Abstract
22 min readThe members of the NF-κB transcription factor family are key regulators of gene expression in the immune response. Different combinations of NF-κB subunits not only diverge in timing to induce transcription but also recognize varying sequences of the NF-κB-binding site of their target genes. The p52 subunit is generated as a result of processing of NF-κB2 p100. Here, we demonstrate that the non-canonical IκB kinase ϵ (IKKϵ) directly interacts with p100. In a transactivation assay, IKKϵ promoted the ability of p52 to transactivate gene expression. This effect was indirect, requiring p65, which was shown to be part of the IKKϵ-p52 complex and to be phosphorylated by IKKϵ. These novel interactions reveal a hitherto unknown function of IKKϵ in the regulation of the alternative NF-κB activation pathway involving p52 and p65. The members of the NF-κB transcription factor family are key regulators of gene expression in the immune response. Different combinations of NF-κB subunits not only diverge in timing to induce transcription but also recognize varying sequences of the NF-κB-binding site of their target genes. The p52 subunit is generated as a result of processing of NF-κB2 p100. Here, we demonstrate that the non-canonical IκB kinase ϵ (IKKϵ) directly interacts with p100. In a transactivation assay, IKKϵ promoted the ability of p52 to transactivate gene expression. This effect was indirect, requiring p65, which was shown to be part of the IKKϵ-p52 complex and to be phosphorylated by IKKϵ. These novel interactions reveal a hitherto unknown function of IKKϵ in the regulation of the alternative NF-κB activation pathway involving p52 and p65. The members of the NF-κB transcription factor family activate defense responses against pathogens and cellular stress. A specific response to a stimulus is achieved through differential activation and dimeric complex formation of the five members of this family: NF-κB1 or p105/p50; NF-κB2 or p100/p52; and the Rel subfamily, p65 (also termed RelA), RelB, and c-Rel. Upon pathway activation, the IκB subunits, which mask the Rel transcription activators, become phosphorylated by the IκB kinases (IKKs) 2The abbreviations used are: IKKs, IκB kinases; IL, interleukin; TNFα, tumor necrosis factor-α; NIK, NF-κB-inducing kinase; IFN, interferon; IRFs, interferon-regulated transcription factors; MEFs, mouse embryonic fibroblasts; GST, glutathione S-transferase; DBD, DNA-binding domain; MS/MS, tandem mass spectrometry; HPLC, high pressure liquid chromatography. and then ubiquitinated and ultimately degraded (1Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2016) Google Scholar, 2Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar). The active NF-κB subunits are consequently released, translocate to the nucleus as dimers to bind their cognate target sites, and undergo further phosphorylation, which promotes their ability to transactivate gene expression. p50 and p52 are different from the other factors in that their activation is not controlled by IκB subunits but rather by the inhibitory ankyrin repeats in the C termini of their full-length forms, p105 and p100. These inhibitory domains have to be phosphorylated and processed to produce the respective active p50 and p52 forms (1Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2016) Google Scholar, 2Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar, 3Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5592) Google Scholar, 4Verma I.M. Stevenson J.K. Schwarz E.M. 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Nuclear shuttling has been linked to regulation of p100 processing (28Liao G. Sun S.C. Oncogene. 