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Molecular and Cellular Biology, February 2007, p. 888-898, Vol. 27, No. 3
0270-7306/07/$08.00+0     doi:10.1128/MCB.02356-05
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Epidermal Growth Factor Receptor Fate Is Controlled by Hrs Tyrosine Phosphorylation Sites That Regulate Hrs Degradation{triangledown}

Kathryn A. Stern,1 Gina D. Visser Smit,1,{dagger} Trenton L. Place,1 Stanley Winistorfer,2 Robert C. Piper,2 and Nancy L. Lill1*

Department of Pharmacology,1 Department of Physiology and Biophysics, The Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 522422

Received 9 December 2005/ Returned for modification 10 January 2006/ Accepted 2 November 2006


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ABSTRACT
 
Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is an endosomal protein essential for the efficient sorting of activated growth factor receptors into the lysosomal degradation pathway. Hrs undergoes ligand-induced tyrosine phosphorylation on residues Y329 and Y334 downstream of epidermal growth factor receptor (EGFR) activation. It has been difficult to investigate the functional roles of phosphoHrs, as only a small proportion of the cellular Hrs pool is detectably phosphorylated. Using an HEK 293 model system, we found that ectopic expression of the protein Cbl enhances Hrs ubiquitination and increases Hrs phosphorylation following cell stimulation with EGF. We exploited Cbl's expansion of the phosphoHrs pool to determine whether Hrs tyrosine phosphorylation controls EGFR fate. In structure-function studies of Cbl and EGFR mutants, the level of Hrs phosphorylation and rapidity of apparent Hrs dephosphorylation correlated directly with EGFR degradation. Differential expression of wild-type versus Y329,334F mutant Hrs in Hrs-depleted cells revealed that one or both tyrosines regulate ligand-dependent Hrs degradation, as well as EGFR degradation. By modulating Hrs ubiquitination, phosphorylation, and protein levels, Cbl may control the composition of the endosomal sorting machinery and its ability to target EGFR for lysosomal degradation.


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INTRODUCTION
 
Tight regulation of receptor tyrosine kinase (RTK) signaling is critical for normal development and growth. This has been clearly demonstrated in studies of a prototype RTK, the epidermal growth factor receptor (EGFR): while an absence of EGFR signaling is lethal, excessive EGFR signaling is associated with the development of a broad range of human cancers. Research aimed at identifying the mechanisms used by normal cells to regulate EGFR suggests that the modulation of receptor trafficking and degradation is essential for appropriate EGFR signaling (8, 14, 15, 22, 39, 40).

The protein c-Cbl modulates EGFR signaling, trafficking, and degradation. Cbl is a RING E3/protein-ubiquitin ligase that is recruited to EGFR through either of two mechanisms. Cbl's N-terminal SH2 domain binds to phosphotyrosine 1045 of ligand-activated receptors (27); C-terminal Cbl sequences associate indirectly with inactive or active EGF receptors via the adaptor protein Grb2 (20, 53). At high but physiological concentrations of EGF (45), the former interaction predominantly effects Cbl-mediated EGFR ubiquitination (19, 27, 29).

The monoubiquitination of a number of plasma membrane proteins correlates with their enhanced endocytosis and lysosomal degradation (16, 18, 34). Extensive investigations have been undertaken to define monoubiquitin-dependent mechanisms that target receptors for degradation. It has been shown that monoubiquitin can bind to the ubiquitin-interacting motifs (UIMs) of endosomal trafficking regulators, such as hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) (4, 41, 44). These associations have been proposed to retain activated receptors at sorting sites on early endosomes and multivesicular bodies, thereby enhancing receptor targeting to lysosomes.

Monoubiquitin-UIM interactions are essential for EGFR degradation (1, 51). Cellular expression of either an Hrs UIM mutant or an EGFR mutant that cannot be efficiently ubiquitinated compromises receptor degradation (27, 51). Furthermore, the covalent attachment of monoubiquitin to the EGFR C terminus enhances ligand-independent receptor internalization and degradation (11, 37). However, the half-life of the EGFR-ubiquitin chimera is significantly longer than that reported for ligand-activated, Cbl-associated EGFR (29, 37). This suggests that receptor monoubiquitination may not be the sole Cbl activity enhancing EGFR degradation.

We hypothesized that additional Cbl-dependent activities regulate receptor fate downstream of EGFR ubiquitination. To address this hypothesis, we used an established human embryonic kidney cell line (HEK 293) model system previously shown to be useful for studies of EGFR signaling and trafficking (29, 52). The system provides several experimental advantages. First, HEK 293 cells have epithelial morphology and are therefore appropriate for studies of EGFR and Cbl function, as the majority of tumors associated with EGFR deregulation are of epithelial origin and the sole EGFR-linked phenotype in Cbl-null mice is manifest in epithelial cells (38). Second, HEK 293 cells express low endogenous levels of EGFR and low to undetectable levels of the related ErbB RTKs. This allows the selective analysis of EGFR homodimers in cells ectopically expressing EGFR with or without recombinant Cbl proteins. Importantly, biochemical and biological readouts obtained in the HEK 293 system have been validated in Cbl knockout cells and other model cell systems (9, 28, 49). The transient-expression system also bypasses a real limitation of some stable expressor cell lines, in which the overexpression of wild-type (wt) Cbl decreases cell surface EGFR levels and/or selects for the outgrowth of cells that have bypassed Cbl-mediated suppression of RTK signaling (36). Using the HEK 293 cell model system, we investigated Cbl-mediated molecular events, downstream of receptor ubiquitination, that determine EGFR fate.

Our results implicate Cbl as a critical regulator of the endosomal protein Hrs. Hrs is an endocytic trafficking regulator that controls the delivery of ubiquitinated cargo to the lumenal vesicles of multivesicular bodies (4, 5). Downstream of EGFR activation by EGF, Hrs is inducibly phosphorylated on tyrosine residues 329 and 334 (48, 51). An E3-deficient Cbl mutant recently was shown to inhibit Hrs tyrosine phosphorylation and EGFR degradation jointly, but it was not established whether the mutant exhibited a loss of wild-type Cbl function or the gain of a novel activity. Hrs phosphorylation might be a normal, Cbl-regulated determinant of receptor fate (43), but no reported results have demonstrated any functional role for Hrs tyrosine phosphorylation.

