Diversity in the control of individual Src‐family kinases by their SH3 domains

Src‐family kinases (SFKs) are non‐receptor protein‐tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix in the SH2‐kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C‐terminal tail. However, growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the activation dynamics of individual SFKs, we used a synthetic SFK SH3 domain‐binding peptide (VSL12) to probe the sensitivity of recombinant, downregulated SFKs to SH3‐based activation in an in vitro kinase assay. We found that Hck and Lyn are much more readily activated by VSL12 than c‐Src and Fyn. Surface plasmon resonance (SPR) studies confirmed VSL12 binding to the isolated recombinant SH3 domains of each of these SFKs. Our results suggest that the differences in SH3‐based activation sensitivity observed in vitro may confer distinct activation thresholds for individual SFKs in vivo despite the similarity of their downregulated conformations. (Supported by grants T32 AI065380 and R21 AI077444 from the National Institute of Allergy and Infectious Diseases.)