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J. Biol. Chem., Vol. 279, Issue 43, 44756-44762, October 22, 2004

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Cell Type-specific Regulation of RhoA Activity during Cytokinesis^*

Hisayoshi Yoshizaki, Yusuke Ohba, Maria-Carla Parrini¶, Natalya G. Dulyaninova||, Anne R. Bresnick||, Naoki Mochizuki, and Michiyuki Matsuda**

From the Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita-shi, Osaka 565-0871, the Department of Structural Analysis, National Cardiovascular Center Research Institute, Fujishirodai, Suita-shi, Osaka 565-8565, Japan, and the ^||Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461

Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), we have shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. Here we show that in Rat1A cells RhoA activity remained suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibited cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibited cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 was suppressed during cytokinesis and started increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression was shown to be essential for cytokinesis because a constitutively active mutant of Rac1 induced a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was shown by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delayed the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells.

Received for publication, March 1, 2004 , and in revised form, August 2, 2004.

^* This work was supported in part by a grant for scientific research on priority areas (special) from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant for comprehensive research on aging and health from the Ministry of Health, Labor, and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a figure.

^¶ Fellow supported by the Japan Society for the Promotion of Science.

^** To whom correspondence should be addressed: Dept. of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita-shi, Osaka 565-0871, Japan. Tel.: 81-6-6879-8316; Fax: 81-6-6879-8314; E-mail: matsudam{at}biken.osaka-u.ac.jp.

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