Research ArticleEndocytosis

Cbl Controls EGFR Fate by Regulating Early Endosome Fusion

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Science Signaling  22 Dec 2009:
Vol. 2, Issue 102, pp. ra86
DOI: 10.1126/scisignal.2000217

Abstract

Amino acid residues 1 to 434 of the E3 ubiquitin ligase Cbl control signaling of the epidermal growth factor receptor (EGFR) by enhancing its ubiquitination, down-regulation, and lysosomal degradation. This region of Cbl comprises a tyrosine kinase–binding domain, a linker region, a really interesting new gene finger (RF), and a subset of the residues of the RF tail. In experiments with full-length alanine substitution mutants, we demonstrated that the RF tail of Cbl regulated biochemically distinct checkpoints in the endocytosis of EGFR. The Cbl- and ubiquitin-dependent degradation of the regulator of internalization hSprouty2 was compromised by the Val431→ Ala mutation, whereas the Cbl- and EGFR-dependent dephosphorylation or degradation of the endosomal trafficking regulator Hrs was compromised by the Phe434→ Ala mutation. Deregulated phosphorylation of Hrs correlated with inhibition of the fusion of early endosomes and of the degradation of EGFR. This study provides the first evidence that Cbl regulates receptor fate by controlling the fusion of sorting endosomes. We postulate that it does so by modulating the abundance of tyrosine-phosphorylated Hrs.

Introduction

The E3 ubiquitin ligase Cbl (for Casitas B-lineage lymphoma protein) suppresses signaling from numerous receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR). Suppression of signaling is due, at least in part, to terminal routing of the kinases to lysosomes, where they are degraded. Cbl controls the degradation of EGFR at a postinternalization trafficking checkpoint that remains ill-defined (14). It is widely accepted that Cbl-mediated monoubiquitination or polyubiquitination of the receptor is critical for the down-regulation and lysosomal degradation of activated EGFR (58); however, receptor fate is also influenced by the posttranslational modification of other Cbl-associated proteins at the cell surface and on endosomes (4, 9, 10).

Several of these modifications require the presence of the really interesting new gene (RING) finger (RF) tail amino acid residues 420 to 434 of Cbl (4). The structure of this region was solved in a co-crystal of residues 47 to 434 of Cbl and the E2 ubiquitin–conjugating enzyme UbcH7 (11). The stability of the RF tail was attributed, at least in part, to crystal packing. Several amino acid residues in the RF tail (Ile429, Val430, Asp432, and Pro433) appear to participate in intramolecular interactions that might affect the folding and function of Cbl. Other residues, including Val431 and Phe434, play no obvious role in intramolecular or intermolecular interactions. Of note, the structural data did not demonstrate a functional role for any amino acid residue of the RF tail in the regulation of EGFR by Cbl.

We hypothesized that the RF tail of Cbl might control the endocytosis and degradation of EGFR at a trafficking checkpoint subsequent to receptor internalization. To test this hypothesis, we used full-length RF tail substitution mutants of Cbl. The strategic benefit of expressing dominant mutants of Cbl is their ability to override the redundant regulation of EGFR by all three endogenous Cbl proteins (c-Cbl, Cbl-b, and Cbl-3) without requiring their simultaneous knockdown. Through our analysis of RF tail substitution mutants, we demonstrate that Cbl enhances the degradation of EGFR by regulating the fusion of sorting endosomes.

Results

The Cbl RF tail mutants V431A and F434A aberrantly regulate EGFR

To investigate how the RF tail might control the fate of EGFR, we performed alanine scanning mutagenesis of amino acid residues 428 to 436 of Cbl (Fig. 1A). The resulting panel of full-length, green fluorescent protein (GFP)–tagged, single amino acid substitution mutants was tested for activity in assays of EGFR down-regulation (receptor loss from the cell surface), ubiquitination, and degradation (Fig. 1, B to D and fig. S1). Most RF tail mutants functioned like wild-type Cbl. Relative to the other mutants, Cbl P428A led to enhanced down-regulation and ubiquitination of EGFR, but this did not result in enhanced degradation of EGFR (fig. S1B). The V430A mutant induced substantially less ubiquitination of EGFR than did wild-type Cbl, yet it retained the ability to enhance the down-regulation and degradation of EGFR. The V431A and F434A mutants of Cbl were functionally compromised in all three assays. Because the results from experiments with the V430A mutant established that a statistically significant decrease in ubiquitination of the receptor was not sufficient to compromise its down-regulation and degradation, this mutant was of minimal interest for further analysis. Instead, we focused on the fully defective mutants V431A and F434A.

