Ampicillin, BSA, acetylated bovine serum albumin, dithiothreitol, EDTA, glutathione, Protein A and Protein G sepharose, HEPES, glutathione agarose were purchased from Sigma. N-α-Fmoc-amino acids were purchased from Novabiochem (San Diego); unlabeled acetyl-CoA, 14C-labelled acetyl-CoA, and 14C-labeled acetylated bovine serum albumin were purchased from Amersham Biosciences. Oligonucleotide primers were obtained from Integrated DNA Technologies (Coralville, Iowa). Phenol, trifluoroacetic acid, and IPTG were obtained from Fisher Scientific. Quickchange mutagenesis and the sequences were confirmed by DNA sequencing. Anti-ATF-2 and anti-acetyl-lysine antibodies were purchased from Santa Cruz Biotechnology, Upstate Biotechnology and Cell signaling Technologies. Dual Glow luciferase kit was obtained from Promega Corporation. Glutathione S-transferase was expressed and purified from a standard GST expression plasmid using glutathione agarose.
p300 C-terminal peptide CMLVELHTQSQDRF for expressed protein ligation and H4-15 peptide Ac-GRGKGGKGLGKGGAK for acetyltransferase assays were prepared using the Fmoc strategy. Peptides were purified (>95% homogeneity) by reversed phase (C-18) high performance liquid chromatography as described previously using a gradient of water-acetontitrile (0.05% trifluoroacetic acid). Electrospray mass spectrometry of peptide confirmed the correct structures.
Preparation of Wt p300 HAT and p300 HAT-ΔLoop
These semisynthetic proteins were prepared and purified following previously described procedures (33
). Briefly, pTYB2 expression plasmids encoding p300 HAT protein (aa 1287-1652) or (aa 1287-1652 with an internal deletion of aa 1523-1554) containing an M1652G mutation fused to VMA intein-chitin binding domain was grown in E. coli
BL21(DE3)-RIL cells to A600
of 0.45 at which point the incubator temperature was reduced to 16°C and media allowed to cool. After 15 min, protein expression was induced by addition of IPTG to a final concentration of 0.5 mM. Cells (1 L) were then grown for 16 h at 16°C, harvested by centrifugation, resuspended in intein lysis buffer (25 mM HEPES (pH 7.9), 500 mM NaCl, 10% glycerol, 1 mM MgSO4
, and 2 mM PMSF) and lysed by two passages through a French press cell. The lysate was cleared by centrifugation and applied to a 12 ml chitin column after extensive washing. Excess buffer was drained and this immobilized fusion protein was treated with 200 mM MESNA to generate the thioester and ligated to 10 mg synthetic peptide aa 1653-1666 (CMLVELHTQSQDRF) over 16 h at room temperature. Fractions containing semisynthetic p300 HAT were pooled and concentrated before being applied to a Mono-S HR5/5 (Amersham Biosciences) strong cation exchange column for further purification. Fractions containing purified protein (>90%), as determined by SDS-PAGE analysis, were pooled and concentrated to ~5 mg/ml as measured by Bradford assay. Following concentration, 5% glycerol was added before flash freezing in liquid N2
and samples were stored at -80°C. Semisynthetic proteins showed the correct molecular weights as determined by MALDI (matrix-assisted laser ddesorption/ionization) TOF (time-of-flight) mass spectrometry.
Purification of GST-ATF-2-b-ZIP
pGEX-4T-3 plasmid encoding basic leucine zipper domain (aa 349-415) was grown in E.coli BL21 (DE3)-RIL cells to A600 of 0.45 at which point the incubator temperature was reduced to 16°C and media allowed to cool. After 15 min, protein expression was induced by addition of IPTG to a final concentration of 1.0 mM. Cells (1 L) were then grown for 16 h at 16°C, harvested by centrifugation, re-suspended in lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1.0% NP-40, 10% glycerol, 5 mM EDTA, 5 mM DTT and 2 mM PMSF) and lysed by two passages through a French press cell. The lysate was cleared by centrifugation and applied to a 10 ml glutathione agarose column. The GST beads were eluted extensively (>5 column volumes) with wash buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1.0% NP-40, 10% glycerol, 5 mM EDTA, 5 mM DTT and 2 mM PMSF). The protein was eluted with elution buffer (20 mM Tris-HCl (pH 8.0), 10 mM reduced glutathione, 5 mM DTT and 2 mM PMSF), fractions were analyzed by 10% (w/v) SDS-PAGE, and fractions containing recombinant GST-ATF-2-b-ZIP (>90% purified) were pooled and dialyzed to remove glutathione and concentrated to 2 mg/mL. GST-ATF-2-b-ZIP was stored in 10% glycerol, 20 mM Tris, pH 7.4, and 1 mM DTT at -80°C.
