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Originally published In Press as doi:10.1074/jbc.M703725200 on June 1, 2007

J. Biol. Chem., Vol. 282, Issue 29, 21278-21284, July 20, 2007
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GAPex-5 Mediates Ubiquitination, Trafficking, and Degradation of Epidermal Growth Factor Receptor*

Xiong Su, Chen Kong, and Philip D. Stahl1

From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, May 4, 2007 , and in revised form, May 25, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Upon ligand stimulation, epidermal growth factor receptor (EGFR) is rapidly ubiquitinated, internalized, and sorted to lysosomes for degradation. Rab5 has been shown to play an important role in the early stages of EGFR trafficking. GAPex-5 is a newly described Rab5 exchange factor. Herein, we investigate the role of GAPex-5 on EGFR trafficking and degradation. Down-regulation of GAPex-5 by RNA interference decreases epidermal growth factor-stimulated EGFR degradation. Moreover, ubiquitination of EGFR is impaired by depletion of GAPex-5. This inhibitory effect is due to a decrease in the interaction between the adapter protein c-Cbl and EGFR, but not the phosphorylation state of EGFR. Consistently, when examined by immunofluorescence microscopy in cells depleted of GAPex-5, ligand-bound EGFR appeared trapped in early endosomes and the trafficking of internalized receptor from early to late endosomes was impaired. In agreement with the depletion studies, EGFR degradation is enhanced by overexpressing GAPex-5 wild type, but not GAPex-5{Delta}GAP, a mutant lacking the Ras GTPase-activating protein (GAP) domain. This is consistent with the finding that c-Cbl binds specifically to the Ras GAP domain. Finally, overexpression of dominant negative Rab5a or depletion of all three isoforms of Rab5 does not inhibit ubiquitination of EGFR, which suggests that GAPex-5-mediated EGFR ubiquitination is independent of Rab5 activation. Collectively, the results suggest a novel mechanism by which EGF-stimulated receptor ubiquitination and trafficking are mediated via GAPex-5.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperactivation of epidermal growth factor receptor (EGFR)2 has been linked to proliferative disorders, including the progression of certain tumors (1, 2). It is critical that healthy cells precisely regulate the activation and attenuation of EGFR signaling (3). The strength and quality of signals generated by EGFR are dictated by both quantity and intracellular localization of the receptor. Although activated EGFR may stimulate downstream signaling molecules either on the plasma membrane or in signaling endosomes, its degradation has been shown to be important in prevention of excessive signaling and cell proliferation (3). The trafficking of EGFR is tightly controlled by chemical modifications of the receptor. Upon ligand binding, the activated, dimerized EGFR autophosphorylates at multiple tyrosines within the cytoplasmic tail. These phosphorylated residues mediate the recruitment of multiple downstream effectors. c-Cbl, a ubiquitin E3 ligase, is recruited to EGFR where it then initiates the ubiquitination of the activated receptor (4, 5). The ubiquitinated EGFR is internalized into early endosomes, from where it is either recycled to plasma membrane or delivered to multivesicular bodies (MVB) and late endosomes for degradation (6, 7). Ubiquitinated EGFR is sorted into the lumen of MVB through interactions with multiple protein complexes required for transport (ESCRTs) (8, 9). The fate of the ESCRT-associated EGFR is subsequently mediated by different deubiquitinating enzymes (e.g. UBPY and AMSH) based on their reaction specificity (10-12).

The small GTPase Rab5 plays important roles in a variety of cellular trafficking and signaling events, including receptor internalization (13), targeting and fusion of endocytic vesicles with early endosomes (14), fusion between early endosomes (15, 16), actin remodeling (17), and signaling to the nucleus (18). Activation of Rab5 is mediated by guanine nucleotide exchange factors that promote the exchange of bound GDP for GTP. A large number of proteins have been identified as potential Rab5 guanine nucleotide exchange factors as they contain a highly conserved domain (Vps9) that catalyzes nucleotide exchange on Rab5. Thus far, in mammalian cells, the tried and proven Rab5 guanine nucleotide exchange factors include Rabex-5 (19), ALS2 (20), Rin proteins (1 through 3) (21-24), and GAPex-5 (25-27). It is likely that the diversity of Rab5 functions is achieved by temporal and spatial regulation of different Rab5 guanine nucleotide exchange factors.

