A direct role for Met endocytosis in tumorigenesis
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ARTICLES A direct role for Met endocytosis in tumorigenesis Carine Joffre1,4 , Rachel Barrow1 , Ludovic Ménard1 , Véronique Calleja2 , Ian R. Hart3 and Stéphanie Kermorgant1,5 Compartmentalization of signals generated by receptor tyrosine kinase (RTK) endocytosis has emerged as a major determinant of various cell functions. Here, using tumour-associated Met-activating mutations, we demonstrate a direct link between endocytosis and tumorigenicity. Met mutants exhibit increased endocytosis/recycling activity and decreased levels of degradation, leading to accumulation on endosomes, activation of the GTPase Rac1, loss of actin stress fibres and increased levels of cell migration. Blocking endocytosis inhibited mutants’ anchorage-independent growth, in vivo tumorigenesis and metastasis while maintaining their activation. One mutant resistant to inhibition by a Met-specific tyrosine kinase inhibitor was sensitive to endocytosis inhibition. Thus, oncogenicity of Met mutants results not only from activation but also from their altered endocytic trafficking, indicating that endosomal signalling may be a crucial mechanism regulating RTK-dependent tumorigenesis. Compartmentalization of signals generated by growth factor endo- utilized because they exhibit constitutive kinase activity and promote cytosis is a key determinant of various cell functions and organ- in vitro and in vivo transformation26–28 . We show that these Met ism development1–3 . Receptors signal on endosomes before their mutants are oncogenic not only because of their activation but degradation4–7 . RTK interactions with adaptor/signalling molecules, for also because of where they signal, such that blocking endocytosis example EGFR (epidermal growth factor receptor) and Grb2 (growth inhibits transformation, tumour growth and metastasis while factor receptor-bound protein 2), were visualized on endosomes8 , and maintaining their activation. functional endocytosis machinery is required for optimal activation of signals, such as ERK1/2 (extracellular signal-regulated kinase RESULTS 1/2; refs 5,9,10) and STAT3 (signal transducer and activator of Active Met mutants accumulate in intracellular compartments transcription 3; ref. 7). We used NIH3T3 cells, producing small amounts of HGF, stably In the ‘signalling endosome’ concept11,12 , the endosome represents transfected with murine wild-type Met or mutant MetD1246N or a platform integrating signalling pathways temporally and spatially, MetM1268T (ref. 26). Both the precursor (p-170) and mature β chain to determine response specificity13–15 for characteristics such as (p-145) forms were detected (Fig. 1a); the expression levels of wild-type cell migration2,5,7,11,16 and cell survival17 , maintaining activated Met and MetM1268T were comparable, and that of D1246N was receptors and signals in close juxtaposition while providing protection elevated slightly (1.6-fold; Fig. 1a and Supplementary Fig. S1a). The against phosphatases18 . By controlling these functions and pathways, phosphorylation levels of Tyr 1234/1235 (kinase domain) and 1349 endocytosis may contribute to oncogenicity, although little direct (docking site) were high in mutants relative to the wild type, consistent evidence supports this possibility19,20 . Overexpression of the clathrin- with enhanced kinase activity27 and total phosphorylation26 (Fig. 1a,b associated protein HIP1 (Huntingtin interacting protein 1) occurs and Supplementary Fig. S1b). Normalization on Met expression in cancers, which possibly alters EGFR trafficking while promoting excluded that this resulted from higher expression levels (Fig. 1b and tumour formation21 . However, no causative role for RTK endocytosis Supplementary Fig. S1b). Exogenous HGF significantly enhanced the in cancer development has yet been established. levels of phosphorylation of wild type and mutant proteins (Fig. 1b Here we address this issue using oncogenic Met mutants. Met, and Supplementary Fig. S1b). the receptor for HGF (hepatocyte growth factor), is implicated The Met-specific TKI (tyrosine kinase inhibitor), PHA-665752 in the growth, survival and spread of various human cancers22–24 . (PHA), reduced the mutant M1268T basal phosphorylation level, Two mutations (M1268T and D1246N) in the kinase domain, whereas D1246N seemed resistant to it (Fig. 1c and Supplementary identified originally in human papillary renal carcinomas25 , are Fig. S1c). Wild-type Met was expressed at the plasma membrane in 1 Spatial Signalling Team, Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK. 2 Cell Biophysics Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK. 3 Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK. 4 Present address: Institut Curie, INSERM U-830, 26 rue d’Ulm, 75248 Paris Cedex 05, France. 5 Correspondence should be addressed to S.K. (e-mail: email@example.com) Received 1 February 2011; accepted 7 April 2011; published online 5 June 2011; DOI: 10.1038/ncb2257 NATURE CELL BIOLOGY VOLUME 13 | NUMBER 7 | JULY 2011 827 © 2011 Macmillan Publishers Limited. All rights reserved.
