IκB Kinases Phosphorylate NF-κB p65 Subunit on Serine 536 in the Transactivation Domain

Recent investigations have elucidated the cytokine-induced NF-κB activation pathway. IκB kinase (IKK) phosphorylates inhibitors of NF-κB (IκBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-κB. We have examined the possibility that IKK can phosphorylate the p65 NF-κB subunit as well as IκB in the cytokine-induced NF-κB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-α in a time dependent manner similar to IκB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKKβ efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-κB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-κB·IκB complex. Recent investigations have elucidated the cytokine-induced NF-κB activation pathway. IκB kinase (IKK) phosphorylates inhibitors of NF-κB (IκBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-κB. We have examined the possibility that IKK can phosphorylate the p65 NF-κB subunit as well as IκB in the cytokine-induced NF-κB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-α in a time dependent manner similar to IκB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKKβ efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-κB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-κB·IκB complex. nuclear factor-κB IκB kinase NF-κB-inducing kinase mitogen-activated protein kinase kinase kinase tumor necrosis factor hemagglutinin polyacrylamide gel electrophoresis glutathione S-transferase N-acetyl-leucyl-leucyl-norleucinal Xpress transcriptional activation domain Rel homology domain transforming growth factor-β activated kinase 1 TAK1-binding protein 1 The transcription factor nuclear factor-κB (NF-κB)1 plays a pivotal role in inflammatory and immune responses (1Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4599) Google Scholar, 2Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1662) Google Scholar, 3Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2931) Google Scholar). NF-κB is composed of a heterodimer of p65 and p50 subunits in most cell types and is sequestered in the cytoplasm by its inhibitor proteins, the IκBs (4Haskill S. Beg A.A. Tompkins S.M. Morris J.S. Yurochko A.D. Sampson-Johannes A. Mondal K. Ralph P. Baldwin Jr., A.S. Cell. 1991; 65: 1281-1289Abstract Full Text PDF PubMed Scopus (586) Google Scholar, 5Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 6Whiteside S.T. Epinat J.C. Rice N.R. Israel A. EMBO J. 1997; 16: 1413-1426Crossref PubMed Scopus (342) Google Scholar, 7Li Z. Nabel G.J. Mol. Cell. Biol. 1997; 17: 6184-6190Crossref PubMed Google Scholar, 8Simeonidis S. Liang S. Chen G. Thanos D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14372-14377Crossref PubMed Scopus (78) Google Scholar). Several NF-κB-activating agents, including pro-inflammatory cytokines, phorbol esters, and bacterial lipopolysaccaride, induce the phosphorylation of IκBs at two NH2-terminal Ser residues. The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, thereby releasing NF-κB to enter the nucleus for gene expression (9Traenckner E.B. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (933) Google Scholar, 10Scherer D.C. Brockman J.A. Chen Z. Maniatis T. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11259-11263Crossref PubMed Scopus (501) Google Scholar, 11Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1170) Google Scholar, 12DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar, 13Rodriguez M.S. Wright J. Thompson J. Thomas D. Baleux F. Virelizier J.L. Hay R.T. Arenzana-Seisdedos F. Oncogene. 