Fig. 1

Biochemical characterization of the alanine substitution mutants of the RF tail of Cbl. (A) The conserved C-terminal flank of the RF in isoforms of human Cbl. Domains relevant to the regulation of EGFR include the TKB domain, the linker region (L); the RING finger domain (RF), the C-terminal flank or “tail” (T) domain following the RF, the proline-rich region (PRO), and the leucine zipper (LZ). Tyr371 lies within the linker region. Sequences of the RF tail of isoforms of human Cbl (c-Cbl, Cbl-b, and Cbl-3) are aligned in the expanded portion of the graphic. Evolutionarily conserved sequences are marked with shaded (sequence identity) or plain (amino acid similarity) boxes. The underlined residues were those selected for substitution in this study. The vertical dashed line marks the C-terminal limit of the evolutionarily conserved sequences of Cbl that are sufficient to enhance the ubiquitination, down-regulation, and degradation of EGFR. (B) Mutants V431A and F434A of the RF tail of Cbl are compromised in their ability to down-regulate EGFR. The abundance of EGFR at the surface that remained at each stimulation time point was calculated relative to the amount of surface receptor in matched unstimulated cells. Results reflect the mean of three independent experiments ± SD of the mean. (C) Mutants V430A, V431A, and F434A of the RF tail of Cbl effect reduced ubiquitination of EGFR after 10-min incubation of cells with EGF. Upper section, 750-μg immunoprecipitates (with an antibody against EGFR, 528). Isotype-matched antibody against Syk 4D10 [C] was the specificity control. Immunoprecipitates (I.P.) and 75-μg lysate protein samples were resolved by SDS-PAGE and sequentially analyzed by Western blotting (I.B.). Lower section: quantitation of results. To compare the amount of ubiquitinated EGFR induced by the wild-type and mutant Cbl proteins, the abundance of ubiquitinated protein in each Western blot was normalized to that of EGFR. Mean values were expressed relative to the signal achieved with GFP-Cbl wild type (WT) (1.00). Results represent data from three independent experiments and are shown as mean ± SD. Only Cbl V431A and Cbl V430A produced significantly less ubiquitination of EGFR than did WT Cbl, as determined by Student’s t test with α = 0.05. (D) The F434A and V431A mutants of Cbl cause reduced and delayed ubiquitination of EGFR and reduced degradation of EGFR. Upper section: 1-mg aliquots of samples immunoprecipitated with an antibody against EGFR (528). I.P.s (top three panels) and 100-μg aliquots of lysates from each sample (bottom panel) were sequentially analyzed by Western blotting with the indicated antibodies. The guillemet marks the position of the predominant species of ubiquitinated EGFR. Lower section: quantitation of the degradation of EGFR that was effected by WT Cbl and RF tail mutant Cbl proteins (100 μg protein per lane).

Cbl V431A is compromised in its ability to induce degradation of hSprouty2

We assessed the ability of the mutant proteins to bind to and enhance the degradation of the protein hSprouty2, which is a direct ubiquitination target of Cbl. In experiments with human embryonic kidney (HEK) 293 cells, other groups have shown that hSprouty2, which inhibits the internalization of EGFR, associates with the RF domain of Cbl under conditions of EGF depletion and then binds to the tyrosine kinase–binding (TKB) domain of Cbl after activation of the receptor by its ligand (12, 13). Translocation of hSprouty2 frees the RF to bind to an E2 ubiquitin–conjugating enzyme, a process that is required for Cbl-mediated ubiquitination of hSprouty2 and all of the other targets of Cbl (14). The subsequent ubiquitination and degradation of TKB domain–associated hSprouty2 then facilitates the binding of the TKB domain of Cbl to activated EGFR, which leads to the ubiquitination of EGFR (1416). Because Cbl V431A and Cbl F434A were defective in their ability to ubiquitinate EGFR, we asked whether either mutant was compromised in its ability to bind to or lead to the degradation of hSprouty2. As in previous investigations of the degradation of hSprouty2 (4, 1216), an ectopically expressed, epitope-tagged hSprouty2 protein was examined to compensate for the lack of commercially available antibodies that can effectively detect endogenous Sprouty2. This strategy also enabled us to track the fate of the Sprouty2 protein in transfected cells only, where the effect of ectopically expressed wild-type and mutant GFP-Cbl proteins should be determined.

