Lipopolysaccharide-mediated Interferon Regulatory Factor Activation Involves TBK1-IKKϵ-dependent Lys63-linked Polyubiquitination and Phosphorylation of TANK/I-TRAF

Type I interferon gene induction relies on IKK-related kinase TBK1 and IKKϵ-mediated phosphorylations of IRF3/7 through the Toll-like receptor-dependent signaling pathways. The scaffold proteins that assemble these kinase complexes are poorly characterized. We show here that TANK/ITRAF is required for the TBK1- and IKKϵ-mediated IRF3/7 phosphorylations through some Toll-like receptor-dependent pathways and is part of a TRAF3-containing complex. Moreover, TANK is dispensable for the early phase of double-stranded RNA-mediated IRF3 phosphorylation. Interestingly, TANK is heavily phosphorylated by TBK1-IKKϵ upon lipopolysaccharide stimulation and is also subject to lipopolysaccharide- and TBK1-IKKϵ-mediated Lys63-linked polyubiquitination, a mechanism that does not require TBK1-IKKϵ kinase activity. Thus, we have identified TANK as a scaffold protein that assembles some but not all IRF3/7-phosphorylating TBK1-IKKϵ complexes and demonstrated that these kinases possess two functions, namely the phosphorylation of both IRF3/7 and TANK as well as the recruitment of an E3 ligase for Lys63-linked polyubiquitination of their scaffold protein, TANK. Type I interferon gene induction relies on IKK-related kinase TBK1 and IKKϵ-mediated phosphorylations of IRF3/7 through the Toll-like receptor-dependent signaling pathways. The scaffold proteins that assemble these kinase complexes are poorly characterized. We show here that TANK/ITRAF is required for the TBK1- and IKKϵ-mediated IRF3/7 phosphorylations through some Toll-like receptor-dependent pathways and is part of a TRAF3-containing complex. Moreover, TANK is dispensable for the early phase of double-stranded RNA-mediated IRF3 phosphorylation. Interestingly, TANK is heavily phosphorylated by TBK1-IKKϵ upon lipopolysaccharide stimulation and is also subject to lipopolysaccharide- and TBK1-IKKϵ-mediated Lys63-linked polyubiquitination, a mechanism that does not require TBK1-IKKϵ kinase activity. Thus, we have identified TANK as a scaffold protein that assembles some but not all IRF3/7-phosphorylating TBK1-IKKϵ complexes and demonstrated that these kinases possess two functions, namely the phosphorylation of both IRF3/7 and TANK as well as the recruitment of an E3 ligase for Lys63-linked polyubiquitination of their scaffold protein, TANK. The innate immunity in response to a variety of pathogen-associated molecular patterns is established upon binding of their molecular components on specific receptors and ultimately leads to the transcriptional induction of type I interferon (IFN) 8The abbreviations used are: HA, hemagglutinin; IFN, interferon; IKK, IκB kinase; IRF, interferon-regulatory factor; ISRE, interferon-stimulated response element; LPS, lipopolysaccharide; Myd88, myeloid differentiation primary response gene 88; NEMO, NF-κB essential modulator; NF-κB, nuclear factor κB; TANK, TRAF family member-associated NF-κB activator; TBK1, TANK-binding kinase-1; TNFα, tumor necrosis factor α; TLR, Toll-like receptor; TRAF, tumor necrosis factor receptor-associated factor; TRIF, Toll-interleukin-1 receptor domain-containing adaptor inducing interferon-β-mediated transcription factor activation; GST, glutathione S-transferase; Ub, ubiquitin; shRNA, short hairpin RNA; dsRNA, double-stranded RNA; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase. genes. Signaling pathways triggered by these viral or bacterial products occur through the Toll-like receptor (TLR) or the cytosolic receptor pathway, and both of them rely on the coordinated activation of transcriptional factors, among which the interferon-regulatory factors (IRFs) are critical for the immune response (1Kato H. Kim S. Yoneyama M. Yamamoto M. Uematsu S. Matsui K. Tsujimura T. Takeda K. Fujita T. Takeuchi O. Akira S. Immunity. 2005; 23: 19-28Abstract Full Text Full Text PDF PubMed Scopus (1113) Google Scholar, 2Honda K. Kim T. Nat. Rev. Immunol. 2006; 6: 644-658Crossref PubMed Scopus (1277) Google Scholar, 3O'Neill L.A. Curr. Opin. Immunol. 