Identification and Functional Analysis of Phosphorylated Tyrosine Residues within EphA2 Receptor Tyrosine Kinase
2008; Elsevier BV; Volume: 283; Issue: 23 Linguagem: Inglês
10.1074/jbc.m709934200
ISSN1083-351X
AutoresWei Bin Fang, Dana M. Brantley‐Sieders, Yoonha Hwang, Amy‐Joan L. Ham, Jin Chen,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoEphA2 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 (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, 22Lawrenson I.D. Wimmer-Kleikamp S.H. Lock P. Schoenwaelder S.M. Down M. Boyd A.W. Alewood P.F. Lackmann M. J. Cell Sci. 2002; 115: 1059-1072Crossref PubMed Google Scholar). However, since these phosphorylated tyrosine residues are not mapped in endothelial cells, their role in signal transduction leading to angiogenic responses is not clear. Moreover, phosphorylated tyrosine residues have not been mapped in EphA2, a major EphA receptor that is critical in mediating tumor angiogenesis. We have previously shown that activation of the EphA2 receptor in endothelial cells recruits Vav GEFs, 2The abbreviations used are: GEF, guanine nucleotide exchange factor; PI, phosphatidylinositol; LC, liquid chromatography; MS, mass spectrometry; SH2, Src homology 2; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; TAg, T-antigen. resulting in up-regulation of GTP-bound activated Rac1 GTPase and endothelial cell migration (23Hunter S.G. Zhuang G. Brantley-Sieders D.M. Swatt W. Cowan C.W. Chen J. Mol. Cell. Biol. 2006; 26: 4830-4842Crossref PubMed Scopus (102) Google Scholar). The Vav GEF/Rac1 pathway appears to be regulated by PI 3-kinase, since PI 3-kinase-specific inhibitors wortmannin and LY294002 or a dominant negative p85 subunit of PI 3-kinase blocks 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). Since the SH2 domains of both Vav GEFs and p85 subunit of the PI 3-kinase are capable of binding to phosphorylated EphA2 receptor (23Hunter S.G. Zhuang G. Brantley-Sieders D.M. Swatt W. Cowan C.W. Chen J. Mol. Cell. Biol. 2006; 26: 4830-4842Crossref PubMed Scopus (102) Google Scholar, 24Pandey A. Lazar D.F. Saltiel A.R. Dixit V.M. J. Biol. Chem. 1994; 269: 30154-30157Abstract Full Text PDF PubMed Google Scholar), we sought to identify critical phosphorylated tyrosine residues that mediate the recruitment of Vav GEFs and p85. As a first step, we have used a combination of mass spectrometry analysis and traditional phosphopeptide mapping to identify the phosphorylated tyrosine residues within the EphA2 receptor. Four phosphorylated tyrosine residues in the cytoplasmic domain of the EphA2 receptor were identified. Changing three of these sites to phenylalanine or glutamic acid resulted in an EphA2 mutant that could not be phosphorylated, failed to interact with p85 or Vav GEFs, and was unable to rescue defects in endothelial assembly in EphA2-deficient cells in vitro and in vivo. Our results suggest that phosphorylation of Tyr587/Tyr593 and Tyr734 is critical in recruitment of Vav and p85, respectively. Phosphorylation of these tyrosines is also essential in activation of Rac1 GTPase and promoting angiogenic responses and tumor neovascularization. Plasmids, 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 anti-Rac1 and anti-Cdc42 (1:1000; Transduction Laboratories). Immunoprecipitation of EphA2 from cell lysates was performed with anti-EphA2 antibody (2 μg; Upstate Biotechnology), and p85 was immunoprecipitated by anti-FLAG (M2)-agarose beads (Sigma). Recombinant ephrin-A1-Fc proteins were purchased from R&D Systems (Minneapolis, MN). Growth factor-reduced Matrigel was purchased from BD Biosciences. Transient transfection was performed using Lipofectamine 2000 (Invitrogen). EphA2 mutations were generated by PCR amplification using EphA2-specific primers containing tyrosine to phenylalanine or glutamic acid mutations. The fragments were digested with AgeI and BsiWI (for tyrosine mutations 593-846) and BamHI and BlpI (for tyrosine mutations 921-959) and ligated into the digested plasmids pcDNA3.0-EphA2 and LZRS-EphA2. All mutations were verified by DNA sequencing. LC-MS Analysis—LC-MS was performed by the Proteomics Laboratory in the Vanderbilt Mass Spectrometry Research Center. Resolved mouse EphA2 was excised from SDS-polyacrylamide gels for in-gel digestion with trypsin (25Ham, A.