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Etk/Bmx Transactivates Vascular Endothelial Growth Factor 2 and Recruits Phosphatidylinositol 3-Kinase to Mediate the Tumor Necrosis Factor-induced Angiogenic Pathway

2003; Elsevier BV; Volume: 278; Issue: 51 Linguagem: Inglês

10.1074/jbc.m310678200

ISSN

1083-351X

Autores

Rong Zhang, Yingqian Xu, Niklas Ekman, Zhenhua Wu, Jiong Wu, Kari Alitalo, Min Wang,

Tópico(s)

PI3K/AKT/mTOR signaling in cancer

Resumo

Tumor necrosis factor (TNF), via its receptor 2 (TNFR2), induces Etk (or Bmx) activation and Etk-dependent endothelial cell (EC) migration and tube formation. Because TNF receptor 2 lacks an intrinsic kinase activity, we examined the kinase(s) mediating TNF-induced Etk activation. TNF induces a coordinated phosphorylation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Etk, which is blocked by VEGFR2-specific inhibitors. In response to TNF, Etk and VEGFR2 form a complex resulting in a reciprocal activation between the two kinases. Subsequently, the downstream phosphatidylinositol 3-kinase (PI3K)-Akt signaling (but not signaling through phospholipase C-γ) was initiated and directly led to TNF-induced EC migration, which was significantly inhibited by VEGFR2-, PI3K-, or Akt-specific inhibitors. Phosphorylation of VEGFR2 at Tyr-801 and Tyr-1175, the critical sites for VEGF-induced PI3K-Akt signaling, was not involved in TNF-mediated Akt activation. However, TNF induces phosphorylation of Etk at Tyr-566, directly mediating the recruitment of the p85 subunit of PI3K. Furthermore, TNF- but not VEGF-induced activation of VEGFR2, Akt, and EC migration are blunted in EC genetically deficient with Etk. Taken together, our data demonstrated that TNF induces transactivation between Etk and VEGFR2, and Etk directly activates PI3K-Akt angiogenic signaling independent of VEGF-induced VEGFR2-PI3K-Akt signaling pathway. Tumor necrosis factor (TNF), via its receptor 2 (TNFR2), induces Etk (or Bmx) activation and Etk-dependent endothelial cell (EC) migration and tube formation. Because TNF receptor 2 lacks an intrinsic kinase activity, we examined the kinase(s) mediating TNF-induced Etk activation. TNF induces a coordinated phosphorylation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Etk, which is blocked by VEGFR2-specific inhibitors. In response to TNF, Etk and VEGFR2 form a complex resulting in a reciprocal activation between the two kinases. Subsequently, the downstream phosphatidylinositol 3-kinase (PI3K)-Akt signaling (but not signaling through phospholipase C-γ) was initiated and directly led to TNF-induced EC migration, which was significantly inhibited by VEGFR2-, PI3K-, or Akt-specific inhibitors. Phosphorylation of VEGFR2 at Tyr-801 and Tyr-1175, the critical sites for VEGF-induced PI3K-Akt signaling, was not involved in TNF-mediated Akt activation. However, TNF induces phosphorylation of Etk at Tyr-566, directly mediating the recruitment of the p85 subunit of PI3K. Furthermore, TNF- but not VEGF-induced activation of VEGFR2, Akt, and EC migration are blunted in EC genetically deficient with Etk. Taken together, our data demonstrated that TNF induces transactivation between Etk and VEGFR2, and Etk directly activates PI3K-Akt angiogenic signaling independent of VEGF-induced VEGFR2-PI3K-Akt signaling pathway. Although angiogenic factors such as vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGFvascular endothelial growth factorVEGFR2VEGF receptor 2ECendothelial cellsHUVEChuman umbilical veinBAECbovine ECJNKc-Jun NH2-terminal kinaseEGFRepidermal growth factor receptorMLECmouse lungTNFtumor necrosis factorPI3Kphosphatidylinositol 3-kinasePLCphospholipase CPHpleckstrin homologyEtkendothelial/epithelial tyrosine kinase. promote angiogenesis in vitro and in vivo, it has been demonstrated that inflammatory responses (as defined by the presence of infiltrated macrophages and proinflammatory cytokines) play an important role in stimulating angiogenesis (1Leibovich S.J. Polverini P.J. Shepard H.M. Wiseman D.M. Shively V. Nuseir N. 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Frausto R. Stupack D.G. Cheresh D.A. Science. 2003; 301: 94-96Crossref PubMed Scopus (293) Google Scholar). vascular endothelial growth factor VEGF receptor 2 endothelial cells human umbilical vein bovine EC c-Jun NH2-terminal kinase epidermal growth factor receptor mouse lung tumor necrosis factor phosphatidylinositol 3-kinase phospholipase C pleckstrin homology endothelial/epithelial tyrosine kinase. Endothelial cell signaling in response to proangiogenic factor VEGF has been extensively studied (14Matsumoto, T., and Claesson-Welsh, L. (2001) Science's STKE, http://www.stke.org/cgi/content/full/OC_sigtrans;2001/112/re21,Google Scholar, 15Ferrara N. Gerber H.P. LeCouter J. Nat. Med. 2003; 9: 669-676Crossref PubMed Scopus (7868) Google Scholar). VEGF primarily utilizes its receptor VEGFR2 (also Flk-1 or KDR) to induce angiogenic responses by activating a variety of signaling cascades including activation of phosphatidylinositol 3-kinase (PI3K)-Akt, phospholipase Cγ (PLC-γ) and mitogen-activated protein kinase. It has been proposed that VEGF induces autophosphorylation of VEGFR2 at several tyrosine residues which serve as docking sites for signaling molecules such as PI3K and PLC-γ. The specific tyrosine residue contributing to VEGF-induced recruitment and activation of PLC-γ by VEGFR2 remains controversial (16Wu L.W. Mayo L.D. Dunbar J.D. Kessler K.M. Baerwald M.R. Jaffe E.A. Wang D. Warren R.S. Donner D.B. J. Biol. Chem. 2000; 275: 5096-5103Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 17Takahashi T. Yamaguchi S. Chida K. Shibuya M. EMBO J. 2001; 20: 2768-2778Crossref PubMed Scopus (608) Google Scholar, 18Meyer R.D. Latz C. Rahimi N. J. Biol. Chem. 2003; 278: 16347-16355Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). However, it has been clearly shown that phosphorylation of VEGFR2 at Tyr-1054 and Tyr-1059 within the activation loop enhances its intrinsic tyrosine kinase activity (19Dougher M. Terman B.I. Oncogene. 1999; 18: 1619-1627Crossref PubMed Scopus (164) Google Scholar) and is required for VEGF-induced intracellular Ca2+ mobilization and the extracellular signal-regulated kinase activation (17Takahashi T. Yamaguchi S. Chida K. Shibuya M. EMBO J. 2001; 20: 2768-2778Crossref PubMed Scopus (608) Google Scholar). Phosphorylation of VEGFR2 at Tyr-801 and Tyr-1175 is required for binding and activation of PI3K, which is critical for subsequent activation of Akt and EC migration (20Dayanir V. Meyer R.D. Lashkari K. Rahimi N. J. Biol. Chem. 2001; 276: 17686-17692Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). In contrast, little is known for the molecular signaling pathways involved in TNF-induced angiogenesis. It has been proposed that TNF promotes angiogenesis in large through its ability to induce gene expression of proangiogenic molecules such as VEGF, ephrins, sphingosine 1-phosphate, and their receptors (11Giraudo E. Primo L. Audero E. Gerber H.P. Koolwijk P. Soker S. Klagsbrun M. Ferrara N. Bussolino F. J. Biol. Chem. 1998; 273: 22128-22135Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 12Ristimaki A. Narko K. Enholm B. Joukov V. Alitalo K. J. Biol. Chem. 1998; 273: 8413-8418Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 21Cheng N. Chen J. J. Biol. Chem. 2001; 276: 13771-13777Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 22Maceyka M. Payne S.G. Milstien S. Spiegel S. Biochim. Biophys. Acta. 2002; 1585: 193-201Crossref PubMed Scopus (503) Google Scholar). However, TNF induces EC migration and tube formation in the absence of proangiogenic factors, suggesting that TNF can directly activate EC migratory pathways (5Sarma V. Wolf F.W. Marks R.M. Shows T.B. Dixit V.M. J. Immunol. 