M-RIP, a 116-kDa human protein, was identified to bind directly to both the myosin binding subunit of myosin phosphatase and RhoA in vascular smooth muscle cells.
The identification of M-RIP provides a molecular mechanism for how RhoA may regulate myosin phosphatase activity in vascular smooth muscle cells, which is relevant to vascular tone and diseases like hypertension.
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. 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. Blood vessel tone is regulated by the contractile state of vascular smooth muscle cells in the blood vessel wall. Diseases characterized by abnormal vascular smooth muscle cell contraction include hypertension, blood vessel spasm, and atherosclerosis (1Goldberg I.D. Stemerman M.B. Schnipper L.E. Ransil B.J. Crooks G.W. Fuhro R.L. Science. 1979; 295: 920-922Crossref Scopus (37) Google Scholar, 2Stemerman M.B. Ross R. J. Exp. Med. 1972; 136: 769-789Crossref PubMed Scopus (190) Google Scholar, 3Stary H.C. Arteriocsclerosis Suppl. I. 1989; 9: I19-I32PubMed Google Scholar, 4Shepard J.T. Vanhoutte P.M. Mayo Clinic Proc. 1985; 60: 33-46Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Fuster V. Badimon L. Badimon J.J. Chesebro J.H. N. Engl. J. Med. 1992; 326: 310-318Crossref PubMed Scopus (1402) Google Scholar). Smooth muscle contraction is tightly coupled to myosin light chain phosphorylation (6Hartshorne D.J. Johnson D.R. Physiology of the Gastrointestinal Tract. Raven, New York1987: 423-482Google Scholar), which in turn is regulated by the relative activities of myosin light chain kinase and myosin phosphatase. Myosin light chain kinase is activated by intracellular calcium and phosphorylates myosin light chains, leading to cell contraction (7Kamm K.E. Stull J.T. Annu. Rev. Pharmacol. Toxicol. 1985; 25: 593Crossref PubMed Google Scholar, 8Taylor D.A. Stull J.T. J. Biol. Chem. 1988; 263: 14456-14462Abstract Full Text PDF PubMed Google Scholar). Myosin phosphatase dephosphorylates myosin light chains, leading to smooth muscle cell relaxation (9Alessi D. MacDougall L.K. Sola M.M. Ikebe M. Cohen P. Eur. J. Biochem. 1992; 210: 1023-1035Crossref PubMed Scopus (328) Google Scholar). Myosin phosphatase activity, once thought to be constitutive, is now known to be highly regulated. Both vasoconstrictor signaling pathways, which lead to inhibition of the phosphatase and cell contraction (reviewed in Ref. 10Somlyo A.P. Somlyo A.V. Acta Physiol. Scand. 1998; 164: 437-448Crossref PubMed Scopus (132) Google Scholar), and vasodilator signaling pathways, which lead to cell relaxation via activation of myosin phosphatase have been recently defined (11Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (440) Google Scholar, 12Khatri J.J. Joyce K.M. Brozovich F.V. Fisher S.A. J. Biol. Chem. 2001; 276: 37250-37257Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 13Etter E.F. Eto M. Wardle R.L. Brautigan D.L. Murphy R.A. J. Biol. Chem. 2001; 276: 34681-34685Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 14Lee M.R. Li L. Kitazawa T. J. Biol. Chem. 1997; 272: 5063-5068Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 15Wu X. Somlyo A.V. Somlyo A.P. Biochem. Biophys. Res. Commun. 1996; 220: 658-663Crossref PubMed Scopus (139) Google Scholar). Myosin phosphatase is a heterotrimer consisting of a PP1 catalytic subunit, a 130-kDa myosin binding subunit (MBS) 1The abbreviations used are: MBSmyosin binding subunitM-RIPmyosin phosphatase-RhoA interacting proteinGSTglutathione S-transferasePMSFphenylmethylsulfonyl fluoridePBSphosphate-buffered salineNi-NTAnickel-nitrilotriacetic acidGTPγSguanosine 5′-3-O-(thio)triphosphateGDPβSguanosine 5′-O-2-(thio)diphosphatecGKcGMP-dependent protein kinaseCCcoiled-coilRBDRho-binding domain.1The abbreviations used are: MBSmyosin binding subunitM-RIPmyosin phosphatase-RhoA interacting proteinGSTglutathione S-transferasePMSFphenylmethylsulfonyl fluoridePBSphosphate-buffered salineNi-NTAnickel-nitrilotriacetic acidGTPγSguanosine 5′-3-O-(thio)triphosphateGDPβSguanosine 5′-O-2-(thio)diphosphatecGKcGMP-dependent protein kinaseCCcoiled-coilRBDRho-binding domain. and a 20-kDa subunit of unknown function (9Alessi D. MacDougall L.K. Sola M.M. Ikebe M. Cohen P. Eur. J. Biochem. 