Cbl-mediated Ubiquitinylation Is Required for Lysosomal Sorting of Epidermal Growth Factor Receptor but Is Dispensable for Endocytosis*

Lei Duan {ddagger} §, Yuko Miura {ddagger} §, Manjari Dimri {ddagger}, Biswanath Majumder {ddagger}, Ingrid L. Dodge {ddagger}, Alagarsamy L. Reddi {ddagger}, Amiya Ghosh {ddagger}, Norvin Fernandes {ddagger} , Pengcheng Zhou {ddagger} , Karen Mullane-Robinson {ddagger}, Navin Rao {ddagger}, Stephen Donoghue {ddagger}, Rick A. Rogers ||, David Bowtell **, Mayumi Naramura {ddagger}{ddagger}, Hua Gu {ddagger}{ddagger}, Vimla Band §§ and Hamid Band {ddagger} ¶¶

From the {ddagger}Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, the ||Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts 02115, **Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne 3000, Parkville 3050, Victoria, Australia, the {ddagger}{ddagger}Laboratory of Immunology, NIAID, National Institutes of Health, Rockville, Maryland 20852, and the §§Division of Radiation and Cancer Biology, and Departments of Radiation Oncology and Biochemistry, New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts 02111

Received for publication, April 29, 2003 , and in revised form, May 17, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl/ mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Growth factor receptor tyrosine kinases (RTKs)1 play crucial roles in cellular proliferation, survival, migration, and differentiation. Epidermal growth factor receptor (EGFR/ErbB1) is a member of the ErbB family (ErbB1–4) of RTKs, which play crucial homeostatic roles and are implicated in oncogenesis. Ligand-induced activation of RTKs leads to the assembly of signaling protein complexes and subsequent activation of downstream signaling pathways. The ligand-activated RTKs also undergo rapid endocytosis (1). The endocytosed receptors then undergo a sorting process, which determines receptor fate and signal intensity. The receptors can be targeted to the lysosome for degradation, which terminates receptor signals. Alternatively, the internalized receptors can be recycled back to the cell surface for continued ligand binding and signaling (25). The relative efficiency of lysosomal sorting versus recycling is a key determinant of the signaling potency of RTKs (6). For example, EGFR is predominantly delivered to lysosomes when activated by EGF. In contrast, heregulin-activated ErbB2 is primarily recycled. The greater efficiency of the recycling process is thought to be a major determinant of the signaling superiority of ErbB2 over EGFR (79).

Despite a critical role of endocytic sorting as a determinant of ErbB receptor down-regulation, the biochemical mechanisms that regulate this process have only recently begun to be elucidated. We, and others, have identified Cbl as one such regulator (1012). Cbl is recruited to the activated EGFR through both direct and indirect binding. Direct Cbl-EGFR interaction is mediated through the N-terminal tyrosine kinase-binding domain of Cbl, which binds to phosphorylated Tyr-1045 on EGFR (13). Indirect Cbl-EGFR interaction is primarily mediated through Grb2: the SH3 domain of Grb2 binds to proline-rich sequences in Cbl, whereas the SH2 domain binds to auto-phosphorylated EGFR (14). The RING finger domain in Cbl binds to ubiquitin-conjugating enzymes (E2s), allowing Cbl to function as a ubiquitin ligase (E3) toward activated EGFR (15, 16). Overexpression studies have demonstrated that Cbl-mediated ubiquitinylation promotes down-regulation of EGFR, whereas Cbl proteins with mutations in the RING finger domain, or in a conserved helix connecting the RING finger and tyrosine kinase-binding domains, reduce the extent of EGFR ubiquitinylation and down-regulation (1719). It has not been established, however, whether endogenous Cbl-mediated ubiquitinylation plays an essential role in EGFR degradation.

Two potential mechanisms for Cbl-mediated, and ubiquitinylation-dependent, EGFR down-regulation have been proposed. First, a role for Cbl-mediated ubiquitinylation in EGFR endocytosis has been postulated based on the well established role of ubiquitinylation in the endocytosis of yeast pheromone receptors (20, 21), and the apparent requirement for the ubiquitin pathway in mammalian growth hormone receptor internalization (22). Consistent with this possibility, Cbl was also recently found to associate with a CIN85-endophilin complex and to facilitate the monoubiquitinylation of CIN85 (23). Because a truncated CIN85 mutant that did not associate with Cbl impaired the rate of EGFR internalization, the investigators concluded that Cbl played a role in EGFR endocytosis by serving as an adaptor to link EGFR with CIN85 and endophilin (24). Another study arrived at a similar conclusion in the context of the RTK c-MET (25). More recently, the EGFR Y1068F/Y1086F mutant, which is unable to bind Grb2, was also found to be impaired for endocytosis (26). Interestingly, this mutant was also unable to efficiently recruit Cbl to EGFR, and showed impaired ubiquitinylation. Collectively, these studies suggest a role for Cbl, and Cbl-regulated ubiquitinylation, in EGFR endocytosis. Other studies, however, argue against a role for Cbl-mediated ubiquitinylation in the initial internalization of EGFR. For example, even though EGFR ubiquitinylation occurs at the cell surface, it did not appear to be required for endocytosis (19, 27). In contrast, both ubiquitinylation and proteasome activity were needed for transferring EGFR into internal vesicles of the multivesicular body (19). In other studies, dominant-negative Cbl mutants were found to prevent down-regulation of EGFR, but EGFR was seen to undergo ligand-induced localization to intracellular vesicular structures (17). Furthermore, the rate of initial internalization did not correlate with the ability of overexpressed Cbl mutants to inhibit EGFR down-regulation (18). These results support a role for Cbl and ubiquitinylation at a late step in the endocytic pathway, rather than at the initial internalization step. The relative contribution of these alternate mechanisms in Cbl-mediated down-regulation of EGFR has not been clarified.

