Replacement of threonine 558, a critical site of phosphorylation of moesinin vivo, with aspartate activates F-actin binding of moesin - Regulation by conformational change

Point and deletion mutants of moesin were examined for F-actin binding by b lot overlay and co-sedimentation, and for intra- and intermolecular interac tions with N- and C-terminal domains with yeast two-hybrid and in vitro bin ding assays, Wild-type moesin molecules interact poorly with F-actin and ea ch other, and bind neither C- nor N-terminal fragments. Interaction with F- actin is strongly enhanced by replacement of Thr(558) With aspartate (T558D ), by deletion of 11 N-terminal residues (DelN11), by deletion of the entir e N-terminal membrane-binding domain of both wild type and T558D mutant mol ecules, and by exposure to phosphatidylinositol 4,5-diphosphate, Activation of F-actin binding is accompanied by changes in inter- and intramolecular domain interactions. The T558D mutation renders moesin capable of binding w ild type but not mutated (T558D) C-terminal or wild type N-terminal fragmen ts. The interaction between the latter two is prevented. DelN11 truncation enables binding of wild type N and C domain fragments. These changes sugges t that the T558D mutation, mimicking phosphorylation of Thr(558), promotes F-actin binding by disruption of interdomain interactions between N and C d omains and exposure of the high affinity F-actin binding site in the C-term inal domain. Oscillation between activated and resting state could thus pro vide the structural basis for transient interactions between moesin and the actin cytoskeleton in protruding and retracting microextensions.