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• W2023409936 abstract "Here we identify IKKϵ as a novel NF-κB p65 kinase that mediates inducible phosphorylation of Ser468 and Ser536 in response to T cell costimulation. In addition, the kinase activity of IKKϵ contributes to the control of p65 nuclear uptake. Serines 468 and 536 are evolutionarily conserved, and the surrounding amino acids display sequence homology. Down-regulation of IKKϵ levels by small interfering RNA does not affect inducible phosphorylation of Ser536 but largely prevents Ser468 phosphorylation induced by T cell costimulation. Ser536-phosphorylated p65 is found predominantly in the cytosol. In contrast, the Ser468 phosphorylated form of this transcription factor occurs mainly in the nucleus, suggesting a function for transactivation. Reconstitution of p65–/– cells with either wild type p65 or point-mutated p65 variants showed that inducible phosphorylation of Ser468 serves to enhance p65-dependent transactivation. These results also provide a mechanistic link that helps to explain the relevance of IKKϵ for the expression of a subset of NF-κB target genes without affecting cytosolic IκBα degradation. Here we identify IKKϵ as a novel NF-κB p65 kinase that mediates inducible phosphorylation of Ser468 and Ser536 in response to T cell costimulation. In addition, the kinase activity of IKKϵ contributes to the control of p65 nuclear uptake. Serines 468 and 536 are evolutionarily conserved, and the surrounding amino acids display sequence homology. Down-regulation of IKKϵ levels by small interfering RNA does not affect inducible phosphorylation of Ser536 but largely prevents Ser468 phosphorylation induced by T cell costimulation. Ser536-phosphorylated p65 is found predominantly in the cytosol. In contrast, the Ser468 phosphorylated form of this transcription factor occurs mainly in the nucleus, suggesting a function for transactivation. Reconstitution of p65–/– cells with either wild type p65 or point-mutated p65 variants showed that inducible phosphorylation of Ser468 serves to enhance p65-dependent transactivation. These results also provide a mechanistic link that helps to explain the relevance of IKKϵ for the expression of a subset of NF-κB target genes without affecting cytosolic IκBα degradation. The NF-κB transcription factor system serves to control the expression of an extraordinarily wide array of genes in response to infections, inflammation, and other harmful situations (1Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3380) Google Scholar, 2Ghosh S. Karin M. Cell. 2002; 109: 81-96Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar). NF-κB target genes (such as immunoreceptors, cytokines, and chemokines) contribute to the innate immune response but also serve to control cell survival and proliferation (3Ben Neriah Y. Schmitz M.L. EMBO Rep. 2004; 5: 668-673Crossref PubMed Scopus (25) Google Scholar). NF-κB is a collective name for homo- or heterodimers composed of five different DNA-binding subunits, with the most frequently detected form being a heterodimer of p50 and p65 (RelA). The p65 subunit contains two strong, acidic transactivation domains called TAD1 3The abbreviations used are: TAD, transactivation domain; TNF, tumor necrosis factor; IL, interleukin; PMA, phorbol-12-myristate-13-acetate; MEF, mouse embryo fibroblast; GFP, green fluorescent protein; RANTES, regulated on activation, normal T cell expressed and secreted; IP-10, interferon-γ-inducible protein 10; CREB, cAMP-response element-binding protein; GST, glutathione S-transferase. and TAD2 in its C-terminal portion (4Schmitz 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). A large variety of different inducers leads to NF-κB activation by activation of numerous cellular and membrane receptors, including toll-like receptors and the T cell receptor. Thus far, three major pathways mediating NF-κB activation have been identified, the so-called canonical and noncanonical pathways and the DNA damage-induced NF-κB pathway. All NF-κB activating events have in common that they lead to the proteasome-dependent generation of DNA-binding dimers (5Viatour P. Merville M.P. Bours V. Chariot A. Trends Biochem. Sci. 2005; 30: 43-52Abstract Full Text Full Text PDF PubMed Scopus (1197) Google Scholar). NF-κB signals activating the canonical pathway funnel into the IKK complex, which is composed of the enzymatically active subunits IKKα and IKKβ and the regulatory subunits IKKγ/NEMO (6Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar, 7Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar) and ELKS (8Ducut Sigala J.L. Bottero V. Young D.B. Shevchenko A. Mercurio F. Verma I.M. Science. 