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J Biol Chem, Vol. 274, Issue 44, 31707-31712, October 29, 1999


Ligand-induced Ubiquitination of the Epidermal Growth Factor Receptor Involves the Interaction of the c-Cbl RING Finger and UbcH7*

Masahiro YokouchiDagger §, Takeshi Kondoparallel , Adam HoughtonDagger §, Marcjanna BartkiewiczDagger §, William C. HorneDagger §, Hui Zhangparallel , Akihiko Yoshimura, and Roland BaronDagger §**

From the Departments of Dagger  Cell Biology, § Orthopaedics, and parallel  Genetics, Yale University School of Medicine, New Haven, Connecticut 06510 and the  Institute of Life Science, Kurume University, Aikawamachi 2432-3, Kurume 839-0861, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. We demonstrate here, using the yeast two-hybrid system and an in vitro binding assay, that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacted with the wild-type c-Cbl RING finger domain but not with a RING finger domain that lacks the amino acids that are deleted in 70Z-Cbl, an oncogenic mutant of c-Cbl. The in vitro interaction was enhanced by sequences on both the N- and C-terminal sides of the RING finger. In vivo and in vitro experiments revealed that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR). In contrast, 70Z-Cbl markedly reduced the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolonged the ligand-induced phosphorylation of both the EGFR and c-Cbl. Thus, c-Cbl plays an essential role in the ligand-induced ubiquitination of the EGFR by a mechanism that involves an interaction of the RING finger domain with UbcH7. This mechanism participates in the down-regulation of tyrosine kinase receptors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

c-Cbl is an adaptor protein which is involved in several signaling pathways, where it interacts with receptor or non-receptor tyrosine kinases via at least one of its functional domains. The exact role of c-Cbl in these signaling pathways is not fully elucidated, however. Recent reports have indicated that in several instances c-Cbl exerts a negative regulatory function on receptor and non-receptor tyrosine kinases (1-7), a hypothesis first suggested by genetic studies in Caenorhabditis elegans (8, 9), but the mechanism by which c-Cbl exerts this negative regulatory function remains to be demonstrated.

The functional domains of c-Cbl include a phosphotyrosine-binding (PTB)1 domain, a RING finger domain, a proline-rich region, and a leucine zipper. In addition, several tyrosine residues can be phosphorylated to generate SH2 binding motifs (for review, see Refs. 10 and 11). The PTB and RING finger domains are evolutionarily conserved (10, 11), and Drosophila Cbl (D-Cbl), which lacks both the proline-rich region and the leucine zipper, can still function as a negative regulator in EGF receptor (EGFR) signaling (12, 13). It is therefore most likely that the negative regulatory function of c-Cbl is dependent upon the PTB and/or RING finger domains. Indeed, previous studies have indicated that the PTB domain plays an essential role in c-Cbl-dependent negative regulation of tyrosine kinases (6, 7). It is, however, likely that the RING finger domain is also involved in negative regulation since deletion of 17 amino acids that overlap the N-terminal boundary of the c-Cbl RING finger domain (amino acids 366-382), as occurs in the 70Z/3 mouse pre-B lymphoma cell line, is sufficient for transformation (14). Andoniou et al. (14) tested a number of deletion mutants of c-Cbl for their transforming activity in NIH3T3 cells and suggested that a truncation that disrupts the RING finger domain is sufficient to activate c-Cbl's tumorigenic potential, although it is not enough to give it full transforming potential. While it is known that the PTB domain interacts with phosphotyrosine residues and is involved in the binding of c-Cbl to tyrosine kinases as well as to tyrosine-phosphorylated adaptor molecules (15, 16), the function of the c-Cbl RING finger domain is not known at present.

