Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.
Subscribe

Logo for

J. Biol. Chem. 275 (6): 3737-3740

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

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 KaziroDagger

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.

* 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.

Dagger 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.

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

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
The Tumor Suppressor DiRas3 Forms a Complex with H-Ras and C-RAF Proteins and Regulates Localization, Dimerization, and Kinase Activity of C-RAF.
A. Baljuls, M. Beck, A. Oenel, A. Robubi, R. Kroschewski, M. Hekman, T. Rudel, and U. R. Rapp (2012)
J. Biol. Chem. 287, 23128-23140
   Abstract »    Full Text »    PDF »
Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging.
R. S. Kasai, K. G. N. Suzuki, E. R. Prossnitz, I. Koyama-Honda, C. Nakada, T. K. Fujiwara, and A. Kusumi (2011)
J. Cell Biol. 192, 463-480
   Abstract »    Full Text »    PDF »
The Rsr1/Bud1 GTPase Interacts with Itself and the Cdc42 GTPase during Bud-Site Selection and Polarity Establishment in Budding Yeast.
P. J. Kang, L. Beven, S. Hariharan, and H.-O. Park (2010)
Mol. Biol. Cell 21, 3007-3016
   Abstract »    Full Text »    PDF »
Amino acid regulation of TOR complex 1.
J. Avruch, X. Long, S. Ortiz-Vega, J. Rapley, A. Papageorgiou, and N. Dai (2009)
Am J Physiol Endocrinol Metab 296, E592-E602
   Abstract »    Full Text »    PDF »
Characterization of Ser338 Phosphorylation for Raf-1 Activation.
M. Zang, J. Gong, L. Luo, J. Zhou, X. Xiang, W. Huang, Q. Huang, X. Luo, M. Olbrot, Y. Peng, et al. (2008)
J. Biol. Chem. 283, 31429-31437
   Abstract »    Full Text »    PDF »
Regulation and Role of Raf-1/B-Raf Heterodimerization.
L. K. Rushworth, A. D. Hindley, E. O'Neill, and W. Kolch (2006)
Mol. Cell. Biol. 26, 2262-2272
   Abstract »    Full Text »    PDF »
Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding.
J. Zhu, V. Balan, A. Bronisz, K. Balan, H. Sun, D. T. Leicht, Z. Luo, J. Qin, J. Avruch, and G. Tzivion (2005)
Mol. Biol. Cell 16, 4733-4744
   Abstract »    Full Text »    PDF »
Single-molecule imaging analysis of Ras activation in living cells.
H. Murakoshi, R. Iino, T. Kobayashi, T. Fujiwara, C. Ohshima, A. Yoshimura, and A. Kusumi (2004)
PNAS 101, 7317-7322
   Abstract »    Full Text »    PDF »
Membrane Localization, Oligomerization, and Phosphorylation Are Required for Optimal Raf Activation.
C. A. Goetz, J. J. O'Neil, and M. A. Farrar (2003)
J. Biol. Chem. 278, 51184-51189
   Abstract »    Full Text »    PDF »
Interaction between a Ras and a Rho GTPase Couples Selection of a Growth Site to the Development of Cell Polarity in Yeast.
K. G. Kozminski, L. Beven, E. Angerman, A. H. Y. Tong, C. Boone, and H.-O. Park (2003)
Mol. Biol. Cell 14, 4958-4970
   Abstract »    Full Text »    PDF »
Alternative Splicing of the Human Proto-oncogene c-H-ras Renders a New Ras Family Protein That Trafficks to Cytoplasm and Nucleus.
S. Guil, N. de La Iglesia, J. Fernandez-Larrea, D. Cifuentes, J. C. Ferrer, J. J. Guinovart, and M. Bach-Elias (2003)
Cancer Res. 63, 5178-5187
   Abstract »    Full Text »    PDF »
Nuclear Export and Plasma Membrane Recruitment of the Ste5 Scaffold Are Coordinated with Oligomerization and Association with Signal Transduction Components.
Y. Wang and E. A. Elion (2003)
Mol. Biol. Cell 14, 2543-2558
   Abstract »    Full Text »    PDF »
Alternative Splicing Controls the Mechanisms of FAK Autophosphorylation.
M. Toutant, A. Costa, J.-M. Studler, G. Kadare, M. Carnaud, and J.-A. Girault (2002)
Mol. Cell. Biol. 22, 7731-7743
   Abstract »    Full Text »    PDF »
Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells.
J. Kato-Stankiewicz, I. Hakimi, G. Zhi, J. Zhang, I. Serebriiskii, L. Guo, H. Edamatsu, H. Koide, S. Menon, R. Eckl, et al. (2002)
PNAS 99, 14398-14403
   Abstract »    Full Text »    PDF »
Ras Participates in the Activation of p38 MAPK by Interleukin-1 by Associating with IRAK, IRAK2, TRAF6, and TAK-1.
E. P. McDermott and L. A. J. O'Neill (2002)
J. Biol. Chem. 277, 7808-7815
   Abstract »    Full Text »    PDF »
Critical Contribution of Linker Proteins to Raf Kinase Activation.
A. N. Anselmo, R. Bumeister, J. M. Thomas, and M. A. White (2002)
J. Biol. Chem. 277, 5940-5943
   Abstract »    Full Text »    PDF »
{beta}1PIX, the PAK-interacting exchange factor, requires localization via a coiled-coil region to promote microvillus-like structures and membrane ruffles.
C.-G. Koh, E. Manser, Z.-S. Zhao, C.-P. Ng, and L. Lim (2001)
J. Cell Sci. 114, 4239-4251
   Abstract »    Full Text »    PDF »
Structures of Yeast ARF2 and ARL1. DISTINCT ROLES FOR THE N TERMINUS IN THE STRUCTURE AND FUNCTION OF ARF FAMILY GTPases.
J. C. Amor, J. R. Horton, X. Zhu, Y. Wang, C. Sullards, D. Ringe, X. Cheng, and R. A. Kahn (2001)
J. Biol. Chem. 276, 42477-42484
   Abstract »    Full Text »    PDF »
Active Ras Induces Heterodimerization of cRaf and BRaf.
C. K. Weber, J. R. Slupsky, H. A. Kalmes, and U. R. Rapp (2001)
Cancer Res. 61, 3595-3598
   Abstract »    Full Text »
The Recruitment of Raf-1 to Membranes Is Mediated by Direct Interaction with Phosphatidic Acid and Is Independent of Association with Ras.
M. A. Rizzo, K. Shome, S. C. Watkins, and G. Romero (2000)
J. Biol. Chem. 275, 23911-23918
   Abstract »    Full Text »    PDF »
Membrane Localization of Raf Assists Engagement of Downstream Effectors.
M. A. Farrar, J. Tian, and R. M. Perlmutter (2000)
J. Biol. Chem. 275, 31318-31324
   Abstract »    Full Text »    PDF »
Oligomerization of Rac1 GTPase Mediated by the Carboxyl-terminal Polybasic Domain.
B. Zhang, Y. Gao, S. Y. Moon, Y. Zhang, and Y. Zheng (2001)
J. Biol. Chem. 276, 8958-8967
   Abstract »    Full Text »    PDF »

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882