2004; 304: 1963-1967Crossref PubMed Scopus (187) Google Scholar). IKKβ-mediated Iκβ phosphorylation allows subsequent ubiquitination and proteolytic destruction of this inhibitory protein. This leads to an unmasking of the p65 nuclear localization sequence and results in NF-κB nuclear immigration, DNA binding, and gene expression. Once activated, inducible post-translational modifications, including phosphorylation, acetylation, ubiquitination, or prolyl isomerization, allow the regulation of NF-κB transcriptional activity (9Chen L.F. Greene W.C. Nat. Rev. Mol. Cell Biol. 2004; 5: 392-401Crossref PubMed Scopus (1049) Google Scholar, 10Schmitz M.L. Mattioli I. Buss H. Kracht M. Chembiochem. 2004; 5: 1348-1358Crossref PubMed Scopus (221) Google Scholar). Thus far, eight different phosphorylation sites have been mapped for the strongly activating NF-κB p65 subunit. Three sites are contained in the N-terminal Rel homology domain, whereas five sites (Ser468, Thr505, Ser529, Ser535, and Ser536) are contained within both C-terminal TADs. Inducible phosphorylation of Ser276 and threonine 311 promotes the interaction of p65 with the coactivating acetylase CREB-binding protein/p300, thus leading to p65 acetylation and stimulating NF-κB-driven transcription. Various experimental approaches revealed that phosphorylation of serines 529, 535, and 536 serve to stimulate NF-κB-dependent transcription. Expression of a p65 protein mutated in Ser529 in p65–/– cells revealed only a minor role of Ser529 in transactivation, since it only contributes to achieve the Tax-induced maximal transcriptional response (11O'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 (94) Google Scholar). Phosphorylation of Ser535 is mediated by the calmodulin-dependent protein kinase IV, which results in an increase of NF-κB-dependent transcription, as revealed by a phosphomimetic mutation where Ser535 was replaced by glutamic acid (12Bae J.S. Jang M.K. Hong S. An W.G. Choi Y.H. Kim H.D. Cheong J. Biochem. Biophys. Res. Commun. 2003; 305: 1094-1098Crossref PubMed Scopus (35) Google Scholar). Basal phosphorylation of Ser468, a recently discovered phosphorylation site within TAD2 (13Mattioli I. Dittrich-Breiholz O. Livingstone M. Kracht M. Schmitz M.L. Blood. 2004; 104: 3302-3304Crossref PubMed Scopus (22) Google Scholar), is exerted by GSK3β (14Buss H. Dorrie A. Schmitz M.L. Frank R. Livingstone M. Resch K. Kracht M. J. Biol. Chem. 2004; 279: 49571-49574Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). The same site can also be phosphorylated by IKKβ in response to TNFα or IL-1 stimulation (15Schwabe R.F. Sakurai H. FASEB J. 2005; 19: 1758-1760Crossref PubMed Scopus (66) Google Scholar). We have previously shown that phosphorylation of p65 Ser468 can be induced by T cell costimulation (13Mattioli I. Dittrich-Breiholz O. Livingstone M. Kracht M. Schmitz M.L. Blood. 2004; 104: 3302-3304Crossref PubMed Scopus (22) Google Scholar), but the responsible kinase(s) is not yet known. Phosphorylation of p65 NF-κB at Ser536 couples p65 to TAFII31-mediated transcription and is mediated, dependent on the stimulus, by various kinases, including IKKα/β, RSK1, TBK1 (TANK-binding kinase-1)/NAK (NF-κB-activating kinase)/T2K (TRAF2-associated kinase), and IKKϵ (also called IKKi) (16Buss H. Dorrie A. Schmitz M.L. Hoffmann E. Resch K. Kracht M. J. Biol. Chem. 2004; 279: 55633-55643Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 17Lawrence T. Bebien M. Liu G.Y. Nizet V. Karin M. Nature. 2005; 434: 1138-1143Crossref PubMed Scopus (556) Google Scholar). TBK1 and IKKϵ show sequence homology to IKKα/β but are not components of the IKK complex (18Pomerantz J.L. Baltimore D. EMBO J. 1999; 18: 6694-6704Crossref PubMed Google Scholar, 19Peters R.T. Liao S.M. Maniatis T. Mol. Cell. 2000; 5: 513-522Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Both kinases have been recently mainly recognized for their ability to phosphorylate interferon regulatory factor proteins in response to viral infection (20McWhirter S.M. Fitzgerald K.A. Rosains J. Rowe D.C. Golenbock D.T. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 233-238Crossref PubMed Scopus (453) Google Scholar, 21Hemmi H. Takeuchi O. Sato S. Yamamoto M. Kaisho T. Sanjo H. Kawai T. Hoshino K. Takeda K. Akira S. J. Exp. Med. 2004; 199: 1641-1650Crossref PubMed Scopus (465) Google Scholar). IKKϵ overexpression promotes dimerization and nuclear translocation of interferon regulatory factor-3 but also enhances the DNA binding activity of C/EBPδ (22Kravchenko 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). IKKϵ is mainly regulated via inducible, NF-κB-dependent expression but is prominently expressed in T cells (19Peters R.T. Liao S.M. Maniatis T. Mol. Cell. 2000; 5: 513-522Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 23Shimada T. Kawai T. Takeda K. Matsumoto M. Inoue J. Tatsumi Y. Kanamaru A. Akira S. Int. Immunol. 1999; 11: 1357-1362Crossref PubMed Scopus (312) Google Scholar). Its kinase activity is triggered in response to T cell costimulation or the phorbol ester phorbol-12-myristate-13-acetate (PMA) but not by the cytokines TNFα or IL-1 (19Peters R.T. Liao S.M. Maniatis T. Mol. Cell. 2000; 5: 513-522Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Whereas NF-κB regulates IKKϵ, the role of IKKϵ for NF-κB activation remains elusive, since IKKϵ–/– cells show unchanged inducible IκBα phosphorylation and DNA binding. On the other hand, lipopolysaccharide-induced expression of some late NF-κB target genes, including COX-2, regulated on activation, normal T cell expressed and secreted (RANTES), and interferon-γ-inducible protein 10 (IP-10) is lost in IKKϵ-deficient cells (22Kravchenko 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), suggesting a role of IKKϵ for the regulation of NF-κB at a later step. Here we reveal an additional function for IKKϵ and show that it serves as a novel p65 kinase that mediates inducible phosphorylation at serines 468 and 536 in vitro and in vivo, thus stimulating gene expression. Cell Culture, Transfections, and Stimulations—Mouse embryonic fibroblasts (MEFs) lacking p65 (24Okazaki 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 (138) Google Scholar) and human 293T and HeLa cells were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, 2 mm l-glutamine, and 1% (v/v) penicillin/streptomycin. Adherent cells were transfected using Rotifect (Roth) according to the manufacturer's instructions. Jurkat T leukemia cells and Jurkat Tet-on cells were grown in supplemented RPMI 1640 medium. Jurkat cells (1.5 × 107) were transfected by electroporation using a gene pulser (Bio-Rad) at 250 V/950 microfarads with constant amounts of DNA. Stable Jurkat cell lines were generated by cotransfection of IKKϵ-encoding plasmids or pSUPER-IKKϵ and a plasmid carrying a selection marker (pEF-Puro). After further growth in the presence of puromycin (1 μg/ml), either pools of stably selected cells were used for further experiments (small interfering RNA), or single cells were isolated by limiting dilution (Jurkat Tet-on cells). Costimulation of Jurkat cells was performed in a final volume of 500 μl (2 × 107 cells) by adding PMA (40 ng/ml) together with ionomycin (1 μm). Proteasome activity was inhibited upon preincubation of cells for 1 h with 50 μm MG132. Antisera, Plasmids, and Reagents—The antibodies recognizing p65 phosphorylated at Ser468 or Ser536 and phosphorylated forms of IκBα and of GSK3α/β were from Cell Signaling Technology. Antibodies recognizing the hemagglutinin or Myc epitope, p65, IκBα, p105/p50, IKKα, NEMO/IKKγ, and HDAC-1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). FLAG antibodies