The Vascular Endothelial Growth Factor Receptor KDR Activates Multiple Signal Transduction Pathways in Porcine Aortic Endothelial Cells
1997; Elsevier BV; Volume: 272; Issue: 51 Linguagem: Inglês
10.1074/jbc.272.51.32521
ISSN1083-351X
AutoresJens Krøll, Johannes Waltenberger,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoVascular endothelial growth factor A (here referred to as VEGF) is an endothelium-specific growth factor that binds to two distinct receptor tyrosine kinases, designated Flt-1 and KDR/Flk-1. VEGF stimulates autophosphorylation of both receptors, but little is known about their signal transduction properties. In this study, we used porcine aortic endothelial (PAE) cells overexpressing KDR (PAE/KDR) to evaluate the interaction of KDR with intracellular proteins and compared them with Flt-1-expressing PAE cells (PAE/Flt-1). VEGF-induced stimulation of KDR results in the association and phosphorylation of the 46-, 52-, and 66-kDa isoforms of Shc and the induction of Shc-Grb2 complex formation. In a similar fashion, KDR associates with Grb2 and Nck in a ligand-dependent fashion, suggesting Shc, Grb2, and Nck as potential candidates involved in the regulation of endothelial function. Another strong candidate is mitogen-activated protein (MAP) kinase, which is strongly activated in response to VEGF stimulation as demonstrated by phosphorylation of the specific substrate myelin basic protein. Inhibition of MAP kinase activation by PD98059, a specific MAP kinase kinase inhibitor, results in inhibition of VEGF-induced proliferation of PAE/KDR cells. In contrast, VEGF-induced stimulation of Flt-1 does not activate MAP kinase in PAE/Flt-1 cells. In this study we provide the first two examples of molecules potentially capable of functionally counteracting the endothelial response to VEGF, namely SHP-1 and SHP-2. These two SH2 protein-tyrosine phosphatases physically associate with KDR secondary to VEGF stimulation, raising the interesting possibility that both molecules participate in the generation and/or modulation of VEGF-induced signals. Taken together, our results substantially broaden the spectrum of KDR-associating molecules, indicating that endothelial function and angiogenesis are regulated by a diverse network of signal transduction cascades. Vascular endothelial growth factor A (here referred to as VEGF) is an endothelium-specific growth factor that binds to two distinct receptor tyrosine kinases, designated Flt-1 and KDR/Flk-1. VEGF stimulates autophosphorylation of both receptors, but little is known about their signal transduction properties. In this study, we used porcine aortic endothelial (PAE) cells overexpressing KDR (PAE/KDR) to evaluate the interaction of KDR with intracellular proteins and compared them with Flt-1-expressing PAE cells (PAE/Flt-1). VEGF-induced stimulation of KDR results in the association and phosphorylation of the 46-, 52-, and 66-kDa isoforms of Shc and the induction of Shc-Grb2 complex formation. In a similar fashion, KDR associates with Grb2 and Nck in a ligand-dependent fashion, suggesting Shc, Grb2, and Nck as potential candidates involved in the regulation of endothelial function. Another strong candidate is mitogen-activated protein (MAP) kinase, which is strongly activated in response to VEGF stimulation as demonstrated by phosphorylation of the specific substrate myelin basic protein. Inhibition of MAP kinase activation by PD98059, a specific MAP kinase kinase inhibitor, results in inhibition of VEGF-induced proliferation of PAE/KDR cells. In contrast, VEGF-induced stimulation of Flt-1 does not activate MAP kinase in PAE/Flt-1 cells. In this study we provide the first two examples of molecules potentially capable of functionally counteracting the endothelial response to VEGF, namely SHP-1 and SHP-2. These two SH2 protein-tyrosine phosphatases physically associate with KDR secondary to VEGF stimulation, raising the interesting possibility that both molecules participate in the generation and/or modulation of VEGF-induced signals. Taken together, our results substantially broaden the spectrum of KDR-associating molecules, indicating that endothelial function and angiogenesis are regulated by a diverse network of signal transduction cascades. Vascular endothelial growth factor A is a dimeric endothelium-specific growth factor (1Leung D.W. Cachianes G. Kuang W.