Constructs and transfection
Full-length cDNA of RNF122
(GenBank accession no. NM_024787.2
) was obtained and pEGFP-N1-RNF122 was constructed according to previously described methods [10
](Wang, Shi et al. 2006). RNF122-myc
was amplified and then ligated into pcDNA.3.1/myc-His(-)B (Invitrogen, USA). The first or the second cysteine residue of the RING finger was substituted with an alanine residue by polymerase chain reaction (PCR)-based site-directed mutagenesis, which resulted in the formation of products designated as RNF122C92A-myc or RNF122C95A-myc, respectively. We get RNF122-ΔTM-GFP and CAML-FLAG by inserting RNF122-ΔTM and CAML into the pEGFP-N1 and pFLAG-CMV2 vectors (Clontech, USA), respectively, using a PCR-based method. The bacterially expressed GST-RNF122ΔTM or GST-mutRNF122ΔTM fusion proteins (GST; glutathione S-transferase) were obtained using PCR fragments encoding residues 61-155 of human RNF122 or RNF122-C92A-myc. The PCR primers contained Xho
I and Bam
HI sites. The resulting PCR fragment was inserted into the pGEX-4T-1 vector (GE Healthcare, USA). All the insertions were confirmed by DNA sequencing.
DNA transfection was performed using VigoFect (Vigorous, China), a non-liposomal cationic formula, according to the manufacturer's instructions.
E. coli strain BL-21(DE3) (Novagen, Madison, WI) were cultured overnight at 37°C and induced with 0.4 mM isopropyl-1-thio-β-D-galactopyranoside for 1 h at room temperature (RT). The bacterial pellets were resuspended in a solution containing 50 mM Tris (pH 7.4), 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 5 mM dithiothreitol (DTT), and 2 mM phenylmethylsulfonyl fluoride (PMSF) (sonication buffer), and lysed by probe sonication using 4 mL of sonication buffer per 100 mL of bacterial culture. The sonicate was clarified by centrifugation at 4°C for 15 min at 18,000 × g, divided into aliquots, and stored at -70°C. To estimate the level of GST fusion proteins expressed, the sonicates were incubated with glutathione-Sepharose (GS) beads, washed, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); subsequently, the gel was stained with Coomassie Brilliant Blue R-250.
Cell lines and reagents
Human embryonic kidney cell line HEK293T and human cervix carcinoma cell line HeLa were obtained from the American Type Culture Collection (Manassas, VA). HEK293T and HeLa cells were cultured (37°C, 5% CO2 humidified atmosphere) in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) and supplemented with 2 mM L-glutamine (Invitrogen, Carlsbad, CA). Monoclonal mouse antibodies against β-actin, c-myc, and FLAG were purchased from Sigma (Sigma-Aldrich, St Louis, MO); horseradish peroxidase (HRP)-streptavidin and HRP-myc antibodies, from Upstate (USA); MG132, from Sigma (Sigma-Aldrich, St Louis, MO); tunicamycin, from Alexis (USA); cycloheximide, from Calbiochem (USA); and a ubiquitin-conjugating enzyme kit (mammalian) from Biomol (USA).
Immunoprecipitation and immunoblotting
HEK293T cells were transiently transfected with the epitope-tagged constructs using VigoFect, as described above. Forty-eight hours after transfection, the cells were washed 3 times in phosphate-buffered saline (PBS), harvested by scraping, and centrifuged (5 min, 500 × g). The pelleted cells were homogenized in a cell lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA-Na2, 1% NP-40; 1 μg/μL pepstain, 1 μg/μL aprotinin, 1 μg/μL leupeptin, 1 mmol/L DTT, and 0.0174 μg/μL PMSF]. The cell lysates thus obtained were centrifuged (20 min, 14,000 × g, 4°C), and the resulting supernatants were combined with 12.5 μL (packed gel) of either anti-c-Myc or anti-FLAG M2 affinity agarose (Sigma-Aldrich, St Louis, MO), mixed, and incubated in 4°C overnight. The immunoadsorbents were recovered by centrifugation (5 min, 700 × g) and washed 3 times with a high-salt buffer [500 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA-Na2, 1% NP-40, 1 μg/μL pepstain, 1 μg/μL aprotinin, 1 μg/μL leupeptin, 1 mmol/L DTT, and 0.0174 μg/μL PMSF], followed by re-suspension in cell lysis centrifugation (5 min, 700 × g), and the same procedure was repeated using PBS. The samples were then eluted using 60 μL of 2 × SDS loading buffer (Sigma-Aldrich, St Louis, MO) and analyzed by western blotting using anti-myc or anti-FLAG antibodies. Subsequently, the bands were detected using Ig-HRP-conjugated antibodies and an enhanced chemiluminescence (ECL) detection system (GE Healthcare, USA).
