Quantitative real-time PCR.

Total RNA was extracted from T47D cells using TRIzol reagent (Invitrogen Life Technologies) following the manufacturer's protocol; 5 μg of RNA was reverse transcribed with oligo(dT) (Invitrogen Life Technologies), and the cDNA was amplified using LightCycler-FastStart reaction mix SYBR Green I (Roche Molecular Biochemicals) and Rotor-Gene 3000 light cycler (Corbett Research, Sydney, Australia). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control to normalize mRNA levels. Primer sequences were as follows: GAPDH, sense, 5′-ACCACAGTCCATGCCATCAC-3′, and antisense, 5′-TCCACCACCCTGTTGCTGTA-3′; Met, sense, 5′-GTTTGTCCACAGAGACTTGGCTG-3′, and antisense, 5′-AGTTCAGAAAAGGATGGGCG-3′. The mean threshold cycle (C_t) value for each transcript was normalized by dividing it by the mean C_t value for the GAPDH transcript for that sample. Normalized transcript levels were expressed relative to sample obtained from T47D expressing wild-type Met.

In vivo tumorigenesis assays.

For tumorigenesis assays, NIH 3T3 fibroblast cells were infected with retroviruses expressing either nothing, wild-type Met or Met Y1003F and selected with 10 μg/ml G418 for 2 weeks. The obtained cell populations were injected subcutaneously (5 × 10⁵ cells per 100 μl) into 4- to 5-week-old female nude mice (CD1 nu/nu; Charles River Breeding Laboratories). Tumors were measured periodically and mice were sacrificed prior to the tumors reaching 1 cm³ or undergoing ulceration.

Cell culture, transfections, and focus formation assays.

T47D breast epithelial cells, Madin-Darby canine kidney (MDCK) cells and HEK 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). MDCK cell lines expressing wild-type CSF-Met and CSF-Met Y1003F were established as described previously (46). T47D cell lines expressing wild-type Met and Met Y1003F were generated by retroviral infection. Following infection, cells were selected for 3 weeks in 4 μg/ml G418. Selected cell populations were then sorted by fluorescence-activated cell sorting (FACS) to select cells expressing Met at their cell surface using a MoFlo flow cytometer (Dako Cytomation, Fort Collins, CO).

Cells were labeled with anti-Met (1:50) (R&D Systems) for 1 h and with Alexa 488 donkey anti-goat (1:250) for 30 min in FACS buffer (DMEM containing 1% heat-inactivated FBS, 1 mM CaCl₂ and 1 mM MgCl₂) and then incubated for 5 min in FACS buffer containing 5 mM EDTA; 10⁵ to 2 × 10⁵ sorted cells were collected and plated in DMEM containing 10% FBS. Transient transfections in T47Ds were performed via electroporation. Cells were trypsinized, resuspended in phosphate-buffered saline (PBS), and electroporated at 0.30 kV and 0.975 μF. Transient transfections in HEK 293 cells were performed using Lipofectamine Plus reagent according to the manufacturer's instructions (Invitrogen Life Technologies).

The focus formation assays were performed as described previously (13). For T47D phase contrast pictures, 8 × 10⁵ cells were seeded in 60-mm dishes and 16 h later were treated where indicated with 1.5 nM HGF for 24 h in the presence of serum. For the MDCK scatter assay, 10⁴ cells/well were seeded in 12-well plates and 18 h later were treated where indicated with CSF (50 ng/ml), U0126 (20 μM), LY294002 (25 μM) in the presence of serum for 24 h. Phase contrast images were then taken with a Zeiss Axiovision 135 microscope with a 25× objective (Carl Ziess Canada Ltd, Toronto, Ontario, Canada). Image analysis was carried out using Northern Eclipse version 6.0 (Empix Imaging, Mississauga, Ontario, Canada).

Immunoprecipitation and Western blotting.

