Phosphorylation of Serine 468 by GSK-3β Negatively Regulates Basal p65 NF-κB Activity

The activity of NF-κB is controlled at several levels including the phosphorylation of the strongly transactivating p65 (RelA) subunit. However, the overall number of phosphorylation sites, the signaling pathways and protein kinases that target p65 NF-κB and the functional role of these phosphorylations are still being uncovered. Using a combination of peptide arrays with in vitro kinase assays we identify serine 468 as a novel phosphorylation site of p65 NF-κB. Serine 468 lies within a GSK-3β consensus site, and recombinant GSK-3β specifically phosphorylates a GST-p65-(354–551) fusion protein at Ser468in vitro. In intact cells, phosphorylation of endogenous Ser468 of p65 is induced by the PP1/PP2A phosphatase inhibitor calyculin A and this effect is inhibited by the GSK-3β inhibitor LiCl. Reconstitution of p65-deficient cells with a p65 protein where serine 468 was mutated to alanine revealed a negative regulatory role of serine 468 for NF-κB activation. Collectively our results suggest that a GSK-3β-PP1-dependent mechanism regulates phosphorylation of p65 NF-κB at Ser468 in unstimulated cells and thereby controls the basal activity of NF-κB. The activity of NF-κB is controlled at several levels including the phosphorylation of the strongly transactivating p65 (RelA) subunit. However, the overall number of phosphorylation sites, the signaling pathways and protein kinases that target p65 NF-κB and the functional role of these phosphorylations are still being uncovered. Using a combination of peptide arrays with in vitro kinase assays we identify serine 468 as a novel phosphorylation site of p65 NF-κB. Serine 468 lies within a GSK-3β consensus site, and recombinant GSK-3β specifically phosphorylates a GST-p65-(354–551) fusion protein at Ser468in vitro. In intact cells, phosphorylation of endogenous Ser468 of p65 is induced by the PP1/PP2A phosphatase inhibitor calyculin A and this effect is inhibited by the GSK-3β inhibitor LiCl. Reconstitution of p65-deficient cells with a p65 protein where serine 468 was mutated to alanine revealed a negative regulatory role of serine 468 for NF-κB activation. Collectively our results suggest that a GSK-3β-PP1-dependent mechanism regulates phosphorylation of p65 NF-κB at Ser468 in unstimulated cells and thereby controls the basal activity of NF-κB. NF-κB is a dimeric transcription factor that plays an important role in the immune response, cell survival, and cancer. NF-κB activity is controlled at two levels: (i) by proteasome-dependent generation of DNA-binding subunits and (ii) by regulation of its nuclear function. In the recent years, evidence has accumulated that post-translational modifications of the DNA-binding subunits add another level of regulation for the function of NF-κB (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar, 2Schmitz M.L. Mattioli I. Buss H. Kracht M. ChemBioChem. 2004; 5: 1348-1358Crossref PubMed Scopus (221) Google Scholar, 3Chen L.F. Greene W.C. Nat. Rev. Mol. Cell. Biol. 2004; 5: 392-401Crossref PubMed Scopus (1049) Google Scholar). A number of protein kinases have been shown to phosphorylate the strongly transactivating subunit p65 at Ser276 (4Zhong H. May M.J. Jimi E. Ghosh S. Mol. Cell. 2002; 9: 625-636Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar), Ser311 (5Duran A. Diaz-Meco M.T. Moscat J. EMBO J. 2003; 22: 3910-3918Crossref PubMed Scopus (272) Google Scholar), Ser529 (6Wang 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 (373) Google Scholar), and Ser536 (7Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar), and, with the exception of Ser529, phospho-specific antibodies have confirmed phosphorylation of endogenous p65 at these sites. The relevance of regulatory phosphorylations is also evident from the analysis of cells lacking the protein kinases GSK-3β (8Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgett J.R. Nature. 