Different point mutations in the met oncogene elicitdistinct biological properties
S. GIORDANO,1 A. MAFFE, T. A. WILLIAMS, S. ARTIGIANI, P. GUAL, A. BARDELLI,C. BASILICO, P. MICHIELI, AND P. M. COMOGLIOUniversity of Torino, School of Medicine, Institute for Cancer Research and Treatment (IRCC),10060 Candiolo, Italy
ABSTRACT The MET proto-oncogene, encodingthe tyrosine kinase receptor for HGF, controls ge-netic programs leading to cell growth, invasiveness,and protection from apoptosis. Recently, MET mu-tations have been identified in hereditary and spo-radic forms of papillary renal carcinoma (PRC).Introduction of different naturally occurring muta-tions into the MET cDNA results in the acquisition ofdistinct biochemical and biological properties oftransfected cells. Some mutations result in a highincrease in tyrosine kinase activity and confer trans-forming ability in focus forming assays. These mu-tants hyperactivate the Ras signaling pathway. Othermutations are devoid of transforming potential butare effective in inducing protection from apoptosisand sustaining anchorage-independent growth.These Met PRC receptors interact more efficientlywith the intracellular transducer Pi3Kinase. The re-ported results show that MET PRC mutations can beresponsible for malignant transformation throughdifferent mechanisms, either by increasing thegrowth ability of cells or by protecting cells fromapoptosis and allowing accumulation of other ge-netic lesions.—Giordano, S., Maffe, A., Williams,T. A., Artigiani, S., Gual, P., Bardelli, A., Basilico, C.,Michieli, P., Comoglio, P. M. Different point muta-tions in the met oncogene elicit distinct biologicalproperties. FASEB J. 14, 401–408 (2000)
Key Words: tyrosine kinase receptor z mutations z invasivegrowth z branching morphogenesis
The MET proto-oncogene encodes the tyrosinekinase receptor for hepatocyte growth factor/scatterfactor (HGF/SF) (1, 2). Receptor activation triggersa unique process of differentiation called ‘branchingmorphogenesis’ that involves the promotion of cellgrowth, protection from apoptosis, control of celldissociation, and migration into extracellular matri-ces (3, 4). In transformed epithelia, deregulatedactivation of the MET proto-oncogene can mediateinvasive growth, a feature of neoplastic progressionin which cancer cells invade surrounding tissues andpenetrate vascular walls, eventually leading to sys-temic metastases (5–10).
The role of MET in human tumors has been welldocumented. Previous studies from this and otherlaboratories have shown that the MET oncogene isoverexpressed in tumors of specific histotypes, in-cluding thyroid (11) and pancreatic carcinomas(12), or is activated through autocrine mechanisms(13, 14). Moreover, the MET gene is amplified inliver metastases of colorectal carcinomas (15). Re-cently, a genetic connection between MET and he-reditary papillary renal carcinoma (HPRC) estab-lished a direct role for this receptor in human cancer(16). Sequencing the MET gene from affected mem-bers of HPRC families and from tumor samples ofpatients with sporadic papillary carcinoma identifiednine different mutations (referred to as Met PRC
mutations) that result in amino acid substitutions inthe kinase domain of the receptor. Three of thesemutations (D1228N, D1228H, and M1250T) arelocated in codons homologous to those mutated inthe tyrosine kinase receptors Kit and Ret. Mutated Kitalleles are found in patients with mastocytosis andacute myeloid leukemia of M2 subtype (17, 18) andmissense mutations in Ret are associated with multi-ple endocrine neoplasia type 2B (MEN2B) (19). Thissuggests that alteration of these residues is a criticalevent in deregulating tyrosine kinase receptors.
HGF binding to Met up-regulates the tyrosinekinase (2, 20) and results in phosphorylation of aunique docking site located in the Met carboxyl-terminal tail, which contains the sequence Y1349VHV-Y1356VNV (21). The two phosphorylated tyrosineswithin this sequence couple the receptor to multipleintracellular effectors, among which are the Grb2/SOS complex, the p85 regulatory subunit of PI-3-kinase, Stat-3, src, and the multiadaptor proteinGab1 (21–28). The mechanism by which mutatedMet drives neoplastic transformation has been shownto involve constitutive receptor coupling to down-stream signal transducers as the substitution of these‘docking tyrosines’ with phenylalanines abrogates
1 Correspondence: Institute for Cancer Research, Depart-ment of Molecular Oncology, University of Torino MedicalSchool, Strada Provinciale 142, Km 3.95, 10060 Candiolo(Torino), Italy. E-mail: [email protected]
transformation (21). Recent studies have shown thatsome of the PRC mutations increase the kinaseactivity and confer transforming ability (29). How-ever, in all cases, MET-mediated transformation re-quires an intact docking site (30).