GAPex-5 is composed of a C-terminal Vps9 domain and an N-terminal Ras GAP domain. It was originally found to mediate both fluid phase and receptor-mediated endocytosis in Caenorhabditis elegans via its interaction with Rab5 and was named RME6 (28). The mammalian ortholog was initially identified by Hunker et al. (27) and called RAP6. The exchange activity of RME6/GAPex-5/RAP6 for Rab5 was demonstrated using purified components (27). Previous studies demonstrated that GAPex-5 mediates insulin-stimulated fluid phase endocytosis and insulin receptor internalization via a Rab5-dependent pathway (25, 27). Because the internalization of both EGFR and insulin receptor is mediated by Rab5, we postulated that GAPex-5 may mediate EGFR internalization via activation of Rab5. However, our recent study demonstrated that Rin1 mediates EGFR trafficking from early endosomes to MVB/late endosomes via interacting with STAM (29). This result suggests that Rab5 exchange factors may mediate EGFR trafficking via a Rab5-independent pathway. Because GAPex-5 has been shown to have plasma membrane localization, it seemed possible that it may also affect the post-ligand binding processing of EGFR (e.g. phosphorylation and ubiquitination) before internalization. Herein, we examined the effects of GAPex-5 on EGFR degradation and uncovered a role for GAPex-5 in the ubiquitination and trafficking of EGFR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Fetal calf serum, Dulbecco's modified Eagle's medium, and LipofectamineTM 2000 were purchased from Invitrogen. Rabbit anti-EGFR and mouse anti-HA antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse anti-ubiquitin and mouse anti-EGFR (AB-5) antibodies were from Invitrogen, and mouse anti-c-Cbl antibody was from Upstate%20Biotechnology">Upstate Biotechnology (Lake Placid, NY). Anti-EGFR (pY1045-specific) antibody was obtained from Cell Signaling Technology (Beverly, MA). Mouse anti-EEA1, anti-phosphotyrosine (PY20), and anti-PLC-{gamma}1 antibodies were purchased from BD Biosciences. Mouse anti-CD63 antibody was from the Developmental Studies Hybridoma Bank at the University of Iowa. Anti-GAPex-5 polyclonal antibody and GAPex-5 constructs were kindly provided by Dr. Alan Saltiel (University of Michigan, Ann Arbor, MI).

Cell Culture and Stimulation with EGF—HeLa and human embryonic kidney 293 cells were cultured in Dulbecco's modified Eagle's medium containing 10% bovine growth serum (Hyclone Laboratories, Logan, UT), 100 units/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate at 37 °C with 5% CO2. CHO cells overexpressing human EGFR (CHO/hEGFR) were cultured in F-12 medium containing 10% fetal calf serum, 100 units/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate at 37 °C with 5% CO2. Cells were serum-starved and stimulated with prewarmed serum-free medium containing EGF (100 ng/ml) for the indicated time at 37 °C.

Protein Extraction, Immunoprecipitation, and Immunoblotting—Whole cell lysates were prepared as previously described (25). For immunoprecipitation, 500 µg of clarified lysate protein was incubated with antibodies (0.5 µg) overnight at 4 °C. The immune complexes were precipitated with protein G-agarose (Sigma) for 1 h at 4 °C, and the beads were washed extensively with lysis buffer before solubilization in SDS sample loading buffer. Released proteins were analyzed by SDS-PAGE and immunoblotting as described (25).

siRNA Construction and Transfection—The siRNA-directed human GAPex-5 were constructed and purified employing the SilencerTM siRNA construction kit (Ambion, Austin, TX) as previously described (25). One day before transfection, HeLa cells were plated in growth medium without antibiotics to obtain 30 ~ 40% confluence at the time of transfection. The transfection of siRNA (20 nM final concentration) was performed using LipofectamineTM 2000 according to the manufacturer's instructions. The sequences specific for human GAPex-5 (5'-AAGAATCGATTACCTATAGCA-3'), human Rab5a (5'-GAGTCCGCTGTTGGCAAATCA-3'), human Rab5b (5'-AAGACAGCTATGAACGTGAAT-3'), and human Rab5c (5'-AATGAACGTGAACGAAATCTT-3') were selected based upon their potency to inhibit the target gene expression. A scrambled siRNA (Ambion) was used as a negative control.

