Activation of Nuclear Factor-κB-dependent Transcription by Tumor Necrosis Factor-α Is Mediated through Phosphorylation of RelA/p65 on Serine 529

Nuclear factor-κB (NF-κB) is an essential transcription factor in the control of expression of genes involved in immune and inflammatory responses. In unstimulated cells, NF-κB complexes are sequestered in the cytoplasm through interactions with IκBα and other IκB proteins. Extracellular stimuli that activate NF-κB, such as tumor necrosis factor α (TNFα), cause rapid phosphorylation of IκBα at serines 32 and 36. The inducible phosphorylation of IκBα is followed by its ubiquitination and degradation, allowing NF-κB complexes to translocate into the nucleus and to activate gene expression. Previously, it has been shown that TNFα as well as other stimuli also lead to the phosphorylation of the RelA/p65 subunit of NF-κB. In this report, we demonstrate that the TNFα-induced phosphorylation of the RelA/p65 subunit occurs on serine 529, which is in the C-terminal (TA1) transactivation domain. Accordingly, the TNFα-induced phosphorylation of Rel/p65 increases NF-κB transcriptional activity but does not affect nuclear translocation or DNA binding affinity. Nuclear factor-κB (NF-κB) is an essential transcription factor in the control of expression of genes involved in immune and inflammatory responses. In unstimulated cells, NF-κB complexes are sequestered in the cytoplasm through interactions with IκBα and other IκB proteins. Extracellular stimuli that activate NF-κB, such as tumor necrosis factor α (TNFα), cause rapid phosphorylation of IκBα at serines 32 and 36. The inducible phosphorylation of IκBα is followed by its ubiquitination and degradation, allowing NF-κB complexes to translocate into the nucleus and to activate gene expression. Previously, it has been shown that TNFα as well as other stimuli also lead to the phosphorylation of the RelA/p65 subunit of NF-κB. In this report, we demonstrate that the TNFα-induced phosphorylation of the RelA/p65 subunit occurs on serine 529, which is in the C-terminal (TA1) transactivation domain. Accordingly, the TNFα-induced phosphorylation of Rel/p65 increases NF-κB transcriptional activity but does not affect nuclear translocation or DNA binding affinity. nuclear factor-κB tumor necrosis factor α inhibitor of kappa B IκB kinase lipopolysaccharide electrophoretic mobility shift assay mitogen-activated protein c-Jun N-terminal kinase. NF-κB1/Rel transcription factors are key regulators of transcription of a variety of genes involved in immune and inflammatory responses, growth, differentiation, development, and cell death (1Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4563) Google Scholar, 2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar, 3Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2910) Google Scholar). NF-κB was originally identified as a nuclear factor that binds to the enhancer element of the immunoglobulin kappa light chain gene (4Sen R. Baltimore D. Cell. 1986; 46: 705-716Abstract Full Text PDF PubMed Scopus (1901) Google Scholar). To date, eight members of the NF-κB/Rel proteins have been cloned and characterized. They are c-Rel, NF-κB1 (p50/p105), NF-κB2 (p52/p100), RelA (p65), RelB, and the Drosophila proteins Dorsal, Dif, and Relish (2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar). These proteins can form homo- or heterodimers through their N-terminal Rel homology domains, which also function in DNA binding and interaction with inhibitor proteins known as IκBs. The prototypical, inducible NF-κB complex is a heterodimer containing p50 and p65. The C-terminal region of p65 contains a potent transactivation domain that is lacking in p50 (1Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4563) Google Scholar, 2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar). In most cells, NF-κB is inactive due to its cytoplasmic sequestration through interactions with inhibitor proteins IκBs (1Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4563) Google Scholar, 2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar). The activation of NF-κB by a wide variety of stimuli such as mitogens, cytokines, bacterial lipopolysaccharide, viral infection, double-stranded RNA, and UV light involves the dissociation of NF-κB from IκB, allowing the nuclear translocation of the transcription factor (1Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4563) Google Scholar). There are seven members of the IκB family identified: IκBα, IκBβ, IκBγ, Bcl3, p105, p100, and IκBε as well asDrosophila IκB protein cactus (2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar, 6Whiteside S.T. Epinat J. Rice N.R. Israël A. EMBO J. 1997; 16: 1413-1426Crossref PubMed Scopus (337) Google Scholar, 7Li Z. Nabel G.J. Mol. Cell. Biol. 1997; 17: 6184-6190Crossref PubMed Google Scholar), each of which contains multiple copies of the ankyrin repeat. Stimulation of cells with inducers such as TNFα leads to rapid phosphorylation, ubiquitination, and degradation of IκBα. NF-κB is therefore released and translocates into the nucleus to activate the expression of target genes (2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar). Early studies implicated IκB phosphorylation as a crucial step for NF-κB activation, and much attention has been focused on the signal transduction pathway involved with induced phosphorylation of IκB. Recently, it was shown that two highly related serine kinases, IKKα and IKKβ, are induced in response to TNFα treatment and phosphorylate IκBα and IkBβ on critical serine residues known to be required for NF-κB activation (8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1890) Google Scholar, 9Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J.W. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1831) Google Scholar, 10Régnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar, 11Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1060) Google Scholar, 12Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1562) Google Scholar). Signals that induce phosphorylation of IκBs can also cause the phosphorylation of NF-κB proteins (13Naumann M. Scheidereit C. EMBO J. 1994; 13: 4597-4607Crossref PubMed Scopus (325) Google Scholar, 14Li C. Dai R. Chen E. Longo D.L. J. Biol. Chem. 1994; 269: 30089-30092Abstract Full Text PDF PubMed Google Scholar, 15Li C.H. Korner M. Ferris D.K. Chen E. Dai R. Longo D.L. Biochem. J. 1994; 303: 499-506Crossref PubMed Scopus (40) Google Scholar, 16Diehl J.A. Tong W. Sun G. Hannink M. J. Biol. Chem. 1995; 270: 2703-2707Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 17Schmitz M.L. dos Santos Silva M.A. Baeuerle P.A. J. Biol. Chem. 1995; 270: 15576-15584Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 18Zhong 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). For example, p50 is hyperphosphorylated in response to phorbol myristate acetate in Jurkat cells (15Li C.H. Korner M. Ferris D.K. Chen E. Dai R. Longo D.L. Biochem. J. 1994; 303: 499-506Crossref PubMed Scopus (40) Google Scholar). In vitro studies suggest that phosphorylation of p50 and p65 enhances NF-κB DNA binding ability (13Naumann M. Scheidereit C. EMBO J. 1994; 13: 4597-4607Crossref PubMed Scopus (325) Google Scholar, 14Li C. Dai R. Chen E. Longo D.L. J. Biol. Chem. 1994; 269: 30089-30092Abstract Full Text PDF PubMed Google Scholar). In vivo, the inducible phosphorylation on NF-κB subunits could also be correlated with dimerization, release from IκBs, nuclear translocation, or activation of transcription function of NF-κB. Recent work by Zhong et al. (18Zhong 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) demonstrated that LPS induced the phosphorylation of the p65/RelA subunit on serine 276 and increased its transactivating potential (18Zhong 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). Consistent with reports of others (16Diehl J.A. Tong W. Sun G. Hannink M. J. Biol. Chem. 1995; 270: 2703-2707Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), we report here that p65 phosphorylation is rapidly induced upon TNFα stimulation. Using phosphopeptide mapping and site-directed mutagenesis, we identified the inducible phosphorylation site as serine 529 in the C-terminal region of p65. A mutant p65 protein that has a serine 529 to alanine substitution cannot be phosphorylated in response to TNFα stimulation when stably expressed in fibroblasts from p65 −/− mice. Our data also demonstrate that inducible phosphorylation on p65 does not affect nuclear translocation or DNA binding ability but functions to increase its transcriptional activity. HeLa cells were grown in Dulbecco's modified Eagle's medium. Cos cells were grown in Iscove's minimal essential medium. All media were supplemented with 10% fetal bovine serum, penicillin, and streptomycin. Stable cell lines that express flag-empty, flag-p65, flag-p65(529A), or flag-p65(276A) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, and hygromycin (450 μg/ml). For 32P metabolic labeling, cells were grown in phosphate-free media with 2% serum for 3 h before32P H3PO4 was added. After 3 h of labeling, the cells were stimulated with TNFα (30 ng/ml) and harvested in cold radioimmunoprecipitation assay