[CANCER RESEARCH 59, 1120–1126, March 1, 1999]

Ret-mediated Mitogenesis Requires Src Kinase Activity1

Rosa Marina Melillo, Maria Vittoria Barone, Gelsy Lupoli, Anna Maria Cirafici, Francesca Carlomagno,Roberta Visconti, Brona Matoskova, Pier Paolo Di Fiore, Giancarlo Vecchio, Alfredo Fusco, and Massimo Santoro2

Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facolta` diMedicina e Chirurgia di Napoli, Universita di Napoli “Federico II,” 80131, Naples [R. M. M., M. V. B., G. L., A. M. C., F. C., R. V., G. V., M. S.]; IEO, European Institute ofOncology, 20141, Milan [P. P. D. F., B. M.]; Istituto di Microbiologia, Universita di Bari, Bari [P. P. D. F.]; and Dipartimento di Medicina Sperimentale e Clinica, Facolta` diMedicina e Chirurgia di Catanzaro, Universita di Catanzaro [A. F.], Italy

ABSTRACT

The proto-oncogeneRET encodes a transmembrane growth neurotro-phic receptor with tyrosine kinase (TK) activity. RET mutations areassociated with several human neoplastic and nonneoplastic diseases,including thyroid papillary carcinoma, multiple endocrine neoplasia type2 syndromes, and Hirschsprung’s disease. Activation of receptor TKsresults in the binding and activation of downstream signaling proteins,among which are nonreceptor TKs of the Src family. To test the involve-ment of c-Src in Ret-mediated signaling, we measured the levels of c-Srcactivity in NIH3T3 cells coexpressing Ret and the accessory GFRa-1receptor or an epidermal growth factor receptor/Ret chimeric receptorwhen the cells were stimulated by glial cell line-derived neurotrophicfactor or epidermal growth factor, respectively. Ret stimulation resultedin the activation of c-Src. We also measured the levels of Src kinaseactivity in cell lines expressing isoforms of the Ret receptor activated bydifferent mutations. These cells showed higher Src kinase activity than thenormal counterpart. Furthermore, we show that Ret is able to associatewith the SH2 domain of Src in a phosphotyrosine-dependent fashion.Microinjection of a kinase inactive mutant of c-Src blocked Ret-mediatedmitogenic effect. These experiments demonstrate that activated Ret is ableto bind and stimulate c-Src kinase and that Src activation is essential forthe mitogenic activity of Ret.

INTRODUCTION

The RETproto-oncogene encodes a TK3 transmembrane receptor,Ret (1). Inactivating mutations ofRET are responsible for Hir-schsprung’s disease (2–5). It has been demonstrated that transforminggrowth factor-b-related neurotrophic factors, including GDNF andneurturin, stimulate TK activity of Ret. A family of GPI-linkedproteins, including GFRa-1, GFR a-2, GFR a-3, and GFRa-4,mediates high-affinity ligand binding and ligand-induced Ret phos-phorylation (6–9). Oncogenic activation ofRET can occur throughmultiple mechanisms. Gene rearrangements leading to the fusion of itsTK-encoding domain with the 59terminal region of other genes,generate theRET/PTC oncogenes in human thyroid papillary carci-nomas (10). Specific point mutations ofRETare responsible for theMEN syndrome types 2A and 2B and familial medullary thyroidcarcinoma. MEN2A mutations, which involve cysteine residues of the

extracellular domain of the protein, induce ligand-independent dimer-ization and constitutive activation of the Ret receptor. MEN2B mu-tation is a Met-9183Thr substitution, which likely activates Retthrough a conformational change of its catalytic core and throughaltering its substrate specificity. Other mutations in the TK domain ofRet have been found that are responsible for a small number ofMEN2B and familial medullary thyroid carcinoma cases (11). Acti-vated RET isoforms can transform NIH3T3 and thyroid cells andinduce the appearance of a neuronal-like phenotype in neuroectoder-mal cells (12–17).

Activation of growth factor receptors leads to the binding andactivation of several molecules that are involved in the signal trans-duction cascade (18). Among the effector proteins that bind directly orindirectly to Ret are PLC-g (19, 20), the Shc and Grb2 adaptor (21,22), the Grb7 and Grb10 proteins (23, 24), and the Enigma protein(25, 26). Ret is able to activate the ras/mitogen-activated proteinkinase pathway by phosphorylation and recruitment of the adaptorsGrb2 and Shc to the plasma membrane (21, 22). Furthermore, acti-vated Ret stimulates Jun kinases in certain cell lines (27).