All of the MET PRC mutations segregate with thedisease; however, only some of them display trans-forming potential in vitro and nothing is knownabout the biological properties of the nontransform-ing MET PRC mutations. The present study investi-gates the different mechanisms linking MET PRC
mutations to the development of human tumors.
MATERIALS AND METHODS
Reagents, antibodies, and cell culture
All reagents used were from Fluka (FlukaChemie AG, Buchs,Switzerland) and Sigma (Sigma Chemicals Co., St. Louis,Mo.). Reagents for sodium dodecyl sulfate-polyacrylamide gelelctrophoresis were from Bio-Rad (Bio-Rad Laboratories,Hercules, Calif.). Recombinant HGF was obtained from Bac-ulovirus-infected Sf9 cells (1 scatter unit 5 0.2 ng). Antiphos-photyrosine antibodies were purchased from UBI. The Metprotein was immunoprecipitated with a monoclonal antibody(DQ13) (31) and detected by Western blotting with poly-clonal antibodies (C-28, Santa Cruz Biotechnology, SantaCruz, Calif.). COS-7 cells were purchased from ATCC (Amer-ican Type Culture Collection, Rockville, Md.). Cultures ofmammalian cells were maintained in DMEM supplementedwith 10% fetal calf serum (FCS) in a humidified atmosphereof 5% CO2-air. MLP29 is an epithelial cell line derived fromliver oval cells (32).
Plasmid constructs
Reproduction of PRC mutations in the human Met cDNA wasmediated 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 subclonedinto the pCEV29.1 expression vector, which carries the resis-tance to G418 (34), and into the pMT2 expression vector.
Transfections and transformation assays
To establish stable transfectants, MLP 29 were transfected bythe calcium phosphate method using 40 mg of carrier DNA(calf thymus high molecular weight DNA; Boehringer Mann-heim, Mannheim, Germany) per 100 mm plate. Each platewas transfected with 2 mg of the pCEV29.1 vector containingeither no insert or a cDNA encoding Met (wild-type ormutant). Selection of stable transfectants was performed inDMEM containing 10% FBS and 750 mg/ml G418. Clonesresistant to G418 were pooled and used for Western blotanalysis and biological assays.
Two days after transfection, cells were split into four platesfor transformation assays: three plates were cultured inDMEM containing 5% FCS and used for measuring focusformation; the fourth was cultured in DMEM containing 10%FCS and the selective drug G418 (750 mg/ml) to establishstable transfectants. Foci were scored 2 wk after transfectionfollowing fixation with p-formaldehyde and Giemsa staining.
For transient transfections, cDNAs cloned in the pMT2
vector were transfected in COS-7 cells by the calcium phos-phate method.
Precipitation experiments
GST fusion proteins were kindly donated by Dr. P. P. DiFiore.GST fused to the amino-terminal SH2 domain of the p85subunit of Pi3Kinase (;500 ng/point) was coupled to gluta-thione-Sepharose beads. Lysates from COS-7 cells (one con-fluent 10 cm dish, lysis conditions as described in ref 21)transfected with wild-type or mutant MET PRC cDNAs wereincubated with the immobilized SH2-GSTs for 90 min at 4°Cin the presence of 1 mM sodium o-vanadate. The beads werewashed and proteins were eluted with boiling Laemmli bufferbefore Western blot analysis. Specific detection of proteins byantibodies was visualized by chemiluminescence (ECL1Plusdetection system, Amersham. Little Chalfont, U.K.).
Soft agar and branching morphogenesis assays
For analysis of colony formation in soft agar, MLP 29 cellswere diluted to a concentration of 25,000 cells/ml in DMEMcontaining 10% FCS, 0.5% Seaplaque agar with or withoutadded HGF (200 scatter units/ml). Cells were seeded in6-well plates (2 ml/well) or 24-well plates (0.5 ml/well)containing a 1% agar underlay and supplemented withDMEM containing 10% FCS three times a week (HGF, 200scatter units/ml). Colonies were scored 2 wk after seeding.
For evaluation of branching morphogenesis, cells werecultured in collagen as described previously (32).
