cytoplasmic domain of the met receptor affect signal trans- duction in epithelial cells in a positive or negative fashion: mutation of the C-terminal tyrosine residues ...
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2597-2601, March 1995 Cell Biology
Mutation of juxtamembrane tyrosine residue 1001 suppresses loss-of-function mutations of the met receptor in epithelial cells (scatter factor/hepatocyte growth factor/cell motility/branching morphogenesis/signal transduction/epithelial-mesenchymal
interactions)
K. MICHAEL WEIDNER, MARTIN SACHS, DIETER RIETHMACHER, AND WALTER BIRCHMEIER Max-Delbruck-Center for Molecular Medicine, Robert-Rossle-Strasse 10, 13125 Berlin, Germany
Communicated by Georg Melchers, Max-Planck-Institut ifir Biologie, Tubingen, Germany, November 30, 1994 (received for review August 12 1994)
ABSTRACT Signals transduced by the met tyrosine kinase, which is the receptor for scatter factor/hepatocyte growth factor, are of major importance for the regulation of epithelial cell motility, morphogenesis, and proliferation. We report here that different sets of tyrosine residues in the cytoplasmic domain of the met receptor affect signal transduction in epithelial cells in a positive or negative fashion: mutation of the C-terminal tyrosine residues 13-16 (Y1311, Y1347, Y1354, and Y1363) reduced or abolished ligandinduced cell motility and branching morphogenesis. In contrast, mutation of the juxtamembrane tyrosine residue 2 (Y1001) produced constitutively mobile, fibroblastoid cells. Furthermore, the gain-of-function mutation of tyrosine residue 2 suppressed the loss-of-function mutations of tyrosine residue 15 or 16. The opposite roles of the juxtamembrane and C-terminal tyrosine residues may explain the suggested dual function of the met receptor in both epithelial-mesenchymal interactions and tumor progression.
of tyrosine residues of met, which potential docking sites for tyrosine kinase substates are implicated in the transmission of various signals in epithelial cells.
MATERIALS AND METHODS Mutagenesis and Expression of the trk-met cDNA. Amino acid substitutions were introduced into the trk-met cDNA (8) by site-directed mutagenesis using mismatched oligonucleotide primers (Clontech; ref. 23). The primers were 23-mers or 26-mers with the mutations in the center. Correct sequences in the mutant receptors were confirmed by DNA sequencing. Stable transfections of the mutant cDNAs in MDCK epithelial cells were performed as described (8). Assays for Cellular Phenotype, Cell Motility, and Morphogenesis. The cellular phenotype of MDCK cells (epithelioid or fibroblastoid) was inspected 24 hr after plating the cells on tissue culture plates. Dissociation and motility of MDCK cells was examined in a colony dissociation assay (3, 13) in the presence or absence of the indicated concentrations of SF/ HGF or nerve growth factor (NGF). Branching morphogenesis of MDCK cells was analyzed as described (5, 8). In brief, MDCK cells were cultured within collagen matrices until cyst formation occurred and then exposed to SF/HGF or NGF for 3-10 days. Recombinant SF/HGF was produced by expression of the SF/HGF cDNA (4) in the pBlueBac vector in Sf9 insect cells using the MaxBac baculovirus system (Invitrogen). A one-step purification on Heparin-Sepharose (Pharmacia) resulted in pure SF/HGF (13). Purified NGF (2.5 S) was purchased from Boehringer Mannheim. Metabolic Labeling, Immunoprecipitation, and Western Blotting. MDCK cells were labeled overnight with 0.2 mCi (1 Ci = 37 GBq) of [35S]methionine (Amersham) per ml in methionine-free DMEM/10% (vol/vol) fetal calf serum, washed with PBS, and extracted for 30 min at 4°C with RIPA-kinase lysis buffer (8). After clearing by ultracentrifugation at 100,000 x g, immunoprecipitation was carried out using a 1:200 dilution of a polyclonal rabbit antiserum directed against