IRAK-2 Participates in Multiple Toll-like Receptor Signaling Pathways to NFκB via Activation of TRAF6 Ubiquitination*

Toll-like receptor (TLR) signaling is known to involve interleukin-1 receptor-associated kinases (IRAKs), however the particular role of IRAK-2 has remained unclear. Further, although IRAK-1 was originally thought to be central for the TLR-NFκB signaling axis, recent data have shown that it is dispensable for NFκB activation for some TLRs and demonstrated an alternative role for it in interferon regulatory factor activation. Here we show that IRAK-2 is critical for the TLR-mediated NFκB activation pathway. The poxviral TLR antagonist A52 inhibited NFκB activation by TLR2, -3, -4, -5, -7, and -9 ligands, via its interaction with IRAK-2, while not affecting interferon regulatory factor activation. Knockdown of IRAK-2 expression by small interfering RNA suppressed TLR3, TLR4, and TLR8 signaling to NFκB in human cell lines, and importantly, TLR4-mediated chemokine production in primary human cells. IRAK-2 usage by different TLRs was distinct, because it acted downstream of the TLR adaptors MyD88 and Mal but upstream of TRIF. Expression of IRAK-2, but not IRAK-1, led to TRAF6 ubiquitination, an event critical for NFκB activation. Further, IRAK-2 loss-of-function mutants, which could not activate NFκB, were incapable of promoting TRAF6 ubiquitination. Thus we propose that IRAK-2 plays a more central role than IRAK-1 in TLR signaling to NFκB.

Toll-like receptor (TLR) signaling is known to involve interleukin-1 receptor-associated kinases (IRAKs), however the particular role of IRAK-2 has remained unclear. Further, although IRAK-1 was originally thought to be central for the TLR-NFB signaling axis, recent data have shown that it is dispensable for NFB activation for some TLRs and demonstrated an alternative role for it in interferon regulatory factor activation. Here we show that IRAK-2 is critical for the TLR-mediated NFB activation pathway. The poxviral TLR antagonist A52 inhibited NFB activation by TLR2, -3, -4, -5, -7, and -9 ligands, via its interaction with IRAK-2, while not affecting interferon regulatory factor activation. Knockdown of IRAK-2 expression by small interfering RNA suppressed TLR3, TLR4, and TLR8 signaling to NFB in human cell lines, and importantly, TLR4-mediated chemokine production in primary human cells. IRAK-2 usage by different TLRs was distinct, because it acted downstream of the TLR adaptors MyD88 and Mal but upstream of TRIF. Expression of IRAK-2, but not IRAK-1, led to TRAF6 ubiquitination, an event critical for NFB activation. Further, IRAK-2 loss-of-function mutants, which could not activate NFB, were incapable of promoting TRAF6 ubiquitination. Thus we propose that IRAK-2 plays a more central role than IRAK-1 in TLR signaling to NFB.
Toll-like receptors (TLRs) 3 recognize specific pathogen-associated molecular patterns found on infectious agents (1). TLRs are part of the larger IL-1R/TLR superfamily, defined by the presence of a cytoplasmic Toll-IL-1R-resistance (TIR) signaling domain, which also includes the IL-1, IL-18, and IL-33 receptors. Upon engagement of distinct TLRs by specific pathogen-associated molecular patterns, such as bacterial lipoprotein (for TLR2), viral dsRNA (for TLR3), or LPS (for TLR4), intracellular signaling cascades mediate activation of transcription factors such as NFB and interferon regulatory factors (IRFs) leading to gene induction and the production of pro-inflammatory cytokines, chemokines and interferons.
Previous studies have implicated IRAK-2 in IL-1R and TLR4 signaling by showing that IRAK-2 can associate with MyD88 and Mal (3,16). Furthermore, the poxvirus protein A52, which interacts with both IRAK-2 and TRAF6, can inhibit TLR-induced NFB activation (17,18). However, the extent of the role of IRAK-2 in TLR signaling and its mechanism of action remain to be established. Here we present evidence that A52 inhibits NFB solely via its interaction with IRAK-2 and not TRAF6, and that IRAK-2 is required for all TLR-mediated pathways to NFB. We show that IRAK-2, but not IRAK-1 causes TRAF6 ubiquitination, a critical step in the activation of NFB by TLRs. Thus we propose that IRAK-2 has a more fundamental role than IRAK-1, in the TLR-NFB axis.