The GFP-Cbl proteins and FLAG-tagged hSprouty2 were similarly abundant in all unstimulated cell cultures (Fig. 2A). hSprouty2 associated constitutively with the Cbl fusion proteins, as demonstrated by its recovery in samples immunoprecipitated with an antibody against GFP (Fig. 2A). EGF enhanced the interactions between Cbl and Sprouty2; this was most apparent where the induced interactions were detected in the absence of degradation of Sprouty2 (Fig. 2A). Most of the hSprouty2 protein appeared as a lower molecular mass doublet of ~42 kD (Fig. 2A, species I and II), which was shown by others to consist of nonphosphorylated and phosphorylated species. A FLAG-reactive band of ~50 kD (species III) was detected only after stimulation of cells with EGF. The apparent mass of the latter protein, its reactivity with an antibody against FLAG, and its disappearance over the time of the experiment suggested that it was monoubiquitinated hSprouty2.

Fig. 2

Aberrant regulation of hSprouty2 and Hrs by RF tail substitution mutants of Cbl. (A) The V431A and F434A mutants of Cbl were grossly and slightly compromised, respectively, in their ability to mediate the ubiquitin-associated degradation of the regulatory protein hSprouty2 (hSpry2), but were not defective in binding to hSprouty2. Immunoprecipitates (1 mg of lysate protein per reaction, upper panels, I.P.) and 100-μg lysate samples (bottom panel) were resolved by SDS-PAGE and sequentially analyzed by Western blotting with the indicated antibodies (I.B.). I and II mark the more abundant species of hSprouty2. Both species underwent rapid EGF-induced degradation in the presence of wild-type (WT) Cbl or of the P433A or D432A mutants of Cbl, which were functionally similar to WT Cbl (bottom panel: WT, P433A, and D432A). Species III may represent ligand-induced, ubiquitinated hSprouty2. The ligand-induced increase in the abundance of Cbl-hSprouty2 complexes is best visualized in lanes 17 to 20, where the presence of Cbl V431A blocked the degradation of associated hSprouty2 (third panel). Equivalent amounts of lysate protein contained comparable amounts of the GFP-Cbl proteins (top panel). (B) The F434A mutant Cbl effected the tyrosine phosphorylation of Hrs with kinetics similar to that of WT Cbl, but was compromised in its ability to mediate the apparent dephosphorylation, degradation, or both of Hrs. For each sample, 100 μg of cell lysate protein was analyzed by Western blotting (I.B.) with the indicated antibodies. The γ-tubulin blot shows comparable protein loading in all lanes. Results shown are representative of three independent experiments.

When cells ectopically expressing wild-type Cbl or the Cbl P433A or Cbl D432A mutant (which were biochemically no different from wild-type Cbl) were stimulated over a 20-min period with EGF, Cbl-hSprouty2 complexes were progressively lost (Fig. 2A) and the total cellular pool of hSprouty2 was reduced (Fig. 2A, bottom panel). This depletion correlated with the appearance of ubiquitinated protein smears in samples immunoprecipitated with an antibody against GFP. The protein smears likely contained Cbl and Cbl-associated proteins bearing various posttranslational modifications. The wild-type activity of mutants Cbl D432A and Cbl P433A in this assay demonstrated that a consensus α-adaptin–binding site (Asp432ProPhe434) within the RF tail (17) was not crucial for Cbl-mediated degradation of hSprouty2. The α-adaptin protein mediates the formation of protein complexes involved in the regulation of clathrin-mediated endocytosis. Our results genetically segregated the regulation of hSprouty2 by the RF tail from any potential contribution by its α-adaptin–binding motif.

