The Kinase Activity of IL-1 Receptor-associated Kinase 4 Is Required for Interleukin-1 Receptor/Toll-like Receptor-induced TAK1-dependent NFκB Activation*

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFκB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKα/β phosphorylation and IKKβ activation, resulting in classic NFκB activation through IκBα phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKγ phosphorylation and IKKα activation, resulting in NFκB activation through dissociation of phosphorylated IκBα from NFκB without IκBα degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFκB activation pathway, but mediated IL-1-induced TAK1-independent NFκB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFκB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFκB activation.

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKK␣/␤ phosphorylation and IKK␤ activation, resulting in classic NFB activation through IB␣ phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKK␥ phosphorylation and IKK␣ activation, resulting in NFB activation through dissociation of phosphorylated IB␣ from NFB without IB␣ degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFB activation pathway, but mediated IL-1-induced TAK1-independent NFB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFB activation.
Toll-like receptors (TLRs) 3 play a critical role in innate immune responses in mammals through the recognition of conserved molecular patterns associated with different microorganisms (1)(2)(3)(4)(5)(6). Upon binding of TLR ligands, all of the TLRs except TLR3 recruit the adaptor molecule MyD88 through the TIR domain, mediating the so-called MyD88-dependent pathway (7). MyD88 then recruits serine-threonine kinases IRAK4 (IL-1 receptor-associated kinase 4) and IRAK (8 -12). Although IRAK4 is the kinase that functions upstream of and phosphorylates IRAK, the phosphorylated IRAK mediates the recruitment of TRAF6 to the receptor complex (13,14). Upon phosphorylation of IRAK, the IRAK⅐TRAF6 complex dissociates from the receptor complex to interact with and activate downstream kinases, leading to the activation of NFB and JNK (14,15).
We recently reported the co-existence of the two parallel IL-1-mediated TAK1-dependent and MEKK3-dependent signaling pathways for NFB activation ( Fig. 1) (16). These two pathways are regulated at the level of IRAK modification. The TAK1-dependent pathway causes IKK␣/␤ phosphorylation and IKK␤ activation, leading to classic NFB activation through IB␣ phosphorylation and degradation. The TAK1independent MEKK3-dependent pathway induces IKK␥ phosphorylation and IKK␣ activation, resulting in NFB activation through IB␣ phosphorylation and subsequent dissociation from NFB but without IB␣ degradation. It is important to note that we recently found that TLR8-mediated NFB and JNK activation are TAK1-independent and MEKK3-dependent, suggesting a regulatory mechanism at the level of receptor complexes that determines the usage of TAK1-dependent versus MEKK3-dependent pathways in IL-1R/TLR signaling.
IRAK4 has been shown to play an essential role in TLR-mediated signaling (9,10). IRAK4 kinase-inactive knock-in mice were completely resistant to LPS-and CpG-induced shock, due to impaired TLR-mediated induction of pro-inflammatory cytokines and chemokines (17)(18)(19)(20). Although inactivation of IRAK4 kinase activity did not affect the levels of TLR/IL-1Rmediated NFB activation, a reduction of LPS-, R848-, and IL-1-mediated mRNA stability contributed to the reduced cytokine and chemokine production in bone marrow (BM)derived macrophages from IRAK4 kinase-inactive knock-in mice (18,20). These in vivo studies indicate that IRAK4 kinase activity plays a critical role in TLR-dependent immune responses (21).
Previous studies suggest that IRAK4 is required for the recruitment and activation of IRAK at the signaling complex. Interestingly, IRAK4 kinase-inactive mutant had similar ability as the wild-type IRAK4 in restoring IL-1-mediated NFB in human IRAK4-deficient cells (22). On the other hand, by reconstituting IRAK4-deficient mouse embryonic fibroblasts, Lye et al. (23) showed that the kinase activity of mouse IRAK4 is required for the optimal transduction of IL-1-induced signals, although they found that IRAK4 is capable of mediating some NFB activation. In support of these previous findings, IL-1-, LPS-, and R848-induced NFB activation was not reduced in the BM-derived macrophages from IRAK4 kinase-inactive knock-in mice as compared with that in the wild-type control cells. Therefore, the kinase activity of IRAK4 seems to be dispensable for TLR/IL-1R-mediated NFB activation.
