Cell Culture and Transfection
HeLa and HEK293T cells were grown in DMEM (Sigma, St. Louis, MO) supplemented with 10% FBS (Invitrogen, Carlsbad, CA) and 4 mM l-glutamine (Invitrogen). For HEK293T cells, collagen (type I)-coated dishes (Asahi Techno Glass, Chiba, Japan) were used. CHO cells were grown in F-12 nutrient mixture (Invitrogen) with 10% FBS. In the following, we refer to DMEM containing 4 mM l-glutamine or F-12 as the serum-free medium. For transfection, the cells were incubated in Opti-MEM I reduced-serum medium (Invitrogen) containing plasmid DNA, Lipofectamine (Invitrogen), and Plus reagent (Invitrogen) for 2 h. Subsequently, the medium was replaced by the serum-containing DMEM or F-12, and the cells were incubated for 12–18 h before the experiment. Overexpression of EGFR tends to significantly delay receptor degradation. Therefore, in , Plus reagent was omitted to avoid high-level expression of the receptor.
Figure 6. Inhibition of polyubiquitination reduces EGFR-Hrs interaction and delays the receptor degradation. (A) Schematic representation of the ubiquitin–GFP fusion protein. Wild-type or lysine-less ubiquitin was fused to GFP via a 13-amino acid linker (more ...)
Plasmids expressing human EGFR, c-Cbl, and c-Cbl(C381A) were kindly provided by Minsoo Kim and Tadashi Yamamoto (The University of Tokyo, Tokyo, Japan). PCR-based mutagenesis was used to create EGFR(Y1045F). Plasmids expressing FLAG-E2s were generous gifts from Noriyuki Matsuda and Keiji Tanaka (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). Plasmids containing wild-type and mutant ubiquitin sequences were kindly provided by Hiroshi Ohno (RIKEN Research Center for Allergy and Immunology, Kanagawa, Japan), and the ubiquitin sequences were inserted into pEGFP-N1 (Clontech Laboratories, Mountain View, CA) to express the ubiquitin-green fluorescent protein (GFP) fusion proteins.
Reagents and Antibodies
All Alexa fluor dyes-conjugated reagents and murine natural EGF were purchased from Invitrogen. The EGFR kinase inhibitor AG1478 was from Calbiochem (San Diego, CA). Bovine ubiquitin was from Sigma (St. Louis, MO). Recombinant human E1, UbcH5C, UbcH7, ubiquitin, and ubiquitin(no lysines) were from Boston Biochem (Cambridge, MA). For fluorescent microscopy, the following antibodies were used. Mouse anti-EEA1 and mouse anti-c-Cbl were from BD Biosciences (San Jose, CA) and rabbit anti-FLAG was from Sigma. For immunoblotting, the following antibodies were used. Goat anti-Ubc4 and rabbit anti-Cbl were from Santa Cruz Biotechnology (Santa Cruz, CA), rabbit anti-UbcH7 was from Chemicon (Temecula, CA), goat anti-GST was from GE Healthcare (Chalfont St. Giles, Buckinghamshire, United Kingdom), rabbit anti-ubiquitin was from DakoCytomation (Kyoto, Japan), HRP-conjugated mouse anti-phosphotyrosine PY20 was from BD Biosciences, FK1 and rabbit anti-GFP were from MBL (Nagoya, Japan), and mouse anti-α-tubulin was from Sigma. HRP-linked anti-mouse IgG, anti-rabbit IgG, HRP-labeled protein A, ECL, and ECL Plus and ECL Advance Western Blotting Detection System were from GE Healthcare. Affinity purified rabbit anti-Hrs (Raiborg et al., 2001
) was used for fluorescent microscopy and immunoblotting. Mouse anti-EGFR Ab-11 (Lab Vision, Fremont, CA) was used for the nondenaturing immunoprecipitation and sheep anti-EGFR (Fitzgerald, Concord, MA) for fluorescent microscopy, immunoblotting, and the denaturing immunoprecipitation.
In Vitro Ubiquitination Assay
A central region of wild-type or C381A c-Cbl (amino acids 359–447) containing the RING finger domain was fused to glutathione S-transferase (GST) using pGEX-6P-1 (GE Healthcare). Expression of the GST-fusion proteins in Escherichia coli BL21 was induced with 0.4 mM IPTG at 30°C for 3 h. The cells were lysed using B-PER Bacterial Protein Extraction Reagent (Pierce, Rockford, IL), and the lysates were incubated with glutathione Sepharose 4B (GE Healthcare) at 4°C for 2 h. The Sepharose beads were washed three times with PBS containing 0.1% Triton X-100. Proteins bound to the beads were eluted with elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione), and applied to PD-10 column (GE Healthcare) for buffer exchange. Aliquots in 50 mM Tris-HCl, pH 8.0, containing 50 mM NaCl, 1 mM DTT, and 10% glycerol were frozen with liquid nitrogen and stored at −80°C.
For autoubiquitination, the reaction mixture (66 μl) containing 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 2.5 mM MgCl2, 4 mM ATP, 10 μg of bovine ubiquitin, 100 ng of E1, 0.75 μg of E2 (UbcH5C or UbcH7), and 1 μg of GST-RING (wild-type or C381A) was incubated at 30°C for 2 h. The reaction was terminated by adding 15 μl of 6× SDS-PAGE sample buffer (without DTT) and 9 μl of 1 M DTT and heating at 95°C for 5 min. After centrifugation, the supernatant was subject to immunoblotting.
