Abl Tyrosine Kinase Regulates Endocytosis of the Epidermal Growth Factor Receptor*

Signal attenuation from ligand-activated epidermal growth factor receptor (EGFR) is mediated in part by receptor endocytosis and trafficking to the lysosomal degradative compartment. Uncoupling the activated EGFR from endocytosis and degradation has emerged as a mechanism for oncogenic activation of the EGFR. The Abl nonreceptor tyrosine kinase is activated by ligand-stimulated EGFR, but the role of Abl in EGFR signaling has not been defined. Here we uncovered a novel role for the activated Abl kinase in the regulation of EGFR endocytosis. We show that activated Abl impairs EGFR internalization. Moreover, we show that activated Abl phosphorylates the EGFR primarily on tyrosine 1173, and that mutation of this site to phenylalanine restores ligand-dependent endocytosis of the EGFR in the presence of activated Abl. Furthermore, we show that activated Abl allows the ligand-activated EGFR to escape Cbl-dependent down-regulation by inhibiting the accumulation of Cbl at the plasma membrane in response to epidermal growth factor stimulation and disrupting the formation of the EGFR·Cbl complex without affecting Cbl protein stability. These findings reveal a novel role for Abl in promoting increased cell-surface expression of the EGFR and suggest that Abl/EGFR signaling may cooperate in human tumors.

Ligand-induced endocytosis of the epidermal growth factor receptor (EGFR) 2 is a critical mechanism for regulating the amplitude and kinetics of EGFR signaling (1). Following endocytosis, the internalized EGFR may recycle back to the plasma membrane for continued signaling or is targeted for degradation in the lysosomal compartment, thereby irreversibly terminating receptor signaling. Defective internalization of ligandactivated EGFR leads to prolonged signals from the cell surface and may result in cellular transformation (1)(2)(3). Oncogenic EGFR signaling is associated with mutations of the EGFR itself or molecules required for receptor endocytosis (1,3).
EGFR endocytosis requires post-translational modifications such as phosphorylation, monoubiquitination, and polyubiquitination at multiple sites (3,4). A critical regulator of liganddependent EGFR down-regulation is the Cbl adaptor protein.
Cbl is a ubiquitin-protein isopeptide ligase that mediates ligand-induced ubiquitination of the EGFR, which is required for targeting the receptor to sorting endosomes and subsequently to the lysosomal degradative compartment. Cbl also forms a complex with CIN85 and endophilin to regulate internalization of ligand-bound EGFR (5). Cbl is recruited to the receptor in response to EGF stimulation by direct binding to phosphotyrosine 1045 of the EGFR (6,7). Cbl also binds indirectly to the EGFR through the Grb2 adaptor protein (8 -10). Cbl has been shown to regulate both endocytosis and targeting of the EGFR for lysosomal degradation following ubiquitination (1,11). A role for Cbl in EGFR internalization is also supported by the observation that a mutant EGFR lacking the Cblbinding site exhibits impaired ligand-induced ubiquitination and endocytosis, enhanced recycling to the plasma membrane, and increased mitogenic signaling (8). Moreover, down-regulation of both c-Cbl and the related Cbl-b by RNA interference resulted in substantial inhibition of EGFR internalization (12). Additionally, EGFR internalization is inhibited in cells depleted of the Grb2 adaptor or by mutation of the Grb2-binding site on the receptor (9,10,12). Interestingly, EGFR endocytosis is restored in Grb2-depleted cells by a chimeric protein that fuses the Grb2 Src homology 2 (2) domain to Cbl (10). These findings show that in addition to a role in lysosomal sorting and degradation of the receptor, Cbl regulates EGFR internalization.
Escape from Cbl-mediated degradation has been linked to increased oncogenicity of the EGFR and other ErbB family members. Highly oncogenic forms of the viral erbB protein kinase have lost the Cbl-docking site (13). An EGFR deletion mutant lacking exons 2-7 (EGFRvIII) found in glioblastoma patients exhibits reduced Cbl recruitment and fails to be ubiquitinated and internalized (14). Furthermore, enhanced destruction of Cbl induced by the activated Src tyrosine kinase, or sequestration of Cbl by activated Cdc42 or Sprouty2, has been shown to inhibit Cbl-mediated down-regulation of the EGFR and may contribute to EGFR oncogenicity (15,16). Thus, deregulation of the EGFR in cancer may occur not only through amplification or point mutations of the receptor resulting in elevated receptor levels or ligand-independent receptor activation, but also by uncoupling the EGFR from ligand-induced down-regulation. The mechanisms that promote the uncoupling of these processes are just beginning to be defined. Here we identify a novel role for the Abl tyrosine kinase in uncoupling ligand-activated EGFR from receptor internalization.
The Abl family kinases, Abl1 (c-Abl) and Abl2 (Arg), play a role in the regulation of cytoskeletal reorganization, cell migration, proliferation, and survival (17). We and others have shown that endogenous Abl kinases are rapidly activated after EGFR stimulation (18 -20). Abl kinases are required for EGF-mediated membrane ruffling and Rac activation at physiological concentrations of the ligand (21). Moreover, the SH2 domains of Abl1 and Abl2 have been shown to bind with high affinity to the EGFR and other ErbB family members by interacting with multiple tyrosines on the phosphorylated receptors (19,22). However, little is known regarding the effects of Abl activation on EGFR physiological functions. Similar to the EGFR, Abl kinase hyperactivation has been linked to the pathogenesis of cancer. Chromosomal translocation events in human leukemias give rise to structurally altered oncogenic Abl fusion proteins such as Bcr-Abl, Tel-Abl, and Tel-Arg (17). Furthermore, enhanced expression of Abl kinases is observed in a subset of primary colon carcinomas and metastatic tumors (23), as well as in pancreatic ductal carcinomas and renal medullary carcinoma (24,25). Also, Abl kinases are constitutively activated downstream of ErbB receptors and Src kinases in breast cancer cell lines (20). Here we show that the activated Abl kinase phosphorylates the EGFR at specific sites and uncouples the receptor from ligand-mediated internalization. Additionally, we show that kinase-active Abl disrupts the Cbl recruitment to the activated EGFR. Thus, Abl and the EGFR may function synergistically in the pathogenesis of human tumors.

MATERIALS AND METHODS
Cell Culture-COS7 and 293T cells (ATCC) and were maintained in DMEM (Invitrogen) containing 10% FBS (Invitrogen). NR6 cells devoid of endogenous EGFR were a kind gift from Dr. Jeremy Rich (Duke University Medical Center, Durham, NC) and were maintained in 1ϫ Zinc Option Medium (Invitrogen) and supplemented with 7.5% FBS (Invitrogen) and 100 units/ml penicillin/streptomycin (Invitrogen). Cells were maintained at 37°C in 5% CO 2 .
Generation of Stable Cell Lines-NR6 cells were transfected with pCDNA vector encoding EGFR-WT or tyrosine to phenylalanine mutants of the EGFR and a neomycin resistance gene. Cells were sorted for the EGFR by FACS and selected with 350 g/ml geneticin (G-418, Invitrogen).
DNA Constructs-Abl wild type (Abl-WT), Abl kinase-defective (Abl-KM), and kinase-active Abl sequences were cloned into pCDNA3 for transfection experiments or into MIGR1 for retroviral transduction experiments as described previously (18,26,27). EGFP-C1 vector was purchased from Clontech. Plasmids encoding pCDNA3-EGFR-WT and pCDNA3-EGFR-KD were the generous gifts from Dr. Sarah J. Parsons (University of Virginia, Charlottesville, VA). pC3-dynamin-WT and dynamin-K44A constructs were the kind gifts from Dr. Robert Lefkowitz (Duke University Medical Center, Durham, NC). The pcDNA-cAbl-1q2q3q, pcDNA-cAbl-1q2q3q-K290M, and pcDNA-Abl-P131L constructs were the generous gifts from Dr. Richard Van Etten. The kinase-active Abl form AblP131L is a transforming c-Abl mutant with an SH3 domain point mutation (P131L) that shows increased tyrosine kinase activity (28). Abl-PP harbors mutations in prolines 242 and 249 in the SH2 kinase linker domain, which are mutated to glutamic acid, rendering Abl in a constitutively active state (29). Abl-KM (Abl K290M) is a kinase-inactive mutant (30). pXJ40-Cbl-WT was a generous gift from Graeme R Guy. EGFR-Y1173F was generated by site-directed mutagenesis with a forward primer (AAA ATG CAG AAT TCC TAA GGG TC) and a reverse primer (GAC CCT TAG GAA TTC TGC ATT TT) with the Quick-Change site-directed mutagenesis kit from Stratagene as indicated by the manufacturer.
