Advertisement

Hepatocyte Growth Factor-induced Scatter of Madin-Darby Canine Kidney Cells Requires Phosphatidylinositol 3-Kinase (*)

  • Isabelle Royal
    Footnotes
    Affiliations
    Molecular Oncology Laboratory, Royal Victoria Hospital, the Department of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada
    Search for articles by this author
  • Morag Park
    Correspondence
    Senior scholar of the National Cancer Institute of Canada. To whom correspondence should be addressed: Molecular Oncology Group, H5.21, 687 Pine Ave. West, Montreal, Quebec H3A 1A1, Canada. Tel.: 514-842-1231, ext. 5834; Fax: 514-843-1478
    Affiliations
    Molecular Oncology Laboratory, Royal Victoria Hospital, the Department of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada

    Departments of Medicine Oncology, McGill University, Montreal, Quebec H3A 1A1, Canada

    Departments of Medicine Biochemistry, McGill University, Montreal, Quebec H3A 1A1, Canada
    Search for articles by this author
  • Author Footnotes
    * This research was supported by operating grants from the National Cancer Institute of Canada and the Medical Research Council of Canada (to M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Recipient of a fellowship from the Fonds de la Recherche en Santé du Québec.
Open AccessPublished:November 17, 1995DOI:https://doi.org/10.1074/jbc.270.46.27780
      Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that induces mitogenesis, motility, invasion, and morphogenesis of several epithelial and endothelial cell lines in culture. The receptor for HGF/SF has been identified as the Met tyrosine kinase. To investigate the signaling pathways that are involved in these events, we have generated chimeric receptors containing the extracellular domain of the colony-stimulating factor-1 (CSF-1) receptor fused to the transmembrane and intracellular domains of the Met receptor (MET). Madin-Darby canine kidney (MDCK) epithelial cells expressing the CSF-MET chimera dissociate and scatter in response to CSF-1. However, cells expressing a mutant CSF-MET receptor containing a phenylalanine substitution for tyrosine 1356 were unable to scatter or form branching tubules following stimulation with CSF-1. Tyrosine 1356 is essential for the recruitment of multiple substrates including the p85 subunit of PI3-kinase, phospholipase Cγ, and Grb2. In this study, we have investigated the role of PI3-kinase and a downstream target of PI3-kinase, pp70S6K, in the induction of MDCK cell scatter in response to HGF/SF. Our results demonstrate that following stimulation with HGF/SF, activation of PI3-kinase but not pp70S6K is essential for MDCK cell scatter.

      INTRODUCTION

      Cell motility is a fundamental process required during normal embryonic development, wound healing, inflammatory responses, and tumor progression toward metastasis(
      • Gherardi E.
      ). Hepatocyte growth factor/scatter factor (HGF/SF)
      The abbreviations used are: HGF/SF
      hepatocyte growth factor/scatter factor
      CSF
      colony-stimulating factor
      MDCK
      Madin-Darby canine kidney
      PI3-kinase
      phosphatidylinositol 3-kinase
      ETOH
      ethanol
      PBS
      phosphate-buffered saline
      PIPES
      1,4-piperazinediethanesulfonic acid
      PDGF
      platelet-derived growth factor
      SH2
      Src homology 2
      PTB
      phosphotyrosine binding.
      is a multifunctional cytokine with activities on a wide variety of normal and neoplastic cells. HGF/SF is a mitogen, dissociation, and motility factor for many epithelial cells (
      • Gherardi E.
      • Gray J.
      • Stoker M.
      • Perryman M.
      • Furlong R.
      ,
      • Nakamura T.
      • Teramoto H.
      • Ichihara A.
      ,
      • Stoker M.
      • Gherardi E.
      • Perryman M.
      • Gray J.
      ) and stimulates tubulogenesis of tubular epithelial cells (
      • Montesano R.
      • Schaller G.
      • Orci L.
      ) as well as the invasion of carcinoma cells(
      • Weidner K.M.
      • Behrens J.
      • Vandekerckhove J.
      • Birchmeier W.
      ). In vivo, HGF/SF is a potent angiogenic factor (
      • Bussolino F.
      • Di Renzo M.F.
      • Ziche M.
      • Bocchietto E.
      • Olivero M.
      • Naldini L.
      • Gaudino G.
      • Tamagnone L.
      • Coffer A.
      • Comoglio P.M.
      ,
      • Grant D.S.
      • Kleinman H.K.
      • Goldberg I.D.
      • Bhargava M.M.
      • Nickoloff B.J.
      • Kinsella J.L.
      • Polverini P.
      • Rosen E.M.
      ) and is involved in organ regeneration (
      • Matsumoto K.
      • Nakamura T.
      ) and tumorigenesis(
      • Bellusci S.
      • Moens G.
      • Gaudino G.
      • Comoglio P.
      • Nakamura T.
      • Thiery J.-P.
      • Jouanneau J.
      ,
      • Rong S.
      • Bodescot M.
      • Blair D.
      • Dunn J.
      • Nakamura T.
      • Mizuno K.
      • Park M.
      • Chan A.
      • Aaronson S.
      • Vande Woude G.F.
      ). A high affinity receptor for HGF/SF has been identified as the product of the met proto-oncogene(
      • Bottaro D.P.
      • Rubin J.S.
      • Faletto D.L.
      • Chan A.M.-L.
      • Kmiecik T.E.
      • Vande Woude G.F.
      • Aaronson S.A.
      ,
      • Naldini L.
      • Vigna E.
      • Ferracini R.
      • Longati P.
      • Gandino L.
      • Prat M.
      • Comoglio P.M.
      ), which encodes a receptor tyrosine kinase originally isolated as the tpr-met oncogene(
      • Cooper C.S.
      • Park M.
      • Blair D.G.
      • Tainsky M.A.
      • Huebner K.
      • Croce C.M.
      • Vande Woude G.
      ,
      • Park M.
      • Dean M.
      • Cooper C.S.
      • Schmidt M.
      • O'Brien S.J.
      • Blair D.G.
      • Vande Woude G.
      ). The Met receptor is predominantly expressed in epithelial cells in culture (
      • Park M.
      • Dean M.
      • Cooper C.S.
      • Schmidt M.
      • O'Brien S.J.
      • Blair D.G.
      • Vande Woude G.
      ,
      • Gonzatti-Haces M.
      • Seth A.
      • Park M.
      • Copeland T.
      • Oroszlan S.
      • Vande Woude G.F.
      ,
      • Liu C.
      • Park M.
      • Tsao S.
      ) and in epithelium in vivo (18, 19).
      X. M. Yang and M. Park(1995) Lab. Invest., in press.
      The mature form of the Met receptor is a heterodimeric protein of 190 kDa, which consists of a 45-kDa extracellular α-subunit linked by disulfide bonds to a 145-kDa β-subunit that spans the membrane and contains the catalytic kinase domain(
      • Gonzatti-Haces M.
      • Seth A.
      • Park M.
      • Copeland T.
      • Oroszlan S.
      • Vande Woude G.F.
      ,
      • Giordano S.
      • Di Renzo M.F.
      • Narsimhan R.P.
      • Cooper C.S.
      • Rosa C.
      • Comoglio P.M.
      ,
      • Rodrigues G.A.
      • Naujokas M.A.
      • Park M.
      ,
      • Tempest P.R.
      • Cooper C.S.
      • Major G.N.
      ). Binding of HGF/SF induces activation of the kinase and auto/transphosphorylation of the receptor (
      • Naldini L.
      • Vigna E.
      • Ferracini R.
      • Longati P.
      • Gandino L.
      • Prat M.
      • Comoglio P.M.
      ) on specific tyrosine residues in the β chain(
      • Bottaro D.P.
      • Rubin J.S.
      • Faletto D.L.
      • Chan A.M.-L.
      • Kmiecik T.E.
      • Vande Woude G.F.
      • Aaronson S.A.
      ,
      • Naldini L.
      • Vigna E.
      • Narsimhan R.P.
      • Gaudino G.
      • Zarnegar R.
      • Michalopoulos G.A.
      • Comoglio P.M.
      ).
      Phosphorylated tyrosine residues within receptor tyrosine kinases provide binding sites for molecules containing SH2 and PTB domains that act to transduce extracellular signals to the interior of the cell (
      • Pawson T.
      ). Following stimulation of cells with HGF/SF, several proteins are phosphorylated, activated, and/or associated with the Met receptor. These include p120 GTPase-activating protein, mitogen activated protein kinase, Src, phospholipase Cγ, phosphatidylinositol 3-kinase (PI3-kinase), Grb2(
      • Graziani A.
      • Gramaglia D.
      • Cantley L.C.
      • Comoglio P.M.
      ,
      • Faletto D.L.
      • Kaplan D.R.
      • Halverson D.O.
      • Rosen E.M.
      • Vande Woude G.F.
      ,
      • Halaban R.
      • Rubin J.S.
      • Funasaka Y.
      • Cobb M.
      • Boulton T.
      • Faletto D.
      • Rosen E.
      • Chan A.
      • Yoko K.
      • White W.
      • Cook C.
      • Moellmann G.
      ,
      • Okano Y.
      • Mizuno K.
      • Osada S.
      • Nakamura T.
      • Nozawa Y.
      ,
      • Zhu H.
      • Naujokas M.A.
      • Fixman E.D.
      • Torossian K.
      • Park M.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Zhen Z.
      • Maina F.
      • dalla Zonca P.
      • Giordano S.
      • Graziani A.
      • Panayotou G.
      • Comoglio P.M.
      ), Ras(
      • Graziani A.
      • Gramaglia D.
      • dalla Zonca P.
      • Comoglio P.M.
      ), focal adhesion kinase(
      • Matsumoto K.
      • Matsumoto K.
      • Nakamura T.
      • Kramer R.H.
      ), β-catenin, plakoglobin(
      • Shibamoto S.
      • Hayakawa M.
      • Takeuchi K.
      • Hori T.
      • Oku N.
      • Miyazawa K.
      • Kitamura N.
      • Takeichi M.
      • Ito F.
      ), and the Shc adaptor protein(
      • Pelicci G.
      • Giordano S.
      • Zhen Z.
      • Salcini A.E.
      • Lanfrancone L.
      • Bardelli A.
      • Panayotou G.
      • Waterfield M.D.
      • Ponzetto C.
      • Pelicci P.G.
      • Comoglio P.M.
      ). The Met receptor tyrosine kinase is highly phosphorylated on two tyrosine residues (1234 and 1235) within the kinase domain (
      • Longati P.
      • Bardelli A.
      • Ponzetto C.
      • Naldini L.
      • Comoglio P.M.
      ,
      • Rodrigues G.A.
      • Park M.
      ) that are essential for the catalytic activity of the receptor(
      • Zhu H.
      • Naujokas M.A.
      • Fixman E.D.
      • Torossian K.
      • Park M.
      ,
      • Longati P.
      • Bardelli A.
      • Ponzetto C.
      • Naldini L.
      • Comoglio P.M.
      ). In addition, tyrosine 1356 within the carboxyl terminus of the β-subunit of the Met receptor is phosphorylated (
      • Zhu H.
      • Naujokas M.A.
      • Fixman E.D.
      • Torossian K.
      • Park M.
      ) and is essential for the recruitment of multiple substrates including the p85 subunit of PI3-kinase, phospholipase Cγ, and Grb2(
      • Zhu H.
      • Naujokas M.A.
      • Fixman E.D.
      • Torossian K.
      • Park M.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Zhen Z.
      • Maina F.
      • dalla Zonca P.
      • Giordano S.
      • Graziani A.
      • Panayotou G.
      • Comoglio P.M.
      ,
      • Fixman E.D.
      • Naujokas M.A.
      • Rodrigues G.A.
      • Moran M.F.
      • Park M.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Maina F.
      • Longati P.
      • Panayotou G.
      • Dhand R.
      • Waterfield M.D.
      • Comoglio P.M.
      ). Although a role for many of these signal transduction pathways has been established in cell mitogenesis, their role in dissociation and scatter of epithelial cells is unknown.
      To investigate the signaling pathways that are involved in these events, we have generated chimeric receptors containing the extracellular domain of the CSF-1 receptor fused to the transmembrane and intracellular domains of the Met receptor. Madin-Darby canine kidney (MDCK) epithelial cells dissociate, scatter, and form branching tubules in response to HGF/SF. MDCK cells expressing the CSF-MET chimera dissociate and scatter in response to CSF-1(
      • Zhu H.
      • Naujokas M.A.
      • Park M.
      ); however, MDCK cells expressing a mutant CSF-MET receptor containing a phenylalanine substitution for tyrosine 1356 (Y1356F) are unable to scatter or form branching tubules following stimulation with CSF-1(
      • Zhu H.
      • Naujokas M.A.
      • Fixman E.D.
      • Torossian K.
      • Park M.
      ). This has suggested that one or more of the substrates that bind to Tyr1356 are required for the induction of MDCK cell scatter following stimulation with HGF/SF. Our results demonstrate that following stimulation with HGF/SF, activation of PI3-kinase but not phospholipase Cγ or pp70S6K, a downstream target of PI3-kinase, is essential for MDCK cell scatter.

