Ezrin mutants affecting dimerization and activation.

Abstract

ERM (ezrin/radixin/moesin) proteins provide a regulated linkage between membrane-associated proteins and the actin cytoskeleton. Previous work has shown that ezrin can exist in a dormant monomeric state in which the N-terminal FERM domain is tightly associated with the C-ERMAD (carboxyl-terminal ERM association domain), masking binding sites for at least some ligands, including F-actin and the scaffolding protein EBP50. Activation of ezrin requires relief of the intramolecular association, and this is believed to involve phosphorylation of threonine 567. Studies have therefore employed the T567D phosphomimetic mutant to explore the consequences of ezrin activation in vivo. Ezrin also exists as a stable dimer, in which the orientation of the two subunits is unknown, but might involve the central alpha-helical region predicted to form a coiled-coil. By characterization of ezrin mutants, we show that relief of the intramolecular association in the monomer results in unmasking of ligand binding sites and a significant conformational change, that the T567D mutation has a small effect on the biochemical activation of ezrin, and that the predicted coiled-coil region does not drive dimer formation. These results provide strong support for the conformational activation model of ezrin, elucidate the basis for dimer formation, and reveal that a mutant generally considered to be fully activated is not.