It is currently thought that inactive tyrosine kinase c-Src in platelets binds to the cytoplasmic tail of the b3 integrin subunit via its SH3 domain.  Although a recent NMR study supports this contention, it is likely that such binding would be precluded in inactive c-Src because an auto-inhibitory linker physically occludes the b3 tail binding site.  Accordingly, we have re-examined c-Src binding to b3 by immunoprecipitation as well as NMR spectroscopy.  In unstimulated platelets, we detected little to no interaction between c-Src and b3.  Following platelet activation, however, c-Src was co-immunopreciptated with b3 in a time-dependent manner and underwent progressive activation as well.  We then measured chemical shift perturbations in the 15N-labeled SH3 domain induced by the C-terminal b3 tail peptide NITYRGT and found that the peptide interacted with the SH3 domain RT loop and surrounding residues.  A control peptide whose last three residues where replaced with those of the b1 cytoplasmic tail induced only small chemical shift perturbations on the opposite face of the SH3 domain.  Next, to mimic inactive c-Src, we found that the canonical polyproline peptide RPLPPLP prevented binding of the b3 peptide to the RT loop. Under these conditions, the b3 peptide induced chemical shift perturbations similar to the negative control.  We conclude that the primary interaction of c-Src  with the b3 tail occurs in its activated state and at a site that overlaps with PPII binding site in its SH3 domain. Interactions of inactive c-Src with b3 are weak and insensitive to b3 tail mutations.

Integrin aIIbb3-mediated c-Src activation: Differential b3 binding to inactive and active c-Src

248th National Meeting (2014)

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