2006; 18: 3-9Crossref PubMed Scopus (527) Google Scholar, 4Stetson D.B. Kim R. Immunity. 2006; 25: 373-381Abstract Full Text Full Text PDF PubMed Scopus (897) Google Scholar). TLRs are members of the so-called pattern recognition receptor family, are mainly expressed on immune system sentinel cells, and specifically sense a variety of molecules produced by bacteria, viruses, fungi, and protozoa (5Akira S. Kim S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8874) Google Scholar, 6West A.P. Kim A.A. Ghosh S. Annu. Rev. Cell Dev. Biol. 2006; 22: 409-437Crossref PubMed Scopus (553) Google Scholar). For example, lipopolysaccharide (LPS) of Gram-negative bacteria binds TLR4, triggering two distinct signaling pathways, namely the Myd88-dependent and TRIF-dependent pathways. The Myd88-dependent pathway relies on the scaffold proteins TAB2, TAB3, and TRAF6 and ultimately leads to TAK1- and IKK-mediated NF-κB activation and subsequent induction of proinflammatory genes (7Sato S. Kim H. Takeda K. Ninomiya-Tsuji J. Yamamoto M. Kawai T. Matsumoto K. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 1087-1095Crossref PubMed Scopus (767) Google Scholar, 8Shim J.H. Kim C. Paschal A.E. Bailey S.T. Rao P. Hayden M.S. Lee K.Y. Bussey C. Steckel M. Tanaka N. Yamada G. Akira S. Matsumoto K. Ghosh S. Genes Dev. 2005; 19: 2668-2681Crossref PubMed Scopus (597) Google Scholar). The TRIF-dependent pathway targets IRF3/7 for phosphorylation through a TBK1-IKKϵ-dependent mechanism, a critical step for type I IFN induction (2Honda K. Kim T. Nat. Rev. Immunol. 2006; 6: 644-658Crossref PubMed Scopus (1277) Google Scholar). More recently, a TRAF3- and TBK1-IKKϵ-dependent pathway has also been characterized and appears to be required for the induction of the type I interferons and the anti-inflammatory cytokine IL-10 (9Hacker H. Kim V. Blagoev B. Kratchmarova I. Hsu L.C. Wang G.G. Kamps M.P. Raz E. Wagner H. Hacker G. Mann M. Karin M. Nature. 2006; 439: 204-207Crossref PubMed Scopus (746) Google Scholar, 10Oganesyan G. Kim S.K. Guo B. He J.Q. Shahangian A. Zarnegar B. Perry A. Cheng G. Nature. 2006; 439: 208-211Crossref PubMed Scopus (711) Google Scholar). How TBK1 and IKKϵ are assembled into functional IRF3/7-phosphorylating complexes is poorly understood. To date, NAP1 is the only NAK/TBK1-interacting scaffold protein identified in the dsRNA-mediated TLR3-dependent, Myd88-independent pathway (11Sasai M. Kim H. Matsumoto M. Inoue N. Fujita F. Nakanishi M. Seya T. J. Immunol. 2005; 174: 27-30Crossref PubMed Scopus (108) Google Scholar), but it is not known whether NAP1 is involved in other TLR-dependent pathways or whether other scaffold proteins are required. TANK (also known as I-TRAF) was initially described as a TRAF2/3-interacting molecule that positively regulates NF-κB (12Cheng G. Kim D. Genes Dev. 1996; 10: 963-973Crossref PubMed Scopus (265) Google Scholar) through an interaction with IKKϵ (also called IKK-I) (13Peters R.T. Kim S.M. Maniatis T. Mol. Cell. 2000; 5: 513-522Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 14Nomura F. Kim T. Nakanishi K. Akira S. Genes Cells. 2000; 5: 191-202Crossref PubMed Scopus (96) Google Scholar) and TBK1 (15Pomerantz J.L. Kim D. EMBO J. 1999; 18: 6694-6704Crossref PubMed Google Scholar). TANK/I-TRAF was also characterized as a signaling molecule that negatively regulates NF-κB through its C-terminal domain, but the underlying mechanism remains unclear (16Kaye K.M. Kim O. Harada J.N. Izumi K.M. Yalamanchili R. Kieff E. Mosialos G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11085-11090Crossref PubMed Scopus (221) Google Scholar, 17Rothe M. Kim J. Shu H.B. Williamson K. Goddard A. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8241-8246Crossref PubMed Scopus (189) Google Scholar). A role for TANK in NF-κB signaling was further supported by the association of TANK with NEMO/IKKγ, a subunit of the IKK complex (18Chariot A. Kim 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). This provides a link by which the TANK-interacting TBK1-IKKϵ kinases are connected to the IKK complex for subsequent phosphorylation of NF-κB proteins, such as p65 or c-Rel (19Adli M. Kim A.S. J. Biol. Chem. 2006; 281: 26976-26984Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 20Harris J. Kim S. Sharma S. Sun Q. Lin R. Hiscott J. Grandvaux N. J. Immunol. 