Constitutive and Interleukin-1-inducible Phosphorylation of p65 NF-κB at Serine 536 Is Mediated by Multiple Protein Kinases Including IκB Kinase (IKK)-α, IKKβ, IKKϵ, TRAF Family Member-associated (TANK)-binding Kinase 1 (TBK1), and an Unknown Kinase and Couples p65 to TATA-binding Protein-associated Factor II31-mediated Interleukin-8 Transcription
2004; Elsevier BV; Volume: 279; Issue: 53 Linguagem: Inglês
10.1074/jbc.m409825200
ISSN1083-351X
AutoresHolger Buss, Anneke Dörrie, M. Lienhard Schmitz, Elke Hoffmann, Klaus Resch, Michael Kracht,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoPhosphorylation of NF-κB p65(RelA) serine 536 is physiologically induced in response to a variety of proinflammatory stimuli, but the responsible pathways have not been conclusively unraveled, and the function of this phosphorylation is largely elusive. In contrast to previous studies, we found no evidence for a role of c-Jun N-terminal kinase, p38 kinase, extracellular signal-regulated kinase, or phosphatidylinositol 3-kinase in interleukin-1- or tumor necrosis factor-induced Ser-536 phosphorylation, as revealed by pharmacological inhibitors. We were not able to suppress Ser-536 phosphorylation by either RNA interference directed at IκB kinase (IKK)-α/β (the best characterized Ser-536 kinases so far) or the IKKβ inhibitor SC-514 or dominant negative mutants of either IKK. A green fluorescent protein p65 fusion protein was phosphorylated at Ser-536 in the absence of IKK activation, suggesting the existence of IKKα/β-independent Ser-536 kinases. Chromatographic fractionation of cell extracts allowed the identification of two distinct enzymatic activities phosphorylating Ser-536. Peak 1 represents an unknown kinase, whereas peak 2 contained IKKα, IKKβ, IKKϵ, and TBK1. Overexpressed IKKϵ and TBK1 phosphorylate Ser-536 in vivo and in vitro. Reconstitution of mutant p65 proteins in p65-deficient fibroblasts that either mimicked phosphorylation (S536D) or preserved a predicted hydrogen bond between Ser-536 and Asp-533 (S536N) revealed that phosphorylation of Ser-536 favors interleukin-8 transcription mediated by TATA-binding protein-associated factor II31, a component of TFIID. In the absence of phosphorylation, the hydrogen bond favors binding of the corepressor amino-terminal enhancer of split to the p65 terminal transactivation domain. Collectively, our results provide evidence for at least five kinases that converge on Ser-536 of p65 and a novel function for this phosphorylation site in the recruitment of components of the basal transcriptional machinery to the interleukin-8 promoter. Phosphorylation of NF-κB p65(RelA) serine 536 is physiologically induced in response to a variety of proinflammatory stimuli, but the responsible pathways have not been conclusively unraveled, and the function of this phosphorylation is largely elusive. In contrast to previous studies, we found no evidence for a role of c-Jun N-terminal kinase, p38 kinase, extracellular signal-regulated kinase, or phosphatidylinositol 3-kinase in interleukin-1- or tumor necrosis factor-induced Ser-536 phosphorylation, as revealed by pharmacological inhibitors. We were not able to suppress Ser-536 phosphorylation by either RNA interference directed at IκB kinase (IKK)-α/β (the best characterized Ser-536 kinases so far) or the IKKβ inhibitor SC-514 or dominant negative mutants of either IKK. A green fluorescent protein p65 fusion protein was phosphorylated at Ser-536 in the absence of IKK activation, suggesting the existence of IKKα/β-independent Ser-536 kinases. Chromatographic fractionation of cell extracts allowed the identification of two distinct enzymatic activities phosphorylating Ser-536. Peak 1 represents an unknown kinase, whereas peak 2 contained IKKα, IKKβ, IKKϵ, and TBK1. Overexpressed IKKϵ and TBK1 phosphorylate Ser-536 in vivo and in vitro. Reconstitution of mutant p65 proteins in p65-deficient fibroblasts that either mimicked phosphorylation (S536D) or preserved a predicted hydrogen bond between Ser-536 and Asp-533 (S536N) revealed that phosphorylation of Ser-536 favors