We recently further examined the role of kinase activity of IRAK4 in IL-1-induced NFB activation. Whereas NFB was activated, IB was only phosphorylated but not degraded in human IRAK4-deficient cells transfected with the IRAK4 kinase-inactive mutant in response to IL-1 stimulation, suggesting that kinase activity of IRAK4 might be important for TAK1-dependent NFB activation pathway. Similar results were observed in BM macrophages from IRAK4 kinase-inactive knock-in mice. IRAK4 kinase-inactive mutant failed to mediate IL-1R-TLR-induced TAK1-dependent NFB activation pathway, evident by greatly reduced IL-1R-TLR-induced TAK1 phosphorylation and activation, IKK␣/␤ phosphorylation, and IB␣ degradation. The fact that the kinase-inactive IRAK4 mutant can still mediate IL-1-induced TAK1-independent NFB activation indicates a structural role of IRAK4 in mediating this alternative NFB activation pathway. Deletion analysis of IRAK4 demonstrates the essential structural role of IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFB activation.
Transfection and Luciferase Assay-Transfection of the human IRAK4-deficient fibroblasts was performed using the FuGENE 6 transfection reagent as recommended by the manufacturer (Roche Applied Science). After 24 h, the cells were stimulated with IL-1 or left untreated the next day for 6 h before harvest. Luciferase activity was determined by using the luciferase assay system and chemiluminescent reagents from Promega (Madison, WI).
Plasmids-IRAK4 deletion mutants and IRAK4/IRAK chimeric constructs were generated by overlapping PCR and cloned in pcDNA 3.1 expression vector. For all PCR reactions high fidelity Pfu Turbo polymerase was used (Stratagene).
Gel Shift Assays-An NFB binding site (5Ј-GAGCAGAGG-GAAATTCCGTAACTT-3Ј) from the IP-10 gene was used as a probe (24). Complementary oligonucleotides, end-labeled with polynucleotide kinase (Boehringer Mannheim) and ␥-32 P-labeled ATP, were annealed by slow cooling. Approximately 20,000 cpm of probe was used per assay. Cytoplasmic extracts were prepared as described by Kessler et al. (25). The binding reaction was carried out at room temperature for 20 min in a total volume of 20 l containing 20 mM Hepes buffer, pH 7.0, 10 mM KCl, 0.1% Nonidet P-40, 0.5 mM dithiothreitol, 0.25 mM phenylmethylsulfonyl fluoride, and 10% glycerol.
Kinase Assays-Cell lysates were immunoprecipitated with anti-TAK1 and collected on protein A-Sepharose beads. Kinase reactions were performed in 50 l of buffer containing 20 mM Hepes (pH 7.0), 20 mM MgCl 2 , 1 mM ATP, 10 mCi of [␥-32 P]ATP at 30°C for 30 min. The substrate for TAK1 kinase assay was 2 g of His-MKK6. Samples were analyzed by 10% SDS-PAGE, followed by autoradiography.
Enzyme-linked Immunosorbent Assay-Supernatants from cell cultures were collected and measured for the level of human cytokines IL-6 and IL-8 using OptEIA ELISA kits II (BD Biosciences) according to the manufacturer's instructions.
Illumina Beadchip Micoarray Analysis-250 ng of RNA was reverse transcribed into cRNA and biotin-UTP labeled using the Illumina (San Diego, CA) TotalPrep RNA amplification kit (Ambion, Austin, TX). cRNA was quantified using a nanodrop spectrophotometer, and the cRNA quality (size distribution) was further analyzed on a 1% agarose gel. cRNA was hybridized to the Illumina MouseRef8 v1.1 Expression BeadChip using standard protocols (provided by Illumina).