Following stimulation with HGF, T47D cells were harvested in TGH lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). HEK 293 transfections were harvested 22 h posttransfection with radioimmunoprecipitation assay (RIPA) buffer (0.05% sodium dodecyl sulfate [SDS], 50 mM Tris, pH 8.0, 150 nM NaCl,1% Nonidet P-40, 0.05% sodium deoxycholate) or modified RIPA buffer (0.1% SDS, 25 mM Tris, pH 8.2, 50 mM NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate) supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 1 mM sodium fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin.

Lysates were incubated with the indicated antibody for 2 h at 4°C with gentle rotation. Proteins collected on either protein A- or G-Sepharose were washed three times in their respective lysis buffers, resolved by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to a nitrocellulose membrane. Membranes were blocked in 3% bovine serum albumin in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 2.5 mM EDTA, 0.1% Tween 20) for 1 h and incubated with primary and secondary antibodies in TBST for 2 h and 1 h, respectively. After four washes with TBST, bound proteins were visualized with an ECL detection kit (Amersham Biosciences). Analyses with the Odyssey infrared imaging system were performed according to the manufacturer's instructions. To detect Met receptor ubiquitination, cells from a 10-cm dish were lysed in 200 μl boiling buffer (2% SDS, 1 mM EDTA). Lysates were boiled for 10 min and diluted to 1 ml with a buffer containing 2.5% Triton X-100, 12.5 mM Tris, pH 7.5, 187.5 mM NaCl, and proteasomal inhibitors (66).

Internalization assays using trypsin and flow cytometry.

Cells were seeded in 60-mm dishes and the following day were stimulated with 3 nM HGF. Stimulations were terminated by placing cells on ice and rinsing them with ice-cold DMEM. On ice, cells were acidified for 10 min with cold DMEM at pH 4.0 containing 1% bovine serum albumin. For the trypsin assays, cells were rinsed once with ice-cold PBS and then incubated with 2 ml of 1 mg/ml trypsin in PBS (pH 7.4) for 30 min on ice. The reaction was stopped by adding 2 ml of 5 mg/ml soybean trypsin inhibitor in ice-cold PBS. Cells were collected, centrifuged for 5 min at 200 g at 4°C, washed once with 1 ml of 5 mg/ml soybean trypsin inhibitor in PBS (ice-cold), and lysed in TGH lysis buffer.

For flow cytometry analysis, cells were washed once in ice-cold FACS buffer (PBS containing 1% heat-inactivated FBS, 1 mM CaCl₂ and 1 mM MgCl₂). On ice, cells were then incubated with Met antibody (AF206) in ice-cold FACS buffer for 45 min, washed three times with FACS buffer, incubated with donkey anti-goat Alexa 488 in FACS buffer for 20 min, washed three times, and incubated 5 min in 0.5 ml FACS buffer containing 5 mM EDTA. The cells were scraped and analyzed with the FACScan flow cytometer (BD Biosciences).

Confocal Immunofluorescence microscopy.

Cells were seeded at 2 × 10⁵ on glass coverslips (Bellco Glass Inc. Vineland, NJ) in 24-well plates (Nalge NUNC, Rochester, NY) and 16 h later were serum starved for 2 h prior to HGF treatment (1.5 nM) where indicated. Coverslips were washed once with PBS and then fixed with 3% paraformaldehyde (PFA; Fisher Scientific) in PBS for 20 min. Coverslips were then washed four times in PBS and residual PFA was removed with three 5-minute washes with 100 mM glycine in PBS. Cells were permeabilized with 0.3% Triton X-100/PBS and blocked for 30 min with blocking buffer (5% bovine serum albumin, 0.2% Triton X-100, 0.05% Tween 20, PBS). Coverslips were incubated with primary and secondary antibodies diluted in blocking buffer for 1 h and 40 min, respectively, at room temperature. Cells stained for Hrs were permeabilized with 0.05% saponin and fixed with 3% PFA as described previously (60). Coverslips were mounted with Immu-mount (Thermo-Shandon, Pittsburgh, PA). Confocal images were taken using a Zeiss 510 Meta laser scanning confocal microscope (Carl Ziess, Canada Ltd, Toronto, Ontario, Canada) with 100× objective and 2× zoom. Image analysis was carried out using the LSM 5 image browser (Empix Imaging, Mississauga, Ontario, Canada).

