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Essential Role of c-Cbl in Amphiregulin-Induced Recycling and Signaling of the Endogenous Epidermal Growth Factor Receptor† Aleksander Baldys,‡,§ Monika Go¨oz,‡,§ Thomas A. Morinelli,‡,§ Mi-Hye Lee,§,| John R. Raymond, Jr.,‡ Louis M. Luttrell,§,| and John R. Raymond, Sr.*,‡,§ Nephrology and Endocrinology DiVisions, Department of Medicine, Medical UniVersity of South Carolina, and Medical and Research SerVices, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425 ReceiVed September 16, 2008; ReVised Manuscript ReceiVed December 7, 2008
ABSTRACT:
The intracellular processing of the epidermal growth factor receptor (EGFR) induced by epidermal growth factor (EGF) and transforming growth factor-R (TGF-R) has been studied meticulously, with the former resulting in EGFR degradation and the latter in EGFR recycling to the plasma membrane. However, little is known about how other EGF family growth factors affect the trafficking of the EGFR. Additionally, although both EGF and TGF-R have been shown to effectively induce initial c-Cbl (ubiquitin ligase)-mediated ubiquitination of the EGFR, limited information is available regarding the role of c-Cbl in the trafficking and signaling of recycling EGFR. Thus, in this study, we investigated the roles of c-Cbl in endogenous EGFR trafficking and signaling after stimulation with amphiregulin (AR). We demonstrated that a physiological concentration of AR induced recycling of the endogenous EGFR to the plasma membrane, which correlated closely with transient association of the EGFR with c-Cbl and transient EGFR ubiquitination. Most importantly, we used c-Cbl small interfering RNA (siRNA) duplexes and a c-Cbl dominant negative mutant to show that c-Cbl is critical for the efficient transition of the EGFR from early endosomes to a recycling pathway and that c-Cbl regulates the duration of extracellular signalregulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) phosphorylation. These data support novel functions of c-Cbl in mediating recycling of EGF receptors to the plasma membrane, as well as in mediating the duration of activation (transient vs sustained) of ERK1/2 MAPK phosphorylation. The epidermal growth factor receptor (EGFR)1 belongs to a family of cell surface receptor tyrosine kinases, which includes four ErbB members, i.e., EGFR/ErbB1, HER2/ ErbB2, HER3/ErbB3, and HER4/ErbB4 (reviewed in ref 1). Many different growth factors can serve as ligands for the EGFR, and these include epidermal growth factor (EGF), transforming growth factor-R (TGF-R), heparin-binding EGF-like growth factor (HB-EGF), betacellulin (BTC), amphiregulin (AR), epiregulin (EPR), and epigen (EPG). All † This work was supported by Department of Veterans Affairs Merit and Research Enhancement Award Program grants (to J. R. Raymond, Sr.), by National Institutes of Health Grants DK052448 and GM063909, by an American Heart Association (Mid-Atlantic) fellowship (to A.B.), and by a laboratory endowment jointly supported by the Medical University of South Carolina, Division of Nephrology, and Dialysis Clinics, Inc. (to J. R. Raymond, Sr.). * To whom correspondence should be addressed: Room 213, Colcock Hall, 179 Ashley Ave., Charleston, SC 29425. Phone: (843) 792-3031. Fax: (843) 792-5110. E-mail:
[email protected]. ‡ Nephrology Division, Department of Medicine, Medical University of South Carolina. § Ralph H. Johnson Veterans Affairs Medical Center. | Endocrinology, Department of Medicine, Medical University of South Carolina. 1 Abbreviations: AR, amphiregulin; BTC, betacellulin; EEA1, early endosome antigen 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; EPG, epigen; EPR, epiregulin; ERK1/2 MAPK, extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase; HEK293, human embryonic kidney 293; HB-EGF, heparinbound EGF-like growth factor; Hrs, hepatocyte growth factor-regulated tyrosine kinase substrate; LAMP, lysosome-associated membrane protein; SE, standard error; TGF-R, transforming growth factor-R.
