The Tyrosine Kinase c-Src Specifically Binds to the Active Integrin ?IIb?3 to Initiate Outside-in Signaling in Platelets.

ABSTRACT: It is currently believed that inactive tyrosine kinase c-Src in platelets binds to the cytoplasmic tail of the ?3 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 ?3 tail binding site. Accordingly, we have re-examined c-Src binding to ?3 by immunoprecipitation as well as NMR spectroscopy. In unstimulated platelets, we detected little to no interaction between c-Src and ?3. Following platelet activation, however, c-Src was co-immunoprecipitated with ?3 in a time-dependent manner and underwent progressive activation as well. We then measured chemical shift perturbations in the (15)N-labeled SH3 domain induced by the C-terminal ?3 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 ?1 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 ?3 peptide to the RT- loop. Under these conditions, the ?3 peptide induced chemical shift perturbations similar to the negative control. We conclude that the primary interaction of c-Src with the ?3 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 ?3 are weak and insensitive to ?3 tail mutations.

SUBMITTER: Wu Y 

PROVIDER: S-EPMC4505490 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature