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 Tyr(587) and Tyr(593) 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.
Fang
Wei Bin
WB
Department of Cancer Biology, Division of Rheumatology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Brantley-Sieders
Dana M
DM
Hwang
Yoonha
Y
Ham
Amy-Joan L
AJ
Chen
Jin
J
D019943:Amino Acid Substitution; D000818:Animals; D002465:Cell Movement / Q000235:genetics; D042783:Endothelial Cells / Q000201:enzymology*; D036382:Ephrin-A1 / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D020125:Mutation, Missense; D009361:Neoplasm Invasiveness; D009389:Neovascularization, Pathologic / Q000201:enzymology / Q000235:genetics; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051567:Proto-Oncogene Proteins c-vav / Q000235:genetics / Q000378:metabolism; D036104:Receptor, EphA2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020830:rac1 GTP-Binding Protein
D036382:Ephrin-A1; D020662:Guanine Nucleotide Exchange Factors; D009479:Neuropeptides; D051567:Proto-Oncogene Proteins c-vav; C484602:Rac1 protein, mouse; C490565:Vav2 protein, mouse; C496622:Vav3 protein, mouse; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D036104:Receptor, EphA2; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein