Different point mutations in the met oncogene elicit distinct biological properties S. GIORDANO,1 A. MAFFE, T. A. WILLIAMS, S. ARTIGIANI, P. GUAL, A. BARDELLI, C. BASILICO, P. MICHIELI, AND P. M. COMOGLIO University of Torino, School of Medicine, Institute for Cancer Research and Treatment (IRCC), 10060 Candiolo, Italy ABSTRACT The MET proto-oncogene, encoding the tyrosine kinase receptor for HGF, controls genetic programs leading to cell growth, invasiveness, and protection from apoptosis. Recently, MET mutations have been identified in hereditary and sporadic forms of papillary renal carcinoma (PRC). Introduction of different naturally occurring mutations into the MET cDNA results in the acquisition of distinct biochemical and biological properties of transfected cells. Some mutations result in a high increase in tyrosine kinase activity and confer transforming ability in focus forming assays. These mutants hyperactivate the Ras signaling pathway. Other mutations are devoid of transforming potential but are effective in inducing protection from apoptosis and sustaining anchorage-independent growth. These Met PRC receptors interact more efficiently with the intracellular transducer Pi3Kinase. The reported results show that MET PRC mutations can be responsible for malignant transformation through different mechanisms, either by increasing the growth ability of cells or by protecting cells from apoptosis and allowing accumulation of other genetic lesions.—Giordano, S., Maffe, A., Williams, T. A., Artigiani, S., Gual, P., Bardelli, A., Basilico, C., Michieli, P., Comoglio, P. M. Different point mutations in the met oncogene elicit distinct biological properties. FASEB J. 14, 401– 408 (2000)
Key Words: tyrosine kinase receptor 䡠 mutations 䡠 invasive growth 䡠 branching morphogenesis
The MET proto-oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF) (1, 2). Receptor activation triggers a unique process of differentiation called ‘branching morphogenesis’ that involves the promotion of cell growth, protection from apoptosis, control of cell dissociation, and migration into extracellular matrices (3, 4). In transformed epithelia, deregulated activation of the MET proto-oncogene can mediate invasive growth, a feature of neoplastic progression in which cancer cells invade surrounding tissues and penetrate vascular walls, eventually leading to systemic metastases (5–10). 0892-6638/00/0014-0399/$02.25 © FASEB
The role of MET in human tumors has been well documented. Previous studies from this and other laboratories have shown that the MET oncogene is overexpressed in tumors of specific histotypes, including thyroid (11) and pancreatic carcinomas (12), or is activated through autocrine mechanisms (13, 14). Moreover, the MET gene is amplified in liver metastases of colorectal carcinomas (15). Recently, a genetic connection between MET and hereditary papillary renal carcinoma (HPRC) established a direct role for this receptor in human cancer (16). Sequencing the MET gene from affected members of HPRC families and from tumor samples of patients with sporadic papillary carcinoma identified nine different mutations (referred to as Met PRC mutations) that result in amino acid substitutions in the kinase domain of the receptor. Three of these mutations (D1228N, D1228H, and M1250T) are located in codons homologous to those mutated in the tyrosine kinase receptors Kit and Ret. Mutated Kit alleles are found in patients with mastocytosis and acute myeloid leukemia of M2 subtype (17, 18) and missense mutations in Ret are associated with multiple endocrine neoplasia type 2B (MEN2B) (19). This suggests that alteration of these residues is a critical event in deregulating tyrosine kinase receptors. HGF binding to Met up-regulates the tyrosine kinase (2, 20) and results in phosphorylation of a unique docking site located in the Met carboxylterminal tail, which contains the sequence Y1349VHVY1356VNV (21). The two phosphorylated tyrosines within this sequence couple the receptor to multiple intracellular effectors, among which are the Grb2/ SOS complex, the p85 regulatory subunit of PI-3kinase, Stat-3, src, and the multiadaptor protein Gab1 (21–28). The mechanism by which mutated Met drives neoplastic transformation has been shown to involve constitutive receptor coupling to downstream signal transducers as the substitution of these ‘docking tyrosines’ with phenylalanines abrogates 1 Correspondence: Institute for Cancer Research, Department of Molecular Oncology, University of Torino Medical School, Strada Provinciale 142, Km 3.95, 10060 Candiolo (Torino), Italy. E-mail:
[email protected]
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transformation (21). Recent studies have shown that some of the PRC mutations increase the kinase activity and confer transforming ability (29). However, in all cases, MET-mediated transformation requires an intact docking site (30). All of the MET PRC mutations segregate with the disease; however, only some of them display transforming potential in vitro and nothing is known about the biological properties of the nontransforming MET PRC mutations. The present study investigates the different mechanisms linking MET PRC mutations to the development of human tumors.