Here we provide evidence that the c-Cbl RING finger domain associates with the human ubiquitin-conjugating enzyme 7 (UbcH7) (17) and that their interaction plays a critical role in the ligand-induced ubiquitination of the EGFR in cells. c-Cbl recruits ubiquitin-conjugating enzyme (E2) via its RING finger domain, allowing ubiquitination of phosphorylated substrates and thereby inducing their degradation via the proteasome pathway. Moreover, the oncogenic 70Z-Cbl fails to bind UbcH7, and exerts a dominant negative effect on ligand-induced ubiquitination of the EGFR, suggesting a possible explanation for its transforming potential.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Construction of Fusion Proteins-- The following c-Cbl mutants were prepared by the PCR technique using the clone of hemagglutinin-tagged human c-Cbl in pGEM-4Z, kindly provided by Dr. W. Langdon (Dept. of Biochemistry, University of Western Australia) as template: RING (codon 374-430), RING-A (codon 358-430, lacks residues 366-382), RING-B (codon 358-430), RING-C (codon 358-445), RING-D (codon 358-472), RING-E (codon 358-472, lacks 366-382), and RING-F (codon 374-472) (see Fig. 1A). Glutathione S-transferase (GST) fusion proteins were created by cloning the PCR fragments containing additional EcoRI and SalI sites into the pGEX-4T-1 vector (Amersham Pharmacia Biotech). Plasmids were transformed into Escherichia coli strain NM522 (Stratagene), and GST fusion proteins were purified with GSH-Sepharose (Amersham Pharmacia Biotech). To create Myc-tagged UbcH7 (codon 2-154), the PCR products were subcloned into pCS-MT+ (18) in-frame. The resulting fusion proteins contained a Myc tag at the N terminus. The coding sequences were transferred to a pcDNA3 expression vector using HindIII/XbaI sites.

Two-hybrid Screening-- To identify a clone interacting with the c-Cbl RING finger domain, a two-hybrid screen was performed as described previously (19). DNA fragments encoding the c-Cbl RING finger domain or the deletion mutant of the RING finger domain lacking 17 amino acids were created by PCR, then subcloned into the pBTM116 vector as fusions to the LexA DNA-binding domain (RING/BD and RING-A/BD, respectively; see Fig. 1A). Human MDM2 (20) and UbcH5 (21) cDNAs were obtained by reverse transcriptase-PCR. The sequence encoding the MDM2-RING finger domain (codon 424-496) was subcloned into pBTM (MDM2-RING/BD). Full-length UbcH5 (codon 1-148) was subcloned into the pACT vector as a fusion to GAL4 DNA-activating domain (UbcH5/AD).

Cell Lines and Culture-- 293 cells were grown in minimal essential medium, alpha -modification containing 10% fetal calf serum. To obtain the stable transformants of c-Cbl, the Myc-tagged version of full-length c-Cbl cDNA (codon 2-906) was subcloned into the pcDNA3/Zeo expression vector. The plasmid was introduced into 293 cells using Lipofectin (Life Technologies, Inc.) and stable transformants were selected with 0.5 mg/ml Zeosin. The cell line stably expressing Myc-c-Cbl was maintained in minimal essential medium, alpha -modification containing 10% fetal calf serum.

Antibodies, Immunoprecipitation, and Immunoblotting-- Anti-EGF receptor, anti-c-Cbl, and anti-UbcH7 antibodies were purchased from Transduction Laboratories and the mouse monoclonal anti-ubiquitin antibody was purchased from CHEMICON. Anti-phosphotyrosine antibody (Tyr(P)99), anti-GST antibody, and the mouse monoclonal anti-c-Myc antibody (9E10) were purchased from Santa Cruz Biotechnology, Inc. Immunoprecipitation and immunoblotting were performed as described previously (22). To increase the immunoblot sensitivity with anti-ubiquitin antibody, the membrane was treated at 100 °C before probing with antibody as previously reported (23). For reprobing, immunoblots were stripped with buffer containing 0.2 M glycine and 0.5 M NaCl, pH 2.8.