2006; 177: 2527-2535Crossref PubMed Scopus (87) Google Scholar). Thus, it is believed that TANK acts as a scaffold protein (14Nomura F. Kim T. Nakanishi K. Akira S. Genes Cells. 2000; 5: 191-202Crossref PubMed Scopus (96) Google Scholar, 18Chariot A. Kim 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). Since the TANK-binding kinases TBK1-IKKϵ are essential for IRF3/7 activation (21Fitzgerald K.A. Kim 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 (2093) Google Scholar, 22Sharma S. Kim B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1369) Google Scholar, 23Hemmi H. Kim 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 suggested that TANK may be involved in this and possibly other signaling pathways. Whereas NF-κB activation through multiple signaling pathways relies on sequentially activated kinases, which ultimately target the inhibitory IκB proteins for phosphorylation and subsequent degradative Lys48-linked polyubiquitination (24Karin M. Kim Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4100) Google Scholar, 25Hayden M.S. Kim S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3377) Google Scholar), evidence is accumulating that Lys63-linked, nondegradative polyubiquitination of critical scaffold proteins is also essential for IKK and subsequent NF-κB activations (26Chen Z.J. Nat. Cell Biol. 2005; 7: 758-765Crossref PubMed Scopus (1025) Google Scholar, 27Sebban H. Kim S. Courtois G. Trends Cell Biol. 2006; 16: 569-577Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Apart from TRAF6 (28Wang C. Kim L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1644) Google Scholar), NEMO/IKKγ is the best known example of an NF-κB-activating scaffold protein subject to Lys63-linked polyubiquitination, and several signaling pathways specifically target distinct lysine residues of NEMO/IKKγ (27Sebban H. Kim S. Courtois G. Trends Cell Biol. 2006; 16: 569-577Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 29Israel A. Trends Immunol. 2006; 27: 395-397Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). This NEMO/IKKγ post-translational modification occurs upon TNFα stimulation and appears to require the E3 ligase c-IAP-1 (30Tang E.D. Kim C.Y. Xiong Y. Guan K.L. J. Biol. Chem. 2003; 278: 37297-37305Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 31Wu C.J. Kim D.B. Li T. Srinivasula S.M. Ashwell J.D. Nat. Cell Biol. 2006; 8: 398-406Crossref PubMed Scopus (508) Google Scholar). It also occurs upon T cell receptor signaling, where it relies on MALT-1, a Bcl10-interacting protein potentially acting as an E3 ligase (32Zhou H. Kim I. O'Rourke K. Ultsch M. Seshagiri S. Eby M. Xiao W. Dixit V.M. Nature. 2004; 427: 167-171Crossref PubMed Scopus (453) Google Scholar). Polyubiquitination of NEMO/IKKγ also occurs through a Nod2-dependent pathway, but the E3 ligase in that signaling cascade remains to be identified (33Abbott D.W. Kim A. Asara J.M. Cantley L.C. Curr. Biol. 2004; 14: 2217-2227Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). This nondegradative polyubiquitination typically requires the E2 protein Ubc13, and although the essential role of this protein in NF-κB activation in vivo has recently been questioned, it appears that other pathways, such as those leading to MAPK activation, also involve Lys63-linked and Ubc13-dependent polyubiquitination (34Yamamoto M. Kim T. Takeda K. Sato S. Sanjo H. Uematsu S. Saitoh T. Yamamoto N. Sakurai H. Ishii K.J. Yamaoka S. Kawai T. Matsuura Y. Takeuchi O. Akira S. Nat. Immunol. 2006; 7: 962-970Crossref PubMed Scopus (228) Google Scholar). Like NF-κB activation, the TLR-dependent pathways leading to IRF3/7 activation also rely on sequentially activated kinases, but the extent to which nondegradative Lys63-linked polyubiquitination is required for these signaling pathways is unknown. To identify scaffold proteins in the TLR- and TBK1-IKKϵ-dependent pathways and concomitantly learn more about the biological functions of TANK, we searched for TANK-interacting proteins in a yeast two-hybrid screen. TANK was found associated with IRF7 and connects TBK1-IKKϵ to this protein for subsequent phosphorylation in the TLR- and Myd88-dependent pathways in macrophages. Moreover, we show that TANK is phosphorylated and also subject to Lys63-linked polyubiquitination, both events requiring TBK1-IKKϵ. Whereas LPS-mediated TANK phosphorylation requires TBK1-IKKϵ kinase domains, the Lys63-linked TANK polyubiquitination does not require these domains but is TRAF3-dependent. Thus, our results identify TANK/I-TRAF as a signaling molecule positively regulating transcription of type I interferons through some TLR-dependent pathways. We also provide evidence for TBK1-IKKϵ acting both as IRF3/7 phosphorylating kinases and also as molecules required for the Lys63-linked polyubiquitination of their scaffold protein TANK. Cell Culture, Biological Reagents, and Mice—Human embryonic kidney 293 and HeLa cells were maintained as described (35Leonardi A. Kim A. Claudio E. Cunningham K. Siebenlist U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10494-10499Crossref PubMed Scopus (132) Google Scholar, 36Leonardi A. Kim H. Franzoso G. Brown K. Siebenlist U. J. Biol. Chem. 2000; 275: 271-278Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), whereas RAW 264.7 macrophages were maintained in Dulbecco's modified Eagle's medium supplemented with 5% low endotoxin fetal bovine serum, glutamine, and antibiotics, respectively. CD14-stably expressing THP1 cells, a gift from Dr. P. Tobias (The Scripps Research Institute, La Jolla, CA) were cultured in RPMI supplemented with fetal bovine serum, glutamine, antibiotics, and G418. LPS (0111:B4), mouse TNFα, poly(I:C), and CpG DNA (ODN 1826) were purchased from Sigma, Roche Applied Science, Amersham Biosciences, and Invivogen (San Diego, CA), respectively, whereas staurosporin and female BALB/c and C57BL/6 mice were maintained and bred in the laboratory of Parasitology (University of Brussels, Belgium). For isolation of peritoneal macrophages from these mice, sterile inflammation was induced by intraperitoneal injection of thioglycollate 3% in phosphate-buffered saline. Inflammatory peritoneal exudate cells were harvested 48 h after injection using 10 ml of ice-cold and sterile phosphate-buffered saline. Peritoneal macrophages were selected by adherence on cell dishes and were cultured in RPMI supplemented with 5% low endotoxin fetal bovine serum and antibiotics. Polyclonal anti-human TANK rabbit antibodies were previously described (18Chariot A. Kim 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). These purified antibodies were used to detect endogenous polyubiquitinated forms of TANK (see below). Anti-Myc, -TRAF3, and -IκBα antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) as were anti-HA beads. Anti-FLAG antibodies and beads were purchased from Sigma. Monoclonal anti-IKKϵ and -TBK1 antibodies were from Imgenex (San Diego, CA), whereas polyclonal anti-TBK1 antibody was from Cell Signaling (Danvers, MA). The polyclonal and the monoclonal anti-ubiquitin antibodies were purchased from BIOMOL International (Exeter, UK) and Santa Cruz Biotechnology, respectively. The recombinant TBK1 kinase used to assess TANK and IRF3 phosphorylations in vitro was from Invitrogen. Human FLAG-TANK and truncation mutants of TANK were previously described, as were FLAG-TANK ΔIKKϵ, FLAG-TANKΔZnF, FLAG-IKKϵ, and the wild type and kinase-dead IKKϵ-Myc constructs (18Chariot A. Kim 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, 37Bonif M. Kim M.A. Close P. Benoit V. Heyninck K. Chapelle J.P. Bours V. Merville M.P. Piette J. Beyaert R. Chariot A. Biochem. J. 2006; 394: 593-603Crossref PubMed Scopus (29) Google Scholar). The ISRE reporter plasmid was kindly provided by Dr. R. Beyaert (Department for Molecular Biomedical Research, Unit of Molecular Signal Transduction in Inflammation, VIB-Ghent University, Belgium). The GST-IRF3/7 and -TANK constructs were subcloned by PCR-amplifying the C-terminal portion of IRF3/7 (22Sharma S. Kim B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1369) Google