The Src cytoplasmic TKs can bind to and are activated by mem-brane receptors. Most of the members of this family are expressed ina cell type-restricted fashion, whereas three of them (Src, Fyn, andYes) are ubiquitous (28). Several studies show that Src family kinasesare implicated in signal transduction mediated by transmembranereceptors, which are devoid of catalytic activity, such as T-cell recep-tor and FcgRII receptor (29). In addition, receptors with intrinsic TKactivity (EGFR, c-erbB2/Neu, PDGF receptor, and colony-stimulatingfactor-1 receptor) have been shown to associate and/or activate Src,Fyn, and Yes through the SH2 domains of Src, Fyn, and Yes (30–33).Activation of Src kinase is required for PDGF- and EGF-mediatedsignaling (34–36).

Here, we show that different cell lines expressing oncogenic formsof Ret possess high Src kinase activity levels. Ligand stimulation ofRet resulted in activation of Src. Src kinase was found to coprecipitatein vivo with activated Ret. The binding between the two molecules ismediated by Src SH2 domain because an isolated Src SH2 was able tobind to Retin vitro. Finally, a dominant negative form of c-Src wasable to inhibit the mitogenic activity of Ret when it was microinjectedin NIH3T3 and PCCl3 cells, indicating that Src (or Src-related) kinaseactivity is required for Ret-mediated mitogenic signaling.

MATERIALS AND METHODS

Cell Lines and Plasmids.NIH3T3, NIH RET/MEN2A, NIHRET/MEN2B(13), NIH RET/PTC1 ((37)), EGFR/RET (19), NIH RET (38), andv-Src-expressing fibroblasts (39) have been described previously. They were grownin DMEM (Life Technologies, Inc.) supplemented with 10% calf serum.PCCl3 and PCCl3RET/PTC1 (12) were maintained in Coon’s modified F12medium supplemented with 5% calf serum (Life Technologies, Inc.) and sixgrowth factors (thyrotropin, hydrocortisone, insulin, transferrin, somatostatin,and glycyl-histidyl-lysine; Sigma Chemical Co.). The long terminal repeatRET/MEN2A D100 was generated by recombinant PCR as described (5). Aforward primer mapping on theRETsequence upstream theBglII site (posi-tion: 2690–2710) was used (59-CCATGGGCGACCTCATCT-39). The down-

Received 5/20/98; accepted 12/31/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Associazione Italiana per la Ricerca sul Cancro, bythe Progetto Biotecnologie “5%” of the Consiglio Nazionale delle Ricerche, by EuropeanCommunity Grant BMH4-CT96-0814, and by the Project MURST “Terapie Antineoplas-tiche Innovative.” The support of Associazione Italiana per la Ricerca sul Cancro,Consiglio Nazionale delle Ricerche, European Community (BIOMED-2 Program), andthe Armenise-Harvard Foundation to P. P. D. F. is also acknowledged. M. V. B. wassupported by a scholarship from Fondazione Italiana per la Ricerca sul Cancro.

2 To whom requests for reprints should be addressed, at Centro di Endocrinologia edOncologia Sperimentale del Consiglio Nazionale delle Ricerche, Facolta di Medicina eChirurgia di Napoli “Federico II,” via Pansini 5, 80131, Naples, Italy. Telephone: 39-81-7463056; Fax: 39-81-7463037; E-mail: masantor@unina.it.

3 The abbreviations used are: TK, tyrosine kinase; GDNF, glial cell line-derivedneurotrophic factor; MEN, multiple endocrine neoplasia; PLC-g, phospholipase C-g;EGF, epidermal growth factor; EGFR, EGF receptor; PDGF, platelet-derived growthfactor; GST, glutathioneS-transferase; BrdUrd, bromodeoxyuridine; PVDF, polyvinyli-dene difluoride.

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stream primer was designed to delete the COOH-terminal residues of Ret,including Tyr-1015, Tyr-1029, and Tyr-1062 (59-ACGCGTCTAGTCTCTGC-CTCTTAA-39) and contained aMluI site for the cloning in the long terminalrepeat vector. The cell line NIH3T3RET/MEN2A D100 was obtained bycalcium phosphate coprecipitation transfection technique. Transfected cellswere subjected to selection by the addition of mycophenolic acid, as describedelsewhere (19). The levels of Ret/MEN2A D100 protein in the mass populationwere similar to those of the wild-type Ret/MEN2A-expressing cells (data notshown). The cell lines F212 T1 and F212 T23 were obtained from twomammary tumors arising inRET/PTC1 transgenic mice (40). They werecultivated in Coon’s modified F12 medium supplemented with 5% calf serum(Life Technologies, Inc). The HC11 is a normal mouse mammary epithelialcell line (41). The plasmids pSG5 Src and pSG5 Src K- were kind gifts of S.Courtneidge (Sugen, Inc., Redwood City, CA).