In vitro motility assay
105 cells were seeded on the upper side of a Transwellchamber on a porous polycarbonate membrane (8.0 mM poresize); the lower chamber of the Transwell was filled withDMEM containing 2% FCS in presence of 100 units/ml ofrecombinant Baculovirus-produced human HGF. After 24 hof incubation, cells attached to the upper side of the filterwere mechanically removed; cells that migrated to the lowerside of the filter were fixed, stained with Toluidine blue, andcounted (10 microscopic fields/sample).
In vitro invasion assay
105 cells were seeded on the upper side of a porous polycar-bonate membrane (8.0 mM pore size) coated with the artifi-cial basement membrane Matrigel (12.5 mg per filter; Collab-orative Biomedical Products; Becton Dickinson Labware,Waltham, Mass.). The lower chamber of the Transwell wasfilled with DMEM containing 2% FCS in the presence of 100units/ml of recombinant Baculovirus-produced human HGF.After 24 h of incubation, the filters were removed and cellsthat invaded the Matrigel and attached to the lower chamberof the transwell were fixed with glutheraldeyde, stained withcrystal violet, and photographed.
Apoptosis assays
10,000 cells were plated in each well of a 96-well Costarmicrotiter plate in either the presence or absence of 100U/ml HGF. Apoptosis was induced with 100 nM Staurosporinfor 12 h. Anoikis assay was performed as described previously(35). Apoptosis detection was monitored by TUNEL reaction(Boehringer). The fluorescence labeling was converted into acolorimetric signal for analysis by light microscopy using
400 Vol. 14 February 2000 GIORDANO ET AL.The FASEB Journal
TUNEL AP (Boehringer). Cells positive for the reaction werescored under the microscope.
Luciferase assay
NIH 3T3 cells were transfected with 1 mg of fos luciferasereporter plasmid and 300 ng of the different Met constructseither in the presence or absence of pRSV-Ras N17 (36). After24 h, the cells were harvested in 200 ml luciferase-lysis buffercontaining 25 mM glycylglycinezNaOH, pH 7.8, 1 mM DTT,15% glycerol, 8 mM MgSO4, 1 mM EDTA, and 1% TritonX-100. Total protein (10 mg) was transferred to microtiterplates and luciferase activity was measured in a luminometerafter injecting 100 ml of luciferin solution containing 25 mMglycylglycinezNaOH, pH 7.8, 10 mM MgSO4, 1.5 mM ATP,and 330 mM luciferin.
RESULTS
Transforming ability of MET PRC mutantscorrelates with activation of the Ras pathway
We and others have previously shown that METreceptors containing the PRC point mutations (Fig.1A) display different abilities to induce transforma-tion in NIH 3T3 cells (29, 30). Only mutations thatalter residues located in the kinase activation loopefficiently transform mouse fibroblasts. Further-
more, the MET PRC mutant with the highest trans-forming ability (MET M1250T) also displays thehighest catalytic activity.
Activation of the Ras pathway is critical for MET-induced transformation (21, 37, 38). To evaluate theability of MET PRC mutants to activate this pathway,we performed a transient transfection assay in NIH3T3 fibroblasts using a luciferase expression system.A luciferase construct containing a fos-responsivepromoter was induced 8- and 7-fold by coexpressionof the transforming mutants MET M1250T andMET D1228H, respectively (Fig. 1B). This increasewas strongly suppressed by a cotransfected dominantnegative mutant of Ras (Ras N17), indicating thatthe increase in luciferase activity is strictly dependenton 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 Raspathway.
MET PRC mutants mediate motility in epithelialcells
The Met receptor is expressed in cells of ectodermalorigin, where it elicits different biological responses
Figure 1. Transforming ability of MET PRC mutants correlates with activation of the Ras pathway. A) Map of MET mutationsfound in Papillary renal carcinomas. Schematic representation of functional domains of MET tyrosine kinase. The black boxdepicts 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 (seetext for further details). Mutations found in PRCs are listed and the homology with residues mutated in RET and KIT receptorsare indicated. B) Upper part: Effects of MET PRC mutants on the induction of a fos promoter in NIH 3T3 cells. Cells weretransiently cotransfected with 1 mg 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 activitywas measured as described in Materials and Methods. The experiments were performed in triplicates and mean values areindicated. Each experiment was performed three times with similar results. Lower part: Transforming ability of MET PRC mutantsevaluated using the focus formation assay. Values reported represent the average of three independent experiments.