Preparation of hyperacetylated p300
Semisynthetic hypoacetylated p300 HAT domain (10 μM) was incubated with acetyl-CoA (125 μM) in reaction buffer (50 mM HEPES pH 7.9, 0.1 mM EDTA, 1 mM DTT and 50 μg/ml bovine serum albumin) for 1 h at 30°C (33
). For comparative analysis of binding studies and acetyltransferase assays (see below), hypoacetylated p300-ΔLoop (10 μM) and p300 HAT (10 μM) were incubated with desulfo-CoA (125μM) in reaction buffer above in the absence of acetyl-CoA.
GST-ATF-2-b-ZIP pull down assays
GST-ATF-2-b-ZIP (1 mg/ml) immobilized on glutathione agarose resin in 16 μl incubation buffer (20 mM HEPES pH 7.9, 5 mM DTT, 1 mM EDTA) was incubated with wt hyper- or hypoacetylated p300 HAT domain or hypoacetylated p300 HAT-ΔLoop in binding buffer (50 mM HEPES pH 7.9, 0.1 mM EDTA, 1 mM DTT and 50 μg/ml bovine serum albumin, 30 μL volumes) at 16°C for 20 min. The resultant samples were centrifuged at 10,000g for 1 min and supernatants were collected. The pellets were washed twice with 0.1 ml of wash buffer (20 mM HEPES pH 7.9, 5 mM DTT, 1 mM EDTA, 50 mM NaCl, 50 μM CoASH). Following washing, the samples were treated separately with 5×SDS gel loading buffer and analyzed using 10% SDSPAGE. The dried gels were scanned and analyzed by Scion Image analysis (Scion Corporation).
For the peptide H4-15 substrate, H4-15 (100 μM) was incubated with or without GST or GST-ATF-2-b-ZIP, in the presence of p300 HAT domain (40 nM) in hyperacetylated or hypoacetylated state in assay buffer (50 mM HEPES, pH 7.9, 0.1 mM EDTA, 1 mM DTT and 50 μg/ml bovine serum albumin, 30 μL) for 10 min at 30°C prior to initiation of reaction by the addition of 14
C-acetyl-CoA (20 μM unless otherwise specified) for 0-1 min whereupon the reaction was quenched with 6 μL of 5X SDS gel loading buffer and analyzed using 16% Tris-Tricine SDSPAGE (34
). For acetylation of GST-ATF-2 b-ZIP, up to 5 μM protein substrate was incubated with 40 nM of p300 HAT domain in presence of 14
C-acetyl-CoA (20 μM) for 0-4 min with and the mixture was quenched with 6X-SDS loading solution. After running out on 10% SDS-PAGE, the gels were dried and the radioactivity quantified by phosphorimage analysis (Molecular Dynamics) relative to a 14
C-bovine serum albumin (Amersham) standard. Assays were performed in duplicates and these generally agreed within 20%.
ATF-2-b-ZIP acetylation for mass spectrometry analysis
GST-ATF-2-b-ZIP (5 μM) was incubated with or without wt p300 HAT domain (250 nM) in presence of acetyl-CoA (2 mM) in assay buffer (50 mM HEPES pH 7.9, 0.1 mM EDTA, 1 mM DTT and 50 μg/ml bovine serum albumin) for 1 h. The proteins were digested with trypsin (20:1 w/w) in 25 mM ammonia bicarbonate at 37°C for 18 h. Digested peptide mixtures were separated by reversed phase HPLC on a C18 column with an acetonitrile/water/0.1% trifluoroacetic acid solvent system. The collected fractions were dried completely and resuspended in 3 μl of water. 0.5 μL of the sample was spotted on a MALDI target followed by the addition of 0.5 μL of α-cyano-4-hydroxycinnamic acid matrix and was allowed to dry at room temperature. The MS and MS/MS spectra were acquired in the positive ion mode using a Kratos Analytical (Manchester, UK) AXIMA-CFR MALDI-TOF mass spectrometer equipped with a pulsed extraction source, a 337-nm pulsed nitrogen laser and a curved-field reflectron. The acceleration voltage was 20 kV. For the MS/MS spectra metastable fragmentation was enhance by increasing the MALDI laser power. No collision gas was used.
