Cysteine-rich Region of Raf-1 Interacts with Activator Domain of Post-translationally Modified Ha-Ras

doi:10.1074/jbc.270.51.30274

Cysteine-rich Region of Raf-1 Interacts with Activator Domain of Post-translationally Modified Ha-Ras (*)

From the (1)Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, the
(2)Department of Biophysics and Biochemistry, School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, and the
(3)Cellular Signaling Laboratory, the Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan

^§ To whom correspondence should be addressed. Tel.: 81-78-341-7451 (ext. 3230); Fax: 81-78-341-3837.

Abstract

The interaction between “switch I/effector domain” of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction. However, the importance of the “activator domain” (approximately corresponding to amino acids 26-28 and 40-49) of Ha-Ras and of the “cysteine-rich region” (CRR, amino acids 152-184) of Raf-1 have also been proposed. Here, we found that Raf-1 CRR interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding. Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally. On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected. Mutants that bound to either RBD or CRR alone failed to activate Raf-1. Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR. These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction.

Footnotes

↵* This investigation was supported by grants-in-aid for cancer research and for scientific research from the Ministry of Education, Science, and Culture of Japan, and by research grants from the Senri Bioscience Foundation, the Uehara Memorial Foundation, and the Yamanouchi Foundation for Research on Metabolic Disease. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

CR

conserved region

RBD

Ras-binding domain

CRR

cysteine-rich region

MBP

maltose-binding protein

GTPS

guanosine 5′-O-(3-thiotriphosphate)

GST

glutathione S-transferase

MEK

mitogen-activated protein kinase kinase/ERK kinase

ERK

extracellular signal-regulated kinase

KNERK

kinase-negative mutant of ERK2.
↵²T. Okada, T. Masuda, M. Shinkai, K. Kariya, and T. Kataoka, submitted for publication.
- Received September 20, 1995.
- Revision received October 25, 1995.