Ri. Kirk et al., Threonine phosphorylation of the beta(3) integrin cytoplasmic tail, at a site recognized by PDK1 and Akt/PKB in vitro, regulates Shc binding, J BIOL CHEM, 275(40), 2000, pp. 30901-30906
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.