Threonine phosphorylation of integrin β 3 in calyculin A-treated platelets is selectively sensitive to 5′-iodotubercidin

Abstract

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 β 3. 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 p42/44 mapk, p38 mapk, Akt/PKB, and LKB1. Using a pharmacologic approach, we assessed whether inhibition of these and other enzymes block phosphorylation of β 3. Inhibitors of p38 mapk, casein kinase, AMP kinase, protein kinase C, and calcium–calmodulin-dependent kinases did not block phosphorylation of β 3 on thr 753. In contrast, 5′-iodotubercidin, at 50 μM, blocks β 3 phosphorylation without affecting the efficacy of calyculin A to inhibit platelet aggregation and spreading. These data dissociate threonine phosphorylation of β 3 molecules and inhibition of platelet responses by protein phosphatase inhibitors.