EGF Internalization Assays in COS7 Cells-Cells were plated in coverslips, transfected, and serum-starved 1 day after transfection for 24 h in DMEM containing 0.1% FBS. Cells were placed in serum-free DMEM 10 min prior to the addition of 50 ng/ml EGF at 37°C for 15 min. After EGF stimulation, cells were placed for 15 min in acidic washing buffer (0.2 M acetic acid, pH 2.8, 0.5 M NaCl) to remove cell surface EGF. The cells were then fixed in 4% paraformaldehyde at room temperature for 15 min. Samples were permeabilized at room temperature with 0.5% Triton X-100 and washed twice with 1ϫ PBS, blocked with 10% normal donkey serum (NDS, Jackson Immu-noResearch), and immunostained with primary antibodies for EGF (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) or Abl (Ab-3) in the presence of 2% NDS. After washing three times with 1ϫ PBS, samples were treated with secondary antibodies donkey anti-rabbit CY3 or donkey anti-mouse CY2 (from Jackson ImmunoResearch) in the presence of 4% NDS. EGF internalization in NR6 cells was analyzed in a similar manner, and the number of cells positive for EGF internalization was determined by counting the number of vesicles/ 2 for each cell. Numbers ranged from 0 to 5. EGFR internalization-negative cells had a ratio lower than 0.5 vesicles/ 2 , whereas cells with ratios ranging between 0.5 and 5 were counted as positive for EGFR internalization. Statistical analysis was carried out with GraphPath Prism program.
EGF-Rhodamine Internalization Assays-COS7 cells were plated in coverslips, transfected, and serum-starved 1 day after transfection for 24 h in DMEM containing 0.1% FBS. Cells were placed in ice-cold binding medium (20 mM HEPES-NaOH, pH 7.5, 130 mM NaCl, 0.1% bovine serum albumin) for 30 min and treated with 40 ng/ml rhodamine-EGF (Molecular Probes, Eugene, OR) in serum-free DMEM for 1 h at 4°C. To examine the internalization of EGF, EGF-containing medium was replaced with warm DMEM, in which the cells were incubated for 15 min in serum-free media followed by addition of acidic washing buffer as indicated above. Cells were permeabilized in 0.5% Triton X-100 and immunostained for Abl. EGFR internalization was analyzed with a confocal microscope. Transfected proteins were detected with fluorescein isothiocyanate, and rhodamine-EGF complexes were detected with a CY3 filter channel and three-dimensional projections of slices throughout the cell. Cell counting was performed with the identity of the sample remaining unknown to the observer.
Analysis of EGFR Endocytosis by Cell-surface Biotinylation-NR6 cells expressing wild type EGFR were retrovirally transduced with vector control MIGR1 or MIGR1-Abl-PP and serum-starved 1 day after infection. Cells were stimulated with 50 ng/ml EGF for the indicated times. EGFR internalization was stopped by placing the cells at 4°C. Cells were washed three times in 1ϫ PBS, and a solution with 1 mg/ml EZ-Link NHS-SS-Biotin (Pierce) was added for 15 min. Unreacted biotin was quenched by the addition of 50 mM glycine, and cells were lysed in 1ϫ NETN Buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 8, 200 mM NaCl, 1 mM EDTA) with the addition of inhibitors (1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 25 mM sodium fluoride, 2 mM sodium pyrophosphate, and 1 g/ml aprotinin, leupeptin, and pepstatin) and rotated for 1 h at 4°C. Lysates were cleared by centrifugation at 14,300 rpm for 10 min at 4°C. After quantification of protein concentration with the Bradford assay (Bio-Rad), equal amounts of protein were used to pull down the cell-surface biotinylated product with Tetralink Tetrameric Avidin Resin, Promega V2591.
Subcellular Fractionation-COS7 cells were serum-starved for 24 h and subjected to EGF stimulation 48 h after transfection at the indicated time points. Cells were washed twice with ice-cold 1ϫ PBS and scraped in Hypotonic Buffer C (10 mM HEPES-K, pH 7.6, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol, 1 mM NaEDTA, 1 mM NaEGTA), which was supplemented with inhibitors as described above. Resuspended cells were placed at Ϫ80°C for 30 min and thawed (twice) in order to break down the membrane fraction. Cell suspensions were spun at 14,300 rpm for 30 min in a microcentrifuge at 4°C to separate the cytosolic fraction (supernatant) from the pellet containing the membrane fraction. Pellets were resuspended in modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 150 mM sodium chloride, 1 mM EDTA, pH 8, 1 mM EGTA) with the addition of the inhibitors described above. Protein concentration was quantified as described above. The purity of the fractions was analyzed by Western blotting for ERK1 in the cytosolic fraction and transferrin receptor in the membrane fraction. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and Western-blotted for the proteins of interest.