ARTICLES a d D1 ype Wild type Wild type + HGF D1246N M1268T T M 6N 68 t 24 12 ild W p-170 Met p-145 Met/DAPI P-Met-Y1234/1235 P-Met-Y1349 HSC70 Met/HSC70: 1 1.59 1.03 ± ± 0.16 0.06 4G10 / DAPI b No HGF HGF # 10 # P-Met-Y1349/Met 8 6 * Merge 4 * ## 2 0 e 6N T p 68 ty 24 12 ild D1 M W c e f lar) 60 * ellu DMSO PHA e) ** n d d d d rfac dow oun oun oun oun intracellular Met trac 120 Percentage of Percentage of P-Met/Met l l l ( = su l Unb Unb Unb 40 Unb Tota Tota Tota Tota Pull (=in 100 80 Wild type 20 60 D1246N 40 * 0 D1 ype 20 M 6N M1268T T 68 24 t 12 ild 0 W Biotin: + – + – + – 6N T 68 24 12 D1 p-145 Met P-Met-Y1234/1235 M Figure 1 Active Met mutants accumulate in intracellular compartments. (n = 3). ∗ P < 0.05. (d) Confocal sections of cells, stimulated, or not, (a) Western blots for Met, phosphorylated Met (Tyr 1234/1235 and with HGF and stained for Met (green) and for phospho-tyrosine clone 1349) and the constitutive heat shock protein 70 (HSC70). Numbers 4G10 (red), and with DAPI (blue). Arrows show plasma membrane Met. represent mean ± s.e.m. (arbitrary units, n = 6) of Met p-145/HSC70 Scale bars, 10 µm. (e,f) Proportion of intracellular pools versus total ratios. (b) P-Met-Y1349/Met ratios identified by densitometric analysis cellular Met as determined from a biotin surface removal assay. (e) Cells of western blots (not shown) in the indicated cells stimulated, or not, were surface-biotinylated and the surface protein fraction was removed with HGF for 15 min. Data are mean (arbitrary units) ± s.e.m. (n = 3). by streptavidin pulldown. The total sample before pulldown (total), the #, ∗ P < 0.05; ∗ compared with wild type, no HGF. (c) The indicated supernatant corresponding to the intracellular fraction (unbound; outlined cells were treated with the Met inhibitor PHA-665752 (PHA, 100 in red) and the surface fractions (bound) were analysed by western nM) or DMSO. The amount of phosphorylated Met (phosphorylated blotting for Met (left panels) and phosphorylated Met (Tyr 1234/1235; at Y1234/1235) was calculated as a percentage of the total Met, right panels). (f) The percentage of intracellular Met was calculated as a as assessed from densitometric analysis of western blots (shown in ratio to the total. Data are mean ± s.e.m. (n = 3). ∗ P < 0.05; ∗∗ P < 0.01. Supplementary Fig. S1c). Data are mean (arbitrary units) ± s.e.m. Uncropped images of blots are shown in Supplementary Fig. S9. unstimulated cells and in intracellular vesicles in HGF-stimulated cells7 . Met mutants shuttle between the plasma membrane and In unstimulated cells, the two mutants were expressed at both the endosomes and exhibit an impaired degradation plasma membrane and in punctate cytoplasmic structures (Fig. 1d). Unstimulated mutants co-localized with EEA1 (early endosome antigen Marked phospho-tyrosine staining occurred in mutant-expressing 1), as did HGF-stimulated wild-type Met (Fig. 2a,b). Significantly cells with co-localizations indicative of Met mutant activation at higher levels of co-localization between EEA1 and Met mutants the plasma membrane and in intracellular pools (Fig. 1d). PHA D1246N and M1268T were detected when compared with wild-type abolished phospho-tyrosine staining of cells expressing the M1268T Met (P < 0.05; Fig. 2b). Met mutants also accumulated in a perinuclear mutant; thus, a high phospho-tyrosine cellular content reflects Met area (Fig. 2a,b) and co-localized more with cyanine-3-coupled activity (Supplementary Fig. S1d). Separation procedures (see Methods; transferrin (Cy3–transferrin) and the small GTPases, Rab4 and Rab11, ref. 29) demonstrated that around 52% of the mutants were present than wild-type Met (Fig. 2b and Supplementary Fig. S2a–d). Little in the intracellular fraction, compared with 18% of the wild-type co-localization was detected with the late endosome/multivesicular Met (P < 0.05; Fig. 1e,f). body marker LAMP1 (lysosomal-associated membrane protein 1) 828 NATURE CELL BIOLOGY VOLUME 13 | NUMBER 7 | JULY 2011 © 2011 Macmillan Publishers Limited. All rights reserved.
ARTICLES a Met/DAPI EEA1 /DAPI Merge b 50 Wild type 70 * ** co-localization with Met D1246N 60 with perinuclear Met 40 Percentage of cells M1268T Wild type Percentage of * 50 30 * 40 * * 20 30 20 10 Wild type + HGF 10 0 0 EEA1 Transferrin Rab11 c Wild type M1268T Remained Total post- 25 90 Percentage of Met * internalization Internalized recycling Met recycling surface Percentage of 20 M1268T 15 60 Wild type 10 M1268T 30 5 p-145 Met 0 0 d GFP–Rab11 wild type e Wild type D1246N M1268T HeLa g TS Int. TS Int. GFP–Rab11S25N PHA: – – – – + + + + 100 * p-145 Met M1268T p-145 Met /HSC70 +20 * 80 p-145 Met Percentage of downregulated Met NS Time (min): P-Y1234/1235 0 15 15 0 15 15 (relative to GFP negative cells) 60 at the plasma membrane +10 4 °C 37 °C 4 °C 37 °C 40 0 20 ** ** 28 NS Met internalization 0 24 Percentage of –10 0 1 4 8 20 HGF (h) 16 –20 f HGF (h): 0 2 8 8 12 8 Wild type 4 –30 Wild type M1268T p-145 Met 0 M1268T DMSO PHA Figure 2 Met mutants shuttle between the plasma membrane and or the dominant-negative mutant GFP–Rab11S25N . Percentage of Met endosomes and are protected from degradation. (a,b) Cells were pretreated downregulation at the plasma membrane in transfected cells, compared with cycloheximide for 4 h. (a) Confocal sections of cells stimulated, or with non-transfected cells (GFP-negative cells; mean ± s.e.m., n = 3). not, with HGF and stained for Met (green) and for EEA1 (red), and with (e) Met/HSC70 ratios obtained by densitometric analysis of western blots DAPI (blue). Scale bars, 10 µm. (b) Cells were incubated with or without and plotted as a percentage of the initial content. The indicated cells were Cy3–transferrin for 30 min before fixation and stained for Met and EEA1 pretreated with cycloheximide and stimulated with HGF for the indicated or Rab11 and stained with DAPI (Supplementary Fig. S2a,c). Percentage times (mean ± s.e.m., n = 3). Statistical results are shown for 8 h versus of co-localization between Met and EEA1 or transferrin (mean ± s.e.m., 0 h. (f) Biotinylation degradation assay. The indicated cells were surface n = 3) or Rab11 (mean, n = 2) (left graph) and percentage of cells that were biotinylated and incubated at 37 ◦ C. At the indicated times, cells were positive for perinuclear Met staining (mean ± s.e.m., n = 3, right graph). lysed and the remaining biotinylated Met, after streptavidin pulldown, was (c) Biotinylation internalization/recycling assay. In the indicated cells, analysed by western blotting for Met. (g) Biotinylation internalization assay. the levels of surface biotinylated Met internalized (15 min) and recycled Cells expressing MetM1268T were pretreated (+), or not (−), with PHA for (15 min) were measured. Experiments were carried out in duplicate. Left, 30 min, surface biotinylated and incubated at 37 ◦ C for 15 min with or Met western blots after streptavidin pulldown. Right, quantification of Met without PHA. Top, Met or phospho-Met western blots after streptavidin internalization and recycling through densitometric analysis of bands in pulldown. Bottom, the graph represents the percentage of internalization western blots. Data are mean ± s.e.m. (n = 4, left graph) or mean (n = 1, (mean ± s.e.m. n = 3). TS, total surface; Int., internalized. ∗ P < 0.05; right graph). (d) Expression of Met at the plasma membrane evaluated by ∗∗ P < 0.01; NS, not significant. Uncropped images of blots are shown in flow cytometry in the indicated cells transfected with wild-type GFP–Rab11 Supplementary Fig. S9. or the lysosome marker, lysotracker (Supplementary Fig. S2e). The The rates of surface-biotinylated Met internalization and subse- level of Cy3–transferrin uptake was similar in all cells (Supplementary quent recycling were measured by streptavidin-agarose pulldown Fig. S2f). Thus, under basal conditions, D1246N and M1268T mutants of surface-biotinylated Met after different incubation periods (bi- localized on early and recycling endosomes. Together with localization otin cleavage at the cell surface allowing determination of in- of Met mutants at the plasma membrane and on endosomes (Fig. 1d), tracellular biotinylated Met; see Methods). After 15 min, around this indicated that they internalize and recycle back to the cell 20% of the MetM1268T had internalized (Fig. 2c), a level com- surface constitutively. parable to wild-type Met stimulated with HGF (Supplementay NATURE CELL BIOLOGY VOLUME 13 | NUMBER 7 | JULY 2011 829 © 2011 Macmillan Publishers Limited. All rights reserved.