were from Sigma, the monoclonal IKKϵ antibodies were purchased from Abcam, and polyclonal GFP antibodies were obtained from BD Biosciences. The expression vectors encoding green fluorescent protein (GFP)-tagged p65 (25Schmid J.A. Birbach A. Hofer-Warbinek R. Pengg M. Burner U. Furtmuller P.G. Binder B.R. de Martin R. J. Biol. Chem. 2000; 275: 17035-17042Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), Gal4-p65 (26Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (666) Google Scholar), GST-p65-(354–551) (27Sakurai 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 (303) Google Scholar), epitope-tagged IKKϵ and IKKβ proteins (19Peters R.T. Liao S.M. Maniatis T. Mol. Cell. 2000; 5: 513-522Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar), pSUPER-IKKϵ (28Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2099) Google Scholar), and the (κB)3-Luc reporter gene (29Plaisance S. Vanden Berghe W. Boone E. Fiers W. Haegeman G. Mol. Cell Biol. 1997; 17: 3733-3743Crossref PubMed Google Scholar) were previously described. Tet-inducible IKKϵ expression vectors were produced upon cloning the coding regions of IKKϵ or IKKϵ kinase-inactive into pBi-EGFP. The pEF-p65 expression vector allowing the expression of p65 with a C-terminal hemagglutinin tag was constructed by standard methods, and point mutants were produced by the QuikChange site-directed mutagenesis protocol using partially overlapping primers (30Zheng L. Baumann U. Reymond J.L. Nucleic Acids Res. 2004; 32: e115Crossref PubMed Scopus (848) Google Scholar). PMA and ionomycin were from Sigma. AS602868 was a kind gift from Serono International S.A. (Geneva, Switzerland). Electrophoretic Mobility Shift Assays (EMSAs)—Cells were lysed in TOTEX buffer (20 mm Hepes/KOH, pH 7.9, 0.35 m NaCl, 20% (v/v) glycerol, 1% (v/v) Nonidet P-40, 1 mm MgCl2, 0.5 mm EDTA, 0.1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 10 mm NaF, and 0.5 mm sodium vanadate) and incubated on ice for 30 min. After centrifugation, equal amounts of supernatants were tested for DNA binding activity to a double-stranded, 32P-labeled oligonucleotide as described (31Hehner S.P. Li-Weber M. Giaisi M. Droge W. Krammer P.H. Schmitz M.L. J. Immunol. 2000; 164: 3829-3836Crossref PubMed Scopus (54) Google Scholar). Subsequently, the free and complexed oligonucleotides were separated by electrophoresis on a native 4% polyacrylamide gel, and the dried gel was exposed to an x-ray film. Cell Extracts, Phosphatase Treatment, and Western Blotting—After washing with ice-cold phosphate-buffered saline, cells were collected by centrifugation. The pellet was resuspended in Nonidet P-40 lysis buffer (20 mm Tris/HCl, pH 7.5, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 10 mm NaF, 0.5 mm sodium vanadate, leupeptin (10 μg/ml), aprotinin (10 μg/ml), 1% (v/v) Nonidet P-40) and incubated on ice for 20 min. Cell debris was removed by centrifugation at 13,000 rpm at 4 °C for 10 min. Phosphatase treatment was performed by incubating the cell extract lacking phosphatase inhibitors with 400 units of λ-phosphatase for 1 h at 30°C according to the instructions of the manufacturer (Biolabs). Equal amounts of protein contained in the supernatant were further analyzed by reducing SDS-PAGE and Western blotting onto a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). After blocking, membranes were incubated with the appropriate primary and horseradish peroxidase-coupled secondary antibodies, followed by protein detection using the Amersham Biosciences enhanced chemiluminescence system. Purification of GST-p65 and Kinase Assays—The GST-p65-(354–551) fusion protein was expressed in Escherichia coli BL21 cells and purified by affinity chromatography on glutathione-Sepharose 4B according to standard protocols. The immune complex kinase assays were done by immunoprecipitation of FLAG-tagged IKKϵ using α-FLAG antibodies or by immunoprecipitation of endogenous IKKϵ. The precipitate was washed three times in Nonidet P-40 lysis buffer and two times in kinase buffer (20 mm Hepes/KOH, pH 7.4, 25 mm β-glycerophosphate, 2 mm dithiothreitol, 20 mm MgCl2). The kinase assay was performed in a final volume of 20 μl of kinase buffer containing 40 μm ATP and 2 μg of the purified GST-p65 substrate protein. After incubation for 20 min at 30 °C, the reaction was stopped, separated by SDS-PAGE, and analyzed by immunoblotting with phosphospecific antibodies. Co-immunoprecipitation Experiments—Human 293 T cells were transiently transfected to express the hemagglutinin-tagged p65 variants. 