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 IKK⑀-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/I-TRAF is required for the TBK1-and IKK⑀-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 doublestranded RNA-mediated IRF3 phosphorylation. Interestingly, TANK is heavily phosphorylated by TBK1-IKK⑀ upon lipopolysaccharide stimulation and is also subject to lipopolysaccharide-and TBK1-IKK⑀-mediated Lys 63 -linked polyubiquitination, a mechanism that does not require TBK1-IKK⑀ kinase activity. Thus, we have identified TANK as a scaffold protein that assembles some but not all IRF3/7-phosphorylating TBK1-IKK⑀ 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 Lys 63 -linked polyubiquitination of their scaffold protein, TANK.
The innate immunity in response to a variety of pathogenassociated 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) 8 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 (1)(2)(3)(4).
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 (5,6). 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-B activation and subsequent induction of proinflammatory genes (7,8). The TRIF-dependent pathway targets IRF3/7 for phosphorylation through a TBK1-IKK⑀-dependent mechanism, a critical step for type I IFN induction (2). More recently, a TRAF3-and TBK1-IKK⑀-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 (9, 10). How TBK1 and IKK⑀ are assembled into functional IRF3/7phosphorylating 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 (11), 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-B (12) through an interaction with IKK⑀ (also called IKK-I) (13,14) and TBK1 (15). TANK/I-TRAF was also characterized as a signaling molecule that negatively regulates NF-B through its C-terminal domain, but the underlying mechanism remains unclear (16,17). A role for TANK in NF-B signaling was further supported by the association of TANK with NEMO/IKK␥, a subunit of the IKK complex (18). This provides a link by which the TANK-interacting TBK1-IKK⑀ kinases are connected to the IKK complex for subsequent phosphorylation of NF-B proteins, such as p65 or c-Rel (19,20). Thus, it is believed that TANK acts as a scaffold protein (14,18). Since the TANKbinding kinases TBK1-IKK⑀ are essential for IRF3/7 activation (21)(22)(23), this suggested that TANK may be involved in this and possibly other signaling pathways.
Whereas NF-B activation through multiple signaling pathways relies on sequentially activated kinases, which ultimately target the inhibitory IB proteins for phosphorylation and subsequent degradative Lys 48 -linked polyubiquitination (24,25), evidence is accumulating that Lys 63 -linked, nondegradative polyubiquitination of critical scaffold proteins is also essential for IKK and subsequent NF-B activations (26,27). Apart from TRAF6 (28), NEMO/IKK␥ is the best known example of an NF-B-activating scaffold protein subject to Lys 63 -linked polyubiquitination, and several signaling pathways specifically target distinct lysine residues of NEMO/IKK␥ (27,29). This NEMO/IKK␥ post-translational modification occurs upon TNF␣ stimulation and appears to require the E3 ligase c-IAP-1 (30,31). It also occurs upon T cell receptor signaling, where it relies on MALT-1, a Bcl10-interacting protein potentially acting as an E3 ligase (32). Polyubiquitination of NEMO/IKK␥ also occurs through a Nod2-dependent pathway, but the E3 ligase in that signaling cascade remains to be identified (33). This nondegradative polyubiquitination typically requires the E2 protein Ubc13, and although the essential role of this protein in NF-B activation in vivo has recently been questioned, it appears that other pathways, such as those leading to MAPK activation, also involve Lys 63 -linked and Ubc13-dependent polyubiquitination (34). Like NF-B activation, the TLR-dependent pathways leading to IRF3/7 activation also rely on sequentially activated kinases, but the extent to which nondegradative Lys 63 -linked polyubiquitination is required for these signaling pathways is unknown.
To identify scaffold proteins in the TLR-and TBK1-IKK⑀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-IKK⑀ 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 Lys 63 -linked polyubiquitination, both events requiring TBK1-IKK⑀. Whereas LPS-mediated TANK phosphorylation requires TBK1-IKK⑀ kinase domains, the Lys 63 -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-IKK⑀ acting both as IRF3/7 phosphorylating kinases and also as molecules required for the Lys 63 -linked polyubiquitination of their scaffold protein TANK.

EXPERIMENTAL PROCEDURES
Cell Culture, Biological Reagents, and Mice-Human embryonic kidney 293 and HeLa cells were maintained as described (35,36), 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 TNF␣, 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 (18). These purified antibodies were used to detect endogenous polyubiquitinated forms of TANK (see below). Anti-Myc, -TRAF3, and -IB␣ 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-IKK⑀ 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 ⌬IKK⑀, FLAG-TANK⌬ZnF, FLAG-IKK⑀, and the wild type and kinase-dead IKK⑀-Myc constructs (18,37). 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 (22) 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 Myctagged 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) (38).
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 ␤-galactosidase activity, and DNA sequences from positive clones were identified by sequencing.
Immunoprecipitations and Kinase Assays-For immunoprecipitations involving overexpressed proteins, 293 cells (3 ϫ 10 6 ) 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 FLAGor 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 (36). To assess TANK or IRF3 phosphorylation in vitro, the corresponding purified GST fusion proteins were incubated with a recombinant TBK1 kinase, as previously described (36).
