GlcNAcstatin was obtained from GlycoBioChem (Dundee, UK). Mouse IL-6 and TNFα Elisa kits were purchased from Peprotech, UK. Human IL-1β was from Sigma, murine IL-1α was from Peprotech and glutathione-sepharose was from GE Healthcare. Luciferase reporter assay kit was from Promega. Click-iT™ O-GlcNAc Enzymatic Labeling System and Click-iT Biotin Glycoprotein Detection Kit were from Invitrogen. Dynabeads® Protein G was from Invitrogen.
Full-length TAB1 was cloned and inserted into pGEX6P1 for recombinant protein production in E. coli
and pEBG6P for transfection in mammalian cells, as described previously (Cheung et al, 2004
). Mutations for putative O
-GlcNAc sites on TAB1 (S391A, S395A and S396A) were created following the Quick Change method (Stratagene) using KOD Hot start Polymerase (Novagen). Recombinant OGT was produced as described previously (Clarke et al, 2008
). GST–TAK1 was obtained from the Division of Signal Transduction Therapy DSTT in Dundee. Cp
OGA was produced as described previously (Rao et al, 2006
). For NFκB reporter assays, DNA encoding a ConA basal promoter incorporating three copies of the NFκB DNA response element (termed ‘3 × κB luciferase reporter construct') was provided by Professor Ron Hay, College of Life Sciences, University of Dundee (Rodriguez et al, 1999
). The pRL-TK vector driving Renilla
luciferase expression was from Promega.
The antibodies that recognize TAK1 phosphorylated at Thr187, total TAB1, TAB1 phosphorylated at Ser423 and TAB1 phosphorylated at Ser438 were used as described previously (Cheung et al, 2003
). Antibodies recognizing the active phosphorylated forms of ERK1/2, JNK1/2, p38α MAPK and total ERK1/2, JNK1/2 were from Cell Signalling Technologies. ExtrAvidin®-Peroxidase was from Sigma. For immunoblotting with the phospho-specific antibodies for TAK1 and TAB1, the antibodies were incubated at 3 μg/ml in the presence of 30 μg/ml of the unphosphorylated peptide immunogen to neutralize any antibodies that recognize the unphosphorylated form of the protein. The anti-O
-GlcNAc antibody CTD110.6 was purchased from Abcam. Secondary antibodies conjugated to horseradish peroxidase were from Pierce.
Generation of O-GlcNAc-specific antibody against S395 on TAB1
-GlcNAc peptide CVSVPYS(O-GlcNAc)SAQSTSKTS, corresponding to residues 389–403 of TAB1, was synthesized on a Liberty microwave-assisted peptide synthesizer (CEM) using MBHA Rink-amide low load resin (Novabiochem) with standard protocols of Fmoc SPS chemistry. The QS dipeptide was introduced as pseudoproline to suppress formation of truncated sequences detected in pilot experiments. 4Ac-GlcNAcSerFmoc was synthesized in-house following a published procedure (Saha and Schmidt, 1997
). After high peformance liquid chromatograpy (HPLC) purification of the peptide, it was conjugated to keyhole limpet haemocyanin and injected into rabbits. Antibodies from the serum were first precipitated with ammonium sulphate followed by a one-step purification by passing the resuspended antibody over a non-GlcNAcylated peptide column. The flowthrough from the column was collected and used for immunoblotting.
In vitro O-GlcNAc assay
In vitro o-GlcNAcylation of TAB1 (1 μM) was performed in 20 μl assay volumes containing 100 nM of OGT, reaction buffer (50 mM Tris–HCl, pH 7.5, 1 mM DTT, 12.5 mM MgCl2), and 1 mM UDP-GlcNAc. The reactions were incubated for 90 min at 37°C, stopped by adding 4 × SDS–PAGE sample buffer, resolved on SDS–PAGE, transferred to PVDF and probed with appropriate antibodies.
