Abstract
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.
Highlights
Ogy and binding affinity, the Eph receptors are divided into two subclasses
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 (19 –21)
Since these phosphorylated tyrosine residues are not mapped in endothelial cells, their role in signal transduction leading to angiogenic responses is not clear
Summary
Antibodies, and Reagents—Antibodies used for immunoblot include anti-EphA2 (1:1000, Upstate Biotechnology), anti-phosphotyrosine (1:250; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-tubulin (1:1000, Sigma), and antiRac and anti-Cdc (1:1000; Transduction Laboratories). Endothelial Cell Culture and Retroviral Infection—Wildtype or EphA2-deficient primary murine pulmonary microvascular endothelial cells were isolated from 1–3-month-old mice derived from H-2Kb-tsA58 transgenic “Immorto-mouse” background [17, 29] These cells were grown at 33 °C in EGM-2 medium supplemented with interferon-␥ (10 ng/ml), a permissive condition that allows the expression of SV40 T-antigen (TAg). Cells were lysed and EphA2 was immunoprecipitated and phosphorylated in the presence of [␥-32P]ATP, as described under “Immunoprecipitation, Western Blot Analysis, and Kinase Assay.”. Kinase assays using EphA2 as substrate were performed as described previously [30]. The branch length in assembled endothelial cell networks was expressed as arbitrary units per ϫ10 field in four random fields from each well, with triplicate samples per condition, using Scion Image version 1.62c software for analysis. Data are a representation of eight independent tumors/conditions from two independent experiments
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