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 (30) 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) (39). 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 ϫ 10 5 cells/well) were seeded in 6-well (35-mm) plates. After 12 h, cells were transfected as described above with 1 g 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-B 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 ("TANK⌬ZnF"), which disrupt a NEMO-binding domain (37), still associated with IRF7, as did TANK⌬C20 and TANK⌬C50 (Fig. 1C, top, lanes [3][4][5]. Therefore, TANK binds IRF7 through a C-terminal region that is distinct from the domain required for association with NEMO/ IKK␥.  TANK Enhances IFN Transcription through TBK1 and IKK⑀mediated IRF3/7 Activation-Because TBK1-IKK⑀-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-IKK⑀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 IKK⑀-mediated activation of the ISRE promoter ( Fig. 1, F and G). Therefore, these results suggest that TANK positively regulates TBK1-and IKK⑀-mediated IRF3/7 activation, presumably by connecting both TANK-interacting kinases to their substrate for its subsequent phosphorylation.

Myc-IRF7
TANK Is Part of an IRF3/7-phosphorylating Complex-To explore the hypothesis that TANK connects TBK1-IKK⑀ to their substrates, we addressed IRF3/7 phosphorylations in cells overexpressing IKK⑀ and either wild type TANK or a mutant lacking the TBK1-IKK⑀-interacting site ("TANK⌬IKK⑀") (18) 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-IKK⑀but not Myc-IKK⑀-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 IKK⑀-Myc ( Fig. 2A, top, lanes 4), no IRF3/7 phosphorylation was detected using immunoprecipitates derived from cells overexpressing a kinase-dead version of IKK⑀ or TANK⌬IKK⑀ ( Fig. 2A, top, lanes 5 and 6, respectively). We conclude, therefore, that TANK connects IKK⑀ to IRF3/7 for subsequent phosphorylation. To further explore the significance of the interaction between TANK and TBK/IKK⑀ for IRF3/7 activation, we first defined the TANK-interacting site on both kinases by co-immunoprecipitations. TBK1 and IKK⑀ harbor a N-terminal kinase domain as well as C-terminal coiled-coil regions (40). C-terminal IKK⑀/TBK1-deletions were generated, and the resulting Myc-tagged wild type and IKK⑀/ TBK1 mutants were tested for interaction with FLAG-TANK (Fig. 2, B and C, respectively). IKK⑀/TBK1 and IKK⑀⌬C6/ TBK1⌬C6-Myc associated with FLAG-TANK ( Fig. 2, B, top, lanes 1 and 3, and C, top, lanes 2 and 4), but IKK⑀/TBK1 mutants lacking 30 or 52 (for IKK⑀) 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 IKK⑀ 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 IKK⑀ in IRF3 activation by assessing IRF3 phosphorylation in kinase assays using extracts of cells with ectopic wild type or IKK⑀ mutants deleted in the coiled-coil domains ("IKK⑀⌬C6" or "IKK⑀⌬C30") by kinase assays. IRF3 phospho-rylation was detected following incubation with anti-FLAG immunoprecipitates derived from cells expressing FLAG-IKK⑀ or both FLAG-TANK and wild type but not kinase-dead IKK⑀-Myc ( Fig. 2D, top, lanes 6, 2, and 5, respectively). Also, whereas IRF3 phosphorylation was detected in cells overexpressing FLAG-TANK and IKK⑀⌬C6, (which still interacts with TANK), no IRF3 phosphorylation was detected upon FLAG-TANK and IKK⑀⌬C30 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 IKK⑀. We next performed a kinase assay with immunoprecipitates of wild type and IKK⑀ mutants rather than TANK. As expected, IRF3 phosphorylation was detected using anti-Myc imunoprecipitates derived from cells overexpressing IKK⑀-Myc but not the kinase-dead mutant or FLAG-IKK⑀ (Fig. 2E, top, lanes 2, 5, and 6, respectively). IRF3 phosphorylation was also detected in extracts of cells overexpressing IKK⑀⌬C6 or IKK⑀⌬C30 (Fig. 2E, top, lanes 3 and 4, respectively). Interestingly, whereas IKK⑀ autophosphorylation was observed upon overexpression of wild type or the IKK⑀⌬C6 mutant, such autophosphorylation was disrupted by deleting the last 30 amino acids of IKK⑀ (Fig. 2E, top, lanes 3 and 4, respectively). These results suggest that the N-terminal IKK⑀ 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⑀ autophosphorylation in vitro.
