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• W2020067532 abstract "Upon hepatocyte growth factor stimulation, its receptor c-Met is rapidly internalized via clathrin-coated vesicles and traffics through an early endosomal compartment. We show here that c-Met accumulates progressively in perinuclear compartments, which in part include the Golgi. The c-Met content in the Golgi is principally the newly synthesized precursor form and, to a lesser extent, the internalized, recycling c-Met. By following the trafficking of c-Met inside the cell using a semi-automatic procedure and using inhibition or activation of protein kinase C (PKC) and microtubule depolymerizing agents, we show that PKC positively controls the trans-cytosolic movement of c-Met along microtubules. In parallel to its traffic, internalized c-Met is progressively degraded by a proteasome-sensitive mechanism; the lysosomal pathway does not play a substantial role. Inhibition or promotion of c-Met traffic to the perinuclear compartment does not alter the kinetics of proteasome-dependent c-Met degradation. Thus susceptibility to proteasomal degradation is not a consequence of post-endocytic traffic. The data define a PKC-controlled traffic pathway for c-Met that operates independently of its degradative pathway. Upon hepatocyte growth factor stimulation, its receptor c-Met is rapidly internalized via clathrin-coated vesicles and traffics through an early endosomal compartment. We show here that c-Met accumulates progressively in perinuclear compartments, which in part include the Golgi. The c-Met content in the Golgi is principally the newly synthesized precursor form and, to a lesser extent, the internalized, recycling c-Met. By following the trafficking of c-Met inside the cell using a semi-automatic procedure and using inhibition or activation of protein kinase C (PKC) and microtubule depolymerizing agents, we show that PKC positively controls the trans-cytosolic movement of c-Met along microtubules. In parallel to its traffic, internalized c-Met is progressively degraded by a proteasome-sensitive mechanism; the lysosomal pathway does not play a substantial role. Inhibition or promotion of c-Met traffic to the perinuclear compartment does not alter the kinetics of proteasome-dependent c-Met degradation. Thus susceptibility to proteasomal degradation is not a consequence of post-endocytic traffic. The data define a PKC-controlled traffic pathway for c-Met that operates independently of its degradative pathway. The tyrosine kinase receptor c-Met is the high affinity receptor for hepatocyte growth factor (HGF). 1The abbreviations used are: HGF, hepatocyte growth factor; EGFR, epidermal growth factor receptor; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; PBS, phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic acid; BIM-I, bisindolylmaleimide I; CMV, cytomegalovirus; ANOVA, analysis of variance. Signaling via this receptor-ligand pair can induce diverse biological events. In vitro, these include scattering, invasion, proliferation, branching morphogenesis, and angiogenesis. In vivo, it is responsible for many processes during embryonic development and a variety of activities in the adult; many of these normal activities have been implicated in its role in tumorigenesis and metastasis (reviewed in Refs. 1Trusolino L. Comoglio P.M. Nat. Rev. Cancer. 2002; 2: 289-300Crossref PubMed Scopus (631) Google Scholar and 2Zhang Y.W. Vande Woude G.F. J. Cell. Biochem. 2003; 88: 408-417Crossref PubMed Scopus (272) Google Scholar). Overexpression of c-Met has been observed in a large number of human tumors, correlating closely with metastatic tendency and poor prognosis (3Di Renzo M.F. Olivero M. Katsaros D. Crepaldi T. Gaglia P. Zola P. Sismondi P. Comoglio P.M. Int. J. Cancer. 1994; 58: 658-662Crossref PubMed Scopus (201) Google Scholar, 4Di Renzo M.F. Olivero M. Giacomini A. Porte H. Chastre E. Mirossay L. Nordlinger B. Bretti S. Bottardi S. Giordano S. Plebani M. Gespach C. Comoglio P.M. Clin Cancer Res. 1995; 1: 147-154PubMed Google Scholar, 5Ghoussoub R.A. Dillon D.A. D'Aquila T. Rimm E.B. Fearon E.R. Rimm D.L. Cancer. 1998; 82: 1513-1520Crossref PubMed Scopus (179) Google Scholar, 6Tsarfaty I. Alvord W.G. Resau J.H. Altstock R.T. Lidereau R. Bieche I. Bertrand F. Horev J. Klabansky R.L. Keydar I. Vande Woude G.F. Anal. Quant. Cytol. Histol. 