Here, we report that wild-type Cbl enhances ligand-induced Hrs tyrosine phosphorylation, thereby facilitating functional studies of an expanded phosphoHrs pool. Our Cbl structure-function studies indicate that phosphorylation of Hrs tyrosine 334 correlates with EGFR degradation. We further show that Hrs phosphorylation is not merely a marker for degradative receptor trafficking: mutation of the primary and secondary Hrs tyrosine phosphorylation sites, Y334 and Y329, suppresses Cbl-enhanced EGFR degradation. Mechanistic studies suggest that this is due to phosphorylation-dependent Hrs protein degradation. Our results thus constitute the first evidence of a functional role for Hrs tyrosine 329/334 phosphorylation in regulating Hrs degradation, and thereby EGFR fate.


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MATERIALS AND METHODS
 
Plasmids. The pAlterMAX-EGFR, pAlterMAX-HACbl, pcDNA3GFP, pcDNA3GFP-Cbl wt, pcDNA3GFP-Cbl-N, and pcDNA3GFP-Cbl G306E expression constructs (kindly provided by Hamid Band) have been described previously (29). The plasmids pcDNA3GFP-Cbl Y371F and pAlterMAX-EGFR Y1045F were generated through oligonucleotide-directed mutagenesis, using primer 5'-GCCCATCTCACAGAATAATTCATATTG-3' with template pAlterMAX-HACbl and primer 5'-GGGTCTGAGCTGAATCGCTGCAAG-3' with pAlterMAX-EGFR, respectively. For development of the HA-mHrs wt and HA-mHrs Y329,334F expression constructs, wild-type and mutant murine Hrs (mHrs) cDNA sequences were amplified using primers 5'-GCGGTACCATGTACCCATACGATGTTCCAGATTACGCTGGGCGAGGCAGCGGCACC-3' and 5'-CCTCTAGACTATCAGTCGAAGGAGATGAGCTGGGC-3'. The sense primer incorporated hemagglutinin (HA) epitope tag coding sequences in frame with and upstream of the Hrs cDNAs. The amplification products were digested with KpnI and XbaI, purified, and ligated to KpnI/XbaI-digested pcDNA3 vector. For the development of the FLAG-ubiquitin expression construct, ubiquitin cDNA sequences were PCR amplified using primers 5'-CCGAATTCACAGATCTTCGTCAAGACGTTAACCG-3' (forward; introduces an EcoRI cloning site) and 5'-GCGACTAGTTTACTAACCACCTCTTAGTCTTAAGAC-3' (reverse; introduces an SpeI cloning site). The product was digested with EcoRI and SpeI and then ligated to EcoRI/XbaI-digested pFLAG-CMV4 to produce the FLAG-Ub expression construct. To generate the Hrs short hairpin RNA (shRNA) expression construct human Hrs (hHrs)-shRNA, annealed forward (5'-GATCCCAAGTGGAGGTAAACGTCCGTA TTCAAGAGATACGGACGTTTACCTCCACTTTTTTTTGGAAA-3'; BamHIcompatible) and reverse (5'-AGCTTTTCCAAAAAAAAGTGGAGGTAAACGTCCGTATCTCTTGAATACGGACGTTTACCTCCACTTGG-3'; HindIII-compatible) oligonucleotides were ligated to BamHI/HindIII-digested pSilencer 3.1-H1 puro. The shRNA control employed for these studies was a scrambled DNA sequence that does not target any identified human coding sequence (Ambion).

Antibodies. Anti-EGFR (sc-120; [528] murine immunoglobulin G2a [IgG2a] for immunoprecipitation) antibody was purchased from Santa Cruz Biotechnology, Inc. Anti-green fluorescent protein (GFP) antibody (Ab290; rabbit polyclonal) was acquired from Abcam Ltd. Anti-ubiquitin antibody (NCL-UBIQ; rabbit polyclonal; Novocastra Laboratories Ltd.) was purchased from Vector Laboratories, Inc. Anti-EGFR antibody (no. 06-129; sheep polyclonal for immunoblotting) and anti-phosphotyrosine antibody (no. 05-321; [4G10] murine IgG2b) were obtained from Upstate Biotechnology, Inc. Horseradish peroxidase-conjugated anti-FLAG M2 antibody was obtained from Sigma-Aldrich. Affinity-purified anti-pY334-Hrs rabbit polyclonal antibody was custom prepared by Bethyl Laboratories, Inc.; the immunogen was the synthetic Hrs phosphopeptide C327ARYLNRNY(PO3)WEKKQEE341. Antibodies reactive with nonphosphorylated Hrs were immunodepleted by passage of the immune serum over a C327ARYLNRNYWEKKQEE341 peptide affinity purification matrix. Flowthrough antibodies specific for pY334-Hrs were then collected on a C327ARYLNRNY(PO3)WEKKQEE341 phosphopeptide affinity purification matrix. Anti-Hrs rabbit polyclonal antiserum 932 (custom prepared by Sigma Genosys, Inc.) was generated against the C-terminal Hrs peptide 764PPAQGSEAQLISFD777. Ascites fluid containing anti-{gamma}-tubulin antibody (no. T6557; [GTU-88] murine IgG1) was acquired from Sigma-Aldrich, Inc. Anti-HA epitope antibody (MMS-101P; [HA.11] murine IgG1/{kappa}) was purchased from Covance Research Products, Inc. Peroxidase-conjugated anti-mouse IgG (no. 55563; goat polyclonal) secondary antibody and peroxidase-conjugated protein A (no. 55901) were obtained from ICN Pharmaceuticals, Inc. Peroxidase-conjugated anti-sheep IgG, H+L (no. 402100; rabbit polyclonal) was acquired from Calbiochem-Novabiochem Corporation.

Cells. The human embryonic kidney epithelial cell line HEK 293 was the kind gift of David Spector (Penn State College of Medicine). The cells were maintained in an atmosphere with 5% CO2 at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 units/ml penicillin-streptomycin, and 20 mM HEPES.