1992; 148: 3302-3312PubMed Google Scholar, 6Frater-Schroder M. Risau W. Hallmann R. Gautschi P. Bohlen P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5277-5281Crossref PubMed Scopus (604) Google Scholar, 23Pan S. An P. Zhang R. He X. Yin G. Min W. Mol. Cell. Biol. 2002; 22: 7512-7523Crossref PubMed Scopus (115) Google Scholar). The mechanism by which TNF induces EC migratory pathways is poorly understood. We have recently shown that Etk is a critical mediator in TNF-induced EC migration and tube formation (23Pan S. An P. Zhang R. He X. Yin G. Min W. Mol. Cell. Biol. 2002; 22: 7512-7523Crossref PubMed Scopus (115) Google Scholar). The endothelial/epithelial tyrosine kinase (Etk or Bmx), a member of the Btk non-receptor tyrosine kinase family, has been implicated in cell adhesion, migration, proliferation, and survival (24Tamagnone L. Lahtinen I. Mustonen T. Virtaneva K. Francis F. Muscatelli F. Alitalo R. Smith C.I. Larsson C. Alitalo K. Oncogene. 1994; 9: 3683-3688PubMed Google Scholar, 25Qiu Y. Kung H.J. Oncogene. 2000; 19: 5651-5661Crossref PubMed Scopus (196) Google Scholar, 26Mano H. Cytokine Growth Factor Rev. 1999; 10: 267-280Crossref PubMed Scopus (118) Google Scholar). Etk and three other members of this family (Btk, Itk, and Tec), participate in signal transduction in response to virtually all types of extracellular stimuli that are transmitted by growth factor receptors, cytokine receptors, G-protein-coupled receptors, antigen receptors, and integrins (27Qiu Y. Robinson D. Pretlow T.G. Kung H.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3644-3649Crossref PubMed Scopus (213) Google Scholar, 28Chau C.H. Chen K.Y. Deng H.T. Kim K.J. Hosoya K. Terasaki T. Shih H.M. Ann D.K. Oncogene. 2002; 21: 8817-8829Crossref PubMed Scopus (51) Google Scholar, 29Tsai Y.T. Su Y.H. Fang S.S. Huang T.N. Qiu Y. Jou Y.S. Shih H.M. Kung H.J. Chen R.H. Mol. Cell. Biol. 2000; 20: 2043-2054Crossref PubMed Scopus (111) Google Scholar, 30Mao J. Xie W. Yuan H. Simon M.I. Mano H. Wu D. EMBO J. 1998; 17: 5638-5646Crossref PubMed Scopus (86) Google Scholar, 31Chen R. Kim O. Li M. Xiong X. Guan J.L. Kung H.J. Chen H. Shimizu Y. Qiu Y. Nat. Cell Biol. 2001; 3: 439-444Crossref PubMed Scopus (133) Google Scholar). They share a common structure domain including a pleckstrin homology (PH) domain, TEC homology domain, which has a PXXP motif (with exception of Etk), an SH3 and an SH2 domain, and a kinase domain. It has been proposed that intramolecular interactions between the PXXP motif in TEC homology domain and the SH3 domain and between the PH domain and the kinase domain fold Btk family kinases into a “closed” form (25Qiu Y. Kung H.J. Oncogene. 2000; 19: 5651-5661Crossref PubMed Scopus (196) Google Scholar, 26Mano H. Cytokine Growth Factor Rev. 1999; 10: 267-280Crossref PubMed Scopus (118) Google Scholar). Based on data from Etk activation by focal adhesion kinase, it has been proposed that integrin-induced binding of Etk to focal adhesion kinase leads to phosphorylation of Etk at Tyr-40 to open up the closed conformation of the inactive Etk and allow the kinase to be phosphorylated by Src family kinases at the highly conserved tyrosine residue Tyr-566 in the catalytic domain (25Qiu Y. Kung H.J. Oncogene. 2000; 19: 5651-5661Crossref PubMed Scopus (196) Google Scholar, 31Chen R. Kim O. Li M. Xiong X. Guan J.L. Kung H.J. Chen H. Shimizu Y. Qiu Y. Nat. Cell Biol. 2001; 3: 439-444Crossref PubMed Scopus (133) Google Scholar). However, TNFR2 has no kinase activity, and it is not clear how Etk is activated in response to TNF. We propose that TNF induces recruitment of additional kinase(s) (23Pan S. An P. Zhang R. He X. Yin G. Min W. Mol. Cell. Biol. 2002; 22: 7512-7523Crossref PubMed Scopus (115) Google Scholar). The aim of this study is to identify the kinase for Etk activation by TNF. We show that TNF uses Etk to transactivate VEGFR2, which is in turn required for Etk activation by TNF. Thus, Etk and VEGFR2 serve as