MATERIALS AND METHODS
All reagents used were from Fluka (FlukaChemie AG, Buchs, Switzerland) and Sigma (Sigma Chemicals Co., St. Louis, Mo.). Reagents for sodium dodecyl sulfate-polyacrylamide gel elctrophoresis were from Bio-Rad (Bio-Rad Laboratories, Hercules, Calif.). Recombinant HGF was obtained from Baculovirus-infected Sf9 cells (1 scatter unit ⫽ 0.2 ng). Antiphosphotyrosine antibodies were purchased from UBI. The Met protein was immunoprecipitated with a monoclonal antibody (DQ13) (31) and detected by Western blotting with polyclonal antibodies (C-28, Santa Cruz Biotechnology, Santa Cruz, Calif.). COS-7 cells were purchased from ATCC (American Type Culture Collection, Rockville, Md.). Cultures of mammalian cells were maintained in DMEM supplemented with 10% fetal calf serum (FCS) in a humidified atmosphere of 5% CO2-air. MLP29 is an epithelial cell line derived from liver oval cells (32). Plasmid constructs Reproduction of PRC mutations in the human Met cDNA was mediated by polymerase chain reaction as described (30). Human Met residues are numbered according to Gene Bank # X54559 (33) Wild-type or mutant Met cDNA was subcloned into the pCEV29.1 expression vector, which carries the resistance to G418 (34), and into the pMT2 expression vector. Transfections and transformation assays To establish stable transfectants, MLP 29 were transfected by the calcium phosphate method using 40 mg of carrier DNA (calf thymus high molecular weight DNA; Boehringer Mannheim, Mannheim, Germany) per 100 mm plate. Each plate was transfected with 2 mg of the pCEV29.1 vector containing either no insert or a cDNA encoding Met (wild-type or mutant). Selection of stable transfectants was performed in DMEM containing 10% FBS and 750 g/ml G418. Clones resistant to G418 were pooled and used for Western blot analysis and biological assays. Two days after transfection, cells were split into four plates for transformation assays: three plates were cultured in DMEM containing 5% FCS and used for measuring focus formation; the fourth was cultured in DMEM containing 10% FCS and the selective drug G418 (750 g/ml) to establish stable transfectants. Foci were scored 2 wk after transfection following fixation with p-formaldehyde and Giemsa staining. For transient transfections, cDNAs cloned in the pMT2 Vol. 14
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Precipitation experiments GST fusion proteins were kindly donated by Dr. P. P. DiFiore. GST fused to the amino-terminal SH2 domain of the p85 subunit of Pi3Kinase (⬃500 ng/point) was coupled to glutathione-Sepharose beads. Lysates from COS-7 cells (one confluent 10 cm dish, lysis conditions as described in ref 21) transfected with wild-type or mutant MET PRC cDNAs were incubated with the immobilized SH2-GSTs for 90 min at 4°C in the presence of 1 mM sodium o-vanadate. The beads were washed and proteins were eluted with boiling Laemmli buffer before Western blot analysis. Specific detection of proteins by antibodies was visualized by chemiluminescence (ECL⫹Plus detection system, Amersham. Little Chalfont, U.K.). Soft agar and branching morphogenesis assays
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vector were transfected in COS-7 cells by the calcium phosphate method.