The RF tail mutant Cbl F434A reduced the abundance of Cbl-associated ubiquitinated proteins and induced the degradation of hSprouty2 to an extent similar to that induced by wild-type Cbl (Fig. 2A and fig. S2A). By contrast, the Cbl V431A mutant induced a severe defect in the ubiquitination of Cbl-associated proteins (Fig. 2A) and in the degradation of hSprouty2; Cbl-hSprouty2 complexes persisted throughout the stimulation period (Fig. 2A) and there was no apparent decrease in the total cellular pool of hSprouty2. Both the Cbl F434A and Cbl V431A mutants retained the ability to associate with activated EGFR (Fig. 1D). Thus, Cbl V431A bound to both hSprouty2 and EGFR and failed to mediate efficient ubiquitin-dependent degradation of either substrate, with a complete defect early in the EGFR-trafficking pathway at the site of degradation of Sprouty2. The functional defect of Cbl F434A occurred after the hSprouty2 regulation checkpoint. These results suggest that residues in close proximity to each other within the RF tail differentially control the ubiquitination and degradation of distinct substrates of Cbl. We wished to determine whether different E2-binding abilities of the wild-type and mutant Cbl proteins might explain their distinct activities, but in repeated experiments, we were unable to detect the association of any Cbl protein with endogenous UbcH7. It remains possible that another endogenous E2, such as Ubc5, binds differentially to the wild-type and mutant Cbl proteins.

Cbl F434A is compromised in its ability to regulate timely dephosphorylation or degradation of Hrs

We have reported that the ectopic expression of wild-type Cbl enhances the degradation of EGFR, at least in part, by regulating phosphorylated hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs) (9). Hrs is an endosomal, 110-kD regulator of EGFR trafficking that is inducibly phosphorylated on Tyr329 and Tyr334 after stimulation of cells with EGF (18, 19). Other studies showed a correlation between the extent of tyrosine phosphorylation of Hrs and the degradation of activated receptor tyrosine kinases (2023). We showed that phosphorylation of Hrs is essential for efficient Cbl-mediated degradation of EGFR (9). We further showed that, in cells overexpressing wild-type Cbl, the abundance of phosphorylated Hrs (pHrs) is greatly enhanced and then rapidly depleted after addition of EGF and that this depletion corresponds to the degradation of pHrs (9). We therefore asked whether the RF tail mutant Cbl F434A, which was only mildly compromised in its ability to cause the ubiquitination and degradation of hSprouty2, was defective at the Hrs checkpoint downstream in the EGFR trafficking pathway.

To compare the effects of wild-type Cbl and Cbl F434A on Hrs, lysate proteins from transfected HEK 293 cells were analyzed by Western blotting with a polyclonal antibody against pHrs at Tyr334 (pY334Hrs). Tyr334 is the principal site in Hrs that is inducibly phosphorylated downstream of EGF-activated EGFR (18, 19). GFP-tagged wild-type Cbl enhanced the EGF-induced increase in the abundance of pY334Hrs compared to that in cells transfected with the GFP null control (Fig. 2B and fig. S2B). In repeated studies of cells ectopically expressing GFP-tagged wild-type Cbl, the abundance of pY334Hrs peaked at 5 to 10 min after the addition of ligand and decreased to near that of unstimulated cells by 40 min after stimulation. The E3 ligase–deficient Cbl mutants Y371F and V431A, which suppressed the down-regulation and degradation of EGFR (Fig. 1, B and D), also suppressed the phosphorylation of Hrs at Tyr334 (Fig. 2B and fig. S2B). These results were consistent with the proposed functional defect of the mutants at the level of internalization of EGFR and regulation of hSprouty2.

Wild-type Cbl and Cbl F434A enhanced the abundance of pY334Hrs to a similar extent after 10 min of stimulation (Fig. 2B), but Cbl F434A was compromised in its ability to mediate the apparent dephosphorylation, degradation, or both of pY334Hrs at the 40-min stimulation time point (Fig. 2B and fig. S2B). This defect correlated with decreased degradation of EGFR, which was evaluated at 90 min after stimulation (Fig. 1D). We conclude that the tyrosine phosphorylation of Hrs and the timely dephosphorylation, destruction, or both of pHrs are critical determinants to target activated EGFRs for degradation and that both events are regulated by the RF tail of Cbl.

For the Hrs experiments, protein extracts were derived from pools containing untransfected cells and cells transfected with plasmids expressing the GFP-tagged proteins of interest. The transfection efficiency of cells ranged from 10 to 30% among experiments. Thus, the amount of total Hrs detected by Western blotting (Fig. 2B) reflected contributions from both transfected and untransfected cells. Because of this, the data cannot be used to determine (i) the relative extents of Cbl-mediated degradation of Hrs or (ii) the relative abundance of pHrs and total Hrs specifically in the cells expressing wild-type Cb1 versus Cbl F434A.