EGFR ligands are synthesized as membrane proteins and are subsequently released from the cell surface by regulated proteolysis. However, little is known about what dictates the cleavage and shedding of different EGFR ligands or, most importantly, about the physiological and pathological relevance of the different cognate ligands. To date, how different EGFR ligands could serve distinct functions despite their shared interactions with the same receptor remains an enigma. Ligand binding to the EGFRs causes the formation of homo- and heterodimers, a process that subsequently induces autophosphorylation through activation of the EGFR tyrosine kinase activity. Following activation, the EGFR undergoes internalization and endocytic trafficking. After endocytosis, some receptors recycle from endosomes back to the plasma membrane, whereas others enter the degradative pathway to late endosomes and lysosomes, a process that results in receptor downregulation. In that regard, it is well established that EGF, but not TGF-R, triggers efficient degradation of the EGF receptors (2, 3). A recent report (4) also demonstrated that AR does not induce significant EGFR degradation. There have been significant advances in the understanding of how receptor trafficking and signaling are functionally interrelated (5), yet this relationship still remains obscure. The signaling of the activated EGFR involves numerous downstream pathways, including mitogen-activated protein kinases, phosphatidylinositol 3-kinase, c-Src, and phospholipase C γ/protein kinase C. These complex signal trans-
10.1021/bi801771g CCC: $40.75 2009 American Chemical Society Published on Web 01/27/2009
c-Cbl in AR-Induced EGFR Trafficking and Signaling duction cascades modulate cell proliferation, differentiation, adhesion, migration, survival, and death. Whereas EGFR signaling is crucial for many normal cellular processes, aberrant EGFR activation has been implicated in the pathophysiology of hyperproliferative diseases such as cancer. The mammalian Cbl proteins constitute a highly conserved family of three ubiquitin ligases, known as c-Cbl, Cbl-b, and Cbl-c (reviewed in ref 6). In recent years, Cbl has emerged as a critical player in regulating EGFR endocytic trafficking (7, 8). Numerous studies have provided direct evidence of the role of EGF-induced, Cbl-mediated, sustained EGFR ubiquitination in receptor targeting to lysosomes (3, 9-15). Importantly, although TGF-R has been shown to induce transient association of the EGFR with Cbl and receptor ubiquitination (3), there have been no reports so far in the literature addressing possible roles of Cbl in receptor recycling. The role of c-Cbl as a regulator of signal transduction, and consequently cell function and development, is now wellestablished (16). Evidence suggests that dysregulation and/ or disruption of the function of c-Cbl contributes to the development of many pathological conditions, including immunological and malignant diseases. The role of c-Cbl in signaling is thought to be based largely on its ubiquitin ligase activity, but many cellular events are dependent on its function as an adaptor molecule (16). Others previously have shown that EGF and TGF-R induce differential fates of the internalized EGFR, with the former resulting in EGFR degradation and the latter in EGFR recycling. However, although c-Cbl has been implicated in the regulation of EGFR degradation, possible roles for c-Cbl in EGFR recycling have not yet been addressed. Therefore, in this study, we examine the roles of c-Cbl in ligand-specific EGFR trafficking and signaling. Concentrating on two members of the EGFR ligand family, i.e., EGF and AR, we show that AR and EGF induced similar patterns of shortterm EGFR and c-Cbl phosphorylation, physical association of c-Cbl with the EGFR, and EGFR ubiquitination; however, as previously reported for TGF-R (3), the effects of AR are much more transient than those of EGF. Most importantly, our new data implicate c-Cbl in the active sorting of the EGFR to recycling endosomes. We also show that c-Cbl regulates the duration of AR-induced