the C terminus of mouse met and protein A-Sepharose (Pharmacia). After washing with RIPA-kinase lysis buffer and high salt buffer, the immunoprecipitates were analyzed on an SDS/8% polyacrylamide gel, followed by fluorography at -70°C (8). For examining tyrosine autophosphorylation of the trk-met hybrid, MDCK transfectants (3 x 106 cells) were incubated for 10 min at 37°C in the presence or absence of NGF (100 ng/ml) in DMEM, 0.5% bovine serum albumin, and 7 AM phenylarsine oxide. The cells were then lysed and immunoprecipitated with anti-met antibody, followed by SDS gel electrophoresis as described above. The proteins were transferred to nitrocellulose membranes (Millipore), probed with anti-phosphotyrosine monoclonal antibody (Upstate Bio-
The met protooncogene product, which is the receptor for scatter factor/hepatocyte growth factor (SF/HGF; refs. 1 and 2), transduces pleiotropic signals that regulate motility, proliferation, and branching morphogenesis of epithelial cells (3-8). In vivo, the SF/HGF-met signaling system plays a key role in tissue regeneration (9, 10), in epithelial-mesenchymal interactions (11, 12), and in tumor progression (13-16). Recent studies addressed the question of downstream targets of the met receptor and possible signal-transduction pathways. The met receptor, when autophosphorylated in response to SF/ HGF, binds a number of substrates containing Src homology 2 domains such as phosphatidylinositol 3-kinase, Ras GTPaseactivating protein, and phospholipase C-y (17-19). A docking site of the tandemly arranged tyrosine residues 1347 and 1354 of met has recently been shown to mediate direct interactions with phosphatidylinositol 3-kinase, phospholipase C--y, pp6osrc, and GRB-2/Sos, and mutation of these two tyrosine residues (in particular Y1354) destroys the transforming capacity of the oncogenic variant tpr-met (20). Little is known, however, regarding pathways and substrates involved in the transmission of the diverse signals triggered by the met receptor tyrosine kinase. It has been shown that SF/HGF activates the protooncogene product Ras-i.e., stimulates guanine nucleotide exchange (21). To examine whether Ras is involved in mediating the dissociation and motility signal of SF/HGF, we have recently expressed in Madin-Darby canine kidney (MDCK) epithelial cells the dominant-negative N17Ras under the control of a metallothionein promoter (22). Induced expression of N17Ras prevented dissociation of the cells by SF/HGF. These data indicate that the Ras pathway is essential to mediate the motility signal of SF/HGF-met. In the present investigation we analyzed, by site-directed mutagenesis The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: SF/HGF, scatter factor/hepatocyte growth factor; NGF, nerve growth factor.
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FIG. 1. Effects of tyrosine mutations on signal transduction by the met receptor. The structure of the trk-met hybrid receptor is illustrated on the left. Tyrosine residues of the cytoplasmic domain are numbered from 1 to 16, and their positions in the mouse met receptor are indicated. The kinase domain is boxed and crosshatched. Receptor mutants with tyrosine (0) to phenylalanine (-) substitutions are shown schematically on the right. (A) Analysis of mutants with single Y -> F changes. (B) Fine analysis of mutants in the C-terminal region. (C) Fine analysis of mutants in the juxtamembrane region. (D) Analysis of combinations of juxtamembrane and C-terminal mutations. The effects of ligand-induced autophosphorylation, epithelioid (e) or fibroblastoid (f) phenotype, ligand-induced motility, and branching morphogenesis are listed. +, Identical response as in the nonmutated receptor; 1, reduced response; -, no response.
technology, Lake Placid, NY), and stained using the enhanced chemiluminescence system (ECL; Amersham).
