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The Role of Phosphatidylinositol 3-Kinase in Vascular Endothelial Growth Factor Signaling*

  • Geeta D. Thakker
    Affiliations
    From the Departments of Cardiothoracic Surgery and
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  • David P. Hajjar
    Affiliations
    Pathology, Weill Medical College of Cornell University, New York, New York 10021
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  • William A. Muller
    Affiliations
    Pathology, Weill Medical College of Cornell University, New York, New York 10021
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  • Todd K. Rosengart
    Correspondence
    To whom all correspondence should be addressed: Dept. of Cardiothoracic Surgery, Rm. F2103, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021. Tel.: 212-746-5155; Fax: 212-746-8828;
    Affiliations
    From the Departments of Cardiothoracic Surgery and
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  • Author Footnotes
    * This work was supported in part by a grant from the Thoracic Surgery Foundation for Research and Education.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.
Open AccessPublished:April 09, 1999DOI:https://doi.org/10.1074/jbc.274.15.10002
      Vascular endothelial growth factor (VEGF) receptor Flk-1/KDR in endothelial cells is activated during vasculogenesis and angiogenesis upon ligand-receptor interaction. Activated Flk-1/KDR has been shown to recruit Src homology 2 domain-containing signaling molecules that are known to serve as links to the activation of the mitogen-activated protein (MAP) kinase signaling pathway. To define the functional significance of phosphatidylinositol (PI) 3-kinase in VEGF signaling, we have examined its role in human umbilical vein endothelial cell (HUVEC) cycle progression. We show herein that p85, the regulatory subunit of PI 3-kinase, is constitutively associated with Flk-1/KDR. The treatment of HUVECs with VEGF promoted tyrosine autophosphorylation of Flk-1/KDR and also induced phosphorylation of p85. This was followed by an increase in the PI 3-kinase activity, which was sensitive to wortmannin, a potent PI 3-kinase inhibitor. VEGF also induced a striking activation of MAP kinase in a time-dependent manner. Inhibition studies with both a dominant-negative p85 mutant and the PI 3-kinase inhibitor, wortmannin, were employed to show for the first time that VEGF-stimulated PI 3-kinase modulates MAP kinase activation and nuclear events such as transcription from c-fos promoter and entry into the synthesis (S)-phase. Our data demonstrate the importance of PI 3-kinase as a necessary signaling component of VEGF-mediated cell cycle progression.
      VEGF
      vascular endothelial growth factor
      MAP kinase
      mitogen-activated protein kinase
      ERK
      extracellular-regulated kinase, HUVEC, human umbilical vein endothelial cell
      PI 3-kinase
      phosphoinositide 3-kinase
      PDGF
      platelet-derived growth factor
      PDGFR
      platelet-derived growth factor receptor
      SRE
      serum response element
      RIPA
      radioimmunoprecipitation assay
      Luc
      luciferase
      CMV
      cytomegalovirus
      PAGE
      polyacrylamide gel electrophoresis
      DN
      dominant negative
      HA
      hemagglutinin
      BrdUrd
      5′-bromodeoxyuridine
      SH2 and SH3
      Src homology-2 and -3
      MBP
      myelin basic protein
      PKC
      protein kinase C
      Grb2
      growth factor receptor binding protein
      Shc
      Src homology/collagen
      X-gal
      5-bromo-4-chloro-3-indolyl β-d-galactopyranoside
      Vascular endothelial growth factor (VEGF)1 is a secreted glycoprotein specific for endothelial cells (
      • Leung D.W.
      • Cachianes G.
      • Kuang W.-J.
      • Goeddel D.V.
      • Ferrara N.
      ,
      • Keck P.J.
      • Hauser S.D.
      • Krivi G.
      • Sanzo K.
      • Warren T.
      • Feder J.
      • Connolly D.T.
      ,
      • Senger D.R.
      • Perruzzi C.A.
      • Feder J.
      • Dvorak H.F.
      ). VEGF is angiogenicin vivo (
      • Plouet J.
      • Schilling J.
      • Gospodarowicz D.
      ,
      • Phillips G.D.
      • Stone A.M.
      • Jones B.D.
      • Schultz J.C.
      • Whitehead R.A.
      • Knighton D.R.
      ) and in vitro (
      • Pepper M.S.
      • Ferrara N.
      • Orci L.
      • Montesano R.
      ); its importance in vasculogenesis and angiogenesis has been established through gene deletion studies (
      • Carmeliet P.
      • Ferreira V.
      • Breier G.
      • Pollefeyt S.
      • Kieckens L.
      • Gertenstein M.