Reporter Gene Assays-NFB, p38 MAP kinase, IRF3, IRF5, and IRF7 activation as well as IL-8 promoter induction were measured by reporter gene assay. HEK293 or RAW264.7 cells were seeded into 96-well plates at 2 ϫ 10 4 and 4 ϫ 10 4 cells per well, respectively, and co-transfected with luciferase reporter gene plasmids and expression plasmids 24 h later using the Genejuice transfection reagent (Novagen) as described previously (20,21). For all assays, 20 ng of phRL-TK reporter (Promega) per transfection was included to normalize data for transfection efficiency, and total DNA concentrations were kept constant at 230 ng (for HEK293) or 200 ng (for RAW264.7) by the addition of pcDNA3.1 empty vector (Invitrogen). For measurement of NFB activity, 60 ng of a B-luciferase reporter gene was used. The Pathdetect System (Stratagene) was used to assess p38 MAP kinase activity with 0.25 ng of a CHOP-Gal4 fusion protein expression vector co-transfected with 60 ng of the pFR-luciferase reporter plasmid. IRF activity was determined by co-transfection of 3 ng of either IRF3-, IRF5-, or IRF7-Gal4 fusion protein expression vectors as required in conjunction with the pFR-luciferase reporter plasmid (60 ng). Cells were stimulated with TLR agonists for 6 h prior to lysis and measurement of luciferase activity 24 h following transfection. All transfections were done in triplicate, and data are expressed as -fold induction (mean Ϯ S.D.) relative to control levels for a representative experiment of a minimum of three separate experiments.
ELISA-IL-8 was measured by ELISA (20). Each experiment was done in triplicate, and data are expressed as picograms/ml IL8 (mean Ϯ S.D.) for a representative of at least three independent experiments.
siRNA Gene Silencing-siRNA duplexes targeting the human Irak-2 gene (Qiagen) targeted the following sites: IRAK-2A, 5Ј-CAGCAACGTCAAGAGCTCTAA; IRAK-2B, 5Ј-CCA-GATCATCCTGAACTGGAA. To confirm knockdown of IRAK-2-Myc expression, HEK293 cells (3 ϫ 10 5 cells) were transfected with siRNA duplexes using Lipofectamine 2000 (Invitrogen), followed by transfection with IRAK-2-Myc and a second siRNA transfection 24 h later. After a further 24 h, cell lysates were prepared and analyzed by SDS-PAGE and immunoblotting. For reporter gene assays, HEK293 cells in 96-well plates (3 ϫ 10 4 cells per well) were transfected with siRNA duplexes and then co-transfected with reporter constructs and a second dose of siRNA 24 h later prior to stimulation with 25 g/ml poly(I:C) or 100 ng/ml LPS for 6 h. For siRNA experiments in primary human cells, peripheral blood mononuclear cells were isolated from healthy donor whole blood using Lymphoprep reagent (Axis-shield), seeded in 96-well plates (8 ϫ 10 4 cells per well) at 37°C for 2 h, and transfected with siRNA as above. A second siRNA transfection was delivered 24 h later, and cells were treated for 24 h with 10 ng/ml LPS 48 h following initial transfection. Cell supernatants were harvested and IL-8 cytokine production analyzed by ELISA.
Co-immunoprecipitation and Immunoblotting-Proteins were co-immunoprecipitated from transfected HEK293T cells as previously described (20). For immunoprecipitation of ubiquitinated proteins, lysis buffer was supplemented with 10 mM iodoacetamide. Target proteins were immunoprecipitated by incubating 400 l of lysate with the relevant antibody, precoupled to protein A-or G-Sepharose beads, for 2 h at 4°C. Immune complexes were analyzed by SDS-PAGE and immunoblotting.
Site-directed Mutagenesis of IRAK-2-Point mutations were introduced into the IRAK-2 gene sequence using the QuikChange TM site-directed mutagenesis kit (Stratagene) according to manufacturer's instructions.