Met Y1003F receptor mutant is transforming in vitro and in vivo.

The enhanced stability of the Met Y1003F mutant is reflected by its enhanced biological activity. T47D cell lines stably expressing the Met Y1003F receptor but not the wild-type Met receptor display loss of epithelial organization, a hallmark of epithelial cell transformation that is enhanced by HGF stimulation (Fig. ​(Fig.2A).2A). Furthermore, NIH 3T3 cells expressing a Met Y1003F receptor, when subcutaneously injected into nude mice, formed solid tumors (0.10 cm³) with short latencies (20 days), which reached a volume of 0.70 cm³ within 35 days. In contrast, control NIH 3T3 cells or cells expressing a wild-type Met receptor formed only a small mass of 0.10 cm³ by 35 days postinjection (Fig. ​(Fig.2B2B and ​andC).C). Although the cell populations injected into nude mice expressed similar amounts of Met proteins, the tumors that developed contained higher levels of Met Y1003F than wild-type Met proteins (Fig. ​(Fig.2C).2C). We conclude that the specific uncoupling of the Met receptor from Cbl-mediated ubiquitination is sufficient to increase both the in vitro and in vivo tumorigenicity of the Met receptor.

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FIG. 2.

NIH 3T3 fibroblast cells expressing the ubiquitination-deficient Met Y1003F receptor are tumorigenic. (A) Met Y1003F-expressing cells have an altered morphology. Phase contrast pictures of T47D cells unstimulated or stimulated for 24 h with 1.5 nM HGF. (B) NIH 3T3 cell populations expressing either wild-type Met or Met Y1003F were injected subcutaneously into nude mice. The results represent the mean tumor volume obtained from eight measurements and are representative of two independent experiments. (C) Pictures of representative tumors as well as Met protein expression in the NIH 3T3 cell populations injected and in excised tumors. The tumors were lysed in TGH buffer and total cell lysates (TCL) were blotted with Met (DL-21) antibodies. Then, the membrane was stained with Coomassie brilliant blue.

Met Y1003F receptor preferentially promotes sustained activation of the Ras-MAPK pathway.

Several studies have indicated that recruitment of Cbl is required for the ubiquitination and ligand-induced down-regulation of the Met and EGF receptors (34, 35, 46, 47). However, reports on Met or EGF receptor mutants, uncoupled from Cbl-mediated ubiquitination, have failed to address the consequence of loss of ubiquitination on receptor signaling. To understand at the mechanistic level why the Met Y1003F receptor mutant is tumorigenic and promotes enhanced epithelial scattering, we examined its cellular signaling and trafficking in T47D cell populations stably expressing the Met wild-type or Y1003F receptor.

Signaling downstream from the Met receptor is mediated through the recruitment of the adaptor proteins Grb2 and Shc, which couple the Met receptor to the Ras signaling pathway, and the scaffold protein Gab1. Gab1 couples the Met receptor to the MAPK pathway as well as to phosphatidylinositol 3-kinase (PI3K), phospholipase Cγ, Crk, and the tyrosine phosphatase SHP2 (29, 30, 41, 42). Consistent with the sustained phosphorylation of the Met Y1003F receptor, tyrosine phosphorylation of downstream signals, such as Gab1 and c-Cbl, are prolonged in cells expressing the Met Y1003F receptor (Fig. ​(Fig.3A).3A). Furthermore, Ras activation, as measured by an in vitro Ras binding assay, is transient (1 h) downstream from the wild-type Met receptor, while it is sustained downstream of the Met Y1003F receptor (4 h, Fig. ​Fig.3B).3B). Activation of signaling pathways downstream from Ras, MEK1/2, and Erk1/2, as detected using phosphorylation site-specific antibodies, showed a similar trend (Fig. ​(Fig.3B3B).

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FIG. 3.