TANK Is Involved in the LPS-mediated IRF3/7-activating Pathway-To demonstrate the existence of an endogenous, signal-responsive TANK-containing protein complex that phosphorylates IRF3/7, we first stimulated RAW 264.7 macrophages with LPS, which is known to activate TBK1-IKK⑀ through the TLR4-dependent pathway (23,41), or with TNF␣ and subjected the cell lysates to anti-TANK immunoprecipitations followed by an anti-IRF3 Western blot. Interestingly, IRF3 physically associated with TANK upon LPS but not TNF␣ stimulation, and this interaction was transient, since such a TANK-IRF3 complex was only detectable upon 15 min of stimulation ( Fig. 3A, top, lane 2). To further investigate whether such signal-responsive interaction causes IRF3/7 phosphorylations, RAW 264.7 macrophages were again stimulated with LPS or TNF␣ and subsequently subjected to anti-TANK or -HA (negative control) immunoprecipitation followed by an in vitro kinase assay using GST-IRF7 as substrate. LPS-dependent IRF7 phosphorylation was observed again upon 15 min of treatment, which perfectly matches with the kinetics of the TANK-IRF3 interaction, and decreased but persisted even after 4 h of stimulation (Fig. 3B, top, lanes 2-7). Of note, the association of IKK⑀ or TBK1 with TANK was detected in unstimulated cells and was not modulated by LPS as judged by anti-IKK⑀ or -TBK1 Western analysis performed on the anti-TANK immunoprecipitates (Fig. 3B, second and third panels from the top, compare lane 2 with lane 7). Moreover, a much weaker IRF7 phosphorylation was detected upon TNF␣ stimulation (Fig. 3C, top,  lanes 3 and 4). A similar experiment was performed using peritoneal macrophages harvested from a thioglycollate-treated mice. This also showed that IRF7 was phosphorylated by a TANK/I-TRAF Assembles TBK1 and IKK⑀ in the TLR-dependent Pathways OCTOBER

TANK/I-TRAF Assembles TBK1 and IKK⑀ in the TLR-dependent Pathways
TANK-containing complex in LPS-stimulated cells (Fig. 3D,  top, lanes 2 and 3). These data shows that TANK is part of an LPS-inducible IRF3/7-phosphorylating protein complex in vivo. Slower migrating forms of TANK due to phosphorylation (see below) were detected upon LPS but not TNF␣ stimulation (Fig. 3, A, left, second panel from the top, lanes 2 and 3, and B, second panel from the bottom, lanes [3][4][5][6]. It is likely that TBK1-IKK⑀ are involved in this post-translational modification of TANK, since both of them were previously described as TANK kinases in vitro (14,15). We next infected LPS-responsive human CD14-stably expressing THP1 cells with an shRNA construct targeting either the TANK transcript or green fluorescent protein (negative control) and assessed LPS-mediated IRF7 phosphorylation in those cells. This signaling pathway was impaired upon TANK depletion, as evidenced by IRF7 phosphorylation (Fig. 3E, top, compare lanes 1-4 with lanes 5-8). Therefore, TANK is required to assemble a functional IRF7phosphorylating TBK1-IKK⑀ complex in the LPS-mediated signaling pathway.
TANK Assembles TBK1-IKK⑀ in the TLR-, TRAF3-dependent Pathways-Because IRF7 phosphorylation also occurs through the TLR9-and Myd88-dependent pathway upon stimulation with unmethylated DNA (42), we asked whether TANK also connects TBK1-IKK⑀ to IRF7 in this pathway and indeed found that anti-TANK immunoprecipitates from CpG-treated RAW 264.7 cells harbored an IRF7-phosphorylating activity (Fig. 3F, top, compare lane 2 with lanes [3][4][5]. Therefore, these results suggest that TANK is a scaffold protein that assembles the TBK1-IKK⑀ complex for IRF3/7 phosphorylation in the TLR4but also the TLR9-dependent signaling pathways. We next stimulated RAW 264.7 cells with poly(I:C), which is known to trigger IRF3 activation through the TLR3 and -TRIF dependent but Myd88-independent pathway and determined whether or not a TANK immune complex can indeed phosphorylate IRF3 through that pathway. As expected, anti-TBK1 immunoprecipitates indeed phosphorylated IRF3 upon LPS and poly(I:C) stimulations (Fig. 3G, second panels from the top). Interestingly, whereas anti-TANK immunoprecipitates caused IRF3 phosphorylation after 15 min of stimulation by LPS, TANKmediated IRF3 phosphorylation upon poly(I:C) only occurred after 60 min of stimulation (Fig. 3G, top left and right panels,  respectively). Therefore, another scaffold protein, potentially NAP1 (see below), may be required for early IRF3 phosphorylation through the TLR3 pathway.
Because TRAF3 has been identified as a critical signaling molecule for type I interferon induction through the TLR-dependent pathways (9, 10), we next investigated whether TANK, TBK1, and TRAF3 are part of a common signaling complex by co-immunoprecipitation studies. 293 cells were transfected with FLAG-TANK with or without TBK1-Myc, and anti-FLAG immunoprecipitations were performed. The immunoprecipitates were released from the beads by incubating them with a FLAG peptide, and the released material was immunoprecipitated with antibodies to endogenous TRAF3, followed by an anti-Myc Western analysis, which revealed the presence of TBK1-Myc (Fig. 4A, lane 3). Therefore, a ternary complex of TANK, TBK1, and TRAF3 must have been formed in these cells. The hypothesis that these latter proteins are part of a common LPS-inducible signaling complex was further supported by the fact that LPS enhanced association of TRAF3 with TBK1 in stimulated RAW 264.7 cells (Fig. 4B, top, compare lane  1 with lanes 2 and 3). Thus, our results suggest that TANK connects TBK1-IKK⑀ for IRF3/7 phosphorylation in the TLR-, Myd88-, and TRAF3-dependent pathways.