RESULTS
A52 Specifically Targets IRAK-2 to Inhibit Signal Transduction from TLRs to NFB-Previously we showed that poxvirus protein A52 could inhibit NFB activation by TLR2, -3, -4, or -5 in human HEK293 cells, and associate with both TRAF6 and IRAK-2 (18). Here, we extended this observation to demonstrate that A52 expression antagonized ligand-induced NFB activation through all TLRs tested, including TLR7 and TLR9, in murine macrophage RAW264.7 cells (Fig. 1a). Further A52 substantially reduced MALP-2-and R848-mediated NFB induction in HEK293 cells stably expressing TLR2 and TLR8, respectively (data not shown). Thus, it appeared that A52 was capable of inhibiting all TLR pathways to NFB, both in human and murine cells. We therefore next determined whether the ability of A52 to inhibit NFB depended on its interaction with TRAF6 and/or IRAK-2. Previously, the A52-TRAF6 interaction has been shown to induce MAP kinase activation, whereas ⌬A52, a truncated A52 protein (comprising amino acids 1-144 of the 190-residue protein) could no longer associate with TRAF6 nor induce MAP kinase activation (19). Because ⌬A52 could still interact with IRAK-2, it provided a useful tool to investigate IRAK-2 involvement in NFB activation. Fig. 1 (bd) shows that A52 substantially reduced the induction of an NFB-dependent reporter gene stimulated by exogenous treatment of IL-1R-expressing cells with IL-1␣, co-expression of TLR3 and simultaneous poly(I:C) treatment or introduction of a constitutively active CD4-TLR4 chimera and compellingly, ⌬A52 was as powerful a suppressor of NFB activation via IL-1R, TLR3, and TLR4 as wild-type A52. Thus the interaction of A52 with TRAF6 is dispensable for inhibition of IL-1R-and TLR-induced NFB activation, suggesting that it is via its association with IRAK-2 that A52 can potently antagonize TLR signaling to NFB, thus pointing to IRAK-2 as a critical downstream component of these signaling cascades.
IRAK-2 Induces NFB and p38 MAP Kinase-As previously shown (16) IRAK-2 expression, like IRAK-1, led to NFB activation (Fig. 1e). To determine the effect of IRAK-2 on p38 MAP kinase activation, another downstream event in TLR sig-naling, a reporter gene assay based on the phosphorylation of the p38 substrate CHOP was utilized (20). IRAK-2 induced substantial transactivation of the reporter, to levels comparable to those seen for MyD88 expression (Fig. 1f). Although IRAK-1 failed to stimulate p38 MAP kinase activation in these assays, IRAK-1 has previously been implicated in the induction of p38 MAP kinase by IL-1 (12), and overexpression of IRAK-1 has been shown to induce phosphorylation of p38 MAP kinase (22). IL-8 gene induction is NFBand MAP kinasedependent, and consistent with this, expression of IRAK-2 resulted in strong IL-8 promoter induction (Fig. 1g) and in the production of IL-8 (Fig. 1h), in both cases to levels comparable to those seen for IRAK-1.
IRF Activation by TLRs Is Not Inhibited by the IRAK-2 Inhibitor A52-We have previously shown that, although the poxvirus protein A46, which antagonizes both MyD88-and TRIF-dependent signaling, could potently block the induction of IRF3 via TLR3, A52 could not (20). This suggested that IRAK-2 is not required for IRF3 stimulation by TLR3. To investigate the role of IRAK-2 in TLR-mediated IRF activation in a broader sense, the ability of A52 to inhibit other TLR pathways to IRFs was examined. In these assays, the IRF subunit of interest was overexpressed as a Gal4 fusion protein, and its phosphorylation, dimerization, and DNA-binding activity was assessed using a luciferase reporter construct regulated by tandem Gal4 DNAbinding sequences. A52 had no effect on the activation of IRF3 by TLR4 (Fig. 2a) or by the TLR3-and TLR4-specific downstream adaptor, TRIF (Fig. 2b). Apart from the TLR3/4-TRIF-IRF3 pathway, TLR7, TLR8, and TLR9 can also induce IRF5 and IRF7 activation via MyD88 (23)(24)(25)(26)(27), and similar to the TRIF-IRF3 pathway, activation of IRF5 and IRF7 by MyD88 was not blocked by A52 expression (Fig. 2, c and d), yet these signals were potently blocked by A46 (Fig. 2, e and f). Thus IRAK-2 is unlikely to be a downstream component of TLR-induced IRF3, IRF5, or