extracellular signalregulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) activation. Taken together, our results shed some light on the new, unexplored aspects of specialized endocytic sorting to recycling pathways. EXPERIMENTAL PROCEDURES Materials. Human embryonic kidney (HEK293) cells were purchased from American Type Culture Collection (Manassas, VA). Human recombinant EGF, human recombinant AR, chloroquine, monensin, and all other biochemical reagents were purchased from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies were from Invitrogen (Carlsbad, CA). Sheep polyclonal anti-EGFR and anti-phosphotyrosine (clone 4G10) antibodies were from Upstate Millipore (Billerica, MA). Rabbit polyclonal anti-Rab11 was from Zymed Laboratories (South San Francisco, CA). Mouse monoclonal anti-early endosome antigen 1 (EEA1) (clone 14) and mouse mono-
Biochemistry, Vol. 48, No. 7, 2009 1463 clonal anti-c-Cbl (clone 17) were from BD Transduction Laboratories (Franklin Lakes, NJ). Mouse monoclonal antilysosome-associated membrane protein (LAMP) (clone H4A3) was from BD Pharmingen (San Diego, CA). Mouse monoclonal anti-Cbl-b (clone G-1) was from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antiubiquitin (clone FK2) was from Biomol (Plymouth Meeting, PA). Rabbit anti-phospho-ERK1/2, mouse anti-ERK1/2, and rabbit anti-phospho-EGFR (Tyr-1173) antibodies were from Cell Signaling Technologies (Danvers, MA). Mouse monoclonal anti-β-actin (clone AC-15) antibody was from SigmaAldrich. Peroxidase-labeled secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA) and Rockland Immunochemicals (Gilbertsville, PA). SDSPAGE molecular mass markers were from Bio-Rad (Hercules, CA). Cell Culture, RNA Interference Experiments, and DNA Transfection. HEK293 cells were grown in Eagle’s minimum essential medium (MEM) supplemented with 10% (v/v) heatinactivated fetal bovine serum (Invitrogen Gibco, Carlsbad, CA) at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Cells were grown to ∼75% confluence following which they were placed in serum-deprived binding medium (MEM supplemented with 0.1% bovine serum albumin) for 24 h prior to treatments. For synchronized ligand pulse experiments, cells at 4 °C were incubated with agonists for 45 min in serum-deprived binding medium containing 20 mM HEPES (pH 7.4). Then, the cells were rinsed with icecold PBS to remove unbound ligand, following which the bound ligand stimulated the EGFR upon exposure to prewarmed ligand-free medium at 37 °C. A mixture of four SMARTselection-designed siRNAs targeting one gene (Thermo Fisher Scientific Dharmacon, Inc., Lafayette, CO) was transfected using oligofectamine (Invitrogen) reagent according to the manufacturer’s instructions. A pool of four siGenome nontargeting siRNAs, designated as scrambled (SCR) siRNA, was used as a control; 72 h following transfection, cell lysates were assayed for silencing effectiveness by Western blotting and immunofluorescence staining. The expression constructs pcDNA3GFP-Cbl-WT and pcDNA3GFP-Cbl-N have been described previously and were kindly provided by H. Band (17). HEK293 cells were transiently transfected with the constructs described above using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). Western Blotting. After treatments, cells were rinsed briefly with PBS and extracted with RIPA buffer (150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1% NP-40, and 0.1% SDS in PBS) containing protease inhibitors. Cells subsequently were sonicated, and protein concentrations were determined by the BCA assay (Pierce, Rockford, IL). Equal amounts of proteins were separated by SDS-PAGE on 4 to 12% polyacrylamide gels (Invitrogen), transferred to PVDF membranes, and blocked with 5% milk in PBS for 1 h at room temperature. Following several washes with PBS containing 0.1% Tween, the membranes were incubated with the appropriate dilutions of primary and peroxidaseconjugated secondary antibodies (as directed by the manufacturer) in blocking solution. Immunoblotted proteins were detected using ECL reagents (GE Healthcare Amersham Biosciences, Piscataway, NJ).