In vivo acetylation of ATF-2, immunoprecipitation and western blotting
293 cells and Cos-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Life technologies, Gaithersburg, MD, USA) and 1% antibiotics. Cells were treated with LPS, cycloheximide and phorbol myristate (PMA) with or without HDAC inhibitors (5 mM sodium butyrate, 10 mM nicotinamide and 100 ng/ml TSA) for 8 h. Cells were then harvested and lysed at 4°C by gentle scrapping in lysis buffer (50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 5 mM EDTA, 1 mM DTT, 0.1% NP-40 (w/v), 0.5 mM AEBSF, 1 mg/ml aprotinin/leupeptin, 2 mM NaF and 0.5 mM Na-vanadate). Lysates were further clarified by centrifugation at 10,000g for 20 min at 4°C. The protein concentrations of lysates were estimated by Bradford reagent. Lysates were pre-cleared for 5 min with 10 μl protein G sepharose and immunoprecipitated at 4°C for overnight with mouse monoclonal anti-ATF-2 (sc-242, Santa Cruz Biotechnology) followed by precipitation with 30 μl protein G for 2 h at 4°C. After two washes with PBS buffer (140 mM NaCl, 2.7 mM KCl,10 mM Na2HPO4, 1.8 mM KH2PO4, 2 mM EDTA, 0.1% (w/v) NP-40, Roche protease inhibitor cocktail “COMPLETE”), the immunoprecipitates were run out on 8% SDS-PAGE, transferred to nitrocellulose membrane, immunoblotted with rabbit monoclonal anti-ATF-2 (9226, Cell Signaling Technology), stripped with Pierce stripping buffer and re-probed with rabbit polyclonal anti-acetyl lysine antibody (Upstate 06-933) and detected using ECL chemiluminescence as per manufacturer instructions (Super-signal, Pierce).
Mutagenesis of full-length human ATF-2 human plasmid
) was subjected to site-directed mutations at K357 and K374 which were created by the QuikChange mutagenesis procedure (Stratagene). Primers containing ~18 nucleotides on either side of the mutation were made. Following PCR assays according to the manufacturer's protocol, the DNA was digested with DpnI and transformed into E. coli
. Positive clones were isolated, and the introduction of the mutation was confirmed by sequence analysis. Using the above methodology KK/RR, KK/AA, KK/QQ, K357R, K357A, K357Q, K374R, K374A, K374Q mutants were generated.
Cos-7 cells were plated in 6-well flat bottomed plates on the day prior to transfection at a density of 2 X 104
cells/well in DMEM. Transfections were performed using Lipofectamine-2000 (Invitrogen). The reporter CRE-Luc containing sequence placed at 5′ to the gene of luciferase was employed (37
). Cells were co-transfected with 1μg pCRE-Luc (stratagene), 30 ng of pRL-TK-Renilla (pRL-TK from Promega), and equal amounts of pACT plasmids expressing wt-ATF-2, KK/RR, KK/AA, KK/QQ, K357R, K357A, K357Q, K374R, K374A, K374Q for 36 h and treated with HDAC inhibitors (5 mM sodium butyrate, 10 mM nicotinamide and 100 ng/ml TSA) for 8 h. Cells were lysed and firefly luciferase and Renilla luciferase activities were measured with luciferase assay reagent (Dual-Glow luciferase Reporter Assay system, Promega) using a luminometer. Three replicates were performed and the standard errors are shown in . The lysates were further analyzed for amount of ATF-2 expressed in different transfection reactions by immunoprecipitation and immunoblotting with anti-ATF-2 antibody (mouse monoclonal anti-ATF-2 (sc-242, Santa Cruz Biotechnology) followed by precipitation with 30 μl protein G for 2 h at 4°C. After two washes with PBS buffer (140 mM NaCl, 2.7 mM KCl,10 mM Na2
, 1.8 mM KH2
, 2 mM EDTA, 0.1% (w/v) NP-40, Roche protease inhibitor cocktail “COMPLETE”), the immunoprecipitates were run out on 8% SDS-PAGE, transferred to nitrocellulose membrane, immunoblotted with rabbit monoclonal anti-ATF-2 (9226, Cell Signaling Technology)).
Fig 8 Mutational Analysis of Lys-357 and Lys-374 in ATF-2 on transcriptional regulation using a CREB luciferase reporter plasmid. A) Cell lysates were analyzed for luciferase activity. The bars from left to right are as follows: C-control; CRE–pCRE-luc (more ...)