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Phosphatidylinositol-3′ Kinase Is Not Required for Mitogenesis or Internalization of the Flt3/Flk2 Receptor Tyrosine Kinase*

  • Nathalie Beslu
    Footnotes
    Affiliations
    Molecular Hematology Laboratory and Unite 119, Institut National de la Santé et de la Recherche Mèdicale, 27 Bd Lei Roure, 13009 Marseille, France
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  • Jose LaRose
    Affiliations
    Wellesley Hospital Research Institute, 160 Wellesley St. East, Toronto, Ontario M4Y1J3, Canada, and the
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  • Nathalie Casteran
    Footnotes
    Affiliations
    Molecular Hematology Laboratory and Unite 119, Institut National de la Santé et de la Recherche Mèdicale, 27 Bd Lei Roure, 13009 Marseille, France
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  • Daniel Birnbaum
    Footnotes
    Affiliations
    Molecular Oncology Laboratory, Unite 119, Institut National de la Santé et de la Recherche Mèdicale, 27 Bd Lei Roure, 13009 Marseille, France, the
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  • Eric Lecocq
    Affiliations
    Molecular Hematology Laboratory and Unite 119, Institut National de la Santé et de la Recherche Mèdicale, 27 Bd Lei Roure, 13009 Marseille, France
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  • Patrice Dubreuil
    Footnotes
    Affiliations
    Molecular Hematology Laboratory and Unite 119, Institut National de la Santé et de la Recherche Mèdicale, 27 Bd Lei Roure, 13009 Marseille, France
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  • Robert Rottapel
    Correspondence
    Research Scholar of the Arthritis Society of Canada. To whom correspondence should be addressed. Tel.: 416-926-4820; Fax: 416-926-5109
    Affiliations
    Wellesley Hospital Research Institute, 160 Wellesley St. East, Toronto, Ontario M4Y1J3, Canada, and the

    Departments of Medicine and Immunology, University of Toronto, Toronto, Ontario M56 1A1, Canada
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  • Author Footnotes
    * This research was supported in part by the National Cancer Institute of Canada. 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.
    § Recipient of a fellowship from Minstère de l'Education de la Santé et de la Recherche.
    Recipient of a fellowship from Association pour la Recherche sur le Cancer.
    ‡‡ Supported by the Ligue Nationale Contre le Cancer, by the Comité des Bouches du Rhône de la Ligue Nationale Contre le Cancer, and by Institut National de la Santé et de la Recherche Mèdicale.
Open AccessPublished:August 16, 1996DOI:https://doi.org/10.1074/jbc.271.33.20075
      Flt3/Flk2 is a receptor tyrosine kinase that is expressed on early hematopoietic progenitor cells. Flt3/Flk2 belongs to a family of receptors, including Kit and colony-stimulating factor-1R, which support growth and differentiation within the hematopoietic system. The Flt3/Flk2 ligand, in combination with other growth factors, stimulates the proliferation of hematopoietic progenitors of both lymphoid and myeloid lineages in vitro. We report that phosphatidylinositol 3′-kinase (PI3K) binds to a unique site in the carboxy tail of murine Flt3/Flk2. In distinction to Kit and colony-stimulating factor-1R, mutant receptors unable to couple to PI3K and expressed in rodent fibroblasts or in the interleukin 3-dependent cell line Ba/F3 provide a mitogenic signal comparable to wild-type receptors. Flt3/Flk2 receptors that do not bind to PI3K also normally down-regulate, a function ascribed to PI3K in the context of other receptor systems. These data point to the existence of other unidentified pathways that, alone or in combination with PI3K, transduce these cellular responses following the activation of Flt3/Flk2.

      INTRODUCTION

      Vertebrate hematopoiesis is maintained throughout the life of the organism by a small number of stem cells that have the capacity to give rise to all mature myeloid and lymphoid lineages and to repopulate their own numbers through a process known as self-renewal (
      • Till J.E.
      • McCulloch E.A.
      ). Murine hematopoietic stem cells (HSCs)
      The abbreviations used are: HSC
      hematopoietic stem cell
      CSF
      colony-stimulating factor
      IL
      interleukin
      RTK
      receptor tyrosine kinase
      PDGF
      platelet-derived growth factor
      PDGFR
      PDGF receptor
      PI3K
      phosphatidylinositol 3′-kinase
      Grb2
      growth factor receptor-bound protein 2
      Gap
      GTPase-activating protein
      Shc
      Src homology and collagen
      SH2
      Src homology 2
      FACS
      fluorescence-activated cell sorter
      FCS
      fetal calf serum.
      express characteristic cell surface proteins (AA4.1+ Sca1+ Thy1.1lo linlo) (
      • McKearn J.P.
      • McCubrey J.
      • Fagg B.
      ,
      • Jordan C.T.
      • McKearn J.P.
      • Lemischka I.R.
      ) and are functionally heterogeneous, based upon their state within the cell cycle (
      • Fleming W.H.
      • Alpern E.J.
      • Uchika N.
      • Ikuta K.
      • Spangrude G.J.
      • Weissman I.L.
      ). The majority of HSCs are in a dormant G0-G1 state and are capable of radioprotection and long term reconstitution, while approximately 20% of the cells are in the S-G2-M phases of the cell cycle and have reduced reconstituting ability. The cycling fraction of HSCs may represent an early step toward the commitment of these cells to differentiate into lineage-restricted progenitor cells (
      • Fleming W.H.
      • Alpern E.J.
      • Uchika N.
      • Ikuta K.
      • Spangrude G.J.
      • Weissman I.L.
      ). Little is currently known about the signal transduction molecules that influence the cell cycle of hematopoietic stem cells and the activation of lineage-specific developmental programs. Kit (
      • Papayannopoulou T.
      • Brice M.
      • Broudy V.C.
      • Zsebo K.M.
      ,
      • Okada S.
      • Nakauchi H.
      • Nagayoshi K.
      • Nishikawa S.-I.
      • Miura Y.
      • Suda T.
      ,
      • Okada S.
      • Nakauchi H.
      • Nagayoshi K.
      • Nishikawa S.
      • Nishikawa S.
      • Miura Y.
      • Suda T.
      ,
      • Ogawa M.
      • Nishikawa S.
      • Yoshinaga K.
      • Hayashi S.-I.
      • Kunisada T.
      • Nakao J.
      • Kina T.
      • Sudo T.
      • Kodama H.
      • Nishikawa S.-I.
      ,
      • Ogawa M.
      • Matsuzaki Y.
      • Nishikawa S.
      • Hayashi S.
      • Kunisada T.
      • Sudo T.
      • Kina T.
      • Nakauchi H.
      • Nishikawa S.
      ,
      • Ikuta K.
      • Weissman I.L.
      ) and Flt3/Flk2, (
      • Matthews W.
      • Jordan C.T.
      • Wiegand G.W.
      • Pardoll D.
      • Lemischka I.R.
      ,
      • Zeigler F.C.
      • Bennett B.D.
      • Jordan C.T.
      • Spencer S.D.
      • Baumhueter S.
      • Carroll K.J.
      • Hooley J.
      • Bauer K.
      • Matthews W.
      ) receptor tyrosine kinases, are expressed on HSCs and may participate in these processes. Kit is expressed on AA4.1+Sca1+ fetal liver cells capable of long term reconstitution of irradiated recipient mice at high frequency (
      • Zeigler F.C.
      • Bennett B.D.
      • Jordan C.T.
      • Spencer S.D.
      • Baumhueter S.
      • Carroll K.J.
      • Hooley J.
      • Bauer K.
      • Matthews W.
      ). Flt3/Flk2 is expressed on cells of similar phenotype, which in distinction to the Kit-positive cells, are actively cycling and have reduced reconstitution capacity (
      • Zeigler F.C.
      • Bennett B.D.
      • Jordan C.T.
      • Spencer S.D.
      • Baumhueter S.
      • Carroll K.J.
      • Hooley J.
      • Bauer K.
      • Matthews W.
      ). W mice, which have loss-of-function mutations within the Kit locus, demonstrate diminished stem cell activity and have developmental defects in the erythroid and mast cell lineages (