-J. (2005) in The Encyclopedia of Mass Spectrometry: Biological Proteins and Peptides (Gross, M. L., and Caprioli, R. M., eds) pp. 10-17, Elsevier Ltd., Kidlington, UKGoogle Scholar). The resulting peptides were separated by reverse phase high pressure liquid chromatography that is coupled directly with automatic tandem MS (LC-MS) using a ThermoFinnigan LTQ ion trap mass spectrometer equipped with a Thermo surveyor autosampler and Thermo Surveyor HPLC pump, nanospray source, and Xcalibur 1.4 instrument control. HPLC separation of the tryptic peptides was achieved with a 100 mm × 11-cm C-18 capillary column (Monitor C18, 5 μm, 100 Å; Column Engineering), at a 0.7 μl min-1 flow rate. Solvent A was H2O with 0.1% formic acid, and solvent B was acetonitrile containing 0.1% formic acid. The gradient program was as follows: 0-3 min, linear gradient from 0-5% B; 3-5 min, 5% B; 5-50 min, linear gradient to 50% B; 50-52 min, linear gradient to 80% B; 52-55 min, linear gradient to 90% B; 55-56 min, 90% B in solvent A. MS/MS scans were acquired using an isolation width of 2 m/z, an activation time of 30 ms, and activation Q of 0.250 and 30% normalized collision energy using one microscan and an ion time of 100 for each scan. The mass spectrometer was tuned prior to analysis using the synthetic peptide TpepK (AVAGKAGAR). Typical tune parameters were as follows: spray voltage of 1.8 kV, a capillary temperature of 160 °C, a capillary voltage of 60 V, and tube lens 120 V. Initial analysis was performed using data-dependent scanning in which one full MS spectrum, using a full mass range of 400-2000 atomic mass units, was followed by three MS/MS spectra. Incorporated into the method was a data-dependent scan for the neutral loss of phosphoric acid or phosphate (-98, -80), such that if these masses were found, an MS/MS/MS of the neutral loss ion was performed. Peptides were identified using a cluster-compatible version of the SEQUEST algorithm (26Eng J.K. McCormack A.L. Yates J.R.I. J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5568) Google Scholar, 27Yates J.R. Eng J.K. McCormack A.L. Schieltz D. Anal. Chem. 1995; 67: 1426-1436Crossref PubMed Scopus (1114) Google Scholar), using a mouse subset of proteins from the nonredundant data base from NCBI downloaded in January, 2004 containing 90, 197 sequences. Sequest searches were done on a high speed, multiprocessor Linux cluster in the Advanced Computing Center for Research. In addition to using the SEQUEST algorithm to search for phosphorylation on serines, threonines, or tyrosines, the data were also analyzed using the Pmod algorithm (28Hansen B.T. Davey S.W. Ham A.-J.L. Liebler D.C. J. Proteome Res. 2005; 4: 358-368Crossref PubMed Scopus (86) Google Scholar). All possible modified peptides were verified by manual inspection of the spectra. Endothelial Cell Culture and Retroviral Infection—Wild-type 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 (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, 29Jat P.S. Noble M.D. Ataliotis P. Tanaka Y. Yannoutsos N. Larsen L. Kioussis D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5096-5100Crossref PubMed Scopus (637) Google Scholar). These cells were grown at 33 °C in EGM-2 medium supplemented with interferon-γ (10 ng/ml), a permissive condition that allows the expression of SV40 T-antigen (TAg). The EphA2-deficient endothelial cells were infected with LZRS retroviruses co-expressing IRES-EphA2 (wild-type or mutant)-green fluorescent protein and sorted by a fluorescence-activated cell sorter for comparable EphA2 receptor levels. Cells were placed at physiologic temperature (37 °C) for 4 days to down-regulate thermolabile TAg before experiments. Phosphopeptide Mapping by Two-dimensional Chromatography—EphA2-null murine pulmonary microvascular endothelial cells