Article |
Address correspondence to Inger H. Madshus, Institute of Pathology, The University of Oslo, Rikshospitalet, N-0027 Oslo, Norway. Tel.: 47-23-073-536. Fax: 47-23-071-511. E-mail: i.h.madshus{at}labmed.uio.no
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Abstract |
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Key Words: EGF; c-Cbl; ubiquitination; proteasome; multivesicular bodies
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Introduction |
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The yeast G proteincoupled receptor Ste2p is accumulated in high molecular weight ubiquitinated forms at the cell surface in response to the binding of -factor in endocytosis-deficient yeast cells (Hicke and Riezman, 1996). This finding established a link between ligand-induced ubiquitination, endocytosis, and eventual targeting for vacuolar degradation in yeast. It has also been reported that the ubiquitin conjugation system is required for ligand-induced endocytosis of the growth hormone receptor (van Kerkhof et al., 2000). Based on these and other observations, the ubiquitin system has been suggested as an important regulatory system for clathrin-dependent endocytosis (Strous and Govers, 1999). This is in accordance with the demonstration that polyubiquitination of the EGFR occurs at the plasma membrane. We demonstrated that in endocytosis-deficient cells the EGFR was transiently polyubiquitinated, but not degraded (Stang et al., 2000).
It is still unclear to what extent proteasomal processing is involved in downregulation of receptors. In the case of the PDGFR, the degradation was reportedly inhibited <20% by proteasome inhibitors (Mori et al., 1995a, 1995b). The receptor tyrosine kinase c-Met consists of a 50-kD extracellular subunit and a 140-kD membrane-spanning ß subunit. Upon ligand binding, the ß subunit was ubiquitinated and subsequently degraded in a proteasome-dependent manner (Jeffers et al., 1997). Also in the case of ErbB2, proteasome inhibitors have been demonstrated to inhibit degradation (Mimnaugh et al., 1996). However, the question of whether or not ErbB2 is in fact endocytosed is still controversial. It was recently reported that the antibiotic geldanamycin induced degradation of ErbB2 and that degradation of the COOH-terminal fragment was prevented by proteasome inhibitors, whereas degradation of the membrane-anchored 135-kD fragment was blocked by inhibitors of the endocytosis-dependent degradation pathway (Tikhomirov and Carpenter, 2000). Therefore, the functional role of ubiquitination in endocytosis and downregulation of receptors is unresolved and challenging.
It has been established that EGF and TGF differ in their ability to induce processing of the EGFR (Ebner and Derynck, 1991). EGF is a more acidic molecule than is TGF
and remains bound to the EGFR upon internalization during acidification of the endocytic vesicle, finally resulting in proteolytic degradation of both ligand and receptor in lysosomes. In contrast, TGF
rapidly dissociates from the receptor upon internalization, resulting in recycling of EGFR to the cell surface (Sorkin and Waters, 1993). We report here that in spite of differential degradation induced by EGF and TGF
, both ligands equally efficiently induce initial ubiquitination and endocytosis of the EGFR. However, EGF, which in contrast to TGF
secures the kinase activity of the EGFR upon endocytosis, maintains a sustained polyubiquitination of the EGFR. Consistently, colocalization of EGFR and c-Cbl on endosomes is observed only upon incubation with EGF. By overexpression of the dominant negative N-Cbl, we confirmed that Cbl-induced ubiquitination is important for degradation of EGF-bound EGFR, but not for endocytosis. By the use of inhibitors of endosomal/lysosomal and proteasomal degradation, we further found that degradation of EGFR and EGF requires proteasomal processing. Therefore, we submit that ubiquitination per se is not a signal for EGFR degradation. However, sustained ubiquitination is required for efficient sorting of the EGFR to a degrading compartment, and proteasomal activity is required for efficient transport of EGF and EGFR to inner membranes of MVBs.