      McCulloch, E. A., Siminovitch, L., Till, J. E., (1964) 144, 844-846.

      ,
      • Bernstein S.
      • Russell E.
      ,
      • Russel E.S.
      ). Mice in which the Flt3/Flk2 locus has been disrupted by homologous recombination have no defect in stem cell self-renewal but are deficient in the steady-state numbers of CD43+ pro-B cells and in repopulating both the lymphoid lineages during competition reconstitutive assays (
      • Mackarehtschian K.
      • Hardin J.D.
      • Moore K.A.
      • Boast S.
      • Goff S.P.
      • Lemischka I.R.
      ). The Flt3/Flk2 ligand, FL, by itself is a weak proliferative stimulus for linloSca-1+ progenitors; however, in combination with other growth factors such as granulocyte-CSF, granulocyte/macrophage-CSF, IL-3, IL-6, IL-7, IL-11, and IL-12, it acts as a potent mitogen (
      • Muench M.O.
      • Roncarolo M.G.
      • Menon S.
      • Xu Y.
      • Kastelein R.
      • Zurawski S.
      • Hannum C.H.
      • Culpepper J.
      • Lee F.
      • Namikawa R.
      ,
      • Lyman S.
      • James L.
      • Bos T.V.
      • de Vries P.
      • Brassel K.
      • Gliniak B.
      • Hollingworth L.T.
      • Picha K.
      • McKenna H.
      • Splett R.
      • Fletcher F.
      • Marashovsky E.
      • Farrah T.
      • Foxworthe D.
      • Williams D.
      • Beckmann M.
      ,
      • Jacobsen S.E.W.
      • Okkenhaug C.
      • Myklebust J.
      • Veiby O.P.
      • Lyman S.D.
      ,
      • Hannum C.
      • Culpepper J.
      • Campbell D.
      • McClanahan T.
      • Zurawski S.
      • Bazan J.F.
      • Kastelein R.
      • Hudak S.
      • Wagner J.
      • Mattson J.
      • Luh J.
      • Duda G.
      • Martina N.
      • Peterson D.
      • Menon S.
      • Shanafelt A.
      • Muench M.
      • Keiner G.
      • Namikawa R.
      • Rennick D.
      • Roncarolo M.-G.
      • Zlotnik A.
      • Rosnet O.
      • Dubreuil P.
      • Birnbaum D.
      • Lee F.
      ). AA4.1+Sca1+B220 progenitor cells from day 12.5 fetal liver when cultured in combination with IL-11 and FL give rise to IL-7-responsive pre-B cells at high frequency (
      • Ray B.
      • Paige C.
      • Furlonger C.
      • Lyman S.
      • Rottapel R.
      ). Although Flt3/Flk2 does not appear to play a role in maintaining the process of HSC self-renewal, Flt3/Flk2 in collaboration with other cytokine factors may function to expand the cycling pool of HSCs destined to commit to the lymphoid lineage.
      To elucidate the biochemical basis for Flt3/Flk2 mitogenic function in early B-cell ontogeny, we set out to determine the proximal signaling molecules that interact with Flt3/Flk2. Flt3/Flk2 belongs to the class III family of RTKs, based on structural similarities that include the PDGF α and β receptors, CSF-1R, Kit, and Flt3/Flk2. These molecules are characterized by a ligand-binding extracellular domain composed of five immunoglobulin-like domains and a kinase domain bisected by a non-catalytic region, known as the kinase insert. The kinase insert contains tyrosine autophosphorylation sites and, in the case of Kit, serine residues that are phosphorylated by protein kinase C (
      • Blume-Jensen P.
      • Wernstedt C.
      • Heldin C.-H.
      • Rönnstrand L.
      ). Phosphorylation of tyrosine residues in the PDGF β receptor kinase insert leads to the creation of high affinity binding sites for SH2-containing signaling proteins such as Grb2 (
      • Arvidsson A.-K.
      • Rupp E.
      • Nånberg E.
      • Downward J.
      • Rönnstrand L.
      • Wennström S.
      • Schlessinger J.
      • Heldin C.-H.
      • Claesson-Welsh L.
      ), phosphatidylinositol 3′-kinase (PI3K) (
      • Kazlauskas A.
      • Cooper J.A.
      ,
      • Escobedo J.A.
      • Kaplan D.R.
      • Kavanaugh W.M.
      • Turck C.W.
      • Williams L.T.
      ), Nck (
      • Nishimura R.
      • Li W.
      • Kashishian A.
      • Mondino A.
      • Zhou M.
      • Cooper J.
      • Schlessinger J.
      ), Gap (
      • Kazlauskas A.
      • Kashishian A.
      • Cooper J.A.
      • Valius M.
      ,
      • Fantl W.J.
      • Escobedo J.A.
      • Martin G.A.
      • Turck C.W.
      • Rosario M.
      • McCormick F.
      • Williams L.T.
      ), and Shc (
      • Yokote K.
      • Mori S.
      • Hansen K.
      • McGlade J.
      • Pawson T.
      • Heldin C.-H.
      • Claesson-Welsh L.
      ). In this report, we demonstrate that PI3K, a lipid kinase that has been implicated in mediating mitogenesis and receptor internalization, binds to Flt3/Flk2 in a region outside of the kinase insert domain. In distinction to PDGFRβ (
      • Kazlauskas A.
      • Kashishian A.
      • Cooper J.A.
      • Valius M.
      ,
      • Fantl W.J.
      • Escobedo J.A.
      • Martin G.A.
      • Turck C.W.
      • Rosario M.
      • McCormick F.
      • Williams L.T.
      ,
      • Valius M.
      • Kazlauskas A.
      ), CSF-1R, and Kit (
      • Serve H.
      • Yee N.S.
      • Stella G.
      • Sepp-Lorenzino L.
      • Tan J.C.
      • Besmer P.
      ), Flt3/Flk2 receptors which are uncoupled from PI3K down-modulate normally and are still capable of providing a full mitogenic signal. These data suggest that Flt3/Flk2 may interact with unique mitogenic signaling pathways that are operative in the absence of PI3K activation.