Cbl F434A induces aberrant clustering of endosomes and impedes the fusion of docked early endosomes

We next determined whether a defect in the trafficking of EGFR was apparent at the microscopic level in cells transfected with plasmids expressing GFP-tagged Cbl F434A. Fixed cell immunofluorescence revealed that EGF-stimulated COS-7 cells expressing endogenous EGFR with either GFP-tagged wild-type Cbl or GFP-tagged Cbl F434A contained enlarged early endosomes that contained EEA1, EGFR, and GFP (Fig. 3). However, greater than 70% of the cells that contained Cbl F434A exhibited endosome clustering, pairing, or both.

Fig. 3

The F434A mutant of Cbl induces the aberrant pairing and clustering of early endosomes carrying activated EGFR. COS-7 cells expressing endogenous EGFR were transfected with the indicated GFP-Cbl expression constructs (4 μg per 10-cm dish). The cultures were stimulated with Alexa Fluor 647–conjugated EGF for 25 min, fixed with paraformaldehyde, and incubated with an antibody against EEA1 and a Texas Red–conjugated secondary antibody before visualization of GFP-Cbl, EGF, and EEA1 signals. In five independent experiments, Cbl F434A increased apparent endosome pairing and clustering compared to that in cells containing WT Cbl. Localization of EEA1 to clustered endosomes that contained EGF and GFP-Cbl identified these compartments as early endosomes. Protein colocalization is indicated by the following colors: top row, red and green yield yellow-orange; middle row, red and blue yield magenta; bottom row, green and blue yield aqua–light blue (compare with the darker blue signal of the upper cell in the bottom left panel). Several examples of vesicles exhibiting protein colocalization are marked by the white arrows.

To determine whether this phenotype reflected the delayed fusion of docked early endosomes, we performed live-cell imaging of GFP-Cbl–expressing, EGF-stimulated COS-7 cells. Vesicle docking events were observed throughout the 40-min imaging period. Some docking events were evident in the first 30 s of image collection, whereas others were initiated much later in the collection period. For consistency, fusion times were calculated as the minutes that elapsed from docking to fusion for individual vesicle pairs, regardless of the stimulation time point at which their docking was initiated. Our results revealed that the fusion of docked early endosomes was rapid in cells that contained wild-type Cbl, with most paired vesicles fusing within 30 s of docking (Fig. 4, A and B, and movie S1). In cells that contained Cbl F434A, many paired endosomes failed to fuse, even after docking for 10 min or more (Fig. 4, A to C, and movie S2).

Fig. 4

Deregulation of the Hrs checkpoint by Cbl F434A impedes the fusion of early endosomes. (A) Select images from live cell collections (movies S1 to S3) contrast the rapid fusion of endosomes bearing WT GFP-Cbl with the delayed fusion of docked endosomes carrying GFP-Cbl F434A and the failure of enlarged vesicles to form in cells expressing GFP–Cbl V431A. Moderate to large endosomes rarely developed in cells that contained Cbl V431A, which are shown here at reduced magnification to illustrate their distinct appearance. Note the difference in elapsed time for the paired images. For WT Cbl and the F434A mutant, Lysotracker Red–positive compartments are lysosomes. Images of cells containing Cbl V431A show red signal for fluor-conjugated EGF. Arrows mark the same paired vesicles at the early and later collection times. (B) Fusion time was determined from all docking events observed in movies for 9 live cells containing GFP-Cbl WT and for 12 live cells containing GFP-Cbl F434A. Time to fusion (x axis) is expressed relative to independent vesicle docking times, rather than from a single time point in the collection process. SDs (error bars) were calculated from pooled experimental data. (C) Cox proportional hazard regression analysis of fusion events for paired docked vesicles. Fusion occurred sooner in the cells that contained GFP-Cbl WT compared to that in cells that contained GFP-Cbl F434A (P < 0.0001). The graph shows the fusion distribution based on the fitted Cox model.

The presence of Cbl V431A typically resulted in a failure to generate even moderate-sized fused vesicles. Instead, we observed the fusion of pinpoint vesicle-like structures in close proximity to the plasma membrane (movie S3). This earliest ligand-induced fusion event was common to cells that contained wild-type Cbl, Cbl V431A, or Cbl F434A (movies S1 to S3). In the absence of ultrastructural studies, it was not possible to establish whether the small structures in each transfected cell population were open plasma membrane invaginations or closed vesicles near the cell surface. Because the tiny structures in cells containing Cbl V431A were so dissimilar to the moderate- to large-sized vesicles detected in cells that contained wild-type Cbl or Cbl F434A, no comparable fusion rate was determined for cells that contained Cbl V431A.