-J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4549) Google Scholar, 2Keck P.J. Hauser S.D. Krivi G. Sanzo K. Warren T. Feder J. Connolly D.T. Science. 1989; 246: 1309-1312Crossref PubMed Scopus (1866) Google Scholar, 3Plouët J. Schilling J. Gospodarowicz D. EMBO J. 1989; 8: 3801-3806Crossref PubMed Scopus (440) Google Scholar). It stimulates mitogenicity (4Connolly D.T. Heuvelman D.M. 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Res. 1994; 28: 1176-1179Crossref PubMed Scopus (416) Google Scholar), indicating that VEGF 1The abbreviations used are: VEGF, vascular endothelial growth factor; GAP, Ras GTPase-activating protein; PAE, porcine aortic endothelial; MAP, mitogen-activated protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PDGF, platelet-derived growth factor. is a major angiogenic factor. The VEGF gene encodes four different proteins (VEGF121, VEGF165, VEGF189, and VEGF206) (10Tischer E. Mitchell R. Hartmann R. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar) as a result of alternative splicing. Recently, two novel and distinct VEGF-A-related genes were identified, namely VEGF-B (11Olofsson B. Pajusola K. Kaipainen A. Voneuler G. Joukov V. Saksela O. Orpana A. Petersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (637) Google Scholar) and VEGF-C (12Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1177) Google Scholar). The functional roles of VEGF-B and VEGF-C remain to be elucidated. VEGF-A binds to two different receptor tyrosine kinases, designated Flt-1 ( f ms-like tyrosine kinase-1; VEGF receptor-1) (13Shibuya M. Yamaguchi S. Yamane A. Ikeda T. Tojo A. Matsushime H. Sato M. Oncogene. 1990; 5: 519-524PubMed Google Scholar, 14de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1903) Google Scholar) and KDR (kinase-insert domain-containingreceptor; VEGF receptor-2) (15Terman B.I. Carrion M.E. Kovacs E. Rasmussen B.A. Eddy R.L. Shows T.B. Oncogene. 1991; 6: 1677-1683PubMed Google Scholar, 16Terman B.I. Dougher-Vermazen M. Carrion M.E. Dimitrov D. Armellino D.C. Gospodarowicz D. Böhlen P. Biochem. Biophys. Res. Commun. 1992; 187: 1579-1586Crossref PubMed Scopus (1418) Google Scholar), the human homologue of mouse Flk-1 (17Matthews W. Jordan C.T. Gavin M. Jenkins N.A. Copeland N.G. Lemischka I.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9026-9030Crossref PubMed Scopus (467) Google Scholar). Flt-4 is a structurally related receptor that binds VEGF-C (12Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1177) Google Scholar), but not VEGF-A (18Aprelikova O. Pajusola K. Partanen J. Armstrong E. Alitalo R. Bailey S.K. McMahon J. Wasmuth J. Huebner K. Alitalo K. Cancer Res. 1992; 52: 746-748PubMed Google Scholar). Each of these receptors consists of an extracellular domain containing seven immunoglobulin-like motifs, a transmembrane domain, a juxtamembrane domain, a carboxyl-terminal tail, and a tyrosine kinase domain, which is interrupted by a long kinase-insert domain consisting of >100 amino acids. Binding of VEGF-A (referred to as VEGF below) induces conformational change in KDR and Flt-1, followed by dimerization and autophosphorylation on tyrosine residues (5Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 19Heldin C.