LPS-mediated TANK Phosphorylation in Macrophages-We observed that LPS stimulation of RAW 264.7 macrophages led to the appearance of slower migrating forms of TANK in Western blot analysis (Fig. 3A). These forms were detected after 15 min of stimulation at two LPS concentrations used to treat these cells (Fig. 5A, top, lanes 2-7). Moreover, these slower migrating forms of TANK were also detected in LPS-stimulated but not in TNF␣-stimulated THP1 cells, which stably express CD14 (Fig. 5B, top, compare lanes 1 with lanes 2-5). To test whether the slower migrating species were due to protein phosphorylation, extracts from LPS-stimulated RAW 264.7 cells were incubated with -phosphatase and subjected to anti-TANK Western analysis. We observed that the phosphatase treatment led to the disappearance of the slower migrating TANK forms (Fig. 5C, compare lanes 2 and 3). Additional evidence for TANK phosphorylation was obtained when RAW 264.7 cells were preincubated with staurosporin, a kinase inhibitor, before stimulation with LPS. The LPS-induced TANK phosphorylation was totally abolished upon staurosporin treatment (Fig. 5D, compare lanes 2-4 and lanes 6 -8). Taken together, our results suggest that TANK is subjected to a massive LPS-mediated phosphorylation. To determine whether TBK1-IKK⑀ are the TANK-phosphorylating kinases, we mapped the domains in TANK required for interaction with and/or phosphorylation by overexpressed IKK⑀ or TBK1. To do so, FLAG-TANK was transiently co-expressed in HeLa cells together with Myc-tagged IKK⑀ or TBK1. Anti-FLAG immunoprecipitations were carried out on the cell extracts, and the resulting immunoprecipitates were subjected to anti-Myc Western blots as well as to in vitro kinase assays using immunoprecipitated FLAG-TANK as substrate (Fig. 5E). Both overexpressed IKK⑀ and TBK1 kinases interacted with (Fig. 5E, sec-

TANK/I-TRAF Assembles TBK1 and IKK⑀ in the TLR-dependent Pathways
ond panels from the top, lanes 3) and phosphorylated TANK (Fig. 5E, top panels, lane 3). These experiments were repeated using various ⌬N and ⌬C TANK expression constructs. When co-expressed with IKK⑀, FLAG-⌬N70, -⌬N110, and -⌬C178 TANK were still phosphorylated, whereas FLAG-⌬C256 and -⌬C234 TANK were not (Fig. 5E, top panel on the left), despite the fact that all of these truncations still interacted with IKK⑀ (Fig. 5E, second panel from the top). FLAG-⌬N169 TANK was not phosphorylated by IKK⑀, but it also no longer interacted with this kinase (Fig. 5E, top panel and second panel from the  top, respectively, lane 6). These results suggest that TANK interacts with IKK⑀ in the domain lying between amino acids 111 and 169, just C-terminal to the first of two domains required for interaction with NEMO/IKK␥ (18) and that TANK is phosphorylated between amino acids 192 and 247. These experiments were repeated with FLAG-tagged TANK and TBK1 expression constructs and led to identical results (Fig. 5E, panels on the right), although the expression levels for TBK1 were somewhat below those for IKK⑀. As a proof for TANK being a direct substrate of TBK1, we subjected a purified GST-TANK fusion protein or the GST-IRF3 (positive control) to an in vitro kinase assay using a recombinant TBK1 kinase. Both IRF3 and TANK were indeed phosphorylated by TBK1 in vitro (Fig. 5F, on the right, lanes 2 and 4, respectively). Our results suggest that IKK⑀ and TBK1 behave similarly in terms of their interaction with and phosphorylation of TANK.