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Results |
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TGF and EGF recruit c-Cbl to the plasma membrane, whereas only EGF recruits c-Cbl to endosomes
Immunocytochemical labeling for confocal microscopy was performed, using antibodies to EGFR and c-Cbl. The specificity of the antibodies was investigated by Western blotting (unpublished data) and further confirmed by ligand-induced changes in localization of EGFR and c-Cbl. The EGFR was mainly localized to the plasma membrane in control cells (Fig. 4 A) and in cells incubated with EGF or TGF on ice (Fig. 4, D and G). Whereas c-Cbl in control cells mainly localized to the cytoplasm (Fig. 4 B), c-Cbl was recruited to the plasma membrane upon incubation both with EGF and with TGF
on ice (Fig. 4, E and H) and appeared to colocalize with the EGFR (Fig. 4, F and I). In cells incubated with EGF, both the EGFR and c-Cbl were redistributed and colocalized on vesicular structures upon chase at 37°C for 15 min (Fig. 4, JL). Upon binding of TGF
and further chase at 37°C for 15 min, the EGFR localized both to the plasma membrane and to vesicular structures (Fig. 4 M). However, c-Cbl, relocalized to the cytoplasm (Fig. 4 N), and there was no clear colocalization of EGFR and c-Cbl (Fig. 4 O).
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The finding that MG132, but not lactacystin, inhibits degradation of very low concentrations of EGF can be explained by the possibility that MG132, which is a peptide aldehyde, does not specifically inhibit proteasomal proteases. We used a Cathepsin B enzyme assay to study the potential effect of MG132 and lactacystin on Cathepsin B, reported to be important for degradation of EGF and EGFR (Authier et al., 1999). We found that MG132 caused an 80% inhibition of Cathepsin B, whereas lactacystin only slightly (<10%) inhibited Cathepsin B. Therefore, the strong inhibitory effect of MG132 on degradation of EGF can be explained by the inhibitory effect of MG132 on Cathepsin B.
The effect of lactacystin on ligand-induced trafficking of the EGFR was further studied by immuno-EM. Control cells or cells pretreated with lactacystin or MG132 were incubated with EGF (10 nM) for 15 min on ice, followed by chase in ligand-free, prewarmed medium with inhibitor. After 1 h chase, both EGF and the EGFR (unpublished data) localized to MVBs. However, both the appearance of MVBs and the distribution of EGF and EGFR inside MVBs were different in control cells compared with in cells treated with lactacystin and MG132. In control cells the labeling for EGF was found in MVBs with a high number of internal vesicles, and >80% of the labeling localized to these internal membranes (Fig. 7 A). In cells treated with lactacystin or MG132 (Fig. 7 B; unpublished data), labeling for EGF was mainly found on MVBs with few internal vesicles, and >60% of the labeling was on the outer limiting membrane. This shows that proteasomal activity is involved in the translocation of EGFR from the limiting membrane to internal membranes within MVBs.
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To characterize the endosomes to which EGF and EGFR localize in the presence of lactacystin more closely, we added Rh-EGF and FITC-transferrin (Tf) to cells in the presence or absence of lactacystin. Upon incubation for 15 min, most EGF colocalized with Tf. Upon incubation for 60 min, there was no significantly increased colocalization of EGF and Tf in lactacystin-treated cells, compared with control cells (unpublished data). Therefore, the endosomes depicted in Fig. 7 B do not appear to be part of the recycling compartment.
Proteasomal inhibitors do not inhibit degradation of EGF-EGFR by depleting cellular ubiquitin
A side effect of incubating cells with proteasomal inhibitors could be depletion of intracellular ubiquitin (Swaminathan et al., 1999). Therefore, we investigated the effect on EGFR ubiquitination of incubating Hep2 cells with lactacystin for 3 and 5 h. Hep2 cells were preincubated with both lactacystin and CHX for 3 and 5 h (the same way degradation of EGFR was studied). As shown in Fig. 8, the EGFR was ubiquitinated equally efficiently by addition of EGF regardless of preincubation with lactacystin. This demonstrates that reduction of intracellular ubiquitin resulting from preincubation of cells with lactacystin was not sufficient to inhibit EGF-induced polyubiquitination of the EGFR and strongly supports the notion that proteasomal activity is essential for efficient transport of EGF-bound EGFR to inner membranes of MVBs.