ARTICLES Fig. S2g), whereas 10% of non-stimulated wild-type Met had in- The cells do not express EGFR (Supplementary Fig. S3k), excluding ternalized (P < 0.05; Fig. 2c). After 5 min, 14% of the MetD1246N effects on this RTK, which is also regulated by c-Cbl (ref. 35). The had internalized, compared with 3% of the wild-type Met (Sup- adaptor protein Grb2 is a major determinant of clathrin-dependent plementary Fig. S2h). On re-incubation at 37 ◦ C for a further RTK endocytosis on ligand stimulation33,34 , and a possible constitutive 15 min (see Methods), the proportion of internalized Met mutant association of the D1246N and M1268T mutants with Grb2 has M1268T was much lower when compared with wild-type Met been reported27,36 . Knocking down Grb2 reduced (threefold) the (Fig. 2c). Degradation was excluded (Supplementary Fig. S2i). Ex- level of constitutive endocytosis of MetM1268T (P < 0.05; Fig. 3c,d). pression of the dominant-negative GFP–Rab11S25N construct sig- Co-immunoprecipitation and immunofluorescence microscopy con- nificantly reduced the level of MetM1268T at the cell surface (mea- firmed the association between MetM1268T and Grb2 at the plasma sured by flow cytometry), but had no effect on wild-type Met membrane and on endosomes (Fig. 3e and Supplementary Fig. S3l,m). (Fig. 2d), indicating that increased mutant recycling is promoted, at Transferrin uptake, monitored as a control, was inhibited with least partially by Rab11. dynasore, or on CHC knockdown, but remained unchanged on Cell-surface wild-type Met and MetM1268T levels were analysed c-Cbl or Grb2 knockdown (Supplementary Fig. S3n). MetM1268T over time using a biotinylation degradation assay (see Methods). was further mutated on N1358H, in position +2 of Tyr 1356 in The level of M1268T mutant degradation was less than the wild the Met docking site, to abrogate the Grb2 consensus sequence type (Supplementary Fig. S2i), indicating that increased constitutive without affecting other effectors37 . For this, a human wild-type Met internalization led not to increased levels of degradation but, complementary DNA (hWt) was mutated on M1268T (hM1268T) or rather, to a delay, consistent with recycling to the cell surface. on both M1268T and N1358H (hM1268T/N1358H). The M1268T HGF stimulation normally induces high levels of degradation of mutation triggered an enhanced level of Met phosphorylation (Sup- endogenous wild-type Met (ref. 30), indicating that differences in plementary Fig. S3o) and co-localization with EEA1 (Fig. 3f). The degradation rates between Met forms would be amplified following level of co-localization of the double mutant with EEA1 was less such treatment. Met protein levels were analysed by western than that of the single mutant (P < 0.05) and was similar to that of blotting in cells pretreated with cycloheximide. Wild-type Met was wild-type Met (Fig. 3f). degraded progressively, with only 20% of the initial Met expression after 8 h of HGF stimulation (Fig. 2e). Conversely, the level of Met mutants require endocytosis to control actin cytoskeleton degradation of D1246N and M1268T Met mutants was reduced remodelling markedly, with Met levels non-significantly different between 0 Using these endocytic blockers, we investigated whether endoso- and 8 h of HGF stimulation (Fig. 2e); the results were confirmed mal accumulation of active Met mutants is responsible for cel- with a biotin degradation assay (Fig. 2f). This defect in degradation lular outcomes. Paxillin staining revealed that the numbers of did not result from impaired endocytosis on HGF stimulation cells with focal adhesions were reduced significantly for mutant- (Supplementary Fig. S2g). expressing cells (Supplementary Fig. S4a). Actin organization (visu- Activated Met mutants therefore accumulate on endosomal alized by phalloidin–Cy3 staining) was modified markedly, under compartments through constitutive shuttling between the plasma basal conditions, in cells expressing Met mutants (Fig. 4a); their membrane and endosomes, and defective degradation. A PHA-induced appearance resembling wild-type cells following HGF treatment decrease in the activation level of the M1268T mutant did not affect (Supplementary Fig. S4b). A total of 65–71% of D1246N- and the endocytosis levels (Fig. 2g). M1268T-expressing cells lacked stress fibres, whereas only 28% of wild-type-expressing cells had a similar morphology (Fig. 4b). Endocytosis of Met mutants is dependent on clathrin, dynamin, In M1268T-expressing cells, PHA significantly restored paxillin c-Cbl and Grb2 patches (Supplementary Fig. S4a) and stress fibres (Fig. 4a,b). Cells Endogenous Met internalization is dependent on dynamin and expressing the D1246N mutant did not respond to this treatment clathrin5,7 . The dynamin inhibitors dynasore31 and dynole 34-2 (ref. 32; (Fig. 4a,b), consistent with there being no reduction in the level of Supplementary Fig. S3a), a dynamin dominant-negative construct Met phosphorylation (Fig. 1c). However, Met knockdown restored (dynamin-2K44A –GFP; Supplementary Fig. S3b,c), clathrin heavy chain stress fibres in both mutant-expressing cells and had no effect in (CHC) transient and stable knockdown by RNA-mediated interference wild-type cells (Fig. 4a–c). (RNAi Supplementary Fig. S3d,e) and