Transient transfection, cell stimulation, and cell lysate preparation. HEK 293 cells were transiently transfected using a modification of the calcium phosphate precipitation method (29) and the amounts of DNA indicated in the figure legends. DNA precipitates were removed from the cells after a 14- to 16-h incubation period at 37°C in an atmosphere of 5% CO2. Approximately 40 to 72 h after DNA precipitate addition, the cells were serum starved (0.5% fetal bovine serum in Dulbecco's modified Eagle's medium) for 4 to 6 h to facilitate the accumulation of inactive EGF receptors at the cell surface. The cells then were stimulated at 37°C with 100 ng/ml EGF (17 nM; Sigma) for the periods indicated. Triton X-100 lysis buffer (50 mM Tris, pH 7.5, 150 mM sodium chloride, 0.5% Triton X-100 [Fluka], 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 0.07 trypsin inhibitor units aprotinin/ml, and 1 µg/ml each of leupeptin, pepstatin, antipain, and chymostatin [Sigma]) was used to harvest cellular proteins as described previously (29, 52). Lysate protein concentrations were determined using the Bio-Rad Protein Assay reagent (Hercules, CA) with bovine serum albumin as the protein standard.

Immunoprecipitation and immunoblotting. Procedures for immunoprecipitation and immunoblotting have been described previously (29). Cell lysate proteins and immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore) in 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer (10 mM CAPS, pH 11 [Sigma], 10% methanol, 0.01% SDS [Bio-Rad Laboratories]). For anti-ubiquitin immunoblots, the filters were rinsed thoroughly in water and then autoclaved for 10 min. Membranes were blocked with 2% gelatin or 2% milk (Bio-Rad Laboratories) in 10 mM Tris, pH 8.0, 150 mM NaCl, and 0.025% sodium azide. The filters were incubated with the antibodies indicated, followed by protein detection with Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer Life Sciences). The PVDF membranes were stripped for reprobing as previously described (29).

Fractionation. At 48 h posttransfection, intact cells were harvested in Tris-buffered saline (8% sodium chloride, 0.2% potassium chloride, 3% Tris base) and then collected by centrifugation for 5 min at 200 x g and 4°C. Cell pellets were dispersed in homogenization medium (250 mM sucrose, 10 mM HEPES, 1 mM EDTA, pH 7.4, plus inhibitors [1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 0.07 trypsin inhibitor units aprotinin/ml, and 1 µg/ml each of leupeptin, pepstatin, antipain, and chymostatin]). The cells were mechanically lysed at 4°C by 10 passages through a 22-gauge needle. Unbroken cells and intact nuclei were removed from the homogenate upon centrifugation at 600 x g for 10 min at 4°C. The resulting postnuclear supernatant was then centrifuged at 250,000 x g at 4°C for 30 min to pellet the total membrane fraction. The cytosolic fraction (supernatant) was decanted and reserved. The membrane fraction (pellet) was resuspended in Triton X-100 lysis buffer, and membrane proteins were extracted by rocking the suspension for 30 min at 4°C. Membrane extracts were centrifuged at 16,000 x g, and the supernatants were collected. The membrane and cytosolic protein fractions were used for immunoprecipitation and immunoblotting analyses.

Peptide competition. At 40 to 48 h after transfection, lysates were prepared from HEK 293 cells. The indicated amounts of lysate protein were incubated at 4°C with anti-pY334-Hrs antibody and increasing amounts of Hrs phosphopeptide C327ARYLNRNY(PO3)WEKKQEE341 or its nonphosphorylated counterpart. Immune complex proteins were collected on Protein A-Sepharose, washed with Triton X-100 lysis buffer, and resolved by SDS-PAGE. Following their transfer to PVDF membranes, specific proteins of interest were detected by immunoblotting as described above.


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RESULTS
 
Cbl increases EGF-dependent Hrs tyrosine phosphorylation. The EGF-induced tyrosine phosphorylation of a number of cellular proteins has been reported (43, 48). Functional roles for these protein modifications have not been universally defined; one or more might contribute to the regulation of EGFR trafficking downstream of receptor activation. To begin to identify Cbl-mediated molecular events that enhance EGFR degradation, we compared the patterns of EGF-induced protein tyrosine phosphorylation in cells without or overexpressing ectopic Cbl.

Replicate cultures of HEK 293 cells expressing EGFR and either a GFP control protein or GFP-Cbl wt were serum starved and then incubated over a 90-minute time course with or without EGF. This period encompasses the time necessary for ligand-induced EGFR ubiquitination, downregulation, and degradation to occur but is less than the period required for the synthesis, glycosylation, and plasma membrane delivery of new receptors (46, 47). As observed previously (29), ectopic expression of GFP-Cbl enhanced the ligand-dependent ubiquitination and degradation of EGFR (Fig. 1A, compare lanes 1 to 4 to lanes 5 to 8). The level and kinetics of EGFR tyrosine phosphorylation were similar in GFP- and GFP-Cbl-expressing cells over an expansive time course of stimulation (Fig. 1C, top). However, GFP-Cbl expression reproducibly enhanced the tyrosine phosphorylation of a cellular protein of 110-kDa approximate mass (Fig. 1C, top).