1999; 21: 397-408PubMed Google Scholar). Furthermore, germ line missense mutations of c-Met, which lead to increased tyrosine kinase activity, have been reported in childhood hepatocellular carcinoma (7Park W.S. Dong S.M. Kim S.Y. Na E.Y. Shin M.S. Pi J.H. Kim B.J. Bae J.H. Hong Y.K. Lee K.S. Lee S.H. Yoo N.J. Jang J.J. Pack S. Zhuang Z. Schmidt L. Zbar B. Lee J.Y. Cancer Res. 1999; 59: 307-310PubMed Google Scholar). A molecular understanding of how this receptor is switched on and off will provide the basis for developing rational interventions in such situations. C-Met, encoded by the c-met proto-oncogene is a disulfide-linked α/β heterodimer, derived by proteolytic processing of the precursor p170met (8Gonzatti-Haces M. Seth A. Park M. Copeland T. Oroszlan S. Vande Woude G.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 21-25Crossref PubMed Scopus (155) Google Scholar). The β-chain, p145met, spans the plasma membrane and includes a cytoplasmic domain endowed with tyrosine kinase activity. The mechanisms by which c-Met triggers its regulatory functions involve the activation of several intracellular signaling pathways trough a unique multisubstrate docking site within the C-terminal end of the receptor (reviewed in Ref. 9Furge K.A. Zhang Y.W. Vande Woude G.F. Oncogene. 2000; 19: 5582-5589Crossref PubMed Scopus (364) Google Scholar). It is well established that many transmembrane receptors become internalized upon ligand stimulation. Although EGFR endocytosis and traffic have been extensively studied and represents probably the best understood receptor trafficking system (10Carpenter G. Bioessays. 2000; 22: 697-707Crossref PubMed Scopus (305) Google Scholar), interest in c-Met and its traffic are just emerging. Several receptors such as EGFR or platelet-derived growth factor receptor are ubiquitinated and then degraded by the lysosomal pathway (reviewed in Refs. 11Clague M.J. Biochem. J. 1998; 336: 271-282Crossref PubMed Scopus (149) Google Scholar and 12Bonifacino J.S. Weissman A.M. Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57Crossref PubMed Scopus (536) Google Scholar). So far, it has been reported that c-Met is polyubiquitinated (13Jeffers M. Taylor G.A. Weidner K.M. Omura S. Vande Woude G.F. Mol. Cell. Biol. 1997; 17: 799-808Crossref PubMed Scopus (202) Google Scholar) via the c-Cbl proto-oncogene (14Peschard P. Fournier T.M. Lamorte L. Naujokas M.A. Band H. Langdon W.Y. Park M. Mol. Cell. 2001; 8: 995-1004Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar), (15Taher T.E. Tjin E.P. Beuling E.A. Borst J. Spaargaren M. Pals S.T. J. Immunol. 2002; 169: 3793-3800Crossref PubMed Scopus (58) Google Scholar) and degraded in a proteasome-dependant manner (13Jeffers M. Taylor G.A. Weidner K.M. Omura S. Vande Woude G.F. Mol. Cell. Biol. 1997; 17: 799-808Crossref PubMed Scopus (202) Google Scholar). However, more recent studies (16Hammond D.E. Urbe S. Vande Woude G.F. Clague M.J. Oncogene. 2001; 20: 2761-2770Crossref PubMed Scopus (148) Google Scholar) indicate that the primary effect of proteasome inhibition on c-Met could occur indirectly through an effect on acute HGF-induced c-Met endocytic traffic. Similar results have been described for growth hormone receptor (17van Kerkhof P. Govers R. Alves dos Santos C.M. Strous G.J. J. Biol. Chem. 2000; 275: 1575-1580Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In view of the critical role played by c-Met in cancer, it is important to know how its endocytosis and trafficking are regulated. The endocytosis of several membrane receptors is activated by PKC. For example this is the case for the γ-aminobutyric type A receptor, the parathyroid hormone receptor 1, and the sst2A somatostatin receptor (18Chapell R. Bueno O.F. Alvarez-Hernandez X. Robinson L.C. Leidenheimer N.J. J. Biol. Chem. 1998; 273: 32595-32601Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 19Ferrari S.L. Behar V. Chorev M. Rosenblatt M. Bisello A. J. Biol. Chem. 1999; 274: 29968-29975Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 20Hipkin R.W. Wang Y. Schonbrunn A. J. Biol. Chem. 2000; 275: 5591-5599Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Prior evidence indicates that PKC plays a negative role in controlling c-Met function (21Sipeki S. Bander E. Farkas G. Gujdar A. Ways D.K. Farago A. Cell. Signal. 