The COOH Terminus of Rho-kinase Negatively Regulates Rho-kinase Activity
Rho-kinase is implicated in the phosphorylation of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fiber formation in non-muscle cells. Here, we examined the mode of action of inhibitors of Rho-kinase. The chemical compounds such as HA1077 and Y-32885 inhibited not only the Rho-kinase activity but also the activity of protein kinase N, one of the targets of Rho, but had less of an effect on the activity of myotonic dystrophy kinase-related Cdc42-binding kinase β (MRCKβ). The COOH-terminal portion of Rho-kinase containing Rho-binding (RB) and pleckstrin homology (PH) domains (RB/PH (TT)), in which point mutations were introduced to abolish the Rho binding activity, interacted with Rho-kinase and thereby inhibited the Rho-kinase activity, whereas RB/PH (TT) had no effect on the activity of protein kinase N or MRCKβ, suggesting that the COOH-terminal region of Rho-kinase is a possible negative regulatory region of Rho-kinase. The expression of RB/PH (TT) specifically blocked the stress fiber and focal adhesion formation induced by the active form of Rho or Rho-kinase in NIH 3T3 cells, but not that induced by the active form of MRCKβ or myosin light chain. Thus, RB/PH (TT) appears to specifically inhibit Rho-kinase in vivo. Rho-kinase is implicated in the phosphorylation of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fiber formation in non-muscle cells. Here, we examined the mode of action of inhibitors of Rho-kinase. The chemical compounds such as HA1077 and Y-32885 inhibited not only the Rho-kinase activity but also the activity of protein kinase N, one of the targets of Rho, but had less of an effect on the activity of myotonic dystrophy kinase-related Cdc42-binding kinase β (MRCKβ). The COOH-terminal portion of Rho-kinase containing Rho-binding (RB) and pleckstrin homology (PH) domains (RB/PH (TT)), in which point mutations were introduced to abolish the Rho binding activity, interacted with Rho-kinase and thereby inhibited the Rho-kinase activity, whereas RB/PH (TT) had no effect on the activity of protein kinase N or MRCKβ, suggesting that the COOH-terminal region of Rho-kinase is a possible negative regulatory region of Rho-kinase. The expression of RB/PH (TT) specifically blocked the stress fiber and focal adhesion formation induced by the active form of Rho or Rho-kinase in NIH 3T3 cells, but not that induced by the active form of MRCKβ or myosin light chain. Thus, RB/PH (TT) appears to specifically inhibit Rho-kinase in vivo. protein kinase N myosin light chain pleckstrin homology catalytic domain Rho-binding domain kinase-deficient catalytic domain glutathione S-transferase myotonic dystrophy kinase-related Cdc42-binding kinase β maltose-binding protein polyacrylamide gel electophoresis Dulbecco's modified Eagle's medium phosphate-buffered saline There is mounting evidence that the small GTPase Rho plays crucial roles in the rearrangements of cytoskeleton and cell adhesion (1Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (884) Google Scholar, 2Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2083) Google Scholar, 3Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar). Rho cycles between GDP-bound inactive and GTP-bound active forms, and the GTP-bound form binds to specific effectors and then exerts its biological functions. Numerous putative Rho effectors have been identified; PKN1 (4Amano M. Mukai H. Ono Y. Chihara K. Matsui T. Hamajima Y. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 271: 648-650Crossref PubMed Scopus (395) Google Scholar, 5Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (348) Google Scholar), Rho-kinase/ROKα/ROCK II (6Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (934) Google Scholar, 7Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar, 8Ishizaki T. Maekawa M. Fujisawa K. Okawa K. Iwamatsu A. Fujita A. Watanabe N. Saito Y. Kakizuka A. Morii N. Narumiya S. EMBO J. 1996; 15: 1885-1893Crossref PubMed Scopus (790) Google Scholar), myosin-binding subunit of myosin phosphatase (9Kimura 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 (2423) Google Scholar), mDia1 (10Watanabe N. Madaule P. Reid T. Ishizaki T. Watanabe G. Kakizuka A. Saito Y. Nakao K. Jockusch B.M. Narumiya S. EMBO J. 1997; 16: 3044-3056Crossref PubMed Scopus (681) Google Scholar), citron (11Madaule P. Furuyashiki T. Reid T. Ishizaki T. Watanabe G. Morii N. Narumiya S. FEBS Lett. 1995; 377: 243-248Crossref PubMed Scopus (147) Google Scholar), citron kinase (12Madaule P. Eda M. Watanabe N. Fujisawa K. Matsuoka T. Bito H. Ishizaki T. Narumiya S. Nature. 1998; 394: 491-494Crossref PubMed Scopus (327) Google Scholar), rhophilin, rhotekin (11Madaule P. Furuyashiki T. Reid T. Ishizaki T. Watanabe G. Morii N. Narumiya S. FEBS Lett. 1995; 377: 243-248Crossref PubMed Scopus (147) Google Scholar), Kv1.2 (13Cachero T.G. Morielli A.D. Peralta E.G. Cell. 1998; 93: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and phospholipase D (14Singer W.D. Brown H.A. Sternweis P.C. Annu Rev. Biochem. 1997; 66: 475-509Crossref PubMed Scopus (347) Google Scholar). ROCK I/ROKβ is an isoform of Rho-kinase (7Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar, 8Ishizaki T. Maekawa M. Fujisawa K. Okawa K. Iwamatsu A. Fujita A. Watanabe N. Saito Y. Kakizuka A. Morii N. Narumiya S. EMBO J. 1996; 15: 1885-1893Crossref PubMed Scopus (790) Google Scholar). Rho-kinase is implicated in many processes downstream of Rho; stress fiber and focal adhesion formation (15Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar, 16Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (945) Google Scholar, 17Ishizaki T. Naito M. Fujisawa K. Maekawa M. Watanabe N. Saito Y. Narumiya S. FEBS Lett. 1997; 404: 118-124Crossref PubMed Scopus (454) Google Scholar), smooth muscle contraction (18Kureishi Y. Kobayashi S. Amano M. Kimura K. Kanaide H. Nakano T. Kaibuchi K. Ito M. J. Biol. Chem. 1997; 272: 12257-12260Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar), intermediate filament disassembly (19Kosako H. Amano M. Yanagida M. Tanabe K. Nishi Y. Kaibuchi K. Inagaki M. J. Biol. Chem. 1997; 272: 10333-10336Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 20Goto H. Kosako H. Tanabe K. Yanagida M. Sakurai M. Amano M. Kaibuchi K. Inagaki M. J. Biol. Chem. 1998; 273: 11728-11736Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), neurite retraction (21Amano M. Chihara K. Nakamura N. Fukata Y. Yano T. Shibata M. Ikebe M. Kaibuchi K. Genes Cells. 1998; 3: 177-188Crossref PubMed Scopus (218) Google Scholar,22Hirose M. Ishizaki T. Watanabe N. Uehata M. Kranenburg O. Moolenaar W.H. Matsumura F. Maekawa M. Bito H. Narumiya S. J. Cell Biol. 1998; 141: 1625-1636Crossref PubMed Scopus (410) Google Scholar), microvilli formation (23Oshiro N. Fukata Y. Kaibuchi K. J. Biol. Chem. 1998; 273: 34663-34666Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), cytokinesis (24Yasui Y. Amano M. Inagaki N. Nagata K. Nakamura H. Saya H. Kaibuchi K. Inagaki M. J. Cell Biol. 1998; 143: 1249-1258Crossref PubMed Scopus (148) Google Scholar), and cell migration (25Fukata Y. Oshiro N. Kinoshita N. Kawano Y. Matsuoka Y. Bennett V. Matsuura Y. Kaibuchi K. J. Cell Biol. 1999; 145: 347-361Crossref PubMed Scopus (252) Google Scholar). Rho-kinase regulates the phosphorylation of MLC by the direct phosphorylation of MLC and by the inactivation of myosin phosphatase through the phosphorylation of myosin-binding subunit (9Kimura 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 (2423) Google Scholar, 26Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1663) Google Scholar). In addition to MLC and myosin-binding subunit, Rho-kinase phosphorylates the ezrin/radixin/moesin family proteins and adducin in vitro (27Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (724) Google Scholar, 28Kimura K. Fukata Y. Matsuoka Y. Bennett V. Matsuura Y. Okawa K. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1998; 273: 5542-5548Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). To unravel in vivo functions of Rho-kinase, it is necessary to develop specific probes for Rho-kinase. Recently, chemical compounds such as Y-27632, Y-32885, and HA1077 have been shown to inhibit the Rho-kinase activity in a manner competitive with ATP (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 (2518) Google Scholar), and to suppress hypertension in model animals. However, the modes of action and specificity of these chemical compounds have not yet been elucidated.Rho-kinase is composed of NH2-terminal catalytic, coiled-coil, Rho-binding, and COOH-terminal PH domains (6Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (934) Google Scholar). When the COOH-terminal portion of Rho-kinase is deleted, the NH2-terminal portion containing the catalytic domain of Rho-kinase (CAT) becomes constitutively active and serves as the dominant active form both in vitro and in vivo(16Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (945) Google Scholar). The Rho-binding domain (RB) of Rho-kinase inhibits Rho-dependent activation of Rho-kinase in vitroand interferes with the Rho pathway in vivo (16Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (945) Google Scholar). The catalytic domain mutated at the ATP-binding site (kinase dead: CAT-KD) and the PH domain (PH) serve as dominant negative forms in vivo in some cases (16Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (945) Google Scholar), while they have no or a very weak effect on the Rho-kinase activity in vitro. It has been also reported that ROCK I, an isoform of Rho-kinase, that is kinase dead and cannot bind to Rho (named as KD-IA) functions as dominant negative form in the cells (17Ishizaki T. Naito M. Fujisawa K. Maekawa M. Watanabe N. Saito Y. Narumiya S. FEBS Lett. 1997; 404: 118-124Crossref PubMed Scopus (454) Google Scholar). Recently, we found that the COOH-terminal portion of Rho-kinase containing Rho-binding and PH domains (RB/PH (TT)), in which point mutations are introduced to abolish the Rho binding activity (15Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar,30Fujisawa K. Fujita A. Ishizaki T. Saito Y. Narumiya S. J. Biol. Chem. 1996; 271: 23022-23028Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), inhibits the lysophosphatidic acid-induced neurite retraction in neuroblastoma cells and cytokinesis in Xenopus eggs or in mammalian cells (21Amano M. Chihara K. Nakamura N. Fukata Y. Yano T. Shibata M. Ikebe M. Kaibuchi K. Genes Cells. 1998; 3: 177-188Crossref PubMed Scopus (218) Google Scholar, 24Yasui Y. Amano M. Inagaki N. Nagata K. Nakamura H. Saya H. Kaibuchi K. Inagaki M. J. Cell Biol. 1998; 143: 1249-1258Crossref PubMed Scopus (148) Google Scholar), although PH has minimal effects in these cases. These results suggest that RB/PH (TT) functions as the dominant negative form of Rho-kinase by the different mechanism of inhibition from those of CAT-KD, RB, and PH.Here, we examined the mode of action and the specificity of inhibitors of Rho-kinase. We found that RB/PH (TT) directly and specifically inhibited the kinase activity of catalytic fragment of Rho-kinasein vitro. The expression of RB/PH (TT) specifically blocked the stress fiber and focal adhesion formation induced by the active form of Rho or Rho-kinase in NIH 3T3 cells, but not that induced by the active form of MLC. There is mounting evidence that the small GTPase Rho plays crucial roles in the rearrangements of cytoskeleton and cell adhesion (1Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (884) Google Scholar, 2Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2083) Google Scholar, 3Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar). Rho cycles between GDP-bound inactive and GTP-bound active forms, and the GTP-bound form binds to specific effectors and then exerts its biological functions. Numerous putative Rho effectors have been identified; PKN1 (4Amano M. Mukai H. Ono Y. Chihara K. Matsui T. Hamajima Y. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 271: 648-650Crossref PubMed Scopus (395) Google Scholar, 5Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (348) Google Scholar), Rho-kinase/ROKα/ROCK II (6Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (934) Google Scholar, 7Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar, 8Ishizaki T. Maekawa M. Fujisawa K. Okawa K. Iwamatsu A. Fujita A. Watanabe N. Saito Y. Kakizuka A. Morii N. Narumiya S. EMBO J. 1996; 15: 1885-1893Crossref PubMed Scopus (790) Google Scholar), myosin-binding subunit of myosin phosphatase (9Kimura 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 (2423) Google Scholar), mDia1 (10Watanabe N. Madaule P. Reid T. Ishizaki T. Watanabe G. Kakizuka A. Saito Y. Nakao K. Jockusch B.M. Narumiya S. EMBO J. 1997; 16: 3044-3056Crossref PubMed Scopus (681) Google Scholar), citron (11Madaule P. Furuyashiki T. Reid T. Ishizaki T. Watanabe G. Morii N. Narumiya S. FEBS Lett. 1995; 377: 243-248Crossref PubMed Scopus (147) Google Scholar), citron kinase (12Madaule P. Eda M. Watanabe N. Fujisawa K. Matsuoka T. Bito H. Ishizaki T. Narumiya S. Nature. 1998; 394: 491-494Crossref PubMed Scopus (327) Google Scholar), rhophilin, rhotekin (11Madaule P. Furuyashiki T. Reid T. Ishizaki T. Watanabe G. Morii N. Narumiya S. FEBS Lett. 1995; 377: 243-248Crossref PubMed Scopus (147) Google Scholar), Kv1.2 (13Cachero T.G. Morielli A.D. Peralta E.G. Cell. 1998; 93: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and phospholipase D (14Singer W.D. Brown H.A. Sternweis P.C. Annu Rev. Biochem. 1997; 66: 475-509Crossref PubMed Scopus (347) Google Scholar). ROCK I/ROKβ is an isoform of Rho-kinase (7Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar, 8Ishizaki T. Maekawa M. Fujisawa K. Okawa K. Iwamatsu A. Fujita A. Watanabe N. Saito Y. Kakizuka A. Morii N. Narumiya S. EMBO J. 1996; 15: 1885-1893Crossref PubMed Scopus (790) Google Scholar). Rho-kinase is implicated in many processes downstream of Rho; stress fiber and focal adhesion formation (15Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar, 16Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (945) Google Scholar, 17Ishizaki T. Naito M. Fujisawa K. Maekawa M. Watanabe N. Saito Y. Narumiya S. FEBS Lett. 1997; 404: 118-124Crossref PubMed Scopus (454) Google Scholar), smooth muscle contraction (18Kureishi Y. Kobayashi S. Amano M. Kimura K. Kanaide H. Nakano T. Kaibuchi K. Ito M. J. Biol. Chem. 1997; 272: 12257-12260Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar), intermediate filament disassembly (19Kosako H. Amano M. Yanagida M. Tanabe K. Nishi Y. Kaibuchi K. Inagaki M. J. Biol. Chem. 1997; 272: 10333-10336Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 20Goto H. Kosako H. Tanabe K. Yanagida M. Sakurai M. Amano M. Kaibuchi K. Inagaki M. J. Biol. Chem. 1998; 273: 11728-11736Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), neurite retraction (21Amano M. Chihara K. Nakamura N. Fukata Y. Yano T. Shibata M. Ikebe M. Kaibuchi K. Genes Cells. 1998; 3: 177-188Crossref PubMed Scopus (218) Google Scholar,22Hirose M. Ishizaki T. Watanabe N. Uehata M. Kranenburg O. Moolenaar W.H. Matsumura F. Maekawa M. Bito H. Narumiya S. J. Cell Biol. 1998; 141: 1625-1636Crossref PubMed Scopus (410) Google Scholar), microvilli formation (23Oshiro N. Fukata Y. Kaibuchi K. J. Biol. Chem. 1998; 273: 34663-34666Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), cytokinesis (24Yasui Y. Amano M. Inagaki N. Nagata K. Nakamura H. Saya H. Kaibuchi K. Inagaki M. J. Cell Biol. 1998; 143: 1249-1258Crossref PubMed Scopus (148) Google Scholar), and cell migration (25Fukata Y. Oshiro N. Kinoshita N. Kawano Y. Matsuoka Y. Bennett V. Matsuura Y. Kaibuchi K. J. Cell Biol. 1999; 145: 347-361Crossref PubMed Scopus (252) Google Scholar). Rho-kinase regulates the phosphorylation of MLC by the direct phosphorylation of MLC and by the inactivation of myosin phosphatase through the phosphorylation of myosin-binding subunit (9Kimura 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 (2423) Google Scholar, 26Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1663) Google Scholar). In addition to MLC and myosin-binding subunit, Rho-kinase phosphorylates the ezrin/radixin/moesin family proteins and adducin in vitro (27Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (724) Google Scholar, 28Kimura K. Fukata Y. Matsuoka Y. Bennett V. Matsuura Y. Okawa K. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1998; 273: 5542-5548Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). To unravel in vivo functions of Rho-kinase, it is necessary to develop specific probes for Rho-kinase. Recently, chemical compounds such as Y-27632, Y-32885, and HA1077 have been shown to inhibit the Rho-kinase activity in a manner competitive with ATP (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 (2518) Google Scholar), and to suppress hypertension in model animals. However, the modes of action and specificity of these chemical compounds have not yet been elucidated. Rho-kinase is composed of NH2-terminal catalytic, coiled-coil, Rho-binding, and COOH-terminal PH domains (6Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (934) Google Scholar). When the COOH-terminal portion of Rho-kinase is deleted, the NH2-terminal portion containing the catalytic domain of Rho-kinase (CAT) becomes constitutively active and serves as the dominant active form both in vitro and in vivo(16Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (945) Google Scholar). The Rho-binding domain (RB) of Rho-kinase inhibits Rho-dependent activation of Rho-kinase in vitroand interferes with the Rho pathway in vivo (16Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (945) Google Scholar). The catalytic domain mutated at the ATP-binding site (kinase dead: CAT-KD) and the PH domain (PH) serve as dominant negative forms in vivo in some cases (16Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (945) Google Scholar), while they have no or a very weak effect on the Rho-kinase activity in vitro. It has been also reported that ROCK I, an isoform of Rho-kinase, that is kinase dead and cannot bind to Rho (named as KD-IA) functions as dominant negative form in the cells (17Ishizaki T. Naito M. Fujisawa K. Maekawa M. Watanabe N. Saito Y. Narumiya S. FEBS Lett. 1997; 404: 118-124Crossref PubMed Scopus (454) Google Scholar). Recently, we found that the COOH-terminal portion of Rho-kinase containing Rho-binding and PH domains (RB/PH (TT)), in which point mutations are introduced to abolish the Rho binding activity (15Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar,30Fujisawa K. Fujita A. Ishizaki T. Saito Y. Narumiya S. J. Biol. Chem. 1996; 271: 23022-23028Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), inhibits the lysophosphatidic acid-induced neurite retraction in neuroblastoma cells and cytokinesis in Xenopus eggs or in mammalian cells (21Amano M. Chihara K. Nakamura N. Fukata Y. Yano T. Shibata M. Ikebe M. Kaibuchi K. Genes Cells. 1998; 3: 177-188Crossref PubMed Scopus (218) Google Scholar, 24Yasui Y. Amano M. Inagaki N. Nagata K. Nakamura H. Saya H. Kaibuchi K. Inagaki M. J. Cell Biol. 1998; 143: 1249-1258Crossref PubMed Scopus (148) Google Scholar), although PH has minimal effects in these cases. These results suggest that RB/PH (TT) functions as the dominant negative form of Rho-kinase by the different mechanism of inhibition from those of CAT-KD, RB, and PH. Here, we examined the mode of action and the specificity of inhibitors of Rho-kinase. We found that RB/PH (TT) directly and specifically inhibited the kinase activity of catalytic fragment of Rho-kinasein vitro. The expression of RB/PH (TT) specifically blocked the stress fiber and focal adhesion formation induced by the active form of Rho or Rho-kinase in NIH 3T3 cells, but not that induced by the active form of MLC. We are grateful to A. Takemura for secretarial assistance. We also thank Dr. M. Ito (Mie University School of Medicine) for providing MLC, and Dr. M. Inagaki (Aichi Cancer Center Research Institute) for helpful discussions.