Immunofluorescence Microscopy—Cells grown on coverslips were fixed with 3% paraformaldehyde (Electron Microscope Sciences, Hatfield, PA) for 20 min and quenched for 10 min with 50 mM ammonium chloride. Cells were permeabilized with 0.1% Triton X-100 for 10 min, blocked with 2% goat serum and 1% bovine serum albumin for 1 h, and incubated with primary antibodies for 1 h, followed by Alexa-Fluor 594- or 488-goat anti-mouse or rabbit secondary antibodies (Invitrogen) for 30 min at room temperature. The coverslips were mounted with Fluorescent Mounting Medium (DakoCytomation, Carpinteria, CA). Imagines were collected using a MRC1024 laser scanning confocal microscope equipped with a x63 objective (Bio-Rad Laboratories).

Expression of Rab5a:S34N in CHO/hEGFR Cells by Recombinant Sindbis Virus—Recombinant Sindbis virus expressing Rab5a:S34N was constructed as previously described (30). CHO/hEGFR cell monolayers in 35-mm dishes (5 x 106 cell/dish) were infected with recombinant Sindbis virus as described (30). Four hours post-infection, cells were starved and stimulated with EGF (100 ng/ml). Whole cell lysates were prepared and immunoprecipitation and immunoblotting were performed as described above.

EGF Internalization Assay—HeLa cells pretreated with siRNAs were serum-starved for 4 h prior to incubation with EGF as described (31). Briefly, cells were washed in cold binding buffer (Dulbecco's modified Eagle's medium supplemented with 25 mM HEPES, pH 7.4, and 2 mg/ml bovine serum albumin) and incubated at 4 °C for 1 h with 200 pM 125I-EGF (Amersham Biosciences). To assess internalization, the cells were incubated at 37 °C in binding buffer lacking 125I-EGF for 5 min. Acid wash samples (surface-bound fractions) and cell lysates (internalized fractions) were counted in a {gamma}-counter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Depletion of GAPex-5 Inhibits EGFR Degradation—GAPex-5 is a newly identified Rab5 exchange factor, and we have recently demonstrated that its depletion inhibits insulin receptor internalization and signaling (25). Because EGFR and insulin receptor follow similar endocytic itineraries upon stimulation, we examined the effect of GAPex-5 depletion on EGFR internalization. The expression of GAPex-5 was effectively inhibited by an siRNA against GAPex-5 in HeLa cells (>90%, Fig. 1A). Down-regulation of GAPex-5 decreased the internalization rate of 125I-EGF by 30 ~ 40% (p < 0.05, Fig. 1B). Previous study demonstrated that depletion of all three Rab5 isoforms in HeLa cells decreased 125I-EGF internalization rate by ~50% (32). Previous studies demonstrated that GAPex-5 is an exchange factor for Rab5 (27) and Rab5 activity is required for EGFR internalization (13). Our results indicate that GAPex-5 may mediate EGF internalization through a Rab5-dependent pathway. Receptor internalization is the first step of EGFR trafficking from plasma membrane to lysosomes for degradation. However, whether a decrease of receptor internalization may affect EGFR degradation depends on whether internalization is the rate-limiting step under particular biological conditions. We therefore examined whether EGFR degradation was affected by down-regulation of GAPex-5. Indeed, EGF-stimulated EGFR degradation was significantly delayed when GAPex-5 was depleted (Fig. 1, C and D). Collectively, these results indicate that GAPex-5 regulates EGFR internalization and its trafficking to degradative compartments.


Figure 1
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  		FIGURE 1.
Depletion of GAPex-5 inhibits both EGFR internalization and degradation. A, HeLa cells were transfected with a scrambled negative control (NC) siRNA or siRNA against GAPex-5 (G-5). 48 h post-transfection, whole cell lystates were prepared and subjected to immunoblot (IB) analysis using antibodies recognizing GAPex-5 and tubulin. B, HeLa cells were transfected with a scrambled negative control (NC) siRNA or siRNA against GAPex-5 (G-5). 48 h post-transfection, cells were serum-starved and labeled with 125I-EGF, and the internalization rate of cell surface 125I-EGF was measured. The results represent the mean ± S.E. of three independent experiments. C, HeLa cells were transfected with a scrambled negative control (NC) siRNA or siRNA against GAPex-5 (G-5). 48 h post-transfection, cells were serum-starved for 3 h prior to EGF stimulation (100 ng/ml) for the times indicated. Whole cell lysates were prepared and subjected to immunoblot (IB) analysis using antibodies recognizing EGFR and tubulin. D, quantification of the mean ± S.E. of three independent experiments in panel C. EGFR levels were normalized to samples without EGF stimulation.