In the present study, we used Chinese hamster ovary (CHO) cells conditionally defective in ubiquitinylation, and Cbl-deficient mouse embryonic fibroblast (MEF) cells, to address the role of Cbl and Cbl-mediated ubiquitinylation in the internalization, endosomal sorting, and degradation of EGFR. We demonstrate that Cbl-mediated EGFR ubiquitinylation is required for efficient sorting of activated EGFR into the lysosome for its degradation. In contrast, neither Cbl nor Cbl-mediated ubiquitinylation are required for initial EGFR endocytosis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Reagents—Biotin-EGF complexed with Alexa Fluor 488-labeled streptavidin, and Alexa Fluor 488-conjugated transferrin were obtained from Molecular Probes Inc. Opti-MEM I reduced serum medium was from Invitrogen. The FuGENE-6 reagent was obtained from Roche Diagnostics.

Antibodies—The antibodies used in this study were: mouse anti-EGFR monoclonal (mAb) 528 (IgG2a) from ATCC; rat anti-LAMP-1 mAb and rabbit polyclonal anti-EGFR antibody (sc-03) from Santa Cruz Biotechnology, Inc.; anti-ubiquitin mAb P4G7 (IgG1) from Covance Research Products Inc.; anti-phosphotyrosine mAb 4G10 (IgG2a) from Dr. Brian Druker (Oregon Health Sciences University, Portland, OR); and polyclonal antibody anti-LAMP-1 (931B) (29) from Dr. Minoru Fukuda (The Burnham Institute, CA). Cy3-conjugated goat anti-mouse IgG and Cy2-conjugated goat anti-rabbit IgG secondary reagents were from Jackson ImmunoResearch Laboratories Inc.

DNA Constructs and Mutants—pAlterMAX-HA-Cbl and pAlterMAX-HA-Cbl-C3AHN (RING finger mutant) (30), and pJZenNeo-HA-Cbl retroviral expression construct (31) have been described previously. The human EGFR cDNA insert from the pAlterMAX-EGFR construct (17) was cloned into pcDNA3 to generate pcDNA3-EGFR, and into pMSCV-puro retroviral vector (Clontech) to generate pMSCV-puro-EGFR.

Cell Lines—The CHO cell line with a temperature-sensitive E1 ubiquitin-activating enzyme, CHO-Ts20 and its wild type control cell line, CHO-E36 (32) (from Dr. Ger Strous, University Medical Center, Utrecht, The Netherlands) were grown in minimal essential-{alpha} medium supplemented with 10% fetal bovine serum, and penicillin/streptomycin (Invitrogen). MEF cells were derived using standard isolation methods (33) from day 13.5 Cbl/ and littermate Cbl+/+ embryos, from two separate Cbl/ mouse backgrounds (34, 35), followed by the 3T3 protocol from passage 3 to 25 (36). These cells were grown in minimal essential-{alpha} medium with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 0.1 mM non-essential amino acids, and penicillin/streptomycin. The MEF cells used here are independent of another pair that we have reported previously (37, 38).

Transfections—Transient or stable transfectants of CHO-Ts20, CHO-E36 expressing human EGFR (or its mutants), were generated by FuGENE-6-mediated transfection of pcDNA3-EGFR, according to the manufacturer's instructions. Transfectants were selected in media containing 0.5 mg/ml G418 (Invitrogen) and clones were analyzed for EGFR expression by immunoprecipitation and immunoblotting (described below). Stable transfectants of Cbl+/+ and Cbl/ MEFs expressing human EGFR were established by retroviral infection as described previously (39). Cells were selected in puromycin (Sigma; 2.5 µg/ml) and bulk transfectants were analyzed for EGFR expression using fluorescence-activated cell sorter (FACS) analysis with mAb 528 and immunoblotting with sc-03 anti-EGFR antibody.

Transfections were carried out with the FuGENE-6 reagent. The amounts of input DNA are indicated in the figure legends. Cells were harvested at 48 h after the addition of DNA precipitates.

EGF Stimulation and Preparation of Cell Lysates—For EGF stimulation, cells were placed in starvation medium (growth medium containing 0.5% fetal bovine serum) for 4–6 h and then incubated with purified murine EGF (catalog number E-4127, Sigma) for various lengths of time and at concentrations indicated in figure legends. Cells were rinsed with ice-cold PBS and lysed in 50 mM Tris (pH 7.5), 150 mM sodium chloride, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.07 trypsin inhibitor units/ml of aprotinin, and 1 µg/ml each of leupeptin, pepstatin, antipain, and chymostatin (Sigma) (40). Lysate protein concentration was determined using the Bradford method (Bio-Rad).

Immunoprecipitation and Immunoblotting—These procedures have been described previously (14).