buffer (25 mm Tris, pH 7.6, 150 mm NaCl, 2 mmEDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) supplemented with phosphatase and protease inhibitors. Whole cell lysates were subjected to immunoprecipitation with p65 antibody, and the precipitated proteins were separated on SDS-PAGE and transferred to nitrocellulose (Schleicher 166: 368-379Crossref PubMed Scopus (10410) Google Scholar). After electrophoresis, the gel was dried and exposed to x-ray film at −80 °C for 3–10 days. For phosphoamino acid analysis, excised phosphorylated p65 from the nitrocellulose was incubated with 6 n HCl at 110 °C for 1 h. The resulting amino acids were lyophilized and applied to thin-layer cellulose plates with cold PAA standards (1.0 mg/ml each phosphoserine, phosphothreonine, and phosphotyrosine). Two-dimensional electrophoresis was carried out for 25 min at 1.5 kV in pH 1.9 buffer (formic acid:acetic acid:H2O = 50:156:1794) followed by 12 min at 1.3 kV in pH 3.5 buffer (pyridine:acetic acid:H2O = 1:10:189). Cold phosphoamino acids were detected by spraying the plates with 0.25% ninhydrin. Hot phosphoamino acids were visualized by exposing the plates to x-ray films at −80 °C for a week. F-p65, F539, F534, FΔ534, and F521 were made by cloning different PCR products into the HindIII and EcoRV sites of pFlag-CMV-2 expression vector. The template for the PCR reactions is CMV-p65. The 5′ primer is 1) 5′ GAC AAG CTT GAC GAA CTG TTC CCC CTC AT 3′. The 3′ primers are 2) GTC GAT ATC TTA GGA GCT GAT CTG ACT C (F-p65); 3) GAA GAT ATC GTC CGC AAT GGA GGA GAA GT (F539); 4) GCG GAT ATC GAA GTC TTC ATC TCC TGA AAG GA (F534); 5) GCG GAT ATC GAA GTC TTC ATC TCC TGC AAG G and GCG GAT ATC CCC and were made by site-directed The template for the PCR is CMV-p65. The primers and 5′ CTT CTC CTG GGA C 5′ CTC CTT GGA GAT GAA G 3′ 5′ CTT CTC CTT C 5′ CTC GAT GAA G 3′ 5′ GCG GCG TGC GGA G and 5′ CTC GTC C the that amino The products of PCR were cloned into the HindIII and EcoRV sites of the vector. Nuclear and cytoplasmic were as Jr., A.S. Mol. Cell. Biol. 13: PubMed Google Scholar). were as S. A. P. Jr., A.S. Cell. Full Text PDF PubMed Scopus Google Scholar). The DNA contains the NF-κB binding site from the For a of of cell lysates or nuclear were separated on a 10% were transferred to and were with and with After the were incubated with antibody, were to the proteins. were D. Jr., A.S. 1997; 14: PubMed Scopus Google Scholar). For TNFα cells were grown in Dulbecco's modified Eagle's medium with 0.1% serum for h after with TNFα ng/ml) was to the cells h were by Jr., A.S. J. 1997; PubMed Scopus Google Scholar). For a the hygromycin protein was with expression The were by hygromycin to the The expression of p65 proteins was by inducible phosphorylation on IκBs an in NF-κB activation, NF-κB/Rel proteins are also phosphorylated (13Naumann M. Scheidereit C. EMBO J. 1994; 13: 4597-4607Crossref PubMed Scopus (325) Google Scholar, 14Li C. Dai R. Chen E. Longo D.L. J. Biol. Chem. 1994; 269: 30089-30092Abstract Full Text PDF PubMed Google Scholar, 15Li C.H. Korner M. Ferris D.K. Chen E. Dai R. Longo D.L. Biochem. J. 1994; 303: 499-506Crossref PubMed Scopus (40) Google Scholar, 16Diehl J.A. Tong W. Sun G. Hannink M. J. Biol. Chem. 1995; 270: 2703-2707Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 17Schmitz M.L. dos Santos Silva M.A. Baeuerle P.A. J. Biol. Chem. 1995; 270: 15576-15584Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 18Zhong 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). Consistent with of others (16Diehl J.A. Tong W. Sun G. Hannink M. J. Biol. Chem. 1995; 270: 2703-2707Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), we that p65 phosphorylation is rapidly induced upon TNFα stimulation in HeLa After treatment with TNFα for cell lysates from 32P HeLa cells were and with A TNFα-induced of was detected electrophoresis The of this protein as p65 was by with the which the was not The of p65 protein not in response to TNFα in p65 cannot the increase in a of phosphorylation of p65 in cells was also p65 from cells or cells with TNFα for min was with 6 n and subjected to phosphoamino acid The inducible p65 phosphorylation was on serine residues phosphorylation of p65 is on serine p65 from or HeLa cells was with 6 n The resulting amino acids were separated on layer cellulose The the of the phosphoamino acid The phosphoamino acids were visualized by To a potential function for inducible phosphorylation of p65 in NF-κB activation, it was for to the site of Tris-Tricine gel electrophoresis to the from HeLa cells or from cells with TNFα for a phosphopeptide of