Phosphorylation sites in the integrin beta(3) cytoplasmic domain in intactplatelets

Protein seryl/threonyl phosphatase inhibitors such as calyculin A block ins ide-out and outside-in platelet signaling. Our studies demonstrate that the addition of calyculin A blocks platelet adhesion and spreading on fibrinog en, responses that depend on integrin alpha(IIb)beta(3) signaling. We hypot hesized that this reflects a change in alpha(IIb)beta(3) structure caused b y a specific state of phosphorylation. We show that addition of calyculin A leads to increased phosphorylation of the beta(3) subunit, and phosphoamin o acid analysis reveals that only threonine residues become phosphorylated; sequence analysis by Edman degradation established that threonine 753 beca me stoichiometrically phosphorylated during inhibition of platelet phosphat ases by calyculin A. This region of beta(3) is linked to outside-in signali ng such as platelet spreading responses. The effect of calyculin A on plate let adhesion and spreading and on the phosphorylation of T-753 in beta(3) i s reversed by the calcium ionophore A23187, demonstrating that these effect s of calyculin A are not generally toxic ones. We propose that phosphorylat ion of beta(3) on threonine 753, a region of beta(3) linked to outside-in s ignaling, may be a mechanism by which integrin alpha(IIb)beta(3) function i s regulated.