PHOSPHORYLATION AND ACTIVATION OF MYOSIN BY RHO-ASSOCIATED KINASE (RHO-KINASE)

The small GTPase Rho is implicated in physiological functions associat ed with actin-myosin filaments such as cytokinesis, cell motility, and smooth muscle contraction. We have recently identified and molecularl y 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, H., Iwamatsu, A., and Kai buchi, K. (1996) EMBO J. 15, 2208-2216). Here we found that Rho-kinase stoichiometrically phosphorylated myosin light chain (MLC). Peptide m apping and phosphoamino acid analyses revealed that the primary phosph orylation site of MLC by Rho-kinase was Ser-19, which is the site phos phorylated by MLC kinase. Rho-kinase phosphorylated recombinant MLC, w hereas it failed to phosphorylate recombinant MLC, which contained Ala substituted for both Thr-18 and Ser-19. We also found that the phosph orylation of MLC by Rho-kinase resulted in the facilitation of the act in activation of myosin ATPase. Thus, it is likely that once Rho is ac tivated, then it can interact with Rho-kinase and activate it. The act ivated Rho-kinase subsequently phosphorylates MLC. This may partly acc ount for the mechanism by which Rho regulates cytokinesis, cell motili ty, or smooth muscle contraction.