Src Homology 2-containing Inositol 5-Phosphatase 1 Binds to the Multifunctional Docking Site of c-Met and Potentiates Hepatocyte Growth Factor-induced Branching Tubulogenesis
2001; Elsevier BV; Volume: 276; Issue: 5 Linguagem: Inglês
10.1074/jbc.m009333200
ISSN1083-351X
AutoresMonica Stefan, Alexandra Koch, Annalisa Mancini, Andrea Mohr, K. Michael Weidner, Heiner Niemann, Teruko Tamura,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoHepatocyte growth factor (HGF)/scatter factor is a multifunctional cytokine that induces mitogenesis, motility, and morphogenesis in epithelial, endothelial, and neuronal cells. The receptor for HGF/scatter factor was identified as c-Met tyrosine kinase, and activation of the receptor induces multiple signaling cascades. To gain further insight into c-Met-mediated multiple events at a molecular level, we isolated several signaling molecules including a novel binding partner of c-Met, SH2 domain-containing inositol 5-phosphatase 1 (SHIP-1). Western blot analysis revealed that SHIP-1 is expressed in the epithelial cell line, Madin-Darby canine kidney (MDCK) cells. SHIP-1 binds at phosphotyrosine 1356 at the multifunctional docking site. Because a number of signaling molecules such as Grb2, phosphatidylinositol 3-kinase, and Gab1 bind to the multifunctional docking site, we further performed an in vitro competition study using glutathioneS-transferase- or His-tagged signaling molecules with c-Met tyrosine kinase. Our binding study revealed that SHIP-1, Grb2, and Gab1 bound preferentially over phosphatidylinositol 3-kinase. Surprisingly, MDCK cells that overexpress SHIP-1 demonstrated branching tubulogenesis within 2 days after HGF treatment, whereas wild-type MDCK cells showed tubulogenesis only after 6 days following treatment without altering cell scattering or cell growth potency. Furthermore, overexpression of a mutant SHIP-1 lacking catalytic activity impaired HGF-mediated branching tubulogenesis. Hepatocyte growth factor (HGF)/scatter factor is a multifunctional cytokine that induces mitogenesis, motility, and morphogenesis in epithelial, endothelial, and neuronal cells. The receptor for HGF/scatter factor was identified as c-Met tyrosine kinase, and activation of the receptor induces multiple signaling cascades. To gain further insight into c-Met-mediated multiple events at a molecular level, we isolated several signaling molecules including a novel binding partner of c-Met, SH2 domain-containing inositol 5-phosphatase 1 (SHIP-1). Western blot analysis revealed that SHIP-1 is expressed in the epithelial cell line, Madin-Darby canine kidney (MDCK) cells. SHIP-1 binds at phosphotyrosine 1356 at the multifunctional docking site. Because a number of signaling molecules such as Grb2, phosphatidylinositol 3-kinase, and Gab1 bind to the multifunctional docking site, we further performed an in vitro competition study using glutathioneS-transferase- or His-tagged signaling molecules with c-Met tyrosine kinase. Our binding study revealed that SHIP-1, Grb2, and Gab1 bound preferentially over phosphatidylinositol 3-kinase. Surprisingly, MDCK cells that overexpress SHIP-1 demonstrated branching tubulogenesis within 2 days after HGF treatment, whereas wild-type MDCK cells showed tubulogenesis only after 6 days following treatment without altering cell scattering or cell growth potency. Furthermore, overexpression of a mutant SHIP-1 lacking catalytic activity impaired HGF-mediated branching tubulogenesis. hepatocyte growth factor Src homology 2 growth factor receptor-bound protein phosphatidylinositol phospholipase C-γ Madin-Darby canine kidney SH2-containing inositol 5-phosphatase phosphotyrosine glutathioneS-transferase fetal calf serum SDS-polyacrylamide gel electrophoresis Met binding domain. Met tyrosine kinase is the receptor for hepatocyte growth factor (HGF)1/scatter factor (1Bottaro D.O. Rubin J.S. Faletto D.L. Chan A.M. Kmiecik T.E. Vande Woude G.F. Aaronson S.A. Science. 