Elucidation of Binding Determinants and Functional Consequences of Ras/Raf-Cysteine-rich Domain Interactions

doi:10.1074/jbc.M000397200

Elucidation of Binding Determinants and Functional Consequences of Ras/Raf-Cysteine-rich Domain Interactions*

From the ^‡Department of Biochemistry and Biophysics,^‖Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599

Abstract

Raf-1 is a critical downstream target of Ras and contains two distinct domains that bind Ras. The first Ras-binding site (RBS1) in Raf-1 has been shown to be essential for Ras-mediated translocation of Raf-1 to the plasma membrane, whereas the second site, in the Raf-1 cysteine-rich domain (Raf-CRD), has been implicated in regulating Raf kinase activity. While recognition elements that promote Ras·RBS1 complex formation have been characterized, relatively little is known about Ras/Raf-CRD interactions. In this study, we have characterized interactions important for Ras binding to the Raf-CRD. Reconciling conflicting reports, we found that these interactions are essentially independent of the guanine nucleotide bound state, but instead, are enhanced by post-translational modification of Ras. Specifically, our findings indicate that Ras farnesylation is sufficient for stable association of Ras with the Raf-CRD. Furthermore, we have also identified a Raf-CRD variant that is impaired specifically in its interactions with Ras. NMR data also suggests that residues proximal to this mutation site on the Raf-CRD form contacts with Ras. This Raf-CRD mutant impairs the ability of Ras to activate Raf kinase, thereby providing additional support that Ras interactions with the Raf-CRD are important for Ras-mediated activation of Raf-1.

Footnotes

↵* This work was supported by National Institues of Health Grants CA72644 and CA72644-10 (to G. J. C.), CA42978, CA55008, and CA67771 (to C. J. D.), and CA70308-01 and CA64569-01 (to S. L. C.).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.
↵§ Current addresses: National Council of Radiation Protection and Measurements, Suite 800, 7910 Woodmont Ave., Bethesda, MD 20814.
↵¶ Current address: Ontario Cancer Institute, Dept. of Medical Biophysics, 610 University Ave., Toronto, Ontario, M5G 2M9 Canada.
↵** Recipient of Department of Defense Career Development Award DAMD17-97-1-7050). Current address: NCI, National Institutes of Health, Dept. Cell and Cancer Biology, 9610 Medical Center Dr., Rockville, MD 20850-3300.
↵‡ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, Campus Box 7260, University of North Carolina, Chapel Hill, NC 27599-7260. Tel.: 919-966-7139; Fax: 919-966-2852; E-mail: campbesl@med.unc.edu.
Published, JBC Papers in Press, April 20, 2000, DOI 10.1074/jbc.M000397200
↵2 G. J. Clark, J. K. Drugan, S. Ghosh, K. L. Rossman, R. M. Bell, C. J. Der, and S. L. Campbell, submitted for publication.
Abbreviations:

MEK

mitogen-activated protein kinase/extracellular signal-regulated kinase kinase

MAPK

mitogen-activated protein kinase

RBS-1

Ras-binding site 1

CRD

cysteine-rich domain

ELISA

enzyme-linked immunosorbent assay

DTT

dithiothreitol

PAGE

polyacrylamide gel electrophoresis

GNP-P(NH)P

guanyl-5′-yl imidodiphosphate

ERK

extracellular signal-regulated kinase

MBP

myelin basic protein

PS

phosphatidylserine

HSQC

heteronuclear single quantum coherence
- Received January 19, 2000.
- Revision received March 22, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.