For analysis of colony formation in soft agar, MLP 29 cells were diluted to a concentration of 25,000 cells/ml in DMEM containing 10% FCS, 0.5% Seaplaque agar with or without added HGF (200 scatter units/ml). Cells were seeded in 6-well plates (2 ml/well) or 24-well plates (0.5 ml/well) containing a 1% agar underlay and supplemented with DMEM containing 10% FCS three times a week (HGF, 200 scatter units/ml). Colonies were scored 2 wk after seeding. For evaluation of branching morphogenesis, cells were cultured in collagen as described previously (32). In vitro motility assay 105 cells were seeded on the upper side of a Transwell chamber on a porous polycarbonate membrane (8.0 M pore size); the lower chamber of the Transwell was filled with DMEM containing 2% FCS in presence of 100 units/ml of recombinant Baculovirus-produced human HGF. After 24 h of incubation, cells attached to the upper side of the filter were mechanically removed; cells that migrated to the lower side of the filter were fixed, stained with Toluidine blue, and counted (10 microscopic fields/sample). In vitro invasion assay 105 cells were seeded on the upper side of a porous polycarbonate membrane (8.0 M pore size) coated with the artificial basement membrane Matrigel (12.5 g per filter; Collaborative Biomedical Products; Becton Dickinson Labware, Waltham, Mass.). The lower chamber of the Transwell was filled with DMEM containing 2% FCS in the presence of 100 units/ml of recombinant Baculovirus-produced human HGF. After 24 h of incubation, the filters were removed and cells that invaded the Matrigel and attached to the lower chamber of the transwell were fixed with glutheraldeyde, stained with crystal violet, and photographed. Apoptosis assays 10,000 cells were plated in each well of a 96-well Costar microtiter plate in either the presence or absence of 100 U/ml HGF. Apoptosis was induced with 100 nM Staurosporin for 12 h. Anoikis assay was performed as described previously (35). Apoptosis detection was monitored by TUNEL reaction (Boehringer). The fluorescence labeling was converted into a colorimetric signal for analysis by light microscopy using
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Figure 1. Transforming ability of MET PRC mutants correlates with activation of the Ras pathway. A) Map of MET mutations found in Papillary renal carcinomas. Schematic representation of functional domains of MET tyrosine kinase. The black box depicts the tyrosine kinase domain (KD), which can be subdivided into amino- and carboxyl-terminal lobes (N-L and C-L, respectively) separated by a large cleft referred to as the activation loop (AL). YY represents the multifunctional docking site (see text for further details). Mutations found in PRCs are listed and the homology with residues mutated in RET and KIT receptors are indicated. B) Upper part: Effects of MET PRC mutants on the induction of a fos promoter in NIH 3T3 cells. Cells were transiently cotransfected with 1 g fos luciferase reporter plasmid and 300 ng of pCEV containing the different MET PRC mutants either in the presence (dashed bars) or in the absence (open bars) of dominant negative Ras N17. Luciferase activity was measured as described in Materials and Methods. The experiments were performed in triplicates and mean values are indicated. Each experiment was performed three times with similar results. Lower part: Transforming ability of MET PRC mutants evaluated using the focus formation assay. Values reported represent the average of three independent experiments. TUNEL AP (Boehringer). Cells positive for the reaction were scored under the microscope. Luciferase assay NIH 3T3 cells were transfected with 1 mg of fos luciferase reporter plasmid and 300 ng of the different Met constructs either in the presence or absence of pRSV-Ras N17 (36). After 24 h, the cells were harvested in 200 ml luciferase-lysis buffer containing 25 mM glycylglycine䡠NaOH, pH 7.8, 1 mM DTT, 15% glycerol, 8 mM MgSO4, 1 mM EDTA, and 1% Triton X-100. Total protein (10 mg) was transferred to microtiter plates and luciferase activity was measured in a luminometer after injecting 100 ml of luciferin solution containing 25 mM glycylglycine䡠NaOH, pH 7.8, 10 mM MgSO4, 1.5 mM ATP, and 330 mM luciferin.