      EXPERIMENTAL PROCEDURES

      Cell Lines

      MDCK epithelial cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 50 μg/ml gentamycin (all from Life Technologies, Inc.). The MDCK stable cell line expressing the CSF-MET mutant receptor Y1356F has been described previously(
      • Zhu H.
      • Naujokas M.A.
      • Fixman E.D.
      • Torossian K.
      • Park M.
      ).

      Immunofluorescence Labeling

      Cells (1 × 104 cells/well) were seeded on glass coverslips (Bellco Glass Inc.) in 24-well culture dishes (Nunc). 24 or 48 h later, cells were serum-starved in 0.2% fetal bovine serum for 24 h and stimulated with HGF/SF (5 or 10 units/ml) or CSF (50 ng/ml) at 37°C for the chosen time intervals. When appropriate, cells were preincubated with wortmannin/Me2SO (0.1%) (Sigma), rapamycin/ETOH (0.1%) (Calbiochem), staurosporine/Me2SO (0.1%) (Sigma), U-73122/Me2SO (0.1%), or U-73343/Me2SO (0.1%) (Biomol, Plymouth Meeting, PA) for 30 min at 37°C at concentrations indicated in the text. Following stimulation with HGF/SF, cells were washed in PBS and treated with CSK buffer (10 mM PIPES, pH 7.0, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml DNase, and 0.1 mg/ml RNase A) for 20 min at 4°C, followed by fixation with 1.75% formaldehyde (in PBS) for 5-10 min at room temperature as described(
      • Pasdar M.
      • Nelson W.J.
      ,
      • Pasdar M.
      • Nelson W.J.
      ). For phase contrast pictures, cells were fixed for 30 min in 0.2% glutaraldehyde (JBS-Chem, Montreal, Canada) at 4°C. Primary antibodies used for staining proteins from junctional complexes were: a 1/50 dilution of a mouse monoclonal antibody, 3G8, directed against E-cadherin; a 1/200 dilution of a rabbit polyclonal antibody, DPI/II, directed against desmoplakins I and II (both kindly provided by Dr. M. Pasdar, University of Alberta, Edmonton, Canada); and a 1/200 dilution of a mouse monoclonal antibody, R26-4C, recognizing ZO-1 (Developmental Studies Hybridoma Bank, University of Iowa). These primary antibody reactions were revealed using the appropriate fluorescein isothiocyanate-labeled anti-rabbit antibodies (1/50 dilution) (Jackson ImmunoResearch Laboratories Inc.) or rhodamine-conjugated anti-mouse antibodies (1/200 dilution) (Boehringer Mannheim). All incubations with antibodies were for 30 min at room temperature, and cells were washed with PBS after each incubation. The cells were mounted onto slides in Immuno-Fluore medium (ICN), and observations were made on a Nikon Labophot-2 epifluorescence microscope. Images were photographed using Kodak TMY-400 films.