a mutual activator and effector in TNF pathway. In the TNF response, phosphorylation of Etk, but not of VEGFR2, is a critical mediator in recruiting PI3K and activation of Akt. Etk acts upstream of VEGFR2 and PI3K-Akt to specifically mediate TNF-induced angiogenic signaling pathway distinct from that induced by VEGF. Materials—Mammalian expression plasmids for Etk/Bmx wild type and mutants were provided by Dr. Dianqing Wu (University of Connecticut) and those for VEGFR2 (Flk-1/KDR) were provided by Dr. Bruce Terman (Albert Einstein College of Medicine, New York). The VEGFR2 and Etk point mutants were generated by the QuikChange™ site-specific mutagenesis kit followed the Manufacturer's instructions (Stratagene). VEGF receptor tyrosine kinase inhibitor (4-[4′-chloro-2′-fluoro)phenylamino]-6,7-dimethoxyquinazoline), SU1498, AG1478, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), LY29294002, SP600125, Akt inhibitor, and U73122 were purchased from Calbiochem. Anti-phospho-Akt (Ser-473), anti-Akt, anti-phospho-PLC-γ (Tyr(P)-783), and anti-PLC-γ were from Cell Signaling (Beverly, MA). Anti-phosphotyrosine 4G10 (Tyr(P)-4G10) was from Upstate Biotechnology. Anti-FLAG M2 antibody was from Sigma, and anti-T7 was from Novagen. Anti-VEGFR2, anti-Etk/Bmx, anti-p85 (PI3K), anti-JNK1, and protein A/G PLUS-agarose were from Santa Cruz (Santa Cruz, CA). Generation of Antibodies against Phospho-VEGFR2 and Phospho-Etk—Polyclonal antibodies directed against specific phospho-VEGFR2 and phospho-ETK were produced by immunizing rabbits with one of three synthetic phospho-peptides corresponding to residues surrounding human VEGFR2 Tyr-1054/1059 and Tyr-1175 and human ETK Tyr-566. The peptide sequences are RDIpYKDPDpYVRKG, QQDGKDpYIVLPISE, and VLDDQpYVSSVGT, respectively, where pY indicates phospho-tyrosine. The peptides were synthesized with N-terminal cysteine residues and coupled to KLH for immunization. The antibodies were affinity-purified from rabbit antisera by affinity chromatography steps using protein A columns to purify immunoglobulins followed by specific phospho-peptide (immunogen) columns to obtain the phospho-VEGFR2(Tyr-1054/1059), phospho-VEGFR2(Tyr-1175), and phospho-ETK(Tyr-566) affinity-purified antibodies employed in this study. Cell and Cytokines—Human umbilical vein EC (HUVEC) and bovine aorta endothelial cells (BAEC) were purchased from Clonetics (San Diego, CA). Lung endothelial cells isolated from Etk/Bmx-deficient mice were isolated according to a procedure described (23Pan S. An P. Zhang R. He X. Yin G. Min W. Mol. Cell. Biol. 2002; 22: 7512-7523Crossref PubMed Scopus (115) Google Scholar). Minced tissue was digested with collagenase. Large tissue fragments were removed by filtration through a 100-mesh nylon screen. The filtrate was collected on a 20-mesh nylon screen, washed, then purified using Percoll gradient centrifugation. EC contained in the 3rd through 10th fractions from the top of the gradient (density 1.00-1.050 g/ml) were collected, washed, and then seeded into dishes containing EC growth medium. Contaminating non-EC were removed by mechanical weeding and by fluorescence activated cell sorting using antibodies to platelet endothelial cell adhesion molecule-1 (CD31) to label the EC. The sorted cells were assessed for EC phenotype, including morphology, and expression of von Willebrand Factor and PECAM-1. EC were used at passages 1-5. Human and murine recombinant TNF and human VEGF165 were from R&D Systems (Minneapolis, MN). Transfection—Transfection of BAEC was performed by LipofectAMINE 2000 according to the manufacturer's protocol (Invitrogen). Cells were cultured at 90% confluence in 6-well plates and transfected with total 4 μg of plasmid constructs as indicated. Cells were harvested at 36-48 h post-transfection, and cell lysates were used for protein assays. JNK Kinase Assay—JNK assays were performed as described previously using glutathione S-transferase-c-Jun-(1-80) fusion protein as a substrate (32Liu Y. Yin G. Surapisitchat J. Berk B.C. Min W. J. Clin. Invest. 