1991; 251: 802-804Crossref PubMed Scopus (2088) Google Scholar, 2Naldini L. Weidner K.M. Vigna E. Gaudino G. Bardelli A. 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Met tyrosine kinase was reported to interact with several substrates including the growth factor receptor-bound protein (Grb) 2 (20Ponzetto C. Bardelli A. Zhen Z. Maina F. dalla Zonca P. Giordano S. Graziani A. Panayotou G. Comoglio P.M. Cell. 1994; 77: 261-271Abstract Full Text PDF PubMed Scopus (894) Google Scholar), STAT3 (21Boccaccio C. Andò M. Tamagnone L. Bardelli A. Michieli P. Battistini C. Comoglio P.M. Nature. 1998; 391: 285-288Crossref PubMed Scopus (452) Google Scholar), the p85 subunit of phosphatidylinositol (PI) 3-kinase (22Graziani A. Gramaglia D. Cantley L.C. Comoglio P.M. J. Biol. Chem. 1991; 266: 22087-22090Abstract Full Text PDF PubMed Google Scholar), Shc (23Pelicci G. Giordano S. Zhen Z. Salcini A.E. Lanfrancone L. Bardelli A. Panayotou G. Waterfield M.D. Ponzetto A. Pelicci P.G. Oncogene. 1995; 10: 1631-1638PubMed Google Scholar), phospholipase C-γ (PLCγ) (20Ponzetto C. Bardelli A. Zhen Z. Maina F. dalla Zonca P. Giordano S. Graziani A. Panayotou G. Comoglio P.M. Cell. 1994; 77: 261-271Abstract Full Text PDF PubMed Scopus (894) Google Scholar), c-Src (20Ponzetto C. Bardelli A. Zhen Z. Maina F. dalla Zonca P. Giordano S. Graziani A. Panayotou G. Comoglio P.M. Cell. 1994; 77: 261-271Abstract Full Text PDF PubMed Scopus (894) Google Scholar), and Gab1 (24Weidner K.M. Di Cesare S. Sachs M. Brinkmann V. Behrens J. Birchmeier W. Nature. 1996; 384: 173-176Crossref PubMed Scopus (506) Google Scholar). Interestingly, all these proteins associate with c-Met via a multiple substrate binding site. The c-Met-mediated signaling was well studied in the epithelial cell system, MDCK cells. On planar culture surfaces, sub-confluent MDCK cells normally form coherent islands of relatively flattened cells. HGF has been shown to alter this morphology by promoting the initial expansion of colonies followed by dispersion of the cells that make up these colonies and an increase in their motility (cell scattering) in liquid cell culture medium (25Stoker M. Perryman M. J. Cell Sci. 1985; 77: 209-223Crossref PubMed Google Scholar). The activation of a number of tyrosine kinase receptors including Neu, Ros epidermal growth factor receptor, and keratinocyte growth factor receptor induced cell scattering in MDCK cells (26Sachs M. Weidner K.M. Brinkmann V. Walther I. Obermeier A. Ullrich A. Birchmeier W. J. Cell Biol. 1996; 133: 1095-1107Crossref PubMed Scopus (90) Google Scholar). Royal and Park (27Royal I. Park M. J. Biol. Chem. 1996; 270: 27780-27787Abstract Full Text Full Text PDF Scopus (229) Google Scholar) reported that PI 3-kinase and Ras are required for cell scattering. In addition, stimulation with HGF induces the formation of tubular structures from MDCK cells grown in a three-dimensional collagen matrix (11Montesano R. Matsumoto K. Nakamura T. Orci L. Cell. 1991; 67: 901-908Abstract Full Text PDF PubMed Scopus (1089) Google Scholar). For this, the epithelial cells are grown for several days in collagen, in which they form cysts. When HGF/scatter factor is added, individual cells dissociate and form continuous tubules (28Weidner K.M. Sachs M. Birchmeier W. J. Cell Biol. 1993; 121: 145-154Crossref PubMed Scopus (374) Google Scholar). It has been reported that a number of signaling molecules including Gab1(24), Grb2 (29Royal I. Fournier T.M. Park M. J. Cell. Physiol. 1997; 173: 196-201Crossref PubMed Scopus (67) Google Scholar), PLCγ (30Gual P. Giordano S. Williams T.A. Rocchi S. Van Obberghen E. Comoglio P.M. Oncogene. 2000; 19: 1509-1518Crossref PubMed Scopus (138) Google Scholar), and STAT3 (21Boccaccio C. Andò M. Tamagnone L. Bardelli A. Michieli P. Battistini C. Comoglio P.M. Nature. 