The Kinase Activity of IRAK4 Is Required for IL-1R-induced TAK1-dependent NFB Activation in Human IRAK4-deficient
Fibroblasts-IRAK is phosphorylated after it is recruited to the receptor, subsequently ubiquitinated, and eventually degraded upon IL-1 stimulation. Through the study of IRAK modification, we uncovered two IL-1-mediated signaling pathways for NFB activation: TAK1-dependent and MEKK3-dependent, respectively ( Fig. 1) (16). As depicted in Fig. 1, IL-1R mediates NFB activation through TAK1-and MEKK3-dependent pathways. The TAK1-dependent pathway leads to IKK␣/␤ phosphorylation and IKK␤ activation, resulting in classic NFB activation through IB␣ phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKK␥ phosphorylation and IKK␣ activation, resulting in NFB activation through IB␣ phosphorylation and subsequent dissociation from NFB but without IB␣ degradation. Impairment of IL-1-induced IRAK phosphorylation and ubiquitination shifts receptor proximal signaling from the TAK1-depend-ent to the MEKK3-dependent pathway. Previous studies suggest that IRAK4 is required for the recruitment and activation of IRAK at the signaling complex. Interestingly, IRAK4 kinase-inactive mutant (lysine 213 to methionine, which is part of the ATP binding pocket of the kinase domain) had similar ability as the wild-type IRAK4 in restoring IL-1-mediated NFB activation in IRAK4-deficient fibroblasts derived from human patient (Fig. 2, A and B) (10,22). In the light of the new discovery of TAK1-and MEKK3-dependent NFB activation pathways ( Fig. 1), we reexamined the ability of IRAK4 kinaseinactive mutant in mediating intermediate signaling events of IL-1-induced ⌵F〉 activation in human IRAK4-deficient fibroblasts. Whereas IRAK4 kinase-inactive mutant had similar ability as the wild-type IRAK4 in mediating IL-1-induced IB␣ phosphorylation and NFB activation, the mutant failed to restore IL-1-induced TAK1 phosphorylation and activation, IKK␣/␤ phosphorylation, and IB␣ degradation (Fig. 2, C and  D). These results suggest that IRAK4 kinase activity may be only required for TAK1-dependent NFB activation. Thus, the kinase-inactive IRAK4 mutant probably activates NFB through a TAK1-independent pathway. Based on our previous findings on the link between TAK1-independent NFB activation and MEKK3 (16), we propose that inactivation of IRAK4 kinase activity might shift receptor proximal signaling from the TAK1-dependent to the MEKK3-dependent pathway, which is further addressed below.  NOVEMBER 14, 2008 • VOLUME 283 • NUMBER 46

JOURNAL OF BIOLOGICAL CHEMISTRY 31699
Inactivation of IRAK4 Kinase Activity Abolished TLR4/7/9mediated TAK1-dependent NFB Activation-To further determine the role of the kinase activity of IRAK4 in IL-1R-TLR-mediated signaling, we generated IRAK4 kinase-inactive knock-in mice by mutating lysine 213 and 214 residues to methionines (20). The inactivation of the kinase activity of the mutant IRAK4 from the knock-in mice was confirmed by immunoprecipitation kinase assay in vitro (20). BM-derived macrophages from wild-type, IRAK4-deficient, and IRAK4 kinase-inactive knock-in mice were examined for IL-1R-TLRmediated NFB activation. IL-1, LPS, R848, and CpG induced phosphorylation of IB␣ and NFB activation in both wild-type and IRAK4 kinase-inactive knock-in BM-derived macrophages (Fig. 3, A and B) (20). However, TLR4/7/9-mediated TAK1 phosphorylation (slower migration abolished by phosphatase treatment (16)), IKK␣/␤ phosphorylation, and IB␣ degradation were significantly attenuated in IRAK4 kinase-inactive knock-in macrophages as compared with that in wild-type cells (Fig. 3, B and C, and data not shown). The impact of inactivation of IRAK4 kinase activity on NFB activation echoes our published results of the TAK1-deficeint mouse embryonic fibroblasts for the IL-1-induced signaling, including abolished IL-1induced TAK1 activation, reduced IL-1-induced IKK␣/␤ and IB␣ degradation, retained IB␣ phosphorylation, and NFB activation (16). Taken together, these findings suggest that the impact of IRAK4 kinase activity on NFB activation might be due to a lack of TAK1 activation. In support of this, TAK1 inhibitor blocked TLR4/7-induced IB␣ degradation and retained IB␣ phosphorylation and NFB activation in BMderived macrophages (Fig. 3E), which is very similar to the impact of IRAK4 kinase activity on IB␣ and NFB activation upon TLR4/7 stimulation in BM-derived macrophages. These results confirm that IRAK4 kinaseinactive mutant probably activates NFB through a TAK1-independent pathway.