-H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1459) Google Scholar). The phosphorylation on tyrosine residues makes them become targets for SH2, SH3, and phosphotyrosine-binding domain-containing molecules, which frequently become phosphorylated themselves. Thereafter, consecutive intracellular signal transduction events are triggered (20Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1335) Google Scholar, 21Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2257) Google Scholar). Three classes of SH2 domain-containing molecules are currently known: (i) molecules without any catalytic activity named adapter molecules; (ii) SH2 domain-containing enzymes including cytoplasmic tyrosine kinases, phospholipase Cγ, GAP, and protein-tyrosine phosphatases SHP-1 and SHP-2 (22Adachi M. Fischer E.H. Ihle J. Imai K. Jirik F. Neel B. Pawson T. Shen S.H. Thomas M. Ullrich A. Zhao Z.Z. Cell. 1996; 85: 15Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) as well as nucleotide exchange factors; and (iii) structural proteins (23Hunter T. Biochem. Soc. Trans. 1996; 24: 307-327Crossref PubMed Scopus (80) Google Scholar). So far, only limited information is available concerning function and signal transduction of VEGF receptors. KDR is able to induce striking changes in cell morphology, actin reorganization, membrane ruffling, mitogenicity, and chemotaxis in porcine aortic endothelial (PAE) cells overexpressing KDR (PAE/KDR) in response to VEGF stimulation (5Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). In molecular terms, VEGF stimulation of KDR-expressing cells results in the phosphorylation of GAP, members of the Src family of protein kinases, and phospholipase Cγ as well as of p42 MAP kinase as determined by an in vitro kinase assay and Western blot analysis (5Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 24Waltenberger J. Mayr U. Pentz S. Hombach V. Circulation. 1996; 94: 1647-1654Crossref PubMed Scopus (235) Google Scholar, 25Takahashi T. Shibuya M. Oncogene. 1997; 14: 2079-2089Crossref PubMed Scopus (275) Google Scholar). In the case of Flt-1, the information about cellular function and mechanism of action is even further limited. Flt-1 mediates chemotactic activity in monocytes and stimulates tissue factor expression in monocytes and endothelial cells (26Clauss M. Weich H. Breier G. Knies U. Rockl W. Waltenberger J. Risau W. J. Biol. Chem. 1996; 271: 17629-17634Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar). However, VEGF stimulation of Flt-1 does not induce mitogenicity and chemotaxis in Flt-1-expressing PAE cells (PAE/Flt-1) (5Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). VEGF stimulation of Flt-1-expressing cells, however, results in autophosphorylation of Flt-1 and phosphorylation of members of the Src family of protein kinases, GAP, phospholipase Cγ, and Shc proteins (5Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 27Seetharam L. Gotoh N. Maru Y. Neufeld G. Yamaguchi S. Shibuya M. Oncogene. 1995; 10: 135-147PubMed Google Scholar). Both VEGF receptors play important roles in developmental angiogenesis. Mouse knockout experiments in KDR/Flk-1 cells and Flt-1 resulted in the generation of distinct developmentally lethal phenotypes. While Flt-1−/− mice showed defects in vessel formation (28Fong G.-H. Rossant J. Gertsenstein M. Breitman M.L. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2236) Google Scholar), the Flk-1−/− knockout did not produce endothelial cells at all (29Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.-F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3395) Google Scholar). In this study we used PAE/KDR cells in culture for the analysis of KDR signal transduction. Using this approach, we are able to demonstrate that VEGF stimulation of KDR results in the activation of multiple intracellular signal transduction pathways. We have shown the ligand-induced phosphorylation of all three isoforms of Shc proteins and the phosphorylation of GAP as well as the strong activation of MAP kinase. In addition, KDR is shown to associate with Shc, Shc-Grb2, Nck, Grb2, and GAP. Moreover, we demonstrate that KDR associates with at least two distinct protein-tyrosine