RESULTS AND DISCUSSION Single tyrosine residues (referred to as Yl-Y16) in the juxtamembrane, catalytic, and C-terminal region of the met receptor were replaced by phenylalanine (referred to as Y1FY16F). The mutations were assembled in a trk-met chimeric receptor that consists of the ligand-binding domain of the NGF receptor and the kinase domain of met. This chimeric receptor has previously been shown to mediate met-specific signals of epithelial cells in response to NGF and allows the analysis of biological responses and signaling pathways without interference of the endogenous met receptors (8). MDCK epithelial cells expressing these mutant receptors were assayed for
autophosphorylation of the met kinase, for the phenotype of the cells, for NGF-dependent motility in tissue culture, and for branching morphogenesis in collagen gels. Substitutions of Y8 and Y9 in the catalytic site of met (tyrosine-1232 and tyrosine1233) abolished autophosphorylation and NGF-induced signaling (Fig. 1A; cf. ref. 24). Remarkably, replacement of the juxtamembrane Y2 resulted in a transition of the epithelial cells toward a constitutive fibroblastoid phenotype (Figs. 1A and 2A). Mutation of the C-terminal Y15 or Y16 strongly reduced NGF-dependent branching morphogenesis (Figs. 1A and 2B). Apparently, substitution of Y2 in the juxtamembrane region of the met receptor is a gain-of-function mutation, whereas substitutions of C-terminal tyrosine residues are loss-of-function mutations. Closer analysis of the role of the four C-terminal tyrosine residues revealed a defect in receptor signaling after combined
Table 1. Mutational analysis of amino acid residues around Y2 (tyrosine-1001) of the met receptor: Effect on the cellular phenotype Position Pheno+1 +2 +4 -4 -2 -1 0 +3 Mutation -3 type F S V Y D R A T Y1001 E e S V E D F R A T F f Y1001F S V S R E D A T F f Y100l S f* S V Y E D A T1004A R A F S V Y R1002A E D A A T F f Y S V D1000A E A R A T F f f* Y S V999A E A D R T F A V Y S998A E A D T F R A e S V Y E997A A T F D R A e Single amino acid residues around Y2 (position 0) in the trk-met receptor were replaced as described in Materials and Methods. Mutated residues are indicated in boldface italic type. Transfection of MDCK cells with receptors harboring Y1001F or Y1001S converts the epithelial cells (e) to a constitutive fibroblastoid (f) phenotype. Similar conversion is seen after substitution of T1004A (+3), R1002A (+1), and D1000A (-1), and V999A (-2) but not S998A (-3) and E997A (-4). Note that the sequence ESVDY in the platelet-derived growth factor ,B receptor plays a role in binding of nck. *The fibroblastoid phenotype was less pronounced when T1004A and V999A were expressed.
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the cellular phenotype of MDCK cells. Expression of the nonmutated trk-met (wt) and the Y16F nor NGF-induced scattering of the cells. In contrast, expression of the Y2F receptor led to a constitutive fibroblastoid phenotype of the cells. The NGF concentration was 50 ng/ml. Cell morphology was analyzed after 24 hr as described (8). (Bar = 100 ,Lm.) Con, control. (B) Effect of the Y15F and Y16F mutations on branching morphogenesis. The nonmutated trk-met receptor (wt) conferred NGF-dependent branching morphogenesis in collagen matrices in a similar manner as activation of endogenous met receptors by SF/HGF. In contrast, NGF-dependent branching morphogenesis was strongly reduced in the Y15F or Y16F mutants. MDCK cell clones were cultured in collagen gels until the formation of smooth cysts and incubated for 5 days with NGF (200 ng/ml), SF/HGF (100 ng/ml), or control medium (8). (Bar = 250 ,um.) on
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mutation of Y14 and Y15 (Fig. 1B): motility was strongly reduced and branching morphogenesis was abolished. After mutation of Y16 and/or Y15 to phenylalanine, only branching
morphogenesis was affected (Figs. 1B and 2B). Y13F slightly enforced the effect of the Y14F/Y15F mutation (data not shown). These data demonstrate that the importance of C-
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terminal tyrosine residues for ligand-dependent motility is Y15 2 Y14 > Y13 and for branching morphogenesis Y16 . Y15 > Y14. Apparently, Y16 is particularly important for mediating the morphogenic signal. Combinations of mutations of the three juxtamembrane tyrosine residues (Y1-Y3) revealed that the epithelioidfibroblastoid transition is selectively due to Y2F (Figs. 1 C and 2A). The Y2F cells formed smooth cysts at increased frequency in collagen gels (data not shown), and the addition of NGF induced branching morphogenesis. Gain-of-function was also observed when Y2 was changed to serine (Y1001S; Table 1). We also analyzed receptors with mutations located around Y2. Substitution of threonine-1004 (+3), arginine-1002 (+1), aspartic acid-1000 (-1), or valine-999 (-2) to alanine all resulted in the constitutive fibroblastoid phenotype of MDCK cells. Substitution of serine-998 (-3) or glutamic acid-997 (-4) to alanine did not influence the epithelial characteristics (Table 1). These results suggest that amino acid residues located N- and C-terminal of Y2 represent possible interaction sites for a met receptor substrate. Since the Y2F and Y15F/Y16F mutations exert opposite effects on the ability of the met receptor to induce cell motility and morphogenesis, we determined whether one type of mutation is dominant over the other. We found that cells harboring Y2F/Y16F or Y2F/Y15F double mutations in the met receptor exhibited a fibroblastoid morphology (Figs. 3 and 1D), as did the Y2F single mutant. Furthermore, ligandinduced branching morphogenesis was not impaired in contrast to the Y16F or Y15F single mutants.AInterestingly, multiple C-terminal mutations were not suppressed by Y2F. The o
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b Fio. 4. Effect of the Y -* F substitutions on the autophosphorylation of the met receptor. (A) Expression of receptors with Y2F (lane a), Y16F (lane b), and Y13/14/15F (lane c) substitutions in MDCK cells was analyzed after metabolic labeling with [35S]methionine. In lanes a-c, immunoprecipitation was performed with an antibody directed against a C-terminal peptide of met (8); in lane a*, immunoprecipitation was performed in the presence of the inhibitory peptide. (B) Autophosphorylation on tyrosine residues of mutant met receptors Y2F (lane a), Y16F (lane b), and Y13/14/15F (lane c) in cells cultured with (+) or without (-) NGF at 100 ng/ml. Immunoprecipitates were analyzed by Western blotting using anti-phosphotyrosine antibodies and enhanced chemiluminescence.
