Ras Interaction with Two Distinct Binding Domains in Raf-1 5 Be Required for Ras Transformation

doi:10.1074/jbc.271.1.233

Ras Interaction with Two Distinct Binding Domains in Raf-1 5 Be Required for Ras Transformation (*)

From the (1)Department of Biochemistry and Biophysics
(2)Protein Engineering and Molecular Genetics Training Program
(3)Department of Pharmacology
(4)Curriculum in Genetics and Molecular Biology, and
(5)Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599
(6)Cold Spring Harbor Laboratories, Cold Spring Harbor, New York 11724, and
(7)Department of Molecular Biology, New York State Institute for Basic Research, Staten Island, New York 10314

^¶ To whom correspondence should be addressed. Tel.: 919-966-7139; Fax: 919-966-2852.

Abstract

Although Raf-1 is a critical Ras effector target, how Ras mediates Raf-1 activation remains unresolved. Raf-1 residues 55-131 define a Ras-binding domain essential for Raf-1 activation. Therefore, our identification of a second Ras-binding site in the Raf-1 cysteine-rich domain (residues 139-184) was unexpected and suggested a more complex role for Ras in Raf-1 activation. Both Ras recognition domains preferentially associate with Ras-GTP. Therefore, mutations that impair Ras activity by perturbing regions that distinguish Ras-GDP from Ras-GTP (switch I and II) may disrupt interactions with either Raf-1-binding domain. We observed that mutations of Ras that impaired Ras transformation by perturbing its switch I (T35A and E37G) or switch II (G60A and Y64W) domain preferentially diminished binding to Raf-1-(55-131) or the Raf-1 cysteine-rich domain, respectively. Thus, these Ras-binding domains recognize distinct Ras-GTP determinants, and both may be essential for Ras transforming activity. Finally, since Ha-Ras T35A and E37G mutations prevent Ras interaction with full-length Raf-1, we suggest that Raf-Cys is a cryptic binding site that is unmasked upon Ras interaction with Raf-1-(55-131).

Footnotes

↵^§ Recipient of National Science Foundation and American Association of University Women fellowships.
↵* These studies were supported by National Institutes of Health Grants CA42978, 55008, and 63071 (to C. J. D.), CA64569 (to S. L. C.), CA53782 (to Y-W. H.), and OIG 5R35CA39829-11 (to M. H. Wigler). 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:

MAPK

mitogen-activated protein kinase

MEK

MAPK kinase

GST

glutathione S-transferase

GMPPCP

guanosine 5′-(β,-methylenetriphosphate).
↵²J. K. Drugan, R. Khosravi-Far, C. J. Der, and S. L. Campbell, manuscript in preparation.
- Received September 27, 1995.
- Revision received November 13, 1995.