The COOH Terminus of Rho-kinase Negatively Regulates Rho-kinase Activity

doi:10.1074/jbc.274.45.32418

The COOH Terminus of Rho-kinase Negatively Regulates Rho-kinase Activity*

From the Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101 and the^‡Department of Virology II, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan

Abstract

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.

Footnotes

↵* This work was supported by grants-in-aid for scientific research and cancer research from the Ministry of Education, Science, and Culture of Japan, by the Research for the Future of Japan Society for the Promotion of Science, and by a grant from Kirin Brewery Company Limited.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.
↵§ To whom correspondence should be addressed: Div. of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan. Tel.: 81-743-72-5440; Fax: 81-743-72-5449; E-mail: kaibuchi@bs.aist-nara.ac.jp.
↵2 N. Nakamura, M. Fukata, Y. Fukata, N. Oshiro, M. Amano, S. Kuroda, T. Yano, M. Shibata, M. Ikebe, Y. Matsuura, K. Ookawa, A. Iwamatsu, and K. Kaibuchi, manuscript in preparation.
↵3 M. Amano, K. Chihara, N. Nakamura, T. Kaneko, Y. Matsuura, and K. Kaibuchi, unpublished data.
Abbreviations:

PKN

protein kinase N

MLC

myosin light chain

PH

pleckstrin homology

CAT

catalytic domain

RB

Rho-binding domain

CAT-KD

kinase-deficient catalytic domain

GST

glutathione S-transferase

MRCKβ

myotonic dystrophy kinase-related Cdc42-binding kinase β

MBP

maltose-binding protein

PAGE

polyacrylamide gel electophoresis

DMEM

Dulbecco's modified Eagle's medium

PBS

phosphate-buffered saline
- Received April 7, 1999.
- Revision received June 28, 1999.
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