RESULTS Transforming ability of MET PRC mutants correlates with activation of the Ras pathway We and others have previously shown that MET receptors containing the PRC point mutations (Fig. 1A) display different abilities to induce transformation in NIH 3T3 cells (29, 30). Only mutations that alter residues located in the kinase activation loop efficiently transform mouse fibroblasts. FurtherBIOLOGICAL PROPERTIES OF METPRC MUTANTS
more, the MET PRC mutant with the highest transforming ability (MET M1250T) also displays the highest catalytic activity. Activation of the Ras pathway is critical for METinduced transformation (21, 37, 38). To evaluate the ability of MET PRC mutants to activate this pathway, we performed a transient transfection assay in NIH 3T3 fibroblasts using a luciferase expression system. A luciferase construct containing a fos-responsive promoter was induced 8- and 7-fold by coexpression of the transforming mutants MET M1250T and MET D1228H, respectively (Fig. 1B). This increase was strongly suppressed by a cotransfected dominant negative mutant of Ras (Ras N17), indicating that the increase in luciferase activity is strictly dependent on activation of the Ras pathway. In the same assay, MET PRC mutants MET L1195V and MET Y1230C, which are almost devoid of transforming ability, induce the luciferase construct to a lower degree, displaying a 60% reduced ability to activate the Ras pathway. MET PRC mutants mediate motility in epithelial cells The Met receptor is expressed in cells of ectodermal origin, where it elicits different biological responses 401
such as cell proliferation, protection from apoptosis, and invasion of surrounding extracellular matrices. To determine whether cells expressing MET PRC mutants show an increase in some of these properties, we transfected the human MET cDNAs harboring the different PRC mutations into MLP 29 murine epithelial liver oval cells. These cells display a full spectrum of biological responses on HGF treatment (39). The selected stable cell lines express comparable amounts of exogenous receptor, which is constitutively tyrosine phosphorylated (Fig. 2A). As expected, tyrosine phosphorylation can be further increased by stimulation with HGF (data not shown). Cells expressing MET PRC mutants display a ‘scattered’ phenotype, disassembling the tightly packed islands and inducing cell detachment and migration (Fig. 2B). The scattering response can be further stimulated by the addition of HGF and this response is enhanced compared to that observed in MOCK transfected cells or in cells expressing normal human MET (data not shown). The increased motility displayed by MLP 29 cells expressing MET PRC mutants was quantified by a Boyden chamber assay. After HGF stimulation, cells expressing MET PRC mutants exhibited increased migration, compared to MOCK or cells expressing WT MET (Fig. 2C). MET PRC mutants induce ‘invasive growth’ The morphogenetic response induced by Met in epithelial cells is unique for this receptor and cannot be elicited by other tyrosine kinases (40). Under physiological conditions, the coordinated activation of multiple signaling pathways underlying this invasive growth leads to the formation of tubular structures by epithelial organs, a response termed branched morphogenesis (41), which plays an essential role during embryogenesis. Deregulated activation of the invasive-growth phenotype by the MET oncogene confers transformed and metastatic properties to cells (37). MLP 29 cells are a sensitive target for signals controlling polarized growth. When stimulated by HGF, they migrate in tridimensional collagen gels and form long and branched tubules. MLP 29 cells expressing MET L1195V and MET Y1230C mutants (which are only weakly transforming in a focus forming assay) showed the best morphogenetic response, developing many long tubular structures (Fig. 3A). Moreover, in the absence of HGF, these cells developed typical cystic structures with outwardly projecting spikes, not observed in MOCK transfected cells. Conversely, MET M1250T and MET D1228H mutants, which display the highest transforming potential in vitro, develop shorter, mainly unbranched tubules. 402
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Figure 2. MET PRC mutants mediate motility in epithelial cells. A) MLP 29 oval liver cells were transfected with either wild type or MET PRC mutants and stable cell lines were generated. The amount of Met protein and the level of phosphorylation were evaluated by immunoprecipitation with antibodies directed against human Met, followed by Western blotting with the indicated antibody. Pr190Met ⫽ Met precursor, p145Met ⫽ mature Met. B) Morphology of MLP 29 cells expressing various MET PRC mutants. Cells stably transfected with the different constructs were cultured in growth media for 2 days and photographed at a magnification of ⫻200. C) Motility of MLP 29 cells expressing MET PRC mutants in Transwell chambers. Cells were plated in the upper chamber; the lower chamber was filled with medium supplemented with 2% FCS in the presence of 100 U/ml of recombinant Baculovirusproduced human HGF. After 24 h incubation, cells that attached to the upper side of the filter were mechanically removed; cells that migrated to the lower side of the filter were fixed, stained, and counted. Experiments were performed in triplicate and 10 microscopic fields were counted for each sample.
To evaluate whether cells expressing MET PRC mutants display an invasive phenotype, we tested their ability to invade in vitro reconstituted basement membranes (Matrigel). As shown in Fig. 3B, cells expressing MET L1195V and MET Y1230C mutants, which also induced branched morphogenesis, were effective in invading the reconstituted basal membrane. Since it has been shown that Pi3Kinase plays a critical role in
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Figure 3. MET PRC mutants induce ‘invasive growth’. A) Branching morphogenesis induced by MET PRC mutants was evaluated by measuring the sprouting and migration into a tridimensional network of collagen type I. 5000 cells of each stably transfected cell line were seeded in collagen gels and grown for 5 days either in the absence or in the presence of 100 U/ml of recombinant Baculovirus-produced human HGF. Micrographs of representative fields were taken after 5 days. B) Invasive potential of cells expressing MET PRC mutants. The ability of cells to invade the reconstituted basal membrane was measured by plating cells on Transwell filters coated with Matrigel. Cells migrated into the lower chamber were fixed with glutheraldeyde, stained with crystal violet, and photographed. C) Left part: GST fusion proteins with the N-SH2 domain of the p85 subunit of Pi3Kinase were immobilized on glutathione-Sepharose and incubated with lysates of COS7 cells transiently expressing MET PRC mutants. Complexes were washed and bound Met was eluted with boiling Laemmli buffer. Western blot was probed with anti-Met antibodies. Pr190Met ⫽ Met precursor, p145Met ⫽ mature Met. Right part: Comparable amounts of proteins used for pull-down experiments were loaded on a gel and probed with anti-Met antibodies.