1998; 391: 285-288Crossref PubMed Scopus (452) Google Scholar) are required for this process. The underlying mechanisms of these multiple molecular events, however, are still poorly understood. In the present study we identified SH2-containing inositol 5-phosphatase (SHIP) 1 as a novel binding partner of c-Met by yeast two-hybrid screening using a rat brain library. SHIP-1 binds to one of the tyrosine residues at the multiple substrate binding site, 1356pYVNV, which is also the binding site for Grb2. Interestingly, the YVNV motif is identical to the common binding site of Shc to SHIP and Grb2. Furthermore, we show that PI 3-kinase was not able to bind c-Met in the presence of Grb2, Gab1, or SHIP-1. Overexpression of SHIP-1 in MDCK cells drastically enhanced the branching potency of c-Met without affecting the mitogenic and scattering potency. Furthermore, overexpression of a mutant SHIP-1 lacking catalytic activity impaired HGF-mediated branching tubulogenesis. The construction of LexA fusion genes encoding the cytoplasmic domains of c-Met, c-Fms, TrkA, insulin receptor, and c-Kit downstream of LexA and the expression inSaccharomyces cerevisiae strain L40 were described previously (24Weidner K.M. Di Cesare S. Sachs M. Brinkmann V. Behrens J. Birchmeier W. Nature. 1996; 384: 173-176Crossref PubMed Scopus (506) Google Scholar, 31Mancini A. Niedenthal R. Joos H. Koch A. Trouliaris S. Niemann H. Tamura T. Oncogene. 1997; 15: 1565-1572Crossref PubMed Scopus (44) Google Scholar, 32Tamura T. Mancini A. Joos H. Koch A. Hakim C. Dumanski J. Weidner K.M. Niemann H. Oncogene. 1999; 18: 6488-6495Crossref PubMed Scopus (42) Google Scholar). Met mutants containing tyrosine/phenylalanine replacements have been described (24Weidner K.M. Di Cesare S. Sachs M. Brinkmann V. Behrens J. Birchmeier W. Nature. 1996; 384: 173-176Crossref PubMed Scopus (506) Google Scholar). A single colony, selected for expression of the LexA fusion protein, was tested for autophosphorylation and used for transformation with the VP16 cDNA library derived from an 8-day rat brain (33Okamoto M. Südhof T.C. J. Biol. Chem. 1997; 272: 31459-31464Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). GST·Grb2, GST·SHIP, GST·Gab1, GST·PI 3-kinase fusion proteins were generated in the pGex system (Amersham Pharmacia Biotech). His-tagged SHIP, Grb2, and PI 3-kinase were generated in pQE30 (Qiagen, Hilden, Germany). Myc-tagged wild-type and mutant SHIP-1 were generated using pcDNA3.1Myc-His (Invitrogen, Carlsbad, CA). The qualitative and quantitative evaluations of various two-hybrid protein/protein interactions were described previously (31Mancini A. Niedenthal R. Joos H. Koch A. Trouliaris S. Niemann H. Tamura T. Oncogene. 1997; 15: 1565-1572Crossref PubMed Scopus (44) Google Scholar). MDCK cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS. HGF was from Sigma. Scattering and branching tubulogenesis assays were performed as described by Gual et al. (30Gual P. Giordano S. Williams T.A. Rocchi S. Van Obberghen E. Comoglio P.M. Oncogene. 2000; 19: 1509-1518Crossref PubMed Scopus (138) Google Scholar). Monoclonal antibodies against phosphotyrosine (4G10) and c-Met were from Upstate Biotechnology Inc. (Lake Placid, NY). Monoclonal antibody against Myc and rabbit IgG against c-Met and SHIP-1 were from Santa Cruz Biotechnology (Santa Cruz, CA). These assays were performed as published (31Mancini A. Niedenthal R. Joos H. Koch A. Trouliaris S. Niemann H. Tamura T. Oncogene. 