IRAK4 Kinase Activity Is Required for IL-1R-and TLR4/7/9-mediated IRAK Modification and Degradation-We recently reported that, although a point mutation changing lysine 134 to arginine (K134R) in IRAK abolished IL-1-induced IRAK ubiquitination and degradation, mutations of serines and threonines adjacent to Lys-134 to alanines ((S/ T)A (131-144)) reduced IL-1-induced IRAK phosphorylation and abolished IRAK ubiquitination (16). The IRAK modification mutants failed to form complex with TAK1 and were unable to mediate TAK1 phosphorylation and activation. Interestingly, we now found that IRAK4 kinase activity is also required for TAK1-dependent NFB activation. These results suggest that IRAK4 and IRAK might mediate TAK1-dependent pathway in a sequential fashion, through a kinase-substrate relationship. Importantly, TLR4/7/ 9-induced IRAK modification and degradation were indeed abolished in BM macrophages from IRAK4 kinase-inactive knock-in mice as compared with that of wild-type mice, demonstrating that the kinase activity of IRAK4 is required for IRAK modification (17,19) (Fig. 3F and data not shown).
The Kinase-inactive IRAK4 Mutant Protein Mediates TLR7/ 9-induced TAK1-independent NFB Activation-The above results indicate that TLR4/7/9 can still mediate TAK1-independent NFB activation in the absence of IRAK4 kinase activity. One important question was whether this TAK1-independent NFB activation pathway requires the presence (structural role) of IRAK4 protein. To address this question, we examined TLR2/4/7/9-mediated NFB activation and related signaling events in IRAK4-deficient macrophages. Whereas TLR2/4-mediated NFB activation and related signaling events were attenuated, TLR7/9-mediated signaling events were abolished or greatly reduced in BM-derived macrophages from IRAK4-deficient mice (Fig. 3, G and H). It should be noted that TLR7/9 can sometimes still slightly induce NFB activation in IRAK4deficient macrophages (which was previously observed by Kawagoe et al. (17). However, the level of NFB activation was much lower in IRAK-deficient macrophages compared with that in IRAK4 kinase-inactive knock-in or wild-type macrophages. These results clearly indicate that most of the TLR7/9mediated signaling pathways are IRAK4-dependent, although TLR7/9-mediated TAK1-independent NFB activation does not require IRAK4 kinase activity. In other words, the kinaseinactive IRAK4 protein probably plays a structural role for the TLR7/9-mediated TAK1-independent NFB activation. Our previous studies have suggested a potential role of MEKK3 in IL-1-induced TAK1-independent NFB activation (16). The IRAK modification mutants that failed to interact with TAK1 retained the ability to interact with MEKK3 and were also able to mediate the interaction between TRAF6 and MEKK3, implicating a specific role of MEKK3 in IL-1-induced TAK1-independent signaling. Furthermore, although the TAK1 inhibitor (5Z-7-oxozeaenol) had partial inhibition on NFB activation in wild-type mouse embryonic fibroblasts, the inhibitor completely impaired IL-1-induced NFB activation in the MEKK3-deficient mouse embryonic fibroblasts. We indeed found that, although TLR7-induced TAK1 phosphorylation was abolished in the absence of IRAK4 kinase activity (Fig. 3C), TLR7 stimulation induced MEKK3 phosphorylation in macrophages from both wild-type and IRAK4 kinase-inactive knock-in mice (Fig. 3D). TLR7-induced TAK1 and MEKK3 phosphorylation was confirmed by treatment with calf intestinal phosphatase (Ref. 16 and data not shown). Taken together, these results suggest the possible participation of MEKK3 in TLR7-induced TAK1-independent NFB activation.
IRAK4 Kinase-inactive Mutant Mediates Substantial Expression of TLR7/9-dependent Genes-One important issue is about whether the observed TAK1-independent NFB activation results in substantial expression of TLR-dependent genes in the IRAK4 kinase-inactive knock-in mice. We recently reported that IRAK4 kinase-inactive knock-in mice were completely resistant to LPS-and CpG-induced shock, due to impaired TLR-mediated induction of pro-inflammatory cytokines and chemokines. Although inactivation of IRAK4 kinase activity did not affect the levels of IL-1R-and TLR4/7/9-mediated NFB activation, a reduction of LPS-, R848-and IL-1-mediated mRNA stability contributed to the reduced cytokine and chemokine production in BM-derived macrophages from IRAK4 kinase-inactive knock-in mice (20).