      Phosphatidylinositol 3-Kinase Assay

      MDCK cells (4 × 105) were seeded in 100-mm dishes and then serum-starved for 48 h in 0.2% fetal bovine serum. Monolayers were generally 70-80% confluent and contained mid-size colonies. When appropriate, cells were preincubated with wortmannin/Me2SO (0.1%) for 30 min at 37°C prior to stimulation with HGF/SF (10 units/ml) on ice for 10 min followed by incubation at 37°C for the chosen time intervals. Following stimulation, cells were lysed in a buffer containing 50 mM HEPES, pH 7.5, 1% Triton X-100, 10% glycerol, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4 10 μg/ml aprotinin, and 10 μg/ml leupeptin on ice for 30 min. The PI3-kinase assay was performed essentially as described with minor modifications (
      • Fixman E.D.
      • Naujokas M.A.
      • Rodrigues G.A.
      • Moran M.F.
      • Park M.
      ). Immunoprecipitation was performed on clarified cell lysates (15,000 rpm for 15 min at 4°C) using the monoclonal antibody PY20 against phosphotyrosine-containing proteins (Signal Transduction Laboratories Inc.). Equal amounts of proteins were incubated for 1.5 h with PY20 alone followed by a 1.5-h incubation in the presence of protein A-Sepharose (20% suspension) (Pharmacia) at 4°C with mixing. The Sepharose-immune complexes were then subjected to several washes with ice-cold solutions: three times with lysis buffer; once with PBS; once with 0.1 M Tris-HCl, pH 7.5, 0.5 M LiCl; once with distilled water; once with 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA; and once with kinase buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.5 mM EGTA). The beads were resuspended in 50 μl of kinase buffer containing 0.2 mg/ml of phosphatidylinositol (sonicated 15 min at 4°C; Sigma) preincubated for 10 min at room temperature, at which time, for in vitro assays, wortmannin or an equivalent amount of Me2SO (0.1%) were added. Then 20 μCi of [γ-32P]ATP and 20 mM MgCl2 were added for 10 min at room temperature. Reactions were terminated by adding 150 μl of chloroform/methanol/11.6 M HCl (50:100:1), and phosphatidylinositol was extracted with 100 μl of chloroform. The organic phase was washed with methanol/1 M HCl (1:1) and lyophilized. Phosphatidylinositol, resuspended in 15 μl of chloroform, was spotted on a silica gel 60 thin layer chromatography plate (Merck) and resolved in chloroform/methanol/28% ammonium hydroxide/water (86:76:10:14) for 45 min. Phosphorylated products were visualized by autoradiography and quantified by the Fujix Bio-Imaging Analyzer Bas 1000.

      S6 Kinase Immunoprecipitation, Western Blotting, and in Vitro Kinase Assay

      MDCK cells (1.2 × 105) were plated in 60-mm dishes and serum-starved as described above. When appropriate, cells were preincubated with rapamycin/ETOH (0.1%) or with wortmannin/Me2SO (0.1%) for 30 min at 37°C prior to stimulation. For immunoprecipitations, cells were rapidly washed with ice-cold PBS and lysed in 0.4 ml of lysis buffer containing 50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 100 mM NaCl, 5 mM EDTA, 50 mM NaF, 40 mM β-glycerophosphate, 1 mM Na3VO4 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin on ice for 30 min. As described above and in (
      • Giasson E.
      • Meloche S.
      ), equal amounts of protein from clarified cell lysates were immunoprecipitated with a rabbit polyclonal antibody recognizing the carboxyl terminus of p70S6K (Santa Cruz Biotechnology, Inc.) for 2 h. Protein A-Sepharose was then added for 2 h. Immune complexes were washed four times with lysis buffer, eluted by boiling in Laemmli sample buffer, subjected to SDS-polyacrylamide (8%) gel electrophoresis, and transferred to nitrocellulose (Schleicher & Schuell). For Western blotting, membranes were blocked for 1 h at room temperature with 3% bovine serum albumin in TBST buffer (10 mM Tris-HCl, pH 7.5, 2.5 mM EDTA, 150 mM NaCl, and 0.1% Tween 20), washed several times in TBST buffer, and probed with anti-p70S6K antibodies (1:200 dilution) for 1 h at room temperature. The primary antibody reaction was detected with horseradish peroxidase-coupled protein A (1:5000 dilution) by the enhanced chemiluminescence method (Amersham Corp.) according to the manufacturer's recommendations. For the in vitro kinase assay, p70S6K was immunoprecipitated as above, and the immune complexes were washed three times with lysis buffer and once with kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol, 10 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). The beads were resuspended in 25 μl of kinase buffer containing 10 μCi of [γ-32P]ATP and 0.2 mM of the S6 peptide RRRLSSLRA (Santa Cruz Biotechnology, Inc.) and incubated at 30°C for 20 min. The reactions were terminated by application to 1 cm2 of phosphocellulose p81 paper (Whatman). The filters were then extensively washed (in 500 ml for 15 min: four times in 1% phosphoric acid, twice with distilled water, and once in ETOH) and counted in a liquid scintillation counter. Blank reactions where the S6 peptide was omitted were also performed to control for background levels of radioactivity.

      RESULTS

      A CSF-MET Mutant Chimera, Y1356F, Fails to Promote Cell Dissociation and Redistribution of Junctional Protein Complexes in Response to CSF-1

      MDCK cells form discrete colonies when sparsely seeded on plastic or on glass coverslips. Following stimulation of MDCK cell colonies maintained in 0.2% serum with HGF/SF, we have observed that MDCK cells first spread (2-3 h), dissociate (4-6 h), and then adopt a fibroblastoid cell morphology as they begin to scatter (from 6 h) (Figure 1:, Figure 2:, Figure 3:A). In MDCK epithelial cells, the lateral plasma membranes are connected by the formation of junctional complexes, including tight junctions, zonula adherens, and desmosomes(
      • Farquhar M.G.
      • Palade G.E.
      ). Adherens type junctions and desmosomes play important roles in maintaining cell-cell adhesion(
      • Garrod D.R.
      ,
      • Takeichi M.
      ). Therefore, scattering of MDCK cells requires the breakdown of these intercellular adhesions. In polarized epithelial cells, proteins of tight junctions and zonula adherens are coupled to the actin filaments, whereas desmosomal proteins are associated with intermediate filaments. In this context, these proteins are insoluble, whereas in the absence of cell-cell contact, they are redistributed into the soluble compartment of the cytoplasm(
      • Pasdar M.
      • Nelson W.J.
      ,
      • Citi S.
      ,
      • Pasdar M.
      • Nelson W.J.
      ,
      • Shore E.M.
      • Nelson W.J.
      ,
      • Siliciano J.D.
      • Goodenough D.A.
      ,
      • Stevenson B.R.
      • Anderson J.M.
      • Bullivant S.
      ). To examine the dissociation of MDCK cells in response to HGF/SF, we have analyzed the localization of junctional complex proteins by indirect immunofluorescence microscopy. We have demonstrated that in colonies of MDCK cells where the membranes were solubilized with CSK buffer, E-cadherin (from zonula adherens), desmoplakins I/II (from desmosomes), and ZO-1 (from tight junctions) were insoluble and distributed uniformly along regions of cell-cell contact (Fig. 1, a-d). However, scatter of MDCK cells in response to HGF/SF resulted in the redistribution of each of these proteins, and as a consequence, they were no longer organized in insoluble complexes associated with the cytoskeleton at the plasma membrane (Fig. 1, e-h).
      Figure thumbnail gr1
      Figure 1:E-cadherin, desmoplakins I/II, and ZO-1 remain insoluble following CSF-1 stimulation of MDCK cells expressing the Y1356F CSF-MET mutant receptor. Colonies of MDCK cells expressing the Y1356F CSF-MET mutant receptor were serum-starved for 24 h and further incubated for 24 h in the same medium (a-d), in medium containing HGF/SF (5 units/ml) (e-h), or in medium containing CSF-1 (50 ng/ml) (i-l). Cells were extracted in situ with CSK buffer and fixed with 1.75% formaldehyde in PBS as described under “Experimental Procedures.” Cells were then processed for indirect immunofluorescence with antibodies against E-cadherin (b, f, and j), desmoplakins I/II (c, g, and k), and ZO-1 (d, h, and l) as primary antibodies, followed by rhodamine-coupled anti-mouse antibodies (for E-cadherin and ZO-1) and fluorescein isothiocyanate-coupled anti-rabbit antibodies (for desmoplakins I/II) as secondary antibodies.
      MDCK cells expressing a CSF-MET chimera dissociate and scatter in a similar manner in response to CSF-1 or HGF/SF(
      • Zhu H.
      • Naujokas M.A.
      • Park M.
      ). However, MDCK cells expressing a mutant CSF-MET receptor containing a phenylalanine substitution for tyrosine 1356 (Y1356F) fail to scatter following stimulation with CSF-1(
      • Zhu H.
      • Naujokas M.A.
      • Fixman E.D.
      • Torossian K.
      • Park M.
      ). We show here that junctional complexes remained intact and in an insoluble form at cell-cell interfaces in CSF-1-stimulated MDCK cells expressing the Y1356F CSF-MET mutant (Fig. 1, i-l).