1997; 15: 1565-1572Crossref PubMed Scopus (44) Google Scholar, 34Joos H. Trouliaris S. Helftenbein G. Niemann H. Tamura T. J. Biol. Chem. 1996; 271: 24476-24481Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). After incubation for 8 h with medium containing 0.02% FCS, MDCK cells were stimulated with HGF (60 ng/ml) for 5, 10, or 30 min. Cells were extracted with lysis buffer containing 50 mm HEPES, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1% trasylol, 10% glycerol, 1 mmphenylmethylsulfonyl fluoride, and 400 μm sodium orthovanadate. One aliquot of each clarified lysate (5 × 106 cells) was incubated for 16 h at 4 °C with anti-SHIP or anti-Met antibody preabsorbed on Staphylococcus aureus Cowan I strain. After washing, materials were analyzed by SDS-PAGE and by immunoblot analysis using the appropriate antibodies. GST fusion proteins and His-tagged proteins were produced as recommended by the manufacturer. Purified GST fusion proteins were bound for 1 h at 4 °C to glutathione-agarose beads (40 μl slurry; Amersham Pharmacia Biotech) suspended in the binding buffer (50 mm HEPES, pH 7.5, 150 mmNaCl, 1% Triton X-100, 1% trasylol, 10% glycerol, 1 mmphenylmethylsulfonyl fluoride, 20 mg/ml bovine serum albumin, and 200 μm sodium orthovanadate). After incubation with 32P-labeled autophosphorylated c-Met overnight, beads were washed five times with binding buffer, and pellets were analyzed by SDS-PAGE. To identify neuronal proteins that specifically interact with the cytoplasmic domain of c-Met, we employed a yeast two-hybrid screening using an 8 day-old rat brain library (24Weidner K.M. Di Cesare S. Sachs M. Brinkmann V. Behrens J. Birchmeier W. Nature. 1996; 384: 173-176Crossref PubMed Scopus (506) Google Scholar, 33Okamoto M. Südhof T.C. J. Biol. Chem. 1997; 272: 31459-31464Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 35Field S. Song O.-K. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4875) Google Scholar). A total of 800 cDNA clones of various length were obtained, together encoding seven different proteins. Five of these proteins were previously identified as c-Met-binding proteins including Gab1, PI 3-kinase, Grb2, PLCγ, and c-Src. Furthermore, we detected the interaction with a novel c-Met-interactive zinc finger-containing protein; however, the biological significance of this association remains to be studied. In addition to these molecules, we isolated SHIP-1 as a novel c-Met binding partner. SHIP-1 was originally identified as a signaling molecule in cytokine-stimulated hematopoietic cells such as macrophage colony-stimulating factor-stimulated cells (36Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold A. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (379) Google Scholar) and has been clearly demonstrated to participate in the signaling pathways in hematopoietic cell systems (37Unkeless J.C. Jin J. Curr. Opin. Immunol. 1997; 9: 338-343Crossref PubMed Scopus (126) Google Scholar). To determine whether SHIP-1 is a relevant signaling molecule in other cell systems, we next analyzed SHIP-1 expression in various cell lines such as colon carcinoma CACO-2; MDCK cells; two human breast carcinoma cell lines, EFM-19 and EFM-192A; and Raji cells. For a control, we applied a cell lysate derived from myeloid projenitor cell line FDC-P1Mac11 cells (38Gliniak S.C. Rohrschneider R.L. Cell. 1990; 63: 1073-1083Abstract Full Text PDF PubMed Scopus (84) Google Scholar). In agreement with a previous report (36Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold A. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (379) Google Scholar), SHIP-1 is expressed at high levels in myeloid progenitor cell line FDC-P1Mac11 cells. In addition, SHIP-1 was also detected in the epithelial cell lines, MDCK, both human breast carcinoma cell lines, and Raji cells (Fig.1). To demonstrate the protein/protein interaction between SHIP and c-Metin vivo, MDCK cells were incubated for 8 h with medium containing 0.02% FCS, then stimulated with HGF (60 ng/ml) for 5, 10, and 30 min, and studied by immunoprecipitation. Aliquots of 5 × 106 cells from each preparation were lysed for immunoprecipitations using the SHIP-specific or c-Met-specific antibodies. Precipitated