Src in Vitro Kinase Assay.Confluent cells were serum starved for 12–24h and, when indicated, stimulated with 100 ng of EGF or GDNF as described(19). After two washes with cold PBS, cells were lysed in a buffer containing50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) NP40, 1 mM EDTA, 50 mM

NaF, 20 mM sodium pyrophosphate, 1 mM sodium vanadate, 2 mM phenyl-methylsulfonyl fluoride, and 0.2 mg/ml each aprotinin and leupeptin. Lysateswere centrifuged at 10,0003 g for 30 min and immunoprecipitated byincubating 1 mg of total proteins with anti-Src (Ab-1; Oncogene Science)antibodies for 60 min at 4°C. Immunocomplexes were recovered by incubationwith protein G-Sepharose beads (GammaBind G Sepharose; Pharmacia) on arotating platform at 4°C for 60 min. After three washes with lysis buffer, theimmunoprecipitates were washed with kinase buffer [20 mM Tris-HCl (pH7.0)-5 mM MgCl2] and resuspended in 30 ml of kinase buffer, 10 mCi of[g-32P]ATP (.10,000 Ci/mmol, Amersham), and 10 mM cold ATP. After 30min of incubation at room temperature, the beads were washed twice with lysisbuffer, and the reaction was terminated by adding an equal volume of SDS-gelloading buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS, 5% glycerol, 0.7M2-mercaptoethanol, and 0.25% bromphenol blue]. When indicated, 5 mg ofacid-denatured enolase were added to the reaction (Boehringer MannheimBiochemicals). The samples were electrophoresed on SDS-10% polyacryl-amide gel. After the run, gels were incubated three times for 30 min in a fixingsolution (20% methanol-10% acetic acid), dried, and processed for autoradiog-raphy with phosphor screens.

Western Blot Analysis. Total cellular proteins were quantitated by amodified Bradford assay (Bio-Rad). To evaluate Src expression, equalamounts of proteins were immunoprecipitated with the anti-Src antibodies(Ab-1; Oncogene Science). Antibodies purchased from Santa Cruz were usedto evaluate p27kip1 (C-19), cyclin E (M-20), and D3 (C-16) expression.Proteins were separated by a 10% SDS polyacrylamide gel and transferredonto a PVDF membrane (Millipore). After a 60-min incubation in 1% nonfatdry milk blocker (Bio-Rad), the membrane was probed with primary antibod-ies. Detection of the immunocomplexes was obtained by an enhanced chemi-luminescence system (ECL; Amersham), following manufacturer’s instruc-tions.

Complex Formation in Vivo and in Vitro. In vivoassociation experimentswere conducted by immunoprecipitating 1 mg of total cell lysate with theanti-Src antibody. The immunocomplexes were electrophoresed on a 7.5%SDS acrylamide gel and transferred onto a PVDF membrane (Millipore),which was then incubated with rabbit polyclonal antibodies directed againstthe TK domain of the Ret (19). Proteins were then visualized by an enhancedchemiluminescence system (ECL; Amersham). Forin vitro binding assays, celllysates were incubated either with GST or with GST-SH2-Src protein (5 mg)linked to agarose beads. Following incubation for 3 h at 4°C on arotatingplatform, the complexes were washed several times in lysis buffer, resus-pended in SDS loading buffer, separated on a 7.5% SDS acrylamide gel, andimmunoblotted with the anti-Ret antibodies, as described above. The plasmidexpressing the GST-SH2-Src recombinant protein was provided by G. Superti-Furga and S. Gonfloni (EMBL, Heidelberg, Germany).

Microinjection of Cells. NIH EGFR/Ret cells were seeded on glass cov-erslips in DMEM containing 10% calf serum and grown to 60% confluence.The medium was then replaced with DMEM supplemented with 0.5% calfserum, and the cells were incubated for another 30–48 h. The purifiedplasmids were injected into cell nuclei at a concentration of 25–50 ng/ml usingan automated microinjection system (AIS; Zeiss). Six h later, cells werestimulated with EGF (Upstate Biotechnology) at 300 ng/ml to induce DNA