Confocal Immunofluorescence Microscopy—Cells seeded on glass coverslips were stimulated with EGF as described above. Cell fixation and immunostaining was described previously (17). Cells were stained with the appropriate primary antibody (4 µg/ml anti-EGFR mAb 528 or 1:500 anti-LAMP-1 pAb in blocking buffer) and followed with 1:300 dilution of goat anti-mouse IgG (H+L) F(ab')2-Cy3 and anti-rabbit IgG F(ab')2-Cy2. Coverslips were mounted on glass slides using Fluoromount-G. Confocal microscopy was carried out using a Leica TCS-NT confocal laser scanning microscope fitted with krypton and argon lasers, as previously described (41).

Assessment of EGFR Internalization—Cells were grown on 10-cm tissue culture dishes to 70–80% confluence. Following serum starvation for 4–6 h, the cells were incubated with 25 ng/ml Alexa Fluor 488-conjugated EGF at 4 °C for 30 min, washed 3 times with cold PBS, and incubated at normal cell growth temperature for the indicated time points to allow internalization. The cells were placed on ice to stop internalization, rinsed 3 times with cold PBS, and subjected to an acid wash (0.2 M acetic acid and 0.5 M NaCl, pH 2.8) for 5 min. Non-internalized EGF was removed by 3 washes with PBS, and the cells were detached from tissue culture dishes using a rubber scraper. Cells were washed and suspended in FACS buffer (2% fetal bovine serum and 0.01% sodium azide in PBS), and fixed by adding an equal volume of 4% formaldehyde/PBS. Fluorescence emission because of internalized EGF was detected by flow cytometry. Mean fluorescence intensity of cells after EGF binding but without the acid wash was set to 100%, percentage internalization was calculated after subtracting background (fluorescence of cells subjected to acid wash without allowing internalization). Flow cytometry, data collection, and analysis were performed on a FACSort machine using CellQuest software (BD Biosciences). Each experiment was done in triplicate 3 times. Values are expressed as a percentage of initial EGF binding. The data was analyzed statistically with Prism (Graphpad Software, Inc.) by using Student's t test and one-way analysis of variance. All of the values from a representative experiment are plotted. The median is connected by a line, and the range from maximum to minimum is expressed as an error bar.

Down-regulation of Cell Surface EGFR—Down-regulation of EGFR from the cell surface was assessed as previously described (17).


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Ligand-induced EGFR Ubiquitinylation and Degradation Are Impaired in Cells Lacking Endogenous Cbl Expression— Previous studies supporting a role for Cbl in EGFR ubiquitinylation and down-regulation have exclusively used overexpression of wild type Cbl or its dominant negative mutants (17, 18, 42). However, the role of endogenous Cbl in EGFR ubiquitinylation and degradation has not been addressed directly. To address this question, we derived two distinct pairs of Cbl+/+ and Cbl/ MEF cell lines, using Cbl/ mouse lines developed independently in the Bowtell and Gu laboratories (34, 35); these cell lines are designated Cbl+/+(DB) and Cbl/(DB), and Cbl+/+(HG) and Cbl/(HG), respectively.

Western blotting of whole cell lysates confirmed the Cbl protein expression in the Cbl+/+ MEFs but not in either of the Cbl/ MEFs (Fig. 1A). The Cbl/(DB) MEFs express low levels of a truncated Cbl protein (data not shown) representing a nonfunctional splice product, as previously reported (34, 37). Because other Cbl family members, such as Cbl-b and Cbl-c, may also play a role in EGFR down-regulation (43, 44), we wished to determine the level of expression of Cbl-b and Cbl-c in these MEFs. To assess the expression of Cbl-b, we used an antibody (H454) that recognizes both Cbl and Cbl-b (determined using HA-tagged Cbl and Cbl-b proteins; data not shown). We found that Cbl-b protein was detectable in the lysates of Cbl+/+ and Cbl/ MEFs from which all Cbl protein had been immunodepleted using a Cbl-specific antibody (Fig. 1B, lanes 5–8); notably, the level of Cbl-b protein was substantially lower in the Cbl/(HG) MEFs. We also assayed for the presence of Cbl-c by Northern blot, as no antibody is currently available; the Cbl-c mRNA levels were nearly undetectable in all MEFs and there was no compensatory increase in Cbl/ cells (data not shown). Whereas the reasons for reduced Cbl-b expression in the Cbl/(HG) MEFs are unknown, this trait was advantageous to assess the contribution of endogenous Cbl to EGFR down-regulation. To analyze the impact of Cbl deficiency on EGFR down-regulation, we used retroviral infection to derive stable transfectants of the Cbl+/+ and Cbl/ MEFs expressing human EGFR on the cell surface (Fig. 1, C and D).



