Properties of an ezrin mutant defective in F-actin binding.
Ezrin, radixin and moesin are a family of proteins that provide a link between the plasma membrane and the cortical actin cytoskeleton. The regulated targeting of ezrin to the plasma membrane and its association with cortical F-actin are more than likely functions necessary for a number of cellular processes, such as cell adhesion, motility, morphogenesis and cell signalling. The interaction with F-actin was originally mapped to the last 34 residues of ezrin, which correspond to the last three helices (alphaB, alphaC and alphaD) of the C-terminal tail. We set out to identify and mutate the ezrin/F-actin binding site in order to pinpoint the role of F-actin interaction in morphological processes as well as signal transduction. We report here the generation of an ezrin mutant defective in F-actin binding. We identified four actin-binding residues, T576, K577, R579 and I580, that form a contiguous patch on the surface of the last helix, alphaD. Interestingly, mutagenesis of R579 also eliminated the interaction of band four-point one, ezrin, radixin, moesin homology domains (FERM) and the C-terminal tail domain, identifying a hotspot of the FERM/tail interaction. In vivo expression of the ezrin mutant defective in F-actin binding and FERM/tail interaction (R579A) altered the normal cell surface structure dramatically and inhibited cell migration. Further, we showed that ezrin/F-actin binding is required for the receptor tyrosine kinase signal transfer to the Ras/MAP kinase signalling pathway. Taken together, these observations highlight the importance of ezrin/F-actin function in the development of dynamic membrane/actin structures critical for cell shape and motility, as well as signal transduction.
Saleh
Henri S
HS
Leibniz Institute of Age Research, Fritz Lipmann Institute, Beutenbergstrasse 11, 07745 Jena, Germany.
Merkel
Ulrike
U
Geissler
Katja J
KJ
Sperka
Tobias
T
Sechi
Antonio
A
Breithaupt
Constanze
C
Morrison
Helen
H
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism*; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D021381:Protein Transport; D034421:Sus scrofa; D000099149:Ezrin
D000199:Actins; D000596:Amino Acids; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D050505:Mutant Proteins; D010769:Phosphothreonine; D000099149:Ezrin; C071058:moesin; C071776:radixin