material was analyzed for the presence of c-Met or SHIP by SDS-PAGE and Western blotting using SHIP-, c-Met-, or phosphotyrosine (4G10)-specific antibodies (Fig.2). Following HGF stimulation, tyrosine phosphorylation of c-Met was observed throughout the experiment. Prior to HGF stimulation (0 min), no c-Met was detectable in the SHIP-specific immune complexes. In contrast, between 5 and 30 min after HGF stimulation, c-Met was coprecipitated with SHIP (Fig. 2). To determine whether the SHIP-1-c-Met interaction relied on the presence of particular phosphotyrosine residues of c-Met, we employed a set of c-Met mutants in which established binding sites for defined binding partners were destroyed (24Weidner K.M. Di Cesare S. Sachs M. Brinkmann V. Behrens J. Birchmeier W. Nature. 1996; 384: 173-176Crossref PubMed Scopus (506) Google Scholar). These mutants included Y1349F (Y14F), Y1356F (Y15F), the double mutant Y14F/Y15F, and the kinase negative mutant K1110A. We examined the binding of these mutants with the SH2 domain of SHIP-1, PI 3-kinase, Grb2, Grb10, PLCγ, and c-Src, the Met binding domain (MBD) of Gab1, and the phosphotyrosine binding domain of Shc. As shown in Fig.3, Grb2, Grb10, and SHIP-1 did not associate with the Y15F mutant, suggesting that phosphotyrosine 1356 at the multifunctional docking site is the only binding site for these proteins. On the other hand, PI 3-kinase, Gab1, PLCγ, c-Src, and Shc bind to both Y14F (Y1349F) and Y15F (Y1356F) (Fig. 3). Signaling molecules that bind to the multifunctional docking site such as PI 3-kinase, Shc, and PLCγ associate with most receptor tyrosine kinases. Because SHIP-1 binds at the same site in c-Met with these molecules, we performed similar binding analyses with four additional activated receptor tyrosine kinases including c-Fms, c-Kit, TrkA, and the insulin receptor. Western blotting with anti-phosphotyrosine antibody clearly demonstrated that these receptor moieties were phosphorylated on tyrosine in the yeast two-hybrid assay (data not shown). As expected, c-Fms binds to SHIP-1; however, SHIP-1 did not bind other tyrosine kinase receptors such as c-Kit, TrkA, or the insulin receptor (Fig.4). In agreement with previous data (39Reedijk M. Liu X. van der Geer P. Letwin K. Waterfield M.D. Hunter T. Pawson T. EMBO J. 1992; 11: 1365-1372Crossref PubMed Scopus (179) Google Scholar, 40Herbst R. Shearman M.S. Jallal B. Schlessinger J. Ullrich A. Biochemistry. 1995; 34: 5971-5979Crossref PubMed Scopus (27) Google Scholar, 41Levy-Toledano R. Taouis M. Blaettler D.H. Gorden P. Taylor S.I. J. Biol. Chem. 1994; 269: 31178-31182Abstract Full Text PDF PubMed Google Scholar), PI 3-kinase binds to c-Fms, c-Kit, and the insulin receptor, the sequences of which contain the typical consensus motif, pYXX M, for the PI 3-kinase binding site (42Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.J. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Catley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2391) Google Scholar). It is noteworthy that both of the PI 3-kinase binding sites of c-Met, tyrosine 1349 and 1356, and their following amino acid sequences are different from this motif (Table I).Table IpYVNV in c-Met and Shc binds to both SHIP and Grb2Tyrosine kinase substrateSHIP binding sitePI 3-kinase binding siteGrb2 binding sitec-MetpYVNVpYVHV (20)pYVNV (20, 56)pYVNV (20)ShcpYVNV (44)pYVNV (57)c-Fms?pYVEM (39)pYKNI (58)pYTNL (31)c-KitpYMDM (40)pYKNL (59)pYSNL (59)Insulin receptorpYHTM (41) Open table in a new tab The multifunctional docking site of c-Met provides binding sites for many substrates including PI 3-kinase, Grb2, Gab1, and SHIP. As shown in Fig. 3, PI 3-kinase and Gab1 bind at both phosphotyrosine 1349 and phosphotyrosine 1356, whereas SHIP and Grb2 bind only at phosphotyrosine 1356. This observation raises the question whether two of these