Scholar) and the full-length TANK coding sequence into the pGex-6P3 (Amersham Biosciences). The corresponding purified fusion proteins were used as substrates for the kinase assays (see below). The pCW7 Myc-tagged wild type and the pCW8 K48R ubiquitin constructs were a gift from R. Kopito (Department of Biological Sciences, Stanford University), whereas both the Myc-tagged K63R and the K48R/K63R ubiquitin expression constructs were generated by mutagenesis according to standard protocols, using primers whose sequences are available upon request. The previously described HA-tagged ubiquitin as well as the HA-Ub (K0) were provided by Dr. Yarden (Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israël) (38Mosesson Y. Kim K. Katz M. Zwang Y. Vereb G. Szollosi J. Yarden Y. J. Biol. Chem. 2003; 278: 21323-21326Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Yeast Two-hybrid Screening—The cDNA encoding TANK (amino acids 306–425) was cloned in frame into the GAL4 DNA-binding vector pGADT7 (Clontech). This plasmid was used as bait in a two-hybrid screen of a human HeLa cDNA library in Saccharomyces cerevisiae Y187, according to the Matchmaker Two-Hybrid System II Protocol (Clontech). Positive yeast clones were selected for their ability to grow in the absence of histidine, leucine, and tryptophan. Colonies were subsequently tested for β-galactosidase activity, and DNA sequences from positive clones were identified by sequencing. Immunoprecipitations and Kinase Assays—For immunoprecipitations involving overexpressed proteins, 293 cells (3 × 106) were transfected via FUGENE 6 (Roche Applied Science) with expression vectors as indicated in the figures. 24 h after transfection, cells were washed with phosphate-buffered saline and lysed in 0.5% Triton lysis buffer. Ectopically expressed FLAG- or Myc-tagged proteins were immunoprecipitated by using anti-FLAG or -Myc antibodies bound to agarose beads for 2 h at 4 °C. For anti-TANK immunoprecipitations, cell lysates were incubated with the polyclonal anti-TANK antibody for 2 h followed by an overnight incubation with protein A-agarose. All of the immunoprecipitates were then washed five times with 0.5% Triton lysis buffer and subjected to SDS-PAGE for subsequent Western blot analyses. To assess IRF3/7 phosphorylation, anti-TANK or -TBK1 immunoprecipitates were used in immune complex kinase assays using a purified GST-IRF3/7 as substrate, as described (36Leonardi A. Kim H. Franzoso G. Brown K. Siebenlist U. J. Biol. Chem. 2000; 275: 271-278Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). To assess TANK or IRF3 phosphorylation in vitro, the corresponding purified GST fusion proteins were incubated with a recombinant TBK1 kinase, as previously described (36Leonardi A. Kim H. Franzoso G. Brown K. Siebenlist U. J. Biol. Chem. 2000; 275: 271-278Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In Vivo Ubiquitin Conjugation Assays—293 cells were transfected with Myc- or HA-ubiquitin and either FLAG-TANK or FLAG-TANK mutants together with the indicated expression vectors, according to the protocol described above. 24 h after transfection, cells were lysed, and total cell extracts were subjected to anti-FLAG immunoprecipitations using the anti-FLAG beads, as described above. The ubiquitin-conjugated TANK proteins were subsequently detected by performing anti-Myc or -HA Western analyses. For detection of endogenous polyubiquitinated forms of TANK, cell extracts were lysed as described (30Tang E.D. Kim C.Y. Xiong Y. Guan K.L. J. Biol. Chem. 2003; 278: 37297-37305Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) and subsequently incubated overnight with the purified anti-TANK antibody followed by a 2-h incubation with protein A-agarose. Immunoprecipitates were subsequently subjected to anti-Ub western analyses. RNA Interference and Luciferase Assays—For RNA interference, decreased Ubc13 expression was obtained by transfecting a SMART POOL of Ubc13 (Dharmacon, CO) using the oligofectamine reagent according to the protocol provided by the manufacturer (Invitrogen). For generation of the shRNA constructs targeting either green fluorescent protein (control) or the TANK transcript, inserts were cloned into the pLL3.7 lentivirus according to the protocol kindly provided by Dr. L. van Parijs (MIT, Boston, MA) (39Rubinson D.A. Kim C.P. Kwiatkowski A.V. Sievers C. Yang L. Kopinja J. Rooney D.L. Ihrig M.M. McManus M.T. Gertler F.B. Scott M.L. Van Parijs L. Nat. Genet. 