Activation of the Ras-MAPK but not the PI3K pathway is sustained downstream from Met Y1003F. (A) T47D cells were stimulated with 1.5 nM HGF for the indicated times and lysed in TGH buffer. In the upper two panels, c-Cbl proteins were immunoprecipitated and immunoblotted with phosphotyrosine antibodies. Membranes were stripped and blotted with Cbl antibodies. In the middle two panels, Gab1 proteins were immunoprecipitated and blotted with both antiphosphotyrosine, 4G10, and Gab1 antibodies using the Odyssey Infrared Imaging System (LI-COR). In the bottom two panels, lysates were blotted with either phospho-Ser473 or total Akt antibodies as indicated using the Odyssey system. (B) T47D cells were stimulated with 1.5 nM HGF and lysed as above. Upper two panels: Ras activation was determined using an in vitro binding assay with the GST-RBD fusion protein, with HEK293T cells expressing V12 H-Ras as a positive control. Panels were blotted with an H-Ras antibody using the Odyssey System. In the bottom four panels, cell lysates were blotted with phospho-Ser217/221 MEK1/2, total MEK1/2, phospho-Thr202/Tyr204 Erk1/2, and total Erk1/2 antibodies as indicated using the Odyssey system. (C) T47D cells were plated on coverslips, serum starved for 16 h, and stimulated with 1.5 nM HGF at 37°C for the indicated time points. Coverslips were fixed in 3% paraformaldehyde (PFA), and stained with phospho-Ser217/221 MEK1/2 (first panel). Cell nuclei were visualized using 4′,6′-diamidino-2-phenylindole (DAPI) (second panel). Confocal images were taken with a 100× objective and 2× zoom. The bar represents 5 μm.

HGF stimulation induces a robust but relatively transient activation of MEK1/2 (1 h) and Erk1/2 (decreased by 60 min) in T47D cells expressing a wild-type Met receptor, whereas activation of these proteins is sustained for 4 h in cells expressing the Met Y1003F receptor mutant (Fig. ​(Fig.3B).3B). Indirect immunofluorescence of activated MEK1/2 in T47D cell populations, using phosphorylation-specific antibodies, revealed a sustained and perinuclear signal in Met Y1003F-expressing cells compared to a transient and perinuclear signal in cells expressing the wild-type receptor (Fig. ​(Fig.3C),3C), demonstrating that the subcellular localization of the pMEK1/2 signal is not detectably different following sustained activation. Alternatively, in these cells, PI3K-dependent activation of Akt/PKB, as detected using a phospho-Ser473 antibody, is similar downstream from both the wild-type and Y1003F receptors (Fig. ​(Fig.3A),3A), revealing a selective enhancement of the Ras signaling pathway.

The requirement of the Ras-MAPK and PI3K-dependent signals for biological activity of the Met Y1003F receptor were examined in T47D and MDCK epithelial cells. These cells undergo an epithelial-mesenchymal like transition following stable expression of the Met Y1003F receptor (Fig. ​(Fig.2A2A and ​and4)4) (46). Treatment of fibroblast-like MDCK and T47D cells expressing Met Y1003F with a pharmacological inhibitor of MEK1/2 (U0126) induces a reversal in cell morphology, and cells begin to form tight colonies 24 h posttreatment, in the presence or absence of ligand (Fig. ​(Fig.44 and data not shown). In contrast, minimal changes in the fibroblast-like morphology of the Met Y1003F-expressing cells were observed after treatment with an inhibitor of PI3K (LY294002) (Fig. ​(Fig.44 and data not shown). This demonstrates a specific requirement for the Ras-MAPK pathway in the maintenance of the epithelial scattered phenotype induced by the Met Y1003F receptor mutant.

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FIG. 4.

Ras-MAPK is required for Met Y1003F increased biological activity. MDCK cells stably expressing either wild-type CSF-Met or CSF-Met Y1003F, where indicated, were treated with CSF (2.7 nM), UO126 (20 μM) and LY294002 (25 μM) for 24 h. After 24 h, phase-contrast pictures of live cells were taken with a Zeiss Axiovision 135 microscope under a 10× objective. The bar represents 100 μm.

Wild-type and Y1003F Met receptors internalize with similar kinetics.