Because the impact of IRAK4 kinase activity is mainly on mRNA stabilization of cytokine and chemokine genes, then one A-D, cell lysates from bone marrowderived macrophages from wild-type, IRAK4 kinaseiinactive knock-in or IRAK4-deficient mice that were either untreated or treated with LPS (1 g/ml), R848 (1 g/ml), or CpG (4 g/ml) for the indicated times were analyzed by electrophoretic mobility shift assay with an NFB-specific probe (A) or by Western analysis with antibodies against pJNK, IRAK4 and actin (A) and antibodies against phospho-IB␣, IB␣, phospho-IKK␣/␤, and IKK␣/␤ (B), TAK1 (C), and MEKK3 (D). E, bone marrow-derived macrophages were pretreated with 600 nM TAK1 inhibitor for 3 h prior to stimulation with LPS (1 g/ml) and R848 (1 g/ml) for the indicated times. The cell lysates were analyzed by electrophoretic mobility shift assay with an NFB-specific probe or by Western analysis with antibodies against phospho-IB␣, IB␣, and actin. F, cell lysates from bone marrow-derived macrophages from wild-type and IRAK4-deficient mice that were either untreated or treated with R848 (1 g/ml) or CpG (4 g/ml) for the indicated times were analyzed by Western analysis with antibodies against IRAK and ␤-actin. G and H, cell lysates from bone marrow-derived macrophages from wild-type and IRAK4-deficient mice that were either untreated or treated with LPS (1 g/ml), R848 (1 g/ml), or Malp2 or CpG (4 g/ml) for the indicated times were analyzed by electrophoretic mobility shift assay with an NFB-specific probe (G) or by Western analysis with antibodies against pJNK, phospho-IKK␣/␤, phospho-IB␣, IB␣, and IRAK4 (H). The levels of NFB activation were analyzed by Scion Image 1.62C alias and presented as -fold induction of the untreated samples. NOVEMBER 14, 2008 • VOLUME 283 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 31701 would predict inactivation of IRAK4 kinase activity should have minimum impact on mRNA levels of the genes that are not regulated at the post-transcription levels. We recently examined gene expression profiles of macrophages from wild-type and IRAK4 kinase-inactive knock-in mice in response to TLR7 and TLR9 stimulation using the Illumina Microarray with probes for 46,000 transcripts. Bone marrow-derived macrophages from wild-type and IRAK4 kinase-inactive knock-in mice were treated with TLR7 (R848) and TLR9 (CpG) ligands for 30 and 240 min. The majority of the genes was induced at similar levels in wild-type and IRAK4 kinase-inactive knock-in macrophages at early time (stimulated for 30 min) (supplemen-tal Fig. S1A and data not shown). Some of those genes were then up-regulated at higher levels in wild type than that in kinaseinactive knock-in macrophages at late time (stimulated for 240 min) (supplemental Fig. S1, B and C), which is consistent with the previously reported role of IRAK4 kinase activity in posttranscriptional regulation (20). Importantly, we have also identified a group of genes that were induced similarly in wild-type and IRAK4 kinase-inactive knock-in macrophages at both early and late time in response to TLR7 or TLR9 ligands (Fig. 4, A-D), which was confirmed by real-time PCR (Fig. 4, C and D). The expression of these IRAK4 kinase-independent genes was abolished in IRAK4-deficient macrophages, indicating a structural

. Illumina Microarray analysis of gene expression induced by TLR7 and TLR9 in wild-type and IRAK4 kinase-inactive knock-in macrophages.
A, heat map of the genes that were induced similarly (Ն3-fold) in both wild-type and IRAK4 kinase-inactive knock-in macrophages upon 240 min of R848 (TLR7 ligand, 1 g/ml) stimulation. B, heat map of the genes that were induced similarly (Ն3-fold) in both wild-type and IRAK4 kinase-inactive knock-in macrophage upon 240 min of CpG oligodeoxynucleotide (TLR9 ligand, 2.5 g/ml) stimulation. C, quantitative Real-time PCR of selected genes from Fig. 4 (A). Wild-type, IRAK4 kinase-inactive knock-in and IRAK4-deficient macrophages were either untreated or stimulated with TLR7 ligand for the indicated time points. The -fold change was calculated compared with the expression of untreated samples (set as value 1). D, quantitative real-time PCR of selected genes from B. Wild-type, IRAK4 kinase-inactive knock-in and IRAK4-deficient macrophages were either untreated or stimulated with CpG oligodeoxynucleotide for the indicated time points. The -fold change was calculated compared with the expression of untreated samples (set as value 1). role of IRAK4 in the induction of these genes (Fig. 4, C and D). Based on these findings, we propose that the TAK1-independent pathway in IRAK4 kinase-inactive macrophages can mediate substantial expression of TLR7/9-dependent genes, especially on the genes that are not regulated at post-transcriptional levels.