Figure 1
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  		FIG. 1. Increased Cbl expression enhances EGFR ubiquitination and degradation, as well as Hrs tyrosine 334 phosphorylation. (A) HEK 293 cells in 10-cm dishes were transiently transfected with pAlterMAX-EGFR (0.05 µg) and either pcDNA3GFP-Cbl wt or pcDNA3GFP (4 µg). At 48 h posttransfection, the cells were serum starved for 4 h and then incubated without or with EGF (100 ng/ml; 17 nM) at 37°C for the times indicated; 500 µg of protein from each cell lysate sample was immunoprecipitated (IP) using 2.5 µg of anti ({alpha})-EGFR antibody 528. The immune complex proteins and lysate proteins (100 µg) were resolved by SDS-PAGE and transferred to a PVDF membrane. Immunoprecipitate proteins were immunoblotted (IB) using {alpha}-ubiquitin and {alpha}-EGFR antibodies. Lysate proteins were immunoblotted using the {alpha}-GFP antibody. The arrows mark the ubiquitinated EGFR in the {alpha}-ubiquitin and {alpha}-EGFR immunoblots. (B) HEK 293 cells in 10-cm dishes were transfected with pAlterMAX-EGFR (0.05 µg), pcDNA3GFP-Cbl wt (4 µg), and either mHrs-HA or mHrs Y329,334F-HA (2 µg; C-terminal epitope tag) constructs to overexpress wild-type and mutant Hrs for antibody characterization. At 48 h after calcium phosphate transfection, the cells were serum starved for 4 h and treated with EGF for the times indicated at 37°C. Equal amounts (800 µg) of lysate protein from each sample were used for immunoprecipitation with {alpha}-HA antibody. The immunoprecipitated proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and sequentially immunoblotted with {alpha}-HA, {alpha}-pY334 Hrs, and {alpha}-phosphotyrosine antibodies. (C) HEK 293 cells were transiently transfected, serum starved, and treated with EGF as described for panel A; 100 µg of protein from each cell lysate sample was gel resolved and transferred to a PVDF membrane. The membrane was immunoblotted using {alpha}-GFP, {alpha}-phosphotyrosine, {alpha}-pY334-Hrs, and {alpha}-{gamma}-tubulin antibodies. Nonubiquitinated and ubiquitinated EGFR species are bracketed. The asterisk marks the position of GFP-Cbl. The arrows indicate the positions of total and pY334 Hrs in sequential {alpha}-phosphotyrosine and {alpha}-pY334 Hrs immunoblots of one filter, shown in the top and middle rows, respectively. The {gamma}-tubulin signal served as the control for lysate sample loading. The results shown are representative of those obtained in three independent experiments.

Others have reported that the 110-kDa protein Hrs undergoes EGF-induced phosphorylation, principally upon tyrosine residues 329 and 334 (43, 48). To determine whether the 110-kDa phosphoprotein detected in our experiments was Hrs, we obtained a polyclonal rabbit antiserum specific for Hrs phosphorylated on tyrosine 334 (Fig. 1B) and used it to reprobe the Fig. 1C filter. The immunoblot confirmed that the 110-kDa protein was Y334-phosphorylated Hrs. The pY334 signal in GFP-Cbl-expressing cells was EGF dependent, peaked after approximately 10 min of cell stimulation, and was greatly reduced by 40 min poststimulation. This time course is consistent with reports of rapid Hrs phosphorylation on undefined residues following the activation of various receptors by their ligands (2, 7, 12, 24, 48). Based on these data, we concluded that the expression of wild-type Cbl enhances EGF-dependent Hrs Y334 phosphorylation.

Cbl expression alters the apparent molecular mass and phosphorylation kinetics of Hrs. Hrs is found in the cytosol and on the limiting membranes of early endosomes (3, 10, 50). Others have proposed that Hrs tyrosine phosphorylation may relocate Hrs from endosomes to the cytosol (50), thereby restricting its regulation of endosomal protein trafficking. To determine whether increased Cbl expression affects the subcellular location of phosphorylated Hrs, we evaluated the distribution of pY334 Hrs in membrane and cytosolic fractions derived from HEK 293 cells expressing EGFR and either the GFP control protein or GFP-Cbl wt.

Phosphopeptide competition confirmed that the pY334 Hrs antibody specifically recognized phosphorylated Hrs in immunoprecipitation applications (Fig. 2A), which were used subsequently for subcellular-fractionation studies. Cell stimulation with EGF over a 40-minute time course produced detectable pY334 Hrs in both the membrane and cytosolic fractions (Fig. 2B, top). However, pY334 Hrs levels were higher in the membrane and cytosolic fractions of cells expressing GFP-Cbl than in those from cells expressing GFP alone (Fig. 2B, top; compare lanes 2 and 5 and lanes 8 and 11). Phosphorylated Hrs did not wholly segregate to either fraction, but an Hrs species with reduced mobility accumulated preferentially in the cytosol of GFP-Cbl-expressing cells (Fig. 2B, top; compare lanes 5, 8, and 11). This band was detectable in the absence of Cbl overexpression, but at much reduced levels (Fig. 2B, top, lanes 8 and 9).


Figure 2
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  		FIG. 2. Cbl expression changes the phosphorylation kinetics of Hrs and induces the accumulation of a higher-mass Hrs species in the cytosol. (A) To characterize the anti ({alpha})-pY334 antibody for use in protein immunoprecipitation, HEK 293 cells in 10-cm dishes were transfected as described for Fig. 1B, serum starved, and then incubated without or with EGF for 10 min at 37°C, as indicated. Equal amounts of sample protein (500 µg) were immunoprecipitated (IP) with {alpha}-pY334 Hrs antibody. Peptide competition was performed using the indicated amounts of phosphorylated or nonphosphorylated Hrs peptides. Immunoprecipitates were gel resolved, transferred to a PVDF membrane, and immunoblotted (IB) with {alpha}-pY334 Hrs antibody. (B) To examine the subcellular distribution of pY334 Hrs species, HEK 293 cells were transfected as described for Fig. 1A. At 48 h after calcium phosphate transfection, the cells were serum starved for 4 h and incubated without or with EGF (100 ng/ml) at 37°C for the times indicated. Following stimulation, the cells were fractionated as described in Materials and Methods; 500 µg of protein from each membrane and cytosolic fraction was immunoprecipitated using {alpha}-pY334-Hrs antibody (2 µg). Lysate proteins (100 µg) and immunoprecipitates were gel resolved, transferred to a PVDF membrane, and probed with the following antibodies: {alpha}-EGFR, {alpha}-GFP, and {alpha}-pY334-Hrs. Lysate immunoblots (middle and bottom rows) confirmed the integrity of the fractionation, with segregation of EGFR to the membrane fraction and the majority of Cbl to the cytosolic fraction.

To determine the molecular nature of the upper Hrs species, cells were transfected to express HA-tagged wild-type Hrs, along with FLAG-tagged ubiquitin. Anti-FLAG immunoblotting of anti-HA immunoprecipitates revealed that the reduced-mobility Hrs species corresponded to ubiquitinated Hrs (Fig. 3). A second ubiquitinated band was readily detectable in anti-FLAG immunoblots, but it was evident in anti-HA or anti-Hrs blots only after prolonged exposure (not shown). Both ubiquitinated species were observed in the presence or absence of EGF. Based on these results, we concluded that phosphorylated, ubiquitinated Hrs preferentially localizes to the cytosol at early times after cell stimulation with EGF.