The enhanced stability of the Met Y1003F receptor may indicate that this receptor is retained on the cell surface. Although several reports support the ability of ubiquitination to promote the internalization of cell surface receptors in yeast (20), whether ubiquitination of RTKs is directly involved in their internalization has only been examined for EGFR and remains controversial (10, 16, 24, 34, 62, 68). To address the requirement for Met receptor ubiquitination for its internalization, we compared internalization of the Met wild-type and Y1003F receptors in T47D cells, using trypsin and flow cytometry-based assays. Following HGF stimulation, the amount of internalized, trypsin-protected, receptors increases at a similar rate for Met wild-type and Y1003F (Fig. ​(Fig.5A).5A). In addition, using flow cytometry, we observed that the wild-type and Y1003F Met receptors are internalized at similar rates following stimulation (Fig. ​(Fig.5B).5B). In agreement with this, by immunofluorescence microscopy, both wild-type and Y1003F Met mutant receptors internalize and colocalize with the early endosome marker EEA1, 5 min post-HGF treatment, and this colocalization is enhanced at 15 and 30 min (Fig. ​(Fig.5C).5C). These results demonstrate that there is no significant difference between the rate of internalization and subcellular localization of wild-type Met and Met Y1003F receptors.

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FIG. 5.

Ubiquitination-deficient Met receptor mutant is internalized. (A) T47D cells were stimulated with 3 nM HGF at 37°C and then transferred on ice. Cells were acidified for 10 min and then treated with trypsin for 30 min. Trypsin was inhibited before cells were lysed. Lysates were blotted with Met (DL-21) and c-Cbl antibodies. (B) T47D cells were stimulated with 3 nM HGF at 37°C and then transferred on ice. Cells were acidified for 10 min and then labeled with Met AF276 antibody as described under Materials and Methods. The mean fluorescence intensity per cell was measured by flow cytometry and the percentage of Met receptors remaining at the cell surface over time is plotted. The graph represents the mean ± standard deviation of three independent experiments. (C) Both wild-type Met and Met Y1003F internalize and traffic to early endosomes. T47D cells were plated on coverslips, and 16 h later stimulated with 1.5 nM HGF at 37°C for the indicated times. Coverslips were fixed in 3% PFA and stained with Met AF276 (red) and EEA1 (green) antibodies. Confocal images were taken with a 100× objective and 2× zoom. Yellow staining represents colocalization between Met and EEA1. The bar represents 5 μm.

Met RTK ubiquitination is required for Hrs phosphorylation but not for trafficking to Hrs-positive endosomes.

The enhanced stability of the Met Y1003F receptor could also indicate that the endosomal sorting machinery does not recognize the poorly ubiquitinated Met Y1003F receptor, a process thought to be required for efficient targeting of the receptor to the sorting endosome. Hrs, one member of the endosomal sorting machinery, was first identified as a tyrosine phosphorylated protein downstream from the Met receptor (18, 27). Therefore, as a readout for Met receptor recruitment by endosomal sorting proteins, we examined whether the Met Y1003F receptor mutant can phosphorylate Hrs. Following stimulation of T47D cells with HGF, the endogenous Hrs protein is tyrosine phosphorylated upon activation of a wild-type Met receptor, but is not detectably phosphorylated in cells expressing the Met Y1003F receptor (Fig. ​(Fig.6A6A).

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FIG. 6.

Ubiquitination-deficient Met receptor mutant is unable to induce Hrs tyrosine phosphorylation. (A) T47D cells were serum starved for 16 h and stimulated where indicated with 1.5 nM HGF for 8 min. Cells were then lysed in TGH buffer and Hrs and Met proteins were immunoprecipitated and first blotted with phosphotyrosine antibodies, stripped, and reblotted with Hrs and Met antibodies as indicated. (B and C) Wild-type Met and Met Y1003F both colocalize with endogenous Hrs and transfected GFP-Rab7. T47D cells, nontransfected (B) and transfected (C), were plated on coverslips and 16 h later stimulated with 1.5 nM HGF at 37°C for the indicated times. Coverslips were fixed with 3% PFA and stained with (B and C) Met AF276 (red) and (B) Hrs (green) antibodies. Yellow staining represents colocalization between Met and Hrs or Met and GFP-Rab7. Confocal images were taken with a 100× objective and 2× zoom. The bar represents 5 μm.