short hairpin RNA (shRNA; Decreasing the level of Met mutant accumulation on endosomes, Supplementary Fig. S3f,g) all inhibited HGF–Alexa 555 cellular uptake using dynasore (Fig. 4d), dynamin-2K44A –GFP expression, CHC stable and, therefore, Met endocytosis7 (Supplementary Fig. S3h). knockdown, c-Cbl knockdown, Grb2 knockdown or expression of Grb2 All blockers inhibited the constitutive internalization of the dominant-negative mutants in the SH2 (Src homology 2; Grb2-89A) mutants: Fig. 3a and Supplementary Fig. S3i show blocking of or SH3 (Grb2-49L/203R) domains38 (Fig. 4e and Supplementary MetM1268T internalization by dynasore or CHC knockdown; Fig. 3b Fig. S4c–i), restored stress fibres. There was no effect on wild-type cells and Supplementary Fig. S3j show increased levels of MetM1268T at the (Supplementary Fig. S4f). In cell lines expressing the human constructs, plasma membrane and its decreased level in intracellular pools in cells the percentage of cells lacking stress fibres was significantly higher in knocked down for CHC. Met mutant hM1268T, than in hWt cells (P < 0.05); stress fibres were The E3 ubiquitin ligase and adaptor protein c-Cbl plays a major role restored by PHA in cells expressing hM1268T (Fig. 4f). Cells expressing in promoting RTK endocytosis33,34 . Knocking down c-Cbl markedly Met hM1268T/N1358H had restored stress fibres and looked similar decreased the level of constitutive endocytosis of MetM1268T (Fig. 3c,d). to wild-type cells (Fig. 4f). 830 NATURE CELL BIOLOGY VOLUME 13 | NUMBER 7 | JULY 2011 © 2011 Macmillan Publishers Limited. All rights reserved.
ARTICLES a Total b Met /DAPI d RNAi RNAi surface Internalized Control c-Cbl Control Grb2 p-145 c-Cbl Grb2 M1268T M1268T Met Dynasore: – – – + Tubulin Tubulin Time (min): 0 15 15 4 °C 37 °C M1268T M1268T RNAi control RNAi CHC c Total surface Internalized 25 RNAi control e 12 e Percentage of Met M ype 12 e 12 e T T W 8T T M yp M typ M typ 68 68 68 RNAi c-Cbl internalization 6 t t RNAi control 20 12 ild ild ild ild RNAi Grb2 W W W p-145 p-170 RNAi c-Cbl 15 Met p-145 Met RNAi Grb2 10 * Grb2 5 Grb2 IP Beads IgG TL Time (min): 0 15 15 4 °C 37 °C 0 M1268T M1268T f Met/DAPI EEA1/ DAPI Merge # Percentage of Met / EEA1 co-localization 30 * hWt 20 10 hM1268T 0 t /N 1 T 58 8T hM hW hM 268 13 26 H 1 hM1268T/ N1358H Figure 3 Endocytosis of Met mutant is dependent on clathrin, dynamin, c-Cbl (d) c-Cbl, Grb2 and tubulin western blots. (e) Grb2 immunoprecipitation and Grb2. (a) Biotinylation internalization assay with cells pretreated with from cells expressing wild-type Met and MetM1268T . Met and Grb2 western DMSO (−) or 80 µM dynasore (+) for 30 min. (b) Confocal sections of cells blots from immunoprecipitates and from total lysates (TL). Controls stained for Met (green) and DAPI (blue). Arrows show plasma membrane Met. include incubation with beads alone or coupled to an isotype-matched IgG. Cells expressing MetM1268T were transfected with control or CHC RNAi. Scale (f) Left, confocal sections of cells expressing human Met constructs, hWt, bars, 10 µm. (c,d) M1268T Met mutant expressing cells were transfected hM1268T and hM1268T/N1358H, pretreated with cycloheximide for 4 h with control, c-Cbl or Grb2 RNAi. (c) Left, biotinylation internalization and stained for Met (green), EEA1 (red) and DAPI (blue). Scale bars, 10 µm. assay. Each experiment was done in duplicate. Right, data are mean Right, percentage of co-localization (mean ± s.e.m., n = 3). #, ∗ P < 0.05. ± s.e.m.for control and Grb2 RNAi (n = 3) and mean for Cbl RNAi (n = 1). Uncropped images of blots are shown in Supplementary Fig. S9. Met mutants induce Rac1 activation, which is dependent and Supplementary Fig. S5f), the percentage of Met-mutant-expressing on endocytosis cells positive for Rac1 staining at membrane protrusions was reduced Rac1 induces actin stress fibre loss39 . Activation was detected in cells to that of wild-type-expressing cells. expressing D1246N and M1268T mutants (Fig. 5a) and was reduced c-Cbl or Grb2 knockdown led to a reduction in the per- significantly by PHA in cells expressing the M1268T mutant (P < 0.05; centage of mutant-expressing cells exhibiting Rac1 at membrane Supplementary Fig. S5a). Inhibition by Rac1 RNAi (Fig. 5b and protrusions, to the level of wild-type-expressing cells, whereas Supplementary Fig. S5b) and Rac inhibitor NSC23766 (Supplementary no such change occurred in wild-type cells (P < 0.05; Fig. 5d Fig. S5c) significantly reduced the percentage of cells lacking stress and Supplementary Fig. S5f). Expression of dynamin-2K44A –GFP fibres in mutant, but not in wild-type, cells. also decreased the level of Rac1 localization to membrane pro- In cells expressing Met mutant, Rac1 was observed at membrane trusions significantly (Supplementary Fig. S5g). CHC or Grb2 protrusions (Fig. 5c,d and Supplementary Fig. S5d), resembling HGF- knockdown reduced the level of Rac1 activation in mutant cells, stimulated wild-type cells (Supplementary Fig. S5e). On treatment with whereas no change occurred in wild-type cells (Fig. 5e and Sup- PHA (Fig. 5c and Supplementary Fig. S5d), or Met knockdown (Fig. 5d plementary Fig. S5h). NATURE CELL BIOLOGY VOLUME 13 | NUMBER 7 | JULY 2011 831 © 2011 Macmillan Publishers Limited. All rights reserved.
