Equilibrium and Kinetic Measurements Reveal Rapidly Reversible Binding of Ras to Raf

doi:10.1074/jbc.271.12.6713

Equilibrium and Kinetic Measurements Reveal Rapidly Reversible Binding of Ras to Raf (*)

From the (1)Wellcome Research Laboratories, Langley Court, South Eden Park Road, Beckenham, Kent BR3 3BS and the
(2)Cancer Research Campaign Biomolecular Structure Unit, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom

^¶ To whom correspondence should be addressed. Tel.: 181-639-6370; Fax: 181-639-6877.

Abstract

Raf is a serine/threonine kinase that binds through its amino-terminal regulatory domain to the GTP form of Ras and thereby activates the mitogen-activated protein kinase pathway. In this study, we have characterized the interaction of the Ras-binding domain of Raf with Ras using equilibrium binding methods (scintillation proximity assay and fluorescence anisotropy), rather than with more widely used nonequilibrium procedures (such as enzyme-linked immunosorbent assay and affinity precipitation). Initial studies using glutathione S-transferase fusion proteins with either residues 1-257 or 1-190 of Raf showed that although it was possible to detect Ras binding using an enzyme-linked immunosorbent assay or affinity precipitation, it was substoichiometric; under equilibrium conditions with only a small excess of Raf almost no binding was detected. This difference was probably due to the presence of a high percentage of inactive Raf protein. Further studies used protein containing residues 51-131 of Raf, which expressed in Escherichia coli as a stable glutathione S-transferase fusion. With this protein, binding with Ras could readily be measured under equilibrium conditions. The catalytic domain of neurofibromin inhibited binding of Ras to Raf, and Raf inhibited the binding of Ras to neurofibromin showing that Raf and neurofibromin cannot be bound simultaneously to Ras. The affinities of interaction of neurofibromin and Raf with Harvey-Ras were similar. The rate constant for dissociation of Raf from Ras was estimated to be >1 min, suggesting that Ras, Raf, and neurofibromin may be in rapid equilibrium in the cell. In contrast to previous reports, under equilibrium conditions there was no evidence for a difference in affinity between the minimal Ras binding domain of Raf (residues 51-131) and a region containing an additional 16 carboxyl-terminal amino acids, suggesting that residues 132-147 do not form a critical binding determinant.

Footnotes

↵^§ Supported by a grant from The Wellcome Foundation Ltd. (to J. V. S., and S. N.).
↵* This work was supported in part by a CRC program grant (to S. N.). 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:

GAP

GTPase activating protein

mant-GTP

2′(3′)-O-(N-methylanthraniloyl) derivative of GTP

GST

glutathione S-transferase from Schistosoma japonicum

SPA

scintillation proximity assay

ELISA

enzyme-linked immunosorbent assay; Raf-(x-y) denotes the protein product of the c-raf-1 gene truncated to express residues from x to y; Ha-Ras and K-Ras refer to the protein products of the Harvey- and Kirsten-ras genes; NF1-334 denotes the Ras GTPase-activating domain of neurofibromin

GTPS

guanosine 5′-3-O- (thio)triphosphate.
↵² C. Gorman, R. H. Skinner, M. J. Page, and P. N. Lowe, unpublished results.
- Received October 23, 1995.
- Revision received January 10, 1996.