TBK1-IKK⑀ but Not IKK␤ Triggers TANK Polyubiquitination through a Phospho-independent Pathway-Because evidence is
accumulating that polyubiquitination critically regulates signal transduction (43,44) and because scaffold proteins, such as NEMO/IKK␥, are subjected to this post-translational modification, we investigated whether the LPS-inducible and TANKphosphorylating kinase IKK⑀ causes TANK polyubiquitination. To address this issue, 293 cells were transfected with FLAG-TANK, HA-Ub, or a mutant where all lysines are mutated, HA-Ub (K0), with or without IKK⑀-Myc. Anti-HA Western analyses were performed on the anti-FLAG immunoprecipitates to detect polyubiquitin-conjugated TANK adducts. Coexpression of FLAG-TANK and HA-Ub did not lead to TANK polyubiquitination (Fig. 6A, top, lane 2). However, polyubiquitination of TANK was detected upon its co-expression with HA-Ub and IKK⑀-Myc (Fig. 6A, lane 3). IKK⑀-mediated TANK polyubiquitination occurred through a lysine-dependent mechanism, since no polyubiquitin-conjugated TANK adducts were detected using the HA-Ub (K0) mutant (Fig. 6A, top, lane  4). A similar experiment was conducted by overexpressing TBK1, the other LPS-inducible and TANK-phosphorylating kinase, and a TBK1-dependent TANK polyubiquitination was detected as well (Fig. 6B, top, lane 3). TANK was the targeted substrate for polyubiquitination as evidenced by the detection  2-7) or anti-HA (negative control; lane 1) immunoprecipitates were subjected to anti-IKK⑀ or -TBK1 Western analysis (second and third panels from the top, respectively) or to in vitro kinase assays using the purified GST-IRF7 as substrate (top). Cell extracts were subjected to anti-TANK or IB␣ Western analysis (bottom) as well. D, same experiment as in A, except that RAW 264.7 cells were replaced by peritoneal macrophages. An anti-TBK1 Western blot was used for normalization purposes (middle). E, TANK-depleted macrophages harbor impaired LPS-mediated IRF7 phosphorylation. CD14-stably expressing THP1 cells were infected with a lentiviral construct targeting either green fluorescent protein (negative control) or the TANK transcript, and the resulting cells were left unstimulated or treated with LPS for the indicated periods of time. Lysates were subjected to anti-TBK1 immunoprecipitations followed by in vitro kinase assays using a purified GST-IRF7 as substrate (top). TBK1 P , autophosphorylated TBK1. Cell extracts were subjected to anti-TANK, -TBK1, and -IB␣ Western analyses as well. F, IRF7 is phosphorylated by a TANK-containing complex in CpG-stimulated macrophages. RAW 264.7 cells were left untreated or stimulated with CpG (1 M) for the indicated periods of time, and anti-TANK immunoprecipitates were subjected to in vitro kinase assays using a purified GST-IRF7 as substrate (top). A positive control from LPS-stimulated cells is shown on lane 1. Anti-TANK and -IB␣ Western analyses were carried out on the lysates (bottom). G, TANK is part of an IRF3-phosphorylating complex upon LPS or poly(I:C) stimulation in macrophages. RAW 264.7 cells were left unstimulated or treated with LPS (10 g/ml) or poly(I:C) (100 g/ml) (left or right panel, respectively) for the indicated periods of time, and anti-TANK or -TBK1 immunoprecipitates (top panels and second panels from the top, respectively) were subjected to in vitro kinase assays using the purified GST-IRF3 as substrate. Anti-TANK, -TBK1, or -IB␣ Western analysis was carried out on the cell extracts as well (bottom).

TANK/I-TRAF Assembles TBK1 and IKK⑀ in the TLR-dependent Pathways
of these polyubiquitin-conjugated adducts when the anti-FLAG immunoprecipitates were subjected to anti-TANK and -HA but not anti-Myc Western analyses (Fig. 6C, lanes 3, compare the top and second panel versus the third panel from the top). We previously reported that IKK␤ is also a TANK kinase that targets the C-terminal part of this scaffold protein (37). We therefore tested whether IKK␤ overexpression also triggers TANK polyubiquitination and concluded that, in contrast to ectopically expressed IKK⑀, this was not the case (Fig. 6D, top, lanes 3 and 5, respectively). IKK⑀-mediated TANK polyubiquitination required binding of the two proteins, since no polyubiquitin-conjugated TANK adducts were detected when IKK⑀ ⌬C30, which does not interact with TANK (Fig. 2B), was overexpressed (Fig. 6E, top, compare lanes 3 and 4). We next asked whether IKK⑀ kinase activity was required in this process by testing the ability of an IKK⑀ kinase-dead version ("IKK⑀ KD") to trigger TANK polyubiquitination. Although overexpressed wild type IKK⑀ phosphorylated and polyubiquitinated TANK (Fig. 6F, lane 3, bottom and top panels, respectively), IKK⑀ KD, which indeed did not phosphorylate TANK, still triggered its polyubiquitination (Fig. 6F, bottom and top panels, respectively, lane 4). Thus, IKK⑀ kinase activity is dispensable for TANK polyubiquitination. In conclusion, because TANK polyubiquitination is detected even in the absence of IKK⑀-mediated phosphorylation, our results suggest that the LPS-inducible TANK phosphorylation and the IKK⑀-mediated TANK polyubiquitination occur through independent mechanisms. We next stimulated macrophages with LPS, which triggers TBK1-IKK⑀ activation and indeed detected endogenous polyubiquitinated adducts of TANK upon 15 min of stimulation (Fig. 6G, top, lanes 2 and 3). TANK harbors a TRAF2/3 interaction domain located downstream from the TBK1-IKK⑀-interacting region (12,18). To determine whether this domain is required for IKK⑀-mediated TANK polyubiquitination, we generated a mutant lacking this region ("TANK⌬TRAF") and confirmed that this mutant indeed failed to interact with endogenous TRAF3, whereas wild type TANK did (Fig. 7A, top, compare lanes 4 and 2, respectively). Although a TANK mutant lacking the TBK1-IKK⑀-interacting site was not subject to IKK⑀-mediated polyubiquitination, the TANK⌬TRAF mutant was still polyubiquitinated upon IKK⑀ overexpression (Fig. 7B, top, lanes 4 and 5, respectively). This result demonstrates that TANK polyubiquitination requires binding to TBK1-IKK⑀ but does not require the TRAF3-interacting site. Still, this result does not rule out the possibility that TRAF3 may be required for TANK polyubiquitination. To address this issue, we depleted TRAF3 in THP1/ CD14 cells through RNA interference and assessed LPS-mediated TANK phosphorylation and polyubiquitination in these cells. As expected, LPS-mediated TANK phosphorylation was observed in THP1/CD14 cells infected with the shRNA control vector (Fig. 7C, third panel from the top, compare lanes 6 and 7  with lane 5). Moreover, TANK polyubiquitination was also detectable after 15 min of stimulation (Fig. 7C, top, lane 6). Interestingly, TRAF3 depletion impaired both LPS-mediated TANK phosphorylation and polyubiquitination (Fig. 7C, third  panel from the top, lanes 2 and 3, and top panel, lane 2, respectively). Thus, our results suggest that TRAF3 is essential for the LPS-mediated post-translational modifications of TANK, even if a direct interaction of TRAF3 with TANK is not required.