      Wortmannin Inhibits PI3-Kinase Activity and Scatter of HGF/SF-stimulated MDCK Cells

      Chemotaxis transduced by the PDGF-β receptor requires the binding sites for PI3-kinase(
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ,
      • Wennstrom S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A.-K.
      • Heldin C.-H.
      • Mori S.
      • Claesson-Welsh L.
      ), suggesting that this substrate may play an important role in this event. PI3-kinase is activated following treatment of A-549 (lung carcinoma) cells with HGF/SF(
      • Graziani A.
      • Gramaglia D.
      • Cantley L.C.
      • Comoglio P.M.
      ). Following HGF/SF stimulation of MDCK cells, PI3-kinase activity was increased 4-fold within 1 min, and this level of activation was maintained over 10 min (Fig. 2A). To investigate the involvement of PI3-kinase in the scatter of MDCK cells in response to HGF/SF, we have used the potent inhibitor wortmannin, which is a fungal metabolite that directly binds to and inhibits the p110 subunit of PI3-kinase(
      • Arcaro A.
      • Wymann M.
      ,
      • Woscholski R.
      • Kodaki T.
      • McKinnon M.
      • Waterfield M.D.
      • Parker P.J.
      ,
      • Yano H.
      • Nakanishi S.
      • Kimura K.
      • Hanai N.
      • Saitoh Y.
      • Fukui Y.
      • Nonomura Y.
      • Matsuda Y.
      ). When assayed in vitro, wortmannin inhibited the canine MDCK cell PI3-kinase immunoprecipitated using antibodies to phosphotyrosine with an IC50 (50% inhibitory concentration) below 10 nM (Fig. 2B).
      Figure thumbnail gr2
      Figure 2:PI3-kinase is activated following HGF/SF stimulation of MDCK cells and is inhibited by wortmannin in vitro and in vivo. A, MDCK cells starved for 48 h were stimulated with HGF/SF (10 units/ml) for different time intervals, and cell lysates were then immunoprecipitated with the anti-phosphotyrosine antibody PY20. Immune complexes were adsorbed using protein A-Sepharose and subjected to a PI3-kinase assay. The products of the reaction were analyzed by thin layer chromatography, visualized by autoradiography, and quantified by a PhosphorImager. B, phosphotyrosine-containing proteins from lysates of MDCK cells were immunoprecipitated with the PY20 antibody and assayed for PI3-kinase activity in vitro in the presence of either 0.1% Me2SO or various concentrations of wortmannin for 10 min prior to the kinase reaction. C, serum-starved MDCK cells were preincubated with either 0.1% Me2SO or wortmannin (10 nM to 10 μM) for 30 min at 37°C, followed by stimulation with HGF/SF (10 units/ml) for 1 min at 37°C. Cells were lysed, and phosphotyrosine-containing proteins were immunoprecipitated with the PY20 antibody. Immune complexes were then processed as described above. The position of phosphatidylinositol 3-phosphate (PI(3)P) is indicated.
      To examine the role of PI3-kinase in vivo serum-starved MDCK cells were pretreated for 30 min at 37°C with either wortmannin or its solvent (Me2SO 0.1%) and stimulated with HGF/SF, and PI3-kinase was immunoprecipitated with anti-phosphotyrosine antibodies and assayed in vitro. Wortmannin, at concentrations of 100 nM and 1 μM, reduced the level of PI3-kinase activity in HGF/SF-stimulated MDCK cells by 35 and 65% respectively (Fig. 2C). To evaluate the effect of wortmannin on HGF/SF-induced cell scatter, colonies of serum-starved MDCK cells were pretreated for 30 min at 37°C with wortmannin (10 nM-1 μM) and then stimulated with HGF/SF. MDCK cells, following stimulation with HGF/SF, changed shape and flattened by 3 h (Fig. 3A, e), whereas MDCK cells in the presence of 500 nM wortmannin remained as tight colonies (Fig. 3A, i). Moreover, in the presence of 1 μM wortmannin, MDCK cells stimulated with HGF/SF for 7 h had flattened (Fig. 3B, c) but were inhibited in their ability to scatter when compared with control HGF/SF-stimulated MDCK cells (Fig. 3B, b). The ability of wortmannin to inhibit both cell spreading and scatter was concentration-dependent and correlated with its ability to inhibit PI3-kinase activity in vivo (data not shown).
      Figure thumbnail gr3
      Figure 3:The redistribution of E-cadherin and desmoplakins I/II, cell spreading, and scatter in HGF/SF-stimulated MDCK cells are inhibited by wortmannin. A, colonies of serum-starved MDCK cells were incubated for 3.5 h in medium containing 0.1% Me2SO (a-d), preincubated in medium containing 0.1% Me2SO for 30 min followed by stimulation with HGF/SF (10 units/ml) for 3 h (e-h), or preincubated in medium containing 500 nM wortmannin (in 0.1% Me2SO) for 30 min followed by stimulation with HGF/SF for 3 h (i-l). Cells were extracted in situ with CSK buffer, fixed with 1.75% formaldehyde in PBS, and processed for indirect immunofluorescence with antibodies against E-cadherin (b, f, and j), desmoplakins I/II (c, g, and k), and ZO-1 (d, h, and l). B, colonies of serum-starved MDCK cells were incubated for 7.5 h in medium containing 0.1% Me2SO (a), preincubated in medium containing 0.1% Me2SO for 30 min followed by stimulation with HGF/SF (10 units/ml) for 7 h (b), preincubated in medium containing 1 μM wortmannin (in 0.1% Me2SO) for 30 min followed by stimulation with HGF/SF for 7 h (c), preincubated in medium containing 5 nM staurosporine (in 0.1% Me2SO) for 30 min followed by stimulation with HGF/SF for 7 h (d), preincubated in medium containing 2 μM U-73122 (e), or 2 μM U-73343 (f) (in 0.1% Me2SO) for 30 min followed by stimulation with HGF/SF for 7 h. Cells were fixed in 0.2% glutaraldehyde in PBS.
      To establish which processes were inhibited by wortmannin, we examined the effect of wortmannin on the redistribution of junctional complex proteins. The spreading and changes in morphology observed in MDCK cells, following HGF/SF stimulation for 3 h, were accompanied by a reduction in the amount of insoluble E-cadherin and desmoplakins I/II at cell-cell interfaces (Fig. 3A, f and g). At this time however, the concentration of insoluble ZO-1 at cell-cell interfaces (Fig. 3A, h) was comparable to that observed in control unstimulated MDCK cells (Fig. 3A, d). A redistribution of ZO-1 was only apparent when cells began to scatter (data not shown). However, in the presence of 500 nM wortmannin, all three proteins from junctional complexes were retained in an insoluble compartment at the plasma membrane (Fig. 3A, j-l). Thus, the ability of wortmannin to inhibit MDCK cell spreading and scatter correlated with the retention of insoluble junctional complexes and tight cell-cell interactions, suggesting that PI3-kinase activity is required for the redistribution of junctional complex proteins and cell dissociation induced by HGF/SF in MDCK cells.
      Chemotaxis of some cell types transduced by the PDGF-β and epidermal growth factor receptors requires phospholipase Cγ(
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ,
      • Chen P.
      • Xie H.
      • Sekar M.C.
      • Gupta K.
      • Wells A.
      ); however, the inhibition of protein kinase C, a downstream target of phospholipase Cγ, did not inhibit MDCK cell scatter in response to HGF/SF(
      • Rosen E.M.
      • Meromsky L.
      • Goldberg I.
      • Bhargava M.
      • Setter E.
      ). To investigate the involvement of phospholipase Cγ in HGF/SF-induced MDCK cell scatter, we used an inhibitor of phospholipase C (U-73122; IC50 of 1-2 μM) (
      • Chen P.
      • Xie H.
      • Sekar M.C.
      • Gupta K.
      • Wells A.
      ,
      • Smith R.J.
      • Sam L.M.
      • Justen J.M.
      • Bundy G.L.
      • Bala G.A.
      • Bleasdale J.E.
      ,
      • Bleasdale J.E.
      • Thakur N.R.
      • Gremban R.S.
      • Bundy G.L.
      • Fitzpatrick F.A.
      • Smith R.J.
      • Bunting S.
      ) and the protein kinase C inhibitor staurosporine (IC50 of 0.7 nM)(
      • Tamaoki T.
      • Nomoto H.
      • Takahashi I.
      • Kato Y.
      • Morimoto M.
      • Tomita F.
      ). Colonies of serum-starved MDCK cells were pretreated with Me2SO (0.1%), staurosporine (1 nM-20 nM), U-73122 (0.25-2 μM), or an inactive analogue of U-73122 (U-73343; 0.25-2 μM) for 30 min at 37°C. MDCK cells in the presence of staurosporine (Fig. 3B, d, and data not shown), the phospholipase C inhibitor U-73122 (Fig. 3B, e, and data not shown), the inactive analogue U-73343 (Fig. 3B, f, and data not shown), or Me2SO (0.1%) (Fig. 3B, b) scattered following stimulation with HGF/SF for 7 h. Therefore, in contrast to PI3-kinase, phospholipase Cγ and protein kinase C are not essential for MDCK cell spreading and scatter.