In Vitro Ubiquitination Assay-- Untransfected 293 cells were serum-starved, then stimulated with 100 ng/ml EGF for 1 min to induce phosphorylation. EGF-treated and untreated cells were lysed in 0.5% IGEPAL CA-630 (Sigma), 20 mM Hepes, pH 7.2, 50 mM sodium fluoride, 1 mM sodium vanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride, and the EGFR was immunoprecipitated from 1.5 mg of lysate. The immune complexes on agarose beads were washed with phosphate-buffered saline three times and with reaction buffer two times. Cell extracts of untransfected 293 cells or 293 cells that overexpressed c-Cbl or 70Z-Cbl were prepared by resuspending pelleted cells in 2 volumes of 20 mM Hepes, pH 7.2, 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 20 µM MG132, and protease inhibitors as described above, and sonicating for two cycles of 30 s. The reaction mixture (150 µl) contained the immunoprecipitates, 20 mM Hepes, pH 7.2, 10 mM MgCl2, 1 mM ATP, 1 mM dithiothreitol, 30 mM creatine phosphate, 0.1 mg/ml creatine kinase, 25 µM MG132, 7.5 µg of GST-ubiquitin, and 300 µg of cell extracts. After incubation at 30 °C for 2 h, the beads were washed with 0.5% IGEPAL CA-630 in phosphate-buffered saline three times, then prepared for Western blotting and analyzed for the presence of GST-ubiquitin linked to the EGFR.

Effect of Proteasome Inhibitor-- MG132 (N-Cbz-Leu-Leu-Leu-AL) was purchased from Sigma and was dissolved in dimethyl sulfoxide before use. Throughout the experiments, the final concentration of dimethyl sulfoxide in cell culture media was kept 0.1% in both treated and control cultures.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Yeast Two-hybrid Screening Reveals a c-Cbl RING Finger-UbcH7 Interaction-- To identify proteins that bind to the c-Cbl RING finger domain we screened several yeast two-hybrid libraries using that protein fragment as bait. In several experiments, this led to the cloning of the ubiquitin conjugating enzyme UbcH7. Three cDNAs were isolated, each containing the complete coding sequence but differing in the length of the 5'-untranslated region. Using the yeast two-hybrid procedure, we found that, in yeast, UbcH7 interacts with the c-Cbl RING finger domain but not with the 70Z 17-amino acid deletion mutant of the c-Cbl RING finger domain (RING-A) (Fig. 1B). Recent studies have demonstrated that the RING finger domain-containing protein MDM2 (24) cooperates specifically with another ubiquitin conjugating enzyme, UbcH5, to ubiquitinate the tumor suppressor protein p53 (25). To determine the relative specificity of such RING-ubiquitin conjugating enzyme interactions, we tested the ability of the MDM2 RING finger to interact with UbcH7 and, reciprocally, the ability of the c-Cbl RING finger to interact with UbcH5. Neither interaction could be detected (Fig. 1B), thereby demonstrating that these RING finger domains interact with E2 molecules in a specific manner.


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  		Fig. 1.   The c-Cbl RING finger domain binds to UbcH7. A, schematic representation of c-Cbl constructs used in this study. The location of the RING finger domain is shown by the white box. Numbers indicate amino acid position. An oncogenic 17-amino acid deletion (70Z) is indicated. B, two-hybrid analysis of the interaction between c-Cbl RING finger and UbcH7. Yeast strains carrying the LexA-binding domain fused to the c-Cbl RING finger domain (RING/BD), a deletion mutant of c-Cbl RING finger domain (RING-A/BD), or the MDM2 RING finger domain (MDM2-RING/BD) were transformed with plasmids carrying a fusion between the GAL4 activating domain (AD) and UbcH7. Transformants were restreaked on a paper filter and assayed by an in situ beta -galactosidase assay. C, in vitro association of the c-Cbl RING finger domain and UbcH7. Myc-tagged UbcH7 in pcDNA3 (5 µg/transfection) was transiently expressed in 293 cells grown in 10-cm dishes. 48 h after transfection, the cells were serum-starved and then incubated with (+) or without (-) 100 ng/ml EGF for 5 min at 37 °C. Cells were lysed in 500 µl of lysis buffer, then cell extracts were incubated with 3 µg of immobilized GST or GST fusion proteins as indicated in A at 4 °C for 2 h. The protein complexes or 2% of total cell extracts (TCL) were resolved on 10% SDS-polyacrylamide gel electrophoresis, then immunoblotted with anti-Myc antibody (lower panel). Purified GST fusion proteins were analyzed with 10% SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining (upper panel; CBB). An arrowhead in C indicates the Myc-tagged UbcH7.