2003; 33: 401-406Crossref PubMed Scopus (1347) Google Scholar). Details are available upon request. For TRAF3 depletion in human macrophages, THP1/CD14 cells were transfected with either the MISSION shRNA lentiviral construct targeting the TRAF3 transcript or the MISSION nontarget shRNA control vector, as described by the manufacturer (Sigma). For luciferase assays, 293 cells (4 × 105 cells/well) were seeded in 6-well (35-mm) plates. After 12 h, cells were transfected as described above with 1 μg of the reporter plasmid and with expression plasmids as indicated. The total amount of transfected DNA was kept constant by adding empty expression vector DNA as needed. Cell extracts were prepared 24 h after transfection, and reporter gene activity was determined by the luciferase assay system (Roche Applied Science). A pGL4.74 plasmid (Promega, Madison, WI) was used to normalize for transfection efficiencies. TANK Associates with IRF3 and IRF7 through its C-terminal Domain—In order to identify TANK-dependent signaling pathways other that those leading to NF-κB activation, we searched for proteins that physically interact with this scaffold protein by means of a yeast two-hybrid screen. The C-terminal domain of TANK (amino acids 306–425) fused to the DNA binding domain of the GAL4 transcription factor (Fig. 1A, left) was used to screen a human HeLa cDNA library that expresses the encoded proteins as fusions with the GAL4 transactivation domain. Among clones that were scored positive for interaction with the bait, one encoded the C-terminal domain and regulatory region of IRF7 (Fig. 1A, right). The interaction between IRF7 and TANK was confirmed by co-immunoprecipitation. Ectopically expressed Myc-tagged IRF7 co-immunoprecipitated with FLAG-TANK in 293 cells (Fig. 1B, top, left, lane 2). Myc-tagged IRF3 also bound FLAG-TANK (Fig. 1B, top, right, lane 2). To confirm that the C-terminal domain of TANK is required for binding to IRF3/7 in mammalian cells, similar coimmunoprecipitations were performed with extracts from 293 cells transfected with Myc-IRF7 and either FLAG-TANK or various TANK mutants deleted in the C-terminal domain. Wild type TANK bound IRF7, but a TANK mutant lacking the 178 C-terminal amino acids did not (Fig. 1C, top, lanes 2 and 6, respectively). A TANK mutant with two point mutations within its zinc finger motif ("TANKΔZnF"), which disrupt a NEMO-binding domain (37Bonif M. Kim M.A. Close P. Benoit V. Heyninck K. Chapelle J.P. Bours V. Merville M.P. Piette J. Beyaert R. Chariot A. Biochem. J. 2006; 394: 593-603Crossref PubMed Scopus (29) Google Scholar), still associated with IRF7, as did TANKΔC20 and TANKΔC50 (Fig. 1C, top, lanes 3–5). Therefore, TANK binds IRF7 through a C-terminal region that is distinct from the domain required for association with NEMO/IKKγ. TANK Enhances IFN Transcription through TBK1 and IKKϵ-mediated IRF3/7 Activation—Because TBK1-IKKϵ-mediated IRF3/7 phosphorylation is essential for the transcriptional induction of type I interferon and subsequent development of the innate response, we next determined whether TANK, as a TBK1-IKKϵ- and IRF3/7-binding protein, is involved in IRF3/7 activation. 