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Identification of Tyrosine Residues in Vascular Endothelial Growth Factor Receptor-2/FLK-1 Involved in Activation of Phosphatidylinositol 3-Kinase and Cell Proliferation*

  • Volkan Dayanir
    Footnotes
    Affiliations
    From the Departments of Ophthalmology and
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  • Rosana D. Meyer
    Affiliations
    Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114
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  • Kameran Lashkari
    Affiliations
    Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114
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  • Nader Rahimi
    Correspondence
    To whom correspondence should be addressed
    Footnotes
    Affiliations
    Biochemistry, School of Medicine, Boston University, Boston, Massachusetts 02118 and the
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  • Author Footnotes
    * This work was supported in part by departmental grants from Research To Prevent Blindness, Inc., the Massachusetts Lions Eye Research Fund Inc., and the American Cancer Society, Massachusetts Division, Inc. (to N. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Funded by TUBITAK (the Scientific and Technical Research Council of Turkey) NATO Science Scholarship and Turkish Education Foundation Scholarship Programs.
Open AccessPublished:May 25, 2001DOI:https://doi.org/10.1074/jbc.M009128200
      Activation of vascular endothelial growth factor receptor-2 (VEGFR-2) plays a critical role in vasculogenesis and angiogenesis. However, the mechanism by which VEGFR-2 activation elicits these cellular events is not fully understood. We recently constructed a chimeric receptor containing the extracellular domain of human CSF-1R/c-fms, fused with the entire transmembrane and cytoplasmic domains of murine VEGFR-2 (Rahimi, N., Dayanir, V., and Lashkari, K. (2000) J. Biol. Chem. 275, 16986–16992). In this study we used VEGFR-2 chimera (herein named CKR) to elucidate the signal transduction relay of VEGFR-2 in porcine aortic endothelial (PAE) cells. Mutation of tyrosines 799 and 1173 individually on CKR resulted in partial loss of CKR's ability to stimulate cell growth. Double mutation of these sites caused total loss of CKR's ability to stimulate cell growth. Interestingly, mutation of these sites had no effect on the ability of CKR to stimulate cell migration. Further analysis revealed that tyrosines 799 and 1173 are docking sites for p85 of phosphatidylinositol 3-kinase (PI3K). Pretreatment of cells with wortmannin, an inhibitor of PI3K, and rapamycin, a potent inhibitor of S6 kinase, abrogated CKR-mediated cell growth. However, expression of a dominant negative form of ras (N17ras) and inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD98059 did not attenuate CKR-stimulated cell growth. Altogether, these results demonstrate that activation of VEGFR-2 results in activation of PI3K and that activation of PI3K/S6kinase pathway, but not Ras/MAPK, is responsible for VEGFR-2-mediated cell growth.
      VEGFR
      vascular endothelial growth factor receptor
      PI3K
      phosphatidylinositol 3-kinase
      PLCγ1
      phospholipase-Cγ1
      VEGF
      vascular endothelial growth factor
      PAE
      porcine aortic endothelial cells
      CKR
      VEGFR-2 chimera
      MAPK
      mitogen-activate protein kinase
      PD98059
      a MAPK inhibitor
      DMEM
      Dulbecco's modified Eagle's medium
      ACE
      adrenal microvascular endothelial cells
      pY
      phosphotyrosine
      GST
      glutathione S-transferase
      PI3P
      phosphatidylinositol 3-phosphate
      Vascular endothelial growth factor receptor-1 (VEGFR-11/FLT-1) and VEGFR-2 (FLK-1/KDR) belong to a subfamily of receptor tyrosine kinases implicated in vasculogenesis and angiogenesis. The important contribution of VEGFR-2 in vasculogenesis and angiogenesis was initially underscored by the observation that homozygous knockout mice lacking VEGFR-2 exhibited severe deficiency in vessel formation (
      • Shalaby F.
      • Rossant J.
      • Yamaguchi T.P.
      • Gertsentein M.
      • Fu Wu X.
      • Breitman M.L.
      • Schuh A.C.
