PHOSPHORYLATION OF THREONINE-558 IN THE CARBOXYL-TERMINAL ACTIN-BINDING DOMAIN OF MOESIN BY THROMBIN ACTIVATION OF HUMAN PLATELETS

The phosphorylation and localization of the membrane-linking protein m oesin was analyzed during early activation of platelets with thrombin, Activated platelets elaborate filopodia and spread to assume flat pan cake-like shapes, and moesin is localized in filopodia and cell body, In resting platelets, approximately 25% of moesin molecules are phosph orylated as shown by metabolic labeling with P-32(i) and by isoelectri c focusing, Within seconds after exposure to thrombin, phosphorylation increases, reaching a maximum of 35% labeled molecules by 1 min, foll owed by a decrease to a new basal level within 5 min. This modificatio n 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 o f serine/threonine kinases and phosphatases. Inhibitors affecting tyro sine phosphorylation also modulate phosphorylation at this site sugges ting that the enzymes involved in the modification of Thr(558) are reg ulated by tyrosine phosphorylation. Platelets respond to both extremes of modification by forming extremely long filopodia and the absence o f spreading on glass. Completely phosphorylated moesin is concentrated together with F-actin in the center of the cell, The rapid modificati on of moesin at or near its actin-binding domain suggests a model for regulated membrane-cytoskeleton interaction during cell activation.