To determine if the inability of the Met Y1003F mutant receptor to induce Hrs phosphorylation reflects its inability to reach an Hrs positive endosome, we examined the subcellular localization of both Met wild-type and Y1003F receptors with either endogenous or transiently transfected Hrs. As observed by indirect immunofluorescence, both the Met wild-type and Y1003F receptors traffic to Hrs-positive endosomes (Fig. ​(Fig.6B6B and data not shown). Moreover, this occurs with similar kinetics, where both the wild-type and Y1003F Met receptors begin to localize with either endogenous or GFP-tagged Hrs as early as 5 min post-HGF stimulation (data not shown) and colocalization increases up to 30 min post-HGF stimulation (Fig. ​(Fig.6B6B and data not shown). At the latter time point (30 min), both receptors also begin to localize to GFP-Rab7-positive endosomes (a marker of multivesicular bodies/late endosomes) (Fig. ​(Fig.6C)6C) (11, 67). Hence, although the ubiquitination-deficient Met Y1003F receptor mutant reaches Hrs and Rab7-positive endosomes, it does not induce Hrs tyrosine phosphorylation. This demonstrates that Cbl-mediated ubiquitination of the Met receptor is not essential for its targeting to Hrs- and Rab7-positive endosomes, but is required to induce Hrs tyrosine phosphorylation.

Monoubiquitination of Met Y1003F decreases its stability and rescues Hrs phosphorylation.

The addition of monoubiquitin to the transferrin receptor results in its partial exclusion from the recycling compartment and its retention within late endosomes (51). Similarly, the fusion of ubiquitin to the carboxy terminus of a severely truncated EGFR was sufficient for this receptor to traffic to late endosomes (17). However, the ability of these proteins to interact with components of the cargo sorting pathway and their stability was not examined. To establish whether the addition of monoubiquitin to the Met Y1003F receptor can rescue its targeting to the lysosomal degradative pathway and if this enhances its ability to be recruited to the endosomal sorting complex containing Hrs, we generated Met-ubiquitin receptor chimeras.

We fused a single ubiquitin moiety that cannot form ubiquitin chains, where the seven lysine residues in ubiquitin are replaced with arginine residues (59), to the carboxy terminus of the full-length wild-type and Y1003F Met receptors (Fig. ​(Fig.7A).7A). Following transient overexpression in HEK 293 cells, where the Met receptor is activated in a ligand-independent manner, both proteins are detected as a discrete band with an antibody raised against ubiquitin (Fig. ​(Fig.7B).7B). Moreover, both proteins associate with known receptor binding proteins to similar levels. Both the Grb2 adaptor protein and the Met binding domain (MBD) of Gab1 associate with the chimeric receptors in in vitro binding assays, demonstrating that the chimeric receptors are properly folded, tyrosine phosphorylated and can interact with signaling proteins (Fig. ​(Fig.7B).7B). In stable T47D cell populations, the chimeric receptors are expressed at the cell surface, as determined by flow cytometry (data not shown) and by immunofluorescence microscopy (Fig. ​(Fig.7C).7C). Upon HGF stimulation, both chimeric receptors internalize and colocalize with EEA1-positive endosomes (Fig. ​(Fig.7C7C).

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FIG. 7.

Fusion of monoubiquitin to the carboxy terminus of the Met receptor does not alter its maturation to the cell surface, recruitment of signaling proteins and its HGF-induced internalization. (A) Schematic representation of ubiquitinated wild-type and Y1003 Met chimeric receptors. Met receptor chimeras were generated with a single ubiquitin moiety fused to the carboxy-terminal end of the full-length wild-type and Y1003F Met receptors. The seven lysine residues within the ubiquitin moiety were replaced with arginine residues to prevent polyubiquitination and the carboxy-terminal glycine residue was replaced with a valine residue to prevent conjugation to free amino groups. (B) The chimeric receptors are properly expressed and tyrosine phosphorylated. HEK 293 cells were transiently transfected with the indicated CSF-Met constructs. In the top two panels, lysates were blotted with Met (antibody 144) and ubiquitin antibodies. In the bottom three panels, lysates were incubated with GST alone, GST-Grb2, or GST-MBD (Met binding domain of Gab1). In vitro binding assays and total cell lysates (TCL) were immunoblotted for Met. (C) T47D cells were plated on coverslips and 16 h later stimulated with 1.5 nM HGF at 37°C for 15 min. Coverslips were fixed in 3% PFA and stained with Met AF276 (red) and EEA1 (green) antibodies. Confocal images were taken with a 100× objective and 2× zoom. Yellow staining represents colocalization between Met and EEA1. The bar represents 5 μm.