      MATERIALS AND METHODS

      Growth Factors, Antibodies, GST Fusion Proteins, and Cell Lines

      Recombinant human CSF-1 was kindly provided by Dale Ando (Chiron Corp.) Supernatant from transfected X63Ag8-653 myeloma cells was used as a source of IL-3 (
      • Karasuyama H.
      • Melchers F.
      ). The anti-Flt3/Flk2 antibodies were produced in rabbits against a TrpE interkinase fusion protein as described previously (
      • Maroc N.
      • Rottapel R.
      • Rosnet O.
      • Marchetto S.
      • Lavezzi C.
      • Mannoni P.
      • Birnbaum D.
      • Dubreuil P.
      ). The Shc and Gap antisera were generously provided by Jane McGlade (Amgen Institute, Toronto, Ontario, Canada) and Mike Moran (Banting and Best Institute, Toronto, Ontario, Canada), respectively. p85 antibodies were produced in rabbits against GST-SH2 domain fusion proteins. 4G10 antiphosphotyrosine antibodies were purchased from UBI. The pGEX vectors encoding the GST-Grb2 and p85(N+C) SH2 domains were made available to us by Mike Moran and Tony Pawson (Lunenfeld Research Institute, Toronto, Ontario, Canada), respectively, and the recombinant proteins were prepared as described previously (
      • McGlade C.J.
      • Ellis C.
      • Reedijk M.
      • Anderson D.
      • Mbamalu G.
      • Reith A.D.
      • Panayotou G.
      • End P.
      • Bernstein A.
      • Kazlauskas A.
      • Waterfield M.D.
      • Pawson T.
      ).

      Cell Transfection and Infection

      Cos7 cells were transfected using 1 µg of vector DNA and Lipofectin (Life Technologies, Inc.) according to manufacturer's protocol. GP+E cell lines producing helper-free retrovirus expressing FF3 or mutant receptors were prepared as described (
      • Markowitz D.
      • Goff S.
      • Bank A.
      ,
      • Dubreuil P.
      • Forrester L.
      • Rottapel R.
      • Reedijk M.
      • Fujita J.
      • Bernstein A.
      ) and used to infect Rat2 cells and BA/F3 cells.

      In Vitro Kinase Assays, PI3K Assays, and Western Blotting

      In Vitro Kinase Assays

      Cells were washed with ice-cold Tris-saline and lysed in 1 ml of RIPA buffer (50 mM Tris-HCL, pH 7.5, 150 mM NaCl, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 100 µM sodium vanadate, 100 µg/ml leupeptin, and 1 mM phenylmethylsulfonylfluoride). Lysates were incubated with 5 µl of anti-Flt3/Flk2 antisera and 50 µl of protein A-Sepharose (Pharmacia Biotech Inc.) for 2 h at 4°C. Immunoprecipitates were washed three times in ice-cold RIPA buffer, two times in ice-cold 50 mM Tris-HCL, pH 7.5, and 1% (v/v) Triton X-100 and resuspended in 10 µl kinase reaction buffer (10 mM MnCl2, 1% (v/v) Triton X-100, 10 µCi [γ-32P]ATP (3000 Ci/mmol; Amersham Corp.)). Following incubation for 10 min at 30°C, an equal volume of SDS sample buffer was added, and samples were subjected to 7.5% SDS-polyacrylamide gel electrophoresis. Following electrophoresis, gels were fixed with 10% acetic acid and 30% methanol and treated with 1 M potassium hydroxide prior to autoradiography.

      PI3K Assays

      Rat2 cells expressing wild-type, F922, and F958 mutant Flt3/Flk2 receptors were induced to quiescence, stimulated with CSF-1, lysed in lysis buffer, and incubated with anti-Flt3/Flk2 antibodies as described above. The immunoprecipitates were washed and assayed for in vitro PI kinase activity as described previously (
      • Fukui Y.
      • Hanafusa H.
      ). The inhibitors, 100 µM adenosine and 0.5% Triton X-100, were added to distinguish between types I and II PI3K (
      • Whitman M.
      • Kaplan D.R.
      • Roberts T.M.
      • Cantley L.
      ). Phospholipid standards were visualized by exposure to I2 vapor, and the radiolabeled lipids were detected by autoradiography after thin-layer chromatography.

      Western Blotting

      Growth factor-deprived cells (5 × 106) were stimulated for 5 min at 37°C with 200 ng/ml recombinant human CSF-1 before lysis in ice-cold lysis buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 200 µM sodium orthovanadate, 10 mM pyrophosphate, and 100 mM sodium fluoride. Cell lysates were clarified at 13,000 rpm for 15 min. Lysates were incubated for 1 h at 4°C either with anti-Flt3/Flk2, anti-p85, anti-Shc, or anti-Gap antisera. The immune complexes were washed three times with HNTG (20 mM HEPES, pH 7.5, 10% glycerol, 0.1% Triton X-100, 150 mM NaCl, and 1 mM sodium orthovanadate), heated in SDS-sample buffer, separated by gel electrophoresis, semi-dry transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore) and immunoblotted with 4G10 antiphosphotyrosine antibodies.

      In Vitro Mixing Experiments with GST-SH2 Fusion Proteins

      GST fusion proteins were recovered from clarified bacterial lysates using glutathione-Sepharose beads (Pharmacia) following isopropyl-1-thio-β-D-galactopyranoside induction, as described by the manufacturer's protocol. Five µg of recombinant protein, as assayed by Bradford protein determination assay, was used in all experiments. Lysates from CSF-1-induced Rat2 cells expressing wild-type, F922, or F958 were incubated with recombinant GST-SH2 domains derived from p85 or Grb2 for 1 h at 4°C. Protein complexes were washed three times with HNTG buffer, resolved by SDS-polyacrylamide gel electrophoresis, transferred to Immobilon, and immunoblotted with anti-Flt3/Flk2 antibodies.