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such as cell proliferation, protection from apoptosis,and invasion of surrounding extracellular matrices.To determine whether cells expressing MET PRC mu-tants show an increase in some of these properties,we transfected the human MET cDNAs harboringthe different PRC mutations into MLP 29 murineepithelial liver oval cells. These cells display a fullspectrum of biological responses on HGF treatment(39). The selected stable cell lines express compara-ble amounts of exogenous receptor, which is consti-tutively tyrosine phosphorylated (Fig. 2A). As ex-pected, tyrosine phosphorylation can be furtherincreased by stimulation with HGF (data not shown).
Cells expressing MET PRC mutants display a ‘scat-tered’ phenotype, disassembling the tightly packedislands and inducing cell detachment and migration(Fig. 2B). The scattering response can be furtherstimulated by the addition of HGF and this responseis enhanced compared to that observed in MOCKtransfected cells or in cells expressing normal hu-man MET (data not shown).
The increased motility displayed by MLP 29 cellsexpressing MET PRC mutants was quantified by aBoyden chamber assay. After HGF stimulation, cellsexpressing MET PRC mutants exhibited increased mi-gration, compared to MOCK or cells expressing WTMET (Fig. 2C).
MET PRC mutants induce ‘invasive growth’
The morphogenetic response induced by Met inepithelial cells is unique for this receptor and cannotbe elicited by other tyrosine kinases (40). Underphysiological conditions, the coordinated activationof multiple signaling pathways underlying this inva-sive growth leads to the formation of tubular struc-tures by epithelial organs, a response termedbranched morphogenesis (41), which plays an essen-tial role during embryogenesis. Deregulated activa-tion of the invasive-growth phenotype by the METoncogene confers transformed and metastatic prop-erties to cells (37).
MLP 29 cells are a sensitive target for signalscontrolling polarized growth. When stimulated byHGF, they migrate in tridimensional collagen gelsand form long and branched tubules. MLP 29 cellsexpressing MET L1195V and MET Y1230C mutants(which are only weakly transforming in a focusforming assay) showed the best morphogenetic re-sponse, developing many long tubular structures(Fig. 3A). Moreover, in the absence of HGF, thesecells developed typical cystic structures with out-wardly projecting spikes, not observed in MOCKtransfected cells. Conversely, MET M1250T andMET D1228H mutants, which display the highesttransforming potential in vitro, develop shorter,mainly unbranched tubules.
To evaluate whether cells expressing MET PRC mu-tants display an invasive phenotype, we tested theirability to invade in vitro reconstituted basement mem-branes (Matrigel). As shown in Fig. 3B, cells expressingMET L1195V and MET Y1230C mutants, which alsoinduced branched morphogenesis, were effective ininvading the reconstituted basal membrane. Since ithas been shown that Pi3Kinase plays a critical role in
Figure 2. MET PRC mutants mediate motility in epithelial cells.A) MLP 29 oval liver cells were transfected with either wildtype or MET PRC mutants and stable cell lines were generated.The amount of Met protein and the level of phosphorylationwere evaluated by immunoprecipitation with antibodies di-rected against human Met, followed by Western blotting withthe indicated antibody. Pr190Met 5 Met precursor, p145Met 5mature Met. B) Morphology of MLP 29 cells expressingvarious MET PRC mutants. Cells stably transfected with thedifferent constructs were cultured in growth media for 2 daysand photographed at a magnification of 3200. C) Motility ofMLP 29 cells expressing MET PRC mutants in Transwell cham-bers. Cells were plated in the upper chamber; the lowerchamber was filled with medium supplemented with 2% FCSin the presence of 100 U/ml of recombinant Baculovirus-produced human HGF. After 24 h incubation, cells thatattached to the upper side of the filter were mechanicallyremoved; cells that migrated to the lower side of the filterwere fixed, stained, and counted. Experiments were per-formed in triplicate and 10 microscopic fields were countedfor each sample.
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invasion and tubulogenesis (42), we evaluated theability of MET PRC mutants to interact with the p85subunit of Pi3Kinase. Pull-down experiments of theMET PRC mutants with the GST protein fused to theamino-terminal SH2 domain of p85 showed thatMET L1195V and MET Y1230C mutants, displaying thebest invasive and tubulogenic ability, interact very effi-ciently with Pi3Kinase (Fig. 3C). This was specificallyobserved with the SH2 domain of Pi3Kinase, becauseadditional pull-down experiments with GST-fusionproteins comprising the SH2 domains of Src, PLCg,and Shc did not reveal any differential binding tothe mutants (data not shown). The decreased invasiveand morphogenetic ability of MET M1250T andMET D1228H mutants, which also interact withPi3Kinase, albeit at a lower level than MET L1195V andMET Y1230C, could be due to an unbalanced activa-tion of the Ras and Pi3Kinase pathways as sustainedactivation of the Ras pathway causes disorganizedgrowth resulting in reduced invasion and tubulogen-esis (42).
