Accumulation of GTP-bound RhoA during Cytokinesis and a Critical Role of ECT2 in This Accumulation*
- From the ‡Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606-8501, Japan and the¶Molecular Tumor Biology Section, Basic Research Laboratory, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255
Abstract
We developed a new pull-down assay for GTP-Rho and examined its level during cell cycle. HeLa cells were arrested in the S phase by thymidine and were enriched in the prometaphase, metaphase, telophase, and G1 phase by collecting at 0, 45, 90, and 180 min after the release from the nocodazole arrest, respectively. The level of GTP-Rho did not change significantly from the S phase to the prometaphase, but increased thereafter, peaking in the telophase, and returned to the original level in the G1phase. The GDP-GTP exchange activity for Rho measured in cell lysates in parallel increased also during the mitosis with a peak in the metaphase. Using this system, we examined a role of ECT2, an exchanger for Rho GTPases, suggested to be involved in cytokinesis (Tatsumoto, T., Xie, X., Blumenthal, R., Okamoto, I., and Miki., T. (1999)J. Cell. Biol., 147, 921–928). Expression of the dominant negative form of ECT2 completely suppressed both the rise of GTP-Rho in the telophase and the increased GDP-GTP exchange activity in the mitotic cell extracts. These results suggest a critical role of ECT2 in Rho activation during cytokinesis.
Footnotes
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↵* This work was supported in part by a grant-in-aid for Specially Promoted Research from the Ministry of Education, Science, Culture and Sports of Japan and grants from the Organization for Pharmaceutical Safety and Research and the Human Frontier Science Program.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.
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↵§ Recipient of the fellowship from the Organization for Pharmaceutical Safety and Research.
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↵‖ To whom correspondence should be addressed: Dept. of Pharmacology, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan. Tel.: 81-75-753-4392; Fax: 81-75-753-4693; E-mail: snaru@mfour.med.kyoto-u.ac.jp.
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Published, JBC Papers in Press, April 12, 2000, DOI 10.1074/jbc.C000212200
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↵1 The abbreviations used are; GEF, guanine nucleotide exchange factor; aa, amino acids; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; DAPI, 4′,6-diamidino-2-phenylindole; RBD, Rho-binding domain; GST, glutathione S-transferase; CHAPS, 3-[3-cholamidopropyl)dimethylammonio[-1-propanesulfonic acid.
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- Received March 29, 2000.
- Revision received April 11, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