2000; 406: 86-90Crossref PubMed Scopus (1228) Google Scholar), TBK1/NAK (9Bonnard 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, 10Tojima Y. Fujimoto A. Delhase M. Chen Y. Hatakeyama S. Nakayama K. Kaneko Y. Nimura Y. Motoyama N. Ikeda K. Karin M. Nakanishi M. Nature. 2000; 404: 778-782Crossref PubMed Scopus (316) Google Scholar), IKKϵ (11Kravchenko V.V. Mathison J.C. Schwamborn K. Mercurio F. Ulevitch R.J. J. Biol. Chem. 2003; 278: 26612-26619Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), NIK (12Yin L. Wu L. Wesche H. Arthur C.D. White J.M. Goeddel D.V. Schreiber R.D. Science. 2001; 291: 2162-2165Crossref PubMed Scopus (351) Google Scholar), and PKCζ (13Leitges 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 (325) Google Scholar), which show an intact IκB phosphorylation but an impaired expression of NF-κB target genes. Nonetheless, the overall number of p65 phosphorylation sites is not yet known as is the number of all potential p65 protein kinases. As an example, we and others (14Buss H. Dörrie A. Schmitz M.L. Hoffmann E. Resch K. Kracht M. J. Biol. Chem. 2004; (October 15, 10.1074/jbc.M409825200)Google Scholar, 15Hu J. Nakano H. Sakurai H. Colburn N.H. Carcinogenesis. 2004; 25: 1991-2003Crossref PubMed Scopus (111) Google Scholar, 16Bohuslav J. Chen L.F. Kwon H. Mu Y. Greene W.C. J. Biol. Chem. 2004; 279: 26115-26125Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 17Mattioli 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 (187) Google Scholar) have recently shown that at least six distinct kinases converge on phosphorylation of p65 at Ser536. The molecular mechanisms and the biological consequences of p65 phosphorylation are currently a focal point of intense research (2Schmitz M.L. Mattioli I. Buss H. Kracht M. ChemBioChem. 2004; 5: 1348-1358Crossref PubMed Scopus (221) Google Scholar, 3Chen L.F. Greene W.C. Nat. Rev. Mol. Cell. Biol. 2004; 5: 392-401Crossref PubMed Scopus (1049) Google Scholar). Most p65 phosphorylation sites are located in the COOH-terminal part of the Rel homology domain and in the COOH-terminal transactivation domains (2Schmitz M.L. Mattioli I. Buss H. Kracht M. ChemBioChem. 2004; 5: 1348-1358Crossref PubMed Scopus (221) Google Scholar, 3Chen L.F. Greene W.C. Nat. Rev. Mol. Cell. Biol. 2004; 5: 392-401Crossref PubMed Scopus (1049) Google Scholar). The involvement of GSK-3β in activation of NF-κB as suggested by gene deletion has been a surprising finding, as NF-κB activating stimuli such as IL-1, TNF, or phorbol ester will inactivate GSK-3β by phosphatidylinositol 3-kinase/AKT-mediated phosphorylation of its NH2 terminus (18Doble B.W. Woodgett J.R. J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1774) Google Scholar, 19Cohen P. Goedert M. Nat. Rev. Drug Discov. 2004; 3: 479-487Crossref PubMed Scopus (677) Google Scholar, 20Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar). Furthermore, it is suggested that in NF-κB activation the role of GSK-3β is non-redundant as the closely related enzyme GSK-3α cannot compensate for the loss of GSK-3β (8Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgett J.R. Nature. 2000; 406: 86-90Crossref PubMed Scopus (1228) Google Scholar, 18Doble B.W. Woodgett J.R. J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1774) Google Scholar). In contrast with the results derived from knock-out mice, in neuronal cells expression of an active form of GSK-3β suppresses NF-κB activity by inhibiting IκB kinase (IKK) 1The abbreviations used are: IKK, IκB kinase; IL, interleukin; TNF, tumor necrosis factor; GST, glutathione S-transferase; PP1 and PP2A, protein phosphatase 1 and 2A, respectively. and stabilizing IκB (21Sanchez J.F. Sniderhan L.F. Williamson A.L. Fan S. Chakraborty-Sett S. Maggirwar S.B. Mol. Cell. Biol. 2003; 23: 4649-4662Crossref PubMed Scopus (123) Google Scholar, 22Bournat J.C. Brown A.M. Soler A.P. J. Neurosci. Res. 2000; 61: 21-32Crossref PubMed Scopus (95) Google Scholar). GSK-3β has also recently been shown to phosphorylate a GST-p65 fusion protein in vitro, but the relevant site(s) have not been determined and it has remained unclear if p65 is a physiological substrate for GSK-3β in vivo (23Schwabe R.F. Brenner D.A. Am. J. Physiol. 