IRF7 activation. In keeping with this, although IRAK-2 overexpression led to NFB induction (Fig. 1e), this failed to FIGURE 1. A52 inhibits TLR-induced NFB activation via IRAK-2. a-e, NFB activation was measured by reporter gene assay. a, RAW 264.7 cells were transfected with pcDNA 3.1 (EV) or 100 ng of A52 expression plasmid. Cells were stimulated with 1 nM Pam 3 Csk 4 (Pam), 1 nM Malp-2, 25 g/ml poly(I:C), 1 g/ml LPS, 250 ng/ml flagellin (Flag), 1 M R-848, or 1 g/ml CpG for 6 h. b, HEK293 cells were transfected with the indicated amounts (nanograms) of A52 or ⌬A52 expression plasmids, and cells were stimulated with 100 ng/ml IL1 for 6 h. c and d, HEK293 cells were transfected with 100 ng of A52 or ⌬A52, 0.5 ng of TLR3 (c) or 50 ng of CD4-TLR4 (d) expression plasmids. In c, cells were stimulated with 25 g/ml poly(I:C) for 6 h. e, HEK293 cells were transfected with 30 ng or 100 ng of IRAK-2, 100 ng of IRAK-1, or 25 ng of MyD88 for 24 h. f, similar to e except p38 MAP kinase activation was measured by reporter assay. g, HEK293 cells were transfected with 10, 30, or 100 ng of IRAK-2 or 100 ng of IRAK-1, and IL-8 promoter induction was measured by reporter gene assay. In a-g, all data are expressed as -fold induction (mean Ϯ S.D. of triplicate samples) relative to control levels for representatives of a minimum of three independent experiments. h, similar to g except IL-8 cytokine production was measured by ELISA. Data are mean Ϯ S.D. of triplicate samples and representative of three experiments.
trigger IRF3 or IRF5 activation in these experiments (Fig. 1, g  and h). Similarly, IRF7 was not stimulated by IRAK2 (data not shown).
Inhibition of IRAK-2 Suppresses TLR-induced NFB Activation-We next took a more direct approach to investigate the potential role of IRAK-2 in the TLR-NFB signaling axis. An IRAK-2-truncated protein (with amino acids 97-590, termed kIRAK-2) has been shown to act as a dominant negative, abolishing the induction of NFB by the IL-1R (16), and here we also found that kIRAK-2 inhibited ligandinduced TLR3-, TLR4-, and TLR8-mediated NFB activation, whereas NFB activation induced through ectopically expressed retinoic acid-inducible gene-I was not blocked (Fig. 3a). Like TLR3, retinoic acid-inducible gene-I is a viral RNA pattern recognition receptor that mediates NFB activation (28).
To obtain independent evidence for a role for IRAK-2 in signal transduction from TLR3, TLR4, and TLR8 to NFB, siRNA gene silencing was used to "knock down" IRAK-2 expression. The efficacy of two IRAK-2 sequence-specific siRNA oligonucleotides was confirmed by showing that IRAK-2 protein expression from a transfected expression vector was completely ablated in the presence of either IRAK-2 siRNA compared with cells treated with a non-silencing control  siRNA (Fig. 3b), and that NFB activation by expression of IRAK-2 was also blocked (Fig. 3c). Crucially, while treatment of cells with the negative control siRNA had no effect on poly(I:C)-, LPS-, or CL075-induced NFB activity, the siRNA sequences targeting IRAK-2 diminished stimulation of the NFB reporter gene by these TLR agonists by almost 50% (Fig. 3, d-f). Importantly, the requirement for IRAK-2 in NFB-dependent TLR4 signaling was confirmed in primary human cells by analyzing the effect of IRAK-2 siRNA on LPS-induced IL-8 cytokine production in human peripheral blood mononuclear cells. Here, negative control siRNA treatment did not alter LPS-induced IL-8 production, whereas IRAK-2 siRNA specifically reduced IL-8 release by almost 40% (Fig. 3g).
IRAK-2 Functions Downstream of MyD88 but Upstream of TRIF-We next utilized IRAK-2 siRNA to determine where in TLR signaling cascades to NFB IRAK-2 functioned. Signaling to NFB for TLR4 involves the adaptors MyD88, Mal, TRIF, and TRAM, whereas TLR3 uses only TRIF and TLR8 signals solely through MyD88 (29). Hence, the effect of IRAK-2 siRNA on the induction of NFB by MyD88, Mal, TRIF, and TRAM was investigated. IRAK-2 siRNA clearly inhibited MyD88 signaling to NFB (Fig. 4a), and in particular IRAK-2B diminished MyD88-induced NFB activity by almost 50%. NFB activity induced by Mal was potently blocked in cells treated with both siRNAs (Fig. 4b).