We performed the Cox proportional hazard regression analysis (24) with the robust sandwich method to account for the correlation of observed outcomes from the same image to test for the difference in the endosome fusion time distribution between cells that contained GFP-tagged wild-type Cbl and those that contained GFP-tagged Cbl F434A. The Cox model is a statistical approach that is used to determine the effect of multiple, independent variables on the time required for an event to occur. For our analysis, the independent variables considered were the plasmid construct identity (GFP-Cbl wild type compared to GFP-Cbl F434A), the number of vesicles docked per cluster within each cell analyzed (two compared to three or more), and the interaction of these factors; the timed event was the period required for docked vesicles to fuse.

From the fitted Cox Model, there was a significant Construct × Number docked interaction effect (P = 0.001), meaning that each construct’s effect on fusion time varied with the number of docked vesicles in a given cluster. For paired vesicles, the median fusion time (25th to 75th percentile) was 0.5 min (range, 0.5 to 1.0 min) for cells that contained GFP-tagged wild-type Cbl and 5.5 min (range, 1.5 to 14 min) for cells that contained GFP-tagged Cbl F434A. Where the number of vesicles docked in a cluster was >3, the construct effect was not statistically significant (P = 0.117). In this case, the median fusion time was 6 min (range, 1.5 to 15 min) for cells that contained GFP-tagged wild-type Cbl and 11 min (range, 3 to >34 min) for cells that expressed GFP-tagged Cbl F434A. Results (Fig. 4C) show the effect of wild-type Cbl on the cumulative extent of vesicle fusions (y axis) at defined times after docking (x axis) compared to that of Cbl F434A only when paired vesicles are used for the analysis. Cells expressing GFP-Cbl wild type exhibited rapid fusion of nearly all of the docked endosomes within 5 min, resulting in a steep slope in the fusion distribution curve (dashed line). Where GFP-Cbl F434A was expressed, the delay in fusion resulted in a more gradual slope and a reduction in cumulative fusion events (solid line). Based on these data and our previous study (9), we conclude that Cbl and its RF tail control the fate of EGFR in the endocytic pathway by enhancing the fusion of paired early endosomes, possibly through regulation of the abundance of pHrs.

Discussion

We have investigated the molecular events that regulate EGFR fate and are modulated by the RF tail of Cbl. Our results reveal that specific amino acid residues of the RF tail control the trafficking of EGFR at biochemically distinct checkpoints. The first checkpoint is early in endocytosis, when Cbl regulates the ubiquitination and degradation of hSprouty2. The RF tail mutant Cbl V431A, which is compromised at this stage, fails to effect the degradation of hSprouty2. Consistent with this result, Cbl V431A induced the accumulation of small GFP- and EGFR-containing puncta that remained at the cell surface, as determined by live-cell imaging analysis (movie S3). The second checkpoint is at a later stage of endocytosis, when Cbl controls the apparent dephosphorylation, degradation, or both of the trafficking regulator Hrs, as well as controlling the fusion of early endosomes (which was defective in cells that contained Cbl F434A). The identification here of a Cbl-regulated checkpoint in endosome fusion constitutes the first cell-biological evidence that Cbl, as endocytic cargo, regulates the fate of the EGFR by controlling the maturation of early endosomes. The correlation between the timely dephosphorylation or degradation of Hrs and the fusion of endosomes implies causation; however, definitive data from experiments with Hrs mutants that either lack or mimic tyrosine phosphorylation at specific sites will be necessary to establish a causal link between the regulation of the dephosphorylation or degradation of Hrs and the fusion of endosomes.