interleukin-8 transcription mediated by TATA-binding protein-associated factor II31, a component of TFIID. In the absence of phosphorylation, the hydrogen bond favors binding of the corepressor amino-terminal enhancer of split to the p65 terminal transactivation domain. Collectively, our results provide evidence for at least five kinases that converge on Ser-536 of p65 and a novel function for this phosphorylation site in the recruitment of components of the basal transcriptional machinery to the interleukin-8 promoter. The transcription factor NF-κB regulates the expression of a large number of genes with important functions in the immune response, inflammation, cellular stress reactions, carcinogenesis, and apoptosis. In resting cells NF-κB is trapped in the cytoplasm by its interaction with the inhibitor IκB. A central step in activation of NF-κB is the stimulus-induced phosphorylation of IκB by IκB kinases (IKK) 1The abbreviations used are: IKK, IκB kinase(s); TNF, tumor necrosis factor; LPS, lipopolysaccharide; TAD, terminal transactivation domain; GST, glutathione S-transferase; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; IL, interleukin; siRNA, small interfering RNA; PI3 kinase, phosphatidylinositol 3-kinase; MAP, mitogen-activated protein; wt, wild type; IP, immunoprecipitation; ChIP, chromatin immunoprecipitation; TBK1, TRAF family member-associated (TANK)-binding kinase 1; TAFII31, TATA-binding protein-associated factor II31; AES, amino-terminal enhancer of split.1The abbreviations used are: IKK, IκB kinase(s); TNF, tumor necrosis factor; LPS, lipopolysaccharide; TAD, terminal transactivation domain; GST, glutathione S-transferase; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; IL, interleukin; siRNA, small interfering RNA; PI3 kinase, phosphatidylinositol 3-kinase; MAP, mitogen-activated protein; wt, wild type; IP, immunoprecipitation; ChIP, chromatin immunoprecipitation; TBK1, TRAF family member-associated (TANK)-binding kinase 1; TAFII31, TATA-binding protein-associated factor II31; AES, amino-terminal enhancer of split. α and β. Both IKKs, IκB, and NF-κB subunits form a large signaling complex (1Schmidt C. Peng B. Li Z. Sclabas G.M. Fujioka S. Niu J. Schmidt-Supprian M. Evans D.B. Abbruzzese J.L. Chiao P.J. Mol. Cell. 2003; 12: 1287-1300Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Phosphorylation of IκB results in targeting of IκB to the proteasome followed by release and nuclear translocation of NF-κB. Recent evidence suggests that NF-κB activity is determined by additional mechanisms. Cells lacking the protein kinases glycogen synthase kinase 3β (2Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgett J.R. Nature. 2000; 406: 86-90Crossref PubMed Scopus (1207) Google Scholar), TBK1/NF-kB-activating kinase (3Bonnard M. Mirtsos C. Suzuki S. Graham K. Huang J. Ng M. Itie A. Wakeham A. Shahinian A. Henzel W.J. Elia A.J. Shillinglaw W. Mak T.W. Cao Z. Yeh W.C. EMBO J. 2000; 19: 4976-4985Crossref PubMed Google Scholar, 4Tojima Y. Fujimoto A. Delhase M. Chen Y. Hatakeyama S. Nakayama K. 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Cell. 2001; 8: 771-780Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar) show a normal IκB degradation pathway but impaired activation of NF-κB-dependent gene expression.Furthermore, biochemical and genetic experiments in cells deficient for IKKα or IKKβ strongly suggest that direct phosphorylation and other modifications of NF-κB are essential for NF-κB function (8Schmitz M.L. Mattioli I. Buss H. Kracht M. Chem. Bio. Chem. 2004; 5: 1348-1358Crossref PubMed Scopus (219) Google Scholar, 9Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3273) Google Scholar, 10Chen L.F. Greene W.C. Nat. Rev. Mol. Cell Biol. 2004; 5: 392-401Crossref PubMed Scopus (1020) Google Scholar). The complexity of this regulation is exemplified by recent studies that identified four serine residues in the p65 NF-κB subunit that are inducibly phosphorylated by TNF or LPS and that are targeted by distinct signaling pathways.Phosphorylation of serine 276 of p65 occurs by catalytic subunit of