1996; 12: 2425-2435PubMed Google Scholar, 14Roff M. Thompson J. Rodriguez M.S. Jacque J.M. Baleux F. Arenzana-Seisdedos F. Hay R.T. J. Biol. Chem. 1996; 271: 7844-7850Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 15Weil R. Laurent-Winter C. Israel A. J. Biol. Chem. 1997; 272: 9942-9949Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Recent investigations have focused on the phosphorylation of IκBs and clearly elucidated the molecular mechanisms of the phosphorylation. In brief, two closely related kinases, designated IκB kinase (IKK) α and IKKβ, have been identified as components of the multiprotein IKK complex (500–900 kDa) that directly phosphorylates the critical Ser residues of IκBs (16DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1913) Google Scholar, 17Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar, 18Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1589) Google Scholar, 19Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar, 20Regnier 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 (1072) Google Scholar). IKKα and IKKβ together form a heterodimer through their COOH-terminal leucine zipper motifs, and the functional IKK complex contains both IKK subunits. NF-κB-inducing kinase (NIK), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, interacts with and activates the IKK complex (21Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1165) Google Scholar). Other MAP3Ks, including transforming growth factor-β activated kinase 1 (TAK1) (22Sakurai H. Shigemori N. Hasegawa K. Sugita T. Biochem. Biophys. Res. Commun. 1998; 243: 545-549Crossref PubMed Scopus (89) Google Scholar, 23Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 24Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1019) Google Scholar), MAPK/extracellular signal-regulated kinase kinase kinases (MEKK1–3) (25Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar, 26Yin M.J. Christerson L.B. Yamamoto Y. Kwak Y.T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 27Nakano H. Shindo M. Sakon S. Nishinaka S. Mihara M. Yagita H. Okumura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3537-3542Crossref PubMed Scopus (471) Google Scholar, 28Zhao Q. Lee F.S. J. Biol. Chem. 1999; 274: 8355-8358Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), and Cot/Tpl2 (29Lin X. Cunningham E.T. Mu Y. Geleziunas R. Greene W.C. Immunity. 1999; 10: 271-280Abstract Full Text Full Text PDF PubMed Google Scholar), have been shown to be involved in the IKK activation pathways, indicating the important roles of MAP3K family kinases in the IKK activation by diverse extracellular stimuli. The activity of several inducible transcription factors, including cAMP response element-binding protein (CREB) (30Nichols M. Weih F. Schmid W. DeVack C. Kowenz-Leutz E. Luckow B. Boshart M. Schutz G. EMBO J. 1992; 11: 3337-3346Crossref PubMed Scopus (276) Google Scholar) and c-Jun (31Smeal T. Binetruy B. Mercola D.A. Birrer M. Karin M. Nature. 1991; 354: 494-496Crossref PubMed Scopus (698) Google Scholar), has been shown to be regulated by phosphorylation. It has been shown that the p65 NF-κB subunit is also phosphorylated during the phosphorylation and degradation of IκBs. However, the cytokine-inducible phosphorylating activity of p65 remains to be characterized. Here we show that IKKs are possible p65 kinases in the TNF-α-induced NF-κB activation, and the phosphorylation site is Ser-536 in the COOH-terminal transactivation domain. HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2. Cells were treated with 20 ng/ml TNF-α (R & D Systems) for the indicated time period. Where indicated, cells were treated with a proteasome inhibitor,N-acetyl-leucyl-leucyl-norleucinal (Nacalai Tesque). After stimulating with TNF-α, cytoplasmic and nuclear extracts were prepared as described previously (23Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). The nuclear translocation of p65 and the degradation of IκBα were determined by Western blotting with an anti-p65 antibody (C-20; Santa Cruz Biotechnology) and an anti-IκBα antibody (C-21; Santa Cruz Biotechnology) using nuclear and cytoplasmic extracts, respectively. For metabolic labeling with [32P]orthophosphate, HeLa cells were washed twice with phosphate-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and subsequently incubated with 1 mCi/ml [32P]orthophosphate for 3 h. After stimulating the cells with TNF-α for a given period, we immunoprecipitated p65 or HA-p65 with the anti-p65 antibody or anti-HA antibody as described previously. The precipitated proteins were separated by 7.5% SDS-PAGE and autoradiographed. The cDNA encoding full-length p65 was obtained from HeLa cells by reverse