These data are consistent with previous proposals of IRAK-2 acting downstream of MyD88 (16) and Mal (3). Surprisingly, although NFB activation by poly(I:C)/TLR3 was sensitive to IRAK-2 siRNAs (Fig.  3d), no inhibition of TRIF-induced NFB activation was observed (Fig.  4c). Equally, TRAM, which is upstream of TRIF in the TLR4 pathway, was unaffected (Fig. 4d). These data suggested alternative usage of IRAK-2 in the MyD88-and TRIFdependent pathways to NFB activation. The fact that TLR3 signals independently of MyD88 and Mal and solely utilizes TRIF, yet has a requirement for IRAK-2, suggested that IRAK-2 might be recruited into the TLR3 signaling complex upstream of TRIF.
IRAK-2 Associates with TLR3-Because IRAK-2 can associate with Mal (3) and form a complex with the IL-1R (16), we reasoned that IRAK-2 could interact with TIR domain-containing proteins, in contrast to IRAK-1, which accesses IL-1R⅐TLR complexes via a death domain interaction with MyD88. We therefore tested whether IRAK-2 could associate with the TLR3 receptor complex. Fig. 4e demonstrates by co-immunoprecipitation an association between ectopically expressed TLR3 and IRAK-2. This complex was detectable in the absence of poly(I:C) treatment, and the intensity of the receptor-IRAK-2 interaction was unchanged following stimulation of TLR3 by poly(I:C). Co-immunoprecipitation experiments carried out with ectopically expressed TLR3 and endogenous IRAK-2 confirmed the ability of IRAK-2 to physically associate with TLR3 (Fig. 4f). Again, the IRAK-2⅐TLR3 receptor complex was detectable in the absence of poly(I:C) treatment, which may reflect the strength of the association between these two molecules.
IRAK-2 Activates NFB by Stimulating TRAF6 Ubiquination-IRAK-2 can also interact with the non-TIR domain-containing protein TRAF6 (16), which is an important downstream effector molecule for transcription factor and MAP kinase activation by IL-1 and TLRs. Fig. 5a shows a sequence alignment of mammalian IRAK-2 orthologs indicating the position of two putative consensus TRAF6 binding motifs within IRAK-2, as previously noted by Ye et al. (30). These motifs were mutated to explore the importance of TRAF6 as an effector of IRAK-2 function. The central Glu residues of both sites were mutated concurrently to Ala, because mutation of this residue within the single TRAF6 binding motif of CD40 severely impaired its function (30). Fig. 5b shows that this mutant, IRAK-2DM, was no longer able to activate NFB (left panel), p38 MAP kinase activation (middle panel), or to induce the IL-8 promoter (right panel). Thus, mutation of the two putative TRAF6 binding motifs within IRAK-2 completely silenced all IRAK-2 functions tested suggesting that TRAF6 is a critical effector for IRAK-2induced NFB and MAP kinase activation. The relative contribution of each TRAF6 binding motif to IRAK-2 function was examined by mutating each central Glu independently to generate two distinct mutants, E528A and E559A. IRAK-2E559A induced signal transduction pathways as potently as the wildtype protein (Fig. 5, compare c to b). Surprisingly, IRAK-2E528A was entirely inert, unable to induce NFB or p38 MAP kinase activation, or to up-regulate the IL-8 promoter (Fig. 5c). Both mutant proteins were expressed at comparable levels (Fig. 5d). Hence a single amino acid (Glu-528), in the first TRAF6 binding motif is essential for IRAK-2 function, whereas the second putative TRAF6 binding motif does not appear to be important for IRAK-2 signaling to NFB or p38 MAP kinase activation.
Because mutation of the central Glu residue within the TRAF6 binding motif of CD40 abolished its ability to interact with TRAF6 in an in vitro binding assay, thus accounting for the ensuing loss of activity (30), we compared the ability of the mutants to interact with TRAF6. Surprisingly, when IRAK-2E528A was expressed in cells it was still capable of interacting with TRAF6 (Fig. 5d, left panels). As expected, the functionally active IRAK-2E559A also associated with TRAF6 (Fig. 5d, mid- dle panels). Notably, the IRAK-2DM mutant similarly bound to TRAF6 in co-immunoprecipitation experiments (Fig. 5d, right panels) confirming that the association of IRAK-2E528A with TRAF6 was not simply through the Glu-559-containing TRAF6 binding motif. Thus, other essential residues within these motifs are likely sufficient to facilitate binding.
