The Proto-Oncogene C-Cbl Is a Positive Regulator of Met-Induced MAP Kinase Activation: a Role for the Adaptor Protein Crk

SHORT REPORT The proto-oncogene c-Cbl is a positive regulator of Met-induced MAP kinase activation: a role for the adaptor protein Crk

Miguel Garcia-Guzman1, Elise Larsen1 and Kristiina Vuori*,1

1Cancer Research Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, California, CA 92037, USA

Hepatocyte growth factor triggers a complex biological (Cooper et al., 1984; Park et al., 1986; Rodrigues and program leading to invasive cell growth by activating the Park, 1993). c-Met receptor tyrosine kinase. Following activation, The cytoplasmic domain of Met contains two Met signaling is elicited via its interactions with SH2- autophosphorylation tyrosine residues, tyrosines 1349 containing proteins, or via the phosphorylation of the and 1356, which form a multi-substrate binding site for docking protein Gab1, and the subsequent interaction of several SH2-domain-containing cytoplasmic eectors Gab1 with additional SH2-containing eector molecules. and are thought to be crucial for Met-induced We have previously shown that the interaction between biological signaling (Ponzetto et al., 1994; Weidner et phosphorylated Gab1 and the adaptor protein Crk al., 1995; Zhu et al., 1994). In addition to the mediates activation of the JNK pathway downstream numerous SH2-containing molecules, the 110-kDa of Met. We report here that c-Cbl, which is a Gab1-like docking protein Gab1 also interacts with the activated docking protein, also becomes tyrosine-phosphorylated in Met receptor, either directly or indirectly via the response to Met activation and serves as a docking adaptor protein Grb2 (Fixman et al., 1997; Nguyen molecule for various SH2-containing molecules, includ- et al., 1997; Weidner et al., 1996). Gab1, which ing Crk. We further show that Cbl is similarly capable becomes tyrosine phosphorylated upon Met stimula- of enhancing Met-induced JNK activation, and several tion, contains multiple domains capable of binding to lines of experimentation suggests that it does so by signaling molecules, such as Grb2, the p85 subunit of interacting with Crk. We also show that both Cbl and PI 3-kinase, phospholipase C-g and SHP2 (see Bardelli Gab1 enhance Met-induced activation of another MAP et al., 1997b). Together, these data support a model in kinase cascade, the ERK pathway, in a Crk-independent which Met signaling could be elicited directly via its manner. Taken together, our studies demonstrate a interactions with SH2-containing proteins or establish- previously unidenti®ed functional role for Cbl in Met ment of a Met/(Grb2)/Gab1 complex and the sub- signaling and suggest that Met utilizes at least two sequent interaction of Gab1 with additional SH2- docking proteins, Gab1 and Cbl, to activate downstream containing eector molecules. signaling pathways. Oncogene (2000) 19, 4058 ± 4065. Among the diverse biochemical pathways triggered by Met is the stimulation of mitogen-activated protein Keywords: kinase; oncogenesis; signal transduction; kinases (MAPK), including c-Jun N-terminal kinases tyrosine phosphorylation (JNK). Importantly, it has been shown that JNK activation is essential for cellular transformation and induction of soft agar growth by Tpr/Met (Rodrigues et al., 1997). Recent ®ndings in our laboratory have Hepatocyte growth factor/scatter factor (HGF/SF) established that the SH2-SH3-domain containing triggers a complex biological program leading to adaptor protein c-Crk is a component in connecting invasive cell growth by activating its high-anity various cellular stimuli to JNK activation (Dol® et al., receptor, the c-Met transmembrane tyrosine kinase 1998). In a previous work, we set forth to determine (for reviews see Bardelli et al., 1997b; Vande Woude et whether a connection between the Met and the Crk al., 1997). In addition to mediating biological responses pathways might play a role in Met-induced JNK in normal cells, Met-HGF/SF signaling has been activation and cellular transformation. We found that implicated in the generation and spread of tumors tyrosine-phosphorylated Gab1 serves as a functional (Jeers et al., 1996). Most notably, missense mutations docking site for the SH2-domain of Crk, the dominant- that deregulate the enzymatic activity of Met have been negative forms of Crk block Met-induced JNK found to be associated with human papillary renal activation and that Gab1-Crk complex formation carcinomas (Fischer et al., 1998; Jeers et al., 1997a; correlates with stimulation of the JNK pathway by Schmidt et al., 1997; Zhuang et al., 1998). Consistent Met (Garcia-Guzman et al., 1999). We report here that with these ®ndings, Met was originally identi®ed as an c-Cbl, which is a proto-oncogene product and a Gab1- oncogene, Tpr/Met, which codes for a constitutively like docking protein, is also a component in Met activated Met kinase possessing transforming activity signaling pathways. Upon Met-induced phosphorylation, Cbl interacts with Crk and other SH2-domain containing molecules, connecting Met to the JNK pathway in a Crk-dependent manner. We also show *Correspondence: Kristiina Vuori that both Cbl and Gab1 enhance Met-induced Received 28 February 2000; revised 8 June 2000; accepted 15 June activation of another MAP kinase cascade, the ERK 2000 pathway, in a Crk-independent manner. Collectively, Cbl-mediated regulation of Met signaling M Garcia-Guzman et al 4059 our studies described below demonstrate a heretofore transient Tpr/Met transfection. Unless otherwise in- unidenti®ed functional role for the docking protein Cbl dicated, the results shown are those obtained with in Met signaling pathways. HeLa cells; similar results were obtained in Cos-7 and A previous report by Fixman et al. (1997) has NIH3T3 cells. As shown in Figure 1a, Cbl readily demonstrated that the 120 kDa c-Cbl protein becomes becomes phosphorylated on tyrosine in HGF/SF- tyrosine phosphorylated in Tpr/Met-transformed cells. treated cells in a dose- and time-dependent manner. To further characterize the potential role of Cbl in Met Phosphorylation of Cbl is induced rapidly, within signaling, we studied in more detail the phosphoryla- minutes of receptor engagement, and it closely parallels tion status of Cbl in cells in which Met signaling has the autophosphorylation status of the Met receptor. been activated either by HGF/SF stimulation or by a Further, Cbl phosphorylation occurs at low (20 ±