reconstituted with either wild-type or mutant EphA2 were stimulated with ephrin-A1 for 15 min. Cells were lysed and EphA2 was immunoprecipitated and phosphorylated in the presence of [γ-32P]ATP, as described under "Immunoprecipitation, Western Blot Analysis, and Kinase Assay." Immunoprecipitates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Polyvinylidene difluoride membrane containing 32P-labeled EphA2 receptor was excised, and proteins were digested in membrane with 1 mg/ml l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin. The resulting peptide mixture was resolved in two dimensions on 20 cm × 20-cm thin layer cellulose plates by electrophoresis followed by ascending chromatography. Electrophoresis was performed at pH 1.9 in 10:1:189 acetic acid/pyridine/water for 3 h at 250 V with ∼10 p.s.i. of pressure. Ascending chromatography was carried out in 625:19:48:29: 279 isobutyric acid/n-butanol/pyridine/acetic acid/water for 11 h or until the buffer was about 1 cm from the top of the TLC plate. The plates were dried and subjected to autoradiography overnight at -70 °C with an intensifying screen. Immunoprecipitation, Western Blot Analysis, and Kinase Assay—Co-immunoprecipitation of EphA2 and Vav2/Vav3 were performed as described (23Hunter S.G. Zhuang G. Brantley-Sieders D.M. Swatt W. Cowan C.W. Chen J. Mol. Cell. Biol. 2006; 26: 4830-4842Crossref PubMed Scopus (102) Google Scholar). For co-immunoprecipitation of EphA2 with p85, COS7 cells were co-transfected with 1 μg each of EphA2 and FLAG-tagged p85 per well in a 6-well dish using Lipofectamine 2000. p85 was immunoprecipitated by FLAG-agarose beads (20 μl of beads/ml of lysate; Sigma). The resulting proteins were resolved on SDS-PAGE and Western blotted using anti-EphA2 (D7; 1:1000). Kinase assays using EphA2 as substrate were performed as described previously (30Fang W.B. Brantley-Sieders D.M. Parker M.A. Reith A.D. Chen J. Oncogene. 2005; 24: 7859-7866Crossref PubMed Scopus (112) Google Scholar). Kinase assays were also performed using a single exogenous substrate, biotinylated poly(Glu-Tyr) (1 μg/reaction) according to the manufacturer's instructions (Millipore). Briefly, following the kinase reaction, the samples were denatured by heating for 5 min. Streptavidin-agarose beads were added to the supernatant to precipitate substrates. Tyrosine phosphorylation of substrates was quantified using a scintillation counter. Vascular Assembly Assay—In vitro vascular assembly assays were performed as described 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). Briefly, 12-well plates were coated with 100 μl of growth factor reduced Matrigel (BD Biosciences). After 24-h starvation in Opti-MEM, 25,000 cells were plated in wells in the presence or absence of ephrin-A1 (1.5 μg/ml; R&D Systems) and photographed after 9 h. Images were acquired on an Olympus CK40 inverted microscope through an Optronics DEI-750C CCD video camera using Scion Image version 1.62c capture software. The degree of assembly was quantified by measuring branch length, the distance from branching point to the tip of assembled cells. Only assembled cells consisting of at least three cells were measured. The branch length in assembled endothelial cell networks was expressed as arbitrary units per ×10 field in four random fields from each well, with triplicate samples per condition, using Scion Image version 1.62c software for analysis. Guanine Nucleotide Exchange Assays—For Rac1 and Cdc42 activation assays, cells were serum-starved for 24 h in Opti-MEM medium, followed by stimulation with ephrin-A1 (1 μg/ml). Lysates were prepared and incubated with Pak-1 binding domain-GST beads according to the manufacturer's instructions (Upstate Biotechnology). Proteins were then separated by SDS-PAGE electrophoresis and transferred to a nitrocellulose filter. Activated Rac1 and Cdc42 (or total Rac1 and Cdc42 in lysates) were detected by immunoblotting using anti-Rac1 or anti-Cdc42 antibodies (BD Transduction Laboratories). Relative levels of GTP-bound Rac1 and Cdc42 were quantified by