Outside-in signaling of β3 integrins induces and requires phosphorylation at tyrosine-747 (Y747) and tyrosine-759 (Y759) 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 tyrosine phosphorylation in the synthetic β3 cytoplasmic domain peptide inhibits calpain cleavage. In platelets, tyrosine phosphates inhibitor, sodium vanadate, enhances thrombin-induced phosphorylation at Y747 and Y759, which is associated with the reduced integrin cleavage by calpain. The effects of sodium vanadate is unlikely to be caused by its effects on calpain activity but is likely to be caused by the susceptibility of integrin cytoplasmic domain, because sodium vanadate did not affect the calpain cleavage of another substrate, fodrin, in platelets. To further support the protective effect of tyrosine phosphorylation against calpain cleavage, we show that mouse β3 (DiYF) with both Y759 and Y747 mutated to phenylalanine is more susceptible to calpain cleavage than wild type during thrombin-induced platelet aggregation. Furthermore, phosphorylation at Y747 and Y759 of β3 in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Y759 is associated with calpain cleavage at Y759. Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the β3 cytoplasmic domain.

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