ARTICLES a D1246N M1268T Wild type b Wild type D1246N M1268T ### ## 90 DMSO NS # 80 (RNAi control) *** 70 *** lacking stress fibres Percentage of cells 60 50 40 NS PHA 30 # 20 10 0 RNAi Met Ai A Ai HA RN PH l RN PH l et et Ai A et tro tro l tro M M M RN P on on on C C C Phalloidin /DAPI e 100 f Percentage of M1268T cells lacking stress hWt c RNAi: Control Met Control Met Control Met hM1268T p170 hM1268T + PHA Met 80 p145 fibres relative to control hM1268T/N1358H ** *** Tubulin ## 60 ** 40 # Wild type D1246N M1268T lacking stress fibres * Percentage of cells *** *** 40 * 30 d D1246N M1268T 20 20 10 Dynasore 0 –m c yc RN –G l G G R ic C 20 –m 2 RN A C FP 49 -8 G l sh 44A tro 2- b2 Ai b 3R y 0 L/ 9A rb A H rb r N -C -2 K Con in Phalloidin/DAPI m na Dy Figure 4 Met mutants require endocytosis to control actin cytoskeleton lacking stress fibres when transfected with dynamin-2K44A –GFP (n = 3), remodelling. (a,b and d-f) Indicated cells were stained with Cy3–phalloidin or transduced with CHC shRNA (n = 3) or transfected with c-Cbl RNAi (red) and DAPI (blue). (a,b) The indicated cells were treated with DMSO (n = 4), Grb2 RNAi (n = 3), Grb2-89A–myc (n = 3) or Grb2-49L/203R–myc or PHA for 90 min or transfected with control or Met RNAi. (a) Confocal (n = 3). Data are normalized to appropriate controls (GFP, shRNA control, sections. Scale bars, 10 µm. (b) Percentage of cells lacking stress fibres. RNAi control, myc–Grb2-Wt) and are mean ± s.e.m. (f) Percentage of cells Data are mean ± s.e.m. (n = 3). Control is the average of DMSO (n = 3) expressing human Met forms, hWt, hM1268T and hM1268T/N1358H, and RNAi control (n = 3). (c) Western blots showing Met and tubulin that lack stress fibres. hM1268T cells were treated, or not, with PHA for expression after control or Met RNAi transfections in the indicated cells. 90 min. Data are mean ± s.e.m. (n = 3). #, ∗ P < 0.05; ##, ∗∗ P < 0.01; ###, (d) Confocal sections of D1246N and M1268T cells treated with dynasore ∗∗∗ P < 0.001; NS: not significant. Uncropped images of blots are shown in for 90 min. Scale bars, 10 µm. (e) Percentage of MetM1268T -expressing cells Supplementary Fig. S9. In cells expressing mutant Met, Rac1 was detected in punctate membrane protrusions, appearing similar to hWt cells (Fig. 5g). Thus, cytoplasmic structures, partially co-localizing with EEA1 (Fig. 5c). The Rac1 is activated by endosome-localised Met mutants; such activation level of Rac1 localization at membrane protrusions was reduced by the is responsible for the observed loss of stress fibres. recycling inhibitor primaquine (data not shown). Activated Rac1 was visualized on endosomes (co-localization of Rac1 and GST–PAK-PBD; Endocytosis is required for Met mutants to stimulate glutathione S-transferase (GST)-tagged p21 activated kinase 1 protein cell migration (PAK) Rac/Cdc42 (p21) binding domain (PBD)) co-localizing with After 2 h of incubation, more cells expressing D1246N and M1268T MetM1268T (Fig. 5f) and MetD1246N (not shown). The percentage of cells mutants (>3-fold) had migrated through Transwell membranes than with Rac1 at membrane protrusions was significantly higher in cells cells expressing wild-type Met (P < 0.05; Fig. 6a and Supplementary expressing the hM1268T Met mutant than in hWt cells (P < 0.05) and Fig. S6a). HGF stimulation further increased the number of mutant this was reduced by PHA in cells expressing hM1268T. Cells expressing cells migrating (Supplementary Fig. S6b). PHA treatment inhibited hM1268T/N1358H Met had reduced levels of Rac1 localization at enhanced migration of the M1268T- (Fig. 6b), but not the D1246N-, 832 NATURE CELL BIOLOGY VOLUME 13 | NUMBER 7 | JULY 2011 © 2011 Macmillan Publishers Limited. All rights reserved.
ARTICLES a b NS c 60 * RNAi control Wild type D1246N with no stress fibres Percentage of cells ep RNAi Rac1 T 6N ty 68 24 ild Rac1/ DAPI 12 40 D1 W M Rac1–GTP Rac1 20 d + EEA1 + EEA1 # # 0 M1268T M1268T + PHA Percentage of cells with Rac1 Wild type M1268T 70 * at membrane protrusions * e 60 RNAi: Control CHC Wild type NS CHC 50 D1246N M1268T Rac1–GTP 40 30 Rac1 + EEA1 Co b2 M ol c-C et M ol c-Cet Co b2 c-Cet b2 M ol Gr bl Gr bl Gr bl RNAi: ntr ntr ntr Co HSC70 M1268T f Met Rac1 GST Merge g ## 1 250 GST–PAK-PBD Percentage of cells with Rac1 ## Intensity 200 Rac1 150 70 at membrane protrusions GST 2 100 ** Met 60 50 0 50 0 1 2 3 Distance (µm) 40 1 30 2 250 Rac1 20 GST Intensity 200 10 150 Met GST 100 0 50 hWt 0 0 1 2 3 4 5 hM1268T Distance (µm) hM1268T + PHA M1268T hM1268T/N1358H Figure 5 Met mutants induce Rac1 activation, which is dependent on GST–PAK-PBD (binds to Rac–GTP). Cells were further stained for Met endocytosis. (a) Western blots showing the levels of Rac1–GTP, measured (blue), Rac1 (green) and GST (red). A representative confocal picture of by GST–CRIB pulldown, and total Rac1 (see Methods). (b) Percentage MetM1268T -expressing cells is shown. Arrows indicate activated Rac1 at the of wild-type and M1268T cells lacking stress fibres when transfected with plasma membrane. Right panels, Magnifications 1 and 2 correspond to control or Rac1 RNAi. Data are mean ± s.e.m. (n = 3). (c) Confocal sections cellular location number 1 and 2 in the merged picture of cells treated of the indicated cells treated with DMSO or PHA for 60 min and stained for with GST–PAK-PBD (upper right picture). Plots of the intensity profiles, at Rac1 (red) and EEA1 (green) and with DAPI (blue). Arrows indicate Rac1 at different emission wavelengths corresponding to the signals of Met, Rac1 membrane protrusions. Scale bars, 10 µm. (d) Percentage of the indicated and GST, of a set of pixels distributed on a line drawn across vesicles (in cells with marked Rac1 staining present at membrane protrusions when the magnifications) are shown. Scale bars, 10 µm. (g) Percentage of cells, transfected with control, Met, c-Cbl or Grb2 RNAi. Data are mean ± s.e.m. expressing human Met forms, hWt, hM1268T and hM1268T/N1358H, (n = 3). (e) Western blots showing the levels of CHC, Rac1–GTP (measured that exhibit Rac1 at membrane protrusions. hM1268T were treated, or not, by GST–CRIB pulldown), total Rac1 and HSC70 in MetM1268T -expressing with PHA for 90 min. Data are mean ± s.e.m. (n = 3). #, * P < 0.05; ##, cells transfected with control or CHC RNAi. (f) The localization of active ∗∗ P < 0.01; NS: not significant. Uncropped images of blots are shown in Rac1 in cells was determined by incubating fixed cells with purified Supplementary Fig. S9. expressing cells (data not shown), whereas Met knockdown inhibited CHC (Supplementary Fig. S3g), but not by transduction with control migration in both cell lines (Fig. 6b and Supplementary Fig. S6c). PHA shRNA (Fig. 6d and Supplementary Fig. S6f). or Met knockdown had no effect on the migration of cells expressing wild-type Met (Supplementary Fig. S6d). Blocking endocytosis reduces tumour transformation, stimulated All endocytic blockers significantly reduced the enhanced migration by Met mutants, both in vitro and in vivo level of both mutant-expressing cells (Fig. 6a,b and Supplementary Growth under anchorage-independent conditions was assessed in Fig. S6c), but not wild-type cell migration (Fig. 6a and Supplementary agar gels. After 6 or 9 days of culture, colony number (Fig. 7a) Fig. S6d). Rac1 inhibition, or knockdown, decreased the level and area (Fig. 7a,b and Supplementary Fig. S7a) were increased of M1268T mutant (Fig. 6c), but not wild-type (Supplementary significantly for mutant-expressing cells, compared with wild-type cells. Fig. S6e), cell migration. M1268T mutant, but not wild-type, cells Met knockdown reduced colony area (Fig. 7b). Dynasore treatment, colonized the lung and formed metastases 10 days after injection into from day five, reduced the colony number (Fig. 7a,c) and area the tail veins of nude mice (data not shown). For the M1268T mutant (Supplementary Fig. S7a) of mutant-expressing cells. Stable CHC cells, lung colonization was totally inhibited by shRNA knockdown of knockdown (Supplementary Fig. S7b) prevented growth of cells NATURE CELL BIOLOGY VOLUME 13 | NUMBER 7 | JULY 2011 833 © 2011 Macmillan Publishers Limited. All rights reserved.
ARTICLES a DMSO Dynasore b a Wild type D1246N M1268T Percentage of M1268T cells that 100 migrated relative to control ## DMSO 35 80 ** Number of migrated cells * * 28 ## 60 * 104 190 *** 214 ** ±7 ± 47 ± 20 ** 21 40 Dynasore 14 20 NS ** 7 0 136 71 124 l 44 i M A RN –G t sh Ai FP RN A C 12 RN c-C C T/ G l 13 2 H ± 10 ± 26 ± 23 tro 68 Ai b A e N rb -2 K NA PH H Ai CH 58 on RN C 0 C R e N 8T p 6 ty 6 24 in 12 hM b c m ild D1 10 100 Percentage of colony number M Wild type na ** W Dy D1246N 8 M1268T 80 Colony area (a.u.) c Percentage of M1268T cells that d * 100 * migrated relative to control 6 60 ** M1268T shRNA control 80 *** 4 40 M1268T *** 60 shRNA CHC *** * 2 20 40 M1268T M1268T shRNA control shRNA CHC 0 0 *** A M l RN NA c-C t Ai i G bl RNcon b2 A iM l RN NA c-C t Ai i G bl R con b2 A M l RN i c et G l 2 RN Ai tro RN Ai ore RN i c- et hM Grb l /N 1 2 2 58 T RN a s l RN A tro RN NA tro Ai -Cb R i e R i e Ai Cb y n ro rb 20 800 13 68 A M r r Lung weight (mg) RNcon D ont H i C Ai 600 RN 0 Control 400 NSC23766 200 * Figure 7 Blocking endocytosis reduces in vitro tumour transformation Rac1 RNAi stimulated by Met mutants. Cells were cultured in soft agar. Experiments 0 were done in triplicate. (a) From day 5, DMSO or dynasore was added daily to the medium. Pictures at day 9. Numbers are the average total number of Figure 6 Met mutants require endocytosis to stimulate cell migration. colonies per plate ± s.e.m. (n = 3; see colony area Supplementary Fig. S7a). (a) Average number of the indicated cells that have migrated through (b) The indicated cells were transfected with control, Met, c-Cbl or Grb2 Transwells over 2 h of incubation in the presence of DMSO or dynasore. RNAi. Data are the average colony areas ± s.e.m. (a.u., arbitrary units). Each experiment was done in triplicate. Data are mean ± s.e.m. (n = 3). Pictures (not shown) were taken at day 6 (n = 3). (c) Data are the mean (b) Percentage of MetM1268T -expressing cells that migrated through of the percentage of reduction ± s.e.m.in colony number formed by cells Transwells when treated with PHA (n = 2), transfected with Met RNAi expressing MetM1268T when treated with dynasore or transfected with Met, (n = 3), dynamin-2K44A –GFP (n = 3) or CHC RNAi (n = 3), transduced c-Cbl or Grb2 RNAi versus their appropriate controls (DMSO, RNAi control; with shRNA CHC (n = 2), transfected with c-Cbl (n = 6) or Grb2 RNAi n = 3). The last column represents cells expressing hM1268T/N1358H Met (n = 3). The last column represents the percentage of cells expressing versus cells expressing hM1268T Met (mean ± s.e.m., n = 3). ∗ P < 0.05; hM1268T/N1358H Met (n = 3) that migrated. Data are normalized to ∗∗ P < 0.01; ∗∗∗ P < 0.001. appropriate controls (DMSO, GFP, shRNA control, RNAi control and cells expressing hM1268T Met, respectively). Each experiment was done in (Fig. 8a). The level of phosphorylation remained high in the mutant triplicate. Data are mean ± s.e.m. (c) Percentage of M1268T cells that have proteins in tumour tissue (Supplementary Fig. S7c). migrated through Transwells when treated with the Rac inhibitor NSC23766 or transfected with Rac1 RNAi over appropriate controls (DMSO and RNAi Dynasore, PHA or control dimethylsulphoxide (DMSO) diluents control, respectively). Each experiment was done in triplicate. Data are mean were applied topically to the skin over tumours that had achieved ± s.e.m. (n = 3). (d) MetM1268T -expressing cells (500,000) transduced with 50 mm3 size (Fig. 8b), or on the day following cell injection control or CHC shRNA were injected intravenously in nude mice and, after (Supplementary Fig. S7d). Tumours derived from mutant-expressing 10 days, their lungs were analysed and weighed. Data are mean ± s.e.m. of