2002; 283: G204-G211Crossref PubMed Scopus (120) Google Scholar). Using a peptide array-based approach (24Himpel S. Tegge W. Frank R. Leder S. Joost H.G. Becker W. J. Biol. Chem. 2000; 275: 2431-2438Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) we detected a protein kinase activity that specifically phosphorylated Ser468 of p65 NF-κB. Experiments presented here strongly suggest that GSK-3β is a physiological Ser468 protein kinase and we imply this phosphorylation site in negative control of NF-κB. In the light of the opposing findings regarding the role of GSK-3β in NF-κB signaling our results close an important gap in the understanding of the role of GSK-3β in regulation of p65 activity. Cells and Materials—HeLa cells stably expressing the tet transactivator protein were a kind gift of H. Bujard, Heidelberg, Germany. p65 –/– cells were a kind gift of H. Nakano, Tokyo, Japan. 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-(Ser32/36) IκBα (9241), phospho-Ser468 NF-κB (3039), phospho-(Ser21/9) GSK-3α/β (9331), all from Cell Signaling Technology and p65 NF-κB (C-20) from Santa Cruz and GSK-3β (610201) from BD Biosciences. Recombinant GSK-3β (P6040S) was from New England Biolabs. Horseradish peroxidase-coupled secondary antibodies were from Sigma. Human recombinant IL-1α was a kind gift of J. Saklatvala, London, UK. calyculin A (9902) was from Cell Signaling. Peptide Arrays—The peptide array containing p65 NF-κB peptide spots was generated following SPOT synthesis (25Frank R. Tetrahedron. 1992; 48: 9217-9232Crossref Scopus (927) Google Scholar). 180 peptide fragments of 15 amino acid residues in length and overlapping by 12 residues were generated such that the entire p65 NF-κB protein sequence was covered. These peptides were chemically synthesized as an array of spots on an amino-polyethylene glycol-modified cellulose membrane (AC-S01, AIMS Scientific Products GmbH, Braunschweig, Germany) as described previously (26Frank R. Overwin H. Methods Mol. Biol. 1996; 66: 149-169PubMed Google Scholar). All peptides are NH2-terminal acetylated and remain covalently attached to the membrane via their carboxyl termini. Plasmids and Transfections—The expression plasmid for the p65 TAD, pGEX-p65-(354–551) was a kind gift of H. Sakurai, Toyama, Japan. GST fusion proteins were expressed in bacteria and purified on GSH-Sepharose using standard procedures. pMT7-p65 NF-κB has been published (27Nourbakhsh 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 (107) Google Scholar) and NF-κB (3)luc contained three NF-κB-binding sites upstream of a luciferase cDNA. pSV-β-gal coding for SV40 promoter driven β-galactosidase was from Promega. p65 –/– cells cells were seeded in 6-well plates and transfected at 70–80% confluence using Rotifect (Roth) according to the manufacturer's instructions. Preparation of Cell Extracts—For the preparation of whole cell extracts cells were lysed directly in SDS-PAGE 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,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, 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 (27Nourbakhsh 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 (107) Google Scholar). The protein concentration of cell extracts was determined by the method of Bradford, and samples were stored at –80 °C. In Vitro Kinase Assays—For the kinase assay shown in Fig. 1A,10 μl of cell lysate (50 μg of protein) was added to 1 μg of recombinant protein substrates (GST-p65-(354–551) or mutants thereof) in 10 μlofH2O and 10 μl of kinase buffer (150 mm Tris, pH 7.4, 30 mm MgCl2, 60 μm ATP, 4 μCi of [γ-32P]ATP). After 15 min at 30 °C in vitro phosphorylated GST-p65 fusion proteins were purified on GSH-Sepharose prior to SDS-PAGE as described by Holtmann et al. (28Holtmann 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 (269) Google Scholar). Then, 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% SDS-PAGE. Phosphorylated proteins were visualized by autoradiography. For in