synthesis. BrdUrd (Sigma) was added to the culture medium to a final con-centration of 100 mM, and the labeling procedure was carried out for 18–20 h.Cells were then fixed for immunostaining. Codetection of Src and BrdUrd inthe cells was essentially performed as described previously (42, 43). In brief,cells were fixed in paraformaldehyde, permeabilized with 0.1% Triton X-100,and incubated with a primary rabbit antiserum directed against c-Src. Afterseveral washes in PBS, rhodamine-conjugated antirabbit IgG was added toidentify the Src expressing cells. A fluorescein-conjugated anti-BrdUrd mousemonoclonal antibody (Boehringer Mannheim Biochemicals) was used to detectthe fraction of cells that were in S phase. All coverslips were finally washedin PBS containing Hoechst 33258 (final concentration: 1 mg/ml; Sigma),rinsed in water, and mounted in Moviol on glass slides. The fluorescent signalwas visualized with an epifluorescent microscope (Axiovert 2; Zeiss). BrdUrdincorporation was measured in injected and uninjected cells, stimulated or notwith EGF. In each experiment, at least 60 Src K1 cells were counted andcompared to 400 nonmicroinjected cells from the same coverslip. The resultsshown are the means of three independent experiments. Variations were,25%of the mean. Microinjection ofRET/PTC1-expressing NIH3T3 and PCCl3RET/PTC1 was performed as described above with a few modifications.SrcK2 and SrcK1plasmids were microinjected in proliferating cells main-tained in DMEM or in Coon’s modified F12 medium, respectively, supple-mented with 5% calf serum (Life Technologies, Inc.). The day after, BrdUrdwas added for 1 h. The cells were then fixed, permeabilized, and stained bothfor Src expression and BrdUrd incorporation as described above. BrdUrdincorporation was evaluated in the injected and uninjected cells. In eachexperiment, at least 100 Src K1 cells were counted and compared to 400nonmicroinjected control cells from the same coverslip. The results shown arethe mean of two independent experiments.

RESULTS

Activation of Src in Cell Lines Expressing Inducible or Onco-genic RET Forms. The GDNF is a functional ligand for Ret. GDNFbinds to GFRa-1, a GPI-linked cell surface molecule, which, in turn,activates Ret. We have shown that NIH3T3 coexpressing Ret andGFR a-1 undergo DNA replication upon GDNF triggering (38). Toevaluate whether stimulation of Ret with its physiological ligand wasable to induce Src activation, Src kinase was immunoprecipitatedusing specific antibodies from unstimulated or GDNF-treated NIH-Ret cells and anin vitro kinase assay was performed. As shown in Fig.1A, GDNF triggering resulted in Src activation. We have previouslyshown that an EGFR/Ret chimeric receptor, when exogenously ex-pressed in NIH3T3 cells, is able to transduce mitogenic and trans-forming signals upon EGF triggering (19). To determine whetherc-Src was involved in Ret signaling, Src kinase was immunoprecipi-tated using specific antibodies from quiescent wild-type and EGFR/Ret-expressing NIH3T3 cells (NIH EGFR/Ret), stimulated or not withEGF. Both Src autophosphorylation and its ability to phosphorylate anexogenous substrate, enolase, were measured. As shown in Fig. 1B,EGF stimulation was able to activate Src in NIH EGFR/Ret but not inthe parental cells, which express negligible levels of endogenousEGFR (19). Immunoprecipitation of the same lysates with a preim-mune serum gave negative results. Subsequently, time course evalu-ation of Ret and Src activation was performed in NIH EGFR/Ret cells(Fig. 1C). Cells were treated with EGF and harvested at differenttimes during stimulation. Src (Fig. 1C, top) and Ret (Fig. 1C, bottom)activation were measured. Ret tyrosine phosphorylation reached apeak at 5 min and started decreasing at 15 min when Src reached itsmaximal activation. Furthermore, we studied molecular markers ofS-phase entry upon EGF triggering of NIH EGFR/Ret cells. In par-ticular, we quantitated the amounts of a cyclin-dependent kinaseinhibitor, p27kip1, cyclin D3, and cyclin E. As shown in Fig. 1D,p27kip1 levels were high in quiescent cells, decreased after 6 h ofstimulation with EGF, and remained low thereafter. Cyclin D3 accu-mulation peaked at 90 min, remained stable until 12 h, and then

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decreased. Cyclin E levels peaked at 12 h and were low after 24 h.These kinetics indicated that Ret-mediated activation of Src kinaseprecedes the early G1 events required for cell cycle progression.