Because Cbl is an established suppressor of the signaling of numerous receptor and non–receptor tyrosine kinases, our finding has broad applicability to the field of regulation of tyrosine kinase signaling. Similar to ligand-stimulated EGFR, activated c-Met, platelet-derived growth factor receptor (PDGFR), interleukin-2 receptor (IL-2R), and granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) modulate the tyrosine phosphorylation status of Hrs (25, 26). We predict that the lysosomal degradation of these proteins is similarly dependent on Cbl-mediated degradation of Hrs and the enhancement of the rate of fusion of early endosomes. Endosomal Hrs inhibits the homotypic fusion of early endosomes in vitro (27) and facilitates the recycling of some receptors (28, 29). Phosphorylation may serve as a critical timing switch for the polyubiquitination (30) and degradation of Hrs, which would facilitate the subsequent association of ubiquitinated EGFR with ubiquitin-binding endosomal proteins that escort receptors further along the degradative pathway. The results presented here are consistent with a need for the degradation of pHrs before the fusion of endosomes and the trafficking of EGFR to lysosomes, because these processes are collectively compromised by Cbl F434A. Hrs itself undergoes EGF-induced ubiquitination (30) that may be mediated by Cbl. Further studies at the molecular level will define how the amino acid residues of the RF tail of Cbl control the dephosphorylation, degradation, or both of Hrs and how this process affects signaling from various activated tyrosine kinases.

Finally, our functional analysis of RF tail alanine substitution mutants warrants that a fresh view be taken of the reported crystal structure of Cbl. The RF tail was relatively well ordered in the solved crystal structure of amino acid residues 47 to 447 of Cbl with the E2 protein UbcH7 (11), which was attributed, at least in part, to crystal packing. Based on the structural results, it was postulated that amino acid residues Ile429, Val430, Asp432, and Pro433 in the RF tail might be determinants of the function of Cbl, because these residues mediate intramolecular interactions with the four-helix bundle of the TKB domain. No structural importance was proposed for residues Val431 and Phe434. We were therefore surprised that substitution of the RF tail residues engaged in intramolecular crystal interactions had no apparent effect in our functional assays, whereas the V431A and F434A substitutions did. Further investigations of the activity of the RF tail may clarify the functional relevance of the reported structure of Cbl.

Materials and Methods

Plasmids

The generation of the plasmids pAlterMAX-HA-Cbl, pAlterMAX-EGFR, pcDNA3GFP-Cbl wild type, and FLAG-hSprouty2 has been reported (2, 4). Single amino acid substitution mutants of the RF tail of Cbl were generated with the pAlterMAX mutagenesis system (Promega) with pAlterMAX-HA-Cbl as the template. Mutant sequences were subcloned into the vector derived from digestion of pcDNA3GFP-Cbl-N with Eco RI and Xba I (2).

Antibodies

Antibodies against Syk [sc-1240; (4D10) murine immunoglobulin G2a (IgG2a)], EGFR [sc-120; (528) murine IgG2a], and Cbl [sc-170; (C-15) rabbit polyclonal] were obtained from Santa Cruz Biotechnology, Inc. Primary antibodies against GFP (Ab290; rabbit polyclonal; Abcam Ltd.), ubiquitin (NCL-UBIQ; rabbit polyclonal; Novocastra Laboratories Ltd. from Vector Laboratories, Inc.), EGFR (#06-129; sheep polyclonal for Western blotting; Upstate Biotechnology, Inc.), phosphotyrosine [#05-321; (4G10) murine IgG2b; Upstate Biotechnology, Inc.], and the FLAG epitope tag [(M2) murine IgG1; Sigma] were purchased from the indicated suppliers. Antibody against human leukocyte antigen class I [(American Type Culture Collection W6/32) murine IgG2a] was prepared by the University of Iowa Hybridoma Facility. Secondary detection reagents included AffiniPure rabbit antibody against mouse IgG1, antibody specific for the Fcγ region (#315-035-046; Jackson ImmunoResearch Laboratories, Inc.), (R)-phycoerythrin–conjugated F(ab′)2 fragment of antibody against mouse IgG (H+L) (#115-116-146; goat polyclonal; Jackson ImmunoResearch Laboratories, Inc.), peroxidase-conjugated protein A (#55901; ICN Pharmaceuticals, Inc.), and peroxidase-conjugated antibody against sheep IgG, H+L (#402100; rabbit polyclonal; Calbiochem-Novabiochem Corporation).

Cells

HEK 293 and COS-7 cells were used within 15 and 30 passages, respectively, of recovery from liquid nitrogen. Cultures were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, penicillin and streptomycin (100 U/ml each), and 20 mM Hepes at 37°C in 5% CO2.

