Tyrosine phosphorylation of the integrin β₃ subunit regulates β₃ cleavage by calpain

Outside-in signaling of β₃ integrins induces and requires phosphorylation at tyrosine 747 (Tyr⁷⁴⁷) and tyrosine 759 (Tyr ⁷⁵⁹) of the β₃ subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of β₃ cytoplasmic domain. Here we show that β₃ tyrosine phosphorylation inhibits calpain cleavage. Mutating both tyrosines to phenylalanine sensitizes β₃ to calpain cleavage. Furthermore, phosphorylation at Tyr⁷⁴⁷ and Tyr⁷⁵⁹ of β₃ in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Tyr⁷⁵⁹ is associated with calpain cleavage at Tyr⁷⁵⁹. Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the β₃ cytoplasmic domain.