Figure 3
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  		FIG. 3. The slower-migrating pY334 Hrs species corresponds to ubiquitinated Hrs. HEK 293 cells, either untransfected (–) or transiently transfected with constructs encoding EGFR (0.05 µg), GFP-Cbl wt (4 µg), hHrs-specific shRNA (2.5 µg), HA-mHrs wt (0.05 µg), and FLAG-Ub (1.5 µg), were incubated for 70 h, serum starved, and then incubated without or with EGF. Anti-HA immunoprecipitates (2,000 µg lysate protein, 2 µg anti ({alpha})-HA antibody) were resolved by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted (IB) with {alpha}-FLAG and {alpha}-Hrs antibodies. The solid arrows mark the positions of monoubiquitinated Hrs; the white arrows mark nonubiquitinated Hrs.

The relocation of ubiquitinated Hrs from endosomal sorting sites to the cytosol may be functionally significant. Hoeller and colleagues recently reported that monoubiquitinated Hrs is compromised for binding ubiquitinated cargo (17). By removing ubiquitinated Hrs from endosomal sorting sites, Hrs phosphorylation could facilitate the clearance of the nonfunctional sorting protein and its replacement at the endosome by nonubiquitinated, sorting-competent Hrs. It is important to note that ubiquitinated Hrs represents only a minor fraction of total cellular Hrs (Fig. 3, bottom). However, a predominance of ubiquitinated Hrs at endosomal sorting sites could significantly inhibit EGFR sorting and trafficking to lysosomes.

Cbl-mediated enhancement of Hrs Y334 phosphorylation requires Cbl's tyrosine kinase-binding and ubiquitin ligase activities. We undertook structure-function studies to define the functional domains of Cbl that are essential to enhance Hrs Y334 phosphorylation. HEK 293 cells were transiently transfected for expression of EGFR and each of the following proteins: GFP-Cbl wt, GFP-Cbl G306E, GFP-Cbl-N, and GFP-Cbl Y371F (Fig. 4A). GFP-Cbl G306E bears a single amino acid substitution that compromises tyrosine kinase-binding (TKB) domain-dependent recruitment of Cbl to activated EGF receptors and thus their ubiquitination (29, 49) (Fig. 4B, middle GFP and top, lanes 1 to 5). GFP-Cbl-N is a transforming truncation mutant that contains a functional TKB domain (Fig. 4B, middle GFP, lanes 11 to 15) but lacks residues 358 to 906, which encompass the linker, RING finger, and RING finger tail regions required for Cbl-enhanced EGFR ubiquitination (26, 28, 29, 52) (Fig. 4B, top, lanes 11 to 15). GFP-Cbl-N also lacks C-terminal Cbl sequences that are dispensable for EGFR regulation in the HEK 293 cell system (29). GFP-Cbl Y371F is a linker region substitution mutant that is efficiently recruited to activated EGFR (Fig. 4B, middle GFP, lanes 16 to 20) but is deficient in ubiquitin ligase activity (Fig. 4B, top, lanes 16 to 20).


Figure 4
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  		FIG. 4. Cbl's tyrosine kinase-binding and ubiquitin ligase activities are required for enhanced Hrs tyrosine 334 phosphorylation and receptor degradation. (A) Schematic representation of the domain structure of wild-type Cbl and Cbl mutants used for this study. (B) HEK 293 cells were transiently transfected as described for Fig. 1A, using the following GFP-Cbl expression constructs: GFP-Cbl G306E, GFP-Cbl wt, GFP-Cbl-N, and GFP-Cbl Y371F. The cells were serum starved and EGF stimulated for the times shown. EGFR immunoprecipitates were prepared using 400 µg of lysate protein and 2 µg antibody. Immune complexes and lysate proteins (100 µg per lane) were gel resolved, transferred to a PVDF membrane, and immunoblotted (IB) using anti ({alpha})-ubiquitin, {alpha}-EGFR, {alpha}-GFP, and {alpha}-pY334 Hrs antibodies.

The use of these mutants allowed us to map specific Cbl activities that are essential for Hrs regulation. As shown in Fig. 4B, each of the Cbl mutants was compromised for the enhancement of Hrs Y334 phosphorylation, as well as for EGFR ubiquitination and degradation (pY334, Ub, and EGFR, compare lanes 6 to 10 with lanes 1 to 5, 11 to 15, and 16 to 20). We concluded that Cbl's tyrosine kinase-binding and ubiquitin ligase activities are essential for enhanced Hrs phosphorylation.

These results were supported by complementary structure-function studies of the EGF receptor. EGFR Y1045F is a single amino acid substitution mutant that lacks the site for direct binding of the Cbl TKB domain (27). As expected, EGFR Y1045F was compromised for ligand-induced EGFR degradation, which was only modestly enhanced by Cbl overexpression (Fig. 5A, EGFR, compare lanes 13 to 18, 19 to 24, and 7 to 12). The modest increase in degradation correlated with (i) nearly undetectable EGFR Y1045F ubiquitination, presumably mediated by EGFR binding to the Cbl C terminus via Grb2 (53), and (ii) limited Y334 h phosphorylation (Fig. 5A, pY334 Hrs, compare lanes 19 to 24 to lanes 7 to 12 and 13 to 18). The kinetics of Y334 phosphorylation also differed in cells expressing EGFR Y1045F and GFP-Cbl wt: low levels were maintained throughout the stimulation period (Fig. 5A, pY334 Hrs, compare lanes 20 to 24 to lanes 8 to 12, and B). This suggested that both the extent and timing of Hrs Y334 phosphorylation may be important for targeting EGF receptors for efficient degradation.


