-pyrrolo[2,3-d]pyrimidin-7-yl}-2-hydroxycyclopentyl)methyl sulfamate, the compound that is MLN4924 (referred to throughout as such), was synthesized as described previously [28
Cell culture and transfection
U20S, HEK (human embryonic kidney)-293 and HeLa cells were grown in DMEM (Dulbecco's modified Eagle's medium; GIBCO) supplemented with 10% FBS (fetal bovine serum; GIBCO) and 100 units/ml penicillin/100 μg/ml streptomycin (Intvitrogen). CHO (Chinese-hamster ovary) TS41 cells were grown at 32°C in F-12K (GIBCO) supplemented with 10% FBS (Gibco) and penicillin/streptomycin (Invitrogen). MG132 and bortezomib were from Sigma–Aldrich and LC Laboratories respectively. All plasmid transfections were performed using Lipofectamine™ LTX PLUS (Invitrogen), following the manufacturer's instructions. For pCMV5-NEDD8/NEDD8ΔGG overexpressions, unless stated otherwise, 1 μg of plasmid was transfected per six-well plate, containing approximately 1.5×105 cells. For HA (haemagglutinin)–UBE1 immunoprecipitations, approximately 1×106 cells per 100 mm dish were co-transfected with 5 μg of pCMV HA-UBE1WT/HA-UBE1C632S (where WT is wild-type) and 5 μg of untagged pCMV5-NEDD8. All UBE1 and UBE1L2 siRNA (small interfering RNA) transfections were performed using Dharmacon ON-TARGET plus SMARTpool siRNA oligos at a final concentration of 20 nM and Lipofectamine™ RNAiMAX (Invitrogen), according to the manufacturer's instructions. All UBE1 and UBA6 (UBE1L2) knockdowns were performed 48 h prior to plasmid transfections, and for a total of 72 h.
In vitro NEDD8/ubiquitin activation/transfer assays
His6–UBE1 (6 pmol; Ubiquigent) was added to 20 μl of reaction buffer (50 mM Hepes, pH 7.5, 100 mM MgCl2 and 2 mM MgATP) containing 2.5 μM ubiquitin E2 (Ubiquigent). For E1 activation assays, E2 enzymes were left out. The reaction was started by addition of either 2 nmol of purified ubiquitin or 2 nmol of purified NEDD8, incubated at 30°C and stopped after 30 min by addition of reducing or non-reducing 3× Laemmli buffer.
HA immunoprecipitations were performed under denaturing conditions. Cells were lysed in 1% SDS, 5 mM EDTA, 10 mM iodoacetamide, 15 units/ml DNase I (Qiagen) and 1× Complete™ protease inhibitor cocktail (Roche). Lysis was performed on ice, followed by immediate heating of the samples to 95°C, after which lysates were diluted 10-fold with 20 mM Tris/HCl, pH 8, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM EDTA, 10 mM iodoacetamide and 1× Complete™ protease inhibitor cocktail. DNA was fragmented by passing lysates through a syringe. Lysates were precleared for 1 h rotating at 4°C with control agarose beads (Pierce, 26150), after which lysates were incubated with anti-HA beads (Sigma–Aldrich, A2095). Immunprecipitation was performed at 4°C for 1 h with rotation. Beads were washed, and bound proteins were eluted by addition of low pH buffer (50 mM glycine, pH 2.8). Eluted samples were split into two, and either reducing or non-reducing 3× Laemmli buffer supplemented with 8 M urea was added 1:1.
Western blotting and antibodies
Anti-NEDD8 antibodies used were: rabbit ALX-210-194 (Alexis Biochemical), rabbit MIL-10 (Millennium Pharmaceuticals), rabbit #2745 (Cell Signaling Technology), rabbit #2754 (Cell Signaling Technology), rabbit BML-PW9340 (ENZO Lifesciences) and rabbit A-812 (BostonBiochem). Anti-ubiquitin antibodies used were: mouse P4D1 (Santa Cruz Biotechnology), mouse MAB1510 (Millipore) and rabbit Z0458 (DakoCytomation). All of the above antibodies were used at a dilution of 1:3000, with the exception of MIL-10, which was used at 1:10000. Rabbit anti-UBE1 Ab34711 (Abcam), anti-UBE1L2 antibody (Abcam) and rabbit anti-actin Ab1801-100 were all used at 1:3000. Mouse anti-HA HA.11 16B12 (Covance) and anti-HA HRP (horseradish peroxidase) clone HA-7 (Sigma–Aldrich) were used at 1:2000. Anti-FLAG HRP (Sigma–Aldrich) was used at 1:2000. The goat anti-mouse 170-5046 and goat anti-rabbit 170-5047 secondary antibodies (Bio-Rad) were used at 1:5000. Western blotting was performed using Amersham™ Hybond™ ECL (enhanced chemiluminescence) nitrocellulose membranes (GE Healthcare) with 5% non-fat dried skimmed milk powder/2% BSA blocking agent and standard laboratory techniques.