      • Fahrig M.
      • Vandenhoeck A.
      • Harpal K.
      • Eberhardt C.
      • Declercq C.
      • Pawling J.
      • Moons L.
      • Collen D.
      • Risau W.
      • Nagy A.
      ,
      • Shalaby F.
      • Rossant J.
      • Yamaguchi T.P.
      • Gertenstein M.
      • Wu X.-F.
      • Breitman M.L.
      • Schuh A.C.
      ). Flk-1/KDR and Flt-1 are the two receptor tyrosine kinases that regulate the actions of VEGF and are expressed in endothelial cells (
      • de Vries C.
      • Escobedo J.A.
      • Ueno H.
      • Houck K.
      • Ferrara N.
      • Williams L.T.
      ,
      • Terman B.I.
      • Dougher-Vermazen M.
      • Carrion M.E.
      • Dimitrov D.
      • Armellino D.C.
      • Gospodarowicz D.
      • Bohlen P.
      ,
      • Oberg C.
      • Waltenberger J.
      • Claesson-Welsh L.
      • Welsh M.
      ,
      • Clauss M.
      • Weich H.
      • Breier G.
      • Knies U.
      • Rockl W.
      • Waltenberger J.
      • Risau W.
      ,
      • Brown L.F.
      • Detmar M.
      • Tognazzi K.
      • Abu-Jawdeh G.
      • Iruela-Arispe M.L.
      ), while the related receptor, Flt-4, is found on lymphatic endothelium. The expression pattern of Flt-4 suggests it may play a role during lymphangiogenesis (
      • Kaipainen A.
      • Korhonen J.
      • Mustonen T.
      • van Hinsbergh V.W.M.
      • Fang G.-H.
      • Dumont D.
      • Breitman M.
      • Alitalo K.
      ).
      All of the three VEGF receptors belong to the PDGFR-β family of receptor tyrosine kinases (
      • Ferrara N.
      • Davis-Smyth T.
      ). Upon activation, these receptors dimerize and/or oligomerize, following which autophosphorylation and transphosphorylation of their tyrosine residues in the intracellular domain occurs. There are four putative tyrosine phosphorylation sites (Tyr-951, Tyr-996, Tyr-1054, and Tyr-1059) in the KDR intracellular domain (
      • Dougher-Vermazen M.
      • Hulmes J.D.
      • Bohlen P.
      • Terman B.I.
      ). These phosphorylated tyrosine molecules act as docking sites for adaptor signaling molecules and non-receptor tyrosine kinases, thereby generating a signal cascade that culminates in a cellular response. The signal transduction pathways involved in mediating the various biological functions of VEGF on endothelial cells such as migration, proliferation, differentiation, or survival remain to be completely defined.
      The Ras-MAP (mitogen-activated protein) kinase pathway is a key component in the transduction of signals leading to growth and transformation. It consists of a linear cascade of protein kinases, Raf, MAP kinase kinase, and MAP kinase, which are also called extracellular-regulated kinases (Erks). Erk-1 and Erk-2 are acutely activated upon growth factor stimulation (
      • Malarkey K.
      • Belham C.M.
      • Paul A.
      • Graham A.
      • McLees A.
      • Scott P.H.
      • Plevin R.
      ).
      Phosphatidylinositol (PI) 3-kinase, a heterodimer of an 85-kDa (p85) adaptor subunit and a 100-kDa (p110) catalytic subunit (
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz L.F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Klippel A.
      • Escobedo J.A.
      • Hu Q.
      • Williams L.T.
      ,
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ,
      • Hu P.
      • Schlessinger J.
      ), is activated by most growth factors and has been implicated as a critical factor in the control of cell proliferation and cell survival. PI 3-kinase phosphorylates the D-3 position of the inositol ring of phosphoinositides, which in turn act as second messengers. The p85 subunit contains two Src homology 2 (SH2) domains, which bind to tyrosine-phosphorylated receptors after stimulation of cells with growth factors and in this manner recruit p110 into the complex at the cell membrane. The region between the two SH2 domains, the iSH2 region, mediates the association with p110, and this interaction is required for the enzymatic activity of p110 (
      • Klippel A.
      • Escobeda J.A.
      • Hirano M.
      • Williams L.T.
      ). Phosphorylation of the p85 subunit of PI 3-kinase upon VEGF stimulation (
      • Guo D.
      • Jia Q.
      • Song H.Y.
      • Warren R.S.
      • Donner D.
