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J. Biol. Chem., Vol. 280, Issue 11, 10244-10252, March 18, 2005

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Src Phosphorylates Ezrin at Tyrosine 477 and Induces a Phosphospecific Association between Ezrin and a Kelch-Repeat Protein Family Member^*

Leena Heiska and Olli Carpén¶

From the Department of Pathology, Neuroscience Program, Biomedicum Helsinki, University of Helsinki and Helsinki University Hospital, FIN-00014 Helsinki, Finland and Department of Pathology, University of Turku and Turku University Hospital, FIN-20520 Turku, Finland

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 Tyr⁴⁷⁷ as the primary target residue. We generated a pTyr⁴⁷⁷-phosphospecific antibody, which confirmed that Tyr⁴⁷⁷ becomes phosphorylated in cells in a Src-dependent manner. Tyr⁴⁷⁷ phosphorylation did not affect ezrin head-to-tail association or phosphorylation of ezrin on threonine 566, indicating that the function of Tyr⁴⁷⁷ 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.

Received for publication, October 5, 2004 , and in revised form, December 1, 2004.

^* This work was supported by grants from the Academy of Finland and the Finnish Cancer Organizations. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pathology, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. Tel.: 358-2-3337217; Fax: 358-2-3337459; E-mail: ocarpen{at}utu.fi.

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