protein kinase A in response to LPS (11Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1021) Google Scholar, 12Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar) or by mitogen- and stress-activated protein kinase in response to TNF (13Vermeulen L. De Wilde G. Van Damme P. Vanden Berghe W. Haegeman G. EMBO J. 2003; 22: 1313-1324Crossref PubMed Scopus (637) Google Scholar). Phosphorylation at Ser-276 promotes its interaction with the coactivator CREB-binding protein (a histone acetylase) and is required for TNF-induced IL-6 expression (14Zhong H. May M.J. Jimi E. Ghosh S. Mol. Cell. 2002; 9: 625-636Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar, 15Okazaki T. Sakon S. Sasazuki T. Sakurai H. Doi T. Yagita H. Okumura K. Nakano H. Biochem. Biophys. Res. Commun. 2003; 300: 807-812Crossref PubMed Scopus (136) Google Scholar). Phosphorylation in the REL homology domain of p65 at Ser-311 is required for efficient p65 transactivation mediated by protein kinase Cζ (7Leitges M. Sanz L. Martin P. Duran A. Braun U. Garcia J.F. Camacho F. Diaz-Meco M.T. Rennert P.D. Moscat J. Mol. Cell. 2001; 8: 771-780Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 16Anrather J. Csizmadia V. Soares M.P. Winkler H. J. Biol. Chem. 1999; 274: 13594-13603Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 17Duran A. Diaz-Meco M.T. Moscat J. EMBO J. 2003; 22: 3910-3918Crossref PubMed Scopus (268) Google Scholar).Serine 529 of p65 is phosphorylated by casein kinase II (18Wang D. Baldwin Jr., A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 19Wang D. Westerheide S.D. Hanson J.L. Baldwin Jr., A.S. J. Biol. Chem. 2000; 275: 32592-32597Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar) and by the TAX-activated IKK complex in vitro (20O'Mahony A.M. Montano M. Van Beneden K. Chen L.F. Greene W.C. J. Biol. Chem. 2004; 279: 18137-18145Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In experiments using reconstituted p65 -/- cells, Ser-529 contributes to p65 transactivation (20O'Mahony A.M. Montano M. Van Beneden K. Chen L.F. Greene W.C. J. Biol. Chem. 2004; 279: 18137-18145Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, others have found no role for Ser-529 in p65 activity in response to TNF treatment or IKKα/IKKβ overexpression (15Okazaki T. Sakon S. Sasazuki T. Sakurai H. Doi T. Yagita H. Okumura K. Nakano H. Biochem. Biophys. Res. Commun. 2003; 300: 807-812Crossref PubMed Scopus (136) Google Scholar, 21Yang F. Tang E. Guan K. Wang C.Y. J. Immunol. 2003; 170: 5630-5635Crossref PubMed Scopus (329) Google Scholar, 22Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar).Regulated phosphorylation of serine 536 of the C-terminal transactivation domain (TAD) of p65 was originally found by Sakurai et al. (22Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar) who searched for kinase activities in extracts from TNF-stimulated cells that would phosphorylate various truncated recombinant p65 proteins in vitro. This group also suggested IKKβ as a p65 TAD kinase (22Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar). Since then several groups have confirmed that both IKKs directly phosphorylate the C-terminal TAD of p65 (20O'Mahony A.M. Montano M. Van Beneden K. Chen L.F. Greene W.C. J. Biol. Chem. 2004; 279: 18137-18145Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 23Sizemore N. Lerner N. Dombrowski N. Sakurai H. Stark G.R. J. Biol. Chem. 2002; 277: 3863-3869Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 24Sakurai H. Suzuki S. Kawasaki N. Nakano H. Okazaki T. Chino A. Doi T. Saiki I. J. Biol. Chem. 2003; 278: 36916-36923Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 25Kishore N. Sommers C. Mathialagan S. Guzova J. Yao M. Hauser S. Huynh K. Bonar S. Mielke C. Albee L. Weier R. Graneto M. Hanau C. Perry T. Tripp C.S. J. Biol. Chem. 2003; 278: 32861-32871Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Recombinant or overexpressed IKKs phosphorylate Ser-536 in vitro, with IKKβ somewhat more efficient, as shown by mutational analysis of GST-p65 fusion proteins (22Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar, 24Sakurai H. Suzuki S. Kawasaki N. Nakano H. Okazaki T. Chino A. Doi T. Saiki I. J. Biol. Chem. 2003; 278: 36916-36923Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar) or by mass spectrometry analysis of p65 peptides (25Kishore N. Sommers C. Mathialagan S. Guzova J. Yao M. Hauser S. Huynh K. Bonar S. Mielke C. Albee L. Weier R. Graneto M. Hanau C. Perry T. Tripp C.S. J. Biol. Chem. 2003; 278: 32861-32871Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar).Ser-536 is physiologically induced in response to a variety of proinflammatory stimuli (21Yang F. Tang E. Guan K. Wang C.Y. J. Immunol. 