2001; 107: 917-923Crossref PubMed Scopus (94) Google Scholar). Briefly, a total of 400 μg of cell lysates were immunoprecipitated with 5 μg of antibody against JNK1 (Santa Cruz). The immunoprecipitates were mixed with 10 μg of glutathione S-transferase-c-Jun-(1-80) suspended in the kinase buffer (20 mm Hepes, pH 7.6, 20 mm MgCl2, 25 mm β-glycerophosphate, 100 μm sodium orthovanadate, 2 mm dithiothreitol, 20 μm ATP) containing 1 μl (10 μCi) of [γ-32P]ATP. The kinase assay was performed at 25 °C for 30 min. The reaction was terminated by the addition of Laemmli sample buffer, the products were resolved by SDS-PAGE (12%), and the phosphorylated glutathione S-transferase-c-Jun (1-80) was visualized by autoradiography. The JNK1 protein was determined by Western blot with anti-JNK1. Immunoprecipitation and Immunoblotting—BAEC after various treatments were washed twice with cold phosphate-buffered saline and harvested in a membrane lysis buffer (30 mm Tris, pH 8, 10 mm NaCl, 5 mm EDTA, 10 g/liter polyoxyethylene-8-lauryl ether, 1 mmO-phenanthroline, 1 mm indoacetamide, 10 mm NaF, 5 mm orthovanadate, 10 mm sodium pyrophosphate). Cells were immediately frozen in liquid nitrogen. Cell lysates were then thaw on ice, scraped, sonicated, and centrifuged at 14,000 × g at 4 °C for 15 min. Supernatants were used immediately for immunoblot or immunoprecipitation. For immunoprecipitation to analyze protein interaction in vivo, supernatants of cell lysates were diluted 3 times with a cold lysis buffer (50 mm Tris-HCl, pH 7.6, 150 mm NaCl, 0.1% Triton X-100, 0.75% Brij 96, 1 mm sodium orthovanadate, 1 mm sodium fluoride, 1 mm sodium pyrophosphate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 mm phenylmethylsulfonyl fluoride, 1 mm EDTA). The lysates were then incubated with the first protein-specific antiserum (e.g. anti-Etk or anti-VEGFR2) on ice for 1.5 h. Then 10 μl of protein A/G PLUS-agarose was added and incubated for 2 h with rotation. Immune complexes were collected after each immunoprecipitation by centrifugation at 13,000 × g for 10 min followed by 3-5 washes with lysis buffer. The immune complexes were subjected to SDS-PAGE followed by immunoblot with the second protein (e.g. phosphotyrosine antibody, Upstate, NY). The chemiluminescence was detected using an ECL kit according to the instructions of the manufacturer (Amersham Biosciences). For detection of FLAG-tagged proteins (Etk mutants) and T7-tagged proteins (e.g. Etk), anti-FLAG M2 antibody and anti-T7 were used for immunoblots, respectively. EC Migration Assay and Image Analysis—EC migration was performed as described previously (23Pan S. An P. Zhang R. He X. Yin G. Min W. Mol. Cell. Biol. 2002; 22: 7512-7523Crossref PubMed Scopus (115) Google Scholar). Briefly, BAEC were cultured in 0.5% fetal bovine serum overnight and subjected to “wound injury” with a yellow tip. Cells were washed with phosphate-buffered saline once, and fresh media (0.5% fetal bovine serum) with or without TNF (1 ng/ml) were added. Cells were further cultured for the indicated times. The EC migration in culture was determined by measuring wound areas in cell monolayers. Three different images from each well along the wound were captured by a digital camera under a microscope (4×). A hemocytometer (1 mm2/grid) was used as a standard. Wound area (mm2) was measured and analyzed by NIH Image 1.60. Statistical analyses were performed with StatView 4.0 package (ABACUS Concepts). Data are presented as means (±S.D.). Differences were analyzed by an unpaired two-tailed Student t test. Values of p < 0.05 were taken as significant. TNF Induces a Coordinated Phosphorylation of VEGFR2 and Etk in a VEGFR2 Kinase-dependent Manner—Because TNFR2 has no intrinsic kinase activity, we reasoned that another tyrosine