In summary, our observations suggest that the IRF3/7-and TANK-phosphorylating TBK1-IKK⑀ kinases are necessary for the polyubiquitination of their scaffold protein TANK in the LPS-dependent pathway. Our data also highlight the critical role of TRAF3 in that pathway.
TBK1-IKK⑀ Triggers TANK Lys 63 -linked Polyubiquitination-By analogy with NEMO/IKK␥, which is subject to Lys 63 -linked polyubiquitination, and because a prior phosphorylation does not appear to be required in that process, we hypothesized that TANK may be subjected to a similar post-translational modification. To test this possibility, we looked for polyubiquitinconjugated TANK adducts in the presence of overexpressed HA-IKK⑀ and Myc-tagged wild type or mutated (Lys 48 , Lys 63 , or both) Ub products (Fig. 8A). As expected, TANK was subjected to IKK⑀-mediated polyubiquitination in the presence of wild type Ub (Fig. 8A, top, lane 3). Polyubiquitinated TANK was also detected when the K48R but not the K63R or the K48R/ K63R Ub mutant was co-transfected (Fig. 8A, lanes 4-6). Thus, IKK⑀ triggers TANK polyubiquitination through a Lys 63 -linked pathway. When these experiments were repeated using various TANK constructs lacking the N-or C-terminal domain, they revealed that the targeted residues on TANK are located between amino acids 71 and 110 (Fig. 8B, top, lanes 4 and 5). Moreover, a TANK mutant ("TANK⌬C178") lacking the C-terminal inhibitory domain (12) was even more strongly polyubiquitinated (Fig. 8B, top, lane 6) through a Lys 63 linkage (Fig. 8C,  top, lane 3). This pathway occurred independently of the IKK⑀mediated TANK phosphorylation process. Indeed, polyubiq-  2-5, left and right, respectively), and cell extracts were subjected to anti-TANK, -IB␣, and -TBK1 Western analyses. C, -phosphatase dephosphorylates TANK from LPS-stimulated macrophages. RAW 264.7 cells were left unstimulated (lane 1) or treated with LPS for 15 min (lanes 2 and 3), and the extracts were untreated (lanes 1 and 2) 2-4 and 6-8). Cell lysates were subjected to anti-TANK, -IB␣, and -TBK1 Western analyses (WB). E, IKK⑀ and TBK1 interact and phosphorylate TANK on identical domains. On the left, a schematic representation of the TANK constructs tested for interaction with TBK1-IKK⑀ and phosphorylation by these kinases is shown. The mapping of the IKK⑀-and TBK1-interacting domains of TANK and the targeted domain of TANK for IKK⑀-and TBK1-mediated phosphorylation is illustrated. HeLa cells were transfected with the indicated expression plasmids, and anti-FLAG immunoprecipitates were subjected to in vitro kinase assays (top panels) or to anti-Myc western analyses (second panel from the top). Cell lysates were subjected to anti-FLAG or -Myc Western analyses as well (bottom panels). F, IRF3 and TANK are phosphorylated by TBK1 in vitro. On the left, a Coomassie Blue-stained gel showing the purified GST-IRF3 or -TANK fusion protein used as substrate in the in vitro kinase assay. On the right, the GST-IRF3 (lanes 1 and 2) and -TANK (lanes 3 and 4) were subjected to an in vitro kinase assay in presence (lanes 2 and 4) or absence (lanes 1 and  3) of the recombinant TBK1 kinase. The figure shows the phosphorylated IRF3 and TANK proteins as well as the autophosphorylated TBK1 kinase. uitination was not detectable on the TANK ⌬N110 mutant, which is still subjected to IKK⑀-mediated phosphorylation (see Fig. 5E, top, lane 5), whereas the Ϫ⌬C234 mutant, which is not subjected to IKK⑀-mediated phosphorylation (Fig. 5E,  top, lane 8), was polyubiquitinated upon IKK⑀ overexpression (Fig. 8, B and D).
Ubc13 is an E2 protein required for Lys 63 -linked polyubiquitination (45). We tested IKK⑀-mediated TANK polyubiquitination in Ubc13-depleted cells and found that this process was severely impaired (Fig. 8E, top, lanes 3 and 4). Taken together, our results suggest that TANK is subject to Lys 63linked polyubiquitination, which is dependent on TBK1-IKK⑀ and Ubc13 and independent of TRAF and of IKK⑀mediated phosphorylation.