Figure 1 Met kinase activity induces Cbl tyrosine phosphorylation and association with SH2-signaling molecules. (a) HeLa cells were treated with various concentrations of HGF/SF for the indicated times. Cell lysates (50 mM HEPES pH=7.9, 1% Triton X- 100, 10% glycerol, 150 mM NaCl, 1 mM EGTA, 200 mM Na3VO4,50mM NaF, 1.5 mM MgCl2,10mg/ml leupeptin, 0.1 U/ml aprotinin and 1 mM phenylmethylsulfonyl ¯uoride) containing equal amounts of protein were subjected to immunoprecipitation using antibodies against Cbl (upper panels, Santa Cruz Biotechnology #sc-170-G) or Met (lower panels, Santa Cruz Biotechnology #sc-161) and the immunoprecipitates were analysed by immunoblotting with anti-phosphotyrosine (anti-PY, PY20 : HRPO, Transduction Laboratories #P11625), anti-Cbl or anti-Met antibodies (ascites from mouse monoclonal hybridoma raised against the C-terminal portion of Met, provided by Dr G Vande Woude) as indicated. (b) HeLa cells were transiently transfected with mammalian expression vectors coding for Tpr/Met wild type (`WT') (obtained from Dr G Vande Woude), Tpr/Met Y482F-Y489F (`YY') or Tpr/Met K243A (`KD') (Garcia-Guzman et al., 1999). Lysates containing equal amounts of protein were subjected to immunoprecipitation using anti-Cbl antibodies (upper and middle panels) and analysed by immunoblotting with anti- phosphotyrosine (upper panel) or anti-Cbl antibodies (middle panel). Total cell lysates were analysed by immunoblotting with anti-Met antibodies to con®rm equal expression levels of the various Tpr/Met constructs. (c) Lysates containing equal amounts of protein from HeLa cells stimulated (+) or not (7) with 50 ng/ml of HGF/SF for 15 min were incubated with the indicated GST- fusion proteins coupled to glutathione agarose as described in (Vuori et al., 1996). The precipitates were analysed by immunoblotting with anti-Cbl antibodies. The protein samples used for the in vitro association experiments contained equal amounts of Cbl, as demonstrated by anti-Cbl immunoblotting of total cell lysates (right panel). (d) HGF/SF stimulation induces association of Cbl with the adaptor protein Crk. HeLa cells were treated (+) or not (7) with 50 ng/ml of HGF/SF for 15 min and lysates containing equal amounts of protein were immunoprecipitated with anti-Cbl antibodies or incubated with protein A- sepharose in the absence of the primary antibody (`prot A', negative control). The presence of Crk in the Cbl immunecomplexes was analysed by immunoblotting with anti-Crk antibodies (left panel). The amount of Crk present in the total protein lysates in the non- stimulated (7) and stimulated (+) cells was shown to be equal by immunoblot analysis (right panel)