vitro phosphorylation of immobilized peptides the peptide arrays were incubated with 2 ml of cell extract (4.7 mg of protein), 2 ml of cell lysis buffer, and 2 ml of kinase buffer (150 mm Tris, pH 7.4, 30 mm MgCl2, 10 μm ATP, 200 μCi of [γ-32P]ATP). After 30 min at 30 °C membranes were washed twice in phosphate-buffered saline, once in 8 m urea, 1% SDS, 0.5% β-mercaptoethanol for 30 min at 40 °C, twice in H2O, and three times in EtOH. Air-dried membranes were autoradiographed at 4 °C or at room temperature. For the experiments shown in Fig. 2A 100 units of recombinant GSK-3β was incubated with 1 μg of GST-p65 fusions proteins in 1× GSK-3β reaction buffer (New England Biolabs) supplemented with 20 μm ATP and 2.5 μCi of [γ-32P]ATP in a total volume of 30 μl for 30 min at 30 °C. Reactions were stopped by the addition of SDS-PAGE sample buffer and phosphorylation of proteins visualized as described above. For detection of phosphorylated proteins by immunoblotting as shown in Fig. 2B 500 units of GSK-3β, 50 ng of GST-p65 fusion proteins, and 135 μm ATP were used in the kinase reaction, and radioactive ATP was omitted. Western blotting and site-directed mutagenesis were performed as described by Buss et al. (14Buss H. Dörrie A. Schmitz M.L. Hoffmann E. Resch K. Kracht M. J. Biol. Chem. 2004; (October 15, 10.1074/jbc.M409825200)Google Scholar). To investigate the occurrence of IL-1-inducible phosphorylation sites within the transactivating COOH terminus of p65, whole cell extracts isolated from unstimulated and IL-1-treated HeLa cells were incubated with a recombinant GST-p65-(354–551) fusion protein in the presence of [γ-32P]ATP. These experiments revealed constitutive and IL-1-inducible protein kinase activities. Mutations in Ser529 and Ser536, the sites that have been found as targets for the hitherto identified p65 TAD kinases IKKα and IKKβ (7Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar), casein kinase II (6Wang 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 (373) Google Scholar), TBK1 (14Buss H. Dörrie A. Schmitz M.L. Hoffmann E. Resch K. Kracht M. J. Biol. Chem. 2004; (October 15, 10.1074/jbc.M409825200)Google Scholar, 29Fujita F. Taniguchi Y. Kato T. Narita Y. Furuya A. Ogawa T. Sakurai H. Joh T. Itoh M. Delhase M. Karin M. Nakanishi M. Mol. Cell. Biol. 2003; 23: 7780-7793Crossref PubMed Scopus (143) Google Scholar), RSK1 (16Bohuslav J. Chen L.F. Kwon H. Mu Y. Greene W.C. J. Biol. Chem. 2004; 279: 26115-26125Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), and IKKϵ (14Buss H. Dörrie A. Schmitz M.L. Hoffmann E. Resch K. Kracht M. J. Biol. Chem. 2004; (October 15, 10.1074/jbc.M409825200)Google Scholar), did not completely abolish in vitro phosphorylation of GST-p65, suggesting the existence of further p65 phosphorylation site(s) and kinases (Fig. 1A). To profile phosphorylation sites in p65 the complete p65 coding sequence arrayed as overlapping 15-mer peptides was subjected to in vitro kinase assays with cell extracts from IL-1 stimulated HeLa cells. This approach revealed more than 20 peptides that were phosphorylated in vitro (Fig. 1B), including some that contained already identified phosphorylation sites such Ser536 (Fig. 1B, peptides H1–H3). 19 peptides that are indicated by white circles in Fig. 1B contained potentially novel phosphorylation sites and were selected for further study. To identify the phosphorylated amino acids within these 19 peptides, all threonine, serine, and tyrosine residues within these sequences were systematically mutated and tested for phosphorylation (Fig. 1C). In many peptides these mutations did not change in vitro phosphorylation (Fig. 1C). Some peptides showed reduced phosphorylation upon mutation, and we are currently analyzing the significance of these sites (Fig. 1C). However, systematic replacement of threonine or serine residues in the peptide containing amino acids 463–477 (corresponding to peptide G5 in Fig. 1B) completely abolished in vitro phosphorylation in each peptide version in which serine 468 was mutated to alanine (Fig. 