molecules bind to c-Met simultaneously or sequentially. To answer this question, we have generated the GST·SH2 domain of Grb2 (Grb2(SH2)), p85 of PI 3-kinase (PI 3-kinase(SH2)), and SHIP (SHIP(SH2)) fusion proteins and GST·MBD of Gab1 (Gab1(MBD)), which were incubated with tyrosine-autophosphorylated c-Met in the presence and absence of His-tagged PI 3-kinase(SH2), Grb2(SH2), or SHIP(SH2). Firstly, we compared the binding of c-Met to PI 3-kinase and SHIP. When equal amounts of both molecules were incubated with c-Met, SHIP bound predominantly to c-Met. Secondly, Grb2 bound to c-Met preferentially over PI 3-kinase and Gab1 (Fig. 5). Thirdly, SHIP did not compete with the c-Met-Gab1 interaction. Taken together, these results indicate that when phosphotyrosine 1356 binds to either Grb2 or SHIP, phosphotyrosine 1349 does not provide a binding site for PI 3-kinase and that when Grb2 binds to phosphotyrosine 1356, even Gab1 does not bind to c-Met, suggesting that in vivo these molecules may bind to a single molecule of c-Met sequentially. To investigate the biological role of SHIP-1 in c-Met-mediated signaling, we overexpressed the Myc-tagged SHIP-1 gene in MDCK cells and isolated 19 different clones, each constitutively expressing distinct levels of an Myc-tagged SHIP-1. The two clones (clones 15 and 21) employed in these studies expressed equally high levels of SHIP-1 (Fig.6 A). Using these mutant cell lines together with the wild-type MDCK cells as control, we examined cell dissociation (scattering), cell proliferation, and branching tubulogenesis induced by HGF stimulation. Firstly, the scattering effect of HGF to both transfectants was indistinguishable from that of the wild type (Fig. 6 B). Secondly, in agreement with data obtained from the fibroblast system (36Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold A. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (379) Google Scholar), the overexpression of SHIP-1 showed no influence on [3H]thymidine incorporation, suggesting that overexpression of SHIP-1 did not affect cell growth of MDCK cells (Fig. 6 C). Finally, we tested the ability of these cells to form tubules in semi-solid collagen. To our surprise, both transfectants that overexpressed Myc-tagged SHIP started to form branching tubules within 24 h after HGF treatment. Two days after HGF stimulation, transfectants formed long branching tubules (Fig.7), whereas wild-type MDCK cells formed branching tubules only 6–7 days after HGF treatment (Fig. 7). Furthermore, in agreement with previous data (43Maroun C.R. Holgada-Madruga M. Royal I. Naujokas M.A. Fournier T.M. Wong A.J. Park M. Mol. Cell. Biol. 1999; 19: 1784-1799Crossref PubMed Scopus (175) Google Scholar), even 7 days after HGF treatment, about 70% of wild-type cells formed tubules; however, almost all transfectants formed tubules. In the absence of HGF, none of the transfectants formed any branching tubules throughout a time period of 2 weeks (data not shown). Taken together, these observations suggest that overexpression of SHIP clearly enhances HGF-mediated tubulogenesis.Figure 7Overexpression of SHIP in MDCK cells potentiates HGF-mediated branching tubulogenesis. Wild-type (wt) and SHIP-overexpressing MDCK cell lines, clones 15 and 21, were grown in collagen for 5 days, and then medium containing HGF (40 ng/ml) was added and changed every 2 days. After 2, 4, and 7 days, the branching tubulogenesis response was evaluated. Photomicrographs by Hoffman modulation contrast microphotometry show a representative experiment that had been performed in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As shown above, SHIP-1 overexpression accelerated tubulogenesis. This fact raised the question whether this phenotype results from enhanced phosphoinositol phosphatase activity or from displacement of protein binding to the receptor at the SHIP-1 binding