 
Depletion of GAPex-5 Inhibits EGFR Ubiquitination, but Not Phosphorylation Status—EGFR internalization is necessary, but not sufficient, for degradation. Previous studies demonstrated the important role of receptor ubiquitination and deubiquitination on intracellular trafficking and degradation of EGFR (12). To examine whether GAPex-5 mediates EGFR ubiquitination, HeLa cells transfected with scrambled negative control siRNA or a siRNA against GAPex-5 were starved and stimulated with EGF. The cells were lysed, and whole cell lysates were immunoprecipitated with antibody recognizing EGFR. The resulting immunocomplexes were analyzed by immunoblotting. In cells transfected with scrambled siRNA, EGF dramatically stimulates EGFR ubiquitination after 5 min (Fig. 2). Twenty minutes later, EGFR ubiquitination was found to be decreased, consistent with the sequential process of deubiquitination and trafficking of EGFR from early to late endosomes (Fig. 2). Interestingly, down-regulation of GAPex-5 dramatically inhibits EGF-stimulated receptor ubiquitination at 5 min (Fig. 2). The interaction between ubiquitin E3 ligase c-Cbl and activated EGFR is known to be required for the ubiquitination of EGFR (8). We therefore examined their interaction and found that depletion of GAPex-5 dramatically inhibits EGF-stimulated recruitment of c-Cbl to the EGFR (Fig. 2).

Because tyrosine phosphorylation of EGFR occurs before the recruitment of c-Cbl (4), we analyzed EGFR phosphorylation after stimulation. Five minutes after stimulation, cells depleted of GAPex-5 had similar amounts of total phospho-tyrosine (as analyzed with PY20 antibody) in EGFR as cells treated with scrambled control (Fig. 2). At 20 min, the amount of phosphotyrosine in control cells decreased. However, this decrease was delayed by depletion of GAPex-5. The delayed decrease in tyrosine phosphorylation may be due to either delayed receptor trafficking (Fig. 1, C and D) or failure to recruit phosphatases to the activated EGFR. Immunoblot analysis of whole cell lysates using an antibody recognizing EGFR specifically phosphorylated at tyrosine 1045 also confirmed the delayed dephosphorylation of EGFR in cells depleted of GAPex-5 (Fig. 2). Given that depletion of GAPex-5 decreases the internalization rate of EGFR (Fig. 1B), these results indicate that intracellular compartments may play an important role in EGFR dephosphorylation and signal attenuation.

Previously, studies suggested that different effectors are recruited to activate EGFR via the interaction between their Src homology 2 domains or phospho-tyrosine binding domains and distinct phosphorylated tyrosines within the tail of the EGFR. Phosphorylated tyrosines 1045, 1068, and 1086 have been shown to play important roles in recruiting c-Cbl (4, 33, 34), whereas phosphorylated tyrosines 992 and 1173 are involved in the binding of phospholipase C-{gamma}1 (35). Recruitment of phospholipase C-{gamma}1 to EGFR results in production of the second messenger molecules (e.g. inositol-1,4,5-triphosphate and diacylglycerol) and plays a significant role in intracellular signaling (36). To further confirm that the effect of GAPex-5 depletion on effector recruitment is specific to Cbl, we examined the recruitment of phospholipase C-{gamma}1. Interestingly, down-regulation of GAPex-5 did not inhibit recruitment of phospholipase C-{gamma}1 to the activated EGFR (Fig. 2). Collectively, these results suggest that GAPex-5 mediates the recruitment of c-Cbl to EGFR via a mechanism independent of receptor phosphorylation.


