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Originally published In Press as doi:10.1074/jbc.M009329200 on March 1, 2001

J. Biol. Chem., Vol. 276, Issue 21, 17653-17662, May 25, 2001
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The v-Src SH3 Domain Facilitates a Cell Adhesion-independent Association with Focal Adhesion Kinase*

Christof R. HauckDagger §, Tony Hunter||, and David D. SchlaepferDagger **

From the Dagger  Department of Immunology, The Scripps Research Institute and  The Salk Institute, La Jolla, California 92037

Integrins facilitate cell attachment to the extracellular matrix, and these interactions generate cell survival, proliferation, and motility signals. Integrin signals are relayed in part by focal adhesion kinase (FAK) activation and the formation of a transient signaling complex initiated by Src homology 2 (SH2)-dependent binding of Src family protein-tyrosine kinases to the FAK Tyr-397 autophosphorylation site. Here we show that in viral Src (v-Src)-transformed NIH3T3 fibroblasts, an adhesion-independent FAK-Src signaling complex occurs. Co-expression studies in human 293T cells showed that v-Src could associate with and phosphorylate a Phe-397 FAK mutant at Tyr-925 promoting Grb2 binding to FAK in suspended cells. In vitro, glutathione S-transferase fusion proteins of the v-Src SH3 but not c-Src SH3 domain bound to FAK in lysates of NIH3T3 fibroblasts. The v-Src SH3-binding sites were mapped to known proline-X-X-proline (PXXP) SH3-binding motifs in the FAK N- (residues 371-377) and C-terminal domains (residues 712-718 and 871-882) by in vitro pull-down assays, and these sites are composed of a PXXPXXPhi (where Phi  is a hydrophobic residue) v-Src SH3 binding consensus. Sequence comparisons show that residues in the RT loop region of the c-Src and v-Src SH3 domains differ. Substitution of c-Src RT loop residues (Arg-97 and Thr-98) for those found in the v-Src SH3 domain (Trp-97 and Ile-98) enhanced the binding of distinct NIH3T3 cellular proteins to a glutathione S-transferase fusion protein of the c-Src (Trp-97 + Ile-98) SH3 domain. FAK was identified as a c-Src (Trp-97 + Ile-98) SH3 domain target in fibroblasts, and co-expression studies in 293T cells showed that full-length c-Src (Trp-97 + Ile-98) could associate in vivo with Phe-397 FAK in an SH2-independent manner. These studies establish a functional role for the v-Src SH3 domain in stabilizing an adhesion-independent signaling complex with FAK.

* This work was supported in part by American Cancer Society Grant RPG-98-109-01-TBE and in part by NCI Grants R29-CA75240 (to D. D. S.) and CA39780 (to T. H.) from the National Institutes of Health. This is manuscript number 13622-IMM from The Scripps Research Institute.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.

§ Supported in part by the Deutsche Forschungsgemeinschaft Grant HA-2856/1-1.

|| Frank and Else Schilling American Cancer Society Research Professor.

** To whom correspondence and requests for materials should be addressed: The Scripps Research Institute, Dept. of Immunology, IMM26, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8207; Fax: 858-784-8227; E-mail: dschlaep@scripps.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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