Cell Lysis and Immunoprecipitations-For phosphorylation experiments cells were lysed and immunoprecipitated in modified RIPA buffer with inhibitors (described above). To analyze Cbl/EGFR interactions, cells were lysed in 1% Nonidet P-40 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1% Nonidet P-40) with the combination of inhibitors described above and the addition of N-ethylmaleimide (10 mM). In both cases, lysates were rotated for 20 min at 4°C prior to centrifugation for 10 min at 14,300 rpm in a microcentrifuge also at 4°C. After centrifugation, the supernatants were utilized for immunoprecipitations, incubated with primary antibodies for 2 h (or overnight for the EGFR) and protein G-Sepharose beads (protein A-Sepharose for the EGFR) (Amersham Biosciences) for 2 h, before washing three times in lysis buffer and resuspending the beads in 2ϫ SDS-sample buffer for analysis by SDS-PAGE. All quantifications for protein levels were carried out with the LI-COR Biosciences Odyssey Infrared Imaging software version 2.
It has been shown that upon EGF stimulation, EGF⅐EGFR complexes are targeted for degradation (31,32). To determine whether Abl-PP affected the levels of EGFR upon EGF stimulation, we examined the total amount of endogenous EGFR protein in COS7 cells before and after EGF stimulation in the absence or presence of Abl-PP. COS7 cells expressing Abl-PP had higher levels of total EGFR upon addition of EGF at all time points examined (30 -90 min) compared with vector-transfected cells, as analyzed by Western blotting for the EGFR (Fig. 1B). These data show that Abl-PP inhibits EGFR endocytosis and leads to an increase in the cellular levels of EGFR after EGF binding.
To further examine the role of Abl in EGFR endocytosis, rhodamine-labeled EGF was used to track endogenous EGFR internalization in the presence of wild type and active forms of the Abl kinase. Expression of two different Abl kinase-active mutants, AblP131L and AblPP, resulted in a significant decrease in the number of cells with internalized EGFR (50%) compared with vector-transfected cells (84%) (Fig. 1C). Expression of kinase-active Src (Src527F), previously shown to lead to the accumulation of EGFR at the cell surface (33), caused a slight reduction of EGFR endocytosis (Fig. 1C). As expected, expression of Dyn K44A, a known inhibitor of endocytosis (2), reduced EGFR internalization to a level (40%) that was comparable with that induced by activated Abl kinase expression. Expression of wild type Abl (Abl-WT) also reduced EGFR internalization (Fig. 1C). The total tyrosine phosphorylation in cells expressing Abl-WT, as well as AblP131L and Abl-PP, was markedly higher compared with vector-transfected cells (Fig.  1D), suggesting that enhanced tyrosine phosphorylation may play a role in mediating the Abl effect on EGFR endocytosis.
To analyze surface levels of EGFR, NR6 cells that are devoid of endogenous EGFR were used in cell-surface biotinylation experiments after retroviral transduction with wild type receptor (EGFR-WT) (Fig. 2). Expression of Abl-PP resulted in a significant increase in surface levels of EGFR after EGF stimulation. Analysis of four independent experiments showed that active Abl kinase effectively inhibits ligand-dependent EGFR endocytosis and that this effect is statistically significant (Fig.  2B). Additionally, we analyzed the rate of EGFR internalization for three independent experiments, and we observed a significant decrease in the surface EGFR after 30 min of EGF treatment in control but not in activated Abl-expressing cells (Fig.  2C). The inhibition of EGFR endocytosis by Abl-PP was also observed by analysis of cell-surface biotinylation in COS7 (supplemental Fig. S1) and 293T cells (data not shown). Thus, oncogenic Abl-PP inhibits EGFR endocytosis in multiple cell lines.
