Threonine phosphorylation of the beta(3) integrin cytoplasmic tail, at a site recognized by PDK1 and Akt/PKB in vitro, regulates Shc binding

The mechanism of outside-in signaling by integrins parallels that for growt h factor receptors, In both pathways, phosphorylation of a cytoplasmic segm ent on tyrosine generates a docking site for proteins containing Src homolo gy 2 (SH2) and phosphotyrosine binding domains. We recently observed that p hosphorylation of a threonine (Thr-753), six amino acids proximal to tyrosi ne 759 in beta(3) of the platelet specific integrin alpha(IIb)beta(3) inhib its outside-in signaling through this receptor. We hypothesized that the pr esence of phosphothreonine 753 either renders beta(3) a poor substrate for tyrosine kinases or inhibits the docking capabilities of the tyrosylphospho rylated form of beta(3). The first alternative was tested by comparing the phosphorylation of beta(3) model peptides by the tyrosine kinase pp60(c-src ) and we found that the presence of a phosphate group on a residue correspo nding to Thr-753 did not detectably alter the kinetics of tyrosine phosphor ylation. However, the presence of phosphate on this threonine inhibited the binding of Shc to tyrosyl-phosphorylated beta(3) peptide. The inhibitory e ffect of the phosphate group could be mimicked by substituting an aspartic acid for Thr-753, suggesting that a negative charge at this position modula tes the binding of Shc and possibly other phosphotyrosine binding domain- a nd SH2-containing proteins. A survey of several protein kinases revealed th at Thr-753 was avidly phosphorylated by PDK1 and Akt/PKB in vitro. These ob servations suggest that activation of PDK1 and/or Akt/PHB in platelets may modulate the binding activity and/or specificity of beta(3) for signaling m olecules.