1992; 210: 1023-1035Crossref PubMed Scopus (328) Google Scholar, 16Shirazi A. Iizuka K. Fadden P. Mosse C. Somlyo A.P. Somlyo A.V. Haystead T.A.J. J. Biol. Chem. 1994; 269: 31598-31606Abstract Full Text PDF PubMed Google Scholar, 17Shimizu H. Ito M. Miyahara M. Ichikawa K. Okubo S. Konishi T. Naka M. Tanaka T. Hirano K. Hartshorne D.J. Nakano T. J. Biol. Chem. 1994; 269: 30407-30411Abstract Full Text PDF PubMed Google Scholar, 18Takahashi N. Ito M. Tanaka J. Nakano T. Kaibuchi K. Odai H. Takemura K. Genomics. 1997; 44: 150-152Crossref PubMed Scopus (45) Google Scholar). The MBS is a regulatory subunit that targets PP1 to its substrate, myosin light chain (9Alessi D. MacDougall L.K. Sola M.M. Ikebe M. Cohen P. Eur. J. Biochem. 1992; 210: 1023-1035Crossref PubMed Scopus (328) Google Scholar), and has multiple protein interaction domains, including ankyrin repeats at its amino terminus, and a leucine zipper domain at its carboxyl terminus. MBS binds PP1 and myosin light chain at its amino terminus and the M20 subunit and cGMP-dependent protein kinase 1α (cGK) at its carboxyl terminus (Ref. 11Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (440) Google Scholar, reviewed in Ref. 19Hartshorne D.J. Acta Physiol. Scand. 1998; 164: 483-493Crossref PubMed Scopus (86) Google Scholar). The MBS-cGK interaction is necessary for NO/cGMP-mediated activation of myosin phosphatase (11Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (440) Google Scholar). myosin binding subunit myosin phosphatase-RhoA interacting protein glutathione S-transferase phenylmethylsulfonyl fluoride phosphate-buffered saline nickel-nitrilotriacetic acid guanosine 5′-3-O-(thio)triphosphate guanosine 5′-O-2-(thio)diphosphate cGMP-dependent protein kinase coiled-coil Rho-binding domain. myosin binding subunit myosin phosphatase-RhoA interacting protein glutathione S-transferase phenylmethylsulfonyl fluoride phosphate-buffered saline nickel-nitrilotriacetic acid guanosine 5′-3-O-(thio)triphosphate guanosine 5′-O-2-(thio)diphosphate cGMP-dependent protein kinase coiled-coil Rho-binding domain. In vascular smooth muscle, G-protein-coupled receptor agonists cause contraction in part by inhibition of myosin phosphatase activity (20Kitazawa T. Gaylinn B.D. Denney G.H. Somlyo A.P. J. Biol. Chem. 1991; 266: 1708-1715Abstract Full Text PDF PubMed Google Scholar). Several downstream signaling pathways that inhibit myosin phosphatase activity have been discovered recently, including RhoA/Rho kinase (21Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2433) Google Scholar), protein kinase C activation of the inhibitory phosphoprotein CPI-17 (22Eto M. Senba S. Morita F. Yazawa M. FEBS Lett. 1997; 410: 356-360Crossref PubMed Scopus (226) Google Scholar), and arachidonic acid (23Gong M.-C. Fuglsang A. Alessi D. Kobayashi S. Cohen P. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1992; 267: 21492-21498Abstract Full Text PDF PubMed Google Scholar, 24Araki S. Ito M. Kureishi Y. Feng J. Machida H. Isaka N. Amano M. Kaibuchi K. Hartshorne D.J. Nakano T. Pflugers Arch. 2001; 441: 596-603Crossref PubMed Scopus (73) Google Scholar). In addition, several kinases copurify with myosin phosphatase, including ZIP-like kinase (25MacDonald J.A. Borman M.A. Murani A. Somlyo A.V. Hartshorne D.J. Haystead T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2419-2424Crossref PubMed Scopus (192) Google Scholar), integrin-linked kinase (26Muranyi A. MacDonald J.A. Deng J.T. Wilson D.P. Haystead T.A. Walsh M.P. Erdodi F. Kiss E. Wu Y. Hartshorne D.J. Biochem. J. 2002; 366: 211-216Crossref PubMed Google Scholar), myotonic dystrophy-related kinase (27Muranyi A. Zhang R. Liu F. Hirano K. Ito M. Epstein H.F. Hartshorne D.J. FEBS Lett. 2001; 493: 80-84Crossref PubMed Scopus (85) Google Scholar), and Raf-1 (28Broustas C.G. Grammatikakis N. Eto M. Dent P. Brautigan D.L. Kasid U. J. Biol. Chem. 2002; 277: 3053-3059Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), each of which can phosphorylate MBS and inhibit myosin phosphatase activity. RhoA/Rho kinase has been the most extensively studied myosin phosphatase inhibitor. RhoA binds to a myosin phosphatase complex in vitro, and GTP-bound RhoA, in combination with its downstream effector Rho kinase, inhibits myosin phosphatase activity (21Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2433) Google Scholar). Specific blockade of Rho kinase has been found to ameliorate hypertension in several rat models (29Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. Narumiya S. Nature. 