The c-Cbl RING Finger Binds UbcH7 in Vitro-- The c-Cbl RING finger domain encompasses amino acids 380 to 425, and c-Cbl constructs encoding only the N-terminal 1-436 amino acids or with deletions of either of Tyr368 or Tyr371 have tumorigenic potential (14). In order to confirm the interaction of the c-Cbl RING finger domain with UbcH7 in vitro and to analyze the role of specific c-Cbl sequences in the interaction, we generated several RING finger constructs that fused GST to the RING finger domain and adjacent regions (illustrated in Fig. 1A). Myc epitope-tagged UbcH7 (Myc-UbcH7) was expressed in 293 cells and the cells were either stimulated with EGF or left untreated. The cells were then lysed and cell extracts were incubated in vitro with the GST-RING fusion proteins. As shown in Fig. 1C, the RING-D (codon 358-472) fusion protein efficiently associated with UbcH7 regardless of EGF stimulation. However, the short RING finger region (374-430), or other constructs lacking adjoining regions of the RING finger domain did not interact well with UbcH7 in vitro, indicating that both the N- and C-terminal proximal regions are necessary for high affinity interaction with UbcH7 (Fig. 1C). The requirement for these N- and C-terminal adjacent regions for detection of the RING-UbcH7 interaction by in vitro binding but not for the yeast two-hybrid may be due to a higher sensitivity of the latter assay.

c-Cbl but Not 70Z-Cbl Enhances Ligand-induced EGFR Ubiquitination-- It has recently become evident that c-Cbl regulates the ligand-induced ubiquitination of the platelet-derived growth factor receptor (26, 27), EGFR (28), and colony-stimulating factor-1 receptor (29, 30). However, the molecular mechanism by which c-Cbl does this has not been clarified. Our yeast two-hybrid assays and in vitro binding results suggested that c-Cbl recruits UbcH7 through its RING finger. Ubiquitin-conjugating enzymes (E2) play an essential role in the ubiquitination of proteins (for review, see Refs. 31 and 32). We therefore hypothesized that c-Cbl could on the one hand bind to the receptor and on the other hand serve as a docking protein for UbcH7, thereby acting as a ubiquitin ligase (E3) to mediate the ubiquitination and subsequent degradation of the phosphorylated receptor. To test this hypothesis, we examined the effects of overexpression of UbcH7 and c-Cbl on the ligand-induced ubiquitination of the EGFR (Fig. 2A). Myc-tagged c-Cbl, 70Z-Cbl (the oncogenic mutant c-Cbl lacking 17 amino acids at the boundary of the RING finger domain), or UbcH7 were expressed in 293 cells, and the cells were stimulated with EGF for various times. As hypothesized, overexpression of either Myc-UbcH7 or Myc-c-Cbl clearly enhanced the ligand-induced ubiquitination of the endogenous EGFR. Interestingly, and in contrast with wild-type c-Cbl, overexpression of Myc-70Z-Cbl did not enhance the ligand-induced ubiquitination of the EGFR, suggesting that 70Z-Cbl lacks the ability to promote ubiquitination of the EGFR, and further supporting the role of the c-Cbl RING finger domain and UbcH7 in the ubiquitination of the EFGR.


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  		Fig. 2.   c-Cbl RING finger and UbcH7 mediate the ligand-induced ubiquitination of the EGFR. A, plasmids carrying Myc-tagged full-length c-Cbl (Cbl; lanes 6-10), 70Z-Cbl (70Z; lanes 11-15), UbcH7 (UbcH7; lanes 16-20), or empty vector (empty; lanes 1-5) were transfected into 293 cells. After serum starvation, cells were stimulated with 100 ng/ml EGF for the indicated time periods, then washed and solubilized. Lysates were subjected to immunoprecipitation with anti-EGFR antibody (alpha -EGFR) (A and B). Total cell extracts (TCL) and immunoprecipitates were resolved on 10 (TCL) or 8% (immunoprecipitates) SDS-polyacrylamide gels, then immunoblotted with anti-ubiquitin antibody (alpha -Ub), anti-EGFR antibody (alpha -EGFR), or anti-Myc antibody (alpha -Myc). B, Myc-tagged UbcH7 was transfected into the cells stably expressing the full-length c-Cbl (293-Cbl) and the ubiquitination of the EGFR analyzed as described A.