To test whether different oncogenic forms ofRET activate Srckinase, endogenous c-Src activity was measured in immunocomplexkinase assays in serum-starved NIH3T3 cells expressingRET/MEN2A(Cys634Tyr), RET/MEN2B(Met918Thr) (13), andRET/PTC1(H4-Ret) (37). Positive control fibroblasts expressing v-Srcwere used. Autophosphorylation of c-Src was higher in NIH3T3 cellsexpressing the activated Ret isoforms than in parental cells (;10-fold;Fig. 2A). The increase in Src autophosphorylation activity was paral-leled by an analogous increase in the phosphorylation of the exoge-nous substrate enolase (data not shown). As shown by the immunoblotreported in Fig. 2A, bottom, this effect was not due to increasedexpression levels of the endogenous Src kinase, which were, indeed,comparable in all of the cell lines tested. We have reported previouslythat the exogenous expression ofRET/PTC1 in a rat thyroid cell line,PCCl3, has a mitogenic effect (12). To evaluate whether Src activa-tion occurred also inRET/PTC1 transformed PCCl3 (PCCl3RET/PTC1), Src kinase activity was measured. Src autophosphorylationwas higher in PCCl3RET/PTC1 cells than in untransfected parentalcells (Fig. 2B,top). The expression levels of endogenous Src werecomparable in the cell lines tested, as shown by the immunoblotreported in Fig. 2B, bottom. PCCl3 cells expressing a constitutivelyactive v-Src oncogene were used as a positive control (39). Transgenicmice expressing theRET/PTC1 oncogene under the transcriptionalcontrol of the H4 gene promoter were generated in our laboratory.Despite the fact that theRET/PTC1 transgene was expressed inseveral tissues, the animals developed a restricted pattern of neopla-

sias: mammary adenocarcinomas and adnexial tumors (40). Two celllines, F212T1 and F212T23, derived from two independent mammaryadenocarcinomas that occurred inRET/PTC1 transgenics, were usedto investigate ifRET/PTC1 tumor inductionin vivo correlated with anincrease in endogenous c-Src activity. Levels of c-Src activity in theF212T1 and F212T23 cells were evaluated and compared to those ofnormal mammary cells and mammary tissue from nontransgeniclittermates. As shown in Fig. 2C, c-Src autophosphorylation washigher in the tumor-derived cell lines than in normal mammary cells(HC11 cells) and in normal mammary tissue, and this event was notdue to increased expression levels of endogenous c-Src, as demon-strated by Western blot analysis.

Association between c-Src and Activated Ret.Some TK recep-tors have been shown to directly interact with and activate membersof the Src family (28). To test if this was also the case of Ret, equalamounts of proteins were immunoprecipitated with anti c-Src anti-bodies from quiescent NIH EGFR/Ret cells stimulated or not withEGF. Immunocomplexes were then separated on a denaturing poly-acrylamide gel, transferred to a PVDF membrane, and probed withantibodies directed against the COOH-terminal portion of the Retprotein (19). Association between c-Src and Ret was readily detect-able (Fig. 3A). A preimmune serum was not able to coprecipitate theRet protein from stimulated cells (Fig. 3A). Src associated only withtyrosine-phosphorylated Ret because unstimulated Ret was not copre-cipitated by the anti-Src antibody (Fig. 3A). The requirement of Retphosphorylation for association with Src, suggested that this interac-tion was mediated by the Src SH2 domain. To test this hypothesis, weused a GST-SH2-Src (GST-SH2-Src) recombinant protein. Fig. 3Bshows that the GST-SH2-Src, but not the GST alone, was able to

Fig. 1. A, Src activation by GDNF. Serum-starved NIH3T3 cells coexpressing wild-type Retand GFRa-1 were (NIH RET1) or were not (NIHRET2) stimulated with GDNF for 10 min, and Srckinase activity was measured. GDNF triggering ofparental NIH3T3 cells gave negative results (datanot shown).B, Src kinase assay in parental NIH3T3and in NIH EGFR/Ret cells. Serum-starved cellswere stimulated with EGF for 10 min, lysates wereimmunoprecipitated with anti-Src antibodies, andkinase assays were performed. The Src kinase (toparrow) and the substrate enolase (bottom arrow) areindicated. These results are typical and representa-tive of at least three independent experiments.C,time course of Ret-mediated Src activation in NIHEGFR/Ret cells. Serum-starved cells were stimu-lated with EGF and harvested at the indicated timepoints. Top, Src kinase assays were performed asdescribed above. Laser densitometry was used toquantitate Src autokinase activity after stimulation.Columns, relative inductions, with the levels of Srcactivation calculated as fold increases above theactivity of unstimulated NIH EGFR/RET cells(5 1), means of three experiments;bars, SD.Bot-tom, tyrosine phosphorylation of EGFR/Ret inducedby EGF is shown. Five hundredmg of total lysateswere immunoprecipitated with anti-Ret antibodies.After protein transfer, filters were immunoblottedwith anti-pTyr antibodies. Equal amounts of Ret arepresent in all of the lanes (data not shown).D, EGFstimulation of NIH EGFR/RET cells induces mo-lecular markers of S-phase entry. Serum-starvedcells were stimulated with EGF and assayed at theindicated times for cyclin D3, cyclin E, and p27kip1

expression.