densitomitry using Scion Image version 1.62c software analysis. Transwell Migration Assay—Endothelial cells were serum-starved for 24 h in Opti-MEM medium. Transwells were coated with growth factor-reduced Matrigel (BD Biosciences; 1:20 dilution with Opti-MEM) for 30 min and blocked with 1% bovine serum albumin solution for an additional 30 min. 200,000 cells were plated in the upper chamber of the transwells, and 600 ml of Opti-MEM medium containing ephrin-A1-Fc (1 μg/ml) was added to the lower chamber. After 5 h, cells were fixed and stained with crystal violet to visualize endothelial cells. Cells that have migrated to the lower surface of transwell filters were counted in four random fields from each well, with triplicate samples per condition. Co-transplantation of Tumor Cells and Endothelial Cells—Tumor-endothelial cell co-transplantation experiments were performed as described 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). Briefly, EphA2-null endothelial cells reconstituted with either wild-type or mutant EphA2 were transduced with 1 × 108 plaque-forming units/ml Ad-β-galactosidase adenovirus. 4T1 tumor cells (50,000 cells) and Ad-β-galactosidase-transduced endothelial cells (5 × 105) were resuspended in 300 μl of growth factor-reduced Matrigel and injected into the subcutaneous dorsal flank of 10-week-old BALB/c nude female mice. Tumors were collected 7 days post-transplantation, and tumor volume was assessed using the following formula: volume = length × width 2The abbreviations used are: GEF, guanine nucleotide exchange factor; PI, phosphatidylinositol; LC, liquid chromatography; MS, mass spectrometry; SH2, Src homology 2; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; TAg, T-antigen. × 0.52. Cryosections were processed first for X-gal staining and then subjected to CD31 immunohistochemistry as described 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). Data are a representation of eight independent tumors/conditions from two independent experiments. Mapping Tyrosine Phosphorylation Sites in EphA2 Receptor—To identify the phosphorylated tyrosine residues in the cytoplasmic domain of EphA2 receptor induced upon binding to ephrin-A1 ligand, we initially expressed EphA2 in COS7 cells. Immunoprecipitated EphA2 proteins were digested with trypsin and subjected to LC-MS mass spectrometric analysis. Greater than 50% of the tryptic peptides were not detected and were therefore not analyzed. Among the remaining tryptic peptides analyzed, three phosphorylated peptides were identified that contained Tyr593 in the juxtamembrane domain as well as Tyr734 and Tyr771 in the kinase domain (Fig. 1 and Table 1).TABLE 1Tryptic peptides from in vivo phosphorylated EphA2 identified by mass spectrometryTyrosineMasscalcaMasscalc, calculated mass.MassmeasuredbMassmeasured, measured mass.ΔcΔ, difference between measured mass and calculated mass.PeptideTyr5932068.92071.12.2TYVDPHTYPO4EDPNQAVLKdOX, oxidized methionine.Tyr7341375.61375.40.2YLANMOXNYPO4VHRTyr7711761.71762.30.6VLEDDPEATYPO4TTSGGKa Masscalc, calculated mass.b Massmeasured, measured mass.c Δ, difference between measured mass and calculated mass.d OX, oxidized methionine. Open table in a new tab To verify phosphorylation sites mapped by mass spectrometry and to identify additional sites not covered by mass spectrometric analysis, we performed phosphopeptide mapping by two-dimensional chromatography in conjunction with site-directed mutagenesis. We chose to use immortalized EphA2-null and wild-type control endothelial cell lines for our analysis, since the EphA2-null background facilitates mutational analysis and subsequent functional assays. These endothelial cells were isolated from EphA2-deficient mice that were bred into the H-2Kb-tsA58 transgenic Immorto-mouse background (29Jat P.S. Noble M.D. Ataliotis P. Tanaka Y. Yannoutsos N. Larsen L. Kioussis D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5096-5100Crossref PubMed Scopus (637) Google Scholar). These Immorto-mice harbor a temperature-sensitive SV40 TAg cassette driven by