Oncogene Cbl-mediated regulation of Met signaling M Garcia-Guzman et al 4060 100 ng/ml) ligand concentrations; these same concen- c-Crk is an adaptor protein that is mainly composed trations have been shown to induce activation of of SH2- and SH3-domains (Feller et al., 1998; Matsuda biologically important signaling events downstream of and Kurata, 1996). We have previously determined Met (Boccaccio et al., 1998; Ponzetto et al., 1994). As that Crk connects multiple cellular stimuli to the JNK shown by Fixman et al. (1997), Cbl also becomes pathway and that intact SH2- and SH3-domains in Crk tyrosine phosphorylated in cells expressing the onco- are required for this function (Dol® et al., 1998). In a genic Tpr/Met (Figure 1b). Interestingly, previous previous report, we identi®ed tyrosine-phosphorylated reports have established a correlation between Tpr/ Gab1 as a Crk-SH2-binding molecule in cells in which Met-induced transformation and Gab1 phosphoryla- Met signaling had been activated. We also found that tion; it has been demonstrated that both the kinase expression of Gab1 enhanced Met-induced JNK activity and the integrity of the multi-substrate binding activation in a Crk-dependent manner (Garcia-Guz- site are required for cellular transformation and Gab1 man et al., 1999). To investigate whether the Met- phosphorylation by the Tpr/Met oncogene (Bardelli et induced Cbl-Crk interaction observed here might be of al., 1997a; Fixman et al., 1997). We ®nd here the same functional signi®cance in Met signaling pathways, we to be true for Tpr/Met-induced Cbl phosphorylation. examined the ability of Cbl to modulate Met-induced As shown in Figure 1b, induction of Cbl phosphoryla- JNK activity. As shown in Figure 2, expression of Tpr/ tion is negligible in HeLa cells transfected with the Met in HeLa cells or stimulation of the cells with kinase-dead form of Tpr/Met (`KD'), or with the form HGF/SF results in activation of the JNK kinase of Tpr/Met in which the tyrosine residues in the multi- cascade as measured by a JNK kinase assay (Figure substrate binding site have been mutated to phenyla- 2a) or by a c-Jun transcriptional activity assay (Figure lanine residues (tyrosines 482 and 489 in Tpr/Met, 2b). There appears to be good correlation between `YY'). Immunoblot analysis con®rmed that the Tpr/ Met-induced JNK activation and Cbl phosphorylation: Met variants were expressed at similar levels in the cells in a previous study, we found that HGF/SF stimulates (Figure 1b). Taken together, activation of Met JNK activation in a dose-dependent manner (Garcia- signaling either by physiological levels of HGF/SF or Guzman et al., 1999) that closely parallels Cbl by expression of the Tpr/Met oncogene results in phosphorylation observed here (Figure 1a). Further, enhanced tyrosine phosphorylation of Cbl. The char- we demonstrated earlier that the kinase-dead form of acteristics of this phosphorylation are similar to those Tpr/Met and the form of Tpr/Met in which the observed for Gab1, an established docking molecule in tyrosine residues in the multi-substrate binding site Met signaling events. have been mutated to phenylalanine residues fail to Cbl is known to become tyrosine phosphorylated in activate JNK (Garcia-Guzman et al., 1999); these response to a number of cellular stimuli, including forms of Tpr/Met also fail to induce Cbl phosphoryla- ligation of T-cell and B-cell receptors, Fc-receptors, tion (Figure 