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Discussion |
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We have taken advantage of the two different EGFR ligands, EGF and TGF, to induce different trafficking of the EGFR upon endocytosis. Interestingly, both ligands initially induced ubiquitination and endocytosis of the EGFR to the same extent, even though TGF
very inefficiently induced transport of the EGFR to internal vesicles of MVBs and lysosomal degradation of the EGFR. This clearly illustrates that the initial ubiquitination induced by activation of the EGFR is not a signal for lysosomal sorting. It should be noted that the ubiquitination induced by EGF lasted longer than the ubiquitination induced by TGF
. The sustained ubiquitination induced by EGF correlated with the sustained tyrosine phosphorylation of the EGFR and c-Cbl, and therefore the sustained kinase activity of the EGFR. Because EGFR kinase activity has been demonstrated to be required for transport of the EGFR from the limiting membrane to the internal vesicles of MVBs (Felder et al., 1990), the sustained ubiquitination seen upon incubation with EGF could be important for trafficking of EGFR to lysosomes. Interestingly, Katzmann et al. (2001) recently reported ubiquitin-dependent sorting of the vacuolar hydrolase carboxypeptidase S to internal membranes of MVBs in yeast. They further characterized a conserved endosomal sorting complex containing the protein Vps23. This protein contains a ubiquitin conjugatinglike domain (Babst et al., 2000). The mammalian homologue of Vps23 is the protein encoded by the tumor susceptibility gene 101 (Tsg101), and this protein has been demonstrated to function in late endosomal trafficking. In fact, in tsg 101 mutant cells, endocytosed EGFR was rapidly recycled back to the cell surface and inefficiently degraded (Babst et al., 2000). Katzmann et al. proposes a model whereby ubiquitinated proteins bind to the sorting complex containing Vps23. Several other class E Vps proteins are suggested to be important for the subsequent sorting, as well as for recruitment of deubiquitinating enzymes, like Doa4 (Katzmann et al., 2001).
The importance of proteasomal activity in lysosomal trafficking of EGFR is supported by our finding that lactacystin inhibits degradation of the EGFR as efficiently as does NH4Cl. Our present immuno-EM data show that inhibition of proteasomal activity with lactacystin causes retention of EGFR in MVBs at an early stage of formation. Consistently, we found, as did Levkowitz et al. (1998), that inhibition of proteasomes promoted recycling and inhibited degradation of EGF. However, the effect of lactacystin on recycling and degradation of EGF was smaller than the effect observed in tsg 101 mutant cells (Babst et al., 2000) and smaller than the effect observed upon overexpression of N-Cbl (transfection efficiency considered). That degradation of EGF is inhibited more efficiently by overexpression of N-Cbl and in tsg 101 mutant cells compared with in cells incubated with lactacystin, suggests the involvement of proteasomes downstream of ubiquitinated EGFR interacting with a complex containing Tsg 101. Our data show that proteasomal activity is not required for the formation of MVBs in general, as lactacystin does not inhibit transport of BSA-gold to MVBs. Morphologically the EGF/EGFR positive compartments found upon inhibition of proteasomal activity resembled the EGFR positive compartments found upon incubation with TGF. We found no significantly increased colocalization of EGF and Tf upon incubating cells with lactacystin.
Although ubiquitinated EGFR can be recognized and degraded by purified 26S proteasomes in vitro (Levkowitz et al., 1999), our results show that the cytosolic part of the EGFR is intact on internal membranes of MVBs, and therefore demonstrate that the EGFR itself is not a direct proteasomal target in vivo. It was earlier also demonstrated that although the ubiquitination machinery and proteasomal activity is needed for degradation of the growth hormone receptor, neither the cytosolic tail of the growth hormone receptor is a proteasomal target (van Kerkhof et al., 2001). Therefore, the nature of the proteasomal target(s) is still unclear.
In conclusion, our data demonstrate that there is an initial ubiquitination of the EGFR at the plasma membrane, but that this ubiquitination per se does not encode EGFR degradation and that initiation of endocytosis of the EGFR does not depend on ubiquitination. Upon conditions where the EGFR kinase activity is secured, the ubiquitination of the EGFR is sustained. Eventually, activated EGFR is efficiently transported to internal vesicles of MVBs in a proteasome dependent process. Therefore, the sustained ubiquitination of the EGFR seems to be a way of engaging ubiquitin-interacting proteins, and proteasomal cleavage eventually facilitates the transport of the EGFR to a highly degrading environment.
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Materials and methods |
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Cell culture and treatment
Hep2 and COS-1 cells were grown in DME (BioWhittaker) containing penicillinstreptomycinfungizone mixture (BioWhittaker), L-glutamine (2 mM) (BioWhittaker), and FBS (BioWhittaker) (5% vol/vol in the case of Hep2 cells and 10% for COS-1 cells). The cells were plated at a density of 15,000 cells/cm2 48 h before experiments. In pulse-chase experiments, cells were incubated with ligand in MEM without bicarbonate with 0.1% BSA on ice for 15 min, followed by washing three times in ice-cold PBS to remove unbound ligand, and subsequent chase in ligand-free MEM without bicarbonate at 37°C.
Antibodies
Sheep anti-EGFR was from Gibco Life Technologies, and Fitzgerald Industries International, Inc., rabbit anti-EGFR, rabbit anti-EGF, and rabbit antic-Cbl from Santa Cruz Biotechnology, Inc., mouse antiactivated EGFR (pY1173) and mouse anti-phosphotyrosine from Upstate Biotechnology, rabbit anticonjugated ubiquitin from Sigma-Aldrich, mouse anti-hemagglutinin (HA) from Roche Diagnostics, rabbit anti-GFP from AbCam Ltd., rabbit anti-mouse IgG from Cappel, ICN Biomedicals. Peroxidase-conjugated donkey antisheep, peroxidase-conjugated donkey antimouse, CyTM2-conjugated donkey antisheep and rabbit antisheep IgG were all from Jackson ImmunoResearch Laboratories. Alexa Fluor 594conjugated goat antirabbit was from Molecular Probes.
Immunoprecipitation
Cells were lysed in immunoprecipitation buffer, as described (Stang et al., 2000). Protein A or protein Gcoupled Sepharose beads (Amersham Pharmacia Biotech) were incubated with antibody for 1 h at room temperature and subsequently washed twice with immunoprecipitation buffer, before the cell lysates were added. Immunoprecipitation was performed at 4°C for 1 h. Immunoprecipitation with antibody to conjugated ubiquitin and SDS-PAGE and immunoblotting was performed as described (Stang et al., 2000).
Biotinylation of surface-localized EGFR
Biotinylation was performed at 4°C, essentially as described (Corbeil et al., 1999), using 2 mM biotin. The EGFR was immunoprecipitated using rabbit anti-EGFR and protein Acoupled Sepharose beads (Amersham Pharmacia Biotech). The immunoprecipitate was subjected to SDS-PAGE and electrotransferred to PVDF transfer membrane (Hybond-P; Amersham Pharmacia Biotech). The membrane was incubated with alkaline phosphataseconjugated streptavidin or with sheep anti-EGFR antibody and alkaline phosphatase-conjugated antisheep antibody. Reactive bands were analyzed by enhanced chemifluoresence (Amersham Pharmacia Biotech). The band intensity was measured using a phosphorofluoroImager (Molecular Imager FX; Bio-Rad Laboratories).
Plasmids and transfection of cells
The plasmid EGFR-GFP, encoding a fusion protein of EGFR and enhanced GFP (Carter and Sorkin, 1998) was provided by Dr. Alexander Sorkin. Hep2 cells were transfected with this plasmid using Fugene 24 h upon plating. HA-tagged c-Cbl in the pJZenNeo vector (Andoniou et al., 1994) was provided by Dr. Robin M. Scaife. By polymerase chain reaction a 1,121-bp fragment, corresponding to the HA tag and the 357 amino acids constituting N-Cbl was subcloned into pCDNA3. This plasmid was transfected into COS-1 cells using Fugene 24 h after plating. Transfected cells were analyzed 48 h after transfection.
Immunocytochemistry and confocal microscopy
Cells were plated on 12-mm coverslips (MENZEL-GLÄSER®). After experiments, cells were washed once in cytoskeleton buffer (NaCl [137 mM], KCl [5 mM], Na2HP04 [1.1 mM], KH2PO4 [0.4 mM], glucose [5.5 mM], NaHCO3 [4 mM], MES [10 mM], EGTA [2 mM], MgCl2 [2 mM]) and subsequently fixed with paraformaldehyde (4% wt/vol) (Riedel-de Haën AG) in Soerensen's phosphate buffer for 20 min on ice. Cells were washed three times with cytoskeleton buffer before permeabilization with Triton X-100 (0.1% wt/vol in cytoskeleton buffer) for 10 min. The fixed and permeabilized cells were preincubated with BSA (1% wt/vol in PBS) for 30 min before incubation with primary antibodies for 1 h. The coverslips were washed with PBS and incubated with secondary antibodies for 30 min before mounting with Dako fluorescent mounting medium with NaN3 (15 mM). The cells were examined using a confocal microscope (Leica TCS).
125I-EGF interaction experiments
The cells were incubated as described in legends to Figs. 5 and 6. Internalization and degradation of EGF was analyzed as previously described (Skarpen et al., 1998). Additionally, recycling of EGF was analyzed essentially as described (Babst et al., 2000), loading cells with 8.5 nM 125I-EGF in MEM without bicarbonate and with 0.1% BSA. Upon loading, the surface-localized radioactivity was removed by a glycine-buffered solution, pH 3.0 (Babst et al., 2000). Upon chase at 37°C, the medium was analyzed for degraded and recycled EGF as described (Skarpen et al., 1998), and the cells were analyzed for recycled EGF (released by the pH 3.0 buffer) and for internalized EGF (cells solubilized by 1 M NaOH after treatment with low pH). The cpm representing recycled EGF in the medium and at the cell surface were combined in one fraction.
Inhibition of lysosomal or proteasomal activity
Hep2 cells were preincubated with either NH4Cl (10 mM) for 10 min, MG132 (10 µM) or lactacystin (50 µM) for 1 or 2 h at 37°C. To measure degradation of the EGFR, EGF (10 nM) and CHX (25 µg/ml) were added and the cells incubated at 37°C for the indicated times. Cells were lysed and subjected to SDS-PAGE and immunoblotting with antibody to EGFR. For immuno-EM, cells preincubated with the different inhibitors were incubated with EGF (10 nM) on ice for 15 min followed by chase at 37°C for the indicated time periods in the presence of inhibitors.
Cathepsin B enzyme assay
The activity of Cathepsin B (Calbiochem) in the presence of lactacystin and MG132 was analyzed by use of Colorimetric Substrate I (Calbiochem) according to the manufacturer's instructions. The enzyme assay was performed three times with three parallels for each condition.
Immuno-EM
The cells were incubated as described in legends to figures. The cells were subsequently washed with PBS, fixed with paraformaldehyde (4% wt/vol) and glutaraldehyde (0.1% wt/vol) in Sorensen's phosphate buffer and processed for cryosectioning and immunolabeling (Griffiths et al., 1984). Bound antibodies were visualized using protein A gold, a gift of Dr. G. Posthuma. When the primary antibody was mouse or sheep IgG, incubation with rabbit antimouse or rabbit antisheep IgG was used as an intermediate step between the primary antibody and protein A gold. The sections were examined using a Philips CM 120 electron microscope. To quantify labeling of MVBs, the total number of gold particles found on MVBs (100%) were divided into two groups, gold particles associated with the outer limiting membrane and gold particles associated with internal membranes, and the percentage distribution of the labeling was calculated. In each experiment a minimum of 100 gold particles were counted on MVBs.
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Footnotes |
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* Abbreviations used in this paper: CHX, cycloheximide; EGFR, EGF receptor; GFP, green fluorescent protein; HA, hemagglutinin; MVBs, multivesicular bodies; PDGFR, PDGF receptor; Tf, transferrin.
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Acknowledgments |
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This work was supported by Medinnova, Novo Nordisk Foundation, Anders Jahre's Foundation for the Promotion of Science, Blix Legacy, and Bruun's Legacy. K.E. Longva was supported by a fellowship from The Norwegian Women's Public Health Association, and E. Stang and L.E. Johannessen by fellowships from The Norwegian Cancer Society.
Submitted: 11 June 2001
Revised: 18 June 2001
Accepted: 23 January 2002
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