Figure 2
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  		FIGURE 2.
Down-regulation of GAPex-5 inhibits EGFR ubiquitination, but not phosphorylation. HeLa cells were transfected with a scrambled negative control (NC) siRNA or siRNA against GAPex-5 (G-5). 48 h post-transfection, whole cell lysates (WCL) were prepared and immunoprecipitated (IP) with anti-EGFR antibody as described under "Experimental Procedures." The resulting immunocomplexes and whole cell lysates were subjected to immunoblot (IB) analysis with the antibodies indicated. Data shown are representative of at least three experiments.

 
Depletion of GAPex-5 Blocks the Trafficking of EGFR from Early Endosomes to MVB and Late Endocytic Compartments—EGFR ubiquitination and deubiquitination are temporally and spatially coordinated as EGFR traverses the degradative pathway (12). Because depletion of GAPex-5 inhibits EGFR recruitment of c-Cbl, we further studied its effect on intracellular trafficking of EGFR by light microscopy. HeLa cells, transfected with scrambled siRNA or siRNA against GAPex-5, were starved and stimulated with EGF. Ten minutes post-stimulation, cells were fixed and EGFR and EEA1 were examined by confocal microscopy. Significant co-localization between EGFR and EEA1 was observed regardless of GAPex-5 depletion (Fig. 3A). However, in cells treated with GAPex-5 siRNA, EEA1-positive endosomes appeared smaller and more peripherally distributed than those in cells transfected with scrambled siRNA. These results may be indicative of impaired Rab5 activity and strongly suggest that GAPex-5 operates as an exchange factor for Rab5. Thirty minutes after EGF stimulation, EGFR co-localizes with CD63 (a marker for MVB and late endosome) significantly (Fig. 3B). However, in cells depleted of GAPex-5, a substantial subset of EGFR does not co-localize with CD63. Collectively, these results demonstrate that GAPex-5 is required for trafficking of EGFR from early endosomes to MVB/late endosome.


Figure 3
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  		FIGURE 3.
Depletion of GAPex-5 blocks EGFR trafficking from early endosomes to MVB/late endosomes. HeLa cells were transfected with a scrambled negative control (NC) siRNA or siRNA against GAPex-5 (G-5). 48 h post-transfection, cells were serum-starved for 3 h prior to EGF stimulation (100 ng/ml) for 10 or 30 min. A, 10 min after EGF stimulation, the cells were fixed and immunostained with antibodies recognizing EGFR or EEA1. B, 30 min after EGF stimulation, the cells were fixed and immunostained with antibodies recognizing EGFR or CD63. Marker overlap is shown in the "merge" panels. Scale bar, 10 µm.

 
GAPex-5-mediated EGFR Degradation Is Regulated via its Ras GAP Domain—One possible mechanism for the inhibitory effects of GAPex-5 depletion on the interaction between c-Cbl and EGFR in the absence of an effect on receptor phosphorylation is through an interaction between GAPex5 and c-Cbl. To test this hypothesis, HeLa cells were transfected with empty vector or vectors encoding either wild type or mutant GAPex-5 epitope tagged with HA. After stimulation with EGF for 5 min, whole cell lysates were prepared and immunoprecipitated with anti-HA antibody. The resulting immunocomplexes were examined by immunoblot analysis using an anti-c-Cbl antibody. Interestingly, overexpressed GAPex-5 pulls down endogenous c-Cbl (Fig. 4A). In contrast, a GAPex-5 mutant depleted of RAS GAP domain (GAPex-5{Delta}GAP) does not interact with c-Cbl (Fig. 4A), suggesting that this domain is normally required for interaction between GAPex-5 and c-Cbl. Moreover, c-Cbl was pulled down by glutathione S-transferase-Ras GAP domain (Fig. 4B). This result suggests that Ras GAP domain of GAPex-5 is sufficient for its interaction with c-Cbl. We need to point out here that we do not consistently observe the interaction between endogenous GAPex-5 and endogenous c-Cbl in HeLa cells. This may be due to the quality of our GAPex-5 antibody and the expression level of endogenous GAPex-5 in HeLa cells. It is also likely that only a fraction of GAPex-5 interacts with c-Cbl.