2000; 12: 549-555Crossref PubMed Scopus (12) Google Scholar), however it is not known how this relates to the internalization or traffic of c-Met. In this study we show that, upon HGF stimulation, the rapidly internalized c-Met traffics and accumulates in a perinuclear compartment, which in part includes the Golgi, corresponding presumably to a recycling compartment. By following the trafficking of c-Met inside the cell using a semiautomatic assessment of vesicle distribution, we demonstrate that this trans-cytosolic movement of c-Met requires an intact microtubule network and is promoted by PKC. After2hofHGF stimulation, half of the internalized c-Met has been degraded by a proteasomal pathway. C-Met is likely a direct target of the proteasome, because we do not detect any effect of inhibition of proteasome activity on internalization of c-Met; furthermore, the lysosomal pathway does not play a substantial role. Inhibition or promotion of c-Met traffic to the perinuclear compartment does not alter the kinetics of proteasome-dependent c-Met degradation. Thus susceptibility to proteasomal degradation follows receptor internalization but is not a consequence of post-endocytic traffic. Cell Culture and Transfection—HeLa cells were cultured in Dulbecco's modified Eagle's medium (Cancer Research UK) supplemented with 10% fetal bovine serum (Sigma) and maintained at 37 °C in a humidified 10% CO2 atmosphere. The cells were seeded in 35-mm plates (for Western blot experiments) or on coverslips in 24-well plates (for immunocytochemistry) and stimulated 24 h later with 100 ng/ml HGF for various time periods. Where indicated, the cells were preincubated with appropriate inhibitors 10 or 15 min before HGF stimulation, and the inhibitors were maintained during the stimulations. The preincubation times were routinely longer for proteasome inhibitors: 2 h for lactacystin and 1 h for MG132; shorter preincubations are as indicated in the text or figure legends. Transfections were performed on cells seeded on coverslips in 24-well plates. For each well, 0.8 μg of DNA was mixed with 1.5 μl of LipofectAMINE™ 2000 (Invitrogen) in 100 μl of OPTIMEM1 with Glutamax (Invitrogen), incubated at room temperature for 20 min to allow the precipitate to form and directly added to the cells in their culture medium. Five hours later the culture medium was changed. Stimulations were performed 24 h post-transfection. Growth Factors, Antibodies, Inhibitors, and Constructs—Purified human recombinant HGF was obtained from R&D Systems. The following antibodies were used: affinity-purified rabbit polyclonal anti-human c-Met intracellular domain (12-amino acid CT, Santa Cruz Biotechnology); goat polyclonal anti-early endosome autoantigen 1 (EEA1; Santa Cruz Biotechnology); mouse monoclonal antibodies anti-tubulin (Santa Cruz Biotechnology for Western blots and Sigma for immunofluorescence); mouse monoclonal anti-c-Met extracellular domain (Upstate Biotechnology Inc); mouse monoclonal antibodies against p115 and GM130 (BD Biosciences); goat polyclonal anti-human EGFR (Santa Cruz Biotechnology); goat anti-human HGF (Sigma); mouse monoclonal anti HGF (R&D System). The secondary antibodies used for Western blot were peroxidase-labeled monkey anti-mouse or anti-rabbit IgG (Amersham Biosciences) and peroxidase-labeled rabbit anti-goat IgG (Dako). The secondary antibodies used for immunofluorescence experiments were Alexa 488-conjugated goat anti-rabbit IgG (Molecular Probes), cy3- or cy5-conjugated affinity-purified donkey anti-mouse IgG and cy5-conjugated affinity-purified donkey anti-goat IgG (Jackson ImmunoResearch). MG132, lactacystin, concanamycin, bafilomycin A1, paclitaxel (or taxol), vinblastine sulfate were obtained from Calbiochem. Nocodazole, colchicine, cycloheximide, propidium iodide, TPA (phorbol 12-myristate 13-acetate), and RNase A were purchased from Sigma. Bisindolylmaleimide I (BIM-I) was obtained from Alexis Biochemicals. LysoTracker was obtained from Molecular Probes. The pCMV-Myc r-AP180 C terminus (residues 530–915) (myc-AP180-C) construct was a generous gift from Dr. Harvey T. McMahon (Cambridge, UK). Immunofluorescence and Confocal Analysis—Cells were washed twice in PBS and fixed in 2% paraformaldehyde for 10 