TRAF6 is an E3 ubiquitin ligase, and TRAF6 autoubiquitination is a critical step in the activation of NFB by TLRs (31). Here we demonstrate that expression of wild-type IRAK-2 is sufficient to trigger polyubiquitination of TRAF6. Further, although the IRAK-2 loss-of-function mutants could still interact with TRAF6 they could no longer stimulate TRAF6 polyubiquitination. Fig. 5e shows an ubiquitination assay where conjugation of HA-ubiquitin to endogenous TRAF6 was clearly induced in the presence of IRAK2 (lane 4, upper panel) and similarly by IRAK-2E559A (lane 6), but not by IRAK-2E528A (lane 5) nor IRAK-2DM (lane 7). Thus the ability of the IRAK-2 mutants to activate downstream signaling directly correlated with their ability to stimulate TRAF6 polyubiquitination. The detection of immunoprecipitated TRAF6 was reduced upon triggering of TRAF6 ubiquitination (second panel), likely due to the presence of conjugated ubiquitin blocking antibody recognition of TRAF6 epitopes. Interestingly, immunoprecipitation of TRAF6 was also reduced in the presence of IRAK-2 alone (second panel, lane 3) likely indicating the conjugation of endogenous ubiquitin to TRAF6 when IRAK-2 is introduced into cells.
Although it has been assumed that IRAK1 causes TRAF6 polyubiquitination, here comparison of IRAK-2 to IRAK-1 in the ubiquitination assay revealed that IRAK-1 lacked the ability of IRAK-2 to initiate TRAF6 polyubiquitination (compare lane 4 to lane 6; Fig. 5f, upper panel). Critically, given the essential requirement for this key step in the induction of NFB in response to TLR signal transduction, this functional difference between IRAK-1 and IRAK-2 provides strong evidence for a more central role for IRAK-2 in TLR signaling to NFB compared with IRAK-1.

DISCUSSION
Although identified 10 years ago (16), the exact role of IRAK-2 in TLR signaling has remained unclear, as has the extent of its involvement in different TLR pathways. Here, we have exploited the ability of the poxvirus protein A52 to inhibit TLR signaling to implicate IRAK-2 as having a central role in NFB activation. Like other known virulence factors, A52 seems to have multiple effects on the host immune response, and the availability of an A52 mutant, which only interacted with IRAK-2 and not its other target TRAF6, allowed us to map the inhibitory effect of A52 on NFB to an association with IRAK-2. How exactly A52 disrupts IRAK-2 function remains to be determined, but previous work suggests that it may sequester IRAK-2 out of signaling complexes and prevent it interacting with other signaling molecules (18). Having shown that A52 inhibited NFB activation induced by TLR2-, -3, -4, -5, -7, -8, and -9, we focused on the TLR3 pathways, because IRAK-1 had been clearly shown to have no role in TLR3-mediated NFB activation. NFB activation by poly(I:C)/TLR3 was normal in IRAK-1-deficient cells, and neither IRAK-1 nor IRAK-4 was recruited to the TLR3 receptor complex following ligand engagement (32). In contrast, we showed here that IRAK-2 is part of the unstimulated TLR3 receptor complex (Fig. 4, e and f). Crucially, knockdown of endogenous IRAK-2 expression by siRNA suppressed poly(I: C)/TLR3-mediated NFB activation (Fig. 3d). Further knockdown experiments showed that, in contrast to MyD88-and FIGURE 6. Model for IRAK2 involvement in TLR signaling pathways to NFB. IRAK2 binds directly to the TLR adaptor molecules Mal and MyD88, allowing it to be present in the membrane complex of IL1R⅐TLRs that uses these adaptors, as illustrated here for TLR4. In the case of TLR3, which does not use MyD88 or Mal, IRAK2 binds the receptor directly. Upon TLR activation, IRAK2 then associates with TRAF6. The interaction of IRAK2 with TRAF6 triggers the polyubiquitination of TRAF6, via a process requiring Glu-528 of IRAK2. This likely involves E1, ubc13/uev1A (E2) and the autoubiquitination activity of TRAF6 itself through its E3 ligase domain. The polyubiquitination of TRAF6 allows the subsequent recruitment of a complex containing TAB2 (which recognizes polyubiquitinated TRAF6), TAB1, and TAK1. This activates the kinase activity of TAK1 leading to the phosphorylation of target kinases such as IKK␤ and MKK6, culminating in the induction of NFB and p38 MAP kinase respectively. IRF activation proceeds independently of IRAK2 and, for TLR4 and TLR3, involves a TRIF-dependent pathway. Although TRIF has been shown to be important for the murine TLR3-NFB pathway, and to interact with TRAF6, the relationship between TRIF and IRAK2 in the human TLR3 pathway to NFB is currently unclear.