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associate to activated Retin vitro. As expected, unstimulated Ret wasunable to bind to Src SH2 (Fig. 3B).

The Ret COOH-terminal tail contains three of these autophospho-rylation sites: Tyr-1062, Tyr-1029, and Tyr-1015. Tyr-1062 is the

binding site for Shc (44–46), whereas Tyr-1015 is the docking site forPLC-g (14, 20). On the basis of the optimal consensus sequence forSrc SH2 binding (47), Tyr-1015 (YLDL) and, less likely, Tyr-1029(YDDG) could represent candidates for Src binding. We then inves-tigated whether these tyrosines were involved in Src binding andactivation by using the mutant Ret/MEN2AD100. In this mutant, thelast three tyrosine residues of Ret/MEN2A have been deleted. Asexpected, this mutant was unable to bind to Shc and PLC-g (data notshown). Ret/MEN2AD100 was still able to coimmunoprecipitate Src(Fig. 4A), bind to GST-SH2-Src (Fig. 4B), and activate Src kinase(Fig. 4C).

Dominant Negative Src Inhibits Ret-mediated S-Phase Entry.To investigate whether c-Src activity was required for Ret-mediatedmitogenesis, we applied a microinjection technique. Serum-starvedNIH EGFR/Ret cells, when stimulated with EGF, enter S phase, asmeasured by thymidine incorporation (19). Serum-deprived NIHEGFR/Ret cells were stimulated or not with EGF in the presence ofBrdUrd, and S-phase entry was measured by counting cells stainedwith anti-BrdUrd-specific antibodies. Arrested NIH EGFR/Ret cellsshowed only a very low level of BrdUrd incorporation. Upon stimu-

Fig. 2.A, Src kinase activity in NIH3T3 cells expressing different activated forms ofRET.Cell lysates from NIH3T3 cells and NIH3T3 cells expressingv-Srcor activatedRETonco-genes were prepared, and Src kinase was immunoprecipitated. Half of the immunoprecipitateswere subjected to kinase assays (top), and half were subjected to Western blot analysis(bottom), as indicated (arrows). B, Src kinase activity in thyroid cells. Parental PCCl3 cellswere compared toRET/PTC1 and v-Src expressing cells for Src kinase activity. Src kinaseassays and immunoblots were performed as described above.C, Src kinase activity inRET/PTC1-induced mammary tumors. Src kinase activity was evaluated in cell lines derivedfrom two independent mammary adenocarcinomas explanted fromRET/PTC1 transgenicanimals,F212T1andF212T23, in a normal mouse mammary epithelial cell line (HC11) andin a normal mouse mammary gland (N), derived from three independent nontransgeniclittermates (one is shown; the other two gave consistent results). Src kinase assays andimmunoblots were performed as described above.Arrows, positions of Src kinase detected byeither kinase assay (top arrow) or Western blot analysis (bottom arrow).

Fig. 3. A, in vivo association of activated Ret proteins and Src kinase. The cells were(NIH EGFR/RET1) or were not (NIH EGFR/RET2) stimulated with EGF and harvested.Their lysates were immunoprecipitated either with preimmune rabbit serum(Lane 3,leftto right) or with anti-Src antibody(Lanes 1and2) and subjected to immunoblotting withanti-Ret antibody.Arrow, the migration of the EGFR/Ret protein.B, in vitro binding ofactivated Ret proteins to the SH2 domain of Src. Lysates from unstimulated (NIHEGFR/RET2) and EGF stimulated (NIH EGFR/RET1) NIH EGFR/Ret cells wereincubated either with GST-SH2-Src(Lanes 1and2, left to right) or with GST proteinsimmobilized on glutathione-agarose beads(Lanes 3and4). Proteins were separated on aSDS-7.5% polyacrylamide gel under reducing conditions and subjected to immunoblot-ting with anti-Ret antibodies. These results are typical and representative of at least threeindependent experiments.