Abl Phosphorylates the EGFR-To define the mechanism whereby the activated Abl kinase inhibits EGFR internalization, we first analyzed whether Abl could phosphorylate the EGFR and whether direct EGFR phosphorylation by Abl might regulate receptor internalization. Abl kinases are activated downstream of receptor tyrosine kinases such as the EGFR and the platelet-derived growth factor receptor (PDGFR) (18,19). Following activation by ligand-bound PDGFR, Abl has been shown to phosphorylate the PDGFR (27). Thus, we examined whether Abl kinases are similarly engaged in bidirectional signaling with the EGFR. Addition of EGF promoted an increase in molecular weight in the band corresponding to endogenous EGFR, which correlates with increased tyrosine phosphorylation of the  OCTOBER 27, 2006 • VOLUME 281 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 32717 receptor (Fig. 3A, lanes 1 and 2). Expression of Abl-PP resulted in an increase in the molecular weight of the endogenous EGFR in the absence of EGF (Fig. 3A, upper panel, lane 3), which was similar to that induced by EGF binding. The shift in mobility is suggestive of Abl-dependent EGFR tyrosine phosphorylation. Analysis of the total phosphotyrosine levels showed that the increased EGFR molecular weight was coincident with higher total tyrosine phosphorylation in the presence of Abl-PP (Fig.  3A, lower panel). Because the EGFR itself is a tyrosine kinase and can be autophosphorylated, we utilized a kinase-defective EGFR construct (EGFR-KD) to analyze whether Abl phosphorylates the EGFR. Tyrosine phosphorylation of EGFR-KD increased upon expression of Abl-WT and was further enhanced by expression of activated Abl-PP (Fig. 3B). No tyrosine phosphorylation of EGFR-KD was observed in the presence of the kinase-defective Abl-KM. As reported previously, EGFR-KD phosphorylation was greatly enhanced upon Src-527F overexpression (Fig. 3B). The Abl-dependent phosphorylation of EGFR-KD was independent of the endogenous EGFR present in 293T cells as increasing doses of an EGFRselective inhibitor AG1478 did not affect EGFR-KD tyrosine phosphorylation in the presence of Abl-PP (Fig. 3C).

Abl Phosphorylates the EGFR Primarily at Tyrosine 1173-
The cytoplasmic tail of the EGFR contains a number of tyrosines that upon phosphorylation lead to differential recruitment of signaling partners (34), including Abl (19). To identify the specific EGFR tyrosines inducibly phosphorylated by coexpression of activated Abl, we utilized phospho-specific antibodies corresponding to the different tyrosines in the EGFR cytoplasmic domain and analyzed the pattern of tyrosine phosphorylation by Western blotting in the presence of kinase-active Abl (Fig. 4A). Activated Abl-PP expression produced a marked increase of EGFR-KD tyrosine phosphorylation on tyrosine 1173 (Fig. 4A). Tyrosine 1173 is the main binding site for phospholipase C␥1 and Shc in the EGFR, and phosphorylation of this site been associated with increased tumorigenicity in a mutant EGFR that is retained at the cell surface (35). EGFR-WT was strongly phosphorylated on tyrosine 1173 and all other sites examined as reported previously (36). Activated Abl kinase also induced phosphorylation of tyrosine 1068 and to a lesser extent tyrosine 992 in the EGFR cytoplasmic domain (Fig. 4A). The phosphorylated tyrosine 1068 recruits the Grb2 adaptor and has been implicated in signaling to Ras (37). Notably, there was no detectable phosphorylation of EGFR-KD at

. Ligand-dependent EGFR internalization is inhibited by activated Abl in NR6 cells by analysis of cell-surface biotinylation.
A, NR6 cells stably expressing the EGFR were transduced with retroviruses encoding vector control (1st to 4th lanes) or kinase-active Abl-PP (5th to 8th lanes). Cells were depleted of serum overnight and stimulated with 50 ng/ml EGF at the indicated time points. Surface EGFR was analyzed by cell-surface biotinylation followed by pulldown with neutravidin beads. Proteins were analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, and analyzed by Western blotting for EGFR. Whole cell lysates show expression of Abl and transferrin receptor as indicated. Equal levels of EGFR for all samples were verified by FACS analysis. B, NR6 cells were retrovirally transduced with either vector control or kinase-active Abl-PP as indicated. Cells were serum-starved overnight and stimulated with 50 ng/ml EGF at the indicated time points as shown. Results represent the mean Ϯ S.E. of four independent experiments. Asterisk represents a statistically significant (*, p Ͻ 0.05) change in surface EGFR (pixels/mm 2 ) between vector control and Abl-PP expressing cells. C, rate of EGFR internalization for three independent experiments at the four indicated time points.
tyrosine 1045 upon expression of Abl-PP (Fig. 4A). Tyrosine 1045 is the docking site for Cbl (31), which has been implicated in EGFR endocytosis and signal attenuation (5,31). In contrast to Abl, Src kinases phosphorylate the EGFR primarily at tyrosine 845 (Fig. 4A) in agreement with previous findings (reviewed in Ref. 38). EGFR-KD phosphorylation on tyrosine 845 was undetectable in the presence of kinase-active Abl-PP (Fig. 4A). Analysis of Abl-dependent phosphorylation on the endogenous EGFR in COS7 cells confirmed that Abl caused an increase in the phosphorylation of tyrosines 1173 and 1068 but did not change phosphorylation of tyrosines 845 and 1045 (supplemental Fig. S2). Notably, EGFR tyrosine 1173 was phosphorylated by active Abl-PP even in the absence of EGF stimulation, and this phosphorylation was further increased following addition of EGF (supplemental Fig. S2).