site. To answer this question, we next generated a mutant SHIP-1 lacking catalytic activity by deleting amino acid residues 666–680 (44Vollenweider P. Clodi M. Martin S.S. Imamura T. Kavanaugh W.M. Olefsky J.M. Mol. Cell. Biol. 1999; 19: 1081-1091Crossref PubMed Scopus (76) Google Scholar) using pcDNA3.1Myc-His, and we overexpressed this mutant in MDCK cells. Twelve different clones were isolated, and the two clones (clones 1 and 4) employed in this study expressed equally high levels of mutant SHIP-1 (Fig. 8 A). Using these mutant cell lines together with the pcDNA3.1Myc-His-transfected MDCK cells as control, we examined branching tubulogenesis induced by the HGF stimulation. In agreement with previous experiments using wild-type MDCK cells, the control transfectant formed branching tubules within 7 days after the HGF treatment. Two clones that overexpressed a mutant SHIP-1, however, failed to form branching tubules over 9 days (Fig. 8 B), suggesting that the effects of SHIP-1 overexpression to accelerate tubulogenesis are due to the enhanced phosphoinositide phosphatase activity. The results presented above can be summarized as follows. Firstly, we have identified SHIP as a novel binding partner of c-Met. Secondly, SHIP binds to tyrosine 1356 at the multifunctional docking site of c-Met. Thirdly, we showed here that this site, which contains two phosphotyrosine residues, binds in vitro to only one signaling molecule at a time. Fourthly, overexpression of SHIP drastically enhanced the tubulogenesis potency of c-Met without altering the c-Met-mediated cell scattering and cell proliferation. Cells that overexpressed a mutant SHIP-1 lacking catalytic activity, however, failed to form branching tubules in the presence of HGF, suggesting that the effects of SHIP-1 overexpression to accelerate tubulogenesis are due to the enhanced phosphoinositide phosphatase activity. c-Met signaling is mediated by a multifunctional docking site comprising two phosphotyrosines arranged in tandem (20Ponzetto C. Bardelli A. Zhen Z. Maina F. dalla Zonca P. Giordano S. Graziani A. Panayotou G. Comoglio P.M. Cell. 1994; 77: 261-271Abstract Full Text PDF PubMed Scopus (894) Google Scholar). Most tyrosine kinase receptors including c-Fms, c-Kit, and epidermal growth factor receptor autophosphorylate at multiple sites that bind several signaling molecules, suggesting that one receptor molecule is able to simultaneously associate with multiple signaling molecules. In addition, we showed here that the multifunctional docking site of c-Met binds to only one signaling molecule and that when several effector molecules are present simultaneously, one can observe in vitro the hierarchical binding of proteins, such as Grb2, Gab1, SHIP, and PI 3-kinase. Our competition assay reveals that PI 3-kinase did not compete with c-Met-Grb2 and c-Met-SHIP interactions; however, Grb2 competed with all of these interactions, suggesting that the binding order of these effector molecules is crucial for the c-Met-mediated multiple signal transduction. In vivo binding of full-length molecules, however, may be dependent upon their allosteric interactions. It is therefore also possible that multiple complexes of signaling molecules are assembled to the activated receptor. Furthermore, we have to take into account that the number and the subcellular distribution of each of the signaling molecules expressed in a given cell are different. For instance, when HGF-treated cells are grown in a three-dimensional collagen matrix, cells form tubules that have a lumen surrounded by well polarized epithelial cells, with a smooth basal surface in contact with the collagen matrix and an apical surface rich in microvilli that faces the lumen (11Montesano R. Matsumoto K. Nakamura T. Orci L. Cell. 1991; 67: 901-908Abstract Full Text PDF PubMed Scopus (1089) Google Scholar). The