DISCUSSION
We report here the identification of TANK/I-TRAF as the scaffold protein that assembles the TBK1-IKK⑀ complex for subsequent IRF3/7 activations through some but not all TLRdependent signaling pathways via binding to TRAF3. Impor- tantly, we also demonstrate that TANK undergoes two distinct post-translational modifications, namely phosphorylation and Lys 63 -linked polyubiquitination, both of which require distinct functional domains of TBK1-IKK⑀. TBK1-IKK⑀ are IRF3/7 kinases, but how these phosphorylating complexes are assembled has hitherto been unknown. Here we show that TANK is required for both LPS and CpGmediated IRF3/7 phosphorylations. The observation that TANK occurs in a triple complex with both TRAF3 and TBK1 combined with its inability to interact with TRAF6 9 or TAK1 (37)doesnotsupportaroleforTANKintheMyd88-andTRAF6dependent pathway of IKK activation. This conclusion is supported by the absence of an effect of TANK depletion on LPSmediated IB␣ degradation. Our results rather suggest that TANK assembles TBK1-IKK⑀ downstream of TRAF3 for subsequent induction of IFN and potentially other target genes in LPS and also CpG-stimulated macrophages. The lack of association of TANK with TRIF, which is required for both the LPSand the dsRNA-mediated IRF3 activations (34,46), suggests that TANK may not act downstream of TRIF for IRF3 phosphorylation. However, such interactions were investigated in unstimulated cells and should thus be reevaluated in cells treated with the appropriate stimulus. Additional and still uncharacterized signaling molecules and/or post-translational modifications of TRAF3 or TANK may be required for proper assembling of these signaling complexes. Alternatively, a different scaffold protein may be required to assemble the IRF3 phosphorylating complex, and if so, NAP1 would be the most likely candidate. Indeed, this protein, initially identified as a TBK1/ NAK-binding protein (47), was proposed to be the TBK1 scaffold protein acting in the TLR3-dependent pathway based on the defects seen in dsRNA-mediated IRF3 phosphorylation upon NAP1 depletion (11). Although we show here that a TANK-containing immune complex can phosphorylate IRF3 upon dsRNA stimulation in macrophages, such phosphorylation only occurs with delayed kinetics (60 min), which may reflect indirect mechanisms. Such an observation strongly suggests that another scaffold protein, most likely NAP1, is required for early IRF3 phosphorylation through the TLR3-dependent pathway. Thus, our present report suggests that TBK1-IKK⑀ may be assembled by distinct but structurally similar scaffold proteins in a pathway-specific manner. In other words, TANK/I-TRAF would be the candidate that assembles the TBK1-IKK⑀ complex upon LPS stimulation, whereas NAP1 would be the one in dsRNA-treated cells. This phenomenon has already been demonstrated for the signaling cascades that make use of distinct IKK complexes for NF-B activation. Indeed, whereas NEMO/IKK␥ is essential for the IKK␣/␤mediated NF-B activation through the so-called classical pathway, this scaffold protein is dispensable for the alternative pathway of NF-B activation, which relies on an unidentified IKK␣ adapter (48). We therefore propose the existence of multiple pools of TBK1-IKK⑀ assembled by distinct scaffold proteins, which endow specificity in the TLR-dependent signaling pathways.   A role for TANK/I-TRAF in the TLR-dependent signaling pathways does not rule out the possibility that the TLR-independent pathways triggered upon viral infection, such as the one that relies on retinoic acid-inducible gene I, for example, also involve this scaffold protein, and a couple of recently published studies actually provided experimental evidence for this (49,50).
We demonstrate here that TANK/I-TRAF is subject to posttranslational modifications, namely phosphorylation and Lys 63 -linked polyubiquitination, both of which require TBK1-IKK⑀. TANK phosphorylation is intimately linked to IRF3/7 phosphorylations through both the TLR4 and the TLR9 pathways. Interestingly, the kinetics of TANK phosphorylation is stimulus-dependent, since it occurs after 15 or 60 min of LPS or poly(I:C) stimulations, respectively. Several hypotheses, which all require experimental validations, may explain this shifted kinetics. Besides the possibility that distinct scaffold proteins may assemble the TBK1-IKK⑀ complexes, distinct and yet to be characterized upstream signaling molecules may be involved in this TANK post-translational modification. In this context, whether Myd88 is involved for TANK phosphorylation may be signal-specific. Indeed, whereas the LPS-mediated IRF3 activation relies on the TIR domain-containing adaptors TRIF-related adaptor molecule and TRIF but not on Myd88, the CpGmediated IRF7 activation requires Myd88 (51). Therefore, and because both pathways ultimately trigger TBK1-IKK⑀ activations and TANK phosphorylation, as shown here, this opens the possibility that TBK1-IKK⑀ kinase activity may be induced through recruitment of distinct upstream signaling complexes in a signal-specific manner. The delayed kinetics seen for TANK phosphorylation upon dsRNA stimulation may also be due to a transcriptionally inducible expression of IKK⑀ but not of TBK1, which implies that TANK would be preferentially targeted for phosphorylation by TBK1 in early phases and subsequently by IKK⑀ in latter times. In any case, whether such posttranslational modification of TANK is required for TBK1-IKK⑀ activation is currently unclear. Remarkably, although LPS and CpG-mediated TANK phosphorylations require TBK1-IKK⑀ kinase function, LPS-mediated Lys 63 -linked TANK polyubiquitination does not. TANK mutants lacking the residues phosphorylated by TBK1-IKK⑀ are still able to undergo Lys 63 -linked polyubiquitination, suggesting that the two modifications occur independently. Therefore, TBK1-IKK⑀ harbor a previously undescribed function, namely the ability to trigger nondegradative polyubiquitination of their scaffold protein TANK. Our results also suggest that nondegradative polyubiquitination may critically regulate the TLR-dependent signaling cascades, leading to IFN type I gene induction.