      Flow Cytometric Analysis

      Cells (2.5 × 105) were washed in a washing buffer (PBS supplemented with 5% FCS and 0.2% sodium azide) and resuspended in 50 µl of the same buffer. Anti-murine CSF-1R 7D6-3D mAb (UBI 05-221, Lake Placid, NY) was diluted 1:10 in the washing buffer and added to the cell suspension. After 45 min at 4°C, cells were washed three times with the washing buffer and incubated for 30 min at 4°C in a 1:50 dilution of polyclonal phycoerythrin-labeled Goat anti Rat (Immunotech, Marseille, France). Cells were then washed three times in the washing buffer, and pellets were resuspended in 50 µl of PBS before the addition of 200 µl of PBS supplemented with 2% formaldehyde.

      Internalization Assay

      BA/F3 cells (2.5 × 105) were washed in PBS supplemented with 5% FCS and incubated for 30 min at 4°C with biotinylated CSF-1. Cells were washed at 4°C in 1 ml PBS and resuspended in 100 µl of PBS supplemented with 5% FCS. Cells were then incubated for different periods of time at 37°C. Internalization was stopped by the addition of 1 ml of PBS supplemented with 0.1% of sodium azide. After three washes with PBS/azide buffer, cells were incubated for 15 min at 4°C with 50 µl of 1:80 phycoerythrin-Streptavidin (Immunotech). Cells were then washed three times in the washing buffer, and pellets were resuspended in 50 µl of PBS before the addition of 200 µl of PBS supplemented with 2% formaldehyde.

