The phosphorylation and localization of the membrane-linking protein moesin was analyzed during early activation of platelets with thrombin. Activated platelets elaborate filopodia and spread to assume flat pancake-like shapes, and moesin is localized in filopodia and cell body. In resting platelets, approximately 25% of moesin molecules are phosphorylated as shown by metabolic labeling with 32 P i and by isoelectric focusing. Within seconds after exposure to thrombin, phosphorylation increases, reaching a maximum of 35% labeled molecules by 1 min, followed by a decrease to a new basal level within 5 min. This modification affects a single residue, Thr
558, which is located within or close to a binding site for F-actin. Rapid shifts (0 -100%) in the number of phosphorylated molecules are observed in the presence of inhibitors of serine/threonine kinases and phosphatases. Inhibitors affecting tyrosine phosphorylation also modulate phosphorylation at this site suggesting that the enzymes involved in the modification of Thr 558 are regulated by tyrosine phosphorylation. Platelets respond to both extremes of modification by forming extremely long filopodia and the absence of spreading on glass. Completely phosphorylated moesin is concentrated together with F-actin in the center of the cell. The rapid modification of moesin at or near its actin-binding domain suggests a model for regulated membrane-cytoskeleton interaction during cell activation.Platelets are anucleate, membrane-bound particles that participate in the process of hemostasis and thrombosis. They can be activated by a wide variety of stimuli and predictably respond to such stimuli by changing shape, by releasing the content of storage granules, by adhesion, and by aggregation (1, 2). These phenomena require drastic changes in the organization of the cytoskeleton and thus make platelets a powerful model system for the study of relationships between activating stimuli and cytoskeletal organization. The signal transduction events that mediate platelet cytoskeletal rearrangements have proven to be largely mediated through phosphorylation and dephosphorylation of cytoskeleton-associated and membrane proteins (3, 4). Such modifications result in changes in proteinprotein interactions and/or subcellular distribution and allow for the formation of new protein complexes between membrane components and the cytoskeleton. Following activation by physiological stimuli, for example, phosphorylation of myosin light chain, actin-binding protein, P-selectin, glycoprotein IIIa, glycoprotein Ib, talin, vinculin, cofilin, pp60 c-src , and focal adhesion kinase (pp125
FAK) is induced and regulates some of the molecular changes involved in platelet aggregation and release (5-22).Moesin is a member of the protein 4.1 family (23, 24) and shares considerable structural homology with ezrin and radixin (25-30). These proteins are often co-expressed in a great variety of cell types, where they are localized at the cytoplasmic face of the membrane in filopodia and other microextens...