invasion and tubulogenesis (42), we evaluated the ability of MET PRC mutants to interact with the p85 subunit of Pi3Kinase. Pull-down experiments of the MET PRC mutants with the GST protein fused to the amino-terminal SH2 domain of p85 showed that MET L1195V and MET Y1230C mutants, displaying the best invasive and tubulogenic ability, interact very efficiently with Pi3Kinase (Fig. 3C). This was specifically observed with the SH2 domain of Pi3Kinase, because additional pull-down experiments with GST-fusion proteins comprising the SH2 domains of Src, PLC␥, and Shc did not reveal any differential binding to the mutants (data not shown). The decreased invasive and morphogenetic ability of MET M1250T and MET D1228H mutants, which also interact with Pi3Kinase, albeit at a lower level than MET L1195V and MET Y1230C, could be due to an unbalanced activation of the Ras and Pi3Kinase pathways as sustained activation of the Ras pathway causes disorganized growth resulting in reduced invasion and tubulogenesis (42). BIOLOGICAL PROPERTIES OF METPRC MUTANTS
MET PRC mutants induce protection from apoptosis and anchorage-independent growth
Protection from apoptosis is a critical step during tumor progression. Cells that are resistant to apoptotic stimuli can undergo multiple lesions that would otherwise lead to cell death. Some of these lesions, affecting genes involved in promoting tumor progression, could thus be stabilized. To evaluate how cells expressing MET PRC mutants respond to apoptotic stimuli, we treated the cells with the apoptosis-inducing drug staurosporin. As shown in Fig. 4A, cells expressing MET L1195V and MET Y1230C mutants display an increased resistance to apoptosis, which is further enhanced by HGF stimulation. Similar results were also obtained when cells were tested for they ability to overcome anoikis (data not shown). Notably, the increased resistance to apoptosis is 403
DISCUSSION
Figure 4. MET PRC mutants induce protection from apoptosis and anchorage-independent growth. A) Stable transfectants were tested for their ability to overcome apoptosis. Cells were treated with 100 nM Staurosporin for 12 h in the absence (gray bars) or presence (black bars) of Baculovirus-produced human HGF (200 scatter units/ml). Cell viability was measured using TUNEL assay. Cells positive for the reaction were scored under the microscope. Experiments were performed in triplicate and 10 microscopic fields were counted for each sample. B) Stable transfectants were tested for their ability to form colonies in soft agar either in the absence (gray bars) or presence (black bars) of Baculovirus-produced human HGF (200 scatter units/ml). Cells were photographed 3 wk after seeding (lower part) and colonies were counted (upper part).