To determine whether c-Cbl and UbcH7 had additive effects, we created a stably transformed 293 cell line that overexpresses c-Cbl (293-Cbl). Although 293 cells express endogenous c-Cbl, ligand-induced ubiquitination of the EGFR was enhanced in c-Cbl transformants compared with parental 293 cells (data not shown), as we had observed in the transiently transfected cells (Fig. 2A). Expression of UbcH7 in 293-Cbl cells resulted in greatly enhanced ligand-induced ubiquitination of the EGFR (Fig. 2B). These data indicated that both the RING finger containing c-Cbl and UbcH7 participate in the ligand-induced ubiquitination of the EGFR in cells.

To further confirm the role of a c-Cbl·UbcH7 complex in the ligand-induced ubiquitination of the EGFR, we examined the effect of exogenous UbcH7 on the in vitro ubiquitination of activated EGFR (Fig. 3). Assays were performed using extracts from untransfected 293 cells, c-Cbl overexpressing 293 cells, or 70Z-Cbl overexpressing 293 cells. The c-Cbl cell extract, but not the 70Z-Cbl cell extract, enhanced the ubiquitination of phosphorylated EGFR relative to that obtained with parental 293 cell extracts (Fig. 3, upper panel, lanes 2, 4, and 6). The addition of exogenous UbcH7 significantly increased the ubiquitination of activated-EGFR in the assay with the extracts of the c-Cbl-overexpressing cells (Fig. 3, upper and middle panel, lanes 4 and 5) but not in the assays with extracts of the parental 293 cells (Fig. 3, upper panel, lanes 2 and 3) or those of the 70Z-Cbl-overexpressing cells (Fig. 3, upper panel, lanes 6 and 7). These findings clearly demonstrate that c-Cbl and UbcH7 together promote the ubiquitination of the EGFR via a mechanism that requires an intact c-Cbl RING finger domain.


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  		Fig. 3.   UbcH7 and c-Cbl but not 70Z-Cbl synergistically enhance the ubiquitination of the EGFR in vitro. Reaction mixtures containing extracts of untransfected 293 cells (293) (lanes 1-3), c-Cbl-overexpressing 293 cells (293-Cbl) (lanes 4 and 5), or 70Z-Cbl-overexpressing cells (293-70Z-Cbl) (lanes 6 and 7) were incubated without (lanes 1, 2, 4, and 6) or with (lanes 3, 5, and 7) purified UbcH7 protein (500 ng) at 30 °C for 2 h. The immune complexes were resolved on 8% SDS-polyacrylamide gels, then immunoblotted with anti-ubiquitin antibody (alpha -Ub), anti-GST antibody (alpha -GST), or the anti-EGFR antibody (alpha -EGFR).

Given the fact that c-Cbl binds stably to the activated EGFR, we sought to demonstrate that UbcH7 was also present in the EGFR·c-Cbl complex isolated from cells stimulated with EGF. We were unable, however, to isolate complexes that contained UbcH7 in addition to the EGFR and c-Cbl (data not shown), suggesting that the association of UbcH7 with the c-Cbl-EGFR complex is not sufficiently long lived to be detected in this manner.

