Integrin αIIbβ3-Mediated c-Src Activation: Differential Binding to Inactive and Active c-Src

Activation of the tyrosine kinase c-Src is a fundamental element of integrin αIIbβ3-mediated outside-in signaling in platelets. Presently, it is thought that c-Src is bound via its SH3 domain to the C-terminal Arg-Gly-Thr (RGT) sequence of the β3 cytoplasmic tail (CT). Although a recent NMR study supports this contention, it is likely that such binding would be precluded in the inactive conformation of c-Src because the linker connecting the SH2 and kinase domains of c-Src physically occludes the putative β3 CT binding site in the SH3 domain. Accordingly, we have re-examined the interaction between c-Src and β3 by immunoprecipitating β3 bound c-Src from resting and agonist-stimulated platelets and then applied the biophysical techniques of surface plasmon resonance (SPR) and NMR spectroscopy to directly characterize the interaction of the SH3 domain with the β3 CT. In unstimulated platelets, there was little or no association between c-Src and β3. However, following platelet stimulation with 1 unit/ml thrombin, c-Src binding to β3 is detectable within 10 sec of stimulation and binding persists for at least the next 90 sec. Phosphorylation of c-Src residue Tyr419 located in its activation loop is an indicator of c-Src activation. We detected the phosphorylation of β3-bound c-Src on Tyr419 at 20 sec following thrombin stimulation. Tyr419 phosphorylation then persisted for the next 40 sec after which it rapidly declined. Thus, platelet stimulation induces the rapid interaction of c-Src with the β3 subunit of αIIbβ3 and this interaction is accompanied by the induction of c-Src kinase activity. Since it has been reported that c-Src binds to β3 via its SH3 domain, we used SPR spectroscopy to measure the strength of this interaction. In initial experiments, we measured binding of the C-terminal β3 heptapeptide NITYRGT to the immobilized SH3 domain and detected NITYRGT binding with a dissociation constant (Kd) of approximately 700 mM. We then repeated the measurements by flowing the SH3 domain over the entire 43 residue β3 CT appended to the SPR chip. Under these conditions, we detected a Kd for SH3 domain binding to the β3 CT of 7.21 ± 2.42 µM. Previously, we reported similar behavior for talin-1 FERM domain binding to the β3 CT. Thus, these experiments suggest that like binding of the talin-1 FERM domain to the β3 CT, the interaction of c-Src with β3 is a two-dimensional ternary interaction in which the membrane surface makes an important contribution. Next, to identify the sites in the SH3 domain that interact with β3, we used NMR spectroscopy. In these experiments, we measured the interaction of the β3 peptide NITYRGT with the ¹⁵N-labeled SH3 domain. Because NITYRGT binding to the SH3 domain is weak, we used chemical shift perturbations (CSP) to identify the SH3 domain residues interacting with NITYRGT. We found that NITYRGT interacted with the SH3 domain RT-loop and surrounding residues. A control peptide whose last three residues where replaced with those of the β1 CT (NITYEGK) induced only small CSP 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 β3 peptide to the RT-loop. Under these conditions, the β3 peptide induced CSP similar to the negative control. Thus, these studies indicate that the primary interaction of c-Src with the β3 CT occurs in its activated state at a site that overlaps with the polyproline binding site in its SH3 domain and suggest that protein-membrane interactions make an important contribution to the strength of binding. By contrast, interactions of inactive c-Src with β3 are weak and insensitive to β3 CT mutations.