The Death Domain of IRAK4 Is Essential for IL-1R-induced NFB Activation-The above results showed that the kinaseinactive IRAK4 mutant can still mediate IL-1R-and TLR7/9induced TAK1-independent NFB activation and gene expression, indicating a structural role of IRAK4 in mediating this alternative NFB activation pathway. We were then interested in determining the structural domain of IRAK4 important for this activity. As shown in Fig. 5A, IRAK4 contains several functional domains, including a death domain at the N terminus (DD, residues 1-100), followed by an undetermined region (UD, residues 100 -179) and a kinase domain at the C terminus (residues 179 -460). A set of IRAK4 deletion mutants were generated to assess the function of these different domains in IL-1R-induced NFB activation (Fig. 5A). The deletion mutants were co-transfected with NFB-dependent luciferase construct into IRAK4-deficient cells, followed by IL-1 treatment and luciferase reporter assay. The IRAK4 deletion mutants without UD(d101-179) or kinase domain (d179 -460) can still restore IL-1-induced NFB activation in IRAK4-deficient cells, whereas the DD deletion mutant completely failed to activate NFB in response to stimulation (Fig. 5B). Intriguingly, the expression construct containing the DD fragment only (d100 -460) could also restore a significant level of IL-1-induced NFB activation in IRAK4-deficent cells, indicating the essential structural role of the IRAK4 death domain in receptor proximal signaling (Fig. 5B).
The DD of IRAK4 Distinguishes IRAK4 from IRAK in Mediating IL-1R-induced NFB Activation-We hypothesize that the IRAK4 DD participates in the formation of receptor complex (Complex I, Fig. 1) to facilitate the recruitment of IRAK. To test this hypothesis, we swapped the death domain between IRAK and IRAK4 and tested the resulting chimeric IRAK4/ IRAK constructs (DD4 ϩ 1 and DD1 ϩ 4) for their ability to restore IL-1-inudced NFB activation in IRAK4versus IRAK-deficient cells. Interestingly, although DD4 ϩ 1 restored IL-1-induced NFB activation in IRAK4-deficient cells, DD1 ϩ 4 failed to activate NFB in these cells (Fig. 5, C,  E, and F). These results indicate that the death domain of IRAK4 is essential and sufficient in the recruitment of IRAK to mediate IL-1-induced NFB activation. Interestingly, the DD4 ϩ 1 also restored IL-1-induced NFB activation in IRAK-deficient cells, confirming the critical role of the death domain of IRAK4 for the recruitment of IRAK in mediating IL-1-induced NFB activation (Fig. 5D).

DISCUSSION
Herein, we report that the kinase activity of IRAK4 is required for IL-1R-TLR-induced TAK1-dependent NFB activation pathway, evident by greatly reduced IL-1R-TLR-induced TAK1 phosphorylation and activation, IKK␣/␤ phosphorylation, and IB␣ degradation. The fact that the kinase-inactive IRAK4 mutant can still mediate IL-1-induced TAK1-independ- FIGURE 5. The death domain of IRAK4 is essential for IL-1R-induced NFB activation. A, IRAK4 constructs. B and C, IRAK4 Ϫ/Ϫ human fibroblasts transfected with indicated IRAK4 constructs were untreated and stimulated with 1 ng/ml IL-1 for 6 h, followed by a luciferase assay. pSV2-␤-galactosidase reporter construct was used as a transfection control. Results are from four independent experiments, normalized against galactosidase activity, presented as -fold induction of luciferase activity induced by IL-1 stimulation in comparison with non-stimulated cells. D, I1A (IRAK-deficient 293IL1R cells) transfected with indicated IRAK4 constructs were untreated and treated with 1 ng/ml of IL-1 for 6 h, followed by luciferase assay. Results are from three independent experiments, presented as -fold induction of luciferase activity induced by IL-1 stimulation in comparison with non-stimulated cells. E, IRAK4 Ϫ/Ϫ fibroblasts transfected with different IRAK4 constructs were analyzed by Western blot with anti-IRAK4 antibody (Stressgene). F, IRAK4 Ϫ/Ϫ human fibroblasts transfected with indicated plasmids untreated and stimulated with IL-1␤ (10 ng/ml) for the indicated times. Cell lysates were analyzed by electrophoretic mobility shift assay with an NFB-specific probe. NOVEMBER 14, 2008 • VOLUME 283 • NUMBER 46 ent NFB activation and gene expression indicates a structural role of IRAK4 in mediating this alternative NFB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFB activation.

