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

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. 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. The Eph receptors belong to a large family of receptor tyrosine kinases that regulate a variety of physiological processes during development and contribute to the pathogenesis of diseases such as cancer (1Brantley-Sieders D. Schmidt S. Parker M. Chen J. Curr. Pharm. Des. 2004; 10: 3431-3442Crossref PubMed Scopus (110) Google Scholar, 2Pasquale E.B. Nat. Rev. Mol. Cell. Biol. 2005; 6: 462-475Crossref PubMed Scopus (865) Google Scholar). One of the key events important both in embryogenesis and pathogenesis in adult organisms is angiogenesis, the process by which new blood vessels are formed from preexisting vasculature. On the basis of sequence homology and binding affinity, the Eph receptors are divided into two subclasses. EphA receptors bind preferentially to the glycosylphosphatidylinositol-linked ephrin-A ligands, whereas EphB receptors bind preferentially to the transmembrane ephrin-B ligands (3Gale N.W. Holland S.J. Valenzuela D.M. Flenniken A. Pan L. Ryan T.E. Henkemeyer M. Strebhardt K. Hirai H. Wilkinson D.G. Neuron. 1996; 17: 9-19Abstract Full Text Full Text PDF PubMed Scopus (763) Google Scholar). Both class A and class B Eph receptors have been implicated in regulation of vascular remodeling and angiogenesis. Targeted disruption of several class B receptor tyrosine kinases and ephrin-B ligands resulted in defects in angiogenic remodeling of the rudimentary embryonic vasculature (4Adams R. Semin. Cell Dev. Biol. 2002; 13: 55-60Crossref PubMed Scopus (98) Google Scholar, 5Brantley-Sieders D. Chen J. Angiogenesis. 2004; 7: 17-28Crossref PubMed Scopus (129) Google Scholar, 6Gerety S.S. Anderson D.J. Development. 2002; 129: 1397-1410Crossref PubMed Google Scholar, 7Foo S.S. Turner C.J. Adams S. Compagni A. Aubyn D. Kogata N. Lindblom P. Shani M. Zicha D. Adams R.H. Cell. 2006; 124: 161-173Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). Manipulation of the level of one receptor, EphB4, in tumor cells also affected tumor angiogenesis in adult animals (8Noren N.K. Lu M. Freeman A.L. Koolpe M. Pasquale E.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5583-5588Crossref PubMed Scopus (221) Google Scholar, 9Erber R. Eichelsbacher U. Powajbo V. Korn T. Djonov V. Lin J. Hammes H.P. Grobholz R. Ullrich A. Vajkoczy P. EMBO J. 2006; 25: 628-641Crossref PubMed Scopus (132) Google Scholar). In the A class, ephrin-A1 stimulates endothelial cell migration and assembly in culture (10Daniel T.O. Stein E. Cerretti D.P. John P.L. Robert B. Abrahamson D.R. Kidney Int. Suppl. 1996; 57: 73-81PubMed Google Scholar, 11Ogawa K. Pasqualini R. Lindberg R.A. Kain R. Freeman A.L. Pasquale E.B. Oncogene. 2000; 19: 6043-6052Crossref PubMed Scopus (336) Google Scholar) and induces corneal angiogenesis in vivo (12Pandey A. Shao H. Marks R.M. Polverini P.J. Dixit V.M. Science. 1995; 268: 567-569Crossref PubMed Scopus (346) Google Scholar, 13Cheng N. Brantley D.M. Liu H. Lin Q. Enriquez M. Gale N.W. Yancopoulos G. Cerretti D.P. Daniel T.O. Chen J. Mol. Cancer Res. 2002; 1: 2-11Crossref PubMed Scopus (37) Google Scholar). More recently, Eph receptors have been detected in tumor blood vessel endothelial cells (1Brantley-Sieders D. Schmidt S. Parker M. Chen J. Curr. Pharm. Des. 2004; 10: 3431-3442Crossref PubMed Scopus (110) Google Scholar, 5Brantley-Sieders D. Chen J. Angiogenesis. 2004; 7: 17-28Crossref PubMed Scopus (129) Google Scholar). Inhibition of class A Eph receptor signaling by soluble EphA2-Fc or EphA3-Fc receptors decreased tumor volume, tumor angiogenesis, and metastatic progression in vivo (14Brantley D.M. Cheng N. Thompson E.J. Lin Q. Brekken R.A. Thorpe P.E. Muraoka R.S. Cerretti D.P. Pozzi A. Jackson D. Lin C. Chen J. Oncogene. 2002; 21: 7011-7026Crossref PubMed Scopus (291) Google Scholar, 15Cheng N. Brantley D. Liu H. Fanslow W. Cerretti D.P. Reith A.D. Jackson D. Chen J. Neoplasia. 