n = 5 mice. ∗ P < 0.05; ##, ∗∗ P < 0.01; ∗∗∗ P < 0.001; NS: not significant. cells treated with dynasore were reduced significantly in size (40–50%, compared with DMSO), after four and five days of treatment (Fig. 8b). expressing the M1268T mutant. c-Cbl and Grb2 knockdown reduced A significant inhibition of M1268T tumour size was also obtained with the colony area (Fig. 7b) and number (Fig. 7c) of mutant-expressing the specific dynamin inhibitor, dynole 34.2 (ref. 32; Supplementary cells to levels attained by wild-type cells, whereas these treatments had Fig. S7e). CHC knockdown in the M1268T cells by shRNA caused a no influence on wild-type cells (Fig. 7b). Finally, cells expressing the significant decrease, of 50%, in tumour volume (Fig. 8c). The Met double mutant hM1268T/N1358H exhibited a reduced colony number TKI PHA induced a similar inhibition of M1268T tumour size as (Fig. 7c) and area (by 50%, not shown) when compared with cells obtained with dynasore (Fig. 8b). D1246N tumours were resistant expressing Met hM1268T (P < 0.05). to PHA (Fig. 8b), consistent with in vitro results (Figs 1c, 4a,b and Cell types were grafted subcutaneously into nude mice and cells Supplementary Fig. S5d). expressing MetM1268T and MetD1246N mutants rapidly formed tumours As wild-type cells can form tumours, although later than that were palpable at one week post-graft. At 11–12 days, all mice mutants, control experiments were carried out in which wild- were killed because the tumour volumes of mutant cells had reached type cells were allowed to form tumours, and dynasore, dynole >500 mm3 , whereas wild-type cells had not formed palpable tumours 34-2, PHA or DMSO was applied once they had achieved 834 NATURE CELL BIOLOGY VOLUME 13 | NUMBER 7 | JULY 2011 © 2011 Macmillan Publishers Limited. All rights reserved.
ARTICLES a 700 Wild type b DMSO PHA Dynasore c d NS Percentage of tumour volume Tumour volume (mm3) 100 600 D124N6 NS NS ** 120 500 M126T8 100 80 DMSO 400 100 80 PHA tumour volume Percentage of tumour volume 60 Percentage of 300 * * ** 80 Dynasore 60 200 40 60 100 40 0 20 40 20 0 2 4 6 8 10 12 0 20 Days post-graft Day of 0 M1268T treatment: 4 5 4 5 Day of 0 D1246N M1268T shRNA control treatment: 4 5 e Met/ EEA1/ DAPI shRNA CHC Wild type Wild type M1268T f M1268T D1246N g M1268T DMSO Grafts: DMSO Dyn DMSO Dyn Grafts: shRNA shRNA Met control CHC Met P-Met-Y1234/1235 HSC70 P-Met-Y1234/1235 Dynasore P-Met/Met: 1 1.12 1 1.46 Tubulin ± ± 0.15 0.20 P-Met/Met: 1 0.91 ± 0.20 Figure 8 Blocking endocytosis reduces in vivo tumour transformation tumours once they had achieved 50 mm3 size. The bars represent the stimulated by Met mutants. The indicated cells (500,000) were injected percentage of tumour volume derived from wild-type Met-expressing cells, subcutaneously into nude mice (minimum 5 mice per group in each after 4 and 5 days of treatment with PHA or dynasore relative to DMSO experiment). (a) Tumour growth curves of the different cell lines over time. treatment. Data are mean ± s.e.m. of n = 10 mice and are pooled from Data are mean ± s.e.m. of n = 10 mice from two independent experiments. two independent experiments. (e) Confocal sections of tumour sections (b) Control DMSO diluents, PHA (100 nM) or dynasore (80 µM) was from wild-type and M1268T-expressing cells (from b and d) stained applied topically to the skin over tumours once they had achieved 50 mm3 for Met (green) and EEA1 (red), and with DAPI (blue). Arrow heads size. Percentage of tumour volume (data are mean ± s.e.m. of n = 10 show plasma membrane Met. Scale bars, 10 µm. (f,g) Western blots for mice from two independent experiments) derived from MetD1246N - and phospho-Met (Tyr 1234/1235), total Met and HSC70 carried out on MetM1268T -expressing cells, after four and five days of treatment with PHA tumour samples from three different mice for each condition (from b or or dynasore relative to DMSO treatment (see Methods). (c) Percentage of c; Dyn, dynasore). The numbers are the fold increases (mean ± s.e.m.) tumour volume derived from MetM1268T -expressing cells transduced with in the level of phosphorylated Met (P-Met/Met) for dynasore treatment control (mean ± s.e.m. of n = 6 mice) or CHC shRNA (mean ± s.e.m. versus DMSO (f) or for CHC shRNA cells versus control shRNA cells (g). of n = 7 mice), after 9 days post-graft. (d) PHA (100 nM), dynasore ∗ P < 0.05; ∗∗ P < 0.01; NS: not significant. Uncropped images of blots (80 µM) or control DMSO diluents were applied topically to the skin over are shown in Supplementary Fig. S9. 50 mm3 size (Fig. 8d and Supplementary Fig. S7e), or on the Generally, the global intensity of activation has been implicated day following cell injection (Supplementary Fig. S7d). No in RTK oncogenicity, with little attention paid to the precise change occurred in tumour size with either treatment. The localization of the generated signals. Thus, it has been assumed efficacy of dynasore in reducing the level of mutant local- that D1246N and M1268T mutants are oncogenic simply because ization in EEA1-positive endosomes and increasing it at the they are highly activated27,40 . Recent studies however have reported plasma membrane was verified by immunofluorescence mi- signalling of mutated activated RTKs, in an immature form, from croscopy (Fig. 8e). intracellular compartments of the secretory pathway41–44 , leading to Unexpectedly, dynasore treatment or CHC knockdown did not tumorigenesis41,45 . We suggest that Met mutants D1246N and M1268T decrease the levels of phosphorylation of the different Met mutants in are oncogenic not only because they are activated but also because tumours (Fig. 8f,g), and in vitro CHC or c-Cbl or Grb2 knockdown also they signal on endosomes. Thus, following inhibition of endocytosis, did not reduce the levels of MetM1268T phosphorylation (Supplementary the mutants remain activated but are not oncogenic. Increased levels Figs. S7f–h). Thus, the high level of