2003; 22: 4868-4874Crossref PubMed Scopus (35) Google Scholar), which has also been described to be a co-translational event (29Mordmuller B. Krappmann D. Esen M. Wegener E. Scheidereit C. EMBO Rep. 2003; 4: 82-87Crossref PubMed Scopus (107) Google Scholar). In contrast to the canonical NF-κB p65 pathway, p100 does not employ IKKβ for activation but has instead been shown to be phosphorylated by IKKα in an NF-κB-inducing kinase (NIK)-dependent manner (30Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar), which is prerequisite for ubiquitination and processing of p100 into p52. Induced and constitutive/pathogenic p100 processing are mechanistically similar. Processing is suppressed by the formation of a presumptive three-dimensional domain consisting of the C terminus, the dimerization domain, and the nuclear translocation domain (31Qing G. Qu Z. Xiao G. J. Biol. Chem. 2005; 280: 18-27Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Inducible processing is crucially controlled by NIK, which activates IKKα, causing it to phosphorylate p100 (32Xiao G. Fong A. Sun S.C. J. Biol. Chem. 2004; 279: 30099-30105Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Constitutive processing also requires phosphorylation by IKKα and is regulated by nuclear shuttling. Different from physiological processing, these pathogenic processing events are NIK-independent (32Xiao G. Fong A. Sun S.C. J. Biol. Chem. 2004; 279: 30099-30105Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 33Qing G. Xiao G. J. Biol. Chem. 2005; 280: 9765-9768Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The non-canonical IKKϵ (IKKi) was first described as a lipopolysaccharide-inducible serine/threonine kinase and was hence thought to be essential in the host response against bacterial pathogens (34Shimada 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, 35Peters R.T. Liao S.M. Maniatis T. Mol. Cell. 2000; 5: 513-522Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Expression of IKKϵ is also induced by proinflammatory cytokines such as TNFα, IL-1β, and IL-6, with preferential expression in thymus, spleen, and peripheral blood lymphocytes (34Shimada 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, 35Peters R.T. Liao S.M. Maniatis T. Mol. Cell. 2000; 5: 513-522Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 36Perry A.K. Chow E.K. Goodnough J.B. Yeh W.C. Cheng G. J. Exp. Med. 2004; 199: 1651-1658Crossref PubMed Scopus (313) Google Scholar). IKKϵ is closely related to the constitutive and ubiquitous TBK-1 (TANK-binding kinase-1) (37Pomerantz J.L. Baltimore D. EMBO J. 1999; 18: 6694-6704Crossref PubMed Google Scholar, 38Tojima 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) and shows structural and sequence homology to IKKα and IKKβ. Both IKKϵ and TBK-1 are downstream kinases in Toll-like receptor pathways and are crucial for regulation of interferon (IFN)-β and IFN-inducible genes (37Pomerantz J.L. Baltimore D. EMBO J. 1999; 18: 6694-6704Crossref PubMed Google Scholar, 39Takeuchi O. Hemmi H. Akira S. J. Endotoxin Res. 2004; 10: 252-256Crossref PubMed Google Scholar, 40Kravchenko V.V. Mathison J.C. Schwamborn K. Mercurio F. Ulevitch R.J. J. Biol. 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In addition, IKKϵ phosphorylates IκBα at Ser36 as well as NF-κB p65 at Ser536 and Ser468 (34Shimada 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, 35Peters R.T. Liao S.M. Maniatis T. Mol. Cell. 2000; 5: 513-522Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 45Buss 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, 46Hemmi 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). However, studies have shown that mice bearing a deletion of the IKKϵ gene respond normally to lipopolysaccharide and double-stranded RNA challenges with respect to activation of IRF-3 and NF-κB (39Takeuchi O. Hemmi H. Akira S. J. Endotoxin Res. 2004; 10: 252-256Crossref PubMed Google Scholar, 46Hemmi 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). This is presumably due to a functional redundancy between IKKϵ and TBK-1. IKKϵ has therefore been linked to the canonical p65/p50 activation pathway and to the Toll-like receptor-3 and -4 pathways to IRFs and IFN-inducible genes (37Pomerantz J.L. Baltimore D. EMBO J. 1999; 18: 6694-6704Crossref PubMed Google Scholar, 39Takeuchi O. Hemmi H. Akira S. J. Endotoxin Res. 2004; 10: 252-256Crossref PubMed Google Scholar, 40Kravchenko 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, 41Civas A. Genin P. Morin P. Lin R. Hiscott J. J. Biol. 