2003; 170: 5630-5635Crossref PubMed Scopus (329) Google Scholar, 24Sakurai H. Suzuki S. Kawasaki N. Nakano H. Okazaki T. Chino A. Doi T. Saiki I. J. Biol. Chem. 2003; 278: 36916-36923Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 26Jiang X. Takahashi N. Matsui N. Tetsuka T. Okamoto T. J. Biol. Chem. 2003; 278: 919-926Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 27Mattioli I. Sebald A. Bucher C. Charles R.P. Nakano H. Doi T. Kracht M. Schmitz M.L. J. Immunol. 2004; 172: 6336-6344Crossref PubMed Scopus (185) Google Scholar), but the responsible pathways and the function of this phosphorylation have not been conclusively unraveled. Sakurai et al. (24Sakurai H. Suzuki S. Kawasaki N. Nakano H. Okazaki T. Chino A. Doi T. Saiki I. J. Biol. Chem. 2003; 278: 36916-36923Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar) identified tumor necrosis factor-associated factor (TRAF)2, TRAF5, TGFβ-activated protein (TAK)1, and IKKα/β as important mediators for TNFα-induced serine 536 phosphorylation, whereas (as shown by us) induction of this phosphorylation by T cell co-stimulation depends on Cot (Tpl2), receptor-interacting protein, protein kinase Cθ, NF-kB-inducing kinase, and IKKβ (27Mattioli I. Sebald A. Bucher C. Charles R.P. Nakano H. Doi T. Kracht M. Schmitz M.L. J. Immunol. 2004; 172: 6336-6344Crossref PubMed Scopus (185) Google Scholar). A further study showed that the LPS- and TNF-induced phosphorylation of endogenous p65 at Ser-536 is unaffected in IKKα -/- fibroblasts. Only the LPS-induced but not the TNF-induced phosphorylation is lost in IKKβ -/- cells (21Yang F. Tang E. Guan K. Wang C.Y. J. Immunol. 2003; 170: 5630-5635Crossref PubMed Scopus (329) Google Scholar).In the light of these contrasting findings and the apparent complexity of p65 regulation by phosphorylation, we have set up experiments to analyze more conclusively the kinases that contribute to Ser-536 phosphorylation of p65 and its function in response to one of the major activators of p65, interleukin-1. We report here that besides IKKα and IKKβ, the IKK-related kinases TBK1 and IKKϵ also phosphorylate Ser-536. We also provide evidence for a novel Ser-536 kinase. Further experiments into the mechanism by which Ser-536 contributes to p65 function suggest that phosphorylation disrupts a hydrogen bond between Ser-536 and Asp-533. As a result, p65 has a lower affinity for the corepressor AES and can interact efficiently with TAFII31, a component of the basal transcription factor machinery, to induce transcription of IL-8, a known p65 target gene. We, thus, identify a hitherto unknown mechanism of p65-mediated gene regulation.EXPERIMENTAL PROCEDURESCells and Materials—HeLa cells stably expressing the tet transactivator protein and HEK293 cells stably expressing the IL-1 receptor (HEK293IL-1R) were kind gifts of H. Bujard and K. Matsumoto, respectively. KB cells were from the American Type Culture Collection, Manassas, VA. p65 -/- cells were a kind gift of H. Nakano. All cells were cultured in Dulbecco's modified Eagle's medium, complemented with 10% fetal calf serum, 2 mm l-glutamine, 1 mm sodium pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin. Antibodies against the following proteins or peptides were used in this study: IκBα (9242), phospho-IκBα (9241), phospho-Ser-536 NF-κB (3031), all from Cell Signaling Technology; p65 NF-κB (C-20), IKKϵ (H-116), TBK1 (M-375), IKKα (H-744), IKKβ (T-20), ERK2 (C-14), all from Santa Cruz; GFP (Roche Applied