PPi exchange and kinetic thioester experiments
P]ATP were obtained from PerkinElmer. Bovine ubiquitin was purchased from Sigma (U6253). NEDD8 was generated in an untagged form in a pDEST vector and was expressed in Escherichia coli
. N-terminal His6
-tagged E1 enzymes (UBE1 and NAE) were expressed in Sf9 insect cells and purified as described previously [30
]. Mouse monoclonal anti-FLAG M2 antibody was purchased from Sigma (F1804). Alexa Fluor® 680-labelled secondary antibodies were purchased from Invitrogen (A21109 for anti-rabbit antibody and A21057 for anti-mouse antibody). The ATP–PPi
exchange assays were performed using an improved protocol developed by Bruzzese et al. [30
]. The final reaction mixture of 50 μl contained 2.5–20 nM UBE1 or NAE, 0.6 μM ubiquitin or 0.2 μM NEDD8 for UBE1 reactions, 0.16 μM NEDD8 for NAE reactions, 100 μM ATP, 0.5 mM PPi
, ~50 c.p.m./pmol [32
, 10 mM MgCl2
and 1 mM TCEP [tris-(2-carboxyethyl)phosphine], in 1× E1 buffer (50 mM Hepes, pH 7.5, 25 mM NaCl, 0.05% BSA and 0.01% Tween 20). Reactions were initiated by adding E1 enzymes and the reaction mixtures were incubated at 37°C. At various time points, the reaction was quenched with 5% (w/v) TCA (trichloroacetic acid) containing 10 mM PPi
. The quenched reaction mixtures were transferred to a Schleicher & Schuell Minifold-I Dot-Blot System (Whatman; 10447900) with activated charcoal filter paper (Whatman; 10320163) pre-rinsed in 2% TCA and 10 mM PPi
, which was then washed for 3×5 min in the same solution. The charcoal filter paper blots were air dried, exposed to an imaging plate for 1 h and visualized using a phosphorimager (Fujifilm FLA-7000; GE Healthcare). Samples from each time point were analysed in duplicate. The spot intensities were converted into the quantity of ATP using a standard curve generated with [α-32
Rate of UBE1-S–NEDD8 and NAE (UBA3) thioester formation
For UBE1, the reaction mixture contained 1.5 μM UBE1, 1 μM NEDD8, 100 μM ATP in 10 mM MgCl2 and 50 mM Hepes, pH 7.5, and was incubated at 37°C. For NAE, the reaction mixture contained 1 μM NAE, 5 μM NEDD8, 25 μM ATP in 5 mM MgCl2 and 20 mM Hepes, pH 7.5. At designated time points, the reaction mixture was quenched with LDS (lithium dodecyl sulfate) sample loading buffer (Invitrogen; the 4× solution contained 40% glycerol, 4%, LDS 4% Ficoll-400, 0.8 M triethanolamine-Cl pH 7.6, 0.025% Phenol Red, 0.025% Coomassie Blue G250 and 2 mM EDTA). The quenched samples were analysed by SDS/PAGE under non-reducing conditions. Samples from each time point were analysed in duplicate. The SDS/PAGE gels were transferred to a 0.2 μm-pore-size Immobilon-P PVDF membrane (Millipore; ISEQ20200) and probed with rabbit anti-NEDD8 or mouse anti-FLAG antibodies. Alexa Fluor® 680-labelled secondary antibody was then used and the intensities of protein bands were quantified on a Li-Cor Odyssey Imaging System (Li-Cor Biosciences).
Competition assay to estimate the affinity of NEDD8 for UBE1
In the competition experiment using the ATP–PPi exchange assay, the reaction mixtures containing 0.5 nM UBE1, 0.6 μM N-terminal FLAG-tagged ubiquitin, 1 mM ATP, 0.5 mM PPi (~100 c.p.m./pmol [32P]PPi) and various amounts of NEDD8 in 1× E1 buffer were incubated at 37°C for 30 min, quenched and analysed as described above. To monitor competition of NEDD8 in UBE1-S–ubiquitin thioester assays, reaction mixtures containing 50 nM UBE1, 0.8 μM ubiquitin, 1 mM ATP, 10 mM MgCl2 and various amounts of NEDD8 (up to 184 μM) in 1× E1 buffer were incubated at 37°C for 15 min and quenched with the LDS loading buffer. The UBE1 thioester levels were analysed using non-reducing SDS/PAGE and Western blot analysis Li-Cor imaging as described above.
Quantification of NEDD8 and ubiquitin
For NEDD8 quantifications, U2OS cells were transfected with the indicated plasmids 24 h prior to harvest. For ubiquitin and NEDD8, cells were treated with 30 μM MG132 or 3 μM MLN4924 for 4 h as indicated. Cells were harvested by trypsination and counted, followed by immediate lysis in reducing or non-reducing Laemmli buffer, supplemented with 8 M urea. A minimum of three independent repeats was performed for all points. Quantification of NEDD8 and ubiquitin was performed by Western blot analysis using standard curves based on known amounts of purified protein. Data acquisition and densitometric analysis was performed using the ChemiDoc™ XRS system and Image Lab software respectively (Bio-Rad). Estimations of NEDD8 and ubiquitin levels were based on interpolation within linear ranges.
Saccharomyces cerevisiae experiments and strains
For overexpression of Rub1, pRD54-HA-Rub1 or pRD54-HARub1ΔGG was transformed into S. cerevisiae S288c using one-step transformation, and expression was induced by addition of 2% final concentration galactose. Extracts were prepared using the TCA precipitation protocol, and resuspended in 3× Laemmli buffer supplemented with 8 M urea. The yeast deletion strains ubc12Δ, rub1Δ and uba3Δ were obtained from the Euroscarf haploid knockout collection. Double deletion mutants were generated by PCR-based knockout methods. Strain uba1-204 and the isogenic WT were from Professor Raymond Deshaies (Division of Biology, California Institute of Technology, Pasadena, CA, U.S.A.). The TRP1 N-end rule reporter strains were obtained from Professor Daniel Finley (Department of Cell Biology, Harvard Medical School, Boston, MA, U.S.A.). Growth assays were performed by serial dilution and spotting on to tryptophan-deficient glucose or galactose agar plates as indicated.