phosphatases, namely SHP-1 and SHP-2, which are potentially involved in either negative regulation (i.e. dephosphorylation) or positive regulation (i.e. signal enhancement) of VEGF-induced signals. PAE cells transfected with a modified pcDNAI vector carrying KDR cDNA or Flt-1 cDNA were used as described previously (5Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). Nontransfected PAE cells served as controls. Cells were cultivated in Ham's F-12 medium (Biochrom) containing 10% fetal calf serum, glutamine, and antibiotics at 37 °C and 5% CO2. For starvation of cells, serum-free medium was supplemented with 0.01 mg/ml bovine serum albumin. The polyclonal antiserum against KDR (NEF) directed against the 25 N-terminal amino acid residues of the kinase-insert region of KDR (amino acids 935–959) was used as described previously (5Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). Recombinant human VEGF165 was obtained from Denis Gospodarowicz (Chiron). The horseradish peroxidase-conjugated monoclonal antibody RC20H (Transduction Laboratories) was used to detect phosphotyrosine residues. The polyclonal antibody recognizing MAP kinase was obtained from Upstate Biotechnology, Inc.; the polyclonal antibody against Shc and the monoclonal antibodies against Grb2, Nck, SHP-1, and SHP-2 were from Transduction Laboratories, as were the human HeLa cell lysate and Jurkat cell lysate that served as positive controls for Nck and for both phosphatases, respectively. The antiserum directed against GAP was obtained from Julian Downward (Imperial Cancer Research Fund, London, United Kingdom.) For detection of primary antibodies in Western blot analyses, peroxidase-conjugated rabbit (Amersham Corp.) and mouse (Dako Corp.) immunoglobulins were used. Anti-mouse IgG (Sigma) was used for binding of primary antibodies to protein A-Sepharose in immunoprecipitation. Myelin basic protein was provided by Fluka, PD98059 was obtained from Calbiochem, and tyrphostin AG1433 was a kind gift from Alexander Levitzki (Hebrew University, Jerusalem, Israel). PD98059 and tyrphostin AG1433 were dissolved in Me2SO. Subconfluent cells were starved overnight in serum-free medium containing 0.01 mg/ml bovine serum albumin and preincubated for 5 min with 100 μm Na3VO4 to inhibit phosphatase activity. Cells were stimulated for 5 min at 37 °C with 50 ng/ml VEGF. After washing with ice-cold phosphate-buffered saline containing 100 μmNa3VO4, cells were solubilized in lysis buffer containing 150 mm NaCl, 20 mm Tris-HCl, pH 7.4, 1% CHAPS (Sigma), 10 mm EDTA, 10% glycerol, 100 μm Na3VO4, 1% Trasylol® (Bayer), and 1 mmphenylmethylsulfonyl fluoride. The cell lysates were centrifuged at 10,000 × g for 15 min, and the supernatants were precleared for 1 h at 4 °C using 25 μl of protein A-Sepharose CL-4B (Pharmacia Biotech Inc.). The supernatants were used for immunoprecipitation either with the receptor-specific antiserum NEF or with antisera recognizing Shc, Grb2, Nck, GAP, MAP kinase, SHP-1, or SHP-2. Immunoprecipitates immobilized on protein A-Sepharose CL-4B were used for the immune complex kinase assay, which was carried out for 7 min at room temperature in 25 μl of 50 mm HEPES, pH 7.4, containing 10 mm MnCl2, 1 mmdithiothreitol, and 5 μCi of [γ-32P]ATP (Amersham Corp.). The MAP kinase assay was carried out under similar conditions; however, the kinase reaction was allowed to stand for 10 min at 30 °C in the presence of 1 μg/μl myelin basic protein. The samples were analyzed by SDS-PAGE using different polyacrylamide concentrations. Following electrophoresis, the gels were incubated for 30 min in 2.5% glutaraldehyde, washed two times for 15 min with 10% acetic acid and 40% methanol, treated for 1 h at 55 °C in 1m KOH to remove serine-bound phosphate (30Cooper J.A. Hunter T. Mol. Cell. Biol. 1981; 1: 165-178Crossref PubMed Scopus (283) Google Scholar), washed three times for 20 min with 10% acetic acid and 40% methanol, dried, and