transcription-polymerase chain reaction. Several deletion cDNAs were inserted into pGEX-5X-3 bacterial expression vector (Amersham Pharmacia Biotech). The GST-fused p65 proteins were expressed inEscherichia coli and purified with glutathione-Sepharose (Amersham Pharmacia Biotech). Point mutations were made by using a QuikChange site-directed mutagenesis kit (Stratagene) and all of the mutations were verified by DNA sequencing analysis. HeLa cells were transfected with expression vectors for Xpress (XP) epitope-tagged IKKs, FLAG epitope-tagged TAK1, HA epitope-tagged TAB1, and HA epitope-tagged p65 using LipofectAMINE reagents (Life Technologies, Inc.). For expression of IKKβ, NIK, and TAK1/TAB1 as 6 × His-tagged proteins in the Baculovirus system, Sf21 insect cells were infected with recombinant viruses generated by co-transfection with the BaculoGold DNA and transfer vectors (pAcHLT-NIK, pAcHLT-IKKβ, or pAcUW51-TAK1/TAB1) (PharMingen). The recombinant kinases were purified by nickel column chromatography (Amersham Pharmacia Biotech). TNF-α-stimulated whole cell lysates, cytoplasmic extracts, immunoprecipitated IKKs, or Baculovirus-expressed recombinant IKKβ were incubated with 1 μg of GST-fused p65 in kinase buffer (20 mm HEPES (pH 7.6), 20 mm MgCl2, 2 mm dithiothreitol, 20 μm ATP, 20 mm β-glycerophosphate, 20 mm disodium p-nitrophenyl phosphate, 0.1 mm sodium orthovanadate, 3 μCi [γ-32P]ATP) at 30 °C for 30 min. The phosphorylated GST-p65 was separated by 10% SDS-PAGE and autoradiographed. Treatment of HeLa cells with TNF-α induced the degradation of IκBα and the subsequent nuclear translocation of p65 NF-κB within 5 min after the treatment (Fig. 1 A). A phosphorylated form of IκBα was detected at 2–5 min, indicating an inducible kinase activity of the endogenous IKK complex (Fig.1 A). Interestingly, an in vivo 32P metabolic labeling immunoprecipitation analysis showed that p65 was also phosphorylated at the time of the IκBα phosphorylation (Fig.1 B). To establish whether the phosphorylation of p65 occurred in the cytoplasm or in the nucleus, the cells were treated with a proteasome inhibitor,N-acetyl-leucyl-leucyl-norleucinal (ALLN). The treatment with ALLN caused an accumulation of the phosphorylated form of IκBα, resulting in an impaired nuclear translocation of p65 (Fig. 1 C). In contrast, the phosphorylated p65 could be detected even in the presence of ALLN, indicating that the phosphorylation occurred in the cytoplasm prior to the nuclear translocation (Fig. 1 D). To characterize the p65 kinase activity in vitro, we generated NH2-terminal (from amino acid 1 to 305) and COOH-terminal (from amino acid 354 to 551) p65 proteins fused with GST. An inducible kinase activity was detected in whole cell lysates of TNF-α-stimulated HeLa cells when the COOH-terminal p65 was used as a substrate (Fig. 2 A). Zhonget al. (32Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar) reported that Ser-276 of p65 was phosphorylated by PKA; however, the TNF-α-induced p65 kinase did not phosphorylate GST-p65-(1–305) containing the Ser residue (Fig. 2 A). Interestingly, the in vitro p65 phosphorylating activity was induced in a time-dependent manner similar to the phosphorylation of p65 in vivo (Fig. 1 B). In addition, the activity was extracted into the cytoplasmic fraction (Fig. 2 B), suggesting that the p65 phosphorylating activity was efficiently extracted from TNF-α-treated HeLa cells. We next determined the phosphorylation site in the COOH-terminal p65 using the TNF-α-treated cytoplasmic extracts as a kinase source. GST-p65-(354–521), a mutant lacking the 30 NH2-terminal amino acids (TA1 domain), failed to be phosphorylated by the activity (Fig. 3 A). Wang et al. (33Wang D. Baldwin A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar) recently reported that TNF-α induced p65 phosphorylation at Ser-529 in this domain. In contrast, our in vitro kinase assays using Ser to Ala substitution mutants indicate that the phosphorylation site is Ser-536 (Fig. 3 B). In contrast to Ser-529, the target Ser residue is conserved in human, mouse, chicken, and Xenopus p65 subunits (Fig.3 