Phosphorylation of Threonine 558 in the Carboxyl-terminal Actin-binding Domain of Moesin by Thrombin Activation of Human Platelets

Abstract

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...

Protein Kinase C-θ Phosphorylation of Moesin in the Actin-binding Sequence

“…PKC-is thus the major moesin kinase directly identifiable in the cell extracts we studied. The single phosphorylation site identified corresponds exactly with that which is now well established to occur in cells (1,33). We are currently extending this work to test further our putative PKC-/moesin link in cellular systems and to analyze its control and effects.…”

“…In this report we present reaction conditions under which PKC-threonine phosphorylates moesin at its previously identified in vivo site within its actin-binding domain (1). PKC-is a recently discovered novel PKC isozyme, which has not previously been shown to phosphorylate moesin or to require the unusual conditions defined here nor has it been linked to any other substrate of likely physiological significance.…”

“…The assay requirement for vesicles correlates with their cellular localization on the cytoplasmic surface of the plasma membrane. The in vitro phosphorylation site of moesin Thr 558 corresponds to that occurring in activated platelet membranes and in macrophages (1,35). We are currently investigating such interactions of moesin and PKCwithin cells to address the physiologic significance of this system.…”

“…ERM proteins have N-terminal membrane-binding domains and Cterminal actin-binding domains. Relatively little is known about their intracellular control, although phosphorylation of moesin Thr 558 by an unidentified kinase in activated platelets has recently been described (1). This residue is within the previously identified actin-binding domain of ezrin, which is highly conserved in moesin (2).…”

The Role of Drosophila Merlin in the Control of Mitosis Exit and Development

“…Phosphorylation on the Thr 576 residue, however, has no effect on merlin's functional activity. In contrast, phosphorylation at the analogous residue is important for the function of the ERM proteins [45,[48][49][50]. Sequence alignment shows that the Ser 518 residue is conserved across all merlin proteins from different taxons with the exception of the fruit fly and worm, which contain a related threonine residue at the corresponding position.…”