subcellular localization of c-Met and substrates differs in these cells from nonpolarized cells that are tested for cell scattering. For the cell-scattering assay, cells were incubated in liquid culture medium without collagen. We show here that the SHIP-1 binding site, tyrosine 1356, is also a binding site for Grb2 with the YX NX motif. Does SHIP-1 regularly share the binding site with Grb2? In the case of c-Fms, tyrosine 696 and tyrosine 921 provide the binding sites for Grb2 with the YX NX motif, 696YKNI and921YTNL, respectively (31Mancini A. Niedenthal R. Joos H. Koch A. Trouliaris S. Niemann H. Tamura T. Oncogene. 1997; 15: 1565-1572Crossref PubMed Scopus (44) Google Scholar). Mutation of both sites, however, did not exert an influence on the SHIP-1-c-Fms association, indicating that none of these sites acts as a binding site for SHIP-1 (data not shown). These data suggest that the second or fourth flanking position is also important for binding. Interestingly, the SH2 domains of SHIP and Grb2 also share the binding site 317pYVNV of Shc (45Wada T. Sasaoka T. Ishii M. Hori H. Haruta T. Ishihara H. Kobayashi M. Endocrinology. 1999; 140: 4585-4594Crossref PubMed Scopus (19) Google Scholar), the sequence of which is identical to the1356pYVNV of c-Met, indicating that YVNV is a consensus sequence for the SHIP binding site (Table I). Interestingly, the point mutation of asparagine 1358 into histidine abolished the branching potential of c-Met (29Royal I. Fournier T.M. Park M. J. Cell. Physiol. 1997; 173: 196-201Crossref PubMed Scopus (67) Google Scholar). A most striking observation of our studies regards the fact that overexpression of SHIP-1 drastically enhanced the HGF-mediated branching tubulogenesis without altering cell growth and cell scattering in response to HGF (Figs. 6 and 7). Here, both transfectants that overexpressed Myc-tagged SHIP started to form branching tubules within 24 h after the HGF treatment. SHIP plays a role in inositol phosphate and phosphatidylinositol phosphate metabolism (46Erneux C. Govaerts C. Communi D. Pesesse X. Biochim. Biophys. Acta. 1998; 1436: 185-199Crossref PubMed Scopus (126) Google Scholar). SHIP-1 displays 5-phosphatase activity specifically with both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetraphosphate as substrate. Phee et al. (47Phee H. Jacob A. Coggeschall K.M. J. Biol. Chem. 2000; 275: 19090-19097Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) reported that enzymatic activity of SHIP is regulated by a plasma membrane localization and, as a result of this regulation, significant reduction in the cellular phosphatidylinositol 3,4,5-trisphosphate level was observed. Furthermore, a striking correlation was observed between phosphatidylinositol 3,4-bisphosphate production and tyrosine phosphorylation of SHIP-1, as well as its relocation to the cytoskeleton upon thrombin stimulation in human blood platelets (48Giuriato S. Payrastre B. Drayer L.A. Plantavid M. Woscholski R. Parker P. Erneux C. Chap H. J. Biol. 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At the moment, the molecular mechanism of potentiation of HGF-mediated branching by SHIP-1 overexpression remains unclear; however, inositol phosphate and/or phosphatidylinositol phosphate metabolisms play a key role in epithelial cell dissociation, reassociation, and formation of continuous tubules. We thank Karsten Heidrich and Regina Wilms (Medizinische Hochschule Hannover) for technical assistance; Walter Birchmeier (Max-Delbruek Center of Molecular Medicine, Berlin, Germany) for generously providing LexA-Met and its mutants, LexA-insulin receptor, LexA-TrkA, and LexA-Kit cDNAs; Thomas Südhof (University of Texas) for the rat brain cDNA library; and Larry Rohrschneider (Fred Hutchinson Cancer Center, Seattle, WA) for providing the SHIP cDNA.
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