To confirm the specificity of Abl-mediated phosphorylation at tyrosine 1173 on the EGFR, we utilized site-directed mutagenesis to substitute tyrosine 1173 to phenylalanine. EGFR-KD or EGFR-KDY1173F was expressed in 293T cells in the absence or presence of kinase-active Abl-PP. Mutation of tyrosine 1173 to phenylalanine markedly decreased Abl-mediated phosphorylation of the EGFR, reducing total EGFR phosphorylation to 40% of the maximal EGFR phosphorylation in the presence of kinase-active Abl-PP. (Fig. 4B), and completely abolished Abl-induced phosphorylation of tyrosine 1173 as detected with the phospho-Tyr-1173-specific antibody (data not shown). Moreover, we found that treatment of metastatic breast cancer cells with the Abl kinase inhibitor STI571 (ima-tinib mesylate, Gleevec TM ) markedly decreased the phosphorylation of tyrosine 1173 on the EGFR in the presence of ligand (Fig. 4C).
Thus, Abl kinases phosphorylate the EGFR primarily at tyrosine 1173 and to a lesser extent at tyrosines 1068 and 992. Tyrosines 1173 and 992 are docking sites for phospholipase C␥1 and Shc, whereas tyrosine 1068 is a binding site for Grb2 and Gab1 (Fig. 4D). Interestingly, Abl kinases have been shown to interact with multiple tyrosines in the cytoplasmic domain of the EGFR, including tyrosine 1173 (19). Our findings suggest that Abl-mediated phosphorylation of the EGFR may regulate EGFR signaling and internalization.

Mutation of Tyrosine 1173 to Phenylalanine Restores Ligand-dependent EGFR Endocytosis in the Presence of Kinase-active Abl-
Activated Abl inhibits endocytosis of wild type EGFR, and Abl phosphorylates the receptor at specific tyrosines, primarily tyrosine 1173. Thus, we sought to test the hypothesis that the Abl-dependent block of EGFR internalization was mediated by phosphorylation of the receptor at tyrosine 1173. To this end we examined whether mutation of tyrosine 1173 to phenylalanine could restore receptor endocytosis in the presence of Abl-PP. NR6 cells, which are devoid of endogenous EGFR, were transfected with EGFR-WT or EGFRY1173F in the presence of activated Abl-PP or kinase-defective Abl-KM. Receptor endocytosis was analyzed in the presence of EGF. Notably, mutation of tyrosine 1173 to phenylalanine restored EGF-dependent EGFR endocytosis in the presence of activated Abl. In contrast to the EGFR-WT, EGF-dependent endocytosis of the Y1173F receptor mutant was observed in cells expressing the activated Abl-PP kinase. The percentage of internalization was comparable with that observed in cells expressing kinase-defective Abl-KM (Fig.  5A). Quantification of cells with internalized EGF in NR6 cells showed that replacement of tyrosine 1173 for phenylalanine in the EGFR restored EGFR internalization in the presence of Abl-PP (Fig. 5B) and that the results are statistically significant. Thus, these data indicate that the effect of Abl on EGFR endocytosis is partially mediated by Abl-dependent phosphorylation of tyrosine 1173.

Kinase-active Abl Reduces Cbl/EGFR Interaction and Accumulation of Cbl to the Plasma Membrane upon EGF Stimulation-
Cbl is recruited to the ligand-activated EGFR (39,40) and accompanies the EGFR throughout the endocytic route (41). To analyze whether active Abl-PP might regulate the Cbl/EGFR interaction, EGFR-WT was expressed in the absence or presence of Abl-PP, and the integrity of Cbl⅐EGFR complex was analyzed upon EGF stimulation. As shown in Fig. 6A, Abl-PP markedly disrupted the interaction between Cbl and the EGFR in the presence of EGF.
Oncogenic Src has been show to affect Cbl recruitment to activated EGFRs by decreasing Cbl protein levels (33). We analyzed whether Abl-PP enhanced the destruction of the Cbl protein in a similar manner. Endogenous Cbl protein levels were examined before and after EGF stimulation in control or Abl-PP-expressing cells. Total Cbl protein levels were unchanged in the presence of Abl-PP compared with vector control transfected cells (Fig. 6B and supplemental Fig. S3). Similarly, the levels of HA-Cbl exogenously expressed in 293T cells were unchanged in the presence of Abl-PP (Fig. 6A). Thus, in contrast to active Src, the constitutively active Abl kinase did not decrease Cbl protein levels ( Fig. 6B and supplemental Fig. S3).
