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      Activation of G 12/G 13 Results in Shape Change and Rho/Rho-Kinase–mediated Myosin Light Chain Phosphorylation in Mouse Platelets

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          Abstract

          Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the α-subunit of the heterotrimeric G protein G q do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A 2 (TXA 2) or thrombin receptors. In contrast to thrombin, the TXA 2 mimetic U46619 led to the selective activation of G 12 and G 13 in Gα q-deficient platelets indicating that these G proteins mediate TXA 2 receptor-induced shape change. TXA 2 receptor-mediated activation of G 12/G 13 resulted in tyrosine phosphorylation of pp72 syk and stimulation of pp60 c-src as well as in phosphorylation of myosin light chain (MLC) in Gα q-deficient platelets. Both MLC phosphorylation and shape change induced through G 12/G 13 in the absence of Gα q were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G 12/G 13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase–mediated regulation of MLC phosphorylation. We provide evidence that G 12/G 13-mediated Rho/Rho-kinase–dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.

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          Most cited references64

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          Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase).

          The small GTPase Rho is implicated in physiological functions associated with actin-myosin filaments such as cytokinesis, cell motility, and smooth muscle contraction. We have recently identified and molecularly cloned Rho-associated serine/threonine kinase (Rho-kinase), which is activated by GTP Rho (Matsui, T., Amano, M., Yamamoto, T., Chihara, K., Nakafuku, M., Ito, M., Nakano, T., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) EMBO J. 15, 2208-2216). Here we found that Rho-kinase stoichiometrically phosphorylated myosin light chain (MLC). Peptide mapping and phosphoamino acid analyses revealed that the primary phosphorylation site of MLC by Rho-kinase was Ser-19, which is the site phosphorylated by MLC kinase. Rho-kinase phosphorylated recombinant MLC, whereas it failed to phosphorylate recombinant MLC, which contained Ala substituted for both Thr-18 and Ser-19. We also found that the phosphorylation of MLC by Rho-kinase resulted in the facilitation of the actin activation of myosin ATPase. Thus, it is likely that once Rho is activated, then it can interact with Rho-kinase and activate it. The activated Rho-kinase subsequently phosphorylates MLC. This may partly account for the mechanism by which Rho regulates cytokinesis, cell motility, or smooth muscle contraction.
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            Focal adhesions, contractility, and signaling.

            Focal adhesions are sites of tight adhesion to the underlying extracellular matrix developed by cells in culture. They provided a structural link between the actin cytoskeleton and the extracellular matrix and are regions of signal transduction that relate to growth control. The assembly of focal adhesions is regulated by the GTP-binding protein Rho. Rho stimulates contractility which, in cells that are tightly adherent to the substrate, generates isometric tension. In turn, this leads to the bundling of actin filaments and the aggregation of integrins (extracellular matrix receptors) in the plane of the membrane. The aggregation of integrins activates the focal adhesion kinase and leads to the assembly of a multicomponent signaling complex.
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              Signal transduction and regulation in smooth muscle.

              A P Somlyo (1994)
              Smooth muscle cells in the walls of many organs are vital for most bodily functions, and their abnormalities contribute to a range of diseases. Although based on a sliding-filament mechanism similar to that of striated muscles, contraction of smooth muscle is regulated by pharmacomechanical as well as by electromechanical coupling mechanisms. Recent studies have revealed previously unrecognized contractile regulatory processes, such as G-protein-coupled inhibition of myosin light-chain phosphatase, regulation of myosin light-chain kinase by other kinases, and the functional effects of smooth muscle myosin isoforms. Abnormalities of these regulatory mechanisms and isoform variations may contribute to diseases of smooth muscle, and the G-protein-coupled inhibition of protein phosphatase is also likely to be important in regulating non-muscle cell functions mediated by cytoplasmic myosin II.
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                Author and article information

                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                22 February 1999
                : 144
                : 4
                : 745-754
                Affiliations
                [* ]Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, 14195 Berlin, Germany; and []Division of Biology, California Institute of Technology, Pasadena, California 91125
                Author notes

                Address correspondence to S. Offermanns, Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Thielallee 67-73, 14195 Berlin, Germany. Tel.: (49) 30-8445-1835. Fax: (49) 30-8445-1818. E-mail: stoff@ 123456zedat.fu-berlin.de

                Article
                10.1083/jcb.144.4.745
                2132941
                10037795
                c67beb06-4cc1-491e-a991-c4383f85148c
                Copyright @ 1999
                History
                : 24 September 1998
                : 19 January 1999
                Categories
                Regular Articles

                Cell biology
                platelet,platelet shape change,g protein,rho-kinase,myosin light chain phosphorylation

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