GC-GAP, a Rho Family GTPase-activating Protein That Interacts with Signaling Adapters Gab1 and Gab2

doi:10.1074/jbc.M304594200

GC-GAP, a Rho Family GTPase-activating Protein That Interacts with Signaling Adapters Gab1 and Gab2*

^‡Burnham Institute, La Jolla, California 92037 and the ^§Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202

∥ To whom the correspondence should be addressed: The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-713-6265; Fax: 858-713-6274; E-mail: gfeng{at}burnham.org.

Abstract

Gab1 and Gab2 are scaffolding proteins acting downstream of cell surface receptors and interact with a variety of cytoplasmic signaling proteins such as Grb2, Shp-2, phosphatidylinositol 3-kinase, Shc, and Crk. To identify new binding partners for GAB proteins and better understand their functions, we performed a yeast two-hybrid screening with hGab2-(120–587) as bait. This work led to identification of a novel GTPase-activating protein (GAP) for Rho family GTPases. The GAP domain shows high similarity to the recently cloned CdGAP and displays activity toward RhoA, Rac1, and Cdc42 in vitro. The protein was named GC-GAP for its ability to interact with GAB proteins and its activity toward Rac and Cdc42. GC-GAP is predominantly expressed in the brain with low levels detected in other tissues. Antibodies directed against GC-GAP recognized a protein of ∼200 kDa. Expression of GC-GAP in 293T cells led to a reduction in active Rac1 and Cdc42 levels but not RhoA. Suppression of GC-GAP expression by siRNA inhibited proliferation of C6 astroglioma cells. In addition, GC-GAP contains several classic proline-rich motifs, and it interacts with the first SH3 domain of Crk and full-length Nck in vitro. We propose that Gab1 and Gab2 in cooperation with other adapter molecules might regulate the cellular localization of GC-GAP under specific stimuli, acting to regulate precisely Rac and Cdc42 activities. Given that GC-GAP is specifically expressed in the nervous system and that it is localized to the dendritic processes of cultured neurons, GC-GAP may play a role in dendritic morphogenesis and also possibly in neural/glial cell proliferation.

Footnotes

↵1 The abbreviations used are: Gab, Grb2-associated binder; CAS, Crk-associated substrate; CSW, Corkscrew; DOS, Daughter of Sevenless; GAP, GTPase-activating protein; IRS, insulin receptor substrate; Erk, extracellular signal-regulated kinase; MDCK, Madin-Darby canine kidney; MAP, mitogen-activated protein; PH, plekstrin homology; PI, phosphatidylinositol; PLCγ, phospholipase Cγ; RNAi, RNA interference; SH, Src homology; Shp-2, SH2 domain containing protein-tyrosine phosphatase-2; SFV, Semliki Forest virus; RBD, Rho binding domain; oligo, oligonucleotide; HA, hemagglutinin; GST, glutathione S-transferase; RACE, rapid amplification of cDNA ends; DAPI, 4,6-diamidino-2-phenylindole; GEFs, guanine nucleotide exchange factors; BrdUrd, 5-bromo-2′-deoxyuridine; EGF, epidermal growth factor; GFP, green fluorescent protein.
↵2 C. Zhao and G. S. Feng, unpublished data.
↵* This work was supported by National Institutes of Health Grants R01HL66208 and R01GM53660 (to G. S. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ Present address: The Salk Institute, 10010 N. Torrey Pines Rd., La Jolla, CA 92037.
- Received May 2, 2003.
- Revision received June 15, 2003.
The American Society for Biochemistry and Molecular Biology, Inc.