Enzymatic labelling of O-GlcNAc sites
GST–TAB1 was bound to glutathione-sepharose beads and was O-GlcNAcylated in vitro. The beads were washed with 10 mM HEPES (pH 7.9) and resuspended in reaction buffer (1% SDS, 20 mM HEPES) then 2 μl of GalT1 Y289L (Invitrogen) and 2 μl of 0.5 mM UDP-GalNAz (Invitrogen) were added in a final reaction volume of 50 μl. The reaction was performed overnight at 4°C. The beads were washed twice with reaction buffer to remove excess UDP-GalNAz. The samples were then reacted with biotin alkyne (Invitrogen) according to the manufacturer's instructions. The proteins were resolved by SDS–PAGE and transferred to PVDF. The blot was incubated with ExtrAvidin-Peroxidase and biotinylated TAB1 was visualized by ECL reaction.
O-GlcNAc site mapping of TAB1
For site mapping analysis of digested TAB1 protein, an Ultimate 3000RSLC nano-HPLC system (Dionex) with a 3D high capacity ion trap mass spectrometer with ETD capability (amaZon ETD; Bruker Daltonics) were used to perform HPLC electrospray MS/MS (ESI-MS/MS). Digested TAB1 samples were reconstituted in 0.1% formic acid, injected and concentrated onto a Dionex PepMap C18 nano-trap column, after a wash step with (2% acetonitrile, 0.1% formic acid (v/v)) peptides were resolved by a Dionex Acclaim PepMap C18 reverse phase column (75 μm i.d. × 15 cm × 2 μm) over a 25-min linear gradient from 4% to 50% buffer B (80% acetonitrile and 0.08% formic acid in Milli-Q water (v/v)), followed by another 2 min to 90% buffer B. The column was then washed by holding the 90% buffer B for 10 min before returning to initial conditions of 96% buffer A (0.1% formic acid in Milli-Q water (v/v)). A typical tandem mass spectrometric (MS/MS) cycle (Alternating CID/ETD) in amaZon ETD happens in the following order: (A) 1 MS full-range scan and precursor ion selection. (B) Accumulation of precursor ion (~10 ms) and fragmentation by CID. (C) (CID-MS/MS spectrum recorded) accumulation of the same precursor ion (~5 ms), which is allowed to mix and react with fluoranthene (50–100 ms; variable) for ETD fragmentation, and ETD-MS/MS spectrum acquired. Steps (B) and (C) are repeated automatically for each precursor ion. In this study, precursor ion selection was set up to five ions per cycle, excluding singly charged ions, with a dynamic exclusion time of 0.5 min for both CID and ETD. Helium gas was used as collision gas (60–200%), collision energy sweep with amplitude 1.0 parameters was used for CID fragmentation. For ETD experiments, the maximum output of ETD reagent ion (202 m
) was achieved with the following nCI source tuned parameters: reagent ion charge control target (ICC) 500 000, maximum emission current 4 μA and ionization energy of 75 eV, reactant remove cutoff
without supplemental energy activation.
Raw MS data were processed using software packages BioTool 3.2 SR1 and DataAnalysis 4.0 (Bruker Daltonic GMBH). In parallel, two database searches were performed for CID and ETD using Mascot v2.3 (Matrix Science Ltd), database used IPI-human 20110731 (91 522 sequences; 36 630 302 residues) with the following Mascot MS/MS ion search parameters: peptide charges considered 2+, 3+ and 4+ ions, peptide tolerance and fragment tolerance was set to ±0.5 Da, # of 13C=1, two missed cleavages allowed, trypsin as proteolysis enzyme, ESI-TRAP (for CID) and ETD-TRAP (for ETD) for instrument type. Carbamidomethyl (C) was used as fixed modification, where Deamidated (NQ), Oxidation (M), Phospho (ST) and HexNAc (ST) (+203.0794 Da) were set as variable modifications.
Cell culture, stimulation and lysis
IL-1R cells HEK293 (cells stably expressing the IL-1 receptor) and immortalized Tab1-deficient MEFs (Tab1−/− MEFs) were provided by Professor Philip Cohen, MRC Unit, University of Dundee. Cells were cultured in DMEM with 1 or 4.5 mg/ml glucose, containing 10% (v/v) heat inactivated fetal calf serum (FCS), and 2 mM L-glutamine. Prior to stimulation with human IL-1β in IL-1R cells or murine IL-1α in mouse cells, the medium was removed and replaced with DMEM from which FCS had been omitted. IL-1R cells were serum deprived for 16 h and MEFs for 6 h. GlcNAcstatin (1 μM) was added to cells during serum starvation, if required. For osmotic shock, cells were treated with either 0.25 or 0.5 M of NaCl by adding it into DMEM for 15 min before harvesting the cells.