Figure 4
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  		FIGURE 4.
GAPex-5 WT, but not GAPex-5 {Delta}GAP, interacts with c-Cbl and enhances EGFR degradation. A, HeLa cells were transfected with empty vector (EV), HA-GAPex-5 WT (WT) construct, or HA-GAPex-5 {Delta}GAP ({Delta}G) construct. 24 h later, whole cell lysates were prepared and immunoprecipitated (IP) with anti-HA antibody. The resulting immunocomplexes were subjected to immunoblot (IB) analysis with antibodies recognizing the HA epitope tag or c-Cbl. B, HeLa cells overexpressing HA-c-Cbl were lysed, and whole cell lysate was pulled down with glutathione beads preloaded with glutathione S-transferase or glutathione S-transferase Ras GAP domain of GAPex-5 (RGD). Proteins pulled down were analyzed with immunoblotting using c-Cbl antibody. C, human embryonic kidney 293 cells were co-transfected with green fluorescet protein-EGFR (0.06 µg/well) and empty vector (EV), HA-GAPex-5 WT (WT) construct, or HA-GAPex-5 {Delta}GAP ({Delta}G) construct (3 µg/well). 24 h post-transfection, cells were serum-starved for 3 h prior to EGF stimulation (100 ng/ml) for 5 min. Whole cell lysates (WCL) were prepared and immunoprecipitated (IP) with mouse anti-EGFR antibody. The immunoprecipitates were analyzed by immunoblotting (IB) using the antibodies indicated. D, human embryonic kidney 293 cells were co-transfected with green fluorescent protein-EGFR and GAPex-5 mutants as described in panel C. 24 h post-transfection, cells were serum-starved for 3 h prior to EGF stimulation (100 ng/ml) for the times indicted. Whole cell lysates were prepared and subjected to immunoblot analysis using antibody recognizing EGFR. E, quantification of the mean ± S.E. of three independent experiments in panel D. EGFR levels were normalized to samples without EGF stimulation.

 
To address the function of the Cbl-GAPex-5 interaction, we examined how or whether overexpressed GAPex-5 WT and GAPex-5{Delta}GAP affect Cbl recruitment, ubiquitination, and degradation of EGFR. Human embryonic kidney 293 cells were co-transfected with green fluorescent protein-EGFR and empty vector, or vector encoding GAPex-5 WT or GAPex-5{Delta}GAP. Cells were starved and stimulated with EGF. The cells were lysed, and the whole cell lysates were immunoprecipitated with antibody recognizing EGFR. The resulting immunocomplexes were analyzed by immunoblotting. Overexpression of GAPex-5 WT, but not GAPex-5{Delta}GAP, enhanced the recruitment of c-Cbl to EGFR and receptor ubiquitination (Fig. 4C). We further examined the effects of GAPex-5 and mutant on EGFR degradation. Consistent with the inhibitory effect of GAPex-5 depletion on EGFR degradation (Fig. 1D), overexpression of GAPex-5 WT substantially enhanced EGFR degradation (Fig. 4, D and E). However, cells overexpressing GAPex-5{Delta}GAP displayed EGFR degradation kinetics similar to cells transfected with the empty vector (Fig. 4, D and E). Collectively, these results suggest that GAPex-5 mediates EGFR degradation via its interaction with c-Cbl through its RAS GAP domain.