      DISCUSSION

      In this study, we showed that PI3K binds to a unique site in the carboxy terminus of the cytoplasmic tail. The location of this binding site distinguishes Flt3/Flk2 from the other four members of the class III family of RTKs, Kit, CSF-1R, PDGFRα, and PDGFRβ, which contain PI3K binding sites within their kinase insert domains (
      • Kazlauskas A.
      • Kashishian A.
      • Cooper J.A.
      • Valius M.
      ,
      • Fantl W.J.
      • Escobedo J.A.
      • Martin G.A.
      • Turck C.W.
      • Rosario M.
      • McCormick F.
      • Williams L.T.
      ,
      • Valius M.
      • Kazlauskas A.
      ,
      • Serve H.
      • Hsu Y.-C.
      • Besmer P.
      ,
      • Reedijk M.
      • Liu X.
      • van der Geer P.
      • Letwin K.
      • Waterfield M.D.
      • Hunter T.
      • Pawson T.
      ). Flt3/Flk2 binds to the p85 subunit of PI3K in an inducible fashion to a sequence surrounding tyrosine 958, YQNM. This sequence conforms to the optimal p85 binding consensus sequence, YXXM, defined by Songyang et al. (
      • Songyang Z.
      • Shoelson S.E.
      • Chaudhuri M.
      • Gish G.
      • Pawson T.
      • Haser W.G.
      • King F.
      • Roberts T.
      • Ratnofsky S.
      • Lechleider R.J.
      • Neel B.G.
      • Birge R.B.
      • Fajardo J.E.
      • Chou M.M.
      • Hanafusa H.
      • Schaffhausen B.
      • Cantley L.C.
      ) and is present in Kit, CSF-1, and PDGFRs. We were unable to detect a secondary p85 binding site, as seen in the case of PDGFR (
      • Escobedo J.A.
      • Kaplan D.R.
      • Kavanaugh W.M.
      • Turck C.W.
      • Williams L.T.
      ,
      • Yu J.-C.
      • Heidaran M.A.
      • Pierce J.H.
      • Gutkind J.S.
      • Lombardi D.
      • Ruggiero M.
      • Aaronson S.A.
      ,
      • Kavanaugh W.M.
      • Klippel A.
      • Escobedo J.A.
      • Williams L.T.
      ,
      • Klippel A.
      • Escobedo J.A.
      • Fantl W.J.
      • Williams L.T.
      ), although another potential binding site, YFVM at codon 922, is present in the carboxy tail. The amino acids neighboring tyrosine 958 contain an asparagine residue at the +2 position and thus conform to a Grb2 SH2 binding sequence (
      • Songyang Z.
      • Shoelson S.E.
      • Chaudhuri M.
      • Gish G.
      • Pawson T.
      • Haser W.G.
      • King F.
      • Roberts T.
      • Ratnofsky S.
      • Lechleider R.J.
      • Neel B.G.
      • Birge R.B.
      • Fajardo J.E.
      • Chou M.M.
      • Hanafusa H.
      • Schaffhausen B.
      • Cantley L.C.
      ). Preliminary data show that Grb2 binds directly to Flt3/Flk2 at Y958 and to least one other site present in the carboxy tail.
      R. Rottapel and P. Dubreuil, unpublished data.
      Tyrosine 958 may, therefore, function as a dual-specific site for binding p85 and Grb2. A similar site, which specifies both p85 and Grb2 binding, is present in the Met RTK (
      • Fixman E.D.
      • Naujokas M.A.
      • Rodrigues G.A.
      • Moran M.F.
      • Park M.
      ).
      PI3K has been linked to a variety of RTK-mediated biological responses including mitogenesis (
      • Serunian L.A.
      • Auger K.R.
      • Roberts T.M.
      • Cantley L.C.
      ), chemotaxis (
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ), cell survival (
      • Yao R.
      • Cooper G.M.
      ), receptor down-modulation (
      • Joly M.
      • Kazlauskas A.
      • Corvera S.
      ,
      • Joly M.
      • Kazlauskas A.
      • Fay F.
      • Corvera S.
      ,
      • Kapeller R.
      • Chakrabarti R.
      • Cantley L.
      • Fay F.
      • Corvera S.
      ), and cell polarization (
      • Stowers L.
      • Yelon D.
      • Berg L.J.
      • Chant J.
      ). The diversity of these biological responses may be related to the complex biochemical interactions with which PI3K is involved. PI3K physically interacts with or lies upstream of several signaling molecules that participate in mitogenic responses, including the Src family kinases (
      • Liu X.
      • Marengere L.M.
      • Koch C.A.
      • Pawson T.
      ,
      • Prasad K.V.S.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ), Ras (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Rodriquez-Viciana P.
      • Warne P.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.
      • Waterfield M.
      • Downward J.
      ,
      • Sjölander A.
      • Yamamoto K.
      • Huber B.E.
      • Lapetina E.G.
      ), the Ras-related GTPase Cdc42HS (
      • Zheng Y.
      • Bagrodia S.
      • Cerione R.A.
      ), the serine/threonine kinase Akt (
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Burgering B.M.T.
      • Coffer P.J.
      ), and pp70/85 S6 kinase (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ). The requirement for PI3K to mediate receptor tyrosine kinase mitogenesis is variable and depends on both the receptor and the cell type. PI3K is both necessary and sufficient for PDGF-dependent mitogenesis through the β receptor (
      • Kazlauskas A.
      • Kashishian A.
      • Cooper J.A.
      • Valius M.
      ,
      • Fantl W.J.
      • Escobedo J.A.
      • Martin G.A.
      • Turck C.W.
      • Rosario M.
      • McCormick F.
      • Williams L.T.
      ) but not the α receptor (
      • Yu J.-C.
      • Heidaran M.A.
      • Pierce J.H.
      • Gutkind J.S.
      • Lombardi D.
      • Ruggiero M.
      • Aaronson S.A.
      ). The CSF-1R mutated at the PI3K binding site and ectopically expressed in rat fibroblasts has a diminished mitogenic response (
      • van der Geer P.
      • Hunter T.
      ), whereas Kit mutant receptors that no longer bind PI3K have only a slight mitogenic defect in mast cells (
      • Serve H.
      • Yee N.S.
      • Stella G.
      • Sepp-Lorenzino L.
      • Tan J.C.
      • Besmer P.
      ). We studied the necessity of PI3K on Flt3/Flk2-dependent mitogenesis in both rat fibroblasts and Ba/F3 cells by analyzing the capacity of the Flt3/Flk2-F958 mutant to induce anchorage-independent cell growth, cell proliferation, and DNA synthesis. Rat2 cells expressing F958 were able to form colonies in soft agar and to proliferate with a doubling time comparable to wild-type receptors in response to ligand. Flt3/Flk2 receptors uncoupled from PI3K expressed in Ba/F3 cells also stimulated a mitogenic response at levels similar to wild-type receptors. We showed that two downstream targets of Flt3/Flk2, Shc and p62, are normally phosphorylated by the F958 Flt3/Flk2 mutant.
      Receptor coupling to Shc may be sufficient to maintain the mitogenic response in some receptor systems. For example, epidermal growth factor receptor mutants with a truncation in the carboxy terminus that removes the major autophosphorylation sites and the binding sites for phospholipase Cγ, Grb-2, PI3K, and Shc are still able to promote the phosphorylation of Shc, induce formation of Grb2-Shc complexes, and provide a mitogenic response (
      • Li N.
      • Schlessinger J.
      • Margolis B.
      ,
      • Gotoh N.
      • Tojo A.
      • Muroya K.
      • Hashimoto Y.
      • Hattori S.
      • Nakamura S.
      • Takenawa T.
      • Yazaki Y.
      • Shibuya M.
      ). Therefore, in distinction to PDGFR and to a lesser degree CSF-1R, the capacity of Flt3/Flk2 to maintain anchorage-independent growth or stimulate proliferative responses does not require PI3K and may be mediated through a Grb2-Shc pathway.
      RTKs rapidly internalize following ligand binding. Internalized receptors are then sorted to distinct subcellular pathways that lead either to degradation or recycling to the cell surface. PDGFR mutants that do not bind to PI3K have attenuated rates of internalization and degradation (
      • Joly M.
      • Kazlauskas A.
      • Corvera S.
      ,
      • Joly M.
      • Kazlauskas A.
      • Fay F.
      • Corvera S.
      ,
      • Kapeller R.
      • Chakrabarti R.
      • Cantley L.
      • Fay F.
      • Corvera S.
      ). These studies have suggested that mammalian PI3K may share with its yeast homologue Vps34, a highly conserved cellular function that directs membrane-associated proteins to sort to postendocytic degradative vesicles (
      • Herman P.K.
      • Stack J.H.
      • DeModena J.A.
      • Emr S.D.
      ). The detailed biochemical basis for this function is not known; however, PI3K has been shown to interact with several GTPases involved in endocytosis and reorganization of the actin cytoskeleton. PI3K binds, via its SH3 domain to dynamin (
      • Gout I.
      • Dhand R.
      • Hiles I.D.
      • Fry M.J.
      • Panayotou G.
      • Das P.
      • Truong O.
      • Totty N.F.
      • Hsuan J.
      • Booker G.W.
      • Campbell I.D.
      • Waterfield M.D.
      ), a large GTPase required for the endocytosis of surface membrane by mediating the fission of invaginated clathrin-coated pits. PI3K, possibly acting through the production of its lipid product phosphoinositide 3,4,5trisphosphate increases the level of the GTP-bound, active form of Rac (
      • Hawkins P.T.
      • Eguinoa A.
      • Rong-Guo Q.
      • Stokoe D.
      • Cooke F.T.
      • Walters R.
      • Wennstrom S.
      • Claesson-Welsh L.
      • Symons M.
      ), which in turn induces reorganization of the membrane-associated actin cytoskeleton to form lamellipodia (
      • Nobes C.D.
      • Hall A.
      ). The Ras-related GTPase, Cdc42, is another target of PI3K (
      • Zheng Y.
      • Bagrodia S.
      • Cerione R.A.
      ) and induces the reorganization of a different subcellular actin compartment, resulting in the formation of filopodia (
      • Nobes C.D.
      • Hall A.
      ). Some or all of these PI3K-related cytoskeletal changes may be required for initiating receptor down-modulation and cellular chemotactic responses. We studied the kinetics of F958 internalization following biotinylated ligand binding by FACS analysis. Both wild-type and F958 Flt3/Flk2 receptors rapidly internalized with comparable kinetics (t1/2 of 10 min). In distinction to PDGFRβ, Flt3/Flk2 internalization was not dependent upon PI3K activation.
      The role of Flt3/Flk2 in early hematopoiesis and B-cell ontogeny may be complex and include diverse cellular responses including mitogenesis, chemotaxis, cell survival, and differentiation. We have begun to study the biochemical basis of some of these responses by identifying and mapping the interaction sites of Flt3/Flk2 targets. We have shown that contrary to other receptor systems, Flt3/Flk2 does not require PI3K to stimulate cell proliferation or receptor down-modulation. These observations point to the existence of other unidentified pathways, which alone or in combination with PI3K, transduce these cellular responses.