min. Free aldehydes were quenched with 50 mm NH4Cl in PBS for 10 min. Fixed cells were permeabilized in 0.1% Triton X-100 in PBS-2% bovine serum albumin for 15 min. For microtubule staining, the fixation and permeabilization were as follows: cells were incubated for 30 s in a buffer containing 1% Nonidet P-40, 100 mm Pipes, 2 mm EDTA, 1 mm MgCl2, and 0.1 mm EDTA. They were then immediately incubated in methanol at –20 °C for 2 min. Fixed cells were incubated at room temperature for 1 h with the primary antibodies at the following concentrations: anti-c-Met (1 μg/ml), anti-EEA1 (2 μg/ml), anti α-tubulin (1/200), anti-p-115 (1/200), and anti-GM130 (1/200). Cells were rinsed and incubated with appropriate secondary antibodies (5 μg/ml) for 30 min. When indicated, nuclei were stained as follows: cells were incubated in RNase A (1 μg/ml) for 15 min and then in propidium iodide (0.2 μg/ml) for 10 min. Cells were washed three times in PBS and once in water and then mounted in Mowiol containing 2.5% DABCO (1,4-diazabicyclo-[2,2,2]octane). Images were acquired using a confocal laser scanning microscope (LSM510, Carl Zeiss Inc.) equipped with a 63×/1.4 Plan-Apochromat oil immersion objective. Alexa 488 was excited with the 488-nm line of an argon laser, Cy3 was excited with a 543-nm HeNe laser and Cy5 was excited with a 633-nm HeNe laser. Each image represents a single section. Western Blot Analysis—Cells were harvested in 100 μl of Laemmli sample buffer (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and boiled for 10 min. Samples were loaded on 7.5% polyacrylamide gels. Separated proteins were transferred to a 0.45-μm nitrocellulose transfer membrane (Schleicher & Schuell). Protein loading was checked by staining with Ponceau Red. Membranes were then blotted with the intracellular domain c-Met antibody and anti-α-tubulin. Blots were then incubated with appropriate peroxidase-linked secondary antibodies. Immunoblots were revealed using the ECL Western blotting detection reagents (Amersham Biosciences). Semiautomatic Assessment of Vesicles in the Cytosol—Fluorescence images of cell nuclei and vesicles were acquired on an Axiovert TM 135 microscope (Carl Zeiss) equipped with a 63× numerical aperture 1.3 objective lens and an Orca ER CCD camera (Hamamatsu) using Acquisition Manager (Kinetic Imaging). Multiple dichroic with excitation and emission filter wheels ensured that there was no lateral shift between the two fluorescent channels. Cell nuclei were automatically detected by thresholding, and their boundaries were interpolated by elliptical curves (see Fig. 1B). c-Met-positive vesicles were detected by Sobel image enhancement followed by thresholding. Cell boundaries were interactively determined. Two distances were calculated for each vesicle located outside the nucleus: the distance from the cell boundary (a) and the distance to the nuclear boundary (b) along a line passing through the center of the nucleus elliptical boundary. The relative distance of a vesicle from the cell boundary was evaluated as a/(a + b). Relative distances can have values between 0 (vesicle at the cell boundary) and 1 (vesicle at the nucleus). The relative distances were weighted according to the total intensity of fluorescence in the vesicles for calculations of mean values, standard deviations, and analysis of variance (ANOVA) with a hierarchical unbalanced model. The following levels and total numbers of data were used in the hierarchical structure: relative distances (78,637), cells (976), observation fields (357), cell cultures (67), experiments (40Santos O.F. Moura L.A. Rosen E.M. Nigam S.K. Dev. Biol. 1993; 159: 535-548Crossref PubMed Scopus (89) Google Scholar), and treatments (12Bonifacino J.S. Weissman A.M. Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57Crossref PubMed Scopus (536) Google Scholar). c-Met Is Rapidly Internalized upon HGF Stimulation and Traffics to a Perinuclear Compartment—In the absence of ligand, c-Met is predominantly distributed around the plasma membrane with some asymmetrically distributed, punctate, perinuclear staining (Fig. 1A, top left image). HGF treatment of HeLa cells leads to the rapid endocytosis of c-Met with the receptor transiently accumulating on vesicular structures, followed by a delayed transcytosolic traffic to and accumulation within a perinuclear compartment. We developed a semiautomatic procedure, as described under “Experimental Procedures,” to follow the migration of fluorescent c-Met vesicles, from the plasma membrane to this perinuclear location (Fig. 1B). This indicated that following internalization the bulk of the immunoreactive protein accumulated within the perinuclear compartment between 30 and 120 min. The initial endocytic event is typical of a clathrin-mediated process, being inhibited by co-transfected myc-AP180-C (Fig. 1C) and by pretreatment with dansylcadaverine (Fig. 1D). Contrary to previous studies (16Hammond D.E. Urbe S. Vande Woude G.F. Clague M.J. Oncogene. 2001; 20: 2761-2770Crossref PubMed Scopus (148) Google Scholar) no inhibition of endocytosis was observed on treatment with the proteasome inhibitors MG132 or lactacystin (Fig. 1E; see further below). At 15 min, the internalized c-Met is found to overlap with EEA1 indicative of an early endocytic compartment; this co-localization has decreased by 120 min (Fig. 1F). HGF co-localizes with c-Met in vesicles and is still present after 120 min of treatment (Fig. 1G). This co-localization has been detected with two distinct HGF-specific antibodies, a polyclonal and a monoclonal antibody. PKC Controls the Movement of c-Met from Early Endosome to a Perinuclear Compartment—It has been shown that PKC can negatively influence c-Met signaling (21Sipeki S. Bander E. Farkas G. Gujdar A. Ways D.K. Farago A. Cell. Signal. 2000; 12: 549-555Crossref PubMed Scopus (12) Google Scholar, 23Gandino L. Longati P. Medico E. Prat M. Comoglio P.M. J. Biol. Chem. 1994; 269: 1815-1820Abstract Full Text PDF PubMed Google Scholar). To establish whether this action is effected through the control of HGF-induced traffic of c-Met, an inhibitor of PKC, BIM-I (bisindolylmaleimide I) was employed. BIM-I did not have a substantial effect upon HGF-induced internalization of c-Met. However, it consistently reduced the rate of accumulation of c-Met in the perinuclear compartment (Fig. 2A). c-Met is consequently retained in an early endosomal compartment at 120 min of stimulation (Fig. 2B, compare with Fig. 1F). Using the semi-automatic assessment of fluorescent vesicle distribution, we quantified this effect at 15 and 120 min of stimulation as shown in Table I. For example, at 15 min, BIM-I reduces the relative distance of c-Met from the cell boundary to nuclear boundary by 14% (p < 0.001). The BIM-I effect is specific to c-Met, because transferrin distribution is not modified after 1 h of treatment (Fig. 2C).Table IVesicle movementTreatmentTimeRelative distance, mean ± S.D.Number of cellsp value (ANOVA)minHGF150.65 ± 0.0429HGF1200.76 ± 0.1031+a+ indicates a significant difference compared to HGF, 5 min.Bim150.56 ± 0.0751***Bim1200.68 ± 0.0424NSVinb.150.58 ± 0.0613NSVinb.1200.57 ± 0.0474***Taxol150.51 ± 0.0571***Taxol1200.59 ± 0.0658**a + indicates a significant difference compared to HGF, 5 min. Open table in a new tab To confirm the effect of PKC on this trans-cytosolic traffic of c-Met the effect of the direct PKC activator TPA was assessed. In combination with HGF, TPA was found to promote the perinuclear accumulation of internalized c-Met (Fig. 2D). This effect was also blocked by BIM-I. The Trans-cytosolic Movement of c-Met Vesicles Is Microtubule-dependent—The effects of PKC inhibition/activation indicate that trans-cytosolic traffic of c-Met is an active, regulated process. The basis of this active traffic was investigated in relation to the requirement for cytoskeletal integrity. Microtubules have been shown to be required for traffic of endocytosed receptors (see Refs. 24Hamm-Alvarez S.F. Alayof B.E. Himmel H.M. Kim P.Y. Crews A.L. Strauss H.C. Sheetz M.