36 h later, cells were lysed in Nonidet P-40 lysis buffer, and one aliquot was used to confirm correct expression of the proteins. Equal amounts of protein contained in the remaining supernatants were immunoprecipitated either with α-hemagglutinin or with control antibodies and 25 μl of protein A/G-Sepharose and rotated for 4 h on a spinning wheel at 4 °C. The immunoprecipitates were washed five times in Nonidet P-40 buffer and then boiled in 1× SDS sample buffer prior to SDS-PAGE and further analysis by Western blotting. Subcellular Fractionation—Nuclear and cytosolic proteins were separated upon resuspending pelleted cells in 400 μl of cold buffer A (10 mm Hepes/KOH, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride) by gentle pipetting. After incubation for 20 min on ice, 10 ml of 10% Nonidet P-40 was added, and cells were lysed by vortexing. The homogenate was centrifuged for 30 s in a microcentrifuge. The supernatant representing the cytosolic fraction was collected, and the pellet containing the cell nuclei was dissolved in 100 μl of buffer C (20 mm Hepes/KOH, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride). The Eppendorf tubes were incubated for 15 min on ice and centrifuged for 10 min with 13,000 rpm at 4 °C. The supernatant representing the nuclear fraction was collected. IKKϵ Directly Phosphorylates NF-κB p65 at Ser468—Basal phosphorylation at Ser468 is mediated by GSK3β, whereas TNFα- and IL-1-induced phosphorylation of this site is maximal already 7.5 min after stimulation and is mediated by IKKβ (15Schwabe R.F. Sakurai H. FASEB J. 2005; 19: 1758-1760Crossref PubMed Scopus (66) Google Scholar). We have previously shown that Ser468 is inducibly phosphorylated by T cell costimulation (13Mattioli I. Dittrich-Breiholz O. Livingstone M. Kracht M. Schmitz M.L. Blood. 2004; 104: 3302-3304Crossref PubMed Scopus (22) Google Scholar), but the kinase mediating inducible phosphorylation in response to this stimulus is not known. Ser468 is contained in a sequence motif called TAD1′ that shows homology to TAD1 (4Schmitz 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 relative positions of Ser536 and Ser468 within the sequence motif are conserved (Fig. 1A), raising the possibility that both sites may employ the same kinase. From all of the kinases known to mediate Ser536 phosphorylation, only IKKα/IKKβ and IKKϵ are known to be induced by T cell costimulation or by PMA and ionomycin, which mimic T cell costimulation upon protein kinase C activation and calcium release, respectively (32Tanahashi M. Yokoyama T. Kobayashi Y. Yamakawa Y. Maeda M. Fujii Y. Hum. Immunol. 2001; 62: 771-781Crossref PubMed Scopus (10) Google Scholar). To compare the relative roles of IKKα, IKKβ, and IKKϵ for p65 phosphorylation, Jurkat T cells were transfected with expression vectors encoding these three IKKs together with very low amounts of a vector directing expression of a GFP-tagged p65 protein, allowing expression of this fusion protein at physiological levels. This experimental approach was taken, because the low transfection efficiency of Jurkat T leukemia cells hampers the analysis of the endogenous p65 protein, thus enabling the detection of the slower migrating GFP-p65 fusion protein, which is fully regulated and functional (25Schmid J.A. Birbach A. Hofer-Warbinek R. Pengg M. Burner U. Furtmuller P.G. Binder B.R. de Martin R. J. Biol. Chem. 2000; 275: 17035-17042Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Whereas expression of IKKα only slightly induced p65 Ser536 phosphorylation, IKKβ strongly triggered Ser536 phosphorylation of GFP-p65 and also of the endogenous p65 protein (Fig. 1B). IKKβ also caused a slight induction of Ser468 