IRAK-mediated Translocation of TRAF6 and TAB2 in the Interleukin-1-induced Activation of NF (cid:1) B*

The interleukin-1 (IL-1) receptor-associated kinase (IRAK) is required for the IL-1-induced activation of nuclear factor (cid:1) B and c-Jun N-terminal kinase. The goal of this study was to understand how IRAK activates the intermediate proteins TRAF6, TAK1, TAB1, and TAB2. When IRAK is phosphorylated in response to IL-1, it binds to the membrane where it forms a complex with TRAF6; TRAF6 then dissociates and translocates to the cytosol. The membrane-bound IRAK similarly mediates the IL-1-induced translocation of TAB2 from the membrane to the cytosol. Different regions of IRAK are required for the translocation of TAB2 and TRAF6, suggesting that IRAK mediates the translocation of each protein separately. The translocation of TAB2 and TRAF6 is needed to form a TRAF6-TAK1-TAB1-TAB2 complex in the cytosol and thus activate TAK1. Our results show that IRAK is required for the IL-1-induced phosphorylation of TAK1, TAB1, and TAB2. The phosphorylation of these three proteins correlates strongly with the activation of nuclear factor (cid:1) NF (cid:1) B activation. IRAK mediates the IL-1-induced translocation of TRAF6 and TAB2, which leads to the formation of a TRAF6-TAK1-TAB1-TAB2 complex in the cytosol, the activation of TAK1, and the phosphorylation of TAK1, TAB1, and TAB2. This eventually results in IL-1-induced activation of NF (cid:1) B. P-TAK1, phospho-TAK1; P-TAB1 , phospho-TAB1; P-TAB2 , phospho-TAB2; P-IRAK-U, phosphorylated and ubiquitinated IRAK.

IRAK, a serine-threonine kinase, is phosphorylated at the receptor complex and then presumably dissociates from that complex to interact with tumor necrosis factor receptor-associated factor 6 (TRAF6) (12)(13)(14)(15)(16). Phosphorylated IRAK is eventually ubiquitinated and degraded (17). Transforming growth factor-␤-activated kinase 1 (TAK1), a member of the mitogenactivated protein kinase kinase kinase family, and two proteins that bind to it, transforming growth factor-␤-activated kinase 1 binding protein 1 and 2 (TAB1 and TAB2), have recently been implicated in IL-1 signaling (18,19). TAB2 is membrane-bound in untreated cells but translocates to the cytosol upon stimulation with IL-1, where it functions as an adaptor, linking TRAF6 to TAK1 and TAB1, thereby activating TAK1 (19). Some investigators (20) have suggested that activated TAK1 triggers the NFB-inducing kinase (NIK) and IB kinase cascade, leading to NFB activation. More recently, studies on NIK-deficient mice have revealed that NIK is required for lymphotoxin-␤ signaling but is dispensable for IL-1 and tumor necrosis factor-␣ signaling (21,22). In light of these contrasting results, it remains an open question whether a NIK-like molecule (not NIK itself) is involved in the IL-1 signaling pathway. Activated IB kinase phosphorylates the inhibitory IB proteins, which are then degraded, releasing NFB, which translocates to the nucleus, where it activates transcription (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). Activated TAK1 has also been implicated in the IL-1-induced activation of c-Jun N-terminal kinase (JNK) and other mitogen-activated protein kinases (18), leading to the phosphorylation and activation of activating transcription factor and activating protein 1, thereby also activating transcription.
By taking a genetic approach to study IL-1 signaling pathways, we used random mutagenesis to generate IL-1-unresponsive cell lines lacking specific components of the pathways, and we have isolated the mutant cell line I1A, which lacks both IRAK protein and mRNA (14,15). IRAK is a multidomain protein (12,17) containing an N-terminal death domain (DD, residues 1-103), followed by a domain of unknown function (UD, residues 104 -198), a kinase domain (KD, residues 199 -522), and a two-part C-terminal domain, also of unknown function (residues 523-618 for C1 and residues 619 -712 for C2). IRAK-deficient I1A cells have been used effectively to study the function of IRAK in IL-1-dependent signaling (14,15). Neither NFB nor JNK is activated in IL-1-treated I1A cells, but these responses are restored in I1A-IRAK cells, indicating that IRAK is required for both. To address the role of the kinase activity of IRAK, its ATP-binding site was mutated (K239A) or the entire kinase domain was deleted (dKD). IRAK-K239A and IRAK-dKD complemented all defects in IL-1-dependent signaling in mutant I1A cells, indicating that the kinase activity of IRAK is not required for these functions (14,15). K239A and dKD were still phosphorylated in I1A cells upon IL-1 stimulation, showing that IRAK must become phosphorylated by another kinase.