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Chem. 2003; 278: 26612-26619Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), suggesting a possible link between the NF-κB and CCAAA/enhancer-binding protein pathways. Here, we demonstrate that IKKϵ can be found in a complex with p52 and p65 and is required for transactivation of gene expression by this complex. We have therefore found a role for IKKϵ in the activation of an NF-κB complex containing p52 and p65. Cell Culture, Plasmids, and Reagents—HEK293 and HeLa cell lines were purchased from the Centre for Applied Microbiology & Research (Salisbury, UK). Mouse embryonic fibroblasts (MEFs) with a deletion of p65 and the corresponding wild-type MEFs were kindly provided by Ron Hay (University of St. Andrews, Fife, Scotland, UK). The cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum and incubated at 37 °C in a humidified atmosphere of 5% CO2. The cells were seeded at 0.5–1 × 105/ml and incubated overnight for experiments. Cells were treated as indicated in the figure legends. Lipopolysaccharide from Escherichia coli serotype O26:B6 and TNFα were purchased from Sigma. Constructs bearing FLAG-tagged IKKϵ and the construct encoding the kinase activity-impaired mutant IKKϵ(K38A) were kindly provided by Shizuo Akira (Osaka University, Osaka, Japan). The TBK-1-encoding plasmid was a kind gift from Dr. Makato Nakanishi (National Institute for Longevity Sciences, Aichi, Japan). The constructs encoding IKKα and IKKβ was kindly donated by Tularik (San Francisco, CA). The construct comprising hemagglutinin-tagged p65 was provided by GlaxoSmithKline (Stevenage, UK). The plasmids encoding FLAG-tagged p100 (pRSV-p100) and p52 (pRSV-NF-p52) and the glutathione S-transferase (GST) fusion constructs GST-p65-(1–306) and GST-p65-(428–551) were a kind gift from Neil Perkins (University of Dundee, Scotland). The NF-κB-luciferase construct was a gift from Robert Hofmeister (Universitaet Regensburg, Regensburg, Germany), and the IFN-stimulated response element-luciferase construct was purchased from Clontech. The Gal4-p65 transactivation construct and the Gal4-dependent reporter construct were kindly provided by Lienhard Schmitz (Deutsches Krebsforschungszentrum, Heidelberg, Germany). For construction of the p52-Gal4 transactivation fusion protein, we used the pAB vector, which was kindly donated by Oliver Schmidt (University of Giessen, Giessen, Germany). p52 was PCR-amplified with a forward primer containing an EcoRI restriction site (5′-AAGAATTCAAGCTTCACCATGG-3′) and a reverse primer containing a BamHI site immediately upstream of the stop codon (5′-CGGGATCCTTCGCGCCCCGCCC-3′). The amplified DNA was then digested with EcoRI and BamHI, and the overhangs were filled with Klenow polymerase (New England Biolabs Ltd., Herts, UK) and ligated with the blunted BamHI site of the pAB vector. Correct orientation was confirmed by sequencing. Overexpressed p52-Gal4 fusion protein was detected in immunoblots. For construction of the GST-p100 fusion protein, p100 was amplified from plasmid pRSV-p100 with primers bearing a BamHI restriction site (p100 for, 5′-GCGGATCCATGGAGAGTTGCTACAACCCAG-3′l and p100rev, 5′-TATTTGGCGGATCCTTATTTGTCCCAACTGAGGGGTG-3′). p100 was then subcloned into the BamHI site of vector pGEXKG. Yeast Two-hybrid Screen—The C-terminal portion of IKK3 (amino acids 541–716) was generated by PCR using Pfu polymerase (Stratagene) and primers 5′-CACCCAGCAGATTCAGTGCTGTTTGG-3′ (forward) and 5′-TCAGACATCAGGAGGTGCTGG-3′ (reverse) against a full-length IKK3 clone (GlaxoSmithKline). The resulting PCR product was subcloned into the TOPO-pENTR vector (Invitrogen) using the directional TOPO-pENTR cloning kit (catalog no. K2400-20, Invitrogen) following the manufacturer's instructions. The resulting entry clones were used in Gateway® LR Clonase™ recombination reactions (catalog no. 11791-019, Invitrogen) with Gateway-converted pYTH9 and pYTH16 destination vectors to clone in-frame with the Gal4 DNA-binding domain (DBD) for yeast two-hybrid studies as described previously (48Fuller K.J. Morse M.A. White J.H. Dowell S.J. Sims M.J. BioTechniques. 