293 cells were transfected with the ISRE reporter plasmid, which harbors an IRF3/7-responsive element, and luciferase assays were performed. As expected, increasing amounts of IRF3/7 led to a dose-dependent activation of the ISRE reporter; TANK overexpression alone did not (Fig. 1, D and E, respectively). The addition of increasing amounts of TANK weakly enhanced IRF3-mediated activation of the ISRE promoter and more strongly induced IRF7 transactivation potential (Fig. 1, D and E, respectively). TANK also enhanced TBK1- and IKKϵ-mediated activation of the ISRE promoter (Fig. 1, F and G). Therefore, these results suggest that TANK positively regulates TBK1- and IKKϵ-mediated IRF3/7 activation, presumably by connecting both TANK-interacting kinases to their substrate for its subsequent phosphorylation. TANK Is Part of an IRF3/7-phosphorylating Complex—To explore the hypothesis that TANK connects TBK1-IKKϵ to their substrates, we addressed IRF3/7 phosphorylations in cells overexpressing IKKϵ and either wild type TANK or a mutant lacking the TBK1-IKKϵ-interacting site ("TANKΔIKKϵ") (18Chariot A. Kim 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) by kinase assays using purified GST-IRF3 or -7 as substrate. In agreement with previous reports, IRF3 and IRF7 were strongly phosphorylated by an anti-FLAG immunoprecipitate derived from FLAG-IKKϵ-but not Myc-IKKϵ-overexpressing cells (Fig. 2A, top, lanes 7 and 3, respectively). Moreover, whereas IRF3/7 phosphorylations were detected by incubating anti-FLAG immunoprecipitates derived from cells expressing both FLAG-TANK and IKKϵ-Myc (Fig. 2A, top, lanes 4), no IRF3/7 phosphorylation was detected using immunoprecipitates derived from cells overexpressing a kinase-dead version of IKKϵ or TANKΔIKKϵ (Fig. 2A, top, lanes 5 and 6, respectively). We conclude, therefore, that TANK connects IKKϵ to IRF3/7 for subsequent phosphorylation. To further explore the significance of the interaction between TANK and TBK/IKKϵ for IRF3/7 activation, we first defined the TANK-interacting site on both kinases by co-immunoprecipitations. TBK1 and IKKϵ harbor a N-terminal kinase domain as well as C-terminal coiled-coil regions (40Huang J. Kim T. Xu L.G. Chen D. Zhai Z. Shu H.B. EMBO J. 2005; 24: 4018-4028Crossref PubMed Scopus (130) Google Scholar). C-terminal IKKϵ/TBK1-deletions were generated, and the resulting Myc-tagged wild type and IKKϵ/TBK1 mutants were tested for interaction with FLAG-TANK (Fig. 2, B and C, respectively). IKKϵ/TBK1 and IKKϵΔC6/TBK1ΔC6-Myc associated with FLAG-TANK (Fig. 2, B, top, lanes 1 and 3, and C, top, lanes 2 and 4), but IKKϵ/TBK1 mutants lacking 30 or 52 (for IKKϵ) and 30 or 55 (for TBK1) C-terminal amino acids failed to interact with FLAG-TANK (Fig. 2, B, top, lanes 5 and 7, and C, top, lanes 6 and 8). This indicates that IKKϵ and TBK1 interact with TANK through their C-terminal regions downstream of the coiled-coil domains. We next defined the role of the TANK-interacting domain of IKKϵ in IRF3 activation by assessing IRF3 phosphorylation in kinase assays using extracts of cells with ectopic wild type or IKKϵ mutants deleted in the coiled-coil domains ("IKKϵΔC6" or "IKKϵΔC30") by kinase assays. IRF3 phosphorylation was detected following incubation with anti-FLAG immunoprecipitates derived from cells expressing FLAG-IKKϵ or both FLAG-TANK and wild type but not kinase-dead IKKϵ-Myc (Fig. 2D, top, lanes 6, 2, and 5, respectively). Also, whereas IRF3 phosphorylation was detected in cells overexpressing FLAG-TANK and IKKϵΔC6, (which still interacts with TANK), no IRF3 phosphorylation was detected upon FLAG-TANK and IKKϵΔC30 overexpression (Fig. 2D, top panel, lanes 3 and 4, respectively). This result provides further support for the hypothesis that TANK is associated with an IRF3 kinase, most likely IKKϵ. We next performed a kinase assay with immunoprecipitates of wild type and IKKϵ mutants rather than TANK. As expected, IRF3 phosphorylation was detected using anti-Myc imunoprecipitates derived from cells overexpressing IKKϵ-Myc but not the kinase-dead mutant or FLAG-IKKϵ (Fig. 2E, top, lanes 2, 5, and 6, respectively). IRF3 phosphorylation was also detected in extracts of cells overexpressing IKKϵΔC6 or IKKϵΔC30 (Fig. 2E, top, lanes 3 and 4, respectively). Interestingly, whereas IKKϵ autophosphorylation was observed upon overexpression of wild type or the IKKϵΔC6 mutant, such autophosphorylation was disrupted by deleting the last 30 amino acids of IKKϵ (Fig. 2E, top, lanes 3 and 4, respectively). These results suggest that the N-terminal IKKϵ kinase domain is sufficient for IRF3 phosphorylation in vitro, whereas the C-terminal coiled coil domain required for TANK interaction is dispensable for IRF3 phosphorylation but required for IKK