      pp70S6KIs Activated Following HGF/SF Stimulation of MDCK Cells But Is Not Required for Scatter

      To further characterize the involvement of PI3-kinase-regulated pathways in HGF/SF-induced cell scatter, we have investigated the role of pp70S6K, a potential downstream target of PI3-kinase(
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ). Activation of pp70S6K is associated with its phosphorylation on Ser/Thr residues (
      • Ballou L.M.
      • Siegmann M.
      • Thomas G.
      ,
      • Ferrari S.
      • Bannwarth W.
      • Morley S.J.
      • Totty N.F.
      • Thomas G.
      ) and its decreased electrophoretic mobility. Phosphorylation of pp70S6K was increased following stimulation of MDCK cells with HGF/SF (Fig. 4). Immunoprecipitation of pp70S6K, followed by immunoblotting with a specific p70S6K antibody, revealed a shift in mobility of pp70S6K that was maximal at 20 min following stimulation with HGF/SF and decreased by 2 h (Fig. 4A, top). In agreement with this result, an increase in pp70S6Kin vitro kinase activity, when assayed against an S6 peptide, was observed following stimulation of MDCK cells by HGF/SF for 20 min (Fig. 4A, bottom). To establish if pp70S6K activity was inhibited by wortmannin in MDCK cells, pp70S6K activation was assayed in serum-starved cells pretreated with wortmannin (10 nM-10 μM) or Me2SO (0.1%) for 30 min and then stimulated with HGF/SF for 20 min. In the presence of 100 nM wortmannin, HGF/SF-induced phosphorylation of pp70S6K was inhibited, and pp70S6K activity was reduced to 30% of control (HGF-stimulated MDCK cells), whereas maximal inhibition, where pp70S6K activity was reduced to 10% of control, was observed in the presence of 1 μM wortmannin (Fig. 4B, bottom).
      Figure thumbnail gr4
      Figure 4:pp70S6K is activated following HGF/SF stimulation of MDCK cells and is inhibited by wortmannin or rapamycin. A, serum-starved MDCK cells were stimulated with HGF/SF (10 units/ml) for different time intervals, and proteins from cell lysates were immunoprecipitated with antibodies against p70S6K. Immune complexes were adsorbed using protein A-Sepharose and subjected to SDS-polyacrylamide (8%) gel electrophoresis, transferred onto nitrocellulose and immunoblotted with an anti-p70S6K antibody (top), or subjected to an in vitro kinase assay using S6 peptide as a substrate as described under “Experimental Procedures” (bottom). The results are reported as counts incorporated into S6 peptide. B, serum-starved MDCK cells were preincubated with either 0.1% Me2SO or various concentrations of wortmannin for 30 min at 37°C, followed by further incubation in Me2SO or by stimulation with HGF/SF (10 units/ml) for 20 min at 37°C. Cells were lysed, pp70S6K was immunoprecipitated, and immune complexes were then processed as described above. C, serum-starved MDCK cells were preincubated with either 0.1% ETOH or various concentrations of rapamycin for 30 min at 37°C, followed by further incubation in ETOH or by stimulation with HGF/SF (10 units/ml) for 20 min at 37°C. Cells were lysed and pp70S6K-immune complexes were processed as described above.
      To establish whether pp70S6K is essential for MDCK cell dissociation and scatter induced by HGF/SF, serum-starved MDCK cells were pretreated with the macrolide antibiotic rapamycin, which inhibits the PI3-kinase-related protein RAFT1 (
      • Sabatini D.M.
      • Erdjument-Bromage H.
      • Lui M.
      • Tempst P.
      • Snyder S.H.
      ) and as a consequence its downstream target pp70S6K(
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ,
      • Kuo C.J.
      • Chung J.
      • Florentino D.F.
      • Flanagan W.M.
      • Blenis J.
      • Crabtree G.R.
      ,
      • Price D.J.
      • Grove J.R.
      • Calvo V.
      • Avruch J.
      • Biere B.E.
      ). HGF/SF-induced pp70S6K phosphorylation (Fig. 4C, top) and in vitro kinase activity (Fig. 4C, bottom) were inhibited equally by rapamycin (1-50 ng/ml) or wortmannin (1-10 μM) (Fig. 4B). However, preincubation of MDCK cells with 20 ng/ml of rapamycin for 30 min followed by stimulation with HGF/SF did not inhibit scattering of MDCK cells (Fig. 5i). Moreover, rapamycin pretreatment did not inhibit the redistribution or solubilization of E-cadherin, desmoplakins I/II, and ZO-1 following stimulation of MDCK cells with HGF/SF (Fig. 5, j-l). Therefore, although pp70S6K is activated following stimulation with HGF/SF and is a possible downstream target of PI3-kinase in MDCK cells, it is not required for scatter of MDCK cells in response to HGF/SF.
      Figure thumbnail gr5
      Figure 5:Inhibition of pp70S6K activation in HGF/SF-stimulated MDCK cells by rapamycin does not block cell spreading and dissociation. Colonies of serum-starved MDCK cells were incubated for 24 h in medium containing 0.1% ETOH (a-d), preincubated in medium containing 0.1% ETOH for 30 min and then stimulated by HGF/SF (10 units/ml) for 24 h (e-h), or preincubated in medium containing 20 ng/ml of rapamycin for 30 min and then stimulated with HGF/SF for 24 h (i-l). Cells were extracted in situ with CSK buffer and fixed with 1.75% formaldehyde in PBS and then processed for indirect immunofluorescence with antibodies against E-cadherin (b, f, and j), desmoplakins I/II (c, g, and k), and ZO-1 (d, h, and l).