      REFERENCES

        • Till J.E.
        • McCulloch E.A.
        Biochim. Biophys. Acta. 1980; 605: 431-459
        • McKearn J.P.
        • McCubrey J.
        • Fagg B.
        Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7414-7418
        • Jordan C.T.
        • McKearn J.P.
        • Lemischka I.R.
        Cell. 1990; 61: 953-962
        • Fleming W.H.
        • Alpern E.J.
        • Uchika N.
        • Ikuta K.
        • Spangrude G.J.
        • Weissman I.L.
        J. Cell Biol. 1993; 122: 897-902
        • Papayannopoulou T.
        • Brice M.
        • Broudy V.C.
        • Zsebo K.M.
        Blood. 1991; 78: 1403-1412
        • Okada S.
        • Nakauchi H.
        • Nagayoshi K.
        • Nishikawa S.-I.
        • Miura Y.
        • Suda T.
        Blood. 1992; 80: 3044-3050
        • Okada S.
        • Nakauchi H.
        • Nagayoshi K.
        • Nishikawa S.
        • Nishikawa S.
        • Miura Y.
        • Suda T.
        Blood. 1991; 78: 1706-1712
        • Ogawa M.
        • Nishikawa S.
        • Yoshinaga K.
        • Hayashi S.-I.
        • Kunisada T.
        • Nakao J.
        • Kina T.
        • Sudo T.
        • Kodama H.
        • Nishikawa S.-I.
        Development (Camb.). 1993; 117: 1089-1098
        • Ogawa M.
        • Matsuzaki Y.
        • Nishikawa S.
        • Hayashi S.
        • Kunisada T.
        • Sudo T.
        • Kina T.
        • Nakauchi H.
        • Nishikawa S.
        J. Exp. Med. 1991; 174: 63-71
        • Ikuta K.
        • Weissman I.L.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1502-1506
        • Matthews W.
        • Jordan C.T.
        • Wiegand G.W.
        • Pardoll D.
        • Lemischka I.R.
        Cell. 1991; 65: 1143-1152
        • Zeigler F.C.
        • Bennett B.D.
        • Jordan C.T.
        • Spencer S.D.
        • Baumhueter S.
        • Carroll K.J.
        • Hooley J.
        • Bauer K.
        • Matthews W.
        Blood. 1994; 8: 2422-2430
      1. McCulloch, E. A., Siminovitch, L., Till, J. E., (1964) 144, 844-846.

