation, regulates smooth muscle cell contraction. Vasoconstrictor and vasodilator pathways inhibit and activate myosin phosphatase, respectively. G-protein-coupled receptor agonists can inhibit myosin phosphatase and cause smooth muscle cell contraction by activating RhoA/Rho kinase, whereas NO/cGMP can activate myosin phosphatase and cause smooth muscle cell relaxation by activation of cGMP-dependent protein kinase. We have used yeast two-hybrid screening to identify a 116-kDa human protein that interacts with both myosin phosphatase and RhoA. This myosin phosphatase-RhoA interacting protein, or M-RIP, is highly homologous to murine p116RIP3, is expressed in vascular smooth muscle, and is localized to actin myofilaments. M-RIP binds directly to the myosin binding subunit of myosin phosphatase in vivo in vascular smooth muscle cells by an interaction between coiled-coil and leucine zipper domains in the two proteins. An adjacent domain of M-RIP directly binds RhoA in a nucleotide-independent manner. M-RIP copurifies with RhoA and Rho kinase, colocalizes on actin stress fibers with RhoA and MBS, and is associated with Rho kinase activity in vascular smooth muscle cells. M-RIP can assemble a complex containing both RhoA and MBS, suggesting that M-RIP may play a role in myosin phosphatase regulation by RhoA.
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PMID:Myosin phosphatase-Rho interacting protein. A new member of the myosin phosphatase complex that directly binds RhoA. 1450 64

Myosin binding protein-C (MyBP-C) is a thick filament-associated protein localized to the crossbridge-containing C zones of striated muscle sarcomeres. The cardiac isoform is composed of eight immunoglobulin I-like domains and three fibronectin 3-like domains and is known to be a physiological substrate of cAMP-dependent protein kinase. MyBP-C contributes to thick filament structure via interactions at its C-terminus with the light meromyosin section of the myosin rod and with titin. The protein also has a role in the regulation of contraction, due to the binding of its N-terminus to the subfragment-2 portion of myosin, which reduces actomyosin ATPase activity; phosphorylation abolishes this interaction, resulting in release of the "brake" on crossbridge cycling. Several structural models of the interaction of MyBP-C with myosin have been proposed, although its precise arrangement on the thick filament remains to be elucidated. Mutations in the gene encoding cardiac MyBP-C are a common cause of hypertrophic cardiomyopathy, and this has led to increased interest in the protein's function. Investigation of disease-causing mutations in domains with unknown function has led to further insights into the mechanism of cMyBP-C action. This Review aims to collate the published data on those aspects of MyBP-C that are well characterized and to consider new and emerging data that further define its structural and regulatory roles and its arrangement in the sarcomere. We also speculate on the mechanisms by which hypertrophic cardiomyopathy-causing truncation and missense mutations affect the normal functioning of the sarcomere.
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PMID:Cardiac myosin binding protein C: its role in physiology and disease. 1516 15

Calponin (CaP), a thin filament-associated protein, is thought to be involved in modulating smooth muscle contractile activity, but the role and mechanism keep unknown. In this study, trace amount of calponin (TAC) was found to obviously influence myosin in different states in Ca(2+)-independent manner, suggesting a high efficient interaction between TAC and myosin. In this assay, the lowest ratio of CaP vs. myosin was 1:10,000, with the concentration of CaP 10,000-fold lower than that used previously. Myosin phosphorylation, myosin Mg(2+)-ATPase activity and protein binding activity were detected to determine the effects of TAC on the myosin in different states. The amount of precipitated myosin that bound to TAC was used as the index to determine the interaction between myosin and TAC in binding assay. Our data showed that in the absence of actin, TAC significantly increased the precipitation of unphosphorylated myosin, Ca(2+)-dependently or independently phosphorylated myosin by MLCK, and stimulated the Mg(2+)-ATPase activities of these myosins slightly but significantly. However, no obvious change of precipitation of myosin phosphorylated by PKA was observed, indicating the relatively selective effect of TAC. In the presence of actin, the increase of myosin precipitations was abolished, and no obvious change of actin precipitations and actin-activated myosin Mg(2+)-ATPase activities were observed implicating the high efficiency of TAC on myosin being present in the absence of actin. Although we can not give conclusive comments to our results, we propose that the high efficiency of TAC-myosin interaction is present when actin is dissociated from myosin, even if CaP/myosin ratio is very low; this high efficient interaction can be abolished by actin. However, why and how TAC can possess such a high efficiency to influence myosin and how the physiological significance of the high efficiency of TAC is in regulating the interaction between myosin and actin remain to be investigated.
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PMID:Influence of trace amount of calponin on smooth muscle myosin in different states. 1529 44


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