Two SH2 Domains of p120 Ras GTPase-activating Protein Bind Synergistically to Tyrosine Phosphorylated p190 Rho GTPase-activating Protein

doi:10.1074/jbc.270.30.17947

Two SH2 Domains of p120 Ras GTPase-activating Protein Bind Synergistically to Tyrosine Phosphorylated p190 Rho GTPase-activating Protein (*)

From the (1) Cellular and Molecular Biology Program, the
(2) Department of Microbiology and Immunology, and the
(3) Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan 48109, the
(4) Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68198,
(5) Onyx Pharmaceuticals, Richmond, California 94806, and the
(6) Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

^¶ To whom correspondence should be addressed. Dept. of Microbiology and Immunology, 6606 Medical Science II, University of Michigan Medical School, Ann Arbor, MI 48109-0620.

Abstract

p120 GTPase-activating protein (GAP) is a negative regulator of Ras that functions at a key relay point in signal transduction pathways that control cell proliferation. Among other proteins, p120 GAP associates with p190, a GAP for the Ras-related protein, Rho. To characterize the p120•p190 interaction further, we used bacterially expressed glutathione S-transferase fusion polypeptides to map the regions of p120 necessary for its interactions with p190. Our results show that both the N-terminal and the C-terminal SH2 domains of p120 are individually capable of binding p190 expressed in a baculovirus/insect cell system. Moreover, the two SH2 domains together on one polypeptide bind synergistically to p190, and this interaction is dependent on tyrosine phosphorylation of p190. In addition, mutation of the highly conserved Arg residues in the critical FLVR sequences of both SH2 domains of full-length p120 reduces binding to tyrosine-phosphorylated p190. The dependence on p190 phosphorylation for complex formation with p120 SH2 domains observed in vitro is consistent with analysis of the native p120•p190 complexes formed in vivo. These findings suggest that SH2-phosphotyrosine interaction is one mechanism by which the cell regulates p120•p190 association and thus may be a means for coordinating the Ras- and Rho-mediated signaling pathways.

Footnotes

↵^§ Fellow of the University of Michigan Cancer Biology Training Program.
↵* This was supported in part by Grants CA58667 (to T. E. S.), CA39438 (to S. J. P.), and CA55652 (to R. J.) from the National Institutes of Health. 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

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis.
↵²R. Roof, J.-H. Chang, and S. J. Parsons, manuscript submitted.
↵³S. Bryant and R. Jove, unpublished data.
↵⁴G. Martin and F. McCormick, unpublished data.
- Received December 21, 1994.
- Revision received April 3, 1995.