Threonine phosphorylation of integrin β3 in calyculin A-treated platelets is selectively sensitive to 5′-iodotubercidin

Exposure of platelets to toxins (calyculin A or okadaic acid) that inhibit protein serine/threonine phosphatases types 1 and 2A, at concentrations that block aggregatory and secretory responses, results in the phosphorylation of several platelet proteins including integrin β3. Since protein phosphorylation represents a balance between kinase and phosphatase activities, this increase in phosphorylation reflects either the removal of phosphatases that oppose constitutively active kinases known to reside in the platelet (e.g., casein kinase 2) or the activation of endogenous kinases. In this study, we demonstrate that the addition of calyculin A promotes the activation of several endogenous platelet protein kinases, including p42/44mapk, p38mapk, Akt/PKB, and LKB1. Using a pharmacologic approach, we assessed whether inhibition of these and other enzymes block phosphorylation of β3. Inhibitors of p38mapk, casein kinase, AMP kinase, protein kinase C, and calcium–calmodulin-dependent kinases did not block phosphorylation of β3 on thr753. In contrast, 5′-iodotubercidin, at 50 μM, blocks β3 phosphorylation without affecting the efficacy of calyculin A to inhibit platelet aggregation and spreading. These data dissociate threonine phosphorylation of β3 molecules and inhibition of platelet responses by protein phosphatase inhibitors.