Src phosphorylates ezrin at tyrosine 477 and induces a phosphospecific association between ezrin and a kelch-repeat protein family member.
Ezrin, a linker between plasma membrane and actin cytoskeleton possesses morphogenic properties and can promote dissemination of tumor cells. Ezrin is phosphorylated on tyrosine, but a detailed picture of the signaling pathways involved in this modification is lacking. The transforming tyrosine kinase Src has various cytoskeletal substrates and is involved in regulation of cellular adhesion. We studied the role of Src in tyrosine phosphorylation of ezrin in adherent cells. We show that ezrin is phosphorylated in human embryonic kidney 293 cells in a Src family-dependent way. In SYF cells lacking Src, Yes, and Fyn, ezrin was not tyrosine-phosphorylated but reintroduction of wild-type Src followed by Src activation or introduction of active Src restored phosphorylation. Mapping of the Src-catalyzed tyrosine in vitro and in vivo by site-directed mutagenesis demonstrated Tyr(477) as the primary target residue. We generated a pTyr(477)-phosphospecific antibody, which confirmed that Tyr(477) becomes phosphorylated in cells in a Src-dependent manner. Tyr(477) phosphorylation did not affect ezrin head-to-tail association or phosphorylation of ezrin on threonine 566, indicating that the function of Tyr(477) phosphorylation is not related to the intramolecular regulation of ezrin. A modified yeast two-hybrid screen in which ezrin bait was phosphorylated by Src identified a novel interaction with a kelch-repeat protein family member, KBTBD2 (Kelch-repeat and BTB/POZ domain containing 2). The Src dependence of the interaction was further verified by affinity precipitation assays. Identification of a functional interplay with Src opens novel avenues for further characterization of the biological activities of ezrin.
Heiska
Leena
L
Department of Pathology, Neuroscience Program, Biomedicum Helsinki, University of Helsinki and Helsinki University Hospital, FIN-00014 Helsinki, Finland.
Carpén
Olli
O
D000199:Actins / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D019169:Jurkat Cells; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000737:chemistry*; D001616:beta-Galactosidase / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*; D000099149:Ezrin
D000199:Actins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D005982:Glutathione Transferase; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014443:Tyrosine; D001616:beta-Galactosidase; D019061:src-Family Kinases; D000099149:Ezrin