MET PRC mutants induce protection fromapoptosis and anchorage-independent growth
Protection from apoptosis is a critical step duringtumor progression. Cells that are resistant to apopto-tic stimuli can undergo multiple lesions that wouldotherwise lead to cell death. Some of these lesions,affecting genes involved in promoting tumor pro-gression, could thus be stabilized.
To evaluate how cells expressing MET PRC mu-tants respond to apoptotic stimuli, we treated thecells with the apoptosis-inducing drug stauro-sporin. As shown in Fig. 4A, cells expressingMET L1195V and MET Y1230C mutants display anincreased resistance to apoptosis, which is furtherenhanced by HGF stimulation. Similar results werealso obtained when cells were tested for theyability to overcome anoikis (data not shown).Notably, the increased resistance to apoptosis is
Figure 3. MET PRC mutants induce ‘invasive growth’. A) Branching morphogenesis induced by MET PRC mutants was evaluatedby measuring the sprouting and migration into a tridimensional network of collagen type I. 5000 cells of each stably transfectedcell line were seeded in collagen gels and grown for 5 days either in the absence or in the presence of 100 U/ml of recombinantBaculovirus-produced human HGF. Micrographs of representative fields were taken after 5 days. B) Invasive potential of cellsexpressing MET PRC mutants. The ability of cells to invade the reconstituted basal membrane was measured by plating cells onTranswell filters coated with Matrigel. Cells migrated into the lower chamber were fixed with glutheraldeyde, stained with crystalviolet, and photographed. C) Left part: GST fusion proteins with the N-SH2 domain of the p85 subunit of Pi3Kinase wereimmobilized 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-Metantibodies. Pr190Met 5 Met precursor, p145Met 5 mature Met. Right part: Comparable amounts of proteins used for pull-downexperiments were loaded on a gel and probed with anti-Met antibodies.
403BIOLOGICAL PROPERTIES OF METPRC MUTANTS
shown by mutants, which display a low transform-ing potential in vitro.
MLP 29 cells expressing MET PRC mutants were alsoassayed for their ability to form colonies in soft agareither in the absence or presence of HGF. As shown inFig. 4B, control cells formed very few small colonies inthe absence or presence of HGF. Cells expressingMET PRC mutants displayed dramatically increased an-chorage-independent growth potential and formednumerous large colonies in the presence of HGF. Themost effective mutants were MET L1195V and MET-Y1230C, which were also more resistant to apoptosis.
DISCUSSION
Identification of the MET PRC mutations provideddirect evidence of an involvement of this tyrosinekinase receptor in human cancer. Although manymutations have been identified in receptor tyrosinekinases, the biological properties displayed by thesemutants have not been studied in detail. As such, it isnot known whether these mutations simply inducean enhancement of the normal behavior of thereceptor or whether they lead to the acquisition orloss of biological properties. In this respect, a quan-titative increase in signal transduction could not onlyresult in a corresponding increased response, butalso lead to the activation of different signal path-ways.
In this study, we show that the MET PRC mutationscan be broadly divided into two groups based on theirbiological properties. Mutants belonging to one group,including MET M1250T and MET D1228H, display in-creased tyrosine kinase activity, stimulate efficiently theRas pathway, and transform recipient cells in focusforming assays. Conversely, the other group of mu-tations, including MET L1195V and MET Y1230C,are almost devoid of in vitro transforming potential butare effective in inducing protection from apoptosis,sustaining anchorage-independent growth, and pro-moting invasion. The transforming mutations of thefirst group are characterized biochemically by prefer-ential activation of the Ras pathway, whereas the sec-ond group is characterized by a more efficient interac-tion with the intracellular transducer Pi3Kinase, whoserole in invasive growth and protection from apoptosis iswell known. We also show that the mutated forms ofMET PRC remain responsive to HGF and the biologicalproperties induced by these mutants are better re-vealed in the presence of the ligand.