exposed to Hyperfilm MP (Amersham Corp.). Subconfluent cells were starved overnight in serum-free medium and preincubated for 5 min at 37 °C with 100 μmNa3VO4 to inhibit phosphatase activity. Cells were stimulated for 5 min at 37 °C with 50 ng/ml VEGF. After washing with ice-cold phosphate-buffered saline containing 100 μmNa3VO4, cells were solubilized in lysis buffer containing 150 mm NaCl, 20 mm Tris-HCl, pH 7.4, 1% Nonidet P-40 (Sigma), 10 mm EDTA, 10% glycerol, 100 μm Na3VO4, 1% Trasylol, 1 mm phenylmethylsulfonyl fluoride, and 1 mm zinc acetate. The cell lysates were centrifuged at 10,000 ×g for 15 min, and the supernatants were precleared for 1 h at 4 °C using 25 μl of protein A-Sepharose CL-4B. The supernatants were used for immunoprecipitation with antisera recognizing Shc, Nck, Grb2, SHP-1, or SHP-2. Immunoprecipitates were subjected to SDS-PAGE and blotted onto a nitrocellulose membrane (Hybond C extra®, Amersham Corp.). Phosphorylated proteins were detected by immunoblotting using the horseradish peroxidase-conjugated anti-phosphotyrosine antibody RC20H. Individual proteins were identified with their specific antibodies. For their detection, peroxidase-conjugated rabbit anti-mouse Ig (Dako Corp.) was used as a secondary antibody, followed by a chemiluminescence-based detection system (ECL, Amersham Corp.). Reprobing of blots was done after incubation of the membrane for 30 min at 50 °C in stripping buffer containing 100 mm β-mercaptoethanol, 62.5 mm Tris-HCl, pH 6.7, and 2% SDS, followed by incubation with another specific antibody. Cells were seeded sparsely in 12-well culture dishes. After 24 h, cells were washed two times with 0.01% bovine serum albumin and Ham's F-12 medium and incubated for an additional 48 h with one renewal of medium. Cells were incubated for 30 min with different concentrations of PD98059 or with the solvent Me2SO alone and stimulated with 1 ng/ml VEGF for 20 h, followed by the addition of 0.25 μCi/ml [3H]thymidine (Amersham Corp.) for 2 h. High molecular mass [3H]-radioactivity was precipitated using 5% trichloroacetic acid at 4 °C for 30 min. After two washes with ice-cold H2O, [3H]-radioactivity was solubilized in 1 m NaOH (400 μl/well) at room temperature for 8 min, neutralized by the addition of 2 m HCl (400 μl/well), and quantitated by liquid scintillation counting. VEGF stimulation of PAE/KDR cells resulted in a strong autophosphorylation of the receptor KDR. Using a receptor-specific antibody, a number of phosphorylated proteins could be immunoprecipitated together with KDR. They migrated at approximate sizes of 44, 52, 60, 65, 85, and 140 kDa (Fig.1). VEGF-activated KDR is able to induce mitogenicity and chemotaxis in PAE/KDR cells in vitro (5Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar), but only limited information is available about the identity and function of molecules that transmit KDR-induced signals. Phosphorylation of Shc proteins is an early event in intracellular signaling in a number of well characterized receptor tyrosine kinases. The ability of KDR to induce phosphorylation of Shc proteins was investigated in PAE/KDR cells using Western blot analysis and in vitro kinase assays. A strong phosphorylation of the 46- and 52-kDa isoforms of Shc was observed upon VEGF stimulation of PAE/KDR cells (Fig. 2 A, upper panel). VEGF-induced tyrosine phosphorylation of the 66-kDa isoform of Shc was demonstrated after a longer exposure of the same membrane (Fig. 2 A, lower panel). In addition, this phosphotyrosine blot was sensitive enough to detect the phosphorylation of three as yet unidentified proteins migrating on SDS-PAGE according to approximate sizes of 110, 140, and 200 kDa (Fig.2 A, lower panel). No VEGF-dependent phosphorylation of Shc proteins was observed in nontransfected PAE cells (data not shown). Grb2 associated with Shc molecules in response to VEGF stimulation of PAE/KDR cells as demonstrated by stripping of