2003; 5: 445-456Crossref PubMed Google Scholar, 16Dobrzanski P. Hunter K. Jones-Bonlin S. Chang H. Robinson C. Pritchard S. Zhao H. Ruggeri B. Cancer Res. 2004; 64: 910-919Crossref PubMed Scopus (144) Google Scholar). A main target of soluble EphA receptors appears to be EphA2, since EphA2-deficient endothelial cells fail to migrate and assemble in vitro (17Brantley-Sieders D. Caughron J. Hicks D. Pozzi A. Ruiz J.C. Chen J. J. Cell Sci. 2004; 117: 2037-2049Crossref PubMed Scopus (168) Google Scholar), and loss of EphA2 receptor resulted in impaired tumor growth and metastasis in vivo (18Brantley-Sieders D.M. Fang W.B. Hicks D. Koyama T. Shyr Y. Chen J. FASEB J. 2005; 19: 1884-1886Crossref PubMed Scopus (114) Google Scholar). The binding of ephrin ligands to Eph receptors induces the transphosphorylation of the cytoplasmic domains and initiates kinase activity. Extensive tyrosine phosphorylation of the activated Eph receptor is not only induced by auto/trans-phosphorylation but is also elicited by receptor-associated protein-tyrosine kinases such as Src family kinases (2Pasquale E.B. Nat. Rev. Mol. Cell. Biol. 2005; 6: 462-475Crossref PubMed Scopus (865) Google Scholar). Many phosphorylated tyrosine residues in the EphB receptors and ephrin-B ligands in neuronal cells/tissues have been mapped by both phosphopeptide mapping using two-dimensional chromatography, and by matrix-assisted laser desorption/ionization mass spectrometry (19Kalo M.S. Pasquale E.B. Biochemistry. 1999; 38: 14396-14408Crossref PubMed Scopus (77) Google Scholar, 20Kalo M.S. Yu H.H. Pasquale E.B. J. Biol. Chem. 2001; 276: 38940-38948Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 21Binns K.L. Taylor P.P. Sicheri F. Pawson T. Holland S.J. Mol. Cell. Biol. 2000; 20: 4791-4805Crossref PubMed Scopus (162) Google Scholar). Several tyrosine phosphorylation sites in EphA3 and EphA4 have also been identified by mutational analysis on sites homologous to those in EphB receptors K.L. Taylor P.P. Sicheri F. Pawson T. Holland S.J. Mol. Cell. Biol. 2000; 20: 4791-4805Crossref PubMed Scopus (162) Google Scholar, P. M. M. J. Cell Sci. 2002; PubMed Google Scholar). since these phosphorylated tyrosine residues are not mapped in endothelial role in to angiogenic is not phosphorylated tyrosine residues have not been mapped in EphA2, a EphA receptor that is critical in tumor angiogenesis. We have previously shown that activation of the EphA2 receptor in endothelial cells Vav guanine nucleotide exchange mass Src homology in of activated Rac1 and endothelial cell migration G. D.M. W. Chen J. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). The Vav appears to be by since and or a p85 subunit of ephrin-A1-induced Rac1 activation and endothelial cell migration (17Brantley-Sieders D. Caughron J. Hicks D. Pozzi A. Ruiz J.C. Chen J. J. Cell Sci. 2004; 117: 2037-2049Crossref PubMed Scopus (168) Google Scholar). the domains of both Vav and p85 subunit of the are of binding to phosphorylated EphA2 receptor G. D.M. W. Chen J. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar, A. Dixit V.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar), we to critical phosphorylated tyrosine residues that the of Vav and a we have a combination of mass spectrometry analysis and phosphopeptide mapping to the phosphorylated tyrosine residues within the EphA2 phosphorylated tyrosine residues in the cytoplasmic domain of the EphA2 receptor were of these sites to or acid resulted in EphA2 that not be to with p85 or Vav and was unable to defects in endothelial assembly in EphA2-deficient cells in vitro and in that phosphorylation of and is critical in of Vav and of these tyrosines is also in activation of Rac1 and angiogenic and tumor and and and of EphA2 from cell was with and p85 was by were from was from was using EphA2 mutations were by using tyrosine to or acid The were with and tyrosine mutations and and tyrosine mutations and into the and mutations were by was by the in the EphA2 was from with in The of and M. and R. Scholar). The were by chromatography that is with using a mass with a and and of the was with a a A was with and B was The was as from to to to B in A. were using of activation of and activation of and using one and of The mass was to analysis using the were as of a of a of and V. analysis was using in which one using a mass of mass was by into the was a the loss