When examined for stability, the Met Y1003F receptor mutant is stable for up to 6 h after stimulation with HGF, whereas the ubiquitinated Met Y1003 chimeric receptor is degraded after 2 h of stimulation (Fig. ​(Fig.8A),8A), in a manner similar to the wild-type Met protein (Fig. ​(Fig.1E).1E). Consistent with this, the prolonged activation of the Ras-MAPK signaling pathway observed downstream of the Met Y1003F receptor (4 h, Fig. ​Fig.8B)8B) is reversed by the addition of monoubiquitin to this receptor. MEK1/2 activation downstream from ubiquitinated Met Y1003 is transient (60 min, Fig. ​Fig.8B),8B), as it is downstream of the wild-type Met receptor (Fig. ​(Fig.3B).3B). These data indicate that the fusion of monoubiquitin to the Met receptor is sufficient to reverse the prolonged stability and signaling of the Met Y1003F receptor mutant. This suggests that monoubiquitination is sufficient to engage the Met Y1003F receptor mutant with components of the cargo-sorting pathway.

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FIG. 8.

Monoubiquitination of the Met Y1003F receptor leads to its degradation and restores HGF-induced Hrs tyrosine phosphorylation. (A) T47D cells treated with 100 μg/ml cycloheximide were stimulated with 3 nM HGF for the indicated amount of time. Total cell lysates (TCL) (30 μg) were immunoblotted for Met (DL-21) and Erk. (B) T47D cells were stimulated with 1.5 nM HGF for the indicated times and lysed in TGH buffer. Cell lysates were blotted with phospho-Ser217/221 MEK1/2 and total MEK1/2. (C) HEK293 cells were cotransfected with GFP-Hrs and vector or the indicated CSF-Met constructs. GFP-Hrs and Met proteins were immunoprecipitated and blotted with phosphotyrosine antibodies, then stripped and reblotted for GFP and Met, respectively.

To test this, we examined its ability to induce phosphorylation of Hrs in HEK 293 transient transfection assays, where Met is constitutively activated due to overexpression. We observed that while the Met Y1003F receptor is severely reduced in its ability to induce robust tyrosine phosphorylation of Hrs compared with a wild-type Met receptor, the ubiquitinated Met Y1003 chimeric receptor induces robust Hrs phosphorylation (Fig. ​(Fig.8C).8C). Hence, this demonstrates that monoubiquitination of the Met Y1003F receptor is sufficient to target the receptor for degradation and for it to phosphorylate Hrs, a component of the cargo-sorting machinery.

We thank Marisa Ponzo and Naila Chughtai for help with transient transfection assays and Dongmei Zuo and Laurent Sansregret for help with confocal imaging and flow cytometry, respectively. We are grateful to members of the Park laboratory for critical comments on the manuscript and Stephane Laporte for his helpful discussion. We thank Harald Stenmark for providing us with Hrs antibodies, Linda Hicke for the ubiquitin (7KtoR) plasmid, George Vande Woude for HGF, and Robert Lodge for GFP-Rab7.

J.A. is a recipient of an Alexander McFee studentship and P. P. is a recipient of a Terry Fox research studentship from National Cancer Institute of Canada. Sylvie Urbé is a Wellcome Trust Career Development Fellow. M.P. is recipient of a Canadian Institute of Health Research (CIHR) senior scientist award. This research was supported by an operating grant to M.P. from the CIHR.