Figure 5
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  		FIG. 5. The direct association of Cbl with EGFR is essential for efficient EGFR ubiquitination and degradation and for enhanced Hrs phosphorylation. (A) HEK 293 cells were transiently transfected as described for Fig. 1A, using either a wild-type or a Y1045F mutant EGFR expression construct. At 48 h after transfection, the cells were serum starved and then incubated without or with EGF at 37°C for the times shown; 500 µg of each cell lysate protein sample was used for immunoprecipitation (IP) with 2.5 µg of anti ({alpha})-EGFR antibody. Immune complexes and lysate protein samples (100 µg) were gel resolved, transferred to a PVDF membrane, and immunoblotted (IB) with the following antibodies: {alpha}-ubiquitin, {alpha}-EGFR, {alpha}-pY334 Hrs, and {alpha}-GFP. (B) Relative intensities of the pY334 Hrs signals from replicate experiments (n = 3) were determined using Scion Image. After 10 min of EGF stimulation, cells expressing EGFR wt and GFP-Cbl wt demonstrated enhanced Hrs Y334 phosphorylation, compared to cells expressing EGFR Y1045F and GFP-Cbl wt or EGFR wt and GFP. This signal peaked by 10 min and sharply decreased at later time points. A different phosphorylation pattern was observed for cells expressing EGFR Y1045F and GFP-Cbl wt: the pY334 signal was modest, peaked at 40 min of EGF stimulation, and did not exhibit the rapid kinetics of apparent dephosphorylation observed with the wild-type receptor. The error bars indicate standard deviations.

EGFR degradation is compromised in cells expressing the phosphorylation-defective Hrs mutant Y329,334F. Our correlative structure-function results, and those reported by others in studies of the EGFR and Met receptor tyrosine kinases, suggested a critical role for Hrs tyrosine phosphorylation in the degradation of ubiquitinated receptors. However, no existing data proved its functional significance. To test the hypothesis that phosphorylation of Hrs Y329 and/or Y334 regulates EGFR fate, we compared the abilities of wild-type and Y329,334F Hrs proteins to mediate Cbl-enhanced EGFR degradation.

Hrs overexpression was used by Urbe and colleagues to examine the impact of the Y329,334F mutation on Hrs localization and EGFR degradation in HeLa cells (51). In their experiments, wild-type and mutant Hrs accumulated on enlarged endosomes. Overexpression of either Hrs protein blocked EGFR degradation. These limitations, combined with other reports of endosome abnormalities and trafficking defects in Hrs-overexpressing cells (6, 25, 42, 50), dictated the use of an alternative experimental approach: shRNA-mediated Hrs knockdown and reconstitution.

The system that we employed was derived from a previous report of Hrs knockdown and reconstitution in HeLa cells (32). For our experiments, HEK 293 cells were depleted of endogenous Hrs through the expression of a human Hrs-specific shRNA (Fig. 6A, top). shRNA-resistant murine Hrs coding sequences were then used to restore protein expression. The murine Hrs expression constructs encoded HA epitope-tagged wild-type and Y329,334F mutant Hrs proteins, which were expressed to the level of the endogenous protein (Fig. 6A, Hrs, compare lanes 3 and 4 with lane 1). Whereas wild-type HA-Hrs effected Cbl-mediated EGFR degradation to the same extent as endogenous Hrs (Fig. 6B, top, compare lanes 3 and 6), the Hrs phosphorylation site mutant Y329,334F was functionally compromised (Fig. 6B, top, lane 9). Quantitative analysis revealed a statistically significant difference in the EGFR levels of Hrs-depleted cells that were reconstituted with wild-type versus Y329,334F mutant Hrs (Fig. 6C). Based on these data, we concluded that phosphorylation of Hrs residue Y329 and/or Y334 is critical for the efficient degradation of ubiquitinated EGF receptors.


Figure 6
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  		FIG. 6. An Hrs mutant lacking the Y329 and Y334 phosphorylation sites inhibits Cbl-enhanced EGFR degradation. (A) HEK 293 cells were transfected with constructs encoding EGFR (0.05 µg), GFP-Cbl wt (3 µg), and either control or hHrs-specific shRNA (2.5 µg). Where indicated, the cells were cotransfected with wild-type or Y329,334F mutant HA-mHrs (0.1 µg). At 48 h after transfection, the GFP-positive cells were selected by flow cytometry and cultured for an additional 18 h. At 66 h posttransfection, the cells were serum starved for 4 hours and harvested for extraction of cellular proteins; 15-µg lysate protein samples were resolved by SDS-PAGE, transferred to a PVDF membrane, and sequentially immunoblotted (IB) with anti ({alpha})-Hrs, {alpha}-HA, and {alpha}-{gamma}-tubulin antibodies. (B) At 66 h posttransfection, identically transfected, unsorted cell pools were serum starved for 4 h and then incubated without or with EGF (100 ng/ml) at 37°C for the indicated times; 100-µg lysate protein samples were gel resolved, transferred to a PVDF membrane, and immunoblotted with the following antibodies: {alpha}-EGFR, {alpha}-HA, and anti-{gamma}-tubulin. (C) The data for the 90-min time point presented in panel B and those derived from two additional experiments were analyzed and graphed. Each bar represents the mean of independent results (n = 3) obtained for the various transfection conditions. The error bars represent the standard deviations. Black bar, cells expressing EGFR, GFP-Cbl, and the control shRNA; white bar, cells expressing EGFR, GFP, and the control shRNA; gray bar, cells expressing EGFR, GFP-Cbl, Hrs-specific shRNA, and the reconstituting construct HA-Hrs wt; hatched bar, cells expressing EGFR, GFP-Cbl, Hrs-specific shRNA, and the reconstituting construct HA-Hrs Y329,334F. The last two transfection conditions yielded means that were statistically different (P < 0.01), as determined by a pooled t test.

Hrs phosphorylation is critical for efficient Hrs degradation. These studies demonstrated an important role for Hrs tyrosine phosphorylation in regulating EGFR fate. However, they did not define a specific functional mechanism for that regulation. Others had proposed that phosphorylation affects the translocation of Hrs from endosomal limiting membranes to the cytosol (50). Our results, shown in Fig. 2B, supported the conclusion that Cbl-enhanced Hrs tyrosine phosphorylation is linked to the cytosolic accumulation of both ubiquitinated and nonubiquitinated phosphoHrs. However, Fig. 2B also indicates that ectopic Cbl expression alters the time course of apparent Y334 h dephosphorylation. Whereas Y334 dephosphorylation or loss from the cytosolic fraction was limited in GFP-expressing cells after 40 min of stimulation, it increased greatly with GFP-Cbl expression (Fig. 2B, top, compare lanes 8 and 9 with lanes 11 and 12). The correlation of Hrs dephosphorylation and/or degradation with Cbl-enhanced EGFR degradation suggested that the former processes might control EGFR fate.