Cells were lysed in ice-cold lysis buffer (50 mM Tris–HCl pH 7.5, 1 mM sodium orthovanadate 1 mM EDTA, 10 mM sodium β-glycerophosphate, 1 mM EGTA, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1% (v/v) Triton X-100, 0.27 M sucrose, 0.1% (v/v) 2-mercaptoethanol, 0.1 mM PMSF, 1 mM benzamidine and 5 μM leupeptin). Lysates were centrifuged at 13 000 g for 15 min at 4°C and the supernatants were used immediately or snap frozen in liquid nitrogen and stored in aliquots at −80°C until use. Protein concentrations were determined using the Bradford assay.
Immunoprecipitation and OGA treatment of TAB1
To immunoprecipitate endogenous TAB1, 1 mg of cell lysate was incubated for 2 h at 4°C with 10 μg of anti-TAB1 antibody coupled with 10 μl of Dynabeads Protein G. The immunoprecipitates were washed twice with 1 ml of lysis buffer containing 0.25 M NaCl, followed by two washes with 1 ml of 50 mM Tris/HCl, pH 7.5, 50 mM NaCl and 0.1 % (v/v) 2-mercaptoethanol and subjected to SDS–PAGE followed by western blotting.
For OGA treatment on immunoprecipitated O-GlcNAcylated TAB1, 3 mg of lysate was used for immunoprecipitation. The samples were divided in three equal volumes. One set of samples was treated with CpOGA (1 μM) for 30 min at room temperature, while the other two sets were left untreated at room temperature. The samples were subjected to SDS–PAGE and western blotting with CTD110.6 antibody. The third set of samples was incubated with CTD110.6 antibody, which was pre-incubated with 500 mM N-acetylglucosamine for 1 h.
IL-1R cells were transfected at 40–50% confluence using poly- ethyleneimine using DNA encoding GST (glutathione transferase)–TAB1, whereas MEFs were transfected at a density of 3 × 106 cells, with the Amaxa MEF2 kit according to the manufacturer's instructions.
NFκB reporter gene assay
For the measurement of NFκB-dependent luciferase gene expression, Tab1−/− MEFs (3 × 106) were co-transfected with either 3 μg of WT TAB1, S395A TAB1 or empty pEBG6 plasmid; 0.5 μg of DNA encoding the 3 × κB luciferase reporter construct; and 0.5 μg pRL-TK and plated 3 × 105 cells per well. After 24 h, the cells were stimulated with 10 ng/ml of IL-1α for 24 h and then the cells were lysed in Passive Lysis Buffer (Promega). The luciferase activity was then measured using a Dual-Luciferase Reporter Assay System (Promega) as per the manufacturer's instructions. Firefly luciferase activity was normalized by Renilla luciferase activity for each transfection.
TAK1 activity assays
TAK1 present in TAB1 immunoprecipitates was assayed by its ability to activate MKK6, as judged by the activation of SAPK2α/p38α. The active SAPK2α/p38α generated in this first stage of assay was then quantitated in a second assay by measuring phosphorylation of myelin basic protein (Cheung et al, 2003
). The TAK1 complexes were pulled down from 1 mg of cell lysate obtained from TAB1 reconstituted MEFs. The cell lysates were incubated for 2 h at 4°C with 20 μl of glutathione-sepharose beads per sample. The beads were washed twice with 1 ml of lysis buffer containing 0.25 M NaCl, followed by two washes with high salt wash buffer (1 ml of 50 mM Tris–HCl pH 7.5, 0.5 M NaCl and 0.1% (v/v) 2-mercaptoethanol). In all, 25% of the TAK1 complex bound to the beads was used to measure TAK1 activity. The remaining 75% of the TAK1 complex was taken for immunoblotting using appropriate antibodies as described earlier.
Cytokine secretion assay
For measuring TNFα and IL-6 secretion into the medium, 3 × 106 MEFs were transfected with the WT or S395A TAB1 and were seeded in 24-well plates at density of 3 × 105 cells/well. At 24 h after transfection, the cells were stimulated with IL-1α for different lengths of time. The media were collected and after brief centrifugation, 100 μl of clear media was used for cytokine ELISA as per the protocol from Peprotech.