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7812-7816Crossref PubMed Scopus (37) Google Scholar and 25Novikoff P.M. Cammer M. Tao L. Oda H. Stockert R.J. Wolkoff A.W. Satir P. J. Cell Sci. 1996; 109: 21-32PubMed Google Scholar). Disruption of the microtubule network with vinblastine or its stabilization with taxol had no substantial effect upon HGF-induced internalization. However, both agents inhibited the movement of c-Met to the perinuclear compartment (Fig. 3A). These effects are quantified in Table I. For example, at 120 min, vinblastine and taxol reduce the relative distance of c-Met from cell boundary to nuclear boundary by 25% (p < 0.001) and 22% (p < 0.01), respectively. A similar inhibitory effect was obtained with the two structurally unrelated microtubule depolymerizing agents colchicine and nocodazole (data not shown). To confirm the action of these agents, the microtubule network was visualized directly (Fig. 3B). The relationship between the microtubule-based transcytosolic traffic of c-Met and the influence of PKC on this process is evidenced by the finding that the promotion of c-Met traffic by TPA, in the presence of HGF, is also blocked by microtubule disruption (Fig. 3C). This indicates that it is the microtubule-based movements of internalized, vesicular c-Met that is positively regulated by PKC. The Accumulating Perinuclear c-Met Compartment in Part Corresponds to the Golgi—Western blot of c-Met demonstrates that, during the time period when c-Met is trafficking, HGF induces a progressive loss of the mature p145 form of c-Met such that at later times the p170 precursor form is roughly at the same concentration as the p145. This has been validated both by the anti-c-Met intracellular domain antibody (Fig. 4A) and the anti-c-Met extracellular domain antibody (data not shown). Densitometric analysis showed that the loss of the p145 form of c-Met reaches 60% by 240–480 min (p < 0.01). The contribution of the immature p170c-Met precursor to the perinuclear pool of immunoreactive receptor was determined by its elimination with the protein synthesis inhibitor cycloheximide. Acute pretreatment with cycloheximide completely inhibited p170c-Met synthesis as evidenced by its steady-state loss (Fig. 4B), however there was no effect upon the rate of HGF-induced p145c-Met degradation. c-Met immunofluorescence of control and cycloheximide treated cells indicated that for controls a substantial overlap with the Golgi compartment was observed at later times following HGF treatment. This was evident at 240 min using the Golgi markers p115 (Fig. 4C) and GM130 (data not shown). In cycloheximide-treated cells, where essentially all the immunoreactivity represents p145c-Met (see above), there was only a weak overlap with the Golgi compartment. This indicates that there is limited recycling of internalized c-Met p145c-Met through the Golgi compartment and that the majority of the internalized receptor is degraded following accumulation in a non-Golgi perinuclear compartment. However, after 240 min of HGF stimulation, only a limited co-localization was detected between c-Met and LBPA (data not shown) or LysoTracker (Fig. 4D) when compared with GM130, indicating that c-Met degradation is not substantially routed through the lysosomal pathway. In addition, co-localized immunofluorescence of LysoTracker is much less evident with c-Met than with EGFR after stimulation with HGF or EGF, respectively (Fig. 4E). It is also notable that perinuclear c-Met displays a distinct distribution compared with γ-tubulin (Fig. 4F). C-Met Degradation Is Proteasome-dependent and Independent of Its Post-endocytic Traffic—To distinguish proteasome and lysosomal directed c-Met degradation, proteasome inhibitors were employed. Both MG132 and lactacystin completely blocked p145c-Met degradation (Fig. 5A). By contrast, MG132 blocks only 24% of EGFR degradation after 120 min of EGF stimulation (Fig. 5B). Previous studies provided evidence that the primary effect of proteasome inhibition on c-Met degradation occurs indirectly through an effect on c-Met endocytosis (16Hammond D.E. Urbe S. Vande Woude G.F. Clague M.J. Oncogene. 2001; 20: 2761-2770Crossref PubMed Scopus (148) Google Scholar). Although no effects of these agents on endocytosis had been observed (see above) the post-endocytic action of the inhibitors was confirmed by application 10 min after HGF, at a time when the bulk of c-Met was already internalized (see Fig. 1A). This post-treatment with MG132 also produced a complete block in HGF-induced p145c-Met degradation (Fig. 5C). In contrast to previous result (16Hammond D.E. Urbe S. Vande Woude G.F. Clague M.J. Oncogene. 