the mouse major histocompatibility complex H-2Kb promoter, which permits expression in a wide array of tissues. In addition, the promoter is responsive to interferon-γ, permitting elevated expression of the TAg in cells derived from these mice when cultured at 33 °C in the presence of interferon-γ. Once cells are placed at physiologic temperature (37 °C), protein levels of the thermolabile TAg are down-regulated, and cells are restored to a nontransformed state over the course of several days (29Jat P.S. Noble M.D. Ataliotis P. Tanaka Y. Yannoutsos N. Larsen L. Kioussis D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5096-5100Crossref PubMed Scopus (637) Google Scholar). Wild-type and a panel of Tyr to Phe mutant EphA2 constructs were stably expressed in endothelial cells via retroviral transduction using the LZRS retroviral system (30Fang W.B. Brantley-Sieders D.M. Parker M.A. Reith A.D. Chen J. Oncogene. 2005; 24: 7859-7866Crossref PubMed Scopus (112) Google Scholar). In vitro kinase assays using an exogenous substrate revealed that Y593E, Y587E/Y593E, Y734F and Y771F mutations do not affect kinase activity significantly. However, Y587F and Y929F inhibited and Y593F abolished EphA2 kinase activity (Fig. 2A). Tyr587/Tyr593 and Tyr771 appeared to be major tyrosine phosphorylation sites in the EphA2 receptor, since phosphorylation of EphA2 was markedly reduced in Y587E/Y593E and Y771F mutants (Fig. 2B), despite the observation that these mutants retained kinase activity. Phosphopeptide mapping by two-dimensional chromatography detected five distinct phosphopeptides in activated wild-type EphA2 (Fig. 3, Experiment #1). To identify the phosphorylated tyrosines within the tryptic peptides, the phosphorylated tyrosines identified by mass spectrometry analysis or those tyrosine residues that were not covered were mutated to phenylalanine. These include tyrosine residues in the juxtamembrane region (Y587F, Y593F), kinase domain (Y685F, Y693F, Y734F, Y771F, Y802F, Y812F, Y816F, and Y846F), and the carboxyl terminal SAM domain (Y921F, Y929F, and Y959F). Because the Y593F could not be analyzed due to defective kinase activity resulting in insufficient γ-32P incorporation, a tyrosine to glutamic acid mutant, Y593E, that retained kinase activity was used for further analysis. Tryptic phosphopeptide maps of wild-type microvascular endothelial cells were similar to those EphA2-null cells reconstituted with wild-type EphA2 receptor (Fig. 3, Experiment #1). Each of the EphA2 mutants was deficient in certain γ-32P-labeled phosphopeptides. The Y587F mutant lacks two major phosphopeptides (a and b). Phosphopeptide c was absent in the Y593E mutant, and phosphopeptide d was absent in the Y771F mutant. Phosphorylation of Tyr734 was identified in a separate experiment when the first dimension chromatography was performed in the reverse direction (Fig. 3, Experiment #2). Taken together, these results suggest that Tyr587, Tyr593, Tyr771, and Tyr734 are likely to be autophospho-rylated in vascular endothelial cells. Vav GEFs Binds to Tyr(P)587/Tyr(P)593 in the Juxtamembrane Region, and p85 Interacts with Tyr(P)734 in the EphA2 Kinase Domain—We have previously shown that guanine nucleotide exchange factors Vav2 and Vav3 are recruited to phosphorylated EphA2 receptor, and the binding is significantly reduced in Y587F/Y593F double mutants (23Hunter S.G. Zhuang G. Brantley-Sieders D.M. Swatt W. Cowan C.W. Chen J. Mol. Cell. Biol. 2006; 26: 4830-4842Crossref PubMed Scopus (102) Google Scholar). To assess which phosphorylated tyrosine residue or whether both Tyr(P) sites in the juxtamembrane region of the EphA2 is/are required for interaction with Vav proteins, we performed a series of co-immunoprecipitation experiments coupled with Western blot analysis. As shown in Fig. 4A, mutation at either Tyr587 or Tyr593 inhibited binding of EphA2 receptor to Vav2 and Vav3 exchange factors, suggesting that both sites are required for optimal binding to Vav GEFs. Interestingly, Tyr92
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