kinase(s) is involved in TNF/TNFR2-induced Etk activation. To define the kinase(s) responsible for Etk activation, we examined the effect of various kinase inhibitors on TNF-induced Etk activation. Human EC (HUVEC) were preincubated with inhibitors specific to EGFR (AG1478, 1 μm), VEGFR (SU1498, 30 μm or VEGF receptor tyrosine kinase inhibitor, 10 μm), or Src family protein kinases (PP2, 10 μm) for 30 min followed by stimulation with TNF (1 ng/ml for 15 min). Etk activation was determined by immunoprecipitation with anti-Etk followed by Western blot with anti-phosphotyrosine (Tyr(P)). Results show that TNF-induced Etk activation was blocked by inhibitors of VEGFR2 but not of EGFR or Src (Fig. 1a). As a control, TNF-induced JNK activation was not blocked by inhibitors to VEGFR2, EGFR, or Src but could be blocked by JNK-specific inhibitor SP600125 (20 μm) (Fig. 1b). It is well known that VEGFR2 activation by VEGF involves receptor tyrosine phosphorylations. To determine whether TNF also induces VEGFR2 phosphorylation, HUVEC were treated with TNF (1 ng/ml) or VEGF (10 ng/ml) for various time points (1, 2, 5, 15 min). VEGFR2 phosphorylation was determined by immunoprecipitation by anti-VEGFR2 followed by Western blot with anti-phosphotyrosine. Consistent with previous reports, VEGF rapidly induces activation of VEGFR2 (peaks at 1 min). In contrast, TNF induces phosphorylation of VEGFR2 in a delayed kinetics (peaks at 15 min) (Fig. 1c) and sustained for 2 h (not shown). To determine whether TNF, like VEGF, induced VEGFR2 autophosphorylation, HUVEC were treated with TNF (1 ng/ml for 15 min) or VEGF (10 ng/ml for 2 min) in the presence of various inhibitors as indicated. VEGFR2 phosphorylation induced by TNF (as well as VEGF) was specifically blocked by VEGFR2 inhibitors (but not by EGFR inhibitor) (Fig. 1d), suggesting that TNF induces a transactivation of VEGFR2 primarily through VEGFR2 autophosphorylation. Similar results were obtained for TNF-induced VEGFR2 and Etk activation in BAEC (data not shown). These data demonstrate a link between VEGFR2 transactivation and Etk activation by TNF. Association of Etk with VEGFR2 Stimulates a Reciprocal Activation between Etk and VEGFR2—To determine the mechanism by which TNF induces VEGFR2 transactivation, which is required for TNF-induced Etk activation, we examined if there is an association of Etk with VEGFR2. HUVEC were treated with TNF (1 ng/ml for 15 min) or VEGF (10 ng/ml for 2 min), and association of VEGFR2 with Etk was determined by immunoprecipitation with anti-VEGFR2 followed by Western blot with anti-Etk. TNF, but not VEGF, strongly induced Etk·VEGFR2 complex formation (Fig. 2a). Association of VEGFR2 and Etk was also observed in BAEC in response to TNF (data not shown). To determine effects of VEGFR2-Etk association on their phosphorylations, Etk and VEGFR2 were co-transfected into BAEC (which has a high transfection efficiency compared with HUVEC). Phosphorylation of VEGFR2 and Etk was determined by immunoprecipitation with anti-VEGFR2 or anti-Etk followed by Western blot with anti-phosphotyrosine (4G10). VEGFR2 showed a basal phosphorylation, likely resulting from an autoactivation upon overexpression. Co-expression of Etk-WT and the constitutively active Etk (Etk-SK containing the SH2 and the kinase domains) enhanced VEGFR2 tyrosine phosphorylation. In contrast, the dominant negative forms of Etk (Etk-KD with a single mutation in the kinase domain or Etk-DK with deletion of kinase domain) decreased autoactivation of VEGFR2 (Fig. 2b). As a control VEGFR2-KM (the kinase-inactive mutant) did not show either the basal or Etk-enhanced phosphorylation (Fig. 2b). These data suggest that Etk induces VEGFR2 autoactivation in Etk kinase-dependent manner. The dominant negative effects of Etk-KD and Etk-DK in EC likely resulted from inhibition of endogenous Etk activity induced by VEGFR2 overexpression. We then examined