Threonine phosphorylation of the β3 integrin cytoplasmic tail, at a site recognized by PDK1 and Akt/PKB in vitro, regulates Shc binding

Abstract

The mechanism of outside-in signaling by integrins parallels that for growth factor receptors. In both pathways, phosphorylation of a cytoplasmic segment on tyrosine generates a docking site for proteins containing Src homology 2 (SH2) and phosphotyrosine binding domains. We recently observed that phosphorylation of a threonine (Thr-753), six amino acids proximal to tyrosine 759 in β3 of the platelet specific integrin α(IIb)β3, inhibits outside-in signaling through this receptor. We hypothesized that the presence of phosphothreonine 753 either renders β3 a poor substrate for tyrosine kinases or inhibits the docking capabilities of the tyrosylphosphorylated form of β3. The first alternative was tested by comparing the phosphorylation of β3 model peptides by the tyrosine kinase pp60(c-src) and we found that the presence of a phosphate group on a residue corresponding to Thr-753 did not detectably alter the kinetics of tyrosine phosphorylation. However, the presence of phosphate on this threonine inhibited the binding of Shc to tyrosyl-phosphorylated β3 peptide. The inhibitory effect of the phosphate group could be mimicked by substituting an aspartic acid for Thr-753, suggesting that a negative charge at this position modulates the binding of Shc and possibly other phosphotyrosine binding domain- and SH2-containing proteins. A survey of several protein kinases revealed that Thr-753 was avidly phosphorylated by PDK1 and Akt/PKB in vitro. These observations suggest that activation of PDK1 and/or Akt/PKB in platelets may modulate the binding activity and/or specificity of β3 for signaling molecules.