of acid or such that these were of the loss was were identified using a of the A.L. J. 5: PubMed Scopus Google Scholar, A.L. D. Chem. 1995; PubMed Scopus Google Scholar), using a of from the from in were on a in the In to using the to phosphorylation on or the were also using the J. Res. 2005; PubMed Scopus Google Scholar). were by of the Cell and or EphA2-deficient endothelial cells were from from (17Brantley-Sieders D. Caughron J. Hicks D. Pozzi A. Ruiz J.C. Chen J. J. Cell Sci. 2004; 117: 2037-2049Crossref PubMed Scopus (168) Google Scholar, P. Y. N. L. D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). cells were in with a that the of The EphA2-deficient endothelial cells were with or and by a cell EphA2 receptor were to by endothelial cells reconstituted with or EphA2 were with ephrin-A1 were and EphA2 was and phosphorylated in the of as and were by and to EphA2 receptor was and were in with The was in two on by by was in with of chromatography was in or the was from the of the The were and to with and of EphA2 and were as G. D.M. W. Chen J. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). of EphA2 with cells were with of EphA2 and p85 in a using p85 was by of of The were on and using using EphA2 as were as previously W.B. D.M. Parker Reith A.D. Chen J. Oncogene. 2005; PubMed Scopus Google Scholar). were also using a to the the kinase the were by were to the to phosphorylation of was using a vitro vascular assembly were as previously (17Brantley-Sieders D. Caughron J. Hicks D. Pozzi A. Ruiz J.C. Chen J. J. Cell Sci. 2004; 117: 2037-2049Crossref PubMed Scopus (168) Google Scholar). were with of growth in cells were in in the or of ephrin-A1 and were on using The of assembly was by the from to the of cells of cells were The in endothelial cell was as in from with using Rac1 and activation cells were in by with ephrin-A1 were and with binding to the were by and to a Rac1 and Rac1 and in were detected by using or of Rac1 and were by using cells were in were with growth with and with cells were in the of the and of was to the cells were and with to endothelial that have to the of were in from with of and cell were as previously (18Brantley-Sieders D.M. Fang W.B. Hicks D. Koyama T. Shyr Y. Chen J. FASEB J. 2005; 19: 1884-1886Crossref PubMed Scopus (114) Google Scholar). EphA2-null endothelial cells reconstituted with or EphA2 were with tumor cells and endothelial cells were in of growth and into the of were and tumor was assessed using the guanine nucleotide exchange mass Src homology were and to as previously (18Brantley-Sieders D.M. Fang W.B. Hicks D. Koyama T. Shyr Y. Chen J. FASEB J. 2005; 19: 1884-1886Crossref PubMed Scopus (114) Google Scholar). are a of from two in EphA2 the phosphorylated tyrosine residues in the cytoplasmic domain of EphA2 receptor induced binding to ephrin-A1 we EphA2 in EphA2 were with and to mass of the were not detected and were not the phosphorylated were identified that Tyr593 in the juxtamembrane domain as as and in the kinase domain and from in vivo phosphorylated EphA2 identified by mass mass and mass and in a new To phosphorylation sites mapped by mass spectrometry and to sites not by mass we phosphopeptide mapping by two-dimensional chromatography in conjunction with site-directed mutagenesis. We to EphA2-null and endothelial cell since the EphA2-null mutational analysis and endothelial cells were from EphA2-deficient that were into the P. Y. N. L. D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). a by the which in a of In addition, the is to of the in cells from these in the of cells are of the are and cells are to a the of several P. Y. N. L. D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and a of to EphA2 were in endothelial cells using the W.B. D.M. Parker Reith A.D. Chen J. Oncogene. 