1998; 25: 85-92Crossref PubMed Scopus (24) Google Scholar). All constructs were confirmed by automated sequencing. Yeast (Saccharomyces cerevisiae) strain Y190 expressing the fusion protein between the Gal4 DBD and the IKK3 C terminus was selected and transformed with a human H9 T-cell cDNA library (GlaxoSmithKline) to give at least a 1-fold representation of the library. Interacting clones were selected with minimal selective dropout medium minus histidine, leucine, and tryptophan and containing 15 mm 3-amino-1,2,4-triazole (catalog no. A8056, Sigma), followed by production of β-galactosidase as determined by a freeze-fracture assay. Plasmid DNA was recovered from yeast using the Yeastmaker yeast plasmid isolation kit (catalog no. 630441, Clontech), and the resulting DNA was transformed into E. coli before sequencing. Mass Spectrometry—Anti-IKK3 antibody was used to immunoprecipitate endogenous IKK3 from HeLa cell extracts (25 × 108) as described below. Eluted endogenous IKK3 complexes were then run on 4–12% gels and separated by gel electrophoresis. Gels were stained overnight with Coomassie Blue. Bands were excised and then reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin using a MassPREP workstation (Waters, Manchester, UK). The resulting peptide mixtures were analyzed by liquid chromatographytandem mass spectrometry (MS/MS) using a CapLC and Q-Tof mass spectrometer (Waters) operating in data-dependent MS/MS mode. Targeted liquid chromatography-MS/MS experiments were carried out by creating a list of expected tryptic fragments for selected proteins. Information on the predicted ions was used to direct sampling of particular peptides derived from mixtures in follow-up liquid chromatography-MS/MS analyses. Peptides and proteins were identified by automated searching of all MS/MS spectra against a GlaxoSmithKline non-redundant protein data base. Immunoprecipitation and Immunoblotting—HeLa cells were seeded in 10- or 15-cm dishes and, if needed, stimulated for 24 h with 20 ng/ml TNFα at 75% confluency. The cells were lysed in 1 ml of low stringency lysis buffer (50 mm HEPES, 100 mm NaCl, 1 mm EDTA, 10% glycerol, 0.5% Nonidet P-40, and protease inhibitors). Anti-IKKϵ polyclonal (catalog no. ab7891, Abcam), anti-p52 monoclonal (catalog no. 05-361, Upstate), or anti-p65 polyclonal (catalog no. sc-8008, Santa Cruz Biotechnology, Inc.) antibody was incubated with cell lysate for 2 h and then for 1 h after addition of protein G-Sepharose. The immune complexes were precipitated, subjected to 10% SDS-PAGE, and subsequently immunoblotted. Anti-FLAG antibody M2 (Sigma) was used for detection of expression levels in luciferase assays. In Vitro Kinase Assay—For kinase assays, immune complexes were washed an additional three times with kinase buffer (20 mm HEPES (pH 7.5), 2 mm dithiothreitol, 10 mm MgCl2, 50 mm NaCl, 100 μm Na3VO4, 20 mm β-glycerol phosphate, 1 mm aprotinin, 1 mm sodium orthovanadate, and 1 mm phenylmethylsulfonyl fluoride). Immune complex kinase reactions were carried out by incubating the immunoprecipitated kinases with GST-p100, GST-p65-(1–306), or GST-p65-(428–551) and 30 μl of kinase buffer plus 2 μCi of [γ-32P]ATP and 0.6 μm nonradioactive ATP. Samples were then incubated at 37 °C for 45 min. Overexpressed and purified GST was used as a negative control. Gels were transferred onto polyvinylidene difluoride membranes and visualized by autoradiography. Gel Filtration—HeLa cells stimulated with 20 ng/ml TNFα for 30 h and lysed in high stringency lysis buffer (50 mm HEPES (pH 7.5), 100 mm NaCl, 1 mm EDTA, 10% glycerol, and 0.5% Nonidet P-40) on ice for 15 min. Approximately 8 μg of protein from the whole cell extracts was loaded onto a Sephacryl S-300 26/60 gel filtration column (Amersham Biosciences) and eluted with 50 mm NaHPO2–4. 3-ml fractions were collected for immunoblotting or precipitation. If not used immediately, the samples were snap-frozen in liquid nitrogen. Transfection-based Reporter Gene Assays—MEFs were seeded at 0.2–0.5 × 105/ml in 24-well plates; incubated overnight; and transfected with GeneJuice transfection reagent (Novagen, Madison, WI) according to the manufacturer's instructions with a total amount of 350–400 μg/well containing 150 ng of p-55UASGLuc and 50 ng of p52-Gal4 fusion construct or of a construct bearing the Gal4 DBD only (used as a negative control). HEK293 cells were seeded at 0.5 × 105/ml in 96-well plates and transfected with a total of 250 ng of DNA containing 100 ng of p-55UASGLuc or 30 ng of p52-Gal4, Gal4-p65, or the