Threonine phosphorylation of the beta 3 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 αIIbβ3, 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 tyrosyl-phosphorylated form of β3. The first alternative was tested by comparing the phosphorylation of β3 model peptides by the tyrosine kinase pp60c-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.