Analysis of other IRAK deletion mutants showed that both the DD and C1C2 domains of IRAK are required for both NFB and JNK activation but that the UD domain was necessary only for NFB, not JNK, activation (15). Furthermore, IL-1-induced phosphorylation of IRAK is required to activate NFB but not JNK (15). Taken together, these results strongly suggest that the IL-1-induced signaling pathways leading to NFB and JNK activation diverge at or upstream of IRAK.
It is clear that IRAK plays a critical role in IL-1 signaling. We now address the mechanisms of the IRAK-mediated activation of the downstream signaling proteins TRAF6, TAK1, TAB1, and TAB2. Previous studies with IRAK-deficient I1A cells have revealed that IRAK is required for the activation of TAK1, the translocation of TAB2 from the membrane to the cytosol upon IL-1 stimulation, and the formation of a TRAF6-TAK1-TAB2 complex (34). Here, we report that stimulation with IL-1 also leads to the translocation of TRAF6 from the membrane to the cytosol and that this process requires IRAK. We have identified the domains of IRAK required for the translocation of TAB2 and TRAF6, and we propose that IRAK mediates those events separately through protein-protein interactions that are modulated by phosphorylation.
Recombinant Plasmids and Transfection-IRAK deletion fragments, generated by polymerase chain reaction using IRAK-K239A as a template, were cloned into an expression vector (15) driven by the thymidine kinase gene promoter.
For stable transfections, 2 ϫ 10 5 cells seeded onto a 10-cm plate were co-transfected the following day by the calcium phosphate method with 10 g of each expression vector and 1 g of pBabePuro. After 48 h, the cells were selected with 1 g/ml puromycin until clones appeared.
Co-immunoprecipitation and Immunoblotting-Samples were separated by SDS-PAGE, and Western blot transfers were analyzed with different antibodies. Membrane fractions used for immunoprecipitation experiments were lysed in lysis buffer containing 0.5% Triton X-100, 20 mM HEPES, pH 7.4, 150 mM NaCl, 12.5 mM ␤-glycerophosphate, 1.5 mM Cell extracts were incubated with 1 l of anti-IRAK or anti-TRAF6 polyclonal antibody (from Zhaodan Cao, Tularik, South San Francisco, CA) or preimmune serum for 4 h, followed by a 1-h incubation with 50 l of packed protein A-Sepharose beads (pre-washed and resuspended in phosphate-buffered saline at a 1:1 ratio). After incubation, the beads were washed five times with lysis buffer, followed by Western analysis with anti-IRAK or anti-TRAF6 antibodies.

IL-1 Induces the Translocation of TRAF6 from the Membrane
to the Cytosol-It has been reported that IL-1 stimulates translocation of TAB2 from the membrane to the cytosol, where it mediates the IL-1-dependent association of TAK1 with TRAF6 (19). We found that TRAF6 also translocates from the membrane to the cytosol upon IL-1 stimulation. To examine the subcellular distribution of the IL-1 signaling proteins, wildtype 293 cells, untreated or treated with IL-1, were fractionated into membranes and cytosol, which were then analyzed with antibodies against IRAK, TRAF6, TAK1, TAB1, and TAB2. The IL-1 receptor and ␣-tubulin were used as markers for the membrane and cytosol fractions, respectively. Whereas unmodified IRAK was found in both membrane and cytosol fractions before IL-1 treatment, the phosphorylated and ubiquitinated IRAK induced by stimulation with IL-1 was found only in the membrane fraction (Fig. 1A). The IL-1-induced phosphorylation of IRAK was confirmed by [ 32 P]orthophos-phate labeling of the wild-type 293 cells untreated or treated with IL-1, followed by immunoprecipitation with anti-IRAK antibody and analysis by SDS-PAGE (Fig. 1B). Since the IRAK deletion mutant dUD, lacking the N-proximal undetermined region, is not phosphorylated upon IL-1 stimulation, IRAKdeficient I1A cells transfected with dUD were used as a negative control (Fig. 1B). To confirm that IRAK is ubiquitinated upon IL-1 stimulation, cell extracts from 293 cells untreated or treated with IL-1 were immunoprecipitated with anti-IRAK antibody followed by Western analysis with anti-ubiquitin antibody (Fig. 1C). As shown in Fig. 1A, IRAK gradually disappeared from the cytosol fraction after stimulation with IL-1; this change probably resulted from the signal-induced modification of IRAK and its membrane localization after being modified. The modified IRAK in the membrane fraction was eventually degraded 6 h after stimulation. 2 TAB2 and TRAF6, mostly in the membrane fraction in untreated cells, translocated to the cytosol upon stimulation with IL-1 (Fig. 1A). The Scion Image 1.62C program was used to quantitate the amount of TRAF6 and TAB2 in the cytosol before and after IL-1 stimulation. From the averages of five experiments, we observed a 5.6-fold increase in TRAF6 and an 8.5-fold increase in TAB2 in the cytosol 30 min after treatment with IL-1. TAK1 and TAB1, on the other hand, were found mostly in the cytosol before and after stimulation (Fig. 1A).