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FIG. 1.
Characterization of Cbl+/+ and Cbl/ MEFs. A, lack of Cbl expression in Cbl/ MEFs. 50-µg aliquots of cell lysates of HG and DB MEFs were resolved by SDS-PAGE and subjected to IB with an anti-Cbl antibody. The faster migrating band in the Cbl/(DB) cell line is a truncated nonfunctional protein. B, expression of Cbl-b relative to Cbl in MEFs, analyzed with a Cbl/Cbl-b cross-reactive antibody H454. 1-mg aliquots of cell lysates were subjected to IP with an anti-Cbl-specific antibody (Santa Cruz C-15, lanes 1–4). Alternatively, the same amounts of lysates were first subjected to two rounds of anti-Cbl IP, and immunodepletion of Cbl was confirmed by IB (not shown). These immunodepleted lysates were subjected to IP with H454 antibody (lanes 5–8) to immunoprecipitate Cbl-b. Both sets of IPs were subjected to IB with H454 antibody (recognizes both Cbl and Cbl-b; data not shown). Note reduced Cbl-b signal in Cbl/(HG) MEFs (lane 8). C and D, analysis of EGFR expression on Cbl+/+ and Cbl/ MEFs. Cbl+/+ and Cbl/ MEFs were stably transfected with EGFR, or vector (–) using retroviral infection. 50-µg aliquots of cell lysate proteins were immunoblotted with an anti-EGFR Ab (sc-03) (C). Alternatively, cells were trypsinized and stained with an anti-EGFR mAb (528, thin line) or an isotype control antibody (anti-Syk; thick line) followed by FACS analysis to quantify the cell surface expression of EGFR (D). The numbers in each box represent mean fluorescence intensity (arbitrary values) of anti-EGFR staining.

 

As Cbl is an EGFR-directed ubiquitin ligase (1012), we first assessed the ligand-induced EGFR ubiquitinylation in EGFR-expressing Cbl+/+ and Cbl/ MEFs. Serum-starved cells were stimulated with EGF for various times, and anti-EGFR immunoprecipitates (IPs) were subjected to immunoblotting (IB) with an anti-ubiquitin antibody. As anticipated, ligand-dependent ubiquitinylation of EGFR was readily detected in both Cbl+/+ MEF lines (DB and HG; Fig. 2A). Notably, ligand-induced EGFR ubiquitinylation in both Cbl/ MEF lines was greatly reduced, indicating that endogenous Cbl is the major ubiquitin ligase involved in EGFR ubiquitinylation in MEFs. Importantly, anti-EGFR blotting of anti-EGFR IPs (Fig. 2A, second panel) or whole cell lysates (Fig. 2B) revealed that the ligand-induced loss of EGFR protein was substantially slower in both Cbl/ MEFs; concomitantly, we observed a slower and less pronounced loss of phosphotyrosine (Tyr(P)) signals on EGFR (Fig. 2A, bottom panel). The pattern of EGFR phosphorylation also indicates that impaired EGFR ubiquitinylation in Cbl/ cells is not because of defective EGFR activation.



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FIG. 2.
Impairment of EGFR ubiquitinylation and degradation in Cbl/ MEFs, and reversal of these defects by reconstitution of Cbl expression. A, impaired EGFR ubiquitinylation in Cbl/ MEFs. The MEFs were stimulated with EGF (100 ng/ml) for the indicated times (min) prior to lysis. Anti-EGFR (mAb 528) IPs from 2-mg aliquots of cell lysates were serially immunoblotted with anti-ubiquitin (upper panel), anti-EGFR (sc-03; middle panel), and anti-Tyr(P) (4G10) (lower panel) antibodies. Ubiquitinylated EGFR is indicated. B, delayed EGFR degradation in Cbl/ MEFs. The cell lysates were prepared as in A, and 500-µg aliquots of cell lysates were immunoblotted with anti-EGFR antibody sc-03. The relative EGFR signals were quantified by densitometry using the Scion Image software (Scion Corp., Frederick, MD), and are depicted as a proportion of signals observed with unstimulated cell lysates (set as 1). CE, reversal of defective EGFR ubiquitinylation and degradation in Cbl/ MEFs by Cbl reconstitution. Retroviral infection was used to derive HA-Cbl and vector-transfected Cbl/–(HG) MEFs. 50-µg aliquots of lysates of these cells, as well as untransfected or vector-transfected Cbl+/+ MEFs, were immunoblotted with an anti-Cbl antibody (C). The relative levels of Cbl expression, as determined by densitometry, are indicated at the bottom. The vector-transfected or Cbl-reconstituted Cbl/(HG) MEFs were stimulated with EGF for the indicated times prior to cell lysis. 2-mg aliquots of lysates were used for anti-EGFR IP followed by serial anti-ubiquitin (upper panel) and anti-EGFR immunoblotting (lower panel) (D). 50-µg aliquots of the same lysates were directly immunoblotted with anti-EGFR antibody (E) to assess EGFR degradation. Relative EGFR signals were determined by densitometry.

 

To establish that defective EGFR ubiquitinylation and degradation in Cbl/ MEFs was because of the lack of Cbl expression, and not an artifact of cell line derivation, we reconstituted Cbl expression in the EGFR-expressing Cbl/(HG) MEFs. IB of whole cell lysates demonstrated that retrovirus-mediated introduction of HA-tagged Cbl led to reconstitution of Cbl expression in Cbl/(HG) MEFs (Fig. 2C). Comparison of the HA-Cbl-transfected versus the vector-transfected cells demonstrated that reconstitution with Cbl fully restored the EGF-induced ubiquitinylation and degradation of EGFR (Fig. 2, D and E). Thus, the defect in EGFR ubiquitinylation and degradation in Cbl/(HG) MEFs is solely because of lack of Cbl expression. Overall, the results with Cbl/ MEFs and their Cbl-reconstituted derivatives establish a clear role for endogenous Cbl in ligand-induced EGFR ubiquitinylation and degradation.