Transient transfections, EGF stimulation, and preparation of cell lysates

Transient transfections, stimulation of cells with EGF, and harvesting of HEK 293 cells were performed as described previously (2). The amounts of input DNA were as follows: For the experiments shown in Figs. 1 and 2, HEK 293 cells were transfected with pAlterMAX-EGFR (0.05 μg per 10-cm dish), a plasmid that expressed FLAG-tagged hSprouty2 (1 μg per 10-cm dish) where indicated, and either a plasmid expressing GFP or plasmids expressing the indicated GFP-Cbl expression constructs (4 μg per 10-cm dish). Calcium phosphate transfection efficiencies for HEK 293 experiments ranged from 10 to 30%. EGF (Sigma) was used at 100 ng/ml (17 nM) to stimulate cells, which is a high concentration of ligand but is within the physiologically relevant range (31).

Immunoprecipitations and Western blotting analysis

The immunoprecipitation procedures used in this study have been reported previously (2, 4). Proteins resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) were transferred to polyvinylidene difluoride (PVDF) membranes. For the analysis of Western blots to detect ubiquitination, PVDF membranes of immunoprecipitated samples were autoclaved in water for 10 min before blocking of the membrane and detection of protein. Incubations of membranes with primary antibodies, secondary reagents, and detection solutions were performed as recommended by the suppliers. Image J and ScionImage software were used to quantify relative signals within experiments.

Down-regulation assays

Assays to detect the down-regulation of EGFR were performed as previously described (2). Transfected cell cultures were left untreated or were stimulated with EGF for the indicated times after which they were harvested and processed at 4°C. To identify surface EGFR, cells were kept intact for staining.

Confocal fluorescence microscopy of fixed cells and live-cell imaging

For confocal microscopy, COS-7 cells transfected with plasmids expressing GFP or GFP-Cbl were serum-starved, stimulated for 25 min with Alexa Fluor 647–labeled EGF, fixed with paraformaldehyde, and incubated with antibody to detect the localization of EEA1 (with a Texas Red–conjugated secondary antibody against mouse IgG). Images were collected on a Zeiss 510 confocal microscope with a PlanApo 63× oil-immersion objective [numerical aperture (NA) 1.4]. Live imaging of transfected COS-7 cells in OptiMEM (Invitrogen) with 0.5% FBS at 37°C in 5% CO2 was performed with an Olympus IX-81 microscope, a PlanApo 100× oil-immersion objective (NA 1.4), and a Hamamatsu Orca ER camera. For the experiments shown in movies S1 and S2, image collection began ~15 min after the addition of Lysotracker Red (Invitrogen) and unlabeled EGF. For the experiment shown in movie S3, the red signal corresponded to Alexa Fluor 647–conjugated EGF. Red and green signals were acquired with SlideBook software at 30-s intervals over a 30- or 40-min collection period. In SlideBook, all images were deconvolved with the no-neighbors deconvolution algorithm. Supplementary movies were generated from sequential image files by Image J. All of the presented still images were processed identically with Adobe Photoshop CS.

Acknowledgments

We thank S. Ramaswamy and R. Friemann for enlightening discussions of the reported Cbl crystal structure. J. Hagen, T. Moninger, and G. Hess provided expert technical advice and assistance. The statistical analysis in Fig. 4C was performed by M. Bridget Zimmerman of the Biostatistics Consulting Center, Department of Biostatistics, University of Iowa. Facilities at the University of Iowa College of Medicine (Flow Cytometry, Crystallography, DNA, Central Microscopy Research, and Tissue Culture/Hybridoma facilities) were used for these studies. This research was funded by the American Cancer Society Research Scholar grant RSG-03-046-01 and NIH award RO1 CA109685 (to N.L.L.); an individual allocation from the American Cancer Society Institutional Research grant IRG-122-V (from G. Weiner at the Holden Comprehensive Cancer Center to N.L.L.); institutional training grants NRSA T32 DEO14678-03 and DEO14678-04 (from National Institute of Dental and Craniofacial Research to Christopher Squier and the Dows Institute and College of Dentistry at the University of Iowa for support of N.L.L. and K.A.C., respectively); and grant BCTR0707177 from Susan G. Komen for the Cure (to J.G.K.).

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/2/102/ra86/DC1

Fig. S1. Quantitation of the ubiquitination and degradation of EGFR.

Fig. S2. Quantitation of data from Fig. 2.

Movies S1 to S3.

References and Notes

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