EGFR Ubiquitination Is Independent of Rab5 Activation—To examine whether GAPex-5-mediated EGFR ubiquitination depends on its ability to activate Rab5, we examined whether a dominant negative form of Rab5 (Rab5a:S34N) affects EGFR ubiquitination. CHO/hEGFR cells were infected with Sindbis virus encoding Rab5a:S34N or empty virus. Consistent with its inhibitory effects on EGFR internalization, Rab5a:S34N dramatically inhibited the degradation of EGFR (Fig. 5A). After starvation and stimulation with EGF for 5 min, cell lysates were prepared and immunoprecipitated with EGFR antibody. Immunoblot analysis of the immunocomplexes demonstrated that cells expressing Rab5a:S34N have higher amounts of ubiquitinated EGFR (Fig. 5B). Previous studies suggested that ubiquitinated EGFRs were deubiquitinated by a variety of deubiquitinating enzymes on early endosomes. Our results indicate that the enhanced ubiquitination of EGFR in cells overexpressing Rab5a:S34N may be due to impaired receptor internalization. We further studied whether Rab5 proteins may be required for EGFR ubiquitination. Accordingly, three Rab5 isoforms were depleted in HeLa cells and EGFR internalization was partially inhibited (~50%, data not shown), which is consistent with previous studies (32). The cells were starved and stimulated with EGF for 5 min. Whole cell lysates were prepared and immunoprecipitated with EGFR antibody and analyzed with immunoblotting. Similar to overexpression of Rab5a:S34N, depletion of all three Rab5 isoforms does not inhibit EGFR ubiquitination (Fig. 5C). Collectively, our results suggest that the effect of GAPex-5 on EGFR ubiquitination is likely independent of Rab5 activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Several recent studies demonstrated that GAPex-5 is a Rab5 exchange factor that mediates multiple endocytic events (25-28). Our present study provides multiple clues regarding a novel mechanism underlying GAPex-5-mediated EGFR trafficking and degradation. First, depletion of GAPex-5 delays EGFR degradation, whereas overexpression of GAPex-5 has the opposite effect (Fig. 4B). Second, depletion of GAPex-5 modestly decreases the rate of EGFR internalization. This effect might be Rab5-dependent. Third, down-regulation of GAPex-5 impairs the recruitment of c-Cbl to EGFR and subsequent receptor ubiquitination, which is not due to any impairment of receptor phosphorylation. Fourth, GAPex-5 depletion blocks the trafficking of EGFR into late, but not early, endocytic compartments. This result is consistent with a requirement for ubiquitination in order for EGFR to be recruited into endosomal sorting complexes required for transport and subsequently delivered into the lumen of MVB (12). Fifth, c-Cbl interacts with GAPex-5 WT, but not GAPex-5{Delta}GAP. Importantly, EGFR degradation is enhanced by GAPex-5 WT, but not by GAPex-5{Delta}GAP. Lastly, EGF-stimulated receptor ubiquitination is independent of Rab5 activity. Together, our present study suggests that GAPex-5 affects EGFR degradation via mediating receptor ubiquitination by its RAS GAP domain.


Figure 5
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  		FIGURE 5.
EGF-stimulated receptor ubiquitination is independent of Rab5 activity. CHO/hEGFR cells were infected with empty virus (EV) or Sindbis virus encoding Rab5a:S34N (S34N) as described under "Experimental Procedures." 4 h post-infection, cells were serum-starved for 2 h and stimulated with EGF (100 ng/ml). A, cells were stimulated for the times indicted, and whole cell lysates were prepared and subjected to immunoblot analysis using antibody recognizing EGFR. B, cells were stimulated with EGF for 5 min. Whole cell lysates were prepared and immunoprecipitated (IP) with mouse anti-EGFR antibody. The immunocomplexes were analyzed by immunoblotting (IB) using the antibodies indicated. C, HeLa cells were transfected with a scrambled negative control (NC) siRNA or siRNAs against Rab5a, -b, and -c (R-5). 48 h post-transfection, whole cell lysates were prepared and immunoprecipitated (IP) with anti-EGFR antibody. The resulting immunocomplexes were subjected to immunoblot (IB) analysis with the antibodies indicated.

 
Upon EGF stimulation, the phosphorylation of the activated receptor provides binding sites for downstream effectors that activate multiple signaling pathways and mediate receptor trafficking (2). The phosphorylated tyrosines are subsequently dephosphorylated to attenuate receptor activity (37). Our study demonstrated that depletion of GAPex-5 delayed the dephosphorylation of EGFR (Fig. 2). Given the finding that down-regulation of GAPex-5 also decreased the internalization of EGFR, our results suggest that dephosphorylation of EGFR phosphotyrosine residues mainly occurred after internalization. This is consistent with a previous study (38) that indicated that protein tyrosine phosphatase 1B-catalyzed dephosphorylation of EGFR required endocytosis and occurred on the surface of the endoplasmic reticulum. Of course, further experiments using independent approaches to inhibit EGFR internalization (e.g. depletion of dynamin 2) are required to confirm this supposition. Although depletion of GAPex-5 sustains the phospho-tyrosine of EGFR (both total phosphor-tyrosine and pY1045), it impairs the recruitment of c-Cbl to EGFR and subsequently inhibits receptor ubiquitination. This result strongly indicates that tyrosine phosphorylation is not sufficient to recruit c-Cbl. Interestingly, GAPex-5 binds to c-Cbl independently of EGF stimulation. It seems likely that GAPex-5 and c-Cbl might form a dynamic complex that facilitates recruitment of Cbl to EGFR and receptor ubiquitination. This model is supported by further experiments using a GAPex-5 mutant, GAPex-5{Delta}GAP. GAPex-5{Delta}GAP does not bind to c-Cbl and cannot enhance EGFR ubiquitination and degradation (Fig. 4).