To determine whether the altered Hrs phosphorylation kinetics in Cbl-expressing cells reflected Hrs degradation, we utilized the experimental Hrs knockdown and reconstitution system employed for our EGFR degradation studies. Endogenous Hrs was knocked down using shRNA and restored by expression of wild-type or Y329,334F murine Hrs. Very low-level Hrs expression was key for these experiments: even modest Hrs overexpression abrogated the Cbl-dependent decrease in Y334 phosphorylation at the 40- and 90-minute stimulation time points (not shown). We assume that reduction of the cellular Hrs pool by knockdown and low-level reconstitution artificially elevated the proportion of Hrs that was recruited to sorting sites and phosphorylated, thereby facilitating analysis of its degradation. As shown in Fig. 7A, cell reconstitution with wild-type Hrs produced phosphorylation kinetics identical to those seen with the endogenous protein (Fig. 1C). The time-dependent decrease in Hrs tyrosine phosphorylation coincided with a reproducible decrease in total Hrs protein (Fig. 7A, top and middle, lanes 2 to 4; quantification provided in Fig. 7B). No decrease in the Hrs level was observed upon expression of the phosphorylation mutant Y329,334F (Fig. 7A, top, lanes 5 to 7; quantification shown in Fig. 7B). Taken together, our data suggested that Hrs degradation is regulated by Hrs tyrosine phosphorylation and that these linked processes control EGFR degradation.


Figure 7
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  		FIG. 7. Hrs phosphorylation sites Y329 and/or Y334 are required for EGF-induced Hrs degradation. (A) HEK 293 cells were transiently transfected with the following expression constructs: EGFR (0.05 µg), GFP-Cbl wt (4 µg), hHrs-specific shRNA (2.5 µg), and either HA-mHrs wt or Y329,334F mutant (0.04 µg); 70 h posttransfection, the cells were serum starved and then left untreated or stimulated with EGF for the indicated times. Lysate proteins (100 µg) were resolved by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted (IB) with anti ({alpha})-HA, {alpha}-pY334 Hrs, and anti-{gamma}-tubulin antibodies. (B) The relative amounts of HA-Hrs remaining after 40 min of EGF treatment were determined for cells expressing either HA-mHrs wt or Y329,334F. Results from three experiments were analyzed using Scion Image. Cells expressing HA-mHrs wt demonstrated a greater reduction in Hrs over a 40-minute time course for EGF stimulation than those expressing HA-mHrs Y329,334F (P < 0.01). The error bars indicate standard deviations. (C) HEK 293 cells were transiently transfected as described for panel A. The cells were serum starved for 4 h. Dimethyl sulfoxide (DMSO) (vehicle) or MG132 (50 µM) was present during the final 2 h of starvation. The cultures were then harvested after a 90-minute incubation without or with EGF. Lysate proteins (100 µg) and immune complexes (1,000 µg protein with 2 µg {alpha}-HA antibody) were gel resolved, transferred to a PVDF membrane, and immunoblotted with {alpha}-HA, {alpha}-EGFR, {alpha}-GFP, and anti-{gamma}-tubulin antibodies. (D) Data from replicate experiments (n = 3), performed as described for panel C, were analyzed to evaluate HA-mHrs levels remaining after MG132 or DMSO treatment. Two-hour MG132 pretreatment induced a statistically significant decrease in total HA-mHrs levels (P < 0.001). This decrease confounded further attempts to address whether phosphorylation-dependent Hrs degradation is proteasome mediated.

To determine whether phosphorylation-associated Hrs degradation was proteasome-dependent, we utilized the proteasome inhibitor MG132. HEK 293 cells expressing low levels of HA-Hrs were serum starved for 4 hours. During the final 2 hours of starvation, the vehicle DMSO or the inhibitor MG132 was added to the cultures. The cells were then harvested without or following EGF stimulation. As shown in Fig. 7C and D, preincubation of the cultures with MG132 dramatically reduced the total pool of cellular Hrs (Fig. 7C, top, compare lanes 1 and 3). This loss of Hrs confounded our assessment of MG132's impact on ligand-induced Hrs degradation. Therefore, it remains possible that phosphorylation-associated Hrs degradation is proteasome mediated and dependent upon Cbl-enhanced Hrs ubiquitination (Fig. 2).


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DISCUSSION
 
We addressed a novel mechanism through which Cbl drives the degradation of ubiquitinated EGFR. Wild-type Cbl enhances Hrs tyrosine phosphorylation and ubiquitination downstream of EGFR activation (Fig. 1). These modifications program Hrs for cytosolic relocation and degradation (Fig. 2 and 7A), thereby sustaining the endosomal sorting and degradation of EGFR (Fig. 6). This report thus constitutes the first evidence that Hrs tyrosine phosphorylation is functionally important for the regulation of receptor tyrosine kinase fate.

Hrs tyrosine phosphorylation and Cbl-enhanced EGFR degradation require Cbl's tyrosine kinase-binding and E3/ubiquitin ligase activities (Fig. 4 and 5). Our results are consistent with previous mechanistic models in which receptor-associated monoubiquitin binds to the Hrs UIM, thereby retaining receptors at endosomal sorting sites (1, 43, 51). Cbl's enhancement of Hrs tyrosine phosphorylation may be entirely due to Cbl-mediated EGFR ubiquitination, which would increase the recruitment of activated EGFR/Src family kinase complexes (3) to endosomal Hrs via ubiquitin-UIM interactions. The present study did not address the nature of the Hrs kinase, nor did it test whether Cbl-mediated EGFR ubiquitination is the sole activity required for Cbl-enhanced Hrs tyrosine phosphorylation.