phosphorylation. In contrast, even faint amounts of IKKϵ potently stimulated p65 phosphorylation at serines 536 and 468 and even caused the appearance of a slower migrating p65 form (Fig. 1B). In contrast to IKKβ, the overexpression of IKKϵ failed to cause the phosphorylation of the endogenous p65, which is in complex with IκB proteins. A similar experimental approach revealed that this strong IKKϵ-induced phosphorylation could not be further enhanced by PMA/ionomycin stimulation (Fig. 1C). Is the slower migrating form of p65 due to IKKϵ-mediated phosphorylation, or is it also caused by other modifications? To address this question, extracts from cells coexpressing GFP-p65 and IKKϵ were incubated with λ-phosphatase. This treatment completely converted the slower migrating form of p65 into the faster migrating version (Fig. 1D), indicating that the upper band represents a phosphorylated form of p65. Of note, phosphorylated Ser536 occurred in the lower and in the upper band, whereas modified Ser468 was found only in the upshifted p65 form. These results raise the possibility that both phosphorylations depend on each other. To test this hypothesis experimentally, IKKϵ-mediated phosphorylation of each individual site was determined with a substrate GFP-p65 protein, where the respective other phosphorylation site was mutated to alanine. These experiments revealed full Ser468 phosphorylation in the presence of a mutated Ser536 and vice versa (Fig. 2A), showing that both sites can be phosphorylated independently from each other. Mutation of the phosphorylatable serines to alanine precluded binding of the phosphospecific antibodies, thus confirming their specificity. Next, we wanted to test whether IKKϵ phosphorylates p65 directly or causes phosphorylation upon activation of a downstream kinase. Cells were transfected to express epitope-tagged wild type or kinase-inactive forms of IKKϵ or the control protein IKKβ, followed by purification via immunoprecipitation and in vitro kinase assays. The GST-p65-(354–551) protein was efficiently phosphorylated at Ser468 and Ser536 by IKKϵ wild type but not by the kinase-inactive IKKϵ point mutant, as revealed by immunoblotting with phosphospecific antibodies (Fig. 2B). These in vitro experiments also revealed that IKKϵ can cause the induction of a slower migrating p65 form. We also found a direct phosphorylation of p65 at both sites when IKKβ was used as a kinase source. In summary, these results identify IKKϵ as a kinase directly mediating p65 phosphorylation at Ser468 and Ser536.FIGURE 2IKKϵ directly phosphorylates p65 in vitro and induces the occurrence of a slower migrating p65 form. A, Jurkat cells were transfected to express GFP-p65 or various GFP-p65 mutants, where Ser468 and/or Ser536 were changed to alanine together with FLAG-tagged IKKϵ at the indicated combinations. Phosphorylation of p65 and expression of IKKϵ was analyzed by immunoblotting as shown. B, Jurkat cells were transfected with expression vectors for epitope-tagged forms of IKKβ, IKKϵ, and kinase-inactive point mutants of these kinases, respectively. After 36 h, cells were lysed, and the IKK proteins were immunoprecipitated (IP) and purified from cell lysates with polyclonal FLAG or α-Myc antibodies, respectively. Subsequently, kinase activity was monitored by immune complex kinase assays (KA) using recombinant GST-p65-(354–551) as substrate. Phosphorylation of p65 was determined by immunoblotting using phosphospecific antibodies as shown. Note that IKKϵ causes the upshift also in these in vitro assays. In the control immunoblot, the α-FLAG antibody was detected first, followed by visualization of IKKβ with αMyc antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT) NF-κB p65 Phosphorylated at Ser468 Is Found Predominantly in the Nucleus—Following T cell costimulation, p65 phosphorylated at Ser536 is predominantly found in the cytosol (33Mattioli 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). We thus asked whether the same holds true for p65 phosphorylated at Ser468 and stimulated Jurkat cells for various time periods with PMA/ionomycin, followed by