The HEK293T or HeLa cells were cotransfected with pEGFP-N1-RNF122 and CAML22-N1 CCAML-FLAG, grown on coverslips, fixed using 3.7% paraformaldehyde at RT for 30 min, and then permeabilized with 0.1% Triton X-100 at RT for a further 10 min. The cover slips were washed 3 times with PBS, treated with a blocking buffer (5% bovine serum albumin in PBS) for 30 min, and incubated with an anti-FLAG primary antibody for 2 h at RT. The cells were then washed 3 times (10 min each) in PBS and incubated with a secondary antibody for 1 h at 37°C. Rhodamine-conjugated goat anti-mouse immunoglobulin G (IgG; ZhongShan Biotechnology) was used as the secondary antibody.
In vivo ubiquitination
In order to analyze ubiquitination, we transfected the cells with a myc-tagged RNF122 vector and hemagglutinin (HA)-tagged ubiquitin, as described previously. Next, we harvested the cells and incubated them with 1 volume of 2% SDS in TBS (10 mM Tris-HCl, pH 8.0) at 95°C for 10 min in order to achieve lysis. Subsequently, we added 9 volumes of 1% Triton X-100 and 2 mM EDTA in TBS to the cell lysates, and incubated the lysates on ice for 1 h. The protein concentrations of the lysates were determined by the bicinchoninic acid (BCA) assay. For immunoprecipitation, 1 mg of protein was incubated with the anti-myc antibody at 4°C overnight; subsequently, this mixture was incubated with protein G beads for 2 h. The beads were washed twice with NaCl (1 M) in TBS supplemented with NP-40 (1%), β-mercaptoethanol (0.05%), and EDTA (1 mM). The proteins were loaded onto a 12% SDS-PAGE gel and analyzed by immunoblotting using the aforementioned antibodies and an ECL detection kit (GE Healthcare, USA).
In vitro ubiquitination
In vitro ubiquitination was performed according to the instructions provided with the ubiquitination kit (BioMol, USA). Briefly, the assays were carried out at 30°C in a 50-μL reaction mixture containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 U/mL of isopentenylpyrophosphate (IPP) (Sigma-Aldrich, St Louis, MO), 50 mM DTT, 50 mM EDTA, 1 mM Mg-ATP (pH 7.5), 100 nM E1 enzyme, 1 μM E2 enzyme, 10 μM GST-RNF122-ΔTM (or mutant GST-RNF122-ΔTM) fusion protein, and 2.5 μM biotin-labeled ubiquitin. After 30-60 min, the reactions were terminated by the addition of a non-reducing gel loading buffer, and subsequently the protein components were separated by SDS-PAGE and analyzed by western blotting using the HRP-streptavidin antibody.
Yeast Two-Hybrid Screening
Yeast Two-Hybrid Screening was performed at Shanghai Genomics (Shanghai, China). The two-hybrid screening system has been previously described [22
]. Briefly, the library consisted of 1500 known genes associated with cell apoptosis, cell proliferation, and cell cycles. Each ORF was amplified by PCR using Pfu DNA polymerase and cloned into pGBK-RC, a Gal4 DNA-binding domain-based bait vector, and pGAD-RC, a Gal4 activation domain-based prey vector, following the MATCHMAKER GAL4 Two-Hybrid System 3 and Libraries User Manual PT3247-1 (PR94575) protocol (Clontech, Mountain View, CA). Plasmids with inserts of expected sizes were confirmed by colony PCR followed by agarose gel electrophoresis. RNF122 bait vector and prey vectors were cotransfected in yeast Y190 and spread into SD/-T-L-H. Formed colonies were picked out, cracked in liquefacient nitrogen, and subsequently utilized in colony lift filter assays.