Etk phosphorylation by VEGFR2. FLAG-tagged Etk was transfected into BAEC in the presence VEGFR2-WT or VEGFR2-KM. Etk tyrosine phosphorylation was determined by immunoprecipitation with anti-FLAG followed by Western blot with anti-phosphotyrosine. Expression of VEGFR2-WT, but not VEGFR2-KM, induces phosphorylation of Etk (Fig. 2c), further confirming that VEGFR2 kinase activity is required for Etk phosphorylation. TNF Induces VEGFR2 and PI3K-dependent Activation of Akt but Not of PLC-γ—VEGF through VEGFR2 activates PI3K-Akt and PLC-γ, two independent signaling pathways were implicated in angiogenesis. To determine whether TNF transactivates VEGFR2, leading to activation of the downstream signaling pathways, BAEC were treated with TNF or VEGF as indicated. The phospho-specific antibodies were used to determine activation of Akt (Ser(P)-384) and PLC-γ (Tyr(P)-783) by Western blot. As a control, VEGF induces activation of both Akt and PLC-γ (Fig. 3a). In contrast, TNF activated Akt but not PLC-γ (Fig. 3a). To examine if TNF-induced Akt is dependent on VEGFR2 and PI3K, BAEC were pretreated with a specific inhibitor to VEGFR2 (SU1498, 10 μm), PI3K (LY294002, 30 μm), or (AG1478, 1 μm) for 30 min followed by TNF treatment (1 ng/ml for 15 min). TNF-induced Akt activation was blocked by the inhibitors specific to VEGFR2 and PI3K but not by the inhibitor to EGFR (Fig. 3b). These data indicate that TNF induces VEGFR2 transactivation sufficient to activate PI3K-Akt but not PLC-γ signaling. Previously we demonstrated that Etk is a critical mediator in TNF-induced EC migration and tube formation (23Pan S. An P. Zhang R. He X. Yin G. Min W. Mol. Cell. Biol. 2002; 22: 7512-7523Crossref PubMed Scopus (115) Google Scholar). To determine whether TNF/Etk-induced VEGFR2-PI3K-Akt signaling contributes to TNF-induced angiogenesis, we determined the effects of VEGFR2, PI3K, and Akt-specific inhibitors on TNF-induced EC migration in an in vitro migration assay. Monolayer culture of BAEC was subjected to wound injury and incubated for indicated times (12-24 h) in the presence of TNF (1 ng/ml) with specific inhibitors to EGFR (AG1478, 1 μm), VEGFR2 (30 μm SU1498 or 10 μm VEGF receptor tyrosine kinase inhibitor), PI3K (LY294002, 30 μm), Akt (Akt inhibitor, 30 μm), or PLC-γ (U73122, 30 μm). EC migration was determined as described previously (23Pan S. An P. Zhang R. He X. Yin G. Min W. Mol. Cell. Biol. 2002; 22: 7512-7523Crossref PubMed Scopus (115) Google Scholar). PI3K inhibitor caused EC death, consistent with the data that inhibition of PI3K synergizes TNF-induced EC apoptosis (33Madge L.A. Pober J.S. J. Biol. Chem. 2000; 275: 15458-15465Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). EGFR inhibitor (AG1478) or PLC-γ inhibitor (U73122) did not block TNF-induced EC migration, consistent with the fact that AG1478 does not block TNF-induced VEGFR2-Akt pathway and that TNF does not induce PLC-γ activation. However, inhibitors to VEGFR2, PI3K, and Akt significantly block BAEC migration (Fig. 3c). These data demonstrate that VEGFR2, PI3K, and Akt are critical for TNF-induced EC migration. The Tyr(P)-1175 of VEGFR2 Is Not Critical for TNF-induced Akt Activation—To define the molecular mechanism by which TNF induces a distinct VEGFR2 downstream pathway from that by VEGF, we reasoned that TNF could induce a different site-specific phosphorylation of VEGFR2. To test this hypothesis, we examined TNF-induced VEGFR2 phosphorylation using the antibodies against site-specific phosphotyrosine (Tyr(P)-1175 and Tyr(P)-1054/1059) on VEGFR2. We first verified the antibody specificity by mutant Flk-1 with mutations at specific phosphotyrosine residues. VEGFR2 mutants (VGFR2-WT, KM, Y1175F, Y801F/Y1175F, Y1054F/Y1059F) were transfected into 293T cells (where no endogenous VEGFR2 was detected), and total phosphorylation of VEGFR2 was detected by

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