the phosphotyrosine blot (shown in Fig. 2 A) and reprobing with an antiserum recognizing Grb2 (Fig. 2 B). Association of KDR with Shc in response to VEGF stimulation was demonstrated using an in vitro kinase assay. KDR was visualized in anti-Shc immunoprecipitates from lysates of VEGF-stimulated PAE/KDR cells on SDS-PAGE as a 210-kDa band following in vitro kinase reaction (Fig. 2 C). In a similar experiment, stimulation of nontransfected PAE cells either with VEGF or with 10% fetal calf serum did not result in the generation of a signal of 210 kDa (data not shown). To characterize the 210-kDa signal, we used tyrphostin AG1433, a specific inhibitor of the PDGF β-receptor and KDR/Flk-1 (31Strawn L.M. McMahon G. App H. Schreck R. Kuchler W.R. Longhi M.P. Hui T.H. Tang C. Levitzki A. Gazit A. Chen I. Keri G. Orfi L. Risau W. Flamme I. Ullrich A. Hirth K.P. Shawver L.K. Cancer Res. 1996; 56: 3540-3545PubMed Google Scholar). In our hands, tyrphostin AG1433 completely inhibited PDGF-BB-induced autophosphorylation of the PDGF β-receptor at a concentration of 10 μm in PAE cells overexpressing the PDGF β-receptor. Complete inhibition of VEGF-induced autophosphorylation of KDR in PAE/KDR cells was found at a concentration of 30 μm (data not shown). Since nontransfected PAE cells do not express the PDGF β-receptor (32Westermark B. Siegbahn A. Heldin C.-H. Claesson-Welsh L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 128-132Crossref PubMed Scopus (132) Google Scholar) and because KDR and the PDGF β-receptor can clearly be distinguished by their different electrophoretic mobilities on SDS-PAGE, tyrphostin AG1433 can be regarded as a specific KDR inhibitor in PAE/KDR cells. Preincubation of PAE/KDR cells with 30 μm tyrphostin AG1433, VEGF stimulation, and Shc immunoprecipitation followed by the in vitro kinase assay did not result in the generation of a phosphorylated signal at 210 kDa (data not shown). KDR associated with the SH2/SH3 domain-containing adapter molecules Grb2 and Nck. KDR associated with Grb2 in response to VEGF stimulation as demonstrated by Grb2 immunoprecipitation followed by the in vitro kinase assay (Fig. 3). Similar results were obtained for the Nck protein (Fig.4 A). Abundant Nck expression was detectable in PAE/KDR cells using Western blot analysis (Fig.4 B). Preincubation of PAE/KDR cells with tyrphostin AG1433 abolished the signal at ∼210 kDa in both Grb2 and Nck immunoprecipitates (Figs. 3 and 4 A). No VEGF-dependent association of either Grb2 or Nck with KDR was observed in nontransfected PAE cells (Figs. 3 and 4 A). We did not, however, observe tyrosine phosphorylation of the Nck or Grb2 protein in response to VEGF stimulation using both the in vitro kinase assay and Western blot analysis (data not shown).Figure 4Nck and KDR signaling. A, association of Nck with KDR. PAE/KDR cells and nontransfected PAE cells were stimulated for 5 min with 50 ng/ml VEGF. The cell lysate was immunoprecipitated (IP) with an antiserum recognizing Nck, followed by the in vitro kinase assay. Samples were analyzed by SDS-PAGE, followed by KOH treatment and autoradiography. Tyrphostin AG1433 was added to PAE/KDR cells 10 min prior to VEGF stimulation at a final concentration of 30 μm. B, identification of Nck in PAE/KDR cells by Western blot analysis. The PAE/KDR cell lysate was subjected to SDS-PAGE and blotted onto a nitrocellulose membrane. Nck was identified by incubation with an anti-Nck antiserum, followed by peroxidase-conjugated rabbit anti-mouse Ig as a secondary antibody and chemiluminescence. The human HeLa cell lysate was used as a positive control (Ctrl) for the Nck protein. IB, immunoblot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previous work has shown that KDR is able to induce weak phosphorylation of GAP (5Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.