Science); FLAG (M2, Eastman Kodak Co.). Horseradish peroxidase-coupled secondary antibodies were from Sigma. Protein A/G-Sepharose was from Amersham Biosciences. Human recombinant IL-1α was a kind gift of J. Saklatvala. PD98059 was from Alexis, SP600125 was from Tocris, and SB203580 and MG132 were from Calbiochem, and SC-514 was a kind gift of Pharmacia Corp. Other reagents were from Sigma-Aldrich or Fisher and were of analytical grade or better.Plasmids and Transfections—The expression plasmids pcDNA3-FLAG-IKKα and pcDNA3-FLAG-IKKβ were kind gifts of David Wallach. pMTNHA-TAFII31 (28Buschmann T. Lin Y. Aithmitti N. Fuchs S.Y. Lu H. Resnick-Silverman L. Manfredi J.J. Ronai Z. Wu X. J. Biol. Chem. 2001; 276: 13852-13857Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) was from X. Wu, pGEX2TK-TAFII31 (29Green V.J. Kokkotou E. Ladias J.A. J. Biol. Chem. 1998; 273: 29950-29957Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) was from A. A. Ladias, pcDNA3.1/HiSA-AES and pGEX-5X-2-AES (30Tetsuka T. Uranishi H. Imai H. Ono T. Sonta S. Takahashi N. Asamitsu K. Okamoto T. J. Biol. Chem. 2000; 275: 4383-4390Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) were from T. Okamoto, and pEGFP-p65 was from Rainer de Martin. Expression vectors for FLAG-IKKαKN, FLAG-IKKβKN, IKKϵ, IKKϵKN, TBK1, TBK1KN, IκBα, and NF-κB(3)luc have been published (27Mattioli I. Sebald A. Bucher C. Charles R.P. Nakano H. Doi T. Kracht M. Schmitz M.L. J. Immunol. 2004; 172: 6336-6344Crossref PubMed Scopus (185) Google Scholar). The expression plasmids for the p65 TAD, pGEX-p65 (354-551), and versions mutated in S529A or S536A were kind gifts of H. Sakurai. pMT7-p65 NF-κB, and the IL-8 promoter-luciferase reporter plasmid pUHC13-3-IL-8pr (nucleotides 1348-1527 of the IL-8 gene) have been described (31Nourbakhsh M. Kalble S. Dorrie A. Hauser H. Resch K. Kracht M. J. Biol. Chem. 2001; 276: 4501-4508Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). pSV-β-gal coding for SV40 promoter-driven β-galactosidase was from Promega. GST fusion proteins were expressed in bacteria and purified on GSH-Sepharose using standard procedures.HEK293IL-1R cells were transiently transfected by the calcium phosphate method, and determination of luciferase reporter gene activity was performed as described (31Nourbakhsh M. Kalble S. Dorrie A. Hauser H. Resch K. Kracht M. J. Biol. Chem. 2001; 276: 4501-4508Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Equal amounts of plasmid DNA within each experiment were obtained by adding empty vector. For determination of promoter activity, cells (seeded at 5 × 105 per well of 6-well plates) were transfected with 0.25 μg of the IL-8-promoter luciferase reporter plasmids and 0.5 μg of pSV-β-gal. β-Galactosidase activity was determined (using reagents from Clontech) to allow normalization of luciferase activity in different transfections. p65 -/- cells were seeded in 6-well plates and transfected using Rotifect (Roth) according to the manufacturer's instructions.Site-directed Mutagenesis—All mutations were performed by a quick change site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol and verified by DNA sequencing on an ABI Prism 310 instrument.siRNA Experiments—Cells were seeded in 24-well plates at 6.5 × 104 cells/ml. At 70% confluency cells were washed 2× in serum-free medium and transfected with a total of 200 nm of a mixture of four double-stranded RNA oligonucleotides directed against human IKKα and IKKβ (Smart Pool, Dharmakon) using 3 μl of Oligofectamine per well. After 72 h cells were treated as indicated and lysed, and protein expression was determined by Western blot. The sequences of the luciferase siRNA oligonucleotides used as a control were as follows (5′-GGCCUUGUGAACAGAUCAGdTdT-3′, 5′-CUGAUCUGUUCACAAGGCCdTdT-3′).Anion Exchange Chromatography—Four T175-cm2 flasks of HeLa cells were stimulated or left untreated as indicated in the figure legend. Cells were washed in ice-cold phosphate-buffered saline and resuspended in 20 mm Tris, pH 8.5, 20 mm β-glycerophosphate, 20 mm NaF, 0.1 mm Na3VO4, 0.5 mm EGTA, 0.5 mm EDTA, 0.1% Nonidet P-40, 2 mm dithiothreitol, 10 μm E64, 2.5 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 1 μm pepstatin, and 400 nm okadaic acid. Cells were broken mechanically by passage three times through a 26-gauge needle. Then the lysate was cleared at 15,000 × g for 20 min at 4 °C. Supernatants were stored frozen at -80 °C or directly used for chromatography. 