1997; 389: 990-994Crossref PubMed Scopus (2538) Google Scholar), to the to vascular and blood vessel in models R. H. Y. S. M. Kaibuchi K. T. 2001; PubMed Scopus Google Scholar, N. H. K. J. K. M. K. S. Y. S. H. N. M. K. PubMed Scopus Google Scholar, Y. H. J. K. T. Y. Amano M. M. Kaibuchi K. A. J. Physiol. Google Scholar, T. H. K. I. Y. Fukata Y. T. K. S. Kaibuchi K. A. PubMed Scopus Google Scholar). inhibitory on MBS that phosphorylation directly with contractile myosin phosphatase inhibition J. Ito M. Ichikawa K. Isaka N. M. Hartshorne D.J. Nakano T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Fukata Y. N. Amano M. T. Ito M. F. M. Kaibuchi K. J. Biol. 1999; PubMed Scopus Google Scholar). a Rho kinase has been found to be in and in A. M. H. L. M. A. 2002; PubMed Scopus Google Scholar, M. H. Y. A. A. J. PubMed Scopus Google Scholar). for RhoA/Rho inhibition of myosin phosphatase, the for contractile is RhoA and Rho kinase to the cell T. E. L. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Amano M. T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. J. 1996; PubMed Scopus Google Scholar), and have been found with actin K. Y. Amano M. H. Kaibuchi K. K. J. Biol. 2001; PubMed Scopus Google Scholar, X. I. C. C. L. T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The RhoA and Rho kinase to and inhibit myosin phosphatase and of myosin light in contractile We that signaling RhoA/Rho inhibition of myosin phosphatase and for that with both myosin phosphatase and RhoA yeast two-hybrid screening the and of a protein that binds both MBS and RhoA. This myosin phosphatase-RhoA interacting protein is a between RhoA signaling and myosin phosphatase and yeast and human New and The and protein kinase and cells in with cells the and smooth muscle cells by the and cells and and for by of the of yeast two-hybrid and MBS with binding and activating domains, respectively. of in yeast by and in yeast with and a human the by on and by The yeast and at the of to the of The on a human that is homologous to the of and rat p116RIP3, The on a yeast two-hybrid The M-RIP the human The the and M-RIP by M-RIP and yeast two-hybrid for the M-RIP and and for The the M-RIP in both M-RIP with an M-RIP M-RIP that a and and the that the of M-RIP the amino terminus of M-RIP by the M-RIP to M-RIP coiled-coil domains M-RIP the and and and and for and respectively. and M-RIP by the and the and the Both and for of in and in respectively. and of RhoA for of RhoA in amino of T. T. Ishizaki T. N. K. N. P. Narumiya S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), by a and (11Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (440) Google Scholar). H.K. Mendelsohn M.E. PubMed Scopus (45) Google Scholar). by human MBS the human by by the domain of with the domain of the in both of the of M-RIP M-RIP and The and The protein of the M-RIP expressed in and (11Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (440) Google Scholar). and at on protein E. D. Scholar). of and in at in with The with and an to a of for and for and the cells and the cell and in of and each of and A. The cell on for and to of and respectively. The and to the of and This for at which the with at to or in and and at the in consisting of each of and to a of and for on The and at for The with of for with at to or the in and and at and of on that of and D. A. PubMed Scopus Google Scholar). for proteins. in and of in and with or for at by the of to a of human smooth muscle cells with and in each of and and or at at for The with protein used for with An of each by human smooth muscle cells in in and each of and for MBS and in and each of and for M-RIP and cGMP-dependent protein kinase at for at The with protein and with or protein kinase and the The with C in and by and of MBS with domains, cells by and with that used for and protein used to protein with cell cells with in for at at for at The with to at the with with and by protein with on in for with protein the with in and by with K. Y. Amano M. H. Kaibuchi K. K. J. Biol. 2001; PubMed Scopus Google Scholar). smooth muscle cells on two to with with of and for with of cell of and in for or of each of and in for for with of or by with of and in for with of in and with a The at for and stress fibers by of the at for The stress in of protein and to and with the of stress human smooth muscle cells on and fibers and human smooth muscle cells and M-RIP for the with The M-RIP and with Rho kinase Feng J. Ito M. Kureishi Y. Ichikawa K. Amano M. Isaka N. Okawa K. Iwamatsu