Domains of IRAK Required for the Translocation of TAB2 and TRAF6 from the Membrane to the Cytosol-By using I1A cells, IRAK was shown to be required for the translocation of TAB2 from the membrane to the cytosol (34). As shown above, IL-1 also induced the translocation of TRAF6, and similar to findings with TAB2, the TRAF6 translocation was abolished in 2 Z. Jiang, Y. Qian, and X. Li, unpublished data.

FIG. 2. IRAK is required for the IL-1-induced translocation of TRAF6 and TAB2.
Membrane and cytosolic fractions were prepared from untreated or IL-1-treated (100 IU/ml) wild-type 293 cells, IRAKnull I1A cells, or I1A cells transfected with wild-type IRAK. The fractions were probed with antibodies against TRAF6 and TAB2 after Western blot transfer. ␤-Catenin and ␣-tubulin were used as controls for the membrane and cytosolic fractions, respectively. (Both IL-1R1 and ␤-catenin behaved the same as markers for the membrane fractions. ␤-Catenin was used in this experiment.) This experiment was repeated four times with consistent results. P100, particulate fraction; S100, soluble fraction.

FIG. 3. Domains of IRAK required for the IL-1-induced translocation of TRAF6 and TAB2.
Membrane and cytosolic fractions were prepared from untreated or IL-1-treated (100 IU/ml) I1A cells transfected with IRAK-K239A or IRAK deletion mutants. These fractions were probed with antibodies against TRAF6 and TAB2 after Western blot transfer. This experiment was repeated three times with consistent results. P100, particulate fraction; S100, soluble fraction.
I1A cells (Fig. 2). Overexpression of wild-type IRAK or IRAK-K239A in I1A cells resulted in some constitutive TRAF6 translocation. Stimulation with IL-1 induced this translocation further (Figs. 2 and 3), indicating that the kinase activity of IRAK is not important for the translocation of TRAF6.
To identify which domains of IRAK are required for the translocation of TRAF6 and TAB2, we studied the subcellular distribution of these proteins in I1A cells transfected with IRAK deletion constructs. Previous studies (14) have shown that overexpression of wild-type IRAK in transfected I1A cells leads to its autophosphorylation, whereas IRAK-K239A is only phosphorylated upon stimulation. The deletion mutant lacking the kinase domain behaved like IRAK-K239A in mediating the translocation of TRAF6 and TAB2 (data not shown). To avoid possible complications from autophosphorylation, the other IRAK deletion constructs were generated from IRAK-K239A. Deletion of DD or C1C2 abolished the ability of IRAK to mediate both the IL-1-induced translocation of TRAF6 and TAB2; deletion of UD was only that of TRAF6 (Fig. 3). The IL-1induced translocation of TAB2 was not affected in I1A-dUD cells (Fig. 3). These results show that different regions of IRAK are required for the translocation of TRAF6 and TAB2, suggesting that IRAK mediates their translocation by separate paths.
Formation of the IRAK-TRAF6 Complex at the Membrane and the TRAF6-TAK1-TAB1-TAB2 Complex in the Cytosol-How does IRAK mediate the translocation of TRAF6? Upon stimulation with IL-1, IRAK was phosphorylated and the modified IRAK was found only in the membrane fraction (Fig. 1). TRAF6 was found mostly in the membrane fraction before stimulation and was translocated to the cytosol after IL-1 treat-ment. Furthermore, previous studies (13) have shown that IRAK interacts with TRAF6 upon IL-1 stimulation. Therefore, it is possible that phosphorylated IRAK, induced by IL-1-stimulation, interacts with TRAF6 at the membrane to facilitate the translocation of TRAF6 to the cytosol, where TRAF6 interacts with downstream proteins.
