Antibodies and reagents
The following commercial antibodies were used: mouse monoclonal anti-ubiquitin (FK2; Affiniti, Devon, UK), mouse monoclonal anti-EEA1, mouse monoclonal anti-CHC and mouse monoclonal anti-GGA3 (BD Transduction Laboratories, San Jose, CA, USA), mouse monoclonal anti-human TGFβRII (R&D Systems Inc., Minneapolis, MN, USA), mouse monoclonal to α-adaptin (AP-6; Affinity Bioreagents, Golden, CO, USA), mouse monoclonal anti-CD63 (H5C6; BD Pharmigen, San Jose, CA, USA), mouse phosphotyrosine antibody (clone 4G10; Upstate Biotechnology, Lake Placid, NY, USA), rabbit anti-GFP (MBL International, Woburn, MA, USA), mouse monoclonal and rat monoclonal anti-EGF receptor (ab3103 and ICR10; Abcam, Cambridge, MA, USA). Rabbit polyclonal antibodies against p38, phospho-p38, p44/42, phospho-p44/42, phospho-JNK, EGF-receptor, phospho-EGF receptor (Tyr845), phospho-EGF receptor (Tyr1068) and phospho-EGF receptor (Tyr1086) were obtained from Cell Signaling Technology (Beverly, MA, USA). Rabbit polyclonal antibodies to µ1, µ2 and µ3 were the kind gift of JS. Bonifacino (NIH, Bethesda, MD, USA).
Anisomycin, EGF, SB203580, AG1478, MG132, bafilomycin A1, ubiquitin aldehyde, mammalian phosphatase inhibitor cocktail and NP40 were obtained from Sigma (St. Louis, MO, USA). Rhodamine-transferrin was supplied by Molecular Probes (Eugene, OR, USA).
Cloning and characterization of the EGFR-GFP chimera has been previously described (34
). Mutations of residues in the cytosolic tail of EGFR-GFP were introduced using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The GFP-tagged dynamin2 K44A construct was a gift from M. McNiven (Mayo Clinic, Rochester, MN, USA).
RNAi of CHC, GGA3 and the µ subunit of the AP2 complex was performed using siRNAs to the following human target sequences: UAAUCCAAUUCGAAGACCAAUU for CHC, GUGGAUGCCUUUCGGGUCA for µ2 and AAAAACGGCUUCCGCAUCCUC for GGA3 (Dharmacon Research, Lafayette, CO). Heterogeneous duplex siRNA pool against p38 MAP kinase and p38 MAP kinase β were obtained from Cell Signaling Technology and Dharmacon, respectively. For each siRNA, two consecutive transfections every 72 h were carried out in HeLa cells according to the manufacturer's instructions.
Transfection and immunofluorescence microscopy
HeLa cells were transfected with a plasmid encoding EGFR-GFP by using FuGENE-6 (Roche Diagnostics, Indianapolis, IN, USA). For immunofluorescence, transfected HeLa cells were grown on coverslips. Twenty-four hours after transfection, the cells were maintained in serum-free medium for 6–8 h, stimulated with EGF (100 ng/mL) or anisomycin (60 µm) for 15 min and fixed in methanol/acetone (1:1, v/v) for 10 min at −20 °C. Coverslips were mounted onto glass slides with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL, USA). Fluorescence images were acquired on an LSM 510 confocal microscope (Carl Zeiss, Thornwood, NY, USA).
To examine the co-localization of EGFR-GFP with endosomal markers, incubation with primary antibodies diluted in PBS, 0.1% (wt/vol) saponin and 0.1% BSA, was carried out for 1 h at room temperature. Unbound antibodies were removed by rinsing with PBS for 5 min, and cells were subsequently incubated with a secondary antibody (Cy3-conjugated donkey anti-rabbit or anti-mouse Ig) diluted in PBS, 0.1% (wt/vol) saponin and 0.1% BSA, for 30–60 min at room temperature. After a final rinse with PBS, coverslips were mounted onto glass slides with Fluoromount G and analyzed by confocal microscopy.
Treatments with SB203580 or AG1478 were carried out by incubation of the cells with the inhibitors for 30 min prior to anisomycin stimulation. Cells were exposed to UV radiation in a UV Stratalinker 2400 (Stratagene).
Immunoprecipitation and immunoblot
HeLa cells were starved for 8 h in serum-free medium and then stimulated with EGF (100 ng/mL) or anisomycin (60 µm) for 15 min. The cells were then washed with ice-cold PBS and lysed for 15 min in lysis buffer (25 mm Tris–Cl, pH 7.4, 150 mm NaCl, 5 mm ethylenediaminetetraacetic acid, 20 mm NaF, 0.5% NP40) supplemented with protease, tyrosine phosphatase and ubiquitin hydrolase inhibitors. The lysate was precleared by centrifugation and incubated with 5 µL of anti-GFP or a non-specific antibody and protein G-Sepharose (Amersham Biosciences, Piscataway, NJ). Immunoprecipitates were collected, washed six times with 1 mL of lysis buffer supplemented with phosphatase inhibitors and analyzed by SDS–PAGE under reducing conditions.
Immunoblotting was carried out according to standard procedures. Proteins were separated by SDS–PAGE and transferred to nitrocellulose. The membrane was then blocked with PBS/0.05% Tween-20/10% BSA and incubated with the various antibodies. Enhanced chemiluminescence reagent (Amersham Biosciences) was used for detection.
Degradation and MAP kinase activation assays
HeLa cells were serum starved for 8 h and stimulated with EGF (100 ng/mL) or anisomycin (60 µm) for the indicated times. For degradation assays, total cell lysates were subjected to immunoblotting with antibodies against EGFR. The amounts of EGFR were quantified at each time-point by using the public domain NIH Image program (1.6–2) and are represented as the percentage of remaining EGFR in comparison with unstimulated cells in the same experiment. The activation of p44/42, JNK and p38 was determined by Western blotting with antibodies specific for phosphorylated, activated forms of these kinases.
Quantitative internalization assay
HeLa cells were grown to 90% confluence. Following serum starvation for 4–5 h, the cells were harvested in Cellstripper™ solution (Mediatech, Inc., Herdon, VA), washed once with cold PBS and incubated with DMSO or SB203580 (10 μM) at 4 °C for 30 min. The cells were then incubated with EGF (100 ng/mL) or anisomycin (10 µm) for the indicated times at 37 °C, and then placed on ice to stop internalization. The cells were fixed with 2% paraformaldehyde, incubated with the FITC-conjugated anti-EGFR antibody for 45 min on ice, washed and analyzed by the FACS-Calibur using cellquest software (BD Bioscience, San Jose, CA). Amounts of EGFR remaining on the cell surface were defined as the specific fluorescence value, which was calculated after subtracting background (fluorescence of non-stained cells). Percent internalization was calculated where the specific mean fluorescence value of cells incubated at 37 °C with SB203580 was treated as 100%.