4.75 mg of lysate was diluted into 2.5 ml of buffer A (20 mm Tris, pH 8.5, 20 mm β-glycerophosphate, 20 mm NaF, 0.1 mm Na3VO4, 0.5 mm EGTA, 0.5 mm EDTA, 0.05% Nonidet P-40, 2 mm dithiothreitol) and loaded onto a 1 ml of ResQ column run by an Aktaprime system (Amersham Biosciences). The column was equilibrated in buffer A, and proteins were eluted with a linear salt gradient (0-0.75 m NaCl in 16 ml). 1-ml fractions were collected and stored frozen at -80 °C until further use.Preparation of Cell Extracts—For the preparation of whole cell extracts cells were lysed directly in SDS sample buffer. DNA was sheared by brief sonification, and soluble proteins were recovered after centrifugation of lysates at 15,000 × g for 15 min at 4 °C. For in vitro kinase assays, cells were lysed in 10 mm Tris, pH 7.05, 30 mm NaPPi, 150 mm NaCl, 1% Triton X-100, 2 mm Na3VO4, 50 mm NaF, 20 mm β-glycerophosphate, and freshly added 0.5 mm phenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, 0.5 μg/ml pepstatin, and 400 nm okadaic acid. After 10 min on ice, lysates were clarified by centrifugation at 10,000 × g for 15 min at 4 °C.Nuclear and cytosolic extracts were prepared as described previously (31Nourbakhsh M. Kalble S. Dorrie A. Hauser H. Resch K. Kracht M. J. Biol. Chem. 2001; 276: 4501-4508Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Protein concentration of cell extracts was determined by the method of Bradford, and samples were stored at -80 °C.Western Blotting—Cell extract proteins were separated on 10% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (Immobilon®, Millipore). After blocking with 5% dried milk in Tris-buffered saline overnight, membranes were incubated for 4-24 h with primary antibodies, washed in Tris-buffered saline, and incubated for 2-4 h with the peroxidase-coupled secondary antibody. Proteins were detected by using the Amersham Biosciences-enhanced chemiluminescence system.In Vitro Kinase Assays—For immune complex kinase assays 500 μg of cell extract protein was diluted in 500 μl of ice-cold immunoprecipitation (IP) buffer (10 mm Tris, pH 7.05, 30 mm NaPPi, 150 mm NaCl, 1% Triton X-100, 2 mm Na3VO4, 50 mm NaF, 20 mm β-glycerophosphate, 2 mm dithiothreitol, and 400 μm phenylmethylsulfonyl fluoride). Samples were incubated for 3 h with 1 μg of antibodies against IKKα or FLAG (to precipitate FLAG-IKKϵ or FLAG-TBK1) followed by the addition of 20 μl of a 50% suspension of protein A- or G-Sepharose beads and incubation for 1-2 h at 4 °C. Beads were spun down, washed 3× in 1 ml of IP buffer A, and resuspended in 10 μl of the same buffer.Then 1 μg of recombinant protein substrates (GST-p65-(354-551) or mutants thereof) in 10 μl of H2O and 10 μl of kinase buffer (150 mm Tris, pH 7.4, 30 mm MgCl2, 60 μm ATP, 4 μCi of [γ-32P]ATP) were added. After 30 min at room temperature SDS-PAGE sample buffer was added, and proteins were eluted from the beads by boiling for 5 min. After centrifugation at 10,000 × g for 5 min, supernatants were separated on 10% or 12.5% SDS-PAGE. Phosphorylated proteins were visualized by autoradiography.Alternatively, 10 μl of ResQ fractions were used in the kinase reactions. For the kinase assay shown in Fig. 4A, 10 μl of cell lysate (50 μg of protein) was added to the kinase reaction, and the in vitro phosphorylated GST-p65 fusion protein was purified on GSH-Sepharose before SDS-PAGE and autoradiography as described in Holtmann et al. (32Holtmann H. Winzen R. Holland P. Eickemeier S. Hoffmann E. Wallach D. Malinin N.L. Cooper J.A. Resch K. Kracht M. Mol. Cell. Biol. 1999; 19: 6742-6753Crossref PubMed Scopus (268) Google Scholar). For the kinase assays shown in Fig. 6A, 10 μl (50 μg) of cytosolic or nuclear proteins were mixed with 10 μl of (150 mm