EGFR Internalization Is Unaltered in Cbl/ MEFs—The precise site(s) of Cbl action in the endocytic trafficking of EGFR has not been clarified. Based on the formation of a Cbl-CIN85-endophilin complex, a recent set of studies concluded that Cbl plays an important role in the initial internalization of EGFR (24, 25). In addition, an EGFR mutant unable to bind Grb2 was impaired in its ability to form an EGFR-Grb2-Cbl complex, and to undergo ligand-induced ubiquitinylation, and internalization, further suggesting a role for Cbl-mediated ubiquitinylation in EGFR internalization (26). However, whether the effects of the CIN85 mutant are because of the loss of a Cbl-CIN85 interaction or of other protein-protein interactions, or whether Cbl-EGFR interaction through Grb2 is required for EGFR internalization, have not been established. In fact, the N-terminal half of Cbl, which included only the tyrosine kinase-binding and RING finger domains and lacked both the CIN85 and Grb2 interaction sites, was sufficient to enhance the ubiquitinylation and degradation of EGFR (13, 17, 47). Moreover, overexpression of Cbl did not enhance the EGFR internalization (42), and dominant-negative Cbl mutants blocked ubiquitinylation and degradation but not the internalization of EGFR (17, 18). These discrepancies suggest that the impairment of EGFR internalization by the dominant interfering forms of CIN85 may not be through an interruption of the Cbl-CIN85 interaction but through another mechanism. Similarly, Cbl-Grb2 interaction may not be the basis for Grb2-regulated EGFR internalization, as Grb2 can also recruit a number of other proteins, such as RN-tre, to activated EGFR (48). To clarify the role of Cbl in EGFR internalization, we used the MEF system characterized above to directly establish if the loss of Cbl function had an obvious impact on ligand-induced internalization of EGFR. For this purpose, we assayed the acid-stable uptake of Alexa Fluor 488-conjugated EGF by MEFs.

To minimize the contribution of recycling and/or lysosomal degradation of internalized EGFR, we quantified the Alexa 488-EGF uptake in MEFs for relatively brief time periods (up to 10 min). We also used EGF at a relatively low concentration (25 ng/ml) that only saturates 40–50% of cell surface EGFR in our system (data not shown). The rate of initial EGFR internalization in Cbl/(HG) MEFs was comparable with that in Cbl+/+(HG) MEFs (Fig. 3A, p > 0.05). Furthermore, the rate of initial internalization of EGFR in Cbl-reconstituted and vector-transfected Cbl/(HG) MEFs was essentially identical (Fig. 3C, left panel, p > 0.05), even though the down-regulation of EGFR at later time points, which reflects a net balance of internalization, recycling, and lysosomal degradation, was dramatically slower in Cbl/(HG) MEFs compared with Cbl-reconstituted MEFs (Fig. 3C, right panel, p < 0.001). Unimpaired EGFR internalization in Cbl/(HG) MEFs was further confirmed by the immunofluorescence staining of EGFR after EGF stimulation. The pattern of EGFR-staining endocytic vesicles 10 min after EGF stimulation was comparable in Cbl/ and Cbl+/+(HG) MEFs (Fig. 3B). The combination of unimpaired initial internalization and reduced EGFR degradation (and down-regulation) in Cbl/ MEFs indicates that Cbl function is critical at a post-internalization step in EGFR down-regulation but not at the internalization step.



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FIG. 3.
Unimpaired internalization but reduced down-regulation of EGFR in Cbl/ MEFs. A, EGFR internalization in Cbl+/+, Cbl/ MEFs. The internalization assay is described under "Materials and Methods." B, internalization of EGFR as determined by immunofluorescence staining as described under "Materials and Methods." Cells were either left unstimulated or stimulated with EGF (25 ng/ml for 10 min). Internalization is indicated by the accumulation of intracellular endocytic vesicles staining for EGFR (negative controls are not shown). C, EGFR internalization and down-regulation in Cbl/ and Cbl-reconstituted Cbl/ MEFs. Internalization assay is described as in A. For down-regulation, cells were either left unstimulated or stimulated with EGF as in B. The levels of EGFR on the cell surface were quantified by FACS analysis after immunostaining with anti-EGFR Ab 528 as described under "Materials and Methods." EGFR levels remaining on the cell surface are represented as a percentage of EGFR levels (mean fluorescence intensity) without EGF stimulation. Every experiment was done in triplicate 3 times. All of the three values from one representative experiment are plotted. The median is connected by a line, and the range from maximum to minimum is expressed as an error bar.

 

CHO-Ts20 Cells Demonstrate That the Ubiquitin Pathway Is Essential for EGFR Degradation but Dispensable for Internalization—Previous analyses using overexpression of wild type or mutant forms of Cbl have demonstrated a tight correlation between the Cbl-regulated ubiquitinylation and degradation of EGFR (12). This correlation is further strengthened by studies of EGFR mutants unable to bind to Cbl (42), and analyses of Cbl-deficient MEFs presented above. However, the questions of whether Cbl-mediated EGFR degradation proceeds through ubiquitinylation and whether the ubiquitin pathway is required to target EGFR for lysosomal degradation have not been directly addressed. To clarify these issues, we utilized a CHO cell line with a temperature-sensitive E1 enzyme, which results in a conditional defect in protein ubiquitinylation (51).