Trafficking of EGFR from plasma membrane to lysosome for degradation may be dissected into multiple steps, including internalization and formation of endocytic vesicles, movement of vesicles on cytoskeletal elements, fusion of endocytic vesicles with early endosomes, and trafficking from early endosomes to late endosomes. Ubiquitination of EGFR occurs on the plasma membrane but is not required for receptor endocytosis (39). However, receptor ubiquitination is critical for trafficking from early endosomes to MVB/late endosomes. In HeLa cells, internalization of EGFR is usually completed within 5-10 min, whereas the half-life of the receptor is ~30 min (40). It seems that the initial step of EGFR internalization may not be the rate-limiting step for receptor degradation. That is, small fluctuations in the rate of EGFR internalization may not affect receptor degradation. Only when EGFR internalization is severely impaired (e.g. by overexpressing Rab5a:S34N, Fig. 5A) will receptor degradation be affected. Thus, the modestly decreased rate of receptor internalization (Fig. 1B) by GAPex-5 depletion may not be a major factor contributing to the impaired EGFR degradation. It seems most likely that depletion of GAPex-5 attenuates EGFR ubiquitination and impairs its trafficking from early endosomes to MVB/late endosomes and that this effect contributes dominantly to delayed receptor degradation. Further experiments using independent approaches to gradually inhibit EGFR internalization by depleting different proteins as described (32) will help to confirm the supposition.

A recent study demonstrated that GAPex-5 is also an exchange factor for Rab31, a Rab5 subfamily GTPase implicated in trafficking from the trans-Golgi network to endosome (26). In the absence of insulin, GAPex-5 activates Rab31 and promotes a futile cycle between GLUT4 storage vesicles and early endosomes, thus retaining GLUT4 inside the cells. Upon insulin stimulation, GAPex-5 is recruited to plasma membrane and releases GLUT4 from intracellular storage vesicles. Given that GAPex-5 also mediates the internalization of insulin receptor and EGFR, GAPex-5 appears to have different Rab targets and to regulate distinct cellular functions. It will be of interest to examine whether GAPex-5-mediated Rab31 activation is involved in EGFR trafficking and how activation of Rab5 and Rab31 by GAPex-5 is coordinated.

The requirement of Ras GAP domain of GAPex-5 for its interaction with c-Cbl raises the question whether Ras play roles in EGFR degradation. Indeed, previous studies demonstrated that dominant positive H-Ras (G12V) delays EGFR degradation (6, 41). Moreover, GAPex-5:Ras GAP domain binds to H-Ras:G12V and has GAP activity toward H-Ras in vitro (27). The model suggested by the current study indicates that G12V H-Ras might compete with GAPex-5 for binding with c-Cbl and thereby inhibit the recruitment of c-Cbl to EGFR.

In summary, the present study demonstrates that GAPex-5 mediates EGFR ubiquitination and degradation via Ras GAP domain. Because Ras mutations significantly contribute to the development of a variety of cancers, our present study suggests that GAPex-5 may be a novel effector for H-Ras and a mediator of carcinogenesis.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant 2R01GM42259-33. 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

1 To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, Campus Box 8228, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-6950; Fax: 314-362-1490; E-mail: pstahl{at}wustl.edu.

2 The abbreviations used are: EGFR, epidermal growth factor receptor; hEGFR, human EGFR; MVB, multivesicular body; siRNA, short interfering RNA; HA, hemagglutinin; GAP, GTPase-activating protein; WT, wild type; CHO, Chinese hamster ovary; E3, ubiquitin-protein isopeptide ligase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Alan R. Saltiel for providing GAPex-5 antibody and constructs. We thank Audra Charron for superb editing and other members of our laboratory for helpful suggestions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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J. Smith, X. Su, R. El-Maghrabi, P. D. Stahl, and N. A. Abumrad
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