1b). Similar to our ®ndings with Gab1 integrins, receptors for GM-CSF, erythropoietin, (Garcia-Guzman et al., 1999), we ®nd here that thrombopoietin, interleukins -3 and -4 and prolactin, exogenous expression of Cbl enhances Tpr/Met- and and stimulation of several receptor tyrosine kinases, HGF/SF-induced JNK activation by about 3 ± 4-fold such as receptors for PDGF, EGF, NGF, insulin and (Figure 2); expression of Cbl in the absence of Met stem cell factor. As a result, Cbl binds to several SH2- signaling had no eect on JNK activity (not shown). domain containing molecules, such as Crk, the p85 Two lines of experimentation suggest that this subunit of PI 3-kinase and Src family tyrosine kinases enhancement is mediated by the Cbl-Crk interaction. (for original references, see Lupher et al., 1998; Miyake First, we found that expression of a c-Crk-I construct et al., 1997). We ®nd that stimulation of cells with in which the SH2-domain has been inactivated by an HGF/SF also enhances the interaction of Cbl with arginine-to-valine mutation at amino acid 38 (Crk- SH2-domain containing proteins. As shown in Figure R38V-mutant) eectively abolishes the enhancing eect 1c, Cbl readily interacts with the SH2-domains of Src of Cbl on Met-induced JNK activation (Figure 2a,b). and p85 in HGF/SF-stimulated HeLa cells, but not in Previous studies by us and others have established that unstimulated cells (Figure 1c). HGF/SF stimulation the Crk-R38V-mutant is a speci®c dominant-negative also enhances the association of Cbl with the SH2- mutant in Crk signaling pathways and functionally domain of Crk, while the tyrosine phosphorylation- dissociates tyrosine-phosphorylated Crk-SH2-binding independent interaction between Cbl and the SH3- proteins from the eector molecules that bind to the domain of Grb2 remains unchanged upon HGF/SF SH3-domain of Crk (Dol® et al., 1998; Garcia-Guzman stimulation; Cbl is known to interact with Grb2 in a et al., 1999; Klemke et al., 1998; Matsuda et al., 1992, constitutive manner by binding to the SH3-domain of 1994; Tanaka et al., 1993, 1995). Second, we found Grb2 (for references, see Miyake et al., 1997). As a that the capability of Cbl to enhance Met-induced JNK control for speci®city, we found that the SH2-domains activation is signi®cantly reduced when the tyrosines at of PLC-g and Shc failed to interact with Cbl in both either position 700 or 774 in Cbl has been mutated to unstimulated and stimulated cells (Figure 1c). The Cbl- phenylalanine (Figure 2c). An even more profoundly Crk interaction was con®rmed by coimmunoprecipita- reduced eect is observed when a Cbl construct tion experiments. As shown in Figure 1d, stimulation harboring mutations in both of the tyrosines was of HeLa cells with HGF/SF results in a signi®cant tested for its capability to enhance Met-induced JNK increase in the amount of c-Crk-I and c-Crk-II, the two activation (Figure 2c). No dierences in the expression cellular forms of c-Crk, associated with Cbl. These levels among the various Cbl-constructs were observed studies suggest that tyrosine-phosphorylated Cbl, (not shown). The sequences around the tyrosine similar to tyrosine-phosphorylated Gab1, may function residues 700 and 774 conform to the consensus SH2- as a docking molecule in Met signaling pathways and binding site for Crk (Songyang et al., 1993), and connect Met to SH2-containing eector molecules. previous reports have established that these two