Overexpression of 70Z-Cbl Reduces Ligand-induced EGFR Ubiquitination-- The transfection and in vitro ubiquitination experiments indicated that 70Z-Cbl, which lacks 17 amino acids at the N-terminal boundary of the RING finger, is unable to enhance ligand-induced ubiquitination of the EGFR (Fig. 2A). Wild type c-Cbl may act as a docking protein, binding to the activated EGFR through its PTB domain and in turn allowing the binding of UbcH7 to its RING finger, thereby bringing the ubiquitin conjugating enzyme to its substrate. We therefore hypothesized that 70Z-Cbl, which lacks residues that are necessary for the Cbl-UbcH7 interaction but is still capable of binding to the activated EGFR (5, 33), would prevent the binding of the c-Cbl·UbcH7 complex to the activated EGFR. According to this hypothesis, overexpression of 70Z-Cbl should not only fail to induce ubiquitination but also prevent the c-Cbl-UbcH7-induced ubiquitination of the EGFR. To test this hypothesis, we examined the effect of overexpressing 70Z-Cbl on UbcH7-mediated ubiquitination of the EGFR. Myc-tagged UbcH7 was expressed in 293 cells with or without 70Z-Cbl, and the cells were stimulated with EGF for various periods of time. UbcH7 again clearly enhanced ubiquitination of the EGFR (Fig. 4, upper panel, compare lanes 1 and 3), and as predicted, the co-expression of the 70Z-Cbl protein inhibited the UbcH7-mediated ubiquitination of the EGFR in a dose-dependent manner. Thus, both c-Cbl and 70Z-Cbl bind to the tyrosine-phosphorylated receptors through their PTB domain (16) but only c-Cbl, with an intact RING finger, can bring ubiquitin to the complex.


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  		Fig. 4.   70Z-Cbl suppresses ligand-induced ubiquitination of the EGFR. Empty vector (lane 1) or Myc-tagged UbcH7 (lanes 2-10) was transfected into 293 cells with 5 µg of 70Z-Cbl (5 µg; lanes 5-7), 10 µg of 70Z-Cbl (10 µg; lanes 8-10), or empty vector (lanes 2-4). After stimulation, total cell lysates (TCL) and immunoprecipitates were resolved on 10 (TCL) or 8% (immunoprecipitates) SDS-polyacrylamide gel, then immunoblotted with anti-ubiquitin antibody (alpha -Ub), the anti-EGFR antibody (alpha -EGFR), or anti-Myc antibody (alpha -Myc).

Proteasome Inhibition Prolongs EGF-induced Tyrosine Phosphorylation of EGFR and c-Cbl-- Ubiquitination of cellular proteins leads to their degradation by the proteasome system, and it has been suggested that this system is involved in ligand-mediated down-regulation of cell surface receptors (for review, see Ref. 31). We therefore examined whether inhibition of ubiquitin/proteasome-dependent degradation would prolong the duration of the ligand-induced phosphorylation of the EGFR. Cells stably expressing c-Cbl (293-Cbl) were preincubated with or without a proteasome inhibitor, MG132, and the cells were stimulated with EGF for various time periods (Fig. 5). In MG132-treated cells, the EGF-induced tyrosine phosphorylation of a number of intracellular proteins was clearly sustained for a longer period of time (Fig. 5A). Immunoprecipitation with anti-EGFR antibody revealed that the duration of the presence of the tyrosine-phosphorylated EGFR was extended when the cells were treated with MG132 (Fig. 5B). Moreover, the ligand-induced phosphorylation of c-Cbl as well as its association with the EGFR were also prolonged in the presence of MG132 (Fig. 5C). Previous studies have demonstrated that overexpression of 70Z-Cbl enhanced tyrosine phosphorylation of the EGFR after stimulation, that tyrosine phosphorylation of 70Z-Cbl in response to EGF stimulation was markedly enhanced compared with wild-type c-Cbl, and that the binding of 70Z-Cbl to the activated EGFR was also enhanced (5, 33). Thus, inhibition of ubiquitin/proteasome-dependent degradation has similar effects on the tyrosine phosphorylation of the EGFR and downstream signaling as does overexpression of 70Z-Cbl. These results suggest that ligand-induced ubiquitination and degradation of the receptor could be an integral part of the mechanism of down-regulation of signaling after activation of the EGFR, and that some or all of 70Z-Cbl's transforming activity may be due to the inability of the defective RING finger to interact with UbcH7, resulting in the dominant negative effect of 70Z-Cbl on the ubiquitination of EGFR.