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Biotinylation of Cell Surface Proteins. HEK293 cells grown on 100 mm dishes were washed one time with icecold PBS and incubated with 0.5 mg/mL sulfo-NHS-biotin (Pierce) for 30 min at 4 °C to label surface proteins. Cells then were washed with 15 mM glycine to quench excess, unreacted biotin. After the indicated treatments, cells were rinsed briefly with ice-cold PBS and extracted with Triton lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, and 5 mM EDTA] supplemented with protease inhibitor cocktail III (EMD Calbiochem, San Diego, CA), 1 mM PMSF, and phosphatase inhibitors [HALT phosphatase inhibitor cocktail (Pierce)]. Equal amounts of proteins (0.5 mg) were precleared by incubation for 30 min at 4 °C with 30 µL of protein A/G Agarose beads (Santa Cruz Biotechnology). After a brief centrifugation, the supernatants were removed and incubated overnight at 4 °C with 50 µL of streptavidin-agarose beads (Novagen, Madison, WI). The samples then were centrifuged and washed three times with 1 mL of Triton lysis buffer. Proteins were eluted from the beads using Laemmli sample buffer. Samples subsequently were analyzed by SDS-PAGE and Western blotting. Immunoprecipitation. After the indicated treatments, HEK293 cells grown in 150 mm dishes were scraped into ice-cold PBS and centrifuged at 200g. Pellets were lysed in 1 mL of Triton lysis buffer supplemented with protease and phosphatase inhibitors, as described above. Equal amounts of proteins (1.5 mg) were precleared by incubation for 30 min at 4 °C with 30 µL of protein A/G Sepharose beads. After a brief centrifugation, the supernatants were removed and incubated overnight at 4 °C with either 8 µg of antiEGFR or 13 µg of anti-Cbl antibodies. Immunoprecipitates were captured with 50 µL of protein A/G beads at 4 °C for 1 h. The samples were then centrifuged and washed three times with 1 mL of Triton lysis buffer. Proteins were eluted from the beads using Laemmli sample buffer. Samples were subsequently analyzed by SDS-PAGE and Western blotting. Immunofluorescence Staining and Confocal Microscopy. Cells were grown on 35 mm lysine-coated, glass-bottom culture dishes (MatTek Corp., Ashland, MA). After treatments, cells were fixed with freshly prepared 3.7% paraformaldehyde in PBS for 15 min at room temperature. Subsequently, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 5 min, following which nonspecific binding sites were blocked with 3% normal serum (Santa Cruz Biotechnology) in PBS for 1 h. Incubations with the appropriate dilutions of primary and Alexa Fluorconjugated secondary antibodies (as directed by the manufacturer) were performed in blocking solution. Confocal microscopy was performed using a Zeiss LSM 510 META laser scanning microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with a 60× objective, using the following laser wavelengths: excitation at 488 nm and emission at 505-530 nm; excitation at 543 nm and emission at 560-615 nm. Quantifications of the colocalization coefficients, derived from measured pixel overlaps between the EGFR and EEA1, Rab11, and LAMP, were performed using Zeiss LSM 510 colocalization analysis software. The mean values were averaged from at least three independent single-cell images. Receptor Recycling Assay. HEK293 cells grown in sixwell plates were incubated with 100 ng/mL AR at 4 °C for 45 min in serum-deprived binding medium containing 20 mM HEPES (pH 7.4). Then, the cells were rinsed with ice-
Baldys et al.
FIGURE 1: Differential fates of the EGFR induced by EGFR ligands. Serum-deprived HEK293 cells were treated at 37 °C with vehicle (NT), 100 ng/mL EGF, HB-EGF, BTC, TGF-R, AR, or EPR for 180 min, extracted with RIPA buffer, and subsequently immunoblotted with anti-EGFR and anti-β-actin antibodies. Data shown are representative of three independent experiments. Results are means ( SE (n ) 3). Asterisks indicate a p of