in and cells 3 that inducible phosphorylation occurs on the as occurs in phosphorylation, and on the serine To the of the phosphopeptide in the was subjected to of immunoprecipitation with C-terminal or N-terminal p65 The could be by a C-terminal p65 but not by an N-terminal p65 3 3 and the phosphopeptide is at the C of p65. trypsin after and residues and the phosphopeptide is it was to that the target of phosphorylation was in the amino C-terminal to 3 The other were to from their was on the of protease not of p65 from cells and cells with V8 a phosphopeptide of 3 could also be by a C-terminal p65 not V8 the after the this protease can a at the C of p65 was by at and 3 The region of the and V8 is from to and in this region is 3 The of phosphopeptide mapping also that the phosphorylation site of p65 is the as the phosphorylation site not These data that serine 529 is the site of phosphorylation on p65 in response to TNFα stimulation. To that serine 529 of p65 is the phosphorylation we therefore made a of expression C-terminal p65 to proteins can be phosphorylated when expressed in Cos After the cells with cell lysates were made and subjected to immunoprecipitation with the p65 was phosphorylated as were and which express p65 proteins from amino acids and FΔ534, which the p65 protein as has a serine to alanine substitution at 529, cannot be In the mutant F521 the amino acids of p65 and was of demonstrated that F521 expressed the of protein expressed the The of phosphorylation of and are due to of expression of proteins to is that serine 529 was or the mutant p65 was serine 529 of p65 is the target for the phosphorylation of p65 in Cos cells was by TNFα was that the kinase that p65 has the potential to function at in Cos cells To demonstrate that serine 529 is the TNFα-induced phosphorylation we made p65 mutant p65 but has an alanine to serine substitution at in Cos cells, p65 has phosphorylation we stably expressed and into p65 −/− The cells that the or were with and stimulated with TNFα for After cells were the cell were subjected to immunoprecipitation with The proteins were separated by SDS-PAGE and visualized by autoradiography The demonstrate that TNFα-induced phosphorylation with p65 3 and phosphorylation in response to TNFα and that the of phosphorylation of was not due to protein Recently, it was that serine 276 of p65 was phosphorylated by protein kinase A after LPS (18Zhong 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). was therefore that different inducers target sites on p65 for To this we made a p65 mutant that has an alanine to serine substitution at stably expressed in p65 −/− cells, this mutant can be phosphorylated in response to TNFα and that and phosphorylation sites on p65 are is that of expression of the alanine 276 mutant leads to phosphorylation, to in To the for TNFα-induced phosphorylation to p65 we the cell lines or The cells were with TNFα for and nuclear were for 6 and with p65 6 The identified two complexes that to the NF-κB The complex can be by a p50 antibody, it is the The complex can be with a p65 antibody, this complex contains the p65 subunit 6 The by the p50 is due to the ability of this to the is that and mutant p65 rapidly to nucleus after TNFα and for at h 6 was in DNA binding for TNFα-induced These demonstrate that the phosphorylation of p65 on serine 529 does not control nuclear translocation or DNA binding affinity. Previously, it has been shown that the C of which contains serine 529, functions as a transactivation domain when to DNA binding M.L. Baeuerle P.A. EMBO J. PubMed Scopus Google Scholar). on serine 529 the transcriptional activity of p65. To this the cells or alanine 529 were with a which contains three copies of binding To assay transcription TNFα was to the cells, and cell lysates were made for shown in 6 TNFα activity in the cells p65 but or in the cells mutant that serine 529 of p65 is the target for TNFα and that the inducible phosphorylation on this site increases p65 transcriptional activity. The transcription in the cells or with the cells be due to nuclear NF-κB in the also the that as a of other members of the NF-κB The NF-κB to a was by TNFα in the cells but not in the cells not of serine 529 the ability of TNFα to activate To that phosphorylation on serine 529 increases p65 transcription we made which has a acid substitution at 529 to phosphorylated p65. For we were to stably express in the p65 cell In and 6 that a that phosphorylation at 529 leads to p65 transcriptional activity. Stimulation of cells with TNFα leads to phosphorylation and degradation of IκBα and to