To determine whether Abl kinase activity affected Cbl accumulation at the plasma membrane, we first analyzed localization of endogenous Cbl before and after EGF stimulation by immunofluorescence in COS7 cells. Expression of active Abl-PP resulted in a marked reduction in the localization of Cbl to the plasma membrane upon EGF stimulation compared with that observed in adjacent untransfected cells. In contrast, expression of kinase-defective Abl-KM did not affect Cbl localization to the plasma membrane in response to EGF, which was comparable with that observed in vector-transfected cells and untransfected cells (Fig. 6C). Moreover, Cbl did not co-localize at the plasma membrane with tyrosine-phosphorylated EGFR in cells expressing active Abl-PP (supplemental Fig. S4). Next, we examined whether the active Abl-PP kinase inhibited Cbl recruitment to the plasma membrane by cellular fractionation. EGF stimulation of vector-transfected cells showed the expected increase in membrane-associated Cbl as early as 30 s after EGF stimulation with little change in cytosolic levels of Cbl (data not shown). In contrast, membrane-associated Cbl was markedly reduced upon expression of kinase-active Abl (Fig. 6D) at all time points examined at 37°C and also for 1 h at 4°C in the presence of EGF. Thus, kinase-active Abl-PP negatively affects the recruitment of Cbl to the activated EGFR and inhibits Cbl accumulation at the plasma membrane.

DISCUSSION
Here we show for the first time that the activated Abl kinase phosphorylates the EGFR and inhibits EGFR internalization. Furthermore, we identify tyrosine 1173 on the EGFR as the primary site phosphorylated by Abl, and we show that mutation of this site to phenylalanine restores normal ligand-induced EGFR internalization in the presence of kinase-active Abl-PP. Moreover, we show that kinase-active Abl disrupts recruitment of Cbl to the activated EGFR and impairs Cbl accumulation at the plasma membrane after EGF stimulation. Together these findings show that active Abl kinase negatively regulates EGFR internalization.
Removal of ligand-bound EGFR from the plasma membrane and subsequent receptor degradation are essential processes for signal attenuation from activated receptors, and disruption of these processes can lead to cellular transformation (2,42). EGFR endocytosis has been shown previously to be regulated by tyrosine phosphorylation in the cytoplasmic tail of the EGFR (7,14,43,44). Oncogenic EGFR mutants such as the EGFRvIII show increased levels of tyrosine 1173 phosphorylation (35,45). The tumorigenic activity of EGFRvIII is attributed to its lack of internalization (35), and mutation of tyrosine 1173 to phenylalanine in the mutant receptor abrogates its mitogenic activity (35). Here we show that mutation of tyrosine 1173 is the major site of Abl-mediated phosphorylation in the EGFR, that activated Abl inhibits EGFR internalization, and that mutation of tyrosine 1173 to phenylalanine restores EGFR internalization in the presence of kinase-active Abl. These data suggest that Ablmediated phosphorylation might play a role in EGFR activation in tumor cells. In addition to tyrosine 1173, the EGFRvIII mutant receptor exhibits constitutive phosphorylation on tyrosines 1068 and 992 (35), which are also sites phosphorylated on the EGFR by Abl. This finding further supports the notion that phosphorylation of the EGFR at these three tyrosines may be mediated by the activated Abl kinases. It is possible that in the absence of ligand stimulation, the EGFR might be phosphorylated on tyrosine 1173 by Abl kinases that are activated by overexpression or by stimulation downstream of Src or other receptor tyrosine kinases. We have shown previously that the Abl nonreceptor tyrosine kinase is activated directly downstream of the EGFR (18). Moreover, it was recently reported that Abl kinases are highly activated downstream of oncogenic ErbB receptors in many breast cancer cell lines and that Abl kinases promote invasion of breast cancer cells (20). Thus, these findings together with our data suggest that Abl and the EGFR could engage in bidirectional signaling. Abl kinase activity might be enhanced in cancer cells with activating EGFR mutations, and in turn the activated Abl kinase might phosphorylate the EGFR thereby inhibiting its internalization and enhancing oncogenic signaling.
Similar to the activated Abl, oncogenic Src kinases inhibit EGFR internalization (33) and phosphorylate the cytoplasmic tail of the receptor at specific tyrosines. Notably, the specific EGFR tyrosines phosphorylated by Abl are different from those phosphorylated by Src, suggesting the existence of a distinct mechanism for the effects of these kinases on EGFR endocytosis.