To test this model, membrane fractions prepared from wild-

FIG. 4. IL-1-induced interaction of TRAF6 and IRAK.
A, interaction of TRAF6 and IRAK at the membrane. The P100 fractions prepared from untreated or IL-1-treated (100 IU/ml) wild-type 293 cells were resuspended in Triton lysis buffer, immunoprecipitated with anti-IRAK, and probed with anti-TRAF6, or vice versa. This experiment was repeated four times with the same results. B, interaction of TRAF6 with TAB2, TAK1, and TAB1 in the cytosol. The S100 fractions were prepared from untreated or IL-1-treated (100 IU/ml) wild-type 293 cells and immunoprecipitated with anti-TRAF6 and probed with antibodies against TAB2, TAK1, TAB1, IRAK, and TRAF6 after Western blot transfer. This experiment was repeated three times with very similar results. P-IRAK, phosphorylated IRAK; U-IRAK, ubiquitinated IRAK; IP, immunoprecipitation; WB, Western blot. type cells untreated or treated with IL-1 were resuspended in lysis buffer, immunoprecipitated with anti-IRAK, and probed with anti-TRAF6 or vice versa. An IRAK-TRAF6 complex was indeed formed in these membrane fractions after IL-1 stimulation (Fig. 4A). Cytosolic fractions were also obtained from wild-type cells untreated or treated with IL-1. Anti-TRAF6 was used to immunoprecipitate these cytosolic fractions, which were analyzed with antibodies against TAB2, TAK1, TAB1, and IRAK after Western blot transfer. TRAF6 formed a complex with TAB2, TAK1, and TAB1 in the IL-1-treated cytosolic fractions (Fig. 4B). IRAK was not detected in the cytosol, indicating that the IRAK-TRAF6 complex dissociates before TRAF6 is translocated. The majority of the IRAK-TRAF6 complex was dissociated at 10 min after IL-1 stimulation (Fig. 4A), which correlates with the time of translocation of TRAF6 to the cytosol (Fig. 1A) and formation of the TRAF6-TAK1-TAB1-TAB2 complex in the cytosol (Fig. 4B).
We then examined whether the IL-1-induced degradation of IRAK plays a role in the dissociation of the IRAK-TRAF6 complex and the subsequent translocation of TRAF6 to the cytosol. Wild-type 293 cells were treated with IL-1 for various times (10 min to 4 h) with or without the presence of protease inhibitor ALLM, followed by Western blot analysis with an anti-IRAK antibody. As shown in Fig. 5A, ALLM inhibited the IL-1-induced degradation of IRAK. However, the inhibition of IRAK degradation had no effect on the IL-1-induced formation or dissociation of the IRAK-TRAF6 complex (data not shown). Likewise, treatment with ALLM did not affect the IL-1-induced IRAK-mediated translocation of TAB2 and TRAF6 (Fig. 5B). These results suggest that the release of TRAF6 and TAB2 from IRAK is probably not through the degradation of IRAK.
IRAK Is Required for the Phosphorylation of TAK1, TAB1, and TAB2-Stimulation with IL-1 leads to the activation of TAK1 (18) and to the phosphorylation of TAK1, TAB1, and TAB2 (19,35). The activation of TAK1 leads to the autophosphorylation of TAK1 and TAB1, but a protein kinase that functions upstream of TAK1 is required for the phosphorylation of TAB2 (19). We have recently shown that IRAK is required for the IL-1-induced activation of TAK1 (34). In the current study, we examined the additional role of IRAK in the IL-1-induced activation of TAK1 and phosphorylation of TAK1, TAB1, and TAB2. The IL-1-induced TAK1 kinase activity was abolished in IRAK-null I1A cells (Fig. 6A). Although TAK1 was constitutively activated in I1A cells transfected with wild-type IRAK, it was only activated upon stimulation in I1A cells transfected with the IRAK kinase-dead mutant (K293A). Western blot analysis revealed that the TAK1, TAB1, and TAB2 proteins from wild-type cells migrated more slowly in SDS-PAGE when cells were treated with IL-1, indicating that they are phosphorylated (Fig. 6B) (19,35). The IL-1-induced phosphorylation of these proteins was abolished in IRAK-null I1A cells (Fig. 6B). Interestingly, whereas TAK1, TAB1, and TAB2 were phosphorylated constitutively in I1A cells transfected with wild-type IRAK, they were also phosphorylated in I1A cells transfected with the kinase-dead K239A upon IL-1 stimulation (Fig. 6B). These results show that, although IRAK is required for the IL-1-induced activation of TAK1 and phosphorylation of TAK1, TAB1, and TAB2, the kinase activity of IRAK is not required for these events. Therefore, IRAK is not the kinase that phosphorylates TAB2.
We then investigated the cause for the constitutive activation of TAK1 and phosphorylation of TAK1, TAB1, and TAB2 in I1A-IRAK cells. IRAK was constitutively autophosphorylated in I1A-IRAK cells, whereas IRAK-K239A was only phosphorylated upon IL-1 stimulation (Fig. 6B). The phosphorylated forms of IRAK were confirmed by both [ 32 P]orthophosphate labeling (Fig. 1B) and treatment with calf intestinal phosphatase (14). The phosphorylated IRAK in I1A-IRAK cells was capable of forming a constitutive complex with TRAF6, whereas IRAK-K239A only formed a complex with TRAF6 upon IL-1 stimulation (Fig. 7). Therefore, it is possible that the phosphorylated IRAK is responsible for the constitutive phosphorylation of TAK1, TAB1, and TAB2 in I1A-IRAK cells through its interaction with TRAF6, thereby activating downstream signaling events.
Domains of IRAK Required for the Phosphorylation of TAK1, TAB1, and TAB2-We examined the phosphorylation of TAK1, TAB1, and TAB2 in I1A cells transfected with different IRAK deletion constructs. Since the expression of wild-type IRAK in I1A cells leads to the constitutive phosphorylation of TAK1, TAB1, and TAB2, deletion constructs derived from the IRAK-K239A mutant were used. When we tested for IL-1-induced phosphorylation of TAK1, TAB1, and TAB2 in the transfected I1A cells, we found that deletion of the DD, UD, or C1C2 regions completely abolished it, whereas deletion of the kinase domain of IRAK had no effect (Fig. 8). Therefore, although the kinase domain of IRAK is dispensable, both the N-terminal and C-terminal regions are required for the activation of TAK1, TAB1, and TAB2. This conclusion was supported further by results obtained with the truncated protein DD ϩ UD ϩ C1, in which the N-terminal domains (DD and UD) were fused with a part of the C-terminal region (C1). DD ϩ UD ϩ C1 behaved like full-length IRAK-K239A in conferring IL-1-induced phosphorylation of TAK1, TAB1, and TAB2 in transfected I1A cells (Fig. 8).
As summarized in Fig. 9, the IRAK deletion mutants that confer IL-1-induced phosphorylation of TAK1, TAB1, and TAB2 in transfected I1A cells are themselves phosphorylated upon IL-1 stimulation (Fig. 8) and form a signal-dependent complex with TRAF6 (15), confirming the correlation between IRAK phosphorylation and phosphorylation of TAK1, TAB1, and TAB2. Fig. 9 also shows that the domains of IRAK (DD, UD, and C1C2) that are required for the IL-1-induced phosphorylation of TAK1, TAB1, and TAB2 are also necessary for the IL-1-induced activation of NFB (15), indicating that the phosphorylation of TAK1, TAB1, and TAB2 correlates with NFB activation. However, the deletion mutant dUD, which fails to activate NFB, TAK1, TAB1, and TAB2, still activates JNK (15) (Fig. 9), implying that the activation of TAK1, TAB1, and TAB2 is not necessary for the IL-1-induced activation of JNK.