Mal-dependent pathways to NFB, IRAK-2 seems to act upstream of the TRIF-dependent pathway (Fig. 4, a-c), consistent with the interaction of IRAK-2 with TLR3. A model for IRAK2 involvement in TLR signaling pathways to NFB is shown in Fig. 6. It is currently unclear how exactly TRIF is involved in IRAK-2-mediated NFB activation by TLR3. Given that, in the absence of TRIF, signaling to NFB from TLR3 is disrupted (6,33), it is unlikely that IRAK-2 controls a dominant TLR3 pathway to NFB independently of TRIF. However, because these studies utilized knock-out mouse models, it remains to be seen whether TRIF functions identically in TLR3 signaling in humans.
Recent studies have questioned the originally proposed central role of IRAK-1 in NFB activation. For TLR9 in dendritic cells, IRAK-1 was dispensable for NFB activation and proinflammatory cytokine production (34). TLR9 is thought to signal in a similar manner to TLR8, and herein, we show that IRAK2 siRNA inhibited TLR8-mediated NFB activation. Further, alternative NFB-independent functions for IRAK-1 have emerged, such as TLR4-mediated STAT3 activation and upregulation of the NFB-independent gene, IL-10 (35). In that study, absence of IRAK-1 had no effect on LPS induction of the NFB-dependent pro-inflammatory cytokine, IL1-␤. In our study, IRAK-2 siRNA inhibited LPS-induced NFB activation, and, in primary human cells, LPS stimulated production of the NFB-dependent chemokine IL-8. Moreover, IRF7 has recently been identified as a target of IRAK-1 kinase activity (34). The formation of an IRF7⅐IRAK-1 complex followed by IRAK-1-mediated phosphorylation of IRF7 appeared to be a prerequisite for interferon-␣ induction by TLR7 and TLR9. In contrast, the lack of any effect of A52 or IRAK2 expression on IRF3, -5, and -7 activation suggests that IRAK-2 has no role in IRF activation. Hence IRAK-1, although involved in some pathways to NFB, may be more fundamentally important for TLR signaling to IRFs. Here, the observation that IRAK-2 but not IRAK-1 could trigger TRAF6 ubiquitination provides direct evidence for the importance of IRAK-2 over IRAK-1 in TLRmediated NFB activation, because the autoubiquitinating E3 ligase activity of TRAF6 is essential for IL-1R/TLR signaling to NFB (36,37), although how exactly this activity of TRAF6 is turned on by IRAK-2 is yet to be established. Although it had been assumed that IRAK-1 could stimulate TRAF6 ubiquitination, a recent study demonstrated that the Epstein-Barr virus protein LMP1 could cause TRAF6 polyubiquitination in the absence of IRAK-1 (38). Thus we have defined a dichotomy in IRAK-1 and IRAK-2 function, which suggests that these proteins are not simply functionally redundant but rather fulfill separate roles in TLR signaling.
Despite two potential TRAF6-binding motifs being previously identified in IRAK-2 (30), their functional significance had not been tested until now. The site containing Glu-528 is conserved among mammalian IRAK-2 orthologs, whereas the second putative TRAF6 binding site containing residue Glu-559 is not, consistent with the functional relevance of the Glu-528-containing site, which when mutated abolished the ability of IRAK-2 expression to induce NFB and MAP kinase activation and IL-8 promoter induction. The fact that IRAK-2E528A still binds to TRAF6 and yet cannot activate NFB suggests that Glu-528 is in fact critical for signal transduction, and consistent with this, IRAK-2E528A no longer induced TRAF6 polyubiquitination.
In conclusion, we have demonstrated that IRAK-2 has a central role in TLR signaling pathways to NFB activation, and in contrast to IRAK-1, is likely utilized by all TLRs for this purpose. IRAK-2 can interact with at least one TLR, namely TLR3, and TRAF6 is a critical downstream mediator of IRAK-2 function. Given that IRAK-2 is unlikely to be involved in IRF activation, selectively targeting IRAK-2 may be a useful therapeutic strategy to block pro-inflammatory TLR signals via NFB, while preserving IRF-regulated pathways.