-H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). In addition to these findings, we were able to demonstrate the VEGF-induced association of GAP with KDR by the in vitrokinase assay (Fig. 5). A functionally important molecule of the Ras pathway is MAP kinase. We therefore tested whether MAP kinase is activated in response to VEGF stimulation of PAE/KDR cells. A strong activation of MAP kinase was found as indicated by the phosphorylation of the MAP kinase-specific substrate myelin basic protein. In contrast, VEGF stimulation of nontransfected PAE cells or of PAE/Flt-1 cells did not induce the activation of MAP kinase (Fig.6 A). Preincubation of PAE/KDR cells with PD98059 at concentrations up to 100 μm, a selective inhibitor of the phosphorylation and activation of MAP kinase kinase (33Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2603) Google Scholar), resulted in a dose-dependent inhibition of MAP kinase activity in PAE/KDR cells (Fig. 6 A). VEGF-induced DNA synthesis of PAE/KDR cells was inhibited by PD98059 in a dose-dependent fashion at concentrations up to 10 μm (Fig. 6 B). Recent data have provided compelling evidence that not only tyrosine kinases, but also tyrosine phosphatases are involved in the signal transduction of receptor tyrosine kinases (34Feng G.-S. Pawson T. Trends Genet. 1994; 10: 54-58Abstract Full Text PDF PubMed Scopus (173) Google Scholar). Expression of the two tyrosine phosphatases SHP-1 and SHP-2 was demonstrated in PAE cells using Western blot analysis and antibodies recognizing SHP-1 (Fig.7 A) and SHP-2 (Fig.7 B). VEGF stimulation of PAE/KDR cells induced a physical association of either phosphatase with KDR as determined by protein-tyrosine phosphatase-specific immunoprecipitation followed by the in vitro kinase assay for visualization of the phosphorylated receptor (Fig. 8). It is likely that the phosphorylated protein migrating at ∼210 kDa on SDS-PAGE in both VEGF-stimulated samples represents the VEGF receptor KDR. Its appearance was dependent on VEGF stimulation, and moreover, treatment of PAE/KDR cells with the KDR-specific tyrphostin AG1433 resulted in a complete inhibition of KDR phosphorylation and in the disappearance of the 210-kDa band. VEGF stimulation of nontransfected PAE cells showed no corresponding signal in a similar experiment (data not shown). To further verify the specificity of this coprecipitation, we repeated the same experiment using an antibody to immunoprecipitate the insulin receptor. In this case, in the in vitro kinase assay, no signal of ∼210 kDa was observed (data not shown). In our hands, tyrosine phosphorylation of SHP-1 or SHP-2 was not detected in PAE/KDR cells following VEGF stimulation using either the in vitro kinase assay or phosphotyrosine blot analysis (data not shown).Figure 8Association of SHP-1 and SHP-2 with KDR.PAE/KDR cells were stimulated for 5 min with 50 ng/ml VEGF. Cell lysates were immunoprecipitated (IP) with antibodies recognizing SHP-1 or SHP-2, followed by the in vitro kinase assay. Samples were analyzed by SDS-PAGE, followed by KOH treatment and autoradiography.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this study, we were able to significantly extend our knowledge on the signal transduction properties of the VEGF receptor KDR. For our in vitro studies, we used porcine aortic endothelial cells overexpressing KDR. VEGF stimulation results in the autophosphorylation of KDR, the phosphorylation of a number of intracellular proteins, and the association of intracellular proteins with phosphorylated KDR. So far, only little information has been available concerning the identity of signal transduction molecules involved in KDR signaling. In this study, we were able to demonstrate that Shc, Nck, and Grb2 as well as SHP-1 and SHP-2 are involved in these processes. The phosphorylation of Shc proteins is an early event in the signal transduction cascade of many activated receptor tyrosine kinases leading to the MAP kin
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