shown by mutants, which display a low transforming potential in vitro. MLP 29 cells expressing MET PRC mutants were also assayed for their ability to form colonies in soft agar either in the absence or presence of HGF. As shown in Fig. 4B, control cells formed very few small colonies in the absence or presence of HGF. Cells expressing MET PRC mutants displayed dramatically increased anchorage-independent growth potential and formed numerous large colonies in the presence of HGF. The most effective mutants were MET L1195V and METY1230C, which were also more resistant to apoptosis. 404
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Identification of the MET PRC mutations provided direct evidence of an involvement of this tyrosine kinase receptor in human cancer. Although many mutations have been identified in receptor tyrosine kinases, the biological properties displayed by these mutants have not been studied in detail. As such, it is not known whether these mutations simply induce an enhancement of the normal behavior of the receptor or whether they lead to the acquisition or loss of biological properties. In this respect, a quantitative increase in signal transduction could not only result in a corresponding increased response, but also lead to the activation of different signal pathways. In this study, we show that the MET PRC mutations can be broadly divided into two groups based on their biological properties. Mutants belonging to one group, including MET M1250T and MET D1228H, display increased tyrosine kinase activity, stimulate efficiently the Ras pathway, and transform recipient cells in focus forming assays. Conversely, the other group of mutations, including MET L1195V and MET Y1230C, are almost devoid of in vitro transforming potential but are effective in inducing protection from apoptosis, sustaining anchorage-independent growth, and promoting invasion. The transforming mutations of the first group are characterized biochemically by preferential activation of the Ras pathway, whereas the second group is characterized by a more efficient interaction with the intracellular transducer Pi3Kinase, whose role in invasive growth and protection from apoptosis is well known. We also show that the mutated forms of MET PRC remain responsive to HGF and the biological properties induced by these mutants are better revealed in the presence of the ligand. The pleiotropic activity of Met results from the concomitant activation of several signal pathways (37). It is known that Ras activation is strictly required and is sufficient for growth and transformation, but not for anchorage-independent growth and invasion. In contrast, Pi3Kinase activation alone does not suffice for these same responses (43). A balance between the activation of Ras and Pi3Kinase is mandatory for anchorage-independent and invasive growth (42). This has been shown in the case of an activated form of Met that preferentially couples to Grb2 and activates the Ras pathway (37). This mutant is highly efficient in transformation but is devoid of invasive properties; however, when one of the two Grb2 binding sites is replaced by a Pi3Kinase binding site, the resulting mutant displays both transforming and invasive potential (43). Similarly, a natural example is provided by the activated form of c-sea, a receptor belonging to the same subfamily of receptors as Met. Activated c-sea, which contains a double Grb2 binding site, transforms but does not invade,
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whereas its viral counterpart v-sea, which contains a point mutation introducing a Pi3Kinase binding site, is effective in both transformation and invasion (37). Several hypotheses could account for the differential ability of the MET mutants to activate the Pi3Kinase pathway. First, some mutations could result in the preferential phosphorylation of Y1349 of the docking site compared to that of Y1356. This could increase the binding of Pi3Kinase, since Y1349 lacks the consensus sequence for Grb2 and would prevent competition with Grb2, whose SH2 domain displays an affinity for Y1356 one log higher than the p85 SH2 domain of Pi3Kinase. Alternatively, a conformational change induced by mutations could result in an alternative phosphorylation of Gab1: preferential phosphorylation of Gab1 tyrosines responsible for Pi3Kinase binding could therefore increase Met-mediated activation of this pathway. Moreover, the increase of Pi3Kinase activation via Met could enhance the localization of Gab1 to the membrane and its further recruitment by Met (44). It has been shown, in fact, that lipid products of Pi3Kinase enhance the membrane localization of Gab1 by interacting with Gab1 PH domain (44). In summary, this is the first report highlighting distinct biological properties elicited by different point mutations in a tyrosine kinase oncogene. In some instances, oncogenic mutations can directly confer a growth advantage by mainly activating the Ras pathway; other mutations activate different intracellular signaling pathways, such as Pi3Kinase, resulting in protection from apoptosis and invasive growth. It could be speculated that in the latter case the affected oncogene may not be directly responsible for transformation, but may allow accumulation of mutations in other genes, which in turn are responsible for transformation. Since the Ret and Kit oncogenes are also activated in human cancers through similar molecular mechanisms, the reported findings are possibly not unique to Met and could provide a general mechanism by which tyrosine kinase receptors are involved in human malignancies. We are grateful to Dr. Bos for the fos luciferase and the RSV-ras-N17 plasmids and to Dr. DiFiore for GST fusion proteins. We thank Dr. Longati for subcloning Met mutants in the pMT2 vector; Drs. Crepaldi, Tamagnone, and Di Renzo for helpful discussion; A. Cignetto for secretarial help, and E. Wright for editing the manuscript. The skilled technical assistance of Mrs. Raffaella Albano, Mrs. Laura Palmas, and Mrs. Giovanna Petruccelli is gratefully acknowledged. T.A.W. and P.G. are both in receipt of Marie-Curie TMR fellowships. This work has been supported by Italian Association for Cancer Research (AIRC), Armenise Harvard Foundation for Advanced Scientific Research, European Commission (EC) grant no. BMH4-CT98 –3852 to P.M.C., National Research Council (CNR) grant no. 98.00430.CT04 to P.M.C., and MURST Cofin 98 grant N.38 to S.G. BIOLOGICAL PROPERTIES OF METPRC MUTANTS
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The FASEB Journal
Received for publication May 6, 1999. Revised for publication August 2, 1999.
GIORDANO ET AL.