        • Bernstein S.
        • Russell E.
        Proc. Exp. Biol. Med. 1959; 101: 769-773
        • Russel E.S.
        Adv. Genet. 1979; 20: 357-458
        • Mackarehtschian K.
        • Hardin J.D.
        • Moore K.A.
        • Boast S.
        • Goff S.P.
        • Lemischka I.R.
        Immunity. 1995; 3: 147-161
        • Muench M.O.
        • Roncarolo M.G.
        • Menon S.
        • Xu Y.
        • Kastelein R.
        • Zurawski S.
        • Hannum C.H.
        • Culpepper J.
        • Lee F.
        • Namikawa R.
        Blood. 1995; 85: 963-972
        • Lyman S.
        • James L.
        • Bos T.V.
        • de Vries P.
        • Brassel K.
        • Gliniak B.
        • Hollingworth L.T.
        • Picha K.
        • McKenna H.
        • Splett R.
        • Fletcher F.
        • Marashovsky E.
        • Farrah T.
        • Foxworthe D.
        • Williams D.
        • Beckmann M.
        Cell. 1993; 75: 1157-1167
        • Jacobsen S.E.W.
        • Okkenhaug C.
        • Myklebust J.
        • Veiby O.P.
        • Lyman S.D.
        J. Exp. Med. 1995; 181: 1357-1363
        • Hannum C.
        • Culpepper J.
        • Campbell D.
        • McClanahan T.
        • Zurawski S.
        • Bazan J.F.
        • Kastelein R.
        • Hudak S.
        • Wagner J.
        • Mattson J.
        • Luh J.
        • Duda G.
        • Martina N.
        • Peterson D.
        • Menon S.
        • Shanafelt A.
        • Muench M.
        • Keiner G.
        • Namikawa R.
        • Rennick D.
        • Roncarolo M.-G.
        • Zlotnik A.
        • Rosnet O.
        • Dubreuil P.
        • Birnbaum D.
        • Lee F.
        Nature. 1994; 368: 643-648
        • Blume-Jensen P.
        • Wernstedt C.
        • Heldin C.-H.
        • Rönnstrand L.
        J. Biol. Chem. 1995; 270: 14192-14200
        • Arvidsson A.-K.
        • Rupp E.
        • Nånberg E.
        • Downward J.
        • Rönnstrand L.
        • Wennström S.
        • Schlessinger J.
        • Heldin C.-H.
        • Claesson-Welsh L.
        Mol. Cell. Biol. 1994; 14: 6715-6726
        • Kazlauskas A.
        • Cooper J.A.
        Cell. 1989; 58: 1121-1133
        • Escobedo J.A.
        • Kaplan D.R.
        • Kavanaugh W.M.
        • Turck C.W.
        • Williams L.T.
        Mol. Cell. Biol. 1991; 11: 1125-1132
        • Nishimura R.
        • Li W.
        • Kashishian A.
        • Mondino A.
        • Zhou M.
        • Cooper J.
        • Schlessinger J.
        Mol. Cell. Biol. 1993; 13: 6889-6896
        • Kazlauskas A.
        • Kashishian A.
        • Cooper J.A.
        • Valius M.
        Mol. Cell. Biol. 1992; 12: 2534-2544
        • Fantl W.J.
        • Escobedo J.A.
        • Martin G.A.
        • Turck C.W.
        • Rosario M.
        • McCormick F.
        • Williams L.T.
        Cell. 1992; 69: 413-423
        • Yokote K.
        • Mori S.
        • Hansen K.
        • McGlade J.
        • Pawson T.
        • Heldin C.-H.
        • Claesson-Welsh L.
        J. Biol. Chem. 1994; 269: 15337-15343
        • Hawkins P.T.
        • Eguinoa A.
        • Rong-Guo Q.
        • Stokoe D.
        • Cooke F.T.
        • Walters R.
        • Wennstrom S.
        • Claesson-Welsh L.
        • Symons M.
        Curr. Biol. 1995; 5: 393-403
        • Hu Q.
        • Klippel A.
        • Muslin A.J.
        • Fantl W.J.
        • Williams L.T.
        Science. 1995; 268: 100-102
        • Rodriquez-Viciana P.
        • Warne P.
        • Dhand R.
        • Vanhaesebroeck B.
        • Gout I.
        • Fry M.
        • Waterfield M.
        • Downward J.
        Nature. 1994; 370: 527-532
        • Sjölander A.
        • Yamamoto K.
        • Huber B.E.
        • Lapetina E.G.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7908-7912
        • Zheng Y.
        • Bagrodia S.
        • Cerione R.A.
        J. Biol. Chem. 1994; 269: 18727-18730
        • Franke T.F.
        • Yang S.-I.
        • Chan T.O.
        • Datta K.
        • Kazlauskas A.
        • Morrison D.K.
        • Kaplan D.R.
        • Tsichlis P.N.
        Cell. 1995; 81: 727-736
        • Burgering B.M.T.
        • Coffer P.J.
        Nature. 1995; 376: 599-602
        • Chung J.
        • Grammer T.C.
        • Lemon K.P.
        • Kazlauskas A.
        • Blenis J.
        Nature. 1994; 370: 71-75
        • Valius M.
        • Kazlauskas A.
        Cell. 1993; 73: 321-334
        • Serve H.
        • Yee N.S.
        • Stella G.
        • Sepp-Lorenzino L.
        • Tan J.C.
        • Besmer P.
        EMBO J. 1995; 14: 473-483
        • Karasuyama H.
        • Melchers F.
        Eur. J. Immunol. 1988; 18: 97-104
        • Maroc N.
        • Rottapel R.
        • Rosnet O.
        • Marchetto S.
        • Lavezzi C.
        • Mannoni P.
        • Birnbaum D.
        • Dubreuil P.
        Oncogene. 1993; 8: 909-918
        • McGlade C.J.
        • Ellis C.
        • Reedijk M.
        • Anderson D.
        • Mbamalu G.
        • Reith A.D.
        • Panayotou G.
        • End P.
        • Bernstein A.
        • Kazlauskas A.
        • Waterfield M.D.
        • Pawson T.
        Mol. Cell. Biol. 1992; 12: 991-997
        • Markowitz D.
        • Goff S.
        • Bank A.
        J. Virol. 1988; 62: 1120-1124
        • Dubreuil P.
        • Forrester L.
        • Rottapel R.
        • Reedijk M.
        • Fujita J.
        • Bernstein A.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2341-2345
        • Fukui Y.
        • Hanafusa H.
        Mol. Cell. Biol. 1989; 9: 1651-1658
        • Whitman M.
        • Kaplan D.R.
        • Roberts T.M.
        • Cantley L.
        Biochemistry. 1987; 247: 165-174
        • Dosil M.
        • Wang S.
        • Lemischka I.R.
        Mol. Cell. Biol. 1993; 13: 6572-6585
        • Rottapel R.
        • Turck C.W.
        • Casteran N.
        • Liu X.
        • Birnbaum D.
        • Pawson T.
        • Dubreuil P.
        Oncogene. 1994; 9: 1755-1765
        • Yu J.-C.
        • Heidaran M.A.
        • Pierce J.H.
        • Gutkind J.S.
        • Lombardi D.
        • Ruggiero M.
        • Aaronson S.A.
        Mol. Cell. Biol. 1991; 11: 3780-3785
        • Reedijk M.
        • Liu X.
        • van der Geer P.
        • Letwin K.
        • Waterfield M.D.
        • Hunter T.
        • Pawson T.
        EMBO J. 1992; 11: 1365-1372
        • Serve H.
        • Hsu Y.-C.
        • Besmer P.
        J. Biol. Chem. 1994; 269: 6026-6030
        • Rottapel R.
        • Reedijk M.
        • Williams D.E.
        • Lyman S.D.
        • Anderson D.M.
        • Pawson T.
        • Bernstein A.
        Mol. Cell. Biol. 1991; 11: 3043-3051
        • Lev S.
        • Givol D.
        • Yarden Y.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 678-682
        • Ponzetto C.
        • Bardelli A.
        • Maina F.
        • Longati P.
        • Panayotou G.
        • Dhand R.
        • Waterfield M.D.
        • Comoglio P.M.
        Mol. Cell. Biol. 1993; 13: 4600-4608
        • He T.C.
        • Zhuang H.
        • Jiang N.
        • Waterfield M.D.
        • Wojchowski D.M.
        Blood. 1993; 82: 3530-3538
        • Mori S.
        • Ronnstrand L.
        • Yokote K.
        • Engstrom A.
        • Courtneidge S.A.
        • Claesson-Welsh L.
        • Heldin C.-H.
        EMBO J. 1993; 12: 2257-2264
        • Myles G.M.
        • Brandt C.S.
        • Carlberg K.
        • Rohrschneider L.R.
        Mol. Cell. Biol. 1994; 14: 4843-4854
        • Fixman E.D.
        • Naujokas M.A.
        • Rodrigues G.A.
        • Moran M.F.
        • Park M.
        Oncogene. 1995; 10: 237-249
        • Pelicci G.
        • Lanfrancone L.
        • Grignani F.
        • McGlade J.
        • Cavallo F.
        • Forni G.
        • Nicoletti I.
        • Grignani F.
        • Pawson T.
        • Pelicci P.G.
        Cell. 1992; 70: 93-104
        • Rozakis-Adcock M.
        • McGlade J.
        • Mbamalu G.
        • Pelicci G.
        • Daly R.
        • Li W.
        • Batzer A.
        • Thomas S.
        • Brugge J.
        • Pelicci P.G.
        • Schlessinger J.
        • Pawson T.
        Nature. 1992; 360: 689-692
        • Liu L.
        • Damen J.E.
        • Cutler R.L.
        • Krystal G.
        Mol. Cell. Biol. 1994; 14: 6926-6935
        • Lioubin M.N.
        • Myles G.M.
        • Carlberg K.
        • Bowtell D.
        • Rohrschneider L.R.
        Mol. Cell. Biol. 1994; 14: 5682-5691
        • Blaikie P.
        • Immanuel D.
        • Wu J.
        • Li N.
        • Yajnik V.
        • Margolis B.
        J. Biol. Chem. 1994; 269: 32031-32034
        • Kavanaugh W.M.
        • Williams L.T.
        Science. 1994; 266: 1862-1865
        • Segatto O.
        • Pelicci G.
        • Giuli S.
        • Digiesi G.
        • Di Fiore P.P.
        • McGlade J.
        • Pawson T.
        • Pelicci P.G.
        Oncogene. 1993; 8: 2105-2112
        • Moran M.F.
        • Koch C.A.
        • Anderson D.
        • Ellis C.
        • England L.
        • Martin G.S.
        • Pawson T.
        Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8622-8626
      2. Ellis, C., Moran, M., McCormick, F., Pawson, T., (1990) 343, 377-381.

