Identification and characterization of cellular proteins that interact with the SH3 domain of Src, Fyn and Lyn

The Src homology 3 (SH3) domain has been identified among members of Src family tyrosine kinases as well as in a variety of proteins otherwise unrelated to protein-tyrosine kinases. To explore possible interactions between the SH3 domain and cellular proteins, we expressed the SH3 domain of c-Src or Src$\sp+$ (a neuronal variant) as a glutathione S-transferase fusion protein and used the fusion proteins as affinity matrices to isolate proteins from mouse fibroblasts. We found that the Src SH3 domain specifically interacted with multiple cellular proteins, whereas Src$\sp+$-SH3 only weakly associated with a small subset of these proteins. The majority of the SH3 binding proteins were recovered from particulate and detergent-insoluble cell fractions, and a serine/threonine kinase activity was associated with these binding proteins. In addition, several Src SH3-binding proteins were phosphorylated in Src-transformed cells and the phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, strongly suggesting a role for the Src SH3 domain in substrates recruitment. By immunoblotting and protein microsequencing, we identified four of these Src SH3-binding proteins: paxillin (a cytoskeleton protein), Shc (a protein coupling membrane tyrosine kinases with Ras), p68 (a Src substrate during mitosis), and hnRNP K (a pre-mRNA-binding protein). Most of these proteins as well as other Src SH3-binding proteins also bound to the SH3 domains of the closely related Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Finally, we examined the importance of Asp99 (a critical residue in the ligand binding pocket of the Src SH3 domain) for determining SH3-binding specificity. We found that there appear to be two Src SH3 binding mechanisms: one requiring Asp99 which plays a dominant role in Src SH3 recognition of cellular proteins, and the other independent of this residue