tumorigenicity in vivo of D1246N of endocytic trafficking of Met mutants influence their oncogenicity, and M1268T mutants is linked directly to enhanced endosomal with activation and altered endocytic trafficking together leading trafficking, leading to their accumulation and signalling on endosomes to transformation. Present models of tumorigenesis stimulated by (Supplementary Fig. S8, model). oncogenic mature forms of RTK may need to be refined to encompass the potential contribution of endosomal signalling. DISCUSSION Initially considered solely as the means whereby RTK desensitization Two distinct activating mutations in the kinase domain of Met, D1246N was achieved, RTK endocytosis has recently been reported to promote and M1268T, lead to increased levels of Met endocytosis/recycling signals2,5,7,9,10 and functions15,46 that play a role in tumour evolution. and reduced levels of Met degradation. This perturbed trafficking Some endocytic proteins, such as HIP1 or Rab25, are overexpressed of activated Met mutants induces neoplastic transformation, directly in human cancers19,20 and mutant p53 drives cell invasion by linking endocytosis and tumorigenicity. promoting α5β1 integrin and EGFR recycling and EGFR constitutive NATURE CELL BIOLOGY VOLUME 13 | NUMBER 7 | JULY 2011 835 © 2011 Macmillan Publishers Limited. All rights reserved.
ARTICLES activation47 . Such studies report on modifications of molecules AUTHOR CONTRIBUTIONS that impinge on RTK trafficking and signalling. Modification of C.J. carried out and analysed most experiments using the NIH3T3 cells both in vitro and in vivo, including characterization of the cell lines, western blots, RTK endocytic trafficking on its own, leading to enhanced levels immunofluorescence analyses, Transwell migration, soft-agar assays, flow cytometry of signalling and transformation, has not been reported; however, analysis, immunoprecipitation and most separation procedures investigating several oncogenic RTKs have been shown to escape degradation35,48,49 . internalization, degradation and recycling. R.B. did most RNAi knockdowns and transfections followed by the subsequent migration assays, soft-agar assays, Here we provide evidence of a direct link between RTK endocytosis western blots and immunofluorescence analysis. R.B. carried out transfection and and cancer development. characterization of hM1268T and hM1268T/N1358H mutants in NIH3T3 cells. The increased level of endocytosis activity of Met mutants is L.M. carried out Rac1 activation assays following Grb2 RNAi and PHA treatment, established the detection of active Rac and helped with immunofluorescence dependent on clathrin, dynamin, c-Cbl and Grb2, but is independent analyses and analysis of hM1268T and hM1268T/N1358H cells. V.C. designed of high activation status, because MetM1268T internalizes just as well and developed the hM1268T and hM1268T/N1358H mutants. S.K. conceived when its activity is strongly reduced by a Met-specific TKI. Previous the project, designed experiments, interpreted the data and carried out some of the immunofluorescence and confocal microscopy analyses. I.R.H. advised on studies showed that the kinase-dead EGFRK721A internalizes50 and that, the design of the in vivo experiments, trained C.J. and carried out inoculations if EGFR kinase activity is partially inhibited, ubiquitylation of the of tumour cells. S.K. and C.J. wrote the manuscript, with additional input receptor can mediate its internalization51 . Dimerization of RTKs has from I.R.H. also been proposed to be required for their internalization, rather than COMPETING FINANCIAL INTERESTS their activation52 . We detected no obvious differences in ubiquitylation The authors declare no competing financial interests. levels in our cells (data not shown). Endosomal signalling of RTK, by spatially and temporally restricting Published online at http://www.nature.com/naturecellbiology signalling, contributes to pathway specificity5,7,13,14 . The basis of Reprints and permissions information is available online at http://www.nature.com/ reprints such selectivity has remained elusive because Met recruits almost all downstream signals through only two tyrosines (Tyr 1349 and Tyr 1356; 1. Teis, D., Wunderlich, W. & Huber, L. A. Localization of the MP1-MAPK scaffold ref. 22). We propose that continuous endocytosis/recycling, associated complex to endosomes is mediated by p14 and required for signal transduction. with an impaired degradation, contributes to the maintenance of Dev. Cell 3, 803–814 (2002). 2. Palamidessi, A. et al. Endocytic trafficking of Rac is required for the spatial activated receptor on endosomes and, therefore, to the persistence restriction of signaling in cell migration. Cell 134, 135–147 (2008). of endosomal signals. Thus, signal specificity (generated through 3. Schenck, A. et al. The endosomal protein Appl1 mediates Akt substrate specificity and cell survival in vertebrate development. Cell 133, 486–497 (2008). endosomal signalling) and persistence together may constitute 4. McPherson, P. S., Kay, B. K. & Hussain, N. K. Signaling on the endocytic pathway. ‘transforming signals’. Traffic 2, 375–384 (2001). 5. Kermorgant, S., Zicha, D. & Parker, P. J. PKC controls HGF-dependent c-Met traffic, The enhanced level of endocytosis/recycling of the two transforming signalling and cell migration. EMBO J. 23, 3721–3734 (2004). mutants, associated with their impaired degradation, indicates that 6. Miaczynska, M., Pelkmans, L. & Zerial, M. Not just a sink: endosomes in control of they undergo a persistent shuttling between the plasma membrane and signal transduction. Curr. Opin. Cell Biol. 16, 400–406 (2004). 7. Kermorgant, S. & Parker, P. J. Receptor trafficking controls weak signal delivery: endosomes. Such shuttling of ligand-stimulated RTKs, and integrins, a strategy used by c-Met for STAT3 nuclear accumulation. J. 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