IRAK4 in TAK1-dependent and -independent NFB Activation
Significant levels of IL-1-, LPS-, and R848-induced NFB activation were retained in the BM-derived macrophages from IRAK4 kinase-inactive knock-in mice as compared with that in the wild-type control cells (17,20). We previously uncovered two parallel IL-1-mediated signaling pathways for NFB activation: TAK1-dependent and MEKK3-dependent, respectively (16). These two pathways bifurcate at the level of IRAK modification. The TAK1-dependent pathway leads to IKK␣/␤ phosphorylation and IKK␤ activation, resulting in classic NFB activation through IB␣ phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKK␥ phosphorylation and IKK␣ activation, resulting in NFB activation through IB␣ phosphorylation and subsequent dissociation from NFB but without IB␣ degradation. These results provide significant insight to our understanding of NFB activation data from the IRAK4 kinase-inactive knock-in cells. LPS and R848 stimulation led to IB␣ phosphorylation; therefore, NFB activation with attenuated IB␣ degradation in IRAK4 kinase-inactive knock-in cells suggests that the kinase activity of IRAK4 is likely to play a more critical role in TLR/IL-1R-induced TAK1-dependent than in the MEKK3-dependent NFB activation pathway. In this report, our results showed that TLR-IL-1R-induced IKK␣/␤ and TAK1 phosphorylation and activation were indeed abolished in the absence of IRAK4 kinase activity. Consistent with the fact that IRAK modification is required for the TAK1-dependent but not the MEKK3-dependent pathway (16), IRAK is not modified in IRAK4 kinaseinactive knock-in macrophages in which TAK1-dependent NFB activation is abolished. Therefore, we concluded that IRAK4 and IRAK function in a sequential fashion, through a kinase-substrate relationship, to mediate TLR-induced TAK1dependent but not MEKK3-dependent NFB activation.
Importantly, we previously reported that IRAK4 kinase-inactive mutant (K213M) had similar ability as the wild-type IRAK4 in restoring IL-1-mediated NFB activation in human IRAK4-deficient fibroblasts (22). In light of the new discovery of TAK1-and MEKK3-dependent NFB activation pathways, we re-examined the ability of IRAK4 kinase-inactive mutant to mediate intermediate signaling events of IL-1-induced NFB activation in human IRAK4-deficient fibroblasts. Consistent with our findings in IRAK4 kinase-inactive knock-in macrophages, human IRAK4 kinase-inactive mutant also failed to activate TAK1-dependent NFB activation in human IRAK4deficient cells. It should be noted that the human fibroblasts are not responsive to TLR ligands. Therefore, one cannot directly compare the role of IRAK4 kinase activity for TLR-mediated TAK1-mediated NFB activation in primary mouse macrophages with that in human fibroblasts. It is intriguing that, although IRAK4 kinase activity is required for TLR7-induced JNK activation, JNK is still activated in IRAK4-deficient cells transfected with IRAK4 kinase-inactive mutant in response to IL-1 stimulation. Overexpression of IRAK4 protein in these human IRAK4-deficient cells might cause hyperactivation of the JNK pathway.
It is interesting that, whereas TLR2/4-mediated NFB activation and related signaling events were attenuated, TLR7/9mediated signaling events were completely abolished in BMderived macrophages from IRAK4-deficient mice. TLR2/4 were able to mediate some NFB activation through one or more IRAK4-independent pathways. Although the TLR4-induced MyD88/IRAK4-independent signaling is known to be mediated by the TRIF-dependent pathway (26 -28), the TLR2induced IRAK4-independent pathway still needs to be defined. Our results are consistent with the studies presented by Kawagoe et al. (17), in which TLR2 signaling was the major focus. Unlike TLR4, TLR2 ligand mediates a unique MyD88-dependent IRAK4-independent pathway. On the other hand, our results clearly indicate that most of the TLR7/9-mediated signaling pathways are IRAK4-dependent, although TLR7/9-mediated TAK1-independent NFB activation does not require IRAK4 kinase activity. IL-1R-and TLR7/9-mediated NFB activation are completely abolished in human patients defective in IRAK4 expression (10), which is consistent with our study in IRAK4-deficient mice.