TRAF6 harbors a so-called RING domain and possesses intrinsic ubiquitin ligase activity, which ultimately targets NEMO/IKK␥ for polyubiquitination upon IL-1␤ stimulation (52). Although TRAF3 harbors a RING domain, which is essential for suppression of the noncanonical NF-B pathway (53), it is currently unclear whether TRAF3 also acts as an E3 ligase similarly to TRAF6. We demonstrate here that TRAF3 is required for LPS-mediated TANK phosphorylation but also for TANK polyubiquitination. Interestingly, TRAF3 does not have to bind TANK for this polyubiquitination to occur. Therefore, TRAF3 may recruit a yet to be identified LPS-inducible E3 ligase to TANK for subsequent polyubiquitination of this scaffold protein. Ongoing experiments are dedicated to the identification of this candidate.
NEMO/IKK␥ is the best known example of a scaffold protein subject to Lys 63 -linked polyubiquitination in response to several stimuli, and this post-translational modification appears to be essential for IKK and subsequent classical NF-B activation. The Lys 63 -linked polyubiquitination of TANK requires TBK1-IKK⑀ and the E2 protein Ubc13 but is TRAF-independent. Even if some analogy with NEMO/IKK␥ can therefore be established, some differences persist. Unlike TANK, which requires binding to TBK1-IKK⑀, NEMO/IKK␥ Lys 63 -linked polyubiquitination does not appear to require IKK␣/␤, the kinase subunits that NEMO/IKK␥ assembles. Although it is unclear how Lys 63linked TANK polyubiquitination regulates IRF3/7 activations, it is noteworthy that the N-terminal part of TANK, which has been described as positively regulating signal transduction (12), harbors the TBK1-IKK⑀-interacting site and is also very strongly Lys 63 -linked polyubiquitinated. It may be speculated that this polyubiquitin linkage of TANK, which does not require prior phosphorylation, positively regulates IRF3/7 activation. In this context, a recent report demonstrated that Lys 63linked polyubiquitination of retinoic acid-inducible gene I by the E3 ligase TRIM25 is critical for the retinoic acid-inducible gene I-dependent antiviral signal transduction (54). Because this latter pathway also involves IRF activation, it is tempting to speculate that optimal activation of these pathways relies on signal-specific E3 ligases whose identities remain unknown in most cases so far.
Recent reports revealed that TBK1 and IKK⑀ have unexpected physiological functions, namely a role in angiogenesis (55) and in linking innate immune signaling and tumor cell survival through a RalB GTPase pathway (for TBK1) (56) as well as a role in actin cytoskeleton organization in Drosophila (for IKK⑀) (57). The upstream signaling molecules such as TRAF3 and TRIF and the scaffold proteins TANK and NAP1 and their regulatory involvement in these new TBK1-IKK⑀-dependent pathways is, as yet, unknown. These reports combined with our study show that a more complete understanding of the varied roles of TBK1-IKK⑀ requires a thorough identification of their substrates and the characterization of their kinase-independent functions. FIGURE 8. IKK⑀-mediated TANK polyubiquitination occurs through an Ubc13-dependent Lys 63 linkage and does not require prior phosphorylation by that kinase. A-D, overexpressed IKK⑀ causes a Lys 63 -linked TANK polyubiquitination through a phospho-independent mechanism. 293 cells were transfected with the indicated expression plasmids, including wild type or Myc-tagged ubiquitin mutants (A, C, and D), whose sequences are illustrated on the left (A). Anti-FLAG immunoprecipitates (IP) were subjected to anti-Myc Western blots (WB)(A, C, and D) and also to anti-HA Western analyses to detect either polyubiquitinated adducts of TANK (B) (top) or HA-tagged IKK⑀ (middle panels in A, C, and D). Anti-FLAG (A-D) or -anti-Myc (A and B) Western blots were also performed with the extracts (bottom). E, Ubc13 is required for IKK⑀-mediated TANK polyubiquitination. 293 cells were transfected with RNA interference targeting either green fluorescent protein (negative control) or the Ubc13 transcript, and the resulting cells were transfected with the indicated expression plasmids. Detection of polyubiquitinated forms was carried out after immunoprecipitation as described here before (top), and extracts were subjected to anti-Myc, FLAG, or Ubc13 Western analyses as well (bottom).