1C). The sequence SVDNS containing Ser468 is located within p65 transactivation domain 2 (TA2) (30Schmitz 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) and also comprises a classical GSK-3β motif (Ser/Thr-Xaa-Xaa-Xaa-phospho-Ser/Thr) (31Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1282) Google Scholar), whereby the COOH-terminal amino acid contains a so-called priming phosphoamino acid (Fig. 1C). To directly address the question whether this consensus sequence is phosphorylated by recombinant GSK-3β in vitro, the purified kinase was incubated with a GST-p65-(354–551) substrate protein and a GST-p65-(354–551-Ser468-Ala) control protein where the phosphorylation site was point mutated. GSK-3β efficiently phosphorylated the GST-p65-(354–551) protein, while the GST-p65-(354–551-Ser468-Ala) mutant was phosphorylated to a minor extent (Fig. 2A), revealing GSK-3β as a serine 468 kinase. To obtain evidence for GSK-3β-mediated p65 serine 468 phosphorylation by an independent experimental approach, the products of the in vitro kinase assays were analyzed using an antibody specifically recognizing the phosphorylated form of serine 468. These experiments clearly confirmed that GSK-3β phosphorylated specifically Ser468, as no signal with the antibody was obtained using the S468A mutant protein as substrate or by omitting the substrate (Fig. 2B). Thus the experiments shown in Fig. 2 identify p65 Ser468 as a specific GSK-3β phosphorylation site in vitro. The residual phosphorylation observed in the radioactive kinase assay (Fig. 2A) and the detection of three proteins bands of different mobility on SDS-PAGE by immunoblotting with the phospho-Ser468-specific antibody (Fig. 2B) leave the possibility that the p65 TAD might contain additional GSK-3β sites as previously suggested by Schwabe and Brenner (23Schwabe R.F. Brenner D.A. Am. J. Physiol. 2002; 283: G204-G211Crossref PubMed Scopus (120) Google Scholar). To investigate whether GSK-3β phosphorylates Ser468in vivo, HeLa cells were stimulated with IL-1, TNF, or phorbol ester plus ionomycin, but phosphorylation of endogenous Ser468 was only faintly activated (data not shown). Intriguingly, treatment of cells with the PP1/PP2A Ser/Thr phosphatase inhibitor calyculin A (32Suganuma M. Fujiki H. Furuya-Suguri H. Yoshizawa S. Yasumoto S. Kato Y. Fusetani N. Sugimura T. Cancer Res. 1990; 50: 3521-3525PubMed Google Scholar) for 30 min strongly induced Ser468 phosphorylation. These results can be reconciled with a recently suggested model whereby PP1 dephosphorylates GSK-3β at the NH2-terminal serine 9, thereby activating the kinase (33Zhang F. Phiel C.J. Spece L. Gurvich N. Klein P.S. J. Biol. Chem. 2003; 278: 33067-33077Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). In this model, mutual control is ensured by GSK-3β-mediated inhibition of the protein I-2, a negative regulator of PP1, thus maintaining GSK-3β in an active state in unstimulated cells. This mechanism also implies that inhibition of GSK-3β accelerates inactivation of PP1 by I-2 (33Zhang F. Phiel C.J. Spece L. Gurvich N. Klein P.S. J. Biol. Chem. 2003; 278: 33067-33077Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). We therefore analyzed if inhibition of PP1 affects phosphorylation of GSK-3β in the cells employed in this study. Incubation of HeLa cells with calyculin A triggered the phosphorylation of GSK-3α at serine 21 and of GSK-3β at serine 9, as revealed by immunoblotting with a phospho-specific antibody that recognizes both enzymes (Fig. 3, compare lanes 1 and 2). In this situation, addition of increasing amounts of LiCl, a specific inhibitor of GSK-3 (33Zhang F. Phiel C.J. Spece L. Gurvich N. Klein P.S. J. Biol. Chem. 2003; 278: 33067-33077Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar), further increased phosphorylation of GSK-3β and in parallel suppressed phosphorylation of Ser468 in a dose-dependent fashion (Fig. 3, lanes 3–5). Simultaneous detection of phosphorylated GSK-3α with the phospho-specific antibody showed that LiCl also impaired phosphorylation of