Oncogene Cbl-mediated regulation of Met signaling M Garcia-Guzman et al 4061

Figure 2 (a) Activation of JNK by Met kinase is enhanced by expression of Cbl and blocked by a dominant-negative Crk construct. HeLa cells were transiently transfected with plasmids (5 mg/dish each) encoding wild-type Tpr/Met (`Tpr/Met'), Cbl, dominant-negative Crk-R38V or Flag-tagged JNK (`JNK') as indicated, and subjected to a JNK immunecomplex kinase assay using GST/c-jun (1 ± 79) (`GST/c-jun') as a substrate; constructs for Tpr/Met, Crk-38V and Flag-tagged JNK, transfections and kinase assay have been described in (Garcia-Guzman et al., 1999). The total amount of DNA used for transfections to study in vitro kinase activity was maintained constant by using a neutral mammalian expression vector (pcDNA3 plasmid, Stratagene). Wild-type Cbl in a pJZenNeo-vector was obtained from Dr WY Langdon. Expression of the transfected proteins was assessed by immunoblotting with anti-Met, anti-Cbl, anti-Crk (Transduction Laboratories #C12620) and anti-Flag antibodies (anti-FLAG M2 mAb, Eastman Kodak). The upper graph shows the normalized median values of three independent experiments with the standard deviation. Where indicated, the cells were stimulated with 50 ng/ml of HFG/SF for 15 min prior to cell lysis. (b) Met-induced transcriptional activation of c-Jun is enhanced by Cbl expression in a Crk-dependent manner. HeLa cells were transfected with pFR-Luc (1 mg) and pFA-c-Jun (1 ± 223) (`Gal4/Jun', 50 ng) plasmids (PathDetect Reporting System, Stratagene) and the expression vectors for Tpr/ Met, Cbl or Crk-R38V at 1 mg/dish each as indicated. Gal4-c-Jun transcriptional activity was determined by measuring activity levels of the luciferase reporter gene as described in (Garcia-Guzman et al., 1999). The total amount of DNA used for transfections to study transcriptional control of luciferase was maintained constant by using a neutral mammalian expression vector (pcDNA3). Transcriptional luciferase assays in this manuscript are indicated as RLU (relative luciferase unit) with the standard deviation (n=3), and were normalized for cotransfected (50 ng) b-galactosidase activity. (c) Mutation of the Crk-binding sites in Cbl reduces the enhancement of JNK activity by Cbl. c-Jun transcription assay was performed as above in cells that had been transfected with Tpr/Met, wild-type Cbl, or Cbl mutants containing point mutations in one (Y700F or Y774F) or both (Y700F/774F) of the c-Crk- SH2 binding motifs present in Cbl, as indicated. These mutations were created by site-directed mutagenesis using wild-type Cbl in a pJZenNeo-vector as a template tyrosines, when phosphorylated, mediate the interac- and later shown to possess constitutive kinase activity tion between Cbl and Crk (Andoniou et al., 1994; and transformation ability (Jeers et al., 1997a). At Reedquist et al., 1996; Sawasdikosol et al., 1996). present, relatively little is known about the molecular Taken together, our studies indicate that Cbl, similar signaling mechanisms downstream of these oncogenic to Gab1, is capable of enhancing Met-induced JNK Met receptors, although some dierences between the activation in a Crk-dependent manner. signaling strategies employed by these receptors and by In order to gain further insight into the roles of Crk, the oncogenic Tpr/Met receptor may exist (Jeers et Cbl and Gab1 in Met signaling pathways, we decided al., 1998). We demonstrate here that similar to Tpr/ to study the capability of oncogenically activated Met Met expression, expression of Met (L1213V/M1268T; to activate the JNK pathway and the potential roles of Met LM in the ®gures) results in a robust activation of Crk, Cbl and Gab1 in mediating this eect. Met the JNK pathway in HeLa cells (Figure 3a). Expression receptor harboring point mutations at sites 1213 and of Met (L1213V/M1268T) also signi®cantly enhances 1268 (Met L1213V/M1268T) is an example of an tyrosine phosphorylation of both Cbl and Gab1 oncogenic Met mutant that was originally identi®ed in (Figure 3a, lower panels). As shown in Figure 3b, human papillary renal carcinoma (Schmidt et al., 1997) activation of JNK and phosphorylation of Cbl in