Tris, pH 7.4, 30 mm MgCl2, 600 μm ATP) and 10 μl (1 μg) of GST-p65-(354-551) fusion proteins for 15 min at 30 °C. Reaction mixtures were separated by SDS-PAGE, and phosphorylation of p65 was detected by immunoblotting with the anti-phospho-Ser-536 antibody.Fig. 6p65 phosphorylated at Ser-536 occurs in the nucleus and is recruited to the endogenous IL-8 promoter. A, HeLa cells were stimulated for 10 min with IL-1 (10 ng/ml) or left untreated. Nuclear and cytosolic extracts were prepared, and activity of kinases that phosphorylate GST-p65-(354-551) or a version in which Ser-536 was mutated to alanine (S536A) was determined by in vitro kinase assay. Phosphorylation of Ser-536 was detected by immunoblotting of reaction mixtures, and the blot membrane was reprobed with anti-p65 antibodies to confirm equal loading. Lanes 9 and 10 contained only GST-p65 fusion proteins without cell extracts. B, HeLa cells were stimulated for the indicated times with IL-1 (10 ng/ml) or left untreated. Nuclear extracts were analyzed for p65 and Ser-536 phosphorylation of p65 by Western blotting. C, nuclear extracts from unstimulated HeLa cells or cells stimulated for 10 min with IL-1 (10 ng/ml) were incubated with a 32P-labeled oligonucleotide containing a consensus NF-κB binding site in the presence or absence of antibodies against p65 or phospho-Ser-536 of p65. Solid arrows indicate the two IL-1-induced protein-DNA complexes, and open arrows indicate forms of these complexes supershifted by the antibodies. D, KB cells were stimulated for the indicated times with IL-1 (10 ng/ml) or left untreated. After in vivo cross-linking soluble chromatin was prepared, and antibodies against p65 and phospho-Ser-536 of p65 were used to immunoprecipitate protein-DNA complexes. IL-8 promoter DNA bound to p65 was amplified by PCR from the immune complexes, separated by agarose gel electrophoresis, and visualized by ethidium bromide staining. In parallel PCR was performed on chromatin before immunoprecipitation (Input).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Electrophoretic Mobility Shift Assay—Electrophoretic mobility shift assay was performed as described in Nourbakhsh et al. (31Nourbakhsh M. Kalble S. Dorrie A. Hauser H. Resch K. Kracht M. J. Biol. Chem. 2001; 276: 4501-4508Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) using an oligonucleotide (5′-tgacagagGGGACTTTCCagaga-3′) containing an NF-κB consensus site (shown in capital letters).GST Pull-down Assays—p65 or AES proteins were in vitro transcribed and translated using the TNT rabbit reticulocyte kit from Promega. Approximately 500 ng of purified GST, GST-AES, or GST-TAFII31 (as judged from Coomassie-stained gels) were immobilized on GSH-Sepharose, resuspended in 70 μl of binding buffer (20 mm Tris, pH 7.4, 0.3 m NaCl, 1% (w/v) bovine serum albumin, 0.1% Nonidet P-40, 2 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride), and incubated with 3.5 μl of in vitro translated p65 or AES proteins for 60 min at 30 °C. Beads were spun down and washed twice in binding buffer without bovine serum albumin, and bound proteins were eluted in SDS sample buffer for 5 min at 95 °C. Bound proteins and 110 of the in vitro translated proteins (used as input control) were separated on SDS-PAGE, and proteins were analyzed by autoradiography or Western blotting.Chromatin Immunoprecipitation (ChIP)—Proteins bound to DNA were cross-linked in vivo by replacement of the medium with warm phosphate-buffered saline including 1% formaldehyde. After 1 min, this solution was replaced by warm phosphate-buffered saline including 0.125 m glycine to stop cross-linking. Cells were washed, collected, and lysed in ChIP-radioimmune precipitation assay buffer (10 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 1 mm EDTA, and freshly added 1% aprotinin). Lysates were cleared by sonification (4× 1 min on ice) followed by centrifugation at 15,000 × g at 4 °C for 20 min. 4-10 μl of antibodies were added to 250-500 μl of lysates, and the mixture was rotated at 4 °C overnight. Then 4
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