      DISCUSSION

      HGF/SF is a multifunctional cytokine that stimulates dissociation, scatter, and morphogenesis of epithelial cells(
      • Gherardi E.
      • Gray J.
      • Stoker M.
      • Perryman M.
      • Furlong R.
      ,
      • Stoker M.
      • Gherardi E.
      • Perryman M.
      • Gray J.
      ,
      • Montesano R.
      • Schaller G.
      • Orci L.
      ). The response of MDCK cells to HGF/SF can be visualized first as cell spreading (after 2-3 h), when cells are still associated and present within the colony (Fig. 3A), followed by cell dissociation (after 4-6 h) and cell scatter (from 6 h) (Fig. 1). Breakdown of cell contacts is a prerequisite for cell dissociation, and we have shown that dissociation of MDCK cells in response to HGF/SF is concomitant with the loss of stable insoluble junctional complexes at sites of cell contact. Interestingly, following HGF/SF stimulation of MDCK cells, a redistribution of E-cadherin and desmoplakins I/II, which are components of adherens junctions and desmosomes, is observed prior to the redistribution of ZO-1, a component of tight junctions (Fig. 1 and 3A). This may reflect the ability of HGF/SF to stimulate the phosphorylation of β-catenin and plakoglobin(
      • Shibamoto S.
      • Hayakawa M.
      • Takeuchi K.
      • Hori T.
      • Oku N.
      • Miyazawa K.
      • Kitamura N.
      • Takeichi M.
      • Ito F.
      ), which could contribute to the destabilization of adherens junctions and desmosomes but not tight junctions, in which these proteins are absent.
      MDCK cells expressing a CSF-MET mutant receptor (Y1356F) fail to stimulate scatter (
      • Zhu H.
      • Naujokas M.A.
      • Fixman E.D.
      • Torossian K.
      • Park M.
      ) or redistribute junctional complexes (Fig. 1). Tyrosine 1356 in the carboxyl terminus of the Met receptor is essential for association with PI3-kinase, phospholipase Cγ, and Grb2(
      • Zhu H.
      • Naujokas M.A.
      • Fixman E.D.
      • Torossian K.
      • Park M.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Zhen Z.
      • Maina F.
      • dalla Zonca P.
      • Giordano S.
      • Graziani A.
      • Panayotou G.
      • Comoglio P.M.
      ,
      • Fixman E.D.
      • Naujokas M.A.
      • Rodrigues G.A.
      • Moran M.F.
      • Park M.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Maina F.
      • Longati P.
      • Panayotou G.
      • Dhand R.
      • Waterfield M.D.
      • Comoglio P.M.
      ), suggesting that at least one of these signaling pathways is required for cell dissociation and scatter. However, a mutant CSF-MET receptor containing a substitution of a histidine residue for the asparagine downstream from tyrosine 1356 (N1358H), which failed to bind only the Grb2 adaptor protein, stimulated MDCK cell scatter in response to ligand,
      T. Fournier and M. Park, manuscript in preparation.
      demonstrating that association of Grb2 with the Met receptor is not essential. Because binding sites for PI3-kinase (
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ,
      • Wennstrom S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A.-K.
      • Heldin C.-H.
      • Mori S.
      • Claesson-Welsh L.
      ) or phospholipase Cγ (
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ) in the PDGF receptor-β are important for PDGF-BB-induced chemotaxis and because phospholipase Cγ is involved in epidermal growth factor-mediated chemotaxis(
      • Chen P.
      • Xie H.
      • Sekar M.C.
      • Gupta K.
      • Wells A.
      ), we have investigated the role of these signaling pathways in HGF/SF-induced cell dissociation and scatter of MDCK epithelial cells. We have shown that PI3-kinase plays an essential role in HGF/SF-mediated scatter of MDCK cells. In the presence of wortmannin, a potent inhibitor for PI3-kinase, MDCK cells stimulated with HGF/SF for 3 h remained as tight colonies and retained insoluble E-cadherin, desmoplakins I/II, and ZO-1 at cell-cell interfaces. Conversely, control MDCK cell colonies contained cells with a flattened appearance, which showed a redistribution of junctional complex proteins involving E-cadherin and desmoplakins I/II, whereas, as discussed above, ZO-1 from tight junctions was maintained while cells were in contact (Fig. 3A)(
      • Dowrick P.G.
      • Warn R.M.
      ). Moreover, concurrent with the ability of wortmannin to inhibit the spreading of MDCK cells 3 h after HGF/SF stimulation, wortmannin also inhibited the scatter of MDCK cells at 7 h (Fig. 3B). In contrast, an inhibitor of phospholipase C (U-73122) or protein kinase C (staurosporine) at concentrations equal or higher than their respective IC50 did not inhibit scatter of MDCK cells in response to HGF/SF (Fig. 3B).
      The ability of wortmannin to inhibit HGF/SF-induced spreading and scatter of MDCK cells correlated directly with the extent of PI3-kinase inhibition in vivo, thus supporting a crucial role for PI3-kinase activity in the dissociation and scatter of MDCK cells. The concentrations of wortmannin required for the inhibition of cell spreading, redistribution and solubilization of junctional complex proteins, and cell scatter were higher than that reported for the inhibition of diverse biological responses involving membrane ruffling (
      • Wennstrom S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ), histamine secretion(
      • Yano H.
      • Nakanishi S.
      • Kimura K.
      • Hanai N.
      • Saitoh Y.
      • Fukui Y.
      • Nonomura Y.
      • Matsuda Y.
      ), respiratory burst(
      • Arcaro A.
      • Wymann M.
      ,
      • Okada T.
      • Sakuma L.
      • Fukui Y.
      • Hazeki O.
      • Ui M.
      ), or glucose transport (
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ) (between 50-100 nM). Although high concentrations of wortmannin (in the μM range in vitro) have been reported to inhibit myosin light chain kinase(
      • Yano H.
      • Nakanishi S.
      • Kimura K.
      • Hanai N.
      • Saitoh Y.
      • Fukui Y.
      • Nonomura Y.
      • Matsuda Y.
      ,
      • Nakanishi S.
      • Kakita S.
      • Takahashi I.
      • Kawahara K.
      • Tsukuda E.
      • Sano T.
      • Yamada K.
      • Yoshida M.
      • Kase H.
      • Matsuda Y.
      • Hashimoto Y.
      • Nonomura Y.
      ), an enzyme thought to be involved in cell motility, treatment of MDCK cells with an inhibitor of myosin light chain kinase (ML-9) had no effect on HGF/SF-induced MDCK cell scatter (
      • Rosen E.M.
      • Meromsky L.
      • Goldberg I.
      • Bhargava M.
      • Setter E.
      ). Moreover, our data demonstrated that the canine PI3-kinase was sensitive to wortmannin in vitro, with an IC50 below 10 nM, which is comparable to other studies(
      • Arcaro A.
      • Wymann M.
      ,
      • Woscholski R.
      • Kodaki T.
      • McKinnon M.
      • Waterfield M.D.
      • Parker P.J.
      ,
      • Yano H.
      • Nakanishi S.
      • Kimura K.
      • Hanai N.
      • Saitoh Y.
      • Fukui Y.
      • Nonomura Y.
      • Matsuda Y.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ). In addition, wortmannin is a lipophilic compound and is expected to inactivate PI3-kinase localized at the plasma membrane. However, the Met receptor is localized to the basolateral surface of MDCK cells(
      • Crepaldi T.
      • Pollack A.L.
      • Prat M.
      • Zborek A.
      • Mostov K.
      • Comoglio P.M.
      ). Thus the application of wortmannin to the apical surface of a colony of tightly associated MDCK cells may be unable to efficiently inactivate the Met-stimulated PI3-kinase localized to the basolateral compartment.
      Wortmannin is also an unstable compound when maintained at 37°C (
      • Woscholski R.
      • Kodaki T.
      • McKinnon M.
      • Waterfield M.D.
      • Parker P.J.
      ). Thus, the ability of MDCK cells treated with wortmannin to begin to spread at 7 h post HGF/SF stimulation (Fig. 3B) may reflect the instability of wortmannin. Consistent with this possibility, MDCK cells stimulated with HGF/SF for 7 h remained as tight cell colonies when the medium containing wortmannin and HGF/SF was replaced at 2-h intervals (data not shown). Although MDCK cells showed some signs of toxicity under these conditions, this suggests that PI3-kinase is essential for cell spreading. We therefore conclude that PI3-kinase is required for MDCK cell dissociation and thus, scatter, following stimulation with HGF/SF, but due to the instability of wortmannin, we cannot rule out that other factors are involved in these events.
      The pp70S6K has been described as a downstream target of PI3-kinase in various cell types(
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ). We show that pp70S6K is activated following stimulation of MDCK cells with HGF/SF and that this activity is also inhibited by wortmannin, suggesting that pp70S6K is a downstream target of PI3-kinase in MDCK cells (Fig. 4). pp70S6K is required for the progression through G1 in response to serum and growth factors in a variety of cells (
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ,
      • Kuo C.J.
      • Chung J.
      • Florentino D.F.
      • Flanagan W.M.
      • Blenis J.
      • Crabtree G.R.
      ,
      • Price D.J.
      • Grove J.R.
      • Calvo V.
      • Avruch J.
      • Biere B.E.
      ,
      • Lane H.A.
      • Fernandez A.
      • Lamb N.J.C.
      • Thomas G.
      ). However, although pretreatment of MDCK cells with rapamycin inhibited the activation of HGF/SF-induced pp70S6K, this had no effect on MDCK cell spreading, redistribution of junctional complex proteins, or cell scatter (Fig. 5). These results suggest that although PI3-kinase is required for cell dissociation and scatter following stimulation of MDCK cells with HGF/SF, this is independent from the activation of pp70S6K.
      The requirement for a functional PI3-kinase has been implicated in actin reorganization at the plasma membrane (membrane ruffling) stimulated by PDGF (
      • Wennstrom S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ,
      • Nobes C.D.
      • Hawkins P.
      • Stephens L.
      • Hall A.
      ) and insulin(
      • Nobes C.D.
      • Hawkins P.
      • Stephens L.
      • Hall A.
      ). Moreover, PI3-kinase is involved in the activation of the small GTP-binding protein Rac(
      • Hawkins P.T.
      • Eguinoa A.
      • Qiu R.-G.
      • Stokoe D.
      • Cooke F.T.
      • Walters R.
      • Wennstrom S.
      • Claesson-Welsh L.
      • Evans T.
      • Symons M.
      • Stephens L.
      ), which is required for membrane ruffling in response to growth factors (
      • Nobes C.D.
      • Hawkins P.
      • Stephens L.
      • Hall A.
      ,
      • Ridley A.J.
      • Paterson H.F.
      • Johnston C.L.
      • Diekmann D.
      • Hall A.
      ). Interestingly, microinjection of MDCK cells with a dominant negative mutant of Rac (N17Rac1) inhibits cell spreading and actin reorganization induced by HGF/SF(
      • Ridley A.J.
      • Comoglio P.M.
      • Hall A.
      ), thus supporting a crucial role for Rac in these events. In addition, Ras is also essential for the dissociation and scatter of MDCK cells(
      • Ridley A.J.
      • Comoglio P.M.
      • Hall A.
      ,
      • Hartmann G.
      • Weidner K.M.
      • Schwarz H.
      • Birchmeier W.
      ). Expression of a dominant negative mutant Ras protein (N17Ras) (
      • Hartmann G.
      • Weidner K.M.
      • Schwarz H.
      • Birchmeier W.
      ) or the injection of a neutralizing antibody for Ras (Y13-259) (
      • Ridley A.J.
      • Comoglio P.M.
      • Hall A.
      ) block HGF/SF-induced cell dissociation and scatter, whereas microinjection of an activated Ras (V12H-Ras) in MDCK cells promotes cell spreading in the absence of HGF/SF(
      • Ridley A.J.
      • Comoglio P.M.
      • Hall A.
      ). Furthermore, GTP-bound Ras interacts with PI3-kinase and may contribute to its activation(
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ). Thus, we propose that activation of PI3-kinase in MDCK cells following stimulation of the Met receptor by HGF/SF promotes cell dissociation, which is independent from the activation of pp70S6K, but may involve the small GTP-binding proteins Rac and Ras. The relationship between PI3-kinase and these proteins in MDCK cells is currently under investigation.