Gal4 DBD. The assays also contained the plasmid DNA of interest, an empty vector as filler DNA, and 20–50 ng of Renilla reniformis luciferase construct (used as internal control to determine transfection efficiency). For NF-κB-luciferase and IFN-stimulated response element-luciferase assays, HEK293 cells were seeded at 1–2 × 104/well in 96-well plates; incubated overnight; and transfected with a total amount of 250 ng of DNA/well comprising 100 ng of reporter construct, the plasmid DNA of interest, 40 ng of R. reniformis luciferase construct, and empty vector as filler DNA. The cells were lysed in passive lysis buffer (Promega Ltd., Southampton, UK) for 15 min. Cell extracts were monitored 24–36 h post-transfection for firefly luciferase activity following standard protocols. Activities are expressed as -fold activation over unstimulated empty vector controls. The experiments were carried out in triplicate. Electrophoretic Mobility Shift Assays—Wild-type MEFs were grown in 15-cm dishes and treated as described in the figure legends. Nuclear extracts were prepared as described previously (49Alkalay I. Yaron A. Hatzubai A. Jung S. Avraham A. Gerlitz O. Pashut-Lavon I. Ben-Neriah Y. Mol. Cell. Biol. 1995; 15: 1294-1301Crossref PubMed Google Scholar). Nuclear extracts were incubated at room temperature for 30 min with 10,000 cpm double-stranded [γ-32P]ATP and NF-κB binding sequence (5′-AGTTGAGGGGACTTTCCCAGGC-3′) in hybridization buffer (10 mm Tris-HCl (pH 7.5), 100 mm NaCl, 1 mm EDTA, 5 mm dithiothreitol, 4% glycerol, and 100 μg/ml nuclease-free bovine serum albumin) containing 2 μg/ml poly(dI-dC) as a nonspecific competitor. Supershift antibodies specific for p65 (catalog no. sc-8008X, Santa Cruz Biotechnology, Inc.) and NF-κB2 (clone K-27, catalog no. sc-298, Santa Cruz Biotechnology, Inc.) were added to the nuclear extracts and kept on ice for 1 h prior to hybridization with the oligonucleotide. Protein-DNA complexes were separated on a 5% native polyacrylamide gel, and complex formation was detected by autoradiography. IKKϵ Interacts with NF-κB2—To gain insight into the function of IKKϵ and to expand our understanding of the IKKϵ signaling cascade, a yeast two-hybrid screen for IKKϵ-interacting proteins was performed. A schematic representation of the IKKϵ protein and the truncation mutant used for yeast two-hybrid screening is shown in Fig. 1A. Using the C-terminal portion of IKKϵ containing a helix-loop-helix motif (amino acids 541–716) as a bait, we screened an H9 T-cell cDNA library and identified 33 positive clones that were able to activate reporter gene expression. These included IKKϵ-interacting fusion proteins containing portions of TANK (TRAF family member-associated NF-κB activator; I-TRAF (TNF receptor-associated factor)) and NF-κB2. The interaction between IKKϵ and TANK has been reported previously by Nomura et al. (50Nomura F. Kawai T. Nakanishi K. Akira S. Genes Cells. 2000; 5: 191-202Crossref PubMed Scopus (96) Google Scholar). More recently, TANK was shown to link IKKϵ with the classical IKK complex (51Chariot A. Leonardi A. Muller J. Bonif M. Brown K. Siebenlist U. J. Biol. Chem. 2002; 277: 37029-37036Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The amino acid sequence of human NF-κB2 (p100/p52) is shown in Fig. 1B, and highlighted in boldface is the sequence incorporated within the positive clone from the yeast two-hybrid screen. The N-terminal region of NF-κB2 contains the Rel homology region (Fig. 1C). As the association with NF-κB2 was novel, additional studies were undertaken to verify the interaction in cells and to investigate the biological relevance in more detail. Confirmation of an IKKϵ Interaction with NF-κB2 (p52)—Based on the interaction in yeast, the association of IKKϵ with NF-κB2 (p100/p52) was investigated and confirmed to occur endogenously in HeLa cells. IKKϵ was immunoprecipitated from HeLa cell lysates prepared before and after stimulation with 10 ng/ml TNFα for 24 h. Gel-separated proteins were then Western-blotted with an antibody that recognizes the full-length NF-κB2 protein (p100/p52) (Fig. 2). After TNFα stimulation, an endogenous interaction could be det
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