Rather, our results identify molecular events that result from Hrs tyrosine phosphorylation. A key finding of this work is that phosphorylation of Hrs Y329 and/or Y334 programs ubiquitinated Hrs for degradation (Fig. 7A). Monoubiquitinated Hrs is compromised for binding ubiquitinated cargo (17). The depletion of ubiquitinated Hrs from endosomal limiting membranes would facilitate its replacement by nonubiquitinated Hrs at sorting sites. In turn, this would permit additional cycles of ubiquitinated cargo recruitment, thereby sustaining degradative sorting of EGFR. This model is compatible with the fact that Hrs tyrosine phosphorylation is critical for efficient EGFR degradation (Fig. 6).

Our findings are consistent with a recent report addressing the potential regulation of another receptor tyrosine kinase by Hrs (1). Abella and colleagues showed that the Met receptor mutant Y1003F, which lacks the binding site for direct recruitment of Cbl, is deficient for degradation (1). The defect was associated with enhanced signaling through the Ras-MAP kinase pathway and with cellular tumorigenicity. Covalent attachment of ubiquitin to the C terminus of Met Y1003F decreased Met stability and MAP kinase signaling and enhanced Hrs tyrosine phosphorylation. It is noteworthy that, under the experimental conditions used, Met was continuously activated by its ligand, hepatocyte growth factor, and was capable of recruiting Cbl indirectly via a Grb2-mediated mechanism. Thus, receptor monoubiquitination enhanced the degradation of activated, Cbl-associated Met. Importantly, the results did not show that receptor monoubiquitination is sufficient to produce efficient receptor degradation without receptor activation, Cbl recruitment, or Hrs tyrosine phosphorylation. As in the EGFR model system, additional effects of the activated receptor and/or receptor-associated Cbl might be important for the regulation of Hrs and receptor fate.

Based on our results in the EGFR system (Fig. 1 and 2B), we propose a role for Cbl's E3 activity in regulating receptor tyrosine kinase trafficking, not only at the cell surface (inducing the receptor ubiquitination needed for endosomal UIM binding), but also at the early endosome. A requirement for Cbl-mediated ubiquitination of endosomal Hrs is consistent with a prior report that Cbl-EGFR interactions must be maintained throughout the endocytic pathway for efficient receptor degradation to occur (31). Our data indicate that two Cbl-dependent posttranslational modifications of Hrs may act through distinct mechanisms to regulate the Hrs trafficking checkpoint. Cbl-mediated Hrs ubiquitination would constitute a switch to inactivate the UIM of cargo-loaded Hrs, thereby facilitating cargo transfer to other ubiquitin-binding proteins downstream in the sorting pathway. Ubiquitination also may enhance the relocation of Hrs to the cytosol (Fig. 2 and unpublished results). Cbl-dependent Hrs phosphorylation would mark the inactive cytosolic Hrs for degradation. Our model accommodates various reports in the literature, in which Hrs has been shown to promote the apparently opposing processes of receptor degradation and receptor recycling (13, 21, 23, 30, 54). With different posttranslational modifications, Hrs may perform both functions. The order in which Hrs phosphorylation and ubiquitination occur at the endosomal limiting membrane is not addressed here. It represents an intriguing subject for future investigations.

A subset of our results is reminiscent of G-protein-coupled receptor regulation by the E3 ubiquitin ligase AIP4. Marchese and colleagues demonstrated that AIP4 promotes the agonist-induced degradation of receptor CXCR4 via ubiquitination of both CXCR4 and Hrs (33). However, AIP4 regulation of CXCR4 differs from Cbl regulation of EGFR at the level of Hrs posttranslational modifications: activation of CXCR4 fails to induce detectable Hrs tyrosine phosphorylation (33). This suggests that, while a variety of activated receptors effect Hrs ubiquitination via a receptor-associated E3, fewer regulate Hrs subcellular location and degradation via Hrs tyrosine phosphorylation.

How might tyrosine phosphorylation target ubiquitinated Hrs for degradation? It is not yet known whether Hrs phosphorylation alters the structure of Hrs, its binding to PI3-P and retention on endosomes, its association with endosomal clathrin domains, or its oligomerization. Such events could induce Hrs relocation from endosomal limiting membranes to the cytosol. They also might facilitate Hrs polyubiquitination and degradation by proteasomes. Further research will be needed to address these possibilities.

Finally, we note that the maintenance of cellular Hrs levels requires proteasome activity and/or a significant pool of free ubiquitin (Fig. 7C), both of which are reduced upon MG132 treatment of cells. This observation may explain, at least in part, prior reports that MG132 blocks EGFR sorting from early to late endosomes (35) and inhibits the translocation of EGFR from the limiting membrane of multivesicular bodies to their lumenal vesicles (31). Both of these outcomes may actually result from MG132-induced decreases in the cellular pool of Hrs.


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ACKNOWLEDGMENTS
 
We thank David Spector and Hamid Band for providing cells and plasmids used for this study and John Koland and his laboratory for helpful discussions of the work. Excellent technical assistance was provided by Jussara Hagen, Soumya Vemuganti, Alicia Taylor, Sarah Brubaker, and Jeremy Hoffmann.

This work was supported by Research Scholar Grant RSG-03-046-01 from the American Cancer Society (to N.L.L.), a Carver Trust Foundation Collaborative Pilot Grant from the Roy J. and Lucille A. Carver College of Medicine (to N.L.L. and R.C.P.), and institutional training grants NRSA T32 DEO14678-04 and DEO14678-03 (from NIDCR to Christopher Squier and the Dows Institute and College of Dentistry at the University of Iowa, for support of K.A.S. and N.L.L., respectively).


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FOOTNOTES
 
* Corresponding author. Mailing address: 2-450 Bowen Science Building, 51 Newton Road, Iowa City, IA 52242. Phone: (319) 384-4651. Fax: (319) 335-8930. E-mail: nancy-lill{at}uiowa.edu. Back

{triangledown} Published ahead of print on 13 November 2006. Back

{dagger} Present address: Weis Center for Research, Geisinger Health System, Danville, PA 17821. Back


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Molecular and Cellular Biology, February 2007, p. 888-898, Vol. 27, No. 3
0270-7306/07/$08.00+0     doi:10.1128/MCB.02356-05
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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