Although we previously reported that the kinase activity of IRAK4 is essential for IL-1R-TLR-mediated mRNA stabilization of cytokines and chemokines in mouse bone marrow-derived macrophages (20), we now find that IRAK4 kinase activity is required for IL-1R-and TLR4/7/9-mediated TAK1-dependent, but not TAK1-independent NFB activation. We propose that IRAK4 mediates IL-1R-TLR-induced receptor proximal signaling events through its kinase activity to coordinately regulate TAK1-dependent NFB activation and mRNA stabilization to ensure robust production of cytokines and chemokines during inflammatory response. In support of this, Illumina Microarray experiments indeed identified a group of inflammatory genes that were induced at similar levels in wild-type and IRAK4 kinase-inactive knock-in macrophages at early time, but with reduced expression in kinase-inactive knock-in macrophages at late time. One important issue is whether the observed TAK1-independent NFB activation results in any substantial expression of TLR-dependent genes in the IRAK4 kinase-inactive knock-in mice. Based on the fact that mRNA stabilization is abolished in the absence of IRAK4 kinase activity, one would predict that the TAK1-independent pathway in IRAK4 kinase-inactive macrophages probably mediate the expression of genes that are not regulated at post-transcriptional levels. Illumina Microarray experiments have now revealed a group of IRAK4 kinase-independent genes that were induced similarly in wild-type and IRAK4 kinase-inactive knock-in macrophages at both early and late time. However, the TLR-induced expression of these genes was abolished in IRAK4-deficient macrophages, indicating a structural role of IRAK4 in the induction of this group of genes.
The fact that the kinase-inactive IRAK4 mutant can still mediate IL-1-induced TAK1-independent NFB activation and gene expression indicates a structural role of IRAK4 in mediating this alternative NFB activation pathway. Importantly, although the IRAK4 death domain (either alone of fused to IRAK as DD4 ϩ 1) restored IL-1-induced NFB activation in IRAK4-deficient cells, DD1 ϩ 4 failed to activate NFB in these cells. These results indicate that the death domain of IRAK4 plays a key role in mediating receptor proximal signaling, which cannot be replaced by the death domain of IRAK. On the other hand, DD4 ϩ 1 was able to restore IL-1-induced NFB activation in IRAK-deficient cells, which indicates the essential and sufficient role of the death domain of IRAK4 for recruitment of IRAK in mediating IL-1-induced NFB activation. The specific functions of IRAK4 and IRAK death domain are probably due to their structural differences. The crystal structure for the IRAK4 death domain reveals a six-helical bundle with a prominent loop (29). Comparing with other IRAK family members (including IRAK), the highly structured loop contained between helices two and three (an 11-amino acid stretch, residues 39 -49), is unique to IRAK4 and may be responsible for its different function in mediating the formation of receptor signaling complex.
Although the detailed signaling mechanism and physiological impact of the TLR-induced IRAK4 kinase-independent pathway are still unclear, the ability of IRAK4 kinase-inactive mutant to mediate signaling is critical for pursuing IRAK4 as a drug target. It has been controversial whether IRAK4 is a feasible drug target for anti-inflammatory therapy. The total blockage of TLR-IL-1R signaling is likely to severely impair innate immunity, which may lead to immune deficiency with a dysfunctional host defense. Importantly, our studies indicate that, although IRAK4 kinase activity is essential for TLR-IL-1R-mediated production of cytokines and chemokines, not all of the TLR-IL-1R signaling events are ablated in IRAK4 kinase-inactive knock-in mice. Significant levels of NFB activation and substantial gene expression are retained upon TLR7/9 and IL-1R stimulation in the absence of IRAK4 kinase activity. The residual TLR7/9-induced signaling event allows production of some cytokines and chemokines at early times after stimulation and induction of a group of TLR7/9-mediated IRAK4 kinaseindependent genes in IRAK4 kinase-inactive knock-in cells. Therefore, it is likely that pharmacological inhibition of IRAK4 kinase activity will not completely impair host defense, while alleviating the levels and duration of inflammatory responses.