A. Kaibuchi K. Hartshorne D.J. Nakano T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), with or the of of the for at The by the of protein the and by and The by an and smooth muscle cells on in with The with in The cells with and in with The cells with in in for in the cells in to and or in for in and on with acid the the for the phosphorylation and and of identify in regulation of myosin phosphatase, of human MBS used in a yeast two-hybrid of a human and of a with to a murine protein, M. B. J. Biol. 1997; PubMed Scopus Google Scholar). both with with domain homologous to and homologous to of murine human for the of human the human a that highly homologous to the of murine The human by yeast two-hybrid and the The human is at the and at the amino acid to murine and rat The domain of human is at the amino acid to the domain on murine This human is and of amino acid of The human M-RIP is with murine domains M-RIP domain. The amino acid to each domain is the of M-RIP by yeast two-hybrid and of the M-RIP a protein of amino with multiple protein interaction domains, including a of domains two and coiled-coil domains for for phosphorylation by protein kinase protein and kinases in the M-RIP and of M-RIP in Smooth M-RIP and by of cells with M-RIP a of in cells that by of M-RIP of the in which with to identify M-RIP used to two human smooth muscle cell a in that M-RIP is expressed in human vascular smooth muscle cells M-RIP vascular smooth muscle cells at be and human smooth muscle cells with and with to actin M-RIP localized in a in the to the of actin of the two that M-RIP with actin In M-RIP with M-RIP to the of a between and M-RIP in the of vascular smooth muscle cells In between M-RIP and Myosin the interaction between M-RIP and MBS in yeast in vascular smooth muscle with human smooth muscle cell of M-RIP or MBS to of both an in vivo interaction between M-RIP and MBS in vascular smooth muscle cells. M-RIP to of and cGMP-dependent protein kinase and that M-RIP is associated with the myosin phosphatase complex in In interaction binding between M-RIP and PP1 or cGMP-dependent protein kinase be suggesting that M-RIP interacts with via known with MBS of between M-RIP and Myosin interaction and protein interaction of M-RIP domains with vascular smooth muscle cell and MBS by The coiled-coil domains of M-RIP for binding to MBS The coiled-coil domain of M-RIP MBS whereas the and domains or interaction with MBS The domain the of the Rho-binding domain for murine M. B. J. Biol. 1997; PubMed Scopus Google Scholar). MBS binds the M-RIP for the M-RIP domain which the carboxyl terminus of and for the M-RIP domain the carboxyl terminus of and for binding of MBS vascular smooth muscle cell to MBS the whereas both and in with for M-RIP domains expressed in cells that MBS binds a domain of M-RIP and to the Rho-binding domain The carboxyl terminus of MBS a leucine zipper domain that binding to coiled-coil domains, including the zipper domain of cGMP-dependent protein kinase 1α (11Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (440) Google Scholar, 12Khatri J.J. Joyce K.M. Brozovich F.V. Fisher S.A. J. Biol. Chem. 2001; 276: 37250-37257Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, H.K. Mendelsohn M.E. PubMed Scopus (45) Google Scholar). domain of MBS binding to the of M-RIP, MBS and MBS in which in the leucine zipper domain to expressed and for binding to with M-RIP, whereas M-RIP the or studied in cells known to both MBS and to the that the interaction is by the of M-RIP and the leucine zipper domain of binding between M-RIP and MBS or that the of M-RIP binds directly to MBS, whereas with of the coiled-coil domains, that the MBS leucine zipper domain the used to M-RIP binds RhoA. Both and M-RIP expressed in cells In binding RhoA, in with or with Rho-binding domain or M.A. Scholar), whereas both and for or binding state of the M-RIP a including the Myosin and that RhoA binds an M-RIP domain that amino whereas MBS binds a domain that amino and An M-RIP containing both domains expressed in cells and used to the that of M-RIP a complex of M-RIP with MBS and RhoA RhoA to and MBS to that amino of M-RIP, via adjacent and domains, binding of both RhoA and MBS, and that MBS binding is for the binding of RhoA to M-RIP M-RIP with RhoA/Rho in and Rho kinase have been to with actin stress and in stress by stress fibers with K. Y. Amano M. H. Kaibuchi K. K. J. Biol. 