CHO or HeLa cells grown on glass coverslips were shifted to the serum-free medium and incubated for 2 h. Then, the cells were stimulated with 100 ng/ml EGF or 1 μg/ml Alexa 647-EGF in the serum-free medium. At the indicated time periods, the cells were washed three times with ice-cold PBS, fixed with 3% paraformaldehyde in PBS for 20 min, and quenched with 50 mM NH4Cl in PBS for 10 min. The cells were permeabilized with 0.2% saponin (ICN Biomedicals, Aurora, OH) in PBS for 10 min. Alternatively, the cells were first permeabilized with 0.05% saponin in PEM buffer (80 mM PIPES-KOH, pH 6.8, 5 mM EGTA, 1 mM MgCl2) on ice for 5 min and then fixed. The permeabilized cells were incubated in blocking solution (0.1% gelatin in PBS) and then stained with primary and secondary antibodies diluted with the blocking solution. The coverslips were mounted in Vectashield Hard Set Mounting Medium (Vector Laboratories, Burlingame, CA), and the cells were observed with an Olympus FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan). Colocalization of GFP-Cbl with Hrs or EEA1 was quantitated using FV1000 analysis software FV10-ASW.
Immunoprecipitation of EGFR
HEK293T or HeLa cells grown in 10-cm dishes were starved for serum for 2 h and stimulated with 100 ng/ml EGF in the serum-free medium for the indicated time periods. The cells were washed once with ice-cold PBS plus 10 mM N-ethylmaleimide (NEM), covered with 425 μl of lysis buffer (50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1% NP-40, 1 mM EDTA, 0.02% NaN3) containing 1 mM Na3VO4, 10 mM NEM, the Complete (EDTA-free) protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland), 10 μM lactacystin (Peptide Institute, Osaka, Japan), and 10 μM MG132 (Peptide Institute), scraped with a rubber policeman and lysed on ice for 30 min. The cell lysate was centrifuged at 1500 × g for 4 min, and 425 μl of the supernatant was collected. Part of the supernatant (66 μl) was withdrawn and used as the whole cell lysate, and the rest was used for nondenaturing immunoprecipitation. The lysate was mixed with 20 μl protein G Sepharose 4 Fast Flow (GE Healthcare) and rotated at 4°C for 30 min. The precleared supernatant corresponding to 1 mg (HeLa) or 2 mg (HEK293T) proteins was filled up to 300 μl, mixed with 10 μl mouse anti-EGFR Ab-11, and rotated at 4°C. After 2 h, 15 μl protein G Sepharose 4 Fast Flow was added and the microtube was rotated at 4°C for 2.5 h. The immune complexes were washed four times with the lysis buffer, resuspended in 100 μl 50 mM Tris-HCl, pH 6.8, plus 1% SDS, and heated at 95°C for 5 min. Part of the supernatant was used for immunoblotting to monitor coimmunoprecipitation. The rest, typically 55 μl, was subject to denaturing immunoprecipitation. It was diluted 10-fold with the lysis buffer containing 10 mM NEM and the protease inhibitor cocktail, mixed with 1.8 μl sheep anti-EGFR and 15 μl protein G Sepharose 4 Fast Flow, and rotated at 4°C overnight. The immune complexes were washed four times with the lysis buffer, resuspended in SDS-PAGE sample buffer, and heated at 95°C for 5 min. Immunoblotting images were acquired with the LAS-3000 imaging system (Fujifilm, Tokyo, Japan), and the band intensities were quantitated using Fujifilm Image Gauge Ver. 4.0 software.
Depletion of E2s
Ubc4/5 and UbcH7 were depleted by transfection with small-interfering RNA. Among the Ubc4/5 family proteins, Ubc4, UbcH5B, and UbcH5C were simultaneously depleted using sense sequence 5′-CAGUAAUGGCAGCAUUUGUTT-3′ and antisense sequence 5′-ACAAAUGCUGCCAUUACUGTT-3′. Note that, as reported previously (Saville et al., 2004
), the target sequence does not deplete UbcH5A. For UbcH7, sense sequence 5′-GGACCGUAAAAAAUUCUGUTT-3′ and antisense sequence 5′-ACAGAAUUUUUUACGGUCCTT-3′ were used according to the previous report (Verma et al., 2004
). As a control, the target sequence for luciferase was used: sense sequence 5′-CAUACGCGGAAUACUUCGATT-3′ and antisense sequence 5′-UCGAAGUAUUCCGAGUACGTT-3′. HeLa cells grown in a 10-cm dish (30–50% confluent) were transfected with 600 pmol of double-strand RNA using Lipofectamine RNAiMAX (Invitrogen). After 1-d incubation, the medium was changed to the serum-containing one, and the cells were further incubated for 1 d. Then, the cells were replated to 10-cm dishes and incubated for 1.5 d. The serum starvation, EGF stimulation, and immunoprecipitation were done as described above. The goat anti-Ubc4 antibody was precleared by absorption with GST-UbcH5A (Boston Biochem), as reported previously (Saville et al., 2004
), and then used for immunoblotting.