subcellular fractionation into cytosolic and nuclear extracts (Fig. 3A). The Ser468-phosphorylated p65 protein was found predominantly in the nucleus, which is in contrast to the Ser536-phosphorylated p65 occurring mainly in the cytosol. Also, the kinetics revealed differences, since Ser536 phosphorylation started to vanish already 30 min after stimulation, whereas Ser468 phosphorylation displayed a delayed kinetics and was unchanged even 45 min after PMA/ionomycin treatment. To test whether the same intracellular distribution occurs when p65 phosphorylation is triggered by IKKϵ, Jurkat cells were transfected to express GFP-p65 in the absence or presence of cotransfected IKKϵ. Two days after transfection, nuclear and cytosolic extracts were prepared and p65 phosphorylation was analyzed by immunoblotting (Fig. 3B). Also in this setting, Ser468-phosphorylated p65 was found predominantly (but not exclusively) in the nucleus. Overexpressed IKKϵ was found in the nucleus and in the cytosol, which reflects the distribution of the endogenous kinase that is also found in both fractions (Fig. 3A). To address the question of whether IKKϵ-mediated phosphorylation of p65 Ser468 can also occur in the nucleus, we tested the effects of IKKϵ on phosphorylation of the Gal4-p65 protein, which is constitutively nuclear (26Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (666) Google Scholar). IKKϵ-triggered Ser468 phosphorylation was further augmented by treatment with PMA/ionomycin (Fig. 3C). These results show that IKKϵ phosphorylates nuclear p65 that is not in complex with IκB but do not exclude the possibility that IKKϵ-mediated p65 phosphorylation can also happen in the cytoplasm. In contrast, coexpression of a kinase-inactive IKKϵ point mutant completely inhibited this inducible phosphorylation, pointing to the relevance of IKKϵ for this pathway. The Kinase Activity of IKKϵ Is Activated in Response to T Cell Costimulation—To address the question of whether the kinetics of IKKϵ activation parallels that of p65 Ser468 phosphorylation, Jurkat cells either containing or lacking the IKKγ/NEMO protein were stimulated with PMA/ionomycin for various periods. Immune complex kinase assays using the immunoprecipitated endogenous IKKϵ protein as a kinase source revealed phosphorylation of the p65 substrate protein 20 and 45 min after stimulation (Fig. 4A). These data show that induction of IKKϵ kinase activity parallels p65 Ser468 phosphorylation and that IKKγ/NEMO, which is essential for the function of the IKK complex, is not important for primary IKKϵ activation at the time points analyzed. The amount of IKKϵ protein is unchanged in response to T cell costimulation and not influenced by the presence of IKKγ/NEMO (Fig. 4B). Phosphorylation of p65 at Ser468 Requires Its Release from IκBα—We next asked whether free or IκB-bound p65 is phosphorylated at Ser468. To address this question, Jurkat cells lacking the IKKγ/NEMO protein and thus being unable to phosphorylate IκBα and to release the p65 protein from the cytosol (6Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar) were stimulated for various periods with PMA/ionomycin. IKKγ/NEMO-deficient cells failed to induce p65 phosphorylation at Ser468 and Ser536, whereas IKKγ/NEMO-retransfected control cells showed full phosphorylation at both sites (Fig. 5A), suggesting that only free and untrapped p65 can be phosphorylated. To substantiate this finding by an independent experimental approach, Ser468 phosphory" @default.
• W2023409936 created "2016-06-24" @default.
• W2023409936 creator A5011893681 @default.
• W2023409936 creator A5012758596 @default.
• W2023409936 creator A5038809023 @default.
• W2023409936 creator A5053904419 @default.
• W2023409936 creator A5069332888 @default.
• W2023409936 creator A5080222187 @default.
• W2023409936 creator A5084139318 @default.
• W2023409936 date "2006-03-01" @default.
• W2023409936 modified "2023-10-18" @default.
• W2023409936 title "Inducible Phosphorylation of NF-κB p65 at Serine 468 by T Cell Costimulation Is Mediated by IKKϵ" @default.
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