C), suggesting a role for the phosphorylation in the transactivation of NF-κB. We previously reported that the endogenous IKK kinase activity was induced by TNF-α in a time-dependent manner similar to the p65 phosphorylating activity (23Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). We therefore investigated whether the p65 kinase is a component of the IKK complex. The endogenous IKK complex was examined for kinase activity against GST-p65-(354–551) and GST-IκBα-(1–54) (Fig.4 A). Activity could be detected for GST-p65-(354–551), which was comparable with the IKK activity for GST-IκBα-(1–54). Moreover, the p65 phosphorylating activity was competed by an excess amount of GST-IκBα-(1–54), but not GST-c-Jun-(1–79) (Fig. 4 B). We further examined whether two IKK subunits can phosphorylate the p65 subunit by using an overexpression experiment (Fig. 4 C). HeLa cells were transfected with expression vectors for Xpress-epitope tagged IKKs (XP-IKKs) with or without expression vectors for FLAG-epitope tagged TAK1 (FLAG-TAK1) and the TAK1 activator, hemagglutinin-epitope-tagged TAB1 (HA-TAB1). An anti-XP immunocomplex kinase assay showed that TAK1-activated IKKs phosphorylated p65 (Fig. 4 C). These results suggest that the p65 kinase is a component of the IKK complex. Some protein kinases, such as TAK1, NIK, IKKα, and IKKβ, have been shown to be components of the IKK complex. We generated recombinant TAK1, NIK, and IKKβ as 6 × His-tagged proteins by using the Baculovirus expression system and examined their abilities to phosphorylate GST-p65-(354–551) (Fig. 4 D). Only IKKβ could phosphorylate GST-p65, whereas TAK1 and NIK showed autophosphorylation activities. The site of phosphorylation by IKKβ was also Ser-536. These results indicate that the p65 phosphorylation may be mediated by IKKs in vitro. We next investigated whether p65 is a substrate for IKKs in vivo by co-transfection and metabolic labeling analyses. HA-p65 was transiently co-expressed in HeLa cells together with XP-IKKα, XP-IKKβ, and FLAG-NIK. Cell lysates were immunoprecipitated with an anti-HA antibody and analyzed by SDS-PAGE (Fig.5 A). The phosphorylation of HA-p65 was detected when XP-IKKs were activated by the co-expression with Flag-NIK. In addition, the TNF-α-induced phosphorylation of p65 occurred at Ser 536, as demonstrated by the reduced phosphorylation of HA-p65 (S536A). Taken together, these results indicate that the p65 NF-κB subunit is phosphorylated by IKKs in the cytokine-induced NF-κB activation pathway. NF-κB p65 has been shown to be phosphorylated along with phosphorylation of IκB. In contrast to the IκB phosphorylation, the p65 phosphorylation has not been well characterized. Here we show that TNF-induced phosphorylation of p65 is mediated by IKKs prior to the nuclear translocation. It is reasonable that p65 and IκB are phosphorylated by the same protein kinases, since they associate in the cytoplasm and are phosphorylated in a similar time-dependent fashion in response to TNF-α. Mercurioet al. (34Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. Pascual G. Motiwala A. Zhu H. Mann M. Manning A.M. Mol. Cell. Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar) recently reported that p65, but not other Rel family members c-Rel and p52, was phosphorylated by a recombinant constitutive active mutant of IKKβ in vitro with a specificity constant similar to that for IκBα, suggesting a physiological role of the phosphorylation. There are two IKK subunits, and they form a homodimer or heterodimer in the IKK complex; however, the physiological role of the dimerization is still unclear. It is possible that one component of the dimer phosphorylates IκB and the other phosphorylates p65. Recently, IKKα- and IKKβ-deficient mice have been developed (35Takeda K. Takeuchi O. Tsujimura T. Itami S. Adachi O. Kawai T. Sanjo H. Yoshikawa K. Terada N. Akira S. Science. 1999; 284: 313-316Crossref PubMed Scopus (539) Google Scholar, 36Hu Y. Baud V. Delhase M. Zhang P. Deerinck T. Ellisman M. Johnson R. Karin M. Science. 