      Acknowledgments

      We thank members of the Park laboratory for critical reading of the manuscript and Dr. Allison Haggarty for the use of the fluorescent microscope.

      REFERENCES

        • Gherardi E.
        Eur. J. Cancer. 1991; 27: 403-405
        • Gherardi E.
        • Gray J.
        • Stoker M.
        • Perryman M.
        • Furlong R.
        Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5844-5848
        • Nakamura T.
        • Teramoto H.
        • Ichihara A.
        Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6489-6493
        • Stoker M.
        • Gherardi E.
        • Perryman M.
        • Gray J.
        Nature. 1987; 327: 239-242
        • Montesano R.
        • Schaller G.
        • Orci L.
        Cell. 1991; 66: 697-711
        • Weidner K.M.
        • Behrens J.
        • Vandekerckhove J.
        • Birchmeier W.
        J. Cell Biol. 1990; 111: 2097-2108
        • Bussolino F.
        • Di Renzo M.F.
        • Ziche M.
        • Bocchietto E.
        • Olivero M.
        • Naldini L.
        • Gaudino G.
        • Tamagnone L.
        • Coffer A.
        • Comoglio P.M.
        J. Cell Biol. 1992; 119: 629-641
        • Grant D.S.
        • Kleinman H.K.
        • Goldberg I.D.
        • Bhargava M.M.
        • Nickoloff B.J.
        • Kinsella J.L.
        • Polverini P.
        • Rosen E.M.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1937-1941
        • Matsumoto K.
        • Nakamura T.
        Goldberg I.D. Rosen E.R. Hepatocyte Growth Factor-Scatter Factor (HGF-SF) and the c-met Receptor. Birkhauser Verlag, Basel1993: 226-248
        • Bellusci S.
        • Moens G.
        • Gaudino G.
        • Comoglio P.
        • Nakamura T.
        • Thiery J.-P.
        • Jouanneau J.
        Oncogene. 1994; 9: 1091-1099
        • Rong S.
        • Bodescot M.
        • Blair D.
        • Dunn J.
        • Nakamura T.
        • Mizuno K.
        • Park M.
        • Chan A.
        • Aaronson S.
        • Vande Woude G.F.
        Mol. Cell. Biol. 1992; 12: 5152-5158
        • Bottaro D.P.
        • Rubin J.S.
        • Faletto D.L.
        • Chan A.M.-L.
        • Kmiecik T.E.
        • Vande Woude G.F.
        • Aaronson S.A.
        Science. 1991; 251: 802-804
        • Naldini L.
        • Vigna E.
        • Ferracini R.
        • Longati P.
        • Gandino L.
        • Prat M.
        • Comoglio P.M.
        Mol. Cell. Biol. 1991; 11: 1793-1803
        • Cooper C.S.
        • Park M.
        • Blair D.G.
        • Tainsky M.A.
        • Huebner K.
        • Croce C.M.
        • Vande Woude G.
        Nature. 1984; 311: 29-33
        • Park M.
        • Dean M.
        • Cooper C.S.
        • Schmidt M.
        • O'Brien S.J.
        • Blair D.G.
        • Vande Woude G.
        Cell. 1986; 45: 895-904
        • Gonzatti-Haces M.
        • Seth A.
        • Park M.
        • Copeland T.
        • Oroszlan S.
        • Vande Woude G.F.
        Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 21-25
        • Liu C.
        • Park M.
        • Tsao S.
        Oncogene. 1992; 7: 181-185
        • Giordano S.
        • Di Renzo M.F.
        • Narsimhan R.P.
        • Cooper C.S.
        • Rosa C.
        • Comoglio P.M.
        Oncogene. 1989; 4: 1983-1988
        • Rodrigues G.A.
        • Naujokas M.A.
        • Park M.
        Mol. Cell. Biol. 1991; 11: 2962-2970
        • Tempest P.R.
        • Cooper C.S.
        • Major G.N.
        FEBS Lett. 1986; 209: 357-361
        • Naldini L.
        • Vigna E.
        • Narsimhan R.P.
        • Gaudino G.
        • Zarnegar R.
        • Michalopoulos G.A.
        • Comoglio P.M.
        Oncogene. 1991; 6: 501-504
        • Pawson T.
        Nature. 1995; 373: 573-580
        • Graziani A.
        • Gramaglia D.
        • Cantley L.C.
        • Comoglio P.M.
        J. Biol. Chem. 1991; 266: 22087-22090
        • Faletto D.L.
        • Kaplan D.R.
        • Halverson D.O.
        • Rosen E.M.
        • Vande Woude G.F.
        Goldberg I.D. Rosen E.M. Hepatocyte Growth Factor-Scatter Factor (HGF-SF) and the c-met Receptor. Birkhauser Verlag, Basel1993: 108-130
        • Halaban R.
        • Rubin J.S.
        • Funasaka Y.
        • Cobb M.
        • Boulton T.
        • Faletto D.
        • Rosen E.
        • Chan A.
        • Yoko K.
        • White W.
        • Cook C.
        • Moellmann G.
        Oncogene. 1992; 7: 2195-2206
        • Okano Y.
        • Mizuno K.
        • Osada S.
        • Nakamura T.
        • Nozawa Y.
        Biochem. Biophys. Res. Commun. 1993; 190: 842-848
        • Zhu H.
        • Naujokas M.A.
        • Fixman E.D.
        • Torossian K.
        • Park M.
        J. Biol. Chem. 1994; 269: 29943-29948
        • Ponzetto C.
        • Bardelli A.
        • Zhen Z.
        • Maina F.
        • dalla Zonca P.
        • Giordano S.
        • Graziani A.
        • Panayotou G.
        • Comoglio P.M.
        Cell. 1994; 77: 261-271
        • Graziani A.
        • Gramaglia D.
        • dalla Zonca P.
        • Comoglio P.M.
        J. Biol. Chem. 1993; 268: 9165-9168
        • Matsumoto K.
        • Matsumoto K.
        • Nakamura T.
        • Kramer R.H.
        J. Biol. Chem. 1994; 269: 31807-31813
        • Shibamoto S.
        • Hayakawa M.
        • Takeuchi K.
        • Hori T.
        • Oku N.
        • Miyazawa K.
        • Kitamura N.
        • Takeichi M.
        • Ito F.
        Cell Adhesion and Communication. 1994; 1: 295-305
        • Pelicci G.
        • Giordano S.
        • Zhen Z.
        • Salcini A.E.
        • Lanfrancone L.
        • Bardelli A.
        • Panayotou G.
        • Waterfield M.D.
        • Ponzetto C.
        • Pelicci P.G.
        • Comoglio P.M.
        Oncogene. 1995; 10: 1631-1638
        • Longati P.
        • Bardelli A.
        • Ponzetto C.
        • Naldini L.
        • Comoglio P.M.
        Oncogene. 1994; 9: 49-57
        • Rodrigues G.A.
        • Park M.
        Oncogene. 1994; 9: 2019-2027
        • Fixman E.D.
        • Naujokas M.A.
        • Rodrigues G.A.
        • Moran M.F.
        • Park M.
        Oncogene. 1995; 10: 237-249
        • 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
        • Zhu H.