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  		Fig. 5.   MG132 prolongs EGF-induced tyrosine phosphorylation of the EGFR and c-Cbl. Equal numbers of 293-Cbl cells were pretreated with 0.1% dimethyl sulfoxide (DMSO; lanes 1-5) or 50 µM MG132 (MG132; lanes 6-10) for 2 h, then stimulated with 50 ng/ml EGF for the indicated time periods. Proteins from cell lysates were immunoprecipitated with either alpha -EGFR (B) or anti-Myc antibody (alpha -Myc) (C). Total cell extracts (TCL) and immunoprecipitates (IP) were resolved on 10 (TCL) or 8% (immunoprecipitates) SDS-polyacrylamide gels, then immunoblotted with anti-phosphotyrosine antibody (alpha -PY), anti-EGFR antibody (alpha -EGFR), or anti-Myc antibody (alpha -Myc).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We interpret our results to show that c-Cbl, in addition to functioning as an adaptor protein and a substrate for tyrosine phosphorylation, is an E3 ubiquitin ligase, and participates in the down-regulation of tyrosine kinase receptors by mediating their ubiquitination. We show here that this function is dependent on the integrity of the RING finger of c-Cbl and its interaction with UbcH7.

Ubiquitination and the consequent degradation of the ubiquitinated protein by the proteasome or lysosomal pathways plays an important role in the control of numerous cellular processes, including signal transduction (34, 35). It is widely assumed that the E2 and E3 classes of ubiquitinating enzymes play a major role in mediating substrate recognition, and E3s are considered to be largely responsible for target specificity (for review, see Ref. 31). Although the molecular mechanisms by which substrates are selected by the ubiquitin-conjugating apparatus are still poorly understood, some E3 proteins may function simply as docking proteins that bind to both a specific substrate protein and a specific E2 (for review, see Ref. 32). Recent reports from several laboratories (25, 36-40) have identified RING finger domains, or R-boxes, in E3 proteins and suggested that this domain serves as the binding site for a number of E2s on the corresponding E3s that determine target specificity. In many cases, the target recognition and E2-binding functions are performed by different subunits of a multimolecular complex (38-41), as illustrated in Fig. 6A for the recently described S. cerevisiae SCF ubiquitin ligase (41). Interestingly, it is now apparent that in some of cases, the domain in the target recognition subunit that binds the RING finger-containing subunit is a SOCS box (38, 41). This motif, named suppressor of cytokine signaling, was first identified in the SOCS/JAB proteins that down-regulate the activity of the JAK-STAT signaling pathway by binding to phosphorylated JAK (19, 42, 43). Our present data suggest that in contrast with these multimeric ubiquitin ligases, c-Cbl performs both the docking functions of such E3 ligase complexes, combining both the substrate targeting function (PTB domain) and the E2-binding RING domain in a single molecule with multiple protein recognition domains (Fig. 6B).


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  		Fig. 6.   c-Cbl combines the substrate recognition and E2-binding functions of ubiquitin ligase in a single molecule. A, model of the Skp1-Cdc53·CUL1-F-box protein complexes (adapted from Ref. 41). B, wild-type c-Cbl binds to autophosphorylated EGFR via its PTB domain and recruits UbcH7 to the complex through the RING finger domain, leading to the ubiquitination and proteasome-catalyzed degradation of the phosphorylated EGFR. c-Cbl combines both the targeting function (PTB domain) and the E2-binding RING domain in a single molecule. C, 70Z-Cbl cannot bind UbcH7 and so fails to recruit the ubiquitin conjugating enzyme to the activated EGFR, resulting in the dominant negative effect of 70Z-Cbl on the ubiquitination of the activated EGFR. Ub, ubiquitin; RF, c-Cbl RING finger domain.