      ). Furthermore, introduction of either a neutralizing antibody against VEGF or the dominant negative form of VEGFR-2 was able to block angiogenesis (
      • Millauer B.
      • Shawver L.K.
      • Plate K.H.
      • Risau W.
      • Ullrich A.
      ,
      • Kim K.J.
      • Li B.
      • Winer J.
      • Armanini M.
      • Gillett N.
      • Phillips H.S.
      • Ferrara N.
      ). On the other hand, VEGFR-1 activation alone appears to play a less significant role in these cellular processes (
      • Fong G.H.
      • Zhang L.
      • Bryce D.M.
      • Peng J.
      ,
      • Rahimi N.
      • Dayanir V.
      • Lashkari K.
      ,
      • Hiratsuka S.
      • Minowa O.
      • Kuno J.
      • Noda T.
      • Shibuya M.
      ).
      The mechanism by which VEGFR-2 activation evokes angiogenesis is not well understood. It is presumed that these events are initiated by binding of VEGF to VEGFR-2 leading to tyrosine phosphorylation of the dimerized VEGFR-2 and subsequent phosphorylation of SH2-containing intracellular signaling proteins, including phospholipase C-γ1 (PLCγ1), Src family tyrosine kinases, and phosphatidylinositol 3-kinase (PI3K), adaptor molecules, SHC, NCK, and Ras GTPase-activating protein (
      • Waltenberger J.
      • Calaesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.-H.
      ,
      • Takahashi T.
      • Shibuya M.
      ,
      • Igarashi K.
      • Isohara T.
      • Kato T.
      • Shigeta K.
      • Yamano T.
      • Uno I.
      ,
      • Guo D.
      • Jia Q.
      • Song H.Y.
      • Warren R.S.
      • Donner D.B.
      ). The contributions of individual signaling molecules to various aspects of angiogenesis and the tyrosine sites on VEGFR-2 that potentially mediate their recruitment and activation have not been fully investigated. Moreover, the data presented in the literature is often inconsistent. Waltenbergeret al. (1994), Abedi and Zachary (1997), and Takahashiet al. (1997) have suggested that stimulation of endothelial cells with VEGF results in no PI3K activation (
      • Waltenberger J.
      • Calaesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.-H.
      ,
      • Takahashi T.
      • Shibuya M.
      ,
      • Abedi H.
      • Zachary I.
      ), whereas others have shown that VEGFR-2 activation does indeed result in PI3K stimulation, which may stimulate endothelial cell growth and survival (
      • Thakker G.D.
      • Hajjar D.P.
      • Muller W.A.
      • Rosengart T.K.
      ,
      • Gerber H.P.
      • McMurtrey A.
      • Kowalski J.
      • Yan M.
      • Keyt B.A.
      • Dixit V.
      • Ferrara N.
      ).
      The reason for the apparent inconsistency in the activation and association of signaling molecules with VEGFR-2 is not known. It may be due to the complexity of VEGFR-2-mediated signal transduction relay in endothelial cells such as expression of VEGFR-1 and neuropilin-1 and -2, which may modify or antagonize VEGFR-2-mediated signal transduction and its final biological responses (
      • Rahimi N.
      • Dayanir V.
      • Lashkari K.
      ,
      • Soker S.
      • Takashima S.
      • Miao H.Q.
      • Neufeld G.
      • Klagsbrun M.
      ,
      • Kendall R.L.
      • Wang G.
      • Thomas K.A.
      ). To circumvent these issues, we have recently constructed a chimeric receptor containing the extracellular domain of human CSF-1R/c-fms, fused with the transmembrane and the cytoplasmic domains of murine VEGFR-2. (
      • Rahimi N.
      • Dayanir V.
      • Lashkari K.