2001; PubMed Scopus Google Scholar, X. I. C. C. L. T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). and stress fibers vascular smooth muscle cells by both MBS, and M-RIP, whereas stress stress RhoA and Rho kinase of vascular smooth muscle cells of M-RIP, MBS, and RhoA with actin stress fibers of the of and for M-RIP RhoA be in the M-RIP by the M-RIP kinase activity that by the Rho kinase an between M-RIP and RhoA/Rho kinase in Myosin phosphatase activity is regulated by both contractile agonists and RhoA and its downstream effector Rho kinase contractile myosin phosphatase the RhoA interacts with myosin phosphatase RhoA and Rho kinase to the cell and a of RhoA/Rho kinase has been on actin stress fibers K. Y. Amano M. H. Kaibuchi K. K. J. Biol. 2001; PubMed Scopus Google Scholar, X. I. C. C. L. T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The RhoA/Rho kinase to actin stress fibers is The protein M-RIP, is expressed in human vascular and is to myosin phosphatase in cells. that M-RIP colocalizes with actin with a binding to actin J. M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). M-RIP is localized to the contractile myosin light chain phosphorylation regulates the contractile suggesting a role for M-RIP in myosin phosphatase The amino terminus of M-RIP adjacent domains and a combination found on kinase binding to both actin and L. P. J. Y. T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Y. T. S. Nature. 1998; PubMed Scopus Google Scholar). This of has recently been to actin binding and actin activity in J. M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). has been to be in the cell M. B. J. Biol. 1997; PubMed Scopus Google Scholar), M-RIP in the of vascular smooth muscle a in between the murine and human or cell The domain of M-RIP interacts with both MBS and RhoA. The domain of M-RIP the amino of which the that M-RIP interacts with MBS via RhoA. have found that both RhoA and MBS M-RIP directly to adjacent domains for RhoA and for a M-RIP binding MBS and RhoA The domain of MBS a leucine zipper domain that binding to a zipper in cGMP-dependent protein kinase 1α H.K. Mendelsohn M.E. PubMed Scopus (45) Google Scholar). The that the MBS leucine zipper domain binds the amino of the M-RIP domain. the domain of MBS binds to both proteins. that MBS can M-RIP and cGMP-dependent protein kinase 1α M-RIP can with cGMP-dependent protein kinase by of suggesting that the interaction with MBS the be of in to the of a protein D. A. PubMed Scopus Google Scholar), a to binding to RhoA J. M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In both protein interaction and cell that M-RIP binds RhoA directly and of binding The and to and M-RIP be to binding to RhoA and Rho kinase K. Y. Amano M. H. Kaibuchi K. K. J. Biol. 2001; PubMed Scopus Google Scholar). a to RhoA and Rho kinase to stress fibers K. Y. Amano M. H. Kaibuchi K. K. J. Biol. 2001; PubMed Scopus Google found that M-RIP with RhoA and Rho kinase. of that stress and of M-RIP, MBS, and RhoA with the of and Rho kinase activity be in the M-RIP of the of with the in binding that M-RIP interacts with RhoA in The M-RIP interaction for the binding state of RhoA, whereas (21Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2433) Google found that with myosin phosphatase. M-RIP RhoA to myosin phosphatase, is of the phosphatase interaction is to a complex interaction between M-RIP, RhoA, and MBS of the or a Rho kinase phosphorylation the Both of M-RIP and a of that with actin J. M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that is with both stress fibers and has actin in vitro, and actin to M-RIP has a on myosin phosphatase activity, and the smooth muscle contractile state by actin and In M-RIP, the human of p116RIP3, binds myosin phosphatase and RhoA in vascular smooth muscle cells. binding a M-RIP MBS and RhoA The binding to myosin phosphatase and RhoA and to actin that M-RIP may RhoA to the myosin phosphatase complex to the myosin phosphorylation We Mochizuki for RhoA and and for with cell and for
Surks et al. (Mon,) conducted a other in Abnormal vascular smooth muscle cell contractility. Yeast two-hybrid screening and protein characterization was evaluated on Protein interactions of M-RIP with myosin phosphatase and RhoA. M-RIP, a 116-kDa human protein, was identified to bind directly to both the myosin binding subunit of myosin phosphatase and RhoA in vascular smooth muscle cells.