Y2F mutation is thus dominant with respect to Y15F or Y16F; i.e., the Y2F gain-of-function'mutation suppresses loss-offunction mutations in the C-terminal domain. It might be speculated that the Y2F mutation leads to constitutive tyrosine autophosphorylation of the trk-met receptor. This was not the case; phosphorylation of Y2F was dependent on ligand addition as in wild-type receptors and in receptors with C-terminal mutations (Figs. 4 and 1). The data presented here demonstrate that Y13-Y16 of the met receptor participate in a cooperative fashion in the transduction of biological signals of epithelial cells. Recently, Y14 and Y15 of met have been shown to represent tandemly arranged docking sites for a number of signaling molecules including phosphatidylinositol 3-kinase, phospholipase C-y, pp6Osrc, and GRB-2, and mutation of these residues in the tpr-met oncogene prevents transformation of fibroblasts (20). We have shown, by expressing a dominant-negative Ras, that the Ras pathway is essential for the motility response of met (22). We now demonstrate that Y14 and Y15 (tyrosine-1347, tyrosine-1354) of met mediate signals relevant for cell motility and branching morphogenesis in epithelial cells. Y16 (tyrosine-1363), to which no substrate binding can yet be assigned, is an additional site important for branching morphogenesis. While tyrosine residues Y13-Y15 are conserved in the met receptor family of tyrosine kinases (met, ron, and sea), Y16 is unique, suggesting that this residue confers met-specific signaling (25, 26). We have furthermore found that Y2 (tyrosine-1001) of met plays a strikingly different role in epithelial cells: substitution results in a constitutive fibroblastoid phenotype and rescues the loss-of-function mutations of tyrosine residues in the C-terminal region. Interestingly, Y2 is conserved in all members of the c-met protooncogene family (met, ron, sea) but is absent in the oncogenes tpr-met and v-sea (26, 27); this is in line with our finding that the presence of Y2 of met affects signaling in a negative fashion. In general, signal transduction by receptor tyrosine kinases is controlled by the interplay of phosphorylation and dephosphorylation reactions (28-30). Thus, Y2 of the met receptor might bind a tyrosine phosphatase that regulates phosphorylation of C-terminal residues
Cell Biology: Weidner et al. or of downstream targets of met. Alternatively, Y2 might be the docking site for a protein that serves as a negative regulator for signal transduction. Interestingly, the motive ESVD preceding Y2 of met is also present in the kinase insert domain of the platelet-derived growth factor f3 receptor and is involved in binding of the substrate nck (31). Detailed mutational analysis of the region around Y2 of met revealed that the residues at positions + 1, +3, -1, and -2, but not -3 and -4, are critical, thus leaving the nature of the bound substrate open. The fact that tyrosine residues with positive and negative roles are present in met may explain the observed dual function of this receptor tyrosine kinase: met mediates both oncogenic and tumor suppressing function (14, 15, 32) and epithelial-mesenchymal and mesenchymal-epithelial conversions (3, 4, 11, 14) as well as growth stimulatory and inhibitory activity (9, 10, 16, 33).
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