      ). This model permitted us to dissect the functions of VEGFR-2 in endothelial cells by selectively stimulating the receptor with CSF-1. In this study we used this chimeric receptor to elucidate the signal transduction relay induced by VEGFR-2 in PAE cells. We show that mutation of tyrosine sites 799 and 1173 individually on CKR result in partial loss of CKR's ability to stimulate cell growth, whereas double mutation of these tyrosine sites result in complete loss of CKR ability to stimulate endothelial cell growth. Mutation of these sites, however, had no effect on CKR's ability to stimulate cell migration. Further analysis showed that tyrosines 799 and 1173 are binding sites for p85 of PI3K and that activation of PI3K is responsible for CKR-mediated endothelial cell growth.

      DISCUSSION

      Our study demonstrates that mutation of tyrosines 799 and 1173 on VEGFR-2 abolishes binding of P85 of PI3K to CKR without impairing CKR's ability to activate PLCγ and Ras/MAPK pathways. A single mutation of 799 and 1173 on CKR partially inhibited PI3K activation and cell proliferation. Additionally, double mutation of tyrosines 799 and 1173 totally abolished CKR's ability to stimulate PI3K activation and endothelial cell growth but not cell migration. These results suggest that distinct signaling pathways are activated by VEGFR-2 and are responsible for the induction of endothelial cell growth and likely for VEGF-induced angiogenesis. Consistent with mutant CKRs, pretreatment of cells with wortmannin, a potent inhibitor of PI3K, blocked CKR's ability to stimulate cell growth. Activation of PI3K results in PIP3 production, which can activate protein kinase C-ζ (
      • Chou M.M.
      • Hou W.
      • Johnson J.
      • Graham L.K.
      • Lee M.H.
      • Chen C.S.
      • Newton A.C.
      • Schaffhausen B.S.
      • Toker A.
      ), Akt (
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      ), and stimulation of p70 S6 kinase (
      • Romanelli A.
      • Martin K.A.
      • Toker A.
      • Blenis J.
      ). Our results show that rapamycin, a potent inhibitor of the S6 kinase pathway (
      • Lane H.A.
      • Fernandez A.
      • Lamb N.J.
      • Thomas G.
      ), abrogates CKR-mediated cell growth, suggesting that the PI3K/S6 kinase pathway is responsible for CKR-mediated cell growth. Interestingly, it appears that activation of the PLCγ and Ras/MAPK pathways is not involved in VEGFR-2-stimulated growth of PAE cells. Nonetheless, activation of these enzymes by CKR strongly suggests that these molecules are likely to participate in the other VEGF-induced cellular processes such as cell migration and cell differentiation.
      A number of groups have investigated the role PLCγ1 in VEGF-dependent signal relay. The initial observations suggested that PLCγ activation is increased in VEGF-stimulated cells (
      • Takahashi T.
      • Shibuya M.
      ,
      • Igarashi K.
      • Isohara T.
      • Kato T.
      • Shigeta K.
      • Yamano T.
      • Uno I.
      ,
      • Cunningham S.A.
      • Arrate M.P.
      • Brock T.A.
      • Waxham M.N.
      ). Subsequent study showed that tyrosine 951 on the human VEGFR-2 is a major binding site for PLCγ1 (
      • Wu 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.
      ). In agreement with this study, our results demonstrate that tyrosines 799 and 1173 of mouse VEGFR-2 are not required for PLCγ1 activation and likely tyrosine 951 is the primary binding site for PLCγ1. In addition, our results demonstrate that, although PLCγ1 is activated by VEGFR-2, its activation is not required for VEGF-mediated endothelial cell growth. Activation of PLCγ1 has been shown to stimulate cell growth and migration in a variety of cellular systems; however, its role in VEGFR-2-mediated signal transduction and endothelial cell function is largely unknown. Recently, it has been suggested that inhibition of PLCγ1 by pharmacological methods blocks VEGF-stimulated sinusoidal endothelial cell growth (
      • Takahashi T.
      • Ueno H.
      • Shibuya M.
      ). Because sinusoidal cells express both VEGFR-1 and VEGFR-2, it is difficult to judge the contributions of each receptor to the observed PLCγ1 activation.