Oncogene Cbl-mediated regulation of Met signaling M Garcia-Guzman et al 4062

Figure 3 Activation of the JNK pathway by an oncogenic mutant of the human Met tyrosine kinase correlates with tyrosine phosphorylation of the docking proteins Gab1 and Cbl and is enhanced by the expression of the two docking proteins in a Crk- dependent manner. (a) Oncogenic Met activates the JNK pathway and enhances tyrosine phosphorylation of Gab1 and Cbl. HeLa cells were transfected with vectors (5 mg/dish each) coding for Flag-tagged JNK, wild-type Met (`WT') or the oncogenic Met-mutant Met (L1213V/M1268T) (`Met LM', obtained from Dr G Vande Woude) and subjected to a JNK immunecomplex kinase assay as in Figure 2 (upper panels show the normalized median values of three independent experiments with the standard deviation). In parallel, cell lysates containing equal amounts of protein from HeLa cells that had been transfected with plasmids coding for wild- type Met or oncogenic Met (L1213V/M1268T) were subjected to immunoprecipitation analysis by anti-Cbl and anti-Gab1 (Upstate Biotechnology #06-5790) antibodies followed by immunoblotting using anti-phosphotyrosine antibodies (lower panels). The amounts of Cbl in the immunoprecipitates and the Met proteins in the total cell lysates were determined by immunoblotting using the speci®c antibodies (lower panels). (b) The activation of JNK by Met (L1213V/M1268T) correlates with the phosphotyrosine levels of Cbl in a dose-dependent manner. JNK immunecomplex kinase assays were performed as in (a) but using increasing amounts of the plasmid encoding the mutant Met (L1213V/M1268T) for transfections; the total amount of DNA used for the transfections was equalized by using a neutral mammalian expression vector, pCDNA3 (upper panel). In parallel experiments, tyrosine phosphorylation of Cbl was examined in cells harboring the various amounts of Met (L1213V/M1268T). The expression levels of Met (L1213V/M1268T) in the various samples were examined by anti-Met immunoblotting of the total cell lysates (lower panels). (c and d) Induction of JNK activation by Met (L1213V/M1268T) is enhanced by co-expression of Cbl (c) and Gab1 (d) and blocked by the dominant-negative Crk-mutant. HeLa cells were transiently transfected by the indicated plasmids, and a JNK immunecomplex kinase assay was carried out as above (upper panels). Immunoblot analysis of the expressed proteins (middle panels) were performed as above; expression levels of the HA-tagged Gab1 (obtained from Drs M Holgado-Madruga and A Wong) were assessed by immunoblotting total cell lysates using anti-HA antibodies (Santa Cruz Biotechnology #sc-805). Similar ®ndings were obtained in experiments in which the eects of Met (L1213V/M1268T), Cbl, Gab1 and Crk-R38V on the JNK pathway were analysed in a c-Jun transcriptional assay (lower panels in c and d)

response to Met (L1213V/M1268T) expression occurs by treatment of cells with HGF/SF, by expression of in a dose-dependent and closely parallel manner; Tpr/Met or by expression of Met (L1213V/M1268T), similar results were obtained with respect to Gab1 results in tyrosine phosphorylation of the docking phosphorylation (not shown). Similar to what we have proteins Cbl and Gab1, association of these proteins observed for Tpr/Met- and HGF/SF-induced Met with Crk, and activation of the JNK pathway in a Crk- signaling, Cbl and Gab1 were found to enhance Met dependent manner (this study and Garcia-Guzman et (L1213V/M1268T)-induced JNK activation in a Crk- al., 1999). dependent manner. Thus, exogenous expression of Cbl Met signaling induced by HGF/SF stimulation or by (Figure 3c) or Gab1 (Figure 3d) enhances Met Tpr/Met expression is known to activate two parallel (L1213V/M1268T)-induced JNK activation by about MAP kinase pathways, the JNK pathway and the 3 ± 4-fold, while a concomitant expression of the ERK pathway (Rodrigues et al., 1997). Our results dominant-negative Crk-R38V-mutant completely depicted in Figure 3 demonstrate that activation of abolishes these eects. Hence, stimulation of Met Met signaling by expression of Met (L1213V/M1268T) signaling pathways in multiple dierent ways, either activates the JNK-arm of the MAP kinase pathway. As

