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J Biol Chem, Vol. 275, Issue 6, 3737-3740, February 11, 2000

ACCELERATED PUBLICATION
Formation of the Ras Dimer Is Essential for Raf-1 Activation^*

Kaoru Inouye, Shin Mizutani, Hiroshi Koide, and Yoshito Kaziro

From the Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

Although it is well established that Ras requires membrane localization for activation of its target molecule, Raf-1, the reason for this requirement is not fully understood. In this study, we found that modified Ras, which is purified from Sf9 cells, could activate Raf-1 in a cell-free system, when incorporated into liposome. Using a bifunctional cross-linker and a protein-fragmentation complementation assay, we detected dimer formation of Ras in the liposome and in the intact cells, respectively. These results suggest that dimerization of Ras in the lipid membrane is essential for activation of Raf-1. To support this, we found that, when fused to glutathione S-transferase (GST), unprocessed Ras expressed in Escherichia coli could bypass the requirement for liposome. A Ras-dependent Raf-1 activator, which we previously reported (Mizutani, S., Koide, H., and Kaziro, Y. (1998) Oncogene 16, 2781-2786), was still required for Raf-1 activation by GST-Ras. Furthermore, an enforced dimerization of unmodified oncogenic Ras mutant in human embryonic kidney (HEK) 293 cells, using a portion of gyrase B or estrogen receptor, also resulted in activation of Raf-1. From these results, we conclude that membrane localization allows Ras to form a dimer, which is essential, although not sufficient, for Raf-1 activation.

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^* This work was supported by Grants-in-aid for Scientific Research on Priority Areas 10680666 and 11160204 from the Ministry of Education, Science, Sports, and Culture of Japan and by Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corp. (JST). Our laboratory at Tokyo Institute of Technology is supported by funds donated by Schering-Plough Corp.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: Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Tel.: 81-45-924-5745; Fax: 81-45-924-5822; E-mail: ykaziro@bio.titech.ac.jp.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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