        • Naujokas M.A.
        • Park M.
        Cell Growth & Differ. 1994; 5: 359-366
        • Pasdar M.
        • Nelson W.J.
        J. Cell Biol. 1988; 106: 677-685
        • Pasdar M.
        • Nelson W.J.
        J. Cell Biol. 1988; 106: 687-695
        • Giasson E.
        • Meloche S.
        J. Biol. Chem. 1995; 270: 5225-5231
        • Farquhar M.G.
        • Palade G.E.
        J. Cell Biol. 1963; 17: 375-412
        • Garrod D.R.
        Curr. Opin. Cell Biol. 1993; 5: 30-40
        • Takeichi M.
        Science. 1991; 251: 1451-1455
        • Citi S.
        J. Cell Biol. 1992; 117: 169-178
        • Pasdar M.
        • Nelson W.J.
        J. Cell Biol. 1989; 109: 163-177
        • Shore E.M.
        • Nelson W.J.
        J. Biol. Chem. 1991; 266: 19672-19680
        • Siliciano J.D.
        • Goodenough D.A.
        J. Cell Biol. 1988; 107: 2389-2399
        • Stevenson B.R.
        • Anderson J.M.
        • Bullivant S.
        Mol. Cell. Biochem. 1988; 83: 129-145
        • Kundra V.
        • Escobedo J.A.
        • Kazlauskas A.
        • Kim H.K.
        • Rhee S.G.
        • Williams L.T.
        • Zetter B.R.
        Nature. 1994; 367: 474-476
        • Wennstrom S.
        • Siegbahn A.
        • Yokote K.
        • Arvidsson A.-K.
        • Heldin C.-H.
        • Mori S.
        • Claesson-Welsh L.
        Oncogene. 1994; 9: 651-660
        • Arcaro A.
        • Wymann M.
        Biochem. J. 1993; 296: 297-301
        • Woscholski R.
        • Kodaki T.
        • McKinnon M.
        • Waterfield M.D.
        • Parker P.J.
        FEBS Lett. 1994; 342: 109-114
        • Yano H.
        • Nakanishi S.
        • Kimura K.
        • Hanai N.
        • Saitoh Y.
        • Fukui Y.
        • Nonomura Y.
        • Matsuda Y.
        J. Biol. Chem. 1993; 268: 25846-25856
        • Chen P.
        • Xie H.
        • Sekar M.C.
        • Gupta K.
        • Wells A.
        J. Cell Biol. 1994; 127: 847-857
        • Rosen E.M.
        • Meromsky L.
        • Goldberg I.
        • Bhargava M.
        • Setter E.
        J. Cell Sci. 1990; 96: 639-649
        • Smith R.J.
        • Sam L.M.
        • Justen J.M.
        • Bundy G.L.
        • Bala G.A.
        • Bleasdale J.E.
        J. Pharmacol. Exp. Ther. 1990; 253: 688-697
        • Bleasdale J.E.
        • Thakur N.R.
        • Gremban R.S.
        • Bundy G.L.
        • Fitzpatrick F.A.
        • Smith R.J.
        • Bunting S.
        J. Pharmacol. Exp. Ther. 1990; 255: 756-768
        • Tamaoki T.
        • Nomoto H.
        • Takahashi I.
        • Kato Y.
        • Morimoto M.
        • Tomita F.
        Biochem. Biophys. Res. Commun. 1986; 135: 397-402
        • Cheatham B.
        • Vlahos C.J.
        • Cheatham L.
        • Wang L.
        • Blenis J.
        • Kahn C.R.
        Mol. Cell. Biol. 1994; 14: 4902-4911
        • Chung J.
        • Grammer T.C.
        • Lemon K.P.
        • Kazlauskas A.
        • Blenis J.
        Nature. 1994; 370: 71-75
        • Ballou L.M.
        • Siegmann M.
        • Thomas G.
        Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7154-7158
        • Ferrari S.
        • Bannwarth W.
        • Morley S.J.
        • Totty N.F.
        • Thomas G.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7282-7286
        • Sabatini D.M.
        • Erdjument-Bromage H.
        • Lui M.
        • Tempst P.
        • Snyder S.H.
        Cell. 1994; 78: 35-43
        • Chung J.
        • Kuo C.J.
        • Crabtree G.R.
        • Blenis J.
        Cell. 1992; 69: 1227-1236
        • Kuo C.J.
        • Chung J.
        • Florentino D.F.
        • Flanagan W.M.
        • Blenis J.
        • Crabtree G.R.
        Nature. 1992; 358: 70-73
        • Price D.J.
        • Grove J.R.
        • Calvo V.
        • Avruch J.
        • Biere B.E.
        Science. 1992; 257: 973-977
        • Dowrick P.G.
        • Warn R.M.
        Goldberg I.D. Rosen E.M. Cell Motility Factors. Birkhauser Verlag, Basel1991: 89-108
        • Wennstrom S.
        • Hawkins P.
        • Cooke F.
        • Hara K.
        • Yonezawa K.
        • Kasuga M.
        • Jackson T.
        • Claesson-Welsh L.
        • Stephens L.
        Curr. Biol. 1994; 4: 385-393
        • Okada T.
        • Sakuma L.
        • Fukui Y.
        • Hazeki O.
        • Ui M.
        J. Biol. Chem. 1994; 269: 3563-3567
        • Okada T.
        • Kawano Y.
        • Sakakibara T.
        • Hazeki O.
        • Ui M.
        J. Biol. Chem. 1994; 269: 3568-3573
        • Nakanishi S.
        • Kakita S.
        • Takahashi I.
        • Kawahara K.
        • Tsukuda E.
        • Sano T.
        • Yamada K.
        • Yoshida M.
        • Kase H.
        • Matsuda Y.
        • Hashimoto Y.
        • Nonomura Y.
        J. Biol. Chem. 1992; 267: 2157-2163
        • Crepaldi T.
        • Pollack A.L.
        • Prat M.
        • Zborek A.
        • Mostov K.
        • Comoglio P.M.
        J. Cell Biol. 1994; 125: 313-320
        • Lane H.A.
        • Fernandez A.
        • Lamb N.J.C.
        • Thomas G.
        Nature. 1993; 363: 170-172
        • Nobes C.D.
        • Hawkins P.
        • Stephens L.
        • Hall A.
        J. Cell Sci. 1995; 108: 225-233
        • Hawkins P.T.
        • Eguinoa A.
        • Qiu R.-G.
        • Stokoe D.
        • Cooke F.T.
        • Walters R.
        • Wennstrom S.
        • Claesson-Welsh L.
        • Evans T.
        • Symons M.
        • Stephens L.
        Curr. Biol. 1995; 5: 393-403
        • Ridley A.J.
        • Paterson H.F.
        • Johnston C.L.
        • Diekmann D.
        • Hall A.
        Cell. 1992; 70: 401-410
        • Ridley A.J.
        • Comoglio P.M.
        • Hall A.
        Mol. Cell. Biol. 1995; 15: 1110-1122
        • Hartmann G.
        • Weidner K.M.
        • Schwarz H.
        • Birchmeier W.
        J. Biol. Chem. 1994; 269: 21936-21939
        • Rodriguez-Viciana P.
        • Warne P.H.
        • Dhand R.
        • Vanhaesebroeck B.
        • Gout I.
        • Fry M.J.
        • Waterfield M.D.
        • Downward J.
        Nature. 1994; 370: 527-532
        • Hu Q.
        • Klippel A.
        • Muslin A.J.
        • Fantl W.J.
        • Williams L.T.
        Science. 1995; 268: 100-102