Very recently, Miyake et al. (27) reported that a point mutation (G306E) that inactivates the PTB domain of c-Cbl abrogated the ability of c-Cbl to enhance the ligand-induced ubiquitination of platelet-derived growth factor receptor. However, G306E-Cbl can still associate with the activated platelet-derived growth factor receptor via its C terminus region, suggesting that the interaction of the PTB domain with the receptor (or another target protein) somehow promotes the interaction of UbcH7 with c-Cbl and/or induces the transfer of ubiquitin from UbcH7 to the receptor. Given the fact that c-Cbl also functions as a major cellular adaptor protein, such a mechanism could prevent the nonspecific ubiquitination of proteins that bind to other domains of c-Cbl. It also suggests a possible explanation of our inability to detect a stable EGFR·c-Cbl·UbcH7 complex. Since the EGFR must be polyubiquitinated, the E2 protein must disengage from the complex as soon as it has transferred its ubiquitin in order to allow the next ubiquitinated E2 to bind to the RING finger. It would therefore not be expected to remain associated during the isolation of the c-Cbl·EGFR complex.

Since c-Cbl binds to a number of tyrosine-phosphorylated proteins, including non-receptor tyrosine kinases, it may also function as an E3 for other signaling molecules. Wang et al. (44) have shown that c-Cbl is transiently ubiquitinated after colony-stimulating factor-1 stimulation, but that it is not degraded, in contrast to the colony-stimulating factor-1 receptor. We also failed to see any reduction of the level of c-Cbl protein after EGF stimulation in this study (Fig. 5C). Thus, c-Cbl may target the ubiquitinating system to the activated receptors, thereby inducing their degradation, but escape from proteasomal degradation itself.

The targeting of the EGFR and other receptor tyrosine kinases for ubiquitination and proteolysis is only one of several mechanisms that contribute to the rapid down-regulation of signaling by the receptor. As recently discussed (45), two additional independent mechanisms exist in Drosophila to terminate signaling by the EGFR. In one, activation of the EGFR induces expression of the transmembrane protein Kekkon1, which binds directly to the activated receptor and apparently interferes with EGFR activation (46). The other mechanism involves the induction of Sprouty, a protein that binds the downstream signaling molecules Drk and Gap, thereby blocking Ras-related signaling (47).

In conclusion, our results suggest that c-Cbl exerts its negative regulatory function on EGFR (and probably on the platelet-derived growth factor receptor and colony-stimulating factor-1 receptor), at least in part, via the ligand-induced RING finger-dependent recruitment of UbcH7 into complexes with the tyrosine-phosphorylated receptor proteins, leading to their subsequent ubiquitination and degradation. Furthermore, a naturally occurring oncogenic mutant in which the RING finger domain has been partially deleted (70Z-Cbl) functions as a dominant negative form of c-Cbl for EGF-dependent ubiquitination and degradation of the receptor, suggesting that the loss of the RING finger-mediated UbcH7 binding may be an important determinant in the oncogenic properties of 70Z-Cbl (Fig. 6C). Interestingly, v-Cbl also contains the PTB domain but lacks the RING finger, suggesting that its oncogenic activity too could be related to the inhibition of the ubiquitination of receptor(s). Given that the v-Cbl and 70Z-Cbl transformed phenotypes differ, further work will be necessary to understand the possible role of the RING finger deletion in v-Cbl-induced transformation.

    ACKNOWLEDGEMENTS

We thank W. Y. Langdon for the gift of human c-Cbl cDNA, D. Finley for the gift of the plasmid encoding the GST-ubiquitin, and the members of our laboratories for many helpful discussions.

    FOOTNOTES

* This work was supported by the Japan Society for the Promotion of Science and by grants from the Nissan Science Foundation (to M. Y.), The Ministry of Education, Science, Sports and Culture, TORAY Research Foundation, Sumitomo Research Foundation (to A. Y.), and by National Institutes of Health Grant AR-42927 and Ariad Pharmaceuticals (to R. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Depts. of Cell Biology and Orthopaedics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Tel.: 203-785-4150; Fax: 203-785-2744; E-mail; roland.baron{at}yale.edu.

    ABBREVIATIONS

The abbreviations used are: PTB, phosphotyrosine-binding domain; SH2, Src homology domain 2; EGFR, epidermal growth factor receptor; UbcH, human ubiquitin-conjugating enzyme; PCR, polymerase chain reaction; GST, glutathione S-transferase; JAK, Janus kinase; SOCS, suppressor of cytokine signaling; STAT, signal transducers and activators of transcription.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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