Phosphorylation and Activation of Myosin by Rho-associated Kinase (Rho-kinase)

doi:10.1074/jbc.271.34.20246

Phosphorylation and Activation of Myosin by Rho-associated Kinase (Rho-kinase)*

From the Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-01, the
^‡ First Department of Internal Medicine, Mie University School of Medicine, Tsu 514, the
^§ Second Department of Anatomy, Kyoto University Faculty of Medicine, Kyoto 606, and the
^¶ Department of Virology II, National Institute of Health, Tokyo 162, Japan

^∥ To whom correspondence should be addressed. Tel.: 81-7437-2-5440; Fax: 81-7437-2-5449; E-mail: kaibuchi{at}bs.aist-nara.ac.jp.

Abstract

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.

Footnotes

↵* This study was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, and Culture, Japan (1995) and by Mitsubishi Foundation (1995). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact
¹ The abbreviations used are:

MBS

myosin-binding subunit

MLC

myosin light chain

GTPγS

guanosine 5′-(3-O-thio)-triphosphate

GST

glutathione S-transferase

DTT

dithiothreitol

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

PAGE

polyacrylamide gel electrophoresis.
↵² A detailed analysis concerning recombinant Rho-kinase will be described elsewhere.
- Received May 15, 1996.
- Revision received June 19, 1996.