      ) is suggestive of a potential role for PI 3-kinase in VEGF-mediated signaling. Given this observation, we hypothesized that PI 3-kinase might play a critical role in VEGF signaling, including the Ras-MAP kinase pathway. In this report, we demonstrate for the first time the functional significance of PI 3-kinase in VEGF signaling from Flk-1/KDR leading to MAP kinase activation, followed by transcriptional activation of the c-Fos serum response element that eventually culminates in endothelial cell proliferation.

      DISCUSSION

      To further our knowledge of the signal transduction pathways from VEGF receptor, we have analyzed the functional importance of PI 3-kinase in VEGF signaling from Flk-1/KDR. It has been shown that p85, the adaptor subunit of PI 3-kinase, is capable of associating with the VEGF receptor (
      • Guo D.
      • Jia Q.
      • Song H.Y.
      • Warren R.S.
      • Donner D.
      ,
      • Cunningham S.A.
      • Waxham M.N.
      • Arrate P.M.
      • Brock T.A.
      ). The functional significance of PI 3-kinase activation in VEGF signaling has, however, not been defined clearly. In our study, we show that phosphorylation of p85 in response to VEGF regulates its catalytic counterpart, i.e. p110, and furthermore demonstrate that PI 3-kinase activation contributes to MAP kinase activation, transcription of c-Fos SRE, and cell cycle progression in human endothelial cells.
      Several reports have shown that activated Flk-1/KDR receptors will associate with signaling intermediates such as phospholipase C-γ, GTPase-activating protein, Nck-Grb2, and Shc-Grb2 (
      • Guo D.
      • Jia Q.
      • Song H.Y.
      • Warren R.S.
      • Donner D.
      ,
      • Waltenberger J.
      • Claesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.-H.
      ,
      • Seetharam L.
      • Gotoh N.
      • Maru Y.
      • Neufeld G.
      • Yamaguchi S.
      • Shibuya M.
      ,
      • Kroll J.
      • Waltenberger J.
      ). These interactions result in tyrosine phosphorylation of some of the potential substrates. The Ras-MAP kinase pathway has been reported to signal for VEGF-induced growth, as has the PKC-MAP kinase pathway (
      • Kroll J.
      • Waltenberger J.
      ,
      • Takhashi T.
      • Shibuya M.
      ). Our results show for the first time that PI 3-kinase not only gets phosphorylated upon VEGF stimulation but also contributes to cell cycle progression following MAP kinase activation.
      One of the early events observed upon interaction of VEGF with its receptor Flk-1/KDR is activation of the receptor's intrinsic tyrosine kinase activity. In agreement with previous studies (
      • Waltenberger J.
      • Claesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.-H.
      ,
      • Kroll J.
      • Waltenberger J.
      ), Flk-1/KDR was strongly phosphorylated in response to VEGF in our study. Two other proteins phosphorylated and recruited to the activated receptor had approximate molecular masses of 46/47 and 67/70 kDa. The 46/47-kDa protein (p46/47) phosphorylated in our study could either be p46Shc or p47Nck, both of which are known to bind to Flk-1/KDR (
      • Kroll J.
      • Waltenberger J.
      ) as well as to other receptor tyrosine kinases like PDGFR and epidermal growth factor receptor (
      • Pelicci G.
      • Lanfrancone L.
      • Grignani F.
      • McGlade J.
      • Cavallo F.
      • Forni G.
      • Nicoletti I.
      • Grignani F.
      • Pawson T.
      • Pelicci P.G.
      ,
      • van der Greer P.
      • Wiley S.
      • Lai V.K.-M.
      • Oliver J.P.
      • Gish G.D.
      • Stephens R.
      • Kaplan D.
      • Shoelson S.
      • Pawson T.
      ,
      • Li W.
      • Hu P.
      • Skolnik E.Y.
      • Ullrich A.
      • Schlessinger J.
      ). Although we did not characterize the identity of these proteins, we subscribed to the idea that these were SH2 domain-containing tyrosine-phosphorylated proteins, which potentially couple to the activation of Ras-MAP kinase pathway (
      • Pawson T.
      ).
      We also show that Flk-1/KDR constitutively associates with p85 as this association was independent of VEGF stimulation. Flk-1/KDR has a candidate motif YXXM in its intracellular region which is a potential site for p85 to bind. It should be noted here that p85 contains two SH2 domain and a SH3 domain, the SH2 domain of p85 is responsible for interaction with phosphorylated tyrosine in the context of above motif. However, mutational analysis of tyrosine residues in Flk-1/KDR would be required to verify this notion. Flt-1, the other receptor for VEGF, has also been shown to associate with p85 in a two-hybrid system, the binding site for which has been identified as a YVNA motif (
      • Cunningham S.A.