      Thus, it seems that tyrosines 799 and 1173 are novel p85 docking sites for p85 of PI3K, although they may represent low affinity binding sites for P85. Amino acid residues surrounding tyrosine 799 (YLSIVM) and 1173 (YIVLPM) of VEGFR-2 do not correspond to conventional (Y(M/V/I/E)XM) p85 binding sites (
      • Rameh L.E.
      • Chen C.S.
      • Cantley L.C.
      ,
      • Carpenter C.L.
      • Cantley L.C.
      ). Although it is generally believed that SH2 domains of p85 preferentially bind to receptor tyrosine kinases through this motif, other binding sites for p85 of PI3K have been described. For instance, p85 binding to the hepatocyte growth factor receptor family, including c-Met, c-Ron, and c-Sea is mediated by the YVHV sequence (
      • Derman M.P.
      • Chen J.Y.
      • Spokes K.C.
      • Songyang Z.
      • Cantley L.G.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Zhen Z.
      • Maina F.
      • dalla Zonca P.
      • Giordano S.
      • Graziani A.
      • Panayotou G.
      • Comoglio P.M.
      ). Similarly, it has been demonstrated that amino acids YVNA on VEGFR-1 is a binding site for p85 (
      • Cunningham S.A.
      • Waxham M.N.
      • Arrate P.M.
      • Brock T.A.
      ).
      Until now, little evidence existed for the involvement of PI3K in VEGFR-2-mediated signal transduction and angiogenesis. The possibility that PI3K may be involved could be inferred only from very indirect evidence. Initial studies about the activation of PI3K by VEGFR-2 suggested that PI3K is not activated by VEGFR-2 stimulation (
      • Waltenberger J.
      • Calaesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.-H.
      ,
      • Takahashi T.
      • Shibuya M.
      ,
      • Abedi H.
      • Zachary I.
      ). However, subsequent studies suggested that VEGF stimulation of endothelial cells results in activation of PI3K and its activation may promote endothelial cell survival (
      • Thakker G.D.
      • Hajjar D.P.
      • Muller W.A.
      • Rosengart T.K.
      ,
      • Gerber H.P.
      • McMurtrey A.
      • Kowalski J.
      • Yan M.
      • Keyt B.A.
      • Dixit V.
      • Ferrara N.
      ). Furthermore, recently it has been shown that viral oncogenic PI3K stimulates angiogenesis in the CAM assay by stimulating VEGF expression (
      • Jiang B.H.
      • Zheng J.Z.
      • Aoki M.
      • Vogt P.K.
      ). During angiogenesis VEGF induces endothelial cell migration, growth, and differentiation in a coordinated manner. Our current study suggests that specific activation of VEGFR-2 in endothelial cells activates a number of signaling molecules, including PI3K, Akt, PLCγ1, and MAPK. Altogether, this suggests that during angiogenesis stimulation of the PI3K/S6 kinase pathway by VEGFR-2 may influence endothelial cell growth and likely endothelial cell survival leading to formation of new blood vessels. Previous studies have suggested that activation of the PI3K/S6 kinase pathway is essential for serum and fibroblast growth factor-stimulated endothelial cell growth (
      • Vinals F.
      • Chambard J.C.
      • Pouyssegur J.
      ,
      • Kanda S.
      • Hodgkin M.N.
      • Woodfield R.J.
      • Wakelam M.J.
      • Thomas G.
      • Claesson-Welsh L.
      ), implying that activation of PI3K by a variety of factors in the endothelial cells serves as a molecular switch to control cell proliferation.
      Regulation of angiogenesis is the most critical step in the development of tumors, ocular neovascularization, and in inflammation (
      • Risau W.
      ,
      • Folkman J.
      • D'Amore P.
      ). The results presented in this work identify tyrosine residues of VEGFR-2 responsible for recruiting and activation of PI3K and its role as a regulator of endothelial cell growth. These findings are important for understanding the different roles of signaling molecules and the different aspects of angiogenesis. Further studies will delineate the contributions of other signaling molecules to different cellular processes involved during angiogenesis.

      Acknowledgments

      We thank Cyrus Vaziri (Cancer Research Center, Boston University) for providing the N17ras construct.

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