Fig. 4. A, in vivo binding of Ret/MEN2AD100 mutant to Src kinase. NIH cellsexpressingRET/MEN2A and the mutantRET/MEN2A D100 were harvested. Proteinswere immunoprecipitated with anti-Src antibody and immunoblotted with anti-Ret anti-bodies.B, in vitro binding of RET/MEN2A D100 mutant to Src kinase. Cell lysates ofNIH3T3 cells expressingRET/MEN2A andRET/MEN2A D100 were incubated withpurified GST-SH2-Src protein. Bound proteins were eluted with SDS sample buffer,subjected to electrophoresis in a SDS 7.5% polyacrylamide gel, and immunoblotted withanti-Ret antibody.Arrow, the migration of the receptor.C, Src kinase activity in NIH3T3cells expressing theRET/MEN2A D100 mutant. Cells expressingRET/MEN2A andRET/MEN2AD100 were harvested, and cellular proteins were immunoprecipitated withthe anti-Src antibody. The immunoprecipitates were subjected to either kinase assays (top)or Western blot analysis (bottom) as described in “Materials and Methods.”

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lation with EGF, almost 50% of the cells entered S phase (Fig. 5A).Arrested NIH EGFR/Ret cells were microinjected with a dominantnegative form of Src (Src K2; Ref. (43). After 6 h, they werestimulated with EGF for 18 h. The average results of three independ-ent experiments, in which at least 60 Src K2 microinjected cells werecounted, are shown in Fig. 5A. The entrance into S phase wasinhibited by Src K2; only 8% of injected cells incorporated BrdUrd.This effect was specific because serum-induced BrdUrd incorporationwas not affected by the expression of Src K2 (data not shown).Moreover, microinjection of a plasmid expressing wild-type Src (SrcK1) did not affect the mitogenic response to EGF. An example ofthese assays is reported in Fig. 5B. Cells expressing Src K2wereidentified by immunostaining with anti-Src (Src). These cells did notincorporate BrdUrd, whereas surrounding cells did, as shown by thestaining with anti-BrdUrd antibody (Fig. 4B, BrdU). These experi-ments strongly support the concept that Src kinase activity is requiredfor Ret-induced mitogenic response.

To investigate the involvement of Src in mitogenic signaling me-

diated by oncogenic Ret proteins, we used NIH3T3 and PCCl3 cellsexpressing the Ret/PTC1 oncoprotein. These experiments were car-ried on asynchronously growing cells: cells were microinjected witheither Src K2or Src K1 plasmids, and BrdUrd incorporation wasmeasured. As shown in Fig. 6, 50% of the cells entered S phase innormal growing conditions. Microinjection of Src K2 but not Src K1strongly inhibited BrdUrd incorporation of both cell types. Microin-jection of Src K2 in asynchronously growing parental NIH3T3 andPCCl3 did not affect the fraction of cells that entered S phase.

DISCUSSION

Here, we showed that Ret triggering is able to stimulate the activityof pp60 c-Src. A 2–3-fold increase of c-Src activity following Rettriggering was reproducibly measured; this is consistent with theincreases observed for activated PDGFR, colony-stimulating factor-1receptor, and erbB2/Neu (30–33). We also show that, in NIH3T3,thyroid and mammary cell lines stably expressing activated forms ofRET, levels of c-Src kinase activity are elevated in comparison towild-type cells. The induction of c-Src activity correlated with theability of Src to interact with tyrosine-phosphorylated Ret, as shownby coimmunoprecipitation experiments. Activated Ret was also ableto bind c-Src in vitro, and the SH2 domain of c-Src alone wassufficient for this association.

Time-course experiments indicated that Src activation follows thepeak of Ret activation. Moreover, Ret-dependent Src activation occursbefore the first events of G0-G1 transition. This is consistent with thepossibility that Src activation may be necessary for the induction ofRet-mediated cell cyle progression. Indeed, in PDGF-stimulatedNIH3T3 fibroblasts, the block of Src activation inhibits cyclin Eaccumulation.4 Accordingly, a dominant inhibitory form of c-Src wasable to inhibit the mitogenic activity of Ret when it was microinjected

4 M. V. Baroneet al., unpublished observations.

Fig. 5. A, quantitation of BrdUrd incorporationin NIH EGFR/Ret cells injected with Src K2 andSrc K1. Quiescent NIH EGFR/Ret cells seeded oncoverslips were microinjected with an expressionplasmid encoding Src K2. Six h later, cells wereincubated in media containing EGF and BrdUrd.After 18–20 h, they were fixed, stained, and pro-cessed for immunofluorescence as described. Asshown, a decreased fraction of the cells injectedwith Src K2 but not with Src K1 incorporatedBrdUrd after stimulation with EGF. In each exper-iment, at least 60 Src K1cells were counted andcompared to at least 400 nonmicroinjected cellsfrom the same coverslip.Columns, mean results ofthree experiments;bars, SD. B, a representativeexperiment is shown. Injected cells are visualizedwith anti-Src antibody (Src) and rhodamine-conju-gated secondary antibodies. BrdUrd incorporationwas visualized with a fluorescein-conjugated anti-BrdUrd monoclonal antibody (BrdU). Cell nucleiare stained with Hoechst dye (Hoechst).