      • Waxham M.N.
      • Arrate P.M.
      • Brock T.A.
      ). However, Flt-1 does not apparently signal for proliferation, which has been underscored by knockout studies wherein only the tyrosine kinase domain of Flt-1 has been deleted (
      • Hiratsuka S.
      • Minowa O.
      • Kuno J.
      • Noda T.
      • Shibuya M.
      ). Flt-1 tyrosine kinase homozygous mice (Flt-1TK−/−) develop normal blood vessels and survive, which suggests that Flt-1 tyrosine kinase domain does not signal for migration, proliferation, and differentiation, essential features of endothelial cells during vasculogenesis. Although activation of the kinase counterpart of PI 3-kinase may or may not require p85 to be phosphorylated (
      • Van der Greer P.
      • Hunter T.
      • Lindberg R.
      ), we found that VEGF treatment promoted p85 phosphorylation that in turn increased PI 3-kinase activity.
      In accordance with published literature (
      • Kroll J.
      • Waltenberger J.
      ,
      • Abedi H.
      • Zachary I.
      ,
      • Parenti A.
      • Morbidelli L.
      • Cui X.-L.
      • Douglas J.G.
      • Hood J.D.
      • Granger H.J.
      • Ledda F.
      • Ziche M.
      ), we have also observed MAP kinase activation in endothelial cells upon VEGF stimulation. We demonstrate for the first time in our study a link between PI 3-kinase and the downstream activation of MAP kinase in VEGF stimulated cells by 1) significantly inhibiting MAP kinase activation with wortmannin, a potent PI 3-kinase inhibitor; and 2) blocking MAP kinase activation with a dominant negative p85 mutant. These observations suggest that the lipid products generated by the PI (
      • Senger D.R.
      • Perruzzi C.A.
      • Feder J.
      • Dvorak H.F.
      ) kinase following VEGF stimulation may bind to a signaling protein which could activate MAP kinase. Candidate proteins could be pleckstrin homology domain-containing proteins that require PI 3-kinase products to be fully activated (
      • Pawson T.
      ,
      • Falasca M.
      • Logan S.K.
      • Lehto U.P.
      • Baccante G.
      • Lemmon M.A.
      • Schlessinger J.
      ).
      Finally, we demonstrate for the first time the critical role of PI 3-kinase activation in generating a maximal mitogenic response to VEGF. These observations are consistent with previous evidence supporting a role for PI 3-kinase in PDGF-induced and granulocyte-macrophage colony-stimulating factor-induced mitogenic signaling in smooth muscle cells and macrophages, respectively (
      • Iwama A.
      • Sawamura M.
      • Nara Y.
      • Yamori Y.
      ,
      • Yusoff P.
      • Hamilton J.
      • Nolan R.
      • Phillips W.
      ). PI 3-kinase activation has been linked to a number of biologically diverse processes such as cell survival, membrane trafficking, and insulin-stimulated glucose transport (
      • Chen H.C.
      • Appeddu P.A.
      • Isoda H.
      • Guan J.L.
      ,
      • Kapeller R.
      • Cantley L.C.
      ,
      • Shepherd P.R.
      • Reaves B.J.
      • Davidson H.W.
      ). Very recently, the constitutively active forms of PI 3-kinase have been ingeniously used to identify and study responses specifically induced by PI 3-kinase, an approach that has shown that PI 3-kinase activation alone is sufficient to promote entry into S phase of the cell cycle (
      • Klippel A.
      • Escobedo M.A.
      • Wachowicz M.S.
      • Apell G.
      • Brown T.W.
      • Giedlin M.A.
      • Kavanaugh W.M.
      • Williams L.T.
      ). The fact that our inhibition studies with either DN p85 or wortmannin did not abolish VEGF-induced MAP kinase activation and mitogenesis suggests the presence of additional PI 3-kinase-independent pathways in VEGF-induced growth promoting effects, such as the PKC-MAP kinase (
      • Takhashi T.
      • Shibuya M.
      ) and the Ras-MAP kinase pathway (
      • Kroll J.
      • Waltenberger J.
      ). In summary, results of our study identify PI 3-kinase as an important mediator of VEGF-induced MAP kinase activation and subsequent endothelial cell proliferation.

      Acknowledgments

      We thank Dr. Axel Ullrich, Dr. Joseph Schlessinger, Dr. Julian Downward, and Dr. E. Skolnik for kindly providing us the cDNA clones used in this study and Dr. Enrique Mesri for useful comments on the manuscript.

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