translocation of NF-κB to the nucleus to activate In this and with reports (16Diehl J.A. Tong W. Sun G. Hannink M. J. Biol. Chem. 1995; 270: 2703-2707Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), we have shown that TNFα also phosphorylation on the p65 subunit of NF-κB. mapping of TNFα-induced phosphorylated p65 that phosphorylation occurs on serine the of p65 −/− fibroblasts stably p65 or the mutant we that TNFα-induced phosphorylation on p65 does not affect its nuclear translocation or DNA binding but increases its transcriptional with also that phosphorylation on serine 529 increases p65 transactivation RelA/p65 contains at two transactivation in its C-terminal region M.L. Baeuerle P.A. EMBO J. PubMed Scopus Google Scholar, P.A. Mol. Cell. Biol. 13: PubMed Google Scholar). 529 is the which the amino acids of p65. to the of it is not that the by the at serine 529 increases its transcriptional The C-terminal transcriptional activation domain of p65 with and such as and M.L. G. H. M. Baeuerle P.A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, Nabel G.J. Science. 1997; PubMed Scopus Google Scholar, A.S. S. T. S. A. 1997; PubMed Scopus Google Scholar). be to the phosphorylation on serine 529 of it that phosphorylation interaction with other transcription factors and with the ability of NF-κB to the inducible phosphorylation of p65 have different on different The most for inducible NF-κB activation is the phosphorylation of IκBs on serines in the N-terminal region of the proteins (2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar). inducers on this step that involves activation, which degradation of IκBs and nuclear translocation of NF-κB (2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar). Our data and of others that is a of on NF-κB of p65 transactivation potential by phosphorylation et al. M.L. dos Santos Silva M.A. Baeuerle P.A. J. Biol. Chem. 1995; 270: 15576-15584Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) that phosphorylation and transcriptional activity of a region the domain amino acids to were stimulated by phorbol myristate acetate treatment of HeLa Recently, al. (18Zhong 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) that upon LPS the transcriptional activity of p65 was increased after phosphorylation on serine which is in the Rel homology domain of p65. we that a mutant p65 protein with an alanine to serine substitution at 276 can be phosphorylated upon TNFα treatment it is that different inducers can activate different to phosphorylate p65 at sites to its transcriptional activity. of p65 at serine 276 enhances the ability of this transcription factor to with the transcriptional H. R. Ghosh S. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). phosphorylation have the or lead to functions is Recent data from Jr., A.S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and others J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) demonstrate that inducers can control the transcriptional function of NF-κB, of induced nuclear does TNFα induce phosphorylation of TNFα and and studies that and kinase are involved in NF-κB has been that can with and activate and X. C. D. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). p65 phosphorylation is to be et al. R. A. W. S. G. P. W. EMBO J. 1996; PubMed Scopus Google Scholar) that kinase pathway was required for transcriptional by NF-κB on nuclear translocation or DNA binding of NF-κB. Recently, and W. S. E. M.L. W. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that and target the transactivation domain of p65 in response to All data suggest that or kinase a of of NF-κB activation by of transcription or can phosphorylate p65 or control other to phosphorylate p65 In we that a not TNFα-induced p65 phosphorylation not or cause phosphorylation of a of the transcription pathway to p65 transactivation Recently, it was that kinase can phosphorylate the p65 subunit and that kinase is with p65 in H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). kinase could be to induce the potential phosphorylation of p65 is p65 gene expression TNFα 6 and data not for this is that in cells cannot IκB protein to p65 in that p65 has phosphorylation on serine 529 it is that the kinase that p65 is kinase phosphorylate p65 when it is released from IκB example, IκB degradation or when p65 is to function as an inducible kinase. The of the kinase that serine 529 of p65 into TNFα NF-κB activity. a kinase activity to be a target in with of NF-κB activity. for in this work and for and for and with the phosphoamino acid analysis, and for critical of the for p65