Replacement of threonine 558, a critical site of phosphorylation of moesin in 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 blot overlay and co-sedimentation, and for intra- and intermolecular interactions with N- and C-terminal domains with yeast two-hybrid and in vitro binding assays. Wild-type moesin molecules interact poorly with F-actin and each other, and bind neither C- nor N-terminal fragments. Interaction with F-actin is strongly enhanced by replacement of Thr558 with aspartate (T558D), by deletion of 11 N-terminal residues (DelN11), by deletion of the entire N-terminal membrane-binding domain of both wild type and T558D mutant molecules, 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 wild type but not mutated (T558D) C-terminal or wild type N-terminal fragments. The interaction between the latter two is prevented. DelN11 truncation enables binding of wild type N and C domain fragments. These changes suggest that the T558D mutation, mimicking phosphorylation of Thr558, promotes F-actin binding by disruption of interdomain interactions between N and C domains and exposure of the high affinity F-actin binding site in the C-terminal domain. Oscillation between activated and resting state could thus provide the structural basis for transient interactions between moesin and the actin cytoskeleton in protruding and retracting microextensions.
Huang
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Molecular Mechanisms of Disease Laboratories, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324, USA.
Wong
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Lin
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Furthmayr
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D000199:Actins / Q000378:metabolism*; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D013912:Threonine / Q000737:chemistry / Q000378:metabolism*
D000199:Actins; D017931:DNA Primers; D008840:Microfilament Proteins; C071058:moesin; D013912:Threonine; D001224:Aspartic Acid