2005; PubMed Scopus Google Scholar). In vitro kinase using that and mutations not kinase and inhibited and EphA2 kinase and to be tyrosine phosphorylation sites in the EphA2 receptor, since phosphorylation of EphA2 was in and mutants the that these mutants kinase activity. mapping by two-dimensional chromatography detected in activated EphA2 To the phosphorylated tyrosines within the the phosphorylated tyrosines identified by mass spectrometry analysis or those tyrosine residues that were not were to tyrosine residues in the juxtamembrane region (Y587F, kinase domain and and the SAM domain and the not be to defective kinase in a tyrosine to acid that kinase was phosphopeptide of endothelial cells were to those EphA2-null cells reconstituted with EphA2 receptor of the EphA2 mutants was in The two and was in the and phosphopeptide was in the of was identified in a the chromatography was in the these that and are to be in vascular endothelial Vav to in the and p85 with Tyr(P)734 in the EphA2 have previously shown that guanine nucleotide exchange factors Vav2 and Vav3 are to phosphorylated EphA2 receptor, and the binding is in mutants G. D.M. W. Chen J. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). To which phosphorylated tyrosine or both sites in the juxtamembrane region of the EphA2 with Vav we a of with shown in Tyr587 or Tyr593 inhibited binding of EphA2 receptor to Vav2 and Vav3 exchange factors, suggesting that both sites are binding to Vav in the SAM domain also appears to binding to Vav3 but not Vav2 phosphorylation of was not detected by in vitro kinase be phosphorylated by tyrosine kinase in In to binding to Vav we and have shown that activated EphA2 receptor also the p85 subunit of the and is ephrin-A1-induced Rac1 activation and endothelial cell migration (17Brantley-Sieders D. Caughron J. Hicks D. Pozzi A. Ruiz J.C. Chen J. J. Cell Sci. 2004; 117: 2037-2049Crossref PubMed Scopus (168) Google Scholar, A. Dixit V.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the domain of the p85 was shown to with the kinase domain of the EphA2 receptor A. Dixit V.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the that mediates is a of EphA2 mutants to mutations was the to bind to shown in p85 binds to and as as EphA2, to bind to and suggesting that p85 with phosphorylated in the kinase domain and in the SAM We have previously shown that the domain of Vav3 binds to EphA2 in a and in in vitro binding G. D.M. W. Chen J. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). To the of EphA2 and in we using the domain of p85 or Vav3 or a shown in the domain of p85 or Vav3 was of binding to EphA2 receptor, whereas the to with EphA2, suggesting that EphA2 and is by binding domains and sites in that Vav and p85 are not the binding of the EphA2 Mutations EphA2 with Vav or p85 Rac1 and regulation of the is critical in cell and family are to be key of process and have been shown to be endothelial cell migration J. Cell Sci. 2001; PubMed Google Scholar). We have previously that ephrin-A1 of endothelial cells induces activation of Rac1 activation of guanine nucleotide exchange factors G. D.M. W. Chen J. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). In addition, ephrin-A1-induced Rac1 activation is on the of (17Brantley-Sieders D. Caughron J. Hicks D. Pozzi A. Ruiz J.C. Chen J. J. Cell Sci. 2004; 117: 2037-2049Crossref PubMed Scopus (168) Google Scholar). and are of Vav and we and ephrin-A1-induced Rac1 activation in endothelial were with and activated Rac1 or was from by with shown in with (17Brantley-Sieders D. Caughron J. Hicks D. Pozzi A. Ruiz J.C. Chen J. J. Cell Sci. 2004; 117: 2037-2049Crossref PubMed Scopus (168) Google Scholar), ephrin-A1 induced Rac1 activation in EphA2-null endothelial cells reconstituted with EphA2 but not in In and mutants to suggesting that of p85 and Vav to the EphA2 receptor is critical ephrin-A1-induced Rac1 was not in to ephrin-A1 in or EphA2 mutants that EphA2 is not in activity. activation of Rac1 is critical ephrin-A1-induced endothelial cell migration (17Brantley-Sieders D. Caughron J. Hicks D. Pozzi A. Ruiz J.C. Chen J. J. Cell Sci. 2004; 117: 2037-2049Crossref PubMed Scopus (168) Google Scholar), we cell migration is impaired in EphA2-null endothelial cells reconstituted with EphA2 mutants. shown in EphA2-null endothelial cells a in ephrin-A1-induced cell migration. of EphA2 receptor migration defects in EphA2 endothelial In of or mutants to ephrin-A1-induced cell suggesting that phosphorylation of and is critical of the p85 subunit of and Vav which signaling to Rac1 and