DISCUSSION
According to current models (10 -13, 19), upon stimulation with IL-1, IRAK is recruited to the receptor complex, where it is phosphorylated. Phosphorylated IRAK then dissociates from the receptor, followed by its interaction with TRAF6 to activate the downstream signaling components. As summarized in Fig.  10, we report here that phosphorylated IRAK, induced by IL-1 treatment, stays at the membrane, where it forms a complex with TRAF6; TRAF6 then dissociates and translocates to the cytosol to form a complex with TAK1-TAB1-TAB2. Previous studies (34) have indicated that IRAK also mediates the IL-1induced translocation of TAB2 from the membrane to the cytosol. We found that different regions of IRAK are required for the translocation of TAB2 and TRAF6, suggesting that IRAK mediates these two events separately. The domains of IRAK required for the IL-1-induced translocation of TRAF6 are the same as those necessary for the IL-1-induced phosphorylation of TAK1, TAB1, and TAB2 and activation of NFB (15) (Fig. 9), revealing a tight correlation between the translocation of TRAF6 and the activation of downstream signaling. It is likely that both TAB2 and TRAF6 need to be translocated to the cytosol for the TRAF6-TAK1-TAB1-TAB2 complex to form and for these proteins to be activated. Furthermore, our studies show that the phosphorylation of TAK1, TAB1, and TAB2 correlates tightly with IL-1-induced NFB activation but is not necessary for JNK activation. We propose that IRAK mediates the IL-1-induced translocation of TRAF6 and TAB2, which leads to the formation of the TRAF6-TAK1-TAB1-TAB2 complex in the cytosol, the activation of TAK1, and the phosphorylation of TAK1, TAB1, and TAB2, resulting eventually in IL-1-induced NFB activation (Fig. 10). At present, it is still not clear exactly how IRAK mediates the translocation of TRAF6 and TAB2. The translocation is likely to occur through the interaction of IRAK with and dissociation from these two proteins. The phosphorylation of IRAK seems to play an important role in its interaction with them. In wildtype cells, both TAB2 and TRAF6 interact with phosphorylated IRAK upon IL-1 stimulation (12,32). Furthermore, the autophosphorylation of IRAK in untreated I1A-IRAK cells leads to its constitutive interaction with TRAF6 (Fig. 7). Therefore, it is possible that TRAF6 forms a transient complex with phosphorylated IRAK at the membrane upon stimulation. Through its interaction with IRAK, TRAF6 may undergo a conformational change, leading to its dissociation from IRAK, translocation to the cytosol, and formation of the TRAF6-TAK1-TAB1-TAB2 complex. Further modification of IRAK or TRAF6 might be required for the conformational change of the TRAF6-IRAK complex and the dissociation of TRAF6 from IRAK.
We tested whether IL-1-induced IRAK degradation plays any role in the release of TRAF6 and TAB2 to the cytosol. We found that ALLM inhibited IRAK degradation, but it did not affect the dissociation of TRAF6 from IRAK nor the translocation of TRAF6 and TAB2 to the cytosol. Therefore, the IL-1induced and IRAK-mediated translocation of TRAF6 and TAB2 probably does not occur through the degradation of IRAK.
We have identified which domains of IRAK are required for the IL-1-induced translocation of TAB2 and TRAF6. The Nterminal (DD) and C-terminal domains (C1C2) of IRAK are required for both TAB2 and TRAF6 translocation. However, the N-proximal undetermined domain (UD) is required for TRAF6, but not TAB2, translocation (Fig. 3). These results suggest that IRAK mediates the translocation of TRAF6 and TAB2 separately. As proposed in our model (Fig. 10), IRAK might interact with TAB2 and TRAF6 simultaneously through its different domains to form a TAB2-IRAK-TRAF6 complex at the membrane, which would subsequently allow their independent disassociation from IRAK and separate translocation to the cytosol. Alternatively, IRAK might form distinct complexes with TAB2 and TRAF6, thereby mediating their translocation individually.
TAK1 and TAB1 were localized primarily in the cytosol with or without stimulation, but small fractions of TAK1 and TAB1 were also detected in the membrane (Fig. 1A). Although the TRAF6-TAK1-TAB1-TAB2 complex was detected in the cytosol (Fig. 4B), we could not exclude the possibility that this complex first forms at the membrane with IRAK and then dissociates from IRAK and translocates to the cytosol.
Previous studies (18) have shown that the overexpression of TAK1 led to the activation of both NFB and JNK. Furthermore, a dominant-negative mutant of TAK1 blocked the IL-1induced activation of both NFB and JNK (18). Based on these studies, it was concluded that TAK1 is required for both IL-1induced NFB and JNK activation. We found that the domains of IRAK required for the IL-1-induced phosphorylation of TAK1, TAB1, and TAB2 are also required for the IL-1-induced activation of NFB (15, Fig. 9), indicating a tight correlation between the phosphorylation of these three molecules and NFB activation. However, the deletion mutant dUD, which failed to activate NFB and to support the IL-1-induced phosphorylation of TAK1, TAB1, and TAB2, still permitted IL-1induced JNK activation in transfected I1A cells (15, Fig. 9), suggesting that the activation of TAK1, TAB1, and TAB2 