2001; 20: 2761-2770Crossref PubMed Scopus (148) Google Scholar), we do not detect any block of c-Met degradation in presence of the vacuolar-type H+-ATPase inhibitor concanamycin A used at the same concentration (Fig. 5D). Similar results have been obtained with bafilomycin A1 (data not shown). To determine whether the engagement of proteasome-mediated degradation was particular to the perinuclear compartment, delivery to this compartment was blocked by pretreating the cells with BIM-I, vinblastine, and taxol or, conversely, was promoted by TPA. None of these agents affected HGF-induced p145c-Met degradation (Fig. 5, E and F). Moreover, we found that proteasomal degradation can still be blocked in the presence of vinblastine (Fig. 5G). This indicates that engagement of the proteasome machinery is not restricted to the perinuclear compartment and that post-endocytic c-Met traffic and degradation are independent. The endocytic and degradative pathway of c-Met has been detailed here for HeLa cells. The studies demonstrate that the bulk of internalized c-Met is delivered in a microtubule-dependent, PKC-controlled manner to a perinuclear compartment, from where it is subsequently degraded via a proteasome-dependent mechanism. Contrary to previous studies (16Hammond D.E. Urbe S. Vande Woude G.F. Clague M.J. Oncogene. 2001; 20: 2761-2770Crossref PubMed Scopus (148) Google Scholar), we find no evidence for proteasome-sensitive endocytosis per se. On prolonged exposure to HGF, the c-Met p170 precursor becomes a significant form, and this correlates with the immunofluorescence data that indicates a Golgi localization. This late phase localization is consistent with the observation by Kamei and colleagues (26Kamei T. Matozaki T. Sakisaka T. Kodama A. Yokoyama S. Peng Y.F. Nakano K. Takaishi K. Takai Y. Oncogene. 1999; 18: 6776-6784Crossref PubMed Scopus (176) Google Scholar) of a perinuclear localization after 8 h of HGF treatment. Internalized c-Met is progressively degraded by a proteasomal pathway, and the lysosomal pathway does not play a substantial role. Although temporally related to its arrival, the degradation of p145-c-Met is not restricted to this perinuclear compartment, because inhibition of trans-cytosolic traffic (PKC inhibitors and microtubule disruption) or its promotion (PKC activation) does not alter the kinetics of degradation. Thus, susceptibility to proteasome degradation is not a direct consequence of traffic. The mechanism of c-Met internalization appears to involve clathrin-mediated endocytosis, being inhibited by a pretreatment of cells with dansylcadaverine or by overexpression of the COOH part of AP180 (myc-AP180-C). It has been shown previously that EGFR endocytosis was inhibited by the full-length AP180 or AP180-C (27Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Science. 2001; 291: 1051-1055Crossref PubMed Scopus (606) Google Scholar) as a consequence of the blocking of clathrin-coated pit formation. The COOH domain of AP180 is able to bind clathrin and to stimulate cage assembly in vitro. These results are consistent with the finding that c-Met endocytosis is impaired by mutant dynamin expression (16Hammond D.E. Urbe S. Vande Woude G.F. Clague M.J. Oncogene. 2001; 20: 2761-2770Crossref PubMed Scopus (148) Google Scholar). c-Met degradation is dependent on its internalization, because no HGF-dependent degradation is seen in the presence of dansylcadaverine (data not shown). Similarly, it has been shown by others that expression of a dominant interfering mutant of endophilin (28Petrelli A. Gilestro G.F. Lanzardo S. Comoglio P.M. Migone N. Giordano S. Nature. 2002; 416: 187-190Crossref PubMed Scopus (381) Google Scholar) or mutant dynamin (16Hammond D.E. Urbe S. Vande Woude G.F. Clague M.J. Oncogene. 2001; 20: 2761-2770Crossref PubMed Scopus (148) Google Scholar) impairs c-Met internalization and down-regulation. In line with the studies by Jeffers et al. (13Jeffers M. Taylor G.A. Weidner K.M. Omura S. Vande Woude G.F. Mol. Cell. Biol. 1997; 17: 799-808Crossref PubMed Scopus (202) Google Scholar) we observe" @default.
• W2020067532 created "2016-06-24" @default.
• W2020067532 creator A5000996602 @default.
• W2020067532 creator A5017656625 @default.
• W2020067532 creator A5069443670 @default.
• W2020067532 date "2003-08-01" @default.
• W2020067532 modified "2023-10-03" @default.
• W2020067532 title "Protein Kinase C Controls Microtubule-based Traffic but Not Proteasomal Degradation of c-Met" @default.
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