Oncogene Cbl-mediated regulation of Met signaling M Garcia-Guzman et al 4063 shown in Figure 4, expression of Met (L1213V/ 1995; Fukazawa et al., 1995; Meisner et al., 1995; Odai M1268T) also results in induction of ERK activation et al., 1995; Ponzetto et al., 1996; Zhu et al., 1994). It as measured by an ERK kinase assay (Figure 4a,c) or was reported recently, however, that dominant-negative by an Elk transcriptional activity assay (Figure 4b). It forms of Grb2 fail to inhibit Met-induced ERK has been reported that overexpression of Gab1 in activation (Rodrigues et al., 1997), and the downstream epithelial cells leads to phosphorylation of ERK1 and pathways leading to ERK kinase cascade activation ERK2 (Weidner et al., 1996), suggesting that Gab1 remain unknown at present. may be mediating the eects of Met on the ERK Our ®ndings described above identify a novel kinase cascade. Along these lines, we ®nd that functional role for the docking protein Cbl in Met expression of Gab1 enhances Met (L1213V/M1268T)- signaling. Similar to another docking protein, Gab1, induced ERK activation (Figure 4c). Similarly, we ®nd Cbl associates with the adaptor protein Crk and that expression of Cbl also enhances activation of the enhances Met-induced JNK activation in a Crk- ERK pathway downstream of Met (L1213V/M1268T) dependent manner. We have previously established by about 2 ± 3-fold (Figure 4a,b). Notably, the eects that Crk-mediated JNK activation takes place via of Cbl and Gab1 on ERK activation were not aected activation of the small GTP-binding protein Rac, by a concomitant expression of the dominant-negative which in turn occurs through the Crk SH3-binding Crk-R38V construct (Figure 4). Taken together, our proteins, such as C3G, Sos or DOCK180 (Dol® et al., studies demonstrate that the docking proteins Gab1 1998). Recently, Scaife and Langdon (2000) have and Cbl modulate Met-induced activation of MAP reported that the Cbl-Crk interaction results in Rac kinase pathways; our ®ndings described here and activation, and our studies (unpublished data) agree earlier (Garcia-Guzman et al., 1999) support a role with these ®ndings. Also, we have found that for Gab1-Crk and Cbl-Crk complex formation in the dominant-negative Rac blocks the enhancement of activation of the JNK kinase cascade, while Met- JNK activation by Cbl (not shown). Rodrigues et al. induced activation appears to take place in a Crk- (1997) in turn have reported that a dominant-negative independent manner. Both Gab1 and Cbl interact with mutant of Rac eciently blocks Tpr/Met-induced JNK Grb2, an adaptor protein that couples activated activation and transformation. Taken together, these receptor tyrosine kinases to SOS, promoting Ras/Raf/ data suggest a linear pathway from Gab1-Crk and Cbl- ERK activation (Donovan et al., 1994; Fixman et al., Crk to Rac activation that ultimately stimulates the

Figure 4 Activation of the MAPK ERK pathway by oncogenic Met (L1213V/M1268T) is modulated by the co-expression of Cbl and Gab1. (a) Activation of ERK by Met (L1213V/M1268T) is enhanced by co-expression of Cbl. HeLa cells were transfected with expression vectors encoding the indicated proteins (5 mg/dish each) and analysed by an ERK immunecomplex kinase assay using myelin basic protein (MBP) as a substrate; the HA-tagged ERK construct and the kinase assay protocol have been described in (Garcia-Guzman et al., 1999). The bar graph represents the mean with the standard deviation. Expression levels of the proteins were determined by immunoblotting analysis as above (lower panels). In (b) Elk-mediated transcriptional activity was determined by a luciferase assay (PathDetect Reporter System, Stratagene) in cells that had been transfected with the indicated plasmids as described in (Garcia-Guzman et al., 1999). (c) Activation of ERK by Met (L1213V/M1268T) is enhanced by co-expression of Gab1. HeLa cells were transfected with the indicated plasmids (5 mg/dish each) and an ERK immunecomplex kinase assay in the transfected cells was carried out as in (a). The expression levels of the transfected proteins were assessed as above

