ABSTRACT
EPI64 is a TBC domain-containing protein that binds the PDZ domains of EBP50, which binds ezrin, a major actin-binding protein of microvilli. High-resolution light microscopy revealed that ezrin and EBP50 localize exclusively to the membrane-surrounded region of microvilli, whereas EPI64 localizes to variable regions in the structures. Overexpressing EPI64 results in its and EBP50's relocalization to the base of microvilli, including to the actin rootlet devoid of ezrin or plasma membrane. Uncoupling EPI64's binding to EBP50, expression of any construct mislocalizing its TBC domain, or knock down of EBP50 results in loss of microvilli. The TBC domain of EPI64 binds directly to Arf6-GTP. Overexpressing the TBC domain increases Arf6-GTP levels, and expressing dominant-active Arf6 results in microvillar loss. These data reveal that microvilli have distinct cytoskeletal subdomains and that EPI64 regulates microvillar structure.
Subject(s)
ADP-Ribosylation Factors/metabolism , Carrier Proteins/genetics , Microvilli/metabolism , Phosphoproteins/metabolism , Sodium-Hydrogen Exchangers/metabolism , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/physiology , Actins/metabolism , Carrier Proteins/metabolism , Carrier Proteins/physiology , Cell Line, Tumor , Cytoskeletal Proteins/metabolism , GTPase-Activating Proteins , HeLa Cells , Humans , Microscopy, Fluorescence/methods , Microvilli/chemistry , Models, Biological , Protein Structure, Tertiary , Transfection , Vacuoles/metabolismABSTRACT
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.
