A direct role for Met endocytosis in tumorigenesis

 
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: s.kermorgant@qmul.ac.uk)

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
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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;
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