In addition to targeting the EGFR directly by phosphorylation of specific tyrosines, Abl kinases regulate EGFR internalization and degradation by affecting Cbl recruitment to the EGFR at the plasma membrane. Noninternalizing EGFR variants have been shown to have impaired interaction with the Cbl adaptor protein, which is required for internalization and degradation of the EGFR (1,7,8,12,46). Escape of Cbl-mediated down-regulation is a common mechanism in cancer (47), and the efficacy of some tumorspecific anti-EGFR antibody therapies has been shown to be dependent on efficient targeting of the EGFR to Cbl-dependent ubiquitination and degradation (48). Uncoupling of the Cbl/EGFR interaction has been shown to occur by different mechanisms. Oncogenic Src kinase inhibits EGFR endocytosis through proteasomal degradation of Cbl (33). Thus, Cbl-mediated EGFR ubiquitination is impaired in Src-transformed cells, and EGFR endocytosis is inhibited (33). In a distinct pathway, activated Cdc42 has been shown to sequester Cbl through p85Cool-1/␤-Pix restricting the interaction of Cbl with the EGFR, and thereby preventing Cbl from catalyzing EGFR ubiquitination and degradation (15). Addi-FIGURE 6. Kinase-active Abl reduces Cbl/EGFR interaction and the accumulation of Cbl to the plasma membrane in response to EGF stimulation. A, EGFR-WT and HA-tagged Cbl were co-expressed in 293T cells, and recruitment of Cbl to the EGFR upon addition of 50 ng/ml EGF for 5 min at 37°C was observed in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of kinase-active Abl-PP. EGFR, Abl, and tubulin protein levels are shown as indicated. This result is representative of four independent experiments. B, COS7 cells were transfected with vector alone or kinase-active Abl-PP, and endogenous Cbl protein levels were analyzed by Western blot of whole cell lysates (WCL). Lysates were blotted for Cbl, Abl, and ␣-tubulin (loading control) as indicated. C, localization of endogenous Cbl to the plasma membrane upon EGF stimulation was analyzed by immunofluorescence in COS7 cells in the absence or presence of activated Abl-PP or kinase-inactive Abl-KM as indicated. Left panels show unstimulated cells, and right panels show cells stimulated with 50 ng/ml EGF for 30 s. Cbl is shown in red, and Abl transfected cells are shown in green. Arrowheads point to Cbl accumulation to the plasma membrane, and arrows correspond to cytoplasmic localization in the presence of EGF. D, recruitment of Cbl to the plasma membrane was analyzed by membrane fractionation at different time points after EGF stimulation in COS7 cells transfected with vector alone or activated Abl-PP. Cells were stimulated with EGF (50 ng/ml) at 37°C for the indicated times or for 1 h at 4°C. Protein levels for Cbl, transferrin receptor (TR), and Abl were analyzed by Western blotting as indicated. Cbl protein levels were quantified with Odyssey software.
tionally, Sprouty2 has also been shown to sequester Cbl away from the EGFR by binding directly to Cbl (16) or by binding to CIN85 and disrupting Cbl binding to the EGFR through the Cbl-CIN85 complex (11). Our data have revealed a novel Cbl-dependent mechanism for inhibiting EGFR down-regulation by the activated Abl kinase through the inhibition of Cbl accumulation at the plasma membrane, which correlates with marked inhibition of Cbl⅐EGFR complex formation. Interestingly, in contrast to Src, expression of activated Abl did not result in decreased levels of Cbl protein. It is likely that Abl-mediated phosphorylation disrupts or promotes the formation of distinct Cbl-containing protein complexes, which may account for the reduced accumulation of Cbl at the plasma membrane and decreased levels of Cbl⅐EGFR complexes. In this regard, oncogenic Abl kinases have been shown to phosphorylate Cbl on tyrosines 700 and 774 (49). However, the physiological consequences of these phosphorylations have not yet been determined.
In summary, our findings have revealed functional cross-talk between Abl and EGFR kinases, which may have a role in promoting cellular transformation. In support of this possibility is the finding that cooperation between Abl and the EGFR is required for transformation of NIH/3T3 fibroblasts and Rat-1 cells (50). Internalization-deficient mutant EGFRs show increased resistance to EGFR-specific inhibitors such as Iressa (AG1478), which fails to decrease phosphorylation of EGFR tyrosine 1173 (51). Together these data and our findings raise the possibility that a combination of Abl and EGFR kinase inhibitors might be used in the treatment of specific cancers expressing constitutively activated EGFRs that are resistant to ligand-induced internalization.