1999; 284: 316-320Crossref PubMed Scopus (714) Google Scholar, 37Li Q. Lu Q. Hwang J.Y. Buscher D. Lee K.F. Izpisua-Belmonte J.C. Verma I.M. Genes Dev. 1999; 13: 1322-1328Crossref PubMed Scopus (417) Google Scholar, 38Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Crossref PubMed Scopus (856) Google Scholar, 39Tanaka M. Fuentes M.E. Yamaguchi K. Durnin M.H. Dalrymple S.A. Hardy K.L. Goeddel D.V. Immunity. 1999; 10: 421-429Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 40Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Crossref PubMed Scopus (822) Google Scholar). The IKK complex derived from these mice may form a homodimer of the counterpart IKK subunit and showed intact kinase activities for p65 in vitro (37Li Q. Lu Q. Hwang J.Y. Buscher D. Lee K.F. Izpisua-Belmonte J.C. Verma I.M. Genes Dev. 1999; 13: 1322-1328Crossref PubMed Scopus (417) Google Scholar, 38Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Crossref PubMed Scopus (856) Google Scholar). In contrast, IKK complex derived from IKK-β deficient mice, but not IKK-α, has impaired phosphorylation of IκBs in vitro, indicating that the IKKα homodimer might recognize p65, but not IκBs, as a substrate. Future analysis of the three-dimensional structure of IKKs·NF-κB·IκB complex will elucidate the spatial localization of these components and the role of dimerization of IKKs in the phosphorylation of the NF-κB·IκB complex. In addition, the development of an IKKα/IKKβ double knockout mouse will provide more information on the p65 phosphorylation. The transcriptional activation domain (TAD) of p65 has been characterized by using fusion protein with the DNA-binding domain of the yeast GAL4 transcription factor (41Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (666) Google Scholar, 42Schmitz M.L. dos Santos Silva M.A. Altmann H. Czisch M. Holak T.A. Baeuerle P.A. J. Biol. Chem. 1994; 269: 25613-25620Abstract Full Text PDF PubMed Google Scholar, 43Schmitz 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). The COOH-terminal 30 amino acids (TA1 domain) comprise the most important transactivation domain, which is predicted to be α-helix. In addition, there are seven Ser residues in the TA1 domain of human p65 and they locate on one face of the presumptive α-helix, suggesting transcriptional regulation by phosphorylation. In fact, Wang et al. (33Wang D. Baldwin A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar) recently reported that p65 was phosphorylated at Ser-529 in the TA1 domain by an undefined protein kinase in response to TNF-α. Here we demonstrated the IKK-mediated phosphorylation of p65 at Ser-536 in the TA1 domain. In addition, Zhong et al. (32Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar) reported that protein kinase A phosphorylated p65 at Ser-276 in the NH2-terminal Rel homology domain (RHD), which promoted an interaction of p65 with the transcriptional co-activator CBP/p300. Furthermore, MAPK cascades that are sensitive to the MAPK kinase (MEK1, MEK2) inhibitor PD98059 and the p38 MAPK inhibitor SB203580 were shown to enhance the TNF-α-induced transactivation of the p65 subunit (44Vanden Berghe W. Plaisance S. Boone E. De Bosscher K. Schmitz M.L. Fiers W. Haegeman G. J. Biol. Chem. 1998; 273: 3285-3290Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar). Thus, these observations indicate that the NF-κB transactivation may be regulated by multiple phosphorylations in TAD and RHD. In summary, we demonstrated the IKK-mediated phosphorylation of p65 in the cytokine-induced NF-κB activation pathway. Previous studies on the characterization of p65 TAD employed the GAL4 system. However, this system does not reflect inducible phosphorylations in the cytoplasm, because the fusion proteins translocated into the nucleus in a stimulus-independent manner. Therefore, the development of a new transactivation assay system evaluating the IKK-mediated phosphorylation is necessary for future characterization of the physiological role of the phosphorylation. We are grateful to Drs. M. Hibi and K. Hasegawa for materials. We are also grateful to Drs. K. Matsumoto and N. Yanaka for helpful discussions on the manuscript. We thank E. Yamada for DNA sequencing.