SH2- and SH3-mediated Interactions between Focal Adhesion Kinase and Src

Abstract

Intramolecular SH2 and SH3 interactions mediate enzymatic repression of the Src kinases. One mechanism of activation is disruption of these interactions by the formation of higher affinity SH2 and SH3 interactions with specific ligands. We show that a consensus Src SH3-binding site residing upstream of the Src SH2-binding site in FAK can function as a ligand for the Src SH3 domain. Surface plasmon resonance experiments indicate that a FAK peptide containing both the Src SH2-and SH3-binding sites exhibits increased affinity for Src. Furthermore, the presence of both sites in vitro more potently activates c-Src. A FAK mutant (FAK Pro-2 ) with substitutions destroying the SH3-binding site shows reduced binding to Src in vivo. This mutation also reduces Src-dependent tyrosine phosphorylation on the mutant itself and downstream substrates, such as paxillin. These observations suggest that an SH3-mediated interaction between Src-like kinases and FAK may be important for complex formation and downstream signaling in vivo.The x-ray crystal structures of Src and Hck (a Src family member) in their inactive form have revealed intramolecular interactions that function to regulate these protein-tyrosine kinases (PTKs) 1 (1, 2). As expected, the tyrosine phosphorylated, negative regulatory element binds to the SH2 domain (1, 2). In addition, the SH3 domain binds to a polypeptide linking the SH2 and catalytic domains which assumes a polyproline type II helix that is structurally similar to SH3-binding sites (3)(4)(5). In this conformation, ␣-helix C in the small lobe of the catalytic domain is displaced altering the conformation of the ATP-binding site. It is noteworthy that the sequences that bind the SH2 and SH3 domains do not conform to high affinity binding sites (3, 6 -8).Dephosphorylation of the negative regulatory element is one mechanism by which these PTKs can be activated. Consequently, this element fails to bind the SH2 domain and the inactive conformation cannot be maintained. A second mechanism by which the Src-like kinases could be activated is by the disruption of the weaker intramolecular SH2-SH3 interactions by the formation of stronger intermolecular SH2-SH3 domain interactions. It has been shown that disruption of the SH2-or SH3-mediated intramolecular interactions in vitro enhances the activity of the enzyme (9 -12). It is likely that similar mechanisms operate in vivo and that complex formation between Src and its binding partners, like FAK, results in its activation. FAK is a 125-kDa PTK that localizes to focal adhesions and functions in integrin signaling (13,14). Integrin-dependent cell adhesion or cross-linking of cell surface integrins induces the tyrosine phosphorylation of FAK and stimulates its activity (14 -18). The major site of FAK autophosphorylation is Tyr-397, whereas other sites of FAK phosphorylation, e.g. Tyr-576, -577, and -925, are phosphorylated by Src family PTKs (19 -21). The sequence flanking Tyr-397 conforms to a high affinity binding site for the Src SH2 domain and serves as a bind...

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