Oncogene Cbl-mediated regulation of Met signaling M Garcia-Guzman et al 4064 JNK kinase and controls biological events downstream 1998, 1999; Miyake et al., 1998; Waterman et al., 1999; of Met. Along these lines, Feller and coworkers have Yokouchi et al., 1999). Ubiquitination of the receptor recently reported on the importance of Crk signaling tyrosine kinases in turn terminates signaling by complexes in connecting Met to the small GTPase marking active receptors for degradation via the Rap1 (Sakkab et al., 2000). The relative signi®cance of proteasome pathway (for a review, see Ciechanover, Gab1 and Cbl in Met signaling pathways needs to be 1998). Consequently, expression of Cbl results in addressed e.g. by utilizing cell lines generated from inhibition of receptor tyrosine kinase activation and knockout mice de®cient in Gab1- and Cbl-family growth factor-mediated cell proliferation. Somewhat signaling proteins. unexpectedly, however, expression of Cbl has not been It remains to be seen whether Gab1 and Cbl might shown to signi®cantly modulate e.g. ERK signaling have a similar function in other signaling pathways. It downstream of receptor tyrosine kinases. Thus, is interesting to note that several common upstream Langdon and coworkers have found that expression stimuli appear to regulate both Gab1 and Cbl of Cbl inhibits autophosphorylation of EGFR in phosphorylation; in addition to c-Met stimulation, response to EGF stimulation, yet EGF-induced ERK these stimuli activate other receptor tyrosine kinases kinase activity remains unaected in these cells such as EGF receptor, insulin receptor, TrkA receptor, (Bowtell and Langdon, 1995; Thien and Langdon, receptors for several cytokines such as interleukins and 1997). Similar ®ndings have been obtained by Ueno et interferons, thrombopoietin and stem cell factor, as al. (1997) and by us (unpublished data). Collectively, well as T- and B-cell receptors (for original references, these results suggest that at least in vitro, c-Cbl has no see Holgado-Madruga et al., 1996; Lupher et al., 1998; eect or only a marginal eect on EGF-induced ERK Miyake et al., 1997; Nishida et al., 1999). Relatively and JNK activation, while it functions as a positive little is known about the functional signi®cance of regulator of Met-induced MAP kinase activation. In Gab1 in these other pathways. It has been reported contrast, the c-Cbl homologs cbl-b and cbl-3 have been that Gab1 mediates PI 3-kinase activation downstream found to function as negative regulators of ERK and of NGF signaling, while in the cytokine signaling JNK pathways downstream of EGFR (Bustelo et al., pathways, Gab1 appears to link the receptors to the 1997; Ettenberg et al., 1999; Keane et al., 1999); their ERK pathway (Holgado-Madruga et al., 1997; Taka- role in e.g. Met signaling remains unknown. We have hashi-Tezuka et al., 1998). Cbl in turn has been shown not explicitly examined the potential role of c-Cbl in to be an important downstream component in Src regulating protein levels or tyrosine kinase activity of signaling pathways in osteoclasts (Tanaka et al., 1996). Met. Interestingly, however, it has been reported that In addition, Cbl has been demonstrated to enhance PI degradation of Met takes place via the ubiquitin- 3-kinase activity and proliferation of cells in the proteasome pathway (Jeers et al., 1997b). The presence of IL-4 and G-CSF (Grishin et al., 2000; possibility that Cbl might function both as a positive Ueno et al., 1998). Similarly, Cbl has been shown to and negative regulator to control intracellular signaling regulate integrin-mediated cell adhesion, spreading and cascades downstream of Met is intriguing and warrants migration in a PI 3-kinase-dependent manner (Fesh- further study. chenko et al., 1999; Meng and Lowell, 1998; Zell et al., 1998). More recently, a wealth of information has accumu- lated on the role of Cbl as a negative regulator of receptor tyrosine kinase signaling. Thus, it has been Acknowledgments demonstrated that Cbl positively regulates ubiquitina- We thank WY Langdon for the Cbl-construct, M Matsuda tion of receptors for EGF, PDGF and CSF-1 in a for the Crk-construct, A Wong and M Holgado-Madruga for the Gab1 cDNA and GF Vande Woude and M Jeers manner that depends on its variant SH2-domain and a for the wild-type Tpr/Met construct, Met (L1213V/ RING ®nger domain; Cbl binds to the tyrosine- M1268T) construct and anti-Met antibodies. This work phosphorylated receptor via its SH2-domain, and was supported by NIH grants CA71560 and CA76037 (to recruits and allosterically activates an E2-ubiquitin- K Vuori). M Garcia-Guzman was supported by a fellow- conjugating enzyme through its RING domain (Joa- ship from the Human Frontier Science Program. K Vuori zeiro et al., 1999; Lee et al., 1999; Levkowitz et al., is a PEW scholar in biomedical sciences.

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