cell migration. of EphA2 Phosphorylated in and is a process by which new blood vessels are formed from preexisting vasculature. critical in process are endothelial cell migration and assembly into new To the roles of phosphorylated tyrosine residues of EphA2 receptor in ephrin-A1-induced angiogenic we the vascular assembly in EphA2-null endothelial cells reconstituted with EphA2 mutants. induced but not endothelial cell assembly into vascular on a of of EphA2, but not by defects in EphA2-null endothelial of or in EphA2-null cells the of cells to assemble and on In EphA2 mutations in the juxtamembrane (Y587F, Y587E/Y593E), kinase domain (Y734F), or SAM domain (Y929F) inhibited ephrin-A1-induced vascular assembly To phosphorylated tyrosines important in vascular assembly in vitro are also critical in tumor angiogenesis in we tumor cell using EphA2-null endothelial cells reconstituted with or EphA2 endothelial cells were with in to from endothelial cells were with cells in into the of cell were and with and to endothelial shown in of endothelial cells reconstituted with or EphA2 have into tumor or vessels In EphA2-deficient endothelial cells as as cells reconstituted with or and to incorporate into tumor vasculature. In addition, tumor was in endothelial cells reconstituted with or EphA2, to EphA2-deficient endothelial cells or cells reconstituted with or these that phosphorylated tyrosine and are critical in EphA2 and tumor angiogenesis in A of that ephrin-A of EphA2 receptors a signaling that and regulates cell-cell and and cell (17Brantley-Sieders D. Caughron J. Hicks D. Pozzi A. Ruiz J.C. Chen J. J. Cell Sci. 2004; 117: 2037-2049Crossref PubMed Scopus (168) Google Scholar, G. D.M. W. Chen J. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar, H. B. J. Cell Biol. 2003; PubMed Scopus Google Scholar). Phosphorylated tyrosine residues on the EphA2 receptor were to a critical role in the of or signaling such as the p85 subunit of A. Dixit V.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar), A. H. Dixit V.M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) and M.S. Oncogene. 2002; 21: PubMed Scopus Google Scholar), tyrosine H. E. M. E. B. Nat. 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Cerretti D.P. Daniel T.O. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, E. Cerretti D.P. A.D. Daniel T.O. Dev. PubMed Scopus Google Scholar) that protein-tyrosine and bind to a in the protein-tyrosine also with in EphA2 receptor, signaling by of EphA2 receptor, as in tumor cells K. D.R. M.S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, P. P. F. G. G. G. G. Res. PubMed Scopus Google Scholar). protein-tyrosine EphA2 signaling by with to regulate the of W.B. G. T. Chen J. J. Cell Sci. PubMed Scopus Google Scholar). to be protein-tyrosine with EphA2 and ephrin-A1-induced angiogenic responses. that phosphorylated tyrosine residues in the EphA2 receptor are not only critical in endothelial cells in vitro but also important these cells to in tumor angiogenesis in is to that in the of endothelial cells EphA2-null cells or cells reconstituted with or mutants. not be to and by new blood since tumor blood to be to vessels from the tumor is that be signaling from endothelial cells to tumor cells to tumor and is or in EphA2-null endothelial we have previously shown that ephrin-A1 regulates soluble growth in tumor cells D. Fang W.B. Y. Hicks D. Chen J. Cancer Res. 2006; PubMed Scopus Google Scholar). The growth that are by EphA2 receptor activation in vascular endothelial cells to be In mapping of phosphorylated tyrosine residues in the EphA2 receptor to tyrosine to mutants that were to binding sites to key EphA2 signaling such as the p85 subunit of kinase and Vav guanine nucleotide exchange mutants the to of these by EphA2 receptor is critical endothelial cell function in shown in and and are sites of and and the of both Vav and p85 to the activated EphA2 receptor is critical ephrin-A1-induced endothelial cell migration and assembly. be to EphA2 such as and or to as ligands in chromatography analysis to binding EphA2 tyrosine be to the of signaling events with EphA2 tyrosine

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