Fig. 6. Quantitation of BrdUrd incorporation in NIHRET/PTC1 and PCRET/PTC1cells injected with Src K2 and Src K1. Asynchronously growing cells were injected withSrc K2 and Src K1 plasmids and processed for immunofluorescence as described. Inthese conditions, Src K2 but not Src K1 inhibited BrdUrd incorporation.

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in EGF-stimulated NIH EGFR/Ret cells and of Ret/PTC1 in NIH3T3and PCCl3 cells.

The SrcK2 mutant used in our study is probably also active onother members of Src kinase family because its SH2 domain couldtheoretically compete with the binding to the receptor mediated bySH2 domains of closely related Src-like kinases. For this reason, wecannot exclude that the activity of other Src-like kinases may berequired for Ret-mediated biological activity. In support of this hy-pothesis, we have observed that the EGFR/Ret chimera is also anefficient inducer of the c-Fyn kinase.5 Similarly, in addition to Src,other Src-like kinases are required for PDGF- and EGF-mediatedsignaling in NIH3T3 fibroblasts. Indeed, microinjection of antibodiesdirected against Src and Fyn or plasmids expressing dominant nega-tive Src and Fyn inhibited PDGF-induced S-phase entry in NIH3T3cells. On the other side, microinjection of Src dominant-negativeplasmids or of neutralizing antibodies does not interfere aspecificallywith cell functions necessary for S-phase entry. Indeed, serum stim-ulation of NIH3T3 cells is unaffected by the block of Src kinaseactivity (data not shown). In fact, only some growth factors requirefunctional Src family kinases to transmit mitogenic responses in thesecells: lysophosphatidic acid- and bombesin-induced DNA synthesis,for instance, is unaffected by anti-Src-neutralizing antibodies (48, 49).

Deletions or substitutions of specific tyrosine residues in the intra-cellular domain of Ret have been performed. Substitution of Tyr-1062with phenylalanine has been shown to severely impair mitogenic andtransforming activity ofRET/PTC2,RET/MEN2A, andRET/MEN2Bby decreasing the ability of Ret to associate with Shc adaptor proteinand to activate the ras/mitogen-activated protein kinase pathway6 (45,46). Replacement of Tyr-1015, identified as the binding site forPLC-g, with phenylalanine, reduced oncogenic activity of Ret/PTC2(20, 50). We show here that a deletion mutant, Ret/MEN2AD100, inwhich these tyrosines have been abolished, retains its ability to bindand activate c-Src. Thus, tyrosines of the COOH-terminal tail are notinvolved in binding Src, and the ability of Ret to bind Src can beseparated from its ability to associate with PLC-g and Shc. Othertyrosine residues mapping either in the catalytic core or in the jux-tamembrane domain of Ret have been demonstrated to be phospho-rylated and/or relevant for Ret-mediated mitogenesis as Y687, Y826,Y900, and Y905 (23, 24, 44, 50); their potential role in mediatingSrc-binding needs to be explored.

Elevated levels of c-Src activity have been detected in many typesof human tumors, including carcinomas of the breast and colon,whereas normal tissues have low activity (51–55). Furthermore, Srckinase levels are significantly increased in highly metastatic cell linesin comparison to primary tumors. It has been reported that, in thesetumors, receptor TKs participate to activation of c-Src above basallevels. In breast cancer, c-Src is found in association with c-erbB2/Neu, and it has been proposed that this activation might be linked tothe development of the metastatic phenotype (32, 56). Our datademonstrate thatRET/PTC1-induced mammary tumors possess anelevated c-Src activity. Similarities between Ret and c-erbB2/Neuhave been reported in previous studies; for instance, the EGFR/Retchimera and an activated erbB2/Neu kinase displayed a poor ability toinduce survival and proliferation of the 32D hematopoietic cells anda strong transforming ability in NIH3T3 cells (19, 57, 58). Our datastrengthen the hypothesis that Ret and c-erbB2 couple with similarpathways and that one of this pathways involves the activation ofc-Src.

ACKNOWLEDGMENTS

We thank Nina A. Dathan for her early contribution to the expression ofRET/MEN2A mutants and generation of cell lines, Giovanni Santelli for hishelp, and Giulio Superti-Furga and Stefania Gonfloni for providing the GST-SH2-Src plasmid. We also thank F. D’agnello and M. Berardone for theartwork and F. Sferratore for excellent technical assistance.

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