The pleiotropic activity of Met results from theconcomitant activation of several signal pathways(37). It is known that Ras activation is strictly re-quired and is sufficient for growth and transforma-tion, but not for anchorage-independent growth andinvasion. In contrast, Pi3Kinase activation alone doesnot suffice for these same responses (43). A balancebetween the activation of Ras and Pi3Kinase is man-datory for anchorage-independent and invasivegrowth (42). This has been shown in the case of anactivated form of Met that preferentially couples toGrb2 and activates the Ras pathway (37). This mu-tant is highly efficient in transformation but is devoidof invasive properties; however, when one of the twoGrb2 binding sites is replaced by a Pi3Kinase bindingsite, the resulting mutant displays both transformingand invasive potential (43). Similarly, a natural ex-ample is provided by the activated form of c-sea, areceptor belonging to the same subfamily of recep-tors as Met. Activated c-sea, which contains a doubleGrb2 binding site, transforms but does not invade,
Figure 4. MET PRC mutants induce protection from apoptosisand anchorage-independent growth. A) Stable transfectantswere tested for their ability to overcome apoptosis. Cells weretreated with 100 nM Staurosporin for 12 h in the absence(gray bars) or presence (black bars) of Baculovirus-producedhuman HGF (200 scatter units/ml). Cell viability was mea-sured using TUNEL assay. Cells positive for the reaction werescored under the microscope. Experiments were performedin triplicate and 10 microscopic fields were counted for eachsample. B) Stable transfectants were tested for their ability toform colonies in soft agar either in the absence (gray bars) orpresence (black bars) of Baculovirus-produced human HGF(200 scatter units/ml). Cells were photographed 3 wk afterseeding (lower part) and colonies were counted (upper part).
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whereas its viral counterpart v-sea, which contains apoint mutation introducing a Pi3Kinase binding site,is effective in both transformation and invasion (37).
Several hypotheses could account for the differen-tial ability of the MET mutants to activate thePi3Kinase pathway. First, some mutations could re-sult in the preferential phosphorylation of Y1349 ofthe docking site compared to that of Y1356. Thiscould increase the binding of Pi3Kinase, since Y1349lacks the consensus sequence for Grb2 and wouldprevent competition with Grb2, whose SH2 domaindisplays an affinity for Y1356 one log higher than thep85 SH2 domain of Pi3Kinase. Alternatively, a con-formational change induced by mutations couldresult in an alternative phosphorylation of Gab1:preferential phosphorylation of Gab1 tyrosines re-sponsible for Pi3Kinase binding could thereforeincrease Met-mediated activation of this pathway.Moreover, the increase of Pi3Kinase activation viaMet could enhance the localization of Gab1 to themembrane and its further recruitment by Met (44). Ithas been shown, in fact, that lipid products ofPi3Kinase enhance the membrane localization ofGab1 by interacting with Gab1 PH domain (44).
In summary, this is the first report highlightingdistinct biological properties elicited by differentpoint mutations in a tyrosine kinase oncogene. Insome instances, oncogenic mutations can directlyconfer a growth advantage by mainly activating theRas pathway; other mutations activate different intra-cellular signaling pathways, such as Pi3Kinase, result-ing in protection from apoptosis and invasivegrowth. It could be speculated that in the latter casethe affected oncogene may not be directly responsi-ble for transformation, but may allow accumulationof mutations in other genes, which in turn areresponsible for transformation. Since the Ret and Kitoncogenes are also activated in human cancersthrough similar molecular mechanisms, the re-ported findings are possibly not unique to Met andcould provide a general mechanism by which ty-rosine kinase receptors are involved in humanmalignancies.
We are grateful to Dr. Bos for the fos luciferase and theRSV-ras-N17 plasmids and to Dr. DiFiore for GST fusionproteins. We thank Dr. Longati for subcloning Met mutantsin the pMT2 vector; Drs. Crepaldi, Tamagnone, and Di Renzofor helpful discussion; A. Cignetto for secretarial help, and E.Wright for editing the manuscript. The skilled technicalassistance of Mrs. Raffaella Albano, Mrs. Laura Palmas, andMrs. 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 forCancer Research (AIRC), Armenise Harvard Foundation forAdvanced Scientific Research, European Commission (EC)grant no. BMH4-CT98–3852 to P.M.C., National ResearchCouncil (CNR) grant no. 98.00430.CT04 to P.M.C., andMURST Cofin 98 grant N.38 to S.G.
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Received for publication May 6, 1999.Revised for publication August 2, 1999.
406 Vol. 14 February 2000 GIORDANO ET AL.The FASEB Journal