        • Richard S.
        • Yu D.
        • Blumer K.J.
        • Hausladen D.
        • Olszowy M.W.
        • Connelly P.A.
        • Shaw A.S.
        Mol. Cell. Biol. 1995; 15: 186-197
        • Weng Z.
        • Thomas S.M.
        • Rickles R.J.
        • Taylor J.A.
        • Brauer A.W.
        • Seidel-Dugan C.
        • Michael W.M.
        • Dreyfuss G.
        • Brugge J.S.
        Mol. Cell. Biol. 1994; 14: 4509-4521
        • Maa M.C.
        • Leu T.H.
        • Trandel B.J.
        • Chang J.H.
        • Parsons S.J.
        Mol. Cell. Biol. 1994; 14: 5466-5473
        • Wang L.L.
        • Richard S.
        • Shaw A.S.
        J. Biol. Chem. 1995; 270: 2010-2013
        • Fumagalli S.
        • Totty N.F.
        • Hsuan J.J.
        • Courtneidge S.A.
        Nature. 1994; 368: 871-874
        • Taylor S.J.
        • Shalloway D.
        Nature. 1994; 368: 867-871
        • Brott B.K.
        • Decker S.
        • Shafer J.
        • Gibbs J.B.
        • Jove R.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 755-759
        • Kazlauskas A.
        • Ellis C.
        • Pawson T.
        • Cooper J.
        Science. 1990; 247: 1578-1581
        • Kaplan D.R.
        • Morrison D.
        • Wong G.
        • McCormick F.
        • Williams L.
        Cell. 1990; 61: 125-133
        • Cantley L.C.
        • Auger K.R.
        • Carpenter C.
        • Duckworth B.
        • Graziani A.
        • Kapeller R.
        • Soltoff S.
        Cell. 1991; 64: 281-302
        • Weng W.-K.
        • Jarvis L.
        • LeBien T.W.
        J. Biol. Chem. 1994; 269: 32514-32521
        • Tuveson D.A.
        • Carter R.H.
        • Soltoff S.P.
        • Fearon D.T.
        Science. 1993; 260: 986-989
        • Pages F.
        • Ragueneau M.
        • Rottapel R.
        • Truneh A.
        • Nunes J.
        • Imbert J.
        • Olive D.
        Nature. 1994; 369: 327-329
        • Taichman R.
        • Merida I.
        • Torigoe T.
        • Gaulton G.N.
        • Reed J.C.
        J. Biol. Chem. 1993; 268: 20031-20036
        • Stephens L.
        • Smrcka A.
        • Cooke F.T.
        • Jackson T.R.
        • Sternweis P.C.
        • Hawkins P.T.
        Cell. 1994; 77: 83-93
        • Stephens L.
        • Eguinoa A.
        • Corey S.
        • Jackson T.
        • Hawkins P.T.
        EMBO J. 1993; 12: 2265-2273
        • Stephens L.
        • Jackson T.
        • Hawkins P.T.
        J. Biol. Chem. 1993; 268: 17162-17172
        • Serunian L.A.
        • Auger K.R.
        • Roberts T.M.
        • Cantley L.C.
        J. Virol. 1990; 64: 4718-4725
        • Ullrich A.
        • Schlessinger J.
        Cell. 1990; 61: 203-212
        • Joly M.
        • Kazlauskas A.
        • Corvera S.
        J. Biol. Chem. 1995; 270: 13225-13230
        • Joly M.
        • Kazlauskas A.
        • Fay F.
        • Corvera S.
        Science. 1994; 263: 684-687
        • Kapeller R.
        • Chakrabarti R.
        • Cantley L.
        • Fay F.
        • Corvera S.
        Mol. Cell. Biol. 1993; 13: 6052-6063
        • Yee N.S.
        • Hsiau C.-W.M.
        • Serve H.
        • Vosseller K.
        • Besmer P.
        J. Biol. Chem. 1994; 269: 31991-31998
        • Reedijk M.
        • Liu X.
        • van der Geer P.
        • Letwin K.
        • Waterfield M.D.
        • Hunter T.
        • Pawson T.
        EMBO J. 1992; 11: 1365-1372
        • Songyang Z.
        • Shoelson S.E.
        • Chaudhuri M.
        • Gish G.
        • Pawson T.
        • Haser W.G.
        • King F.
        • Roberts T.
        • Ratnofsky S.
        • Lechleider R.J.
        • Neel B.G.
        • Birge R.B.
        • Fajardo J.E.
        • Chou M.M.
        • Hanafusa H.
        • Schaffhausen B.
        • Cantley L.C.
        Cell. 1993; 72: 767-778
        • Kavanaugh W.M.
        • Klippel A.
        • Escobedo J.A.
        • Williams L.T.
        Mol. Cell. Biol. 1992; 12: 3415-3424
        • Klippel A.
        • Escobedo J.A.
        • Fantl W.J.
        • Williams L.T.
        Mol. Cell. Biol. 1992; 12: 1451-1459
        • Kundra V.
        • Escobedo J.A.
        • Kazlauskas A.
        • Kim H.K.
        • Rhee S.G.
        • Williams L.T.
        • Zetter B.R.
        Nature. 1994; 367: 474-476
        • Yao R.
        • Cooper G.M.
        Science. 1995; 267: 2003-2006
        • Stowers L.
        • Yelon D.
        • Berg L.J.
        • Chant J.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5027-5031
        • Liu X.
        • Marengere L.M.
        • Koch C.A.
        • Pawson T.
        Mol. Cell. Biol. 1993; 13: 5225-5232
        • Prasad K.V.S.
        • Janssen O.
        • Kapeller R.
        • Raab M.
        • Cantley L.C.
        • Rudd C.E.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7366-7370
        • van der Geer P.
        • Hunter T.
        EMBO J. 1993; 12: 5161-5172
        • Li N.
        • Schlessinger J.
        • Margolis B.
        Oncogene. 1994; 9: 3457-3465
        • Gotoh N.
        • Tojo A.
        • Muroya K.
        • Hashimoto Y.
        • Hattori S.
        • Nakamura S.
        • Takenawa T.
        • Yazaki Y.
        • Shibuya M.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 167-171
        • Herman P.K.
        • Stack J.H.
        • DeModena J.A.
        • Emr S.D.
        Cell. 1991; 64: 425-437
        • Gout I.
        • Dhand R.
        • Hiles I.D.
        • Fry M.J.
        • Panayotou G.
        • Das P.
        • Truong O.
        • Totty N.F.
        • Hsuan J.
        • Booker G.W.
        • Campbell I.D.
        • Waterfield M.D.
        Cell. 1993; 75: 25-36
        • Nobes C.D.
        • Hall A.
        Cell. 1995; 81: 53-62
        • Ray B.
        • Paige C.
        • Furlonger C.
        • Lyman S.
        • Rottapel R.
        Eur. J. Immunol. 1996; 26: 1504-1510
        • Liobin M.N.
        • Algate P.A.
        • Tsai S.
        • Carlberg K.
        • Aebersold R.
        • Rohrschneider L.R.
        Genes & Dev. 1996; 10: 1084-1095
        • Damen J.E.
        • Liu L.
        • Rosten P.
        • Humphries R.K.
        • Jefferson A.B.
        • Majerus P.W.
        • Krystal G.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693