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J. Biol. Chem., Vol. 283, Issue 23, 16017-16026, June 6, 2008

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Identification and Functional Analysis of Phosphorylated Tyrosine Residues within EphA2 Receptor Tyrosine Kinase^*

Wei Bin Fang, Dana M. Brantley-Sieders, Yoonha Hwang, Amy-Joan L. Ham^¶, and Jin Chen^||**1

From the Department of Cancer Biology, Department of Medicine, Division of Rheumatology and Immunology, ^¶Proteomics Laboratory, Mass Spectrometry Research Center, ^||Department of Cell and Developmental Biology, and ^**Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

EphA2 is a member of the Eph family of receptor tyrosine kinases. EphA2 mediates cell-cell communication and plays critical roles in a number of physiological and pathologic responses. We have previously shown that EphA2 is a key regulator of tumor angiogenesis and that tyrosine phosphorylation regulates EphA2 signaling. To understand the role of EphA2 phosphorylation, we have mapped phosphorylated tyrosines within the intracellular region of EphA2 by a combination of mass spectrometry analysis and phosphopeptide mapping using two-dimensional chromatography in conjunction with site-directed mutagenesis. The function of these phosphorylated tyrosine residues was assessed by mutational analysis using EphA2-null endothelial cells reconstituted with EphA2 tyrosine-to-phenylalanine or tyrosine-to-glutamic acid substitution mutants. Phosphorylated Tyr⁵⁸⁷ and Tyr⁵⁹³ bind to Vav2 and Vav3 guanine nucleotide exchange factors, whereas Tyr(P)⁷³⁴ binds to the p85 regulatory subunit of phosphatidylinositol 3-kinase. Mutations that uncouple EphA2 with Vav guanine nucleotide exchange factors or p85 are defective in Rac1 activation and cell migration. Finally, EphA2 mutations in the juxtamembrane region (Y587F, Y593F, Y587E/Y593E), kinase domain (Y734F), or SAM domain (Y929F) inhibited ephrin-A1-induced vascular assembly. In addition, EphA2-null endothelial cells reconstituted with these mutants were unable to incorporate into tumor vasculature, suggesting a critical role of these phosphorylation tyrosine residues in transducing EphA2 signaling in vascular endothelial cells during tumor angiogenesis.

Received for publication, December 5, 2007 , and in revised form, March 14, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants CA95004 and CA114301 (to J. C.) and CA1179151 to (D. M. B.-S.). This work was also supported by Department of Defense Predoctoral Fellowship W81XWH-05-1-0254 (to W. B. F.). 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.

¹ To whom correspondence should be addressed: Dept. of Medicine, Vanderbilt University School of Medicine, A-4323 MCN, 1161 21st Ave. S., Nashville, TN 37232-2363. Tel.: 615-343-3819; Fax: 615-343-7392; E-mail: jin.chen{at}vanderbilt.edu.

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