GSK-3α (Fig. 3). However, as GSK-3α has not been implicated in in NF-κB activation (18Doble B.W. Woodgett J.R. J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1774) Google Scholar), its role in p65 phosphorylation was not further investigated in our study. In parallel, calyculin A also induced phosphorylation and degradation of IκBα (Fig. 3, lane 2), which is in accordance with earlier studies (34Sun S.C. Maggirwar S.B. Harhaj E. J. Biol. Chem. 1995; 270: 18347-18351Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). These effects support the conclusion that a low level of kinase activity of GSK-3 and of IKKs is maintained in unstimulated cells whose effect on substrate phosphorylation is counteracted by high levels of phosphatase activity. With respect to p65, these experiments strongly implicate GSK-3β in phosphorylation of endogenous Ser468 and also suggest that a calyculin A-sensitive protein phosphatase regulates both NH2-terminal phosphorylation of GSK-3β and Ser468 phosphorylation of p65. Based on the experiments reported by Zhang et al. (33Zhang F. Phiel C.J. Spece L. Gurvich N. Klein P.S. J. Biol. Chem. 2003; 278: 33067-33077Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar) we suggest that PP1 is the phospho-Ser468 phosphatase. In our model a high level of active, dephosphorylated GSK-3β not only increases phosphorylation of Ser468 of p65 but at the same time also activates PP1, which dephosphorylates Ser468. To characterize the role of Ser468 functionally we ectopically expressed wild type p65 or the S468A mutant in p65-deficient cells. Under these conditions both proteins were expressed to comparable levels and were detectable within the cytoplasm and the nucleus (Fig. 4A). Interestingly, in p65-deficient cells the S468A mutant showed a slightly faster mobility on SDS-PAGE compared with wild type p65 (Fig. 4A), a phenomen that was not observed when both proteins were transcribed and translated in vitro (data not shown). This observation is in line with a loss of GSK-3β-mediated phosphorylation. When expressed in p65-negative cells, the S468A mutant displayed an about 4-fold increase in activation of a cotransfected NF-κB reporter gene as compared with wild type p65 (Fig. 4B). Expression of the S468A mutant also enhanced p65 activity induced by IL-1, TNF, or phorbol 12-myristate 13-acetate (data not shown). These experiments suggest that Ser468 phosphorylation by GSK-3β has a negative regulatory role for p65 activity the mechanism of which awaits further investigation. In summary, we provide strong evidence that Ser468 is a new phosphorylation site of p65 NF-κB and identify GSK-3β as the kinase that phosphorylates this residue in the absence of extracellular ligands. Ser468 phosphorylation may also play a role in response to certain specific stimuli as we found during the course of this work that T-cell costimulation induces Ser468 phosphorylation. However, the kinase that mediates this effect and its biological significance remain to be identified (35Schmitz M.L. Mattioli I. Dittrich-Breiholz O. Kracht M. Livingstone M. Blood. 2004; (in press)Google Scholar). Here, we suggest a function for GSK-3β-mediated phosphorylation of Ser468 in negative regulation of p65. Collectively, these results predict that the balance of active GSK-3β and PP1 determines the phosphorylation status of Ser468 in un-stimulated cells (see Fig. 5). In conjunction with other potential GSK-3β phosphorylation sites Ser468 may contribute to the altered constitutive activity of NF-κB that has been observed in chronic inflammatory disease (36Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Crossref PubMed Scopus (3353) Google Scholar) and in different tumors (37Karin M. Cao Y. Greten F.R. Li Z.W. Nat. Rev. Cancer. 2002; 2: 301-310Crossref PubMed Scopus (2265) Google Scholar). Thus Ser468 phosphorylation maybe another crucially important determinant for the outcome of NF-κB activation in inflammation and cancer. We thank Mahtab Nourbakhsh for helpful suggestions in the initial phase of this work.