As CHO cells lack EGFR, the CHO cell line with mutant E1 (CHO-Ts20) and its wild type counterpart (CHO-E36) were stably transfected with human EGFR. Transient overexpression of wild type Cbl, in comparison with vector control, enhanced the ubiquitinylation of EGFR in Ts20-EGFR cells (Fig. 4A, compare lanes 2 and 4). In contrast, the overexpression of a Cbl RING finger domain mutant, Cbl-C3AHN, suppressed the ligand-induced ubiquitinylation (Fig. 4A, lane 6) and degradation of EGFR (Fig. 4B, lanes 7–9). Thus, both ubiquitinylation and degradation of EGFR in CHO-Ts20 cells are dependent on endogenous Cbl.



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FIG. 4.
Ubiquitinylation and degradation of EGFR in CHO-Ts20 cells is mediated by Cbl. A, wild type Cbl enhances, whereas the Cbl RING finger mutant inhibits EGFR ubiquitinylation. CHO-Ts20-EGFR cells were transiently transfected with the pAlterMAX vector or constructs encoding HA-Cbl or HA-Cbl-C3AHN (RING finger mutant). Cells were grown at 30 °C and either left unstimulated or stimulated for 10 min prior to lysis. 1-mg aliquots of cell lysates were subjected to anti-EGFR IPs followed by serial anti-ubiquitin (top panel) and anti-EGFR (bottom panel) IB. B, wild type Cbl enhances, whereas the Cbl-C3AHN inhibits EGFR degradation. Cell lysates at various times following EGF stimulation were prepared as in A, and 50-µg aliquots of lysates were subjected to anti-EGFR IB. The relative EGFR signals (no EGF = 1) were determined by densitometry and are indicated at the bottom.

 

To directly assess the impact of inhibiting ubiquitinylation in CHO-Ts20 cells on EGFR degradation, we compared Ts20-EGFR and E36-EGFR (control) cell lines at 30 °C, the permissive temperature for ubiquitinylation, versus 42 °C, the nonpermissive temperature. When assayed at 30 °C, the ligand-induced EGFR ubiquitinylation was observed in both Ts20-EGFR and E36-EGFR cells (Fig. 5, A and B, lanes 1–3 and 7–9). In contrast, ligand-induced EGFR ubiquitinylation was markedly attenuated in Ts20-EGFR cells shifted to 42 °C (Fig. 5A, lanes 10–12); as a control, the ubiquitinylation of EGFR in E36-EGFR cells was unaffected by the temperature shift (Fig. 5A, lanes 4–6). Analysis of EGFR protein levels over a longer time course of EGF stimulation demonstrated that the kinetics of EGFR degradation in E36-EGFR cells was similar at 30 versus 42 °C (Fig. 5B, compare lanes 1–4 and 5–8). In contrast, the degradation of EGFR in Ts20-EGFR cells was retarded when these cells were shifted to 42 °C to block ubiquitinylation (Fig. 5B, compare lanes 9–12 and 13–16). The requirement of Cbl for EGFR degradation, together with the inhibition of EGFR degradation upon blockade of the ubiquitinylation pathway in Ts20-EGFR cells, supports the view that it is the ubiquitin ligase activity of Cbl rather than another function that is essential in Cbl-mediated EGFR degradation. Interestingly, in both Cbl/ MEF cell lines that we utilized, we observed a low level of residual EGFR ubiquitinylation upon ligand stimulation. Whether this is because of a low level of Cbl-b expression or because of an unrelated ubiquitin ligase capable of targeting EGFR for ligand-induced ubiquitinylation, remains to be investigated. The residual ubiquitinylation could account for the EGFR degradation that is still observed. Alternatively, the continued EGFR degradation could reflect the contribution of other mechanisms, such as those mediated by the dileucine motifs, to endosomal sorting of EGFR (52).



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FIG. 5.
Conditional impairment of EGFR ubiquitinylation and degradation in CHO-Ts20 cells. A, lack of EGFR ubiquitinylation at 42 °C in CHO-Ts20-EGFR cells. Control CHO-E36 cells (left panel) and mutant E1-expressing CHO-Ts20 cells (right panel) were stably transfected with human EGFR and clones expressing EGFR were identified by IB. The cells were plated at 30 °C and then either continued at the same temperature (lanes 1–3 and 7–9) or shifted to 42 °C(lanes 4–6 and 10–12) for 4 h, prior to stimulation with 100 ng/ml EGF for the indicated times and then lysed. 1-mg aliquots of cell lysates were subjected to anti-EGFR IPs and serial immunoblotting with anti-ubiquitin (top panel), anti-EGFR (middle panel), and anti-Tyr(P) (bottom panel) Abs. B, reduced EGFR degradation at 42 °C in CHO-Ts20-EGFR cells. Cell lysates at various times following EGF stimulation were prepared as in A, and 50-µg aliquots of lysates were subjected to anti-EGFR IB. The relative EGFR signals (no EGF = 1) were determined by densitometry and are indicated at the bottom.

 

Our finding that the initial internalization of EGFR was intact in Cbl/ MEFs suggested that Cbl and Cbl-mediated EGFR ubiquitinylation was dispensable for EGFR internalization, but left open the possibility that ubiquitinylation of another protein(s) may mediate the internalization. In this regard, EGF-inducible ubiquitinylation of endocytic proteins, such as Eps15 (53) and CIN85 (23), has been previously demonstrated, although the role of the ubiquitinylation in EGFR internalization remains to be established. Ubiquitin has been clearly recognized as a receptor internalization motif in yeast (20, 21), whereas studies of the growth hormone receptor in mammalian cells indicate that ubiquitinylation of an unknown non-receptor component may be crucial for internalization (22). The Ts20-EGFR system provided a suitable system to test if the ubiquitin pathway was also essential for initial internalization of EGFR. Therefore, we also examined the EGFR internalization in Ts20-EGFR and E36-EGFR cells, using the assay described above. Similar to control E36-EGFR cells (Fig. 6, left panel, p > 0.05), the rate of initial EGFR internalization in Ts20-EGFR cells grown at 30 and 42 °C was comparable (Fig. 6, right panel, p > 0.05). Thus, the ubiquitinylation machinery is dispensable for the initial internalization step in the endocytic traffic of EGFR.



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FIG. 6.
Unimpaired EGFR internalization in CHO-Ts20 cells. Cells were grown at 30 °C, and then either continued at the same temperature or shifted to 42 C, as indicated. Cells were allowed to bind Alexa Fluor 488-conjugated EGF at 4 °C for 30 min, and then returned to 37 °C to allow EGF internalization. Every experiment was done in triplicate 3 times. The percentage of internalized EGF was calculated and plotted as in Fig. 3.

 

Our results indicate that neither EGFR ubiquitinylation nor the monoubiquitinylation of other proteins, such as Eps15 and CIN85, is required for EGFR internalization. Dikic and colleagues have, however, observed that a dominant negative mutant of CIN85, which is not monoubiquitinylated by Cbl upon EGF stimulation, impairs the internalization and degradation of EGFR (23, 24). It is likely that the dominant negative form of CIN85 fails to interact with other partners, which may relate to its role at the internalization step. Furthermore, Dikic and colleagues (23) observed that the Cbl-dependent monoubiquitinylation of CIN85 primarily occurred after EGFR endocytosis, which suggests a role for ubiquitinylated CIN85 at a post-endocytic step, consistent with our conclusions.

Our results do not exclude the possibility that ubiquitinylation can function as an endocytic signal. In fact, monoubiquitinylation of EGFR is an enough signal for EGFR internalization (55). Our results do indicate, although, that this cannot be the sole mechanism for initial internalization, and that other mechanisms can fully support the internalization process in the absence of the ubiquitinylation of EGFR and any accessory proteins. The molecular nature of these additional signals remains to be fully elucidated. Several endocytic motifs have been identified in EGFR, including the dileucine-based motif (56) and the tyrosine-based AP-2 binding motif (57). However, mutational analyses suggest that none of these motifs is essential for internalization (56, 57). It is likely that a complex RTK such as EGFR has evolved multiple redundant mechanisms to ensure internalization, as this is a key regulatory process.

The internally controlled Ts20-EGFR and E36-EGFR cell pair also provided an opportunity to assess the nature of the endocytic compartment(s) where Cbl functions and where ubiquitinylation plays a decisive role in the endocytic trafficking of internalized EGFR. For this purpose, we assessed the colocalization of the internalized EGFR with selected endocytic markers, using confocal microscopy. In both E36-EGFR and Ts20-EGFR cells grown at 30 °C, the EGFR-containing vesicles were peripherally distributed at 5 min after EGF stimulation (Fig. 7A, B1 and E1) and gradually moved near the center of the cells, forming larger clusters by 30 min (Fig. 7A, C1 and F1). When E36-EGFR and Ts20-EGFR cells were compared at 42 °C, the EGFR staining pattern at 5 min of EGF stimulation was similar (Fig. 7B, compare B1 and E1), consistent with the unaltered EGFR internalization seen in the internalization assay. Notably, however, the distribution of EGFR containing vesicles in Ts20-EGFR versus E36-EGFR cells grown at 42 °C and stimulated for 30 min was quite distinct; these vesicles failed to move near the center and remained near the periphery in Ts20-EGFR cells (Fig. 7B, compare C1 and F1).



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FIG. 7.
Conditional alteration of internalized EGFR localization and its colocalization with transferrin receptor in CHO-Ts20 cells. Cells were either maintained at 30 °C (A) or were shifted to 42 °C (B), as described in the legend to Fig. 6, and stimulated with EGF for the indicated times. Cells were loaded with Alexa Fluor 488-conjugated transferrin (36) for 5 min prior to harvesting to visualize the early endosome/recycling endosome compartment. Cells were fixed, permeabilized, and stained with anti-EGFR (mAb 528) followed by Cy3-conjugated anti-mouse antibody (red), and analyzed by confocal microscopy. Colocalization is indicated by yellow coloration in the merged images.

 


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FIG. 7.
Conditional alteration of internalized EGFR localization and its colocalization with transferrin receptor in CHO-Ts20 cells. Cells were either maintained at 30 °C (A) or were shifted to 42 °C (B), as described in the legend to Fig. 6, and stimulated with EGF for the indicated times. Cells were loaded with Alexa Fluor 488-conjugated transferrin (36) for 5 min prior to harvesting to visualize the early endosome/recycling endosome compartment. Cells were fixed, permeabilized, and stained with anti-EGFR (mAb 528) followed by Cy3-conjugated anti-mouse antibody (red), and analyzed by confocal microscopy. Colocalization is indicated by yellow coloration in the merged images.

 

To characterize the EGFR-containing endosomal compartments, we either loaded the cells with fluorescent transferrin to mark the early/recycling endosomes, or carried out double staining for EGFR (red) and LAMP-1 (36), a late endosome/lysosome marker; the cells were then analyzed using confocal microscopy. At 5 min of EGF stimulation at 30 °C, the internalized EGFR in peripherally distributed vesicles mostly colocalized with transferrin (Fig. 7A, B3 and B4; E3 and E4; yellow); this colocalization was lost by 30 min of stimulation, when the EGFR was predominantly centrally clustered (Fig. 7A, C3 and C4; F3 and F4; red). However, EGFR in these vesicles colocalized with LAMP-1 (Fig. 8, B3 and B4; E3 and E4; yellow), indicating that a proportion of internalized EGFR under-went a time-dependent endocytic transport from early to late endosomes. This pattern remained unaltered when the E36-EGFR control cells were examined at 42 °C (Fig. 7B, C3 and C4). In contrast, when Ts20-EGFR cells were grown at 42 °C to disrupt ubiquitinylation, the internalized EGFR remained colocalized with transferrin at 30 min of EGF stimulation (Fig. 7B, F3 and F4; yellow), and showed a lower degree of colocalization with LAMP-1 (Fig. 8, F3 and F4). These results indicate that ubiquitinylation is crucial for an early endosome to late endosome sorting step in the endocytic trafficking of ligand-stimulated EGFR.



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FIG. 8.
Conditional impairment of EGFR localization to late endosome/lysosome in CHO-Ts20 cells. Cells were processed as described in the legend to Fig. 7A, 7B and stained with anti-EGFR antibody (visualized with Cy3-conjugated anti-mouse antibody; red) and rabbit anti-LAMP-1 antibody (late endosome/lysosome marker; visualized with Cy2-conjugated goat anti-rabbit antibody; green), and analyzed by confocal microscopy.

 

The simplest interpretation of these results is that Cbl-mediated ubiquitinylation of EGFR (and/or other accessory proteins) serves as an endosomal sorting signal for EGFR delivery from early to late endosome/lysosome, and that ubiquitin modification serves as an essential signal at this step to ensure the efficient delivery of EGFR into the degradative compartments of the endocytic machinery. This model is consistent with the previous finding that ubiquitinylation and proteasome activity is needed for EGFR transfer into internal vesicle of MVBs (19). It is also compatible with the genetically defined role of ubiquitinylation of yeast transmembrane receptors for sorting into the inner vesicles of the MVB (28, 58). In fact, yeast studies have identified endosomal sorting of receptor traffic (ESCRTs) complexes, such as ESCRT-1 (45), which function as ubiquitin recognizing proteins within the endocytic system. Deletion of TSG-101, one of the ESCRT-1 complex components in mammals, retards EGFR degradation and causes accumulation of ubiquitinylated proteins on endosomes (46). Furthermore, the hepatocyte growth factor receptor substrate, the mammalian counterpart of the yeast ESCRT-2 protein also has a ubiquitin interacting motif (49, 50, 53). A truncated hepatocyte growth factor receptor substrate protein lacking the ubiquitin interacting motif impaired EGFR degradation in Drosophila, by affecting endosome membrane invagination and MVB formation (54). Thus, Cbl-mediated ubiquitinylation could represent the receptor modification required for recognition by mammalian ESCRT proteins to facilitate endosomal sorting of EGFR to lysosomes. Whether or not the ubiquitin modification provides the signal for delivering EGFR into the inner vesicles of the MVB in mammalian cells, and the regulatory control of this process, is an important area of future investigation.

In conclusion, our studies utilizing two distinct and independent experimental systems provide strong evidence that Cbl-mediated ubiquitinylation is essential for efficient ligand-induced degradation of EGFR, but is dispensable for initial receptor internalization. Future studies should address whether this dichotomy is a general feature of Cbl regulation of RTKs and other transmembrane receptors.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grants CA 99163, CA 87986, CA 75075, and CA 76118 (to H. B.), CA 81076 and CA 70195 (to V. B.), National Institutes of Health Training Grant T32ARO7530 (to I. L. D.), and United States Army Fellowships DAMD17-99-1-9086 (to L. D. and K. M.-R.) and DAMD17-991-9085 (to S. D. and A. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to the results of this article. Back

Scholars of the Massachusetts Department of Public Health Breast Cancer Research Program. Back

¶¶ To whom correspondence should be addressed: Brigham and Women's Hospital, Smith Building, Rm. 538C, One Jimmy Fund Way, Boston, MA 02115. Tel.: 617-525-1101; Fax: 617-525-1010; E-mail: hband{at}rics.bwh.harvard.edu.

1 The abbreviations used are: RTK, receptor tyrosine kinase; CHO, Chinese hamster ovary; EGFR, epidermal growth factor receptor; IP, immunoprecipitate; IB, immunoblot; mAb, monoclonal antibody; LAMP, lysosome-associated membrane protein; MEF, mouse embryonic fibroblast; PBS, phosphate-buffered saline; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; FACS, fluorescence-activated cell sorter; HA, hemagglutinin; ESCRT, endosomal sorting of receptor traffic. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Ger Strous (University Medical Center, Utrecht, The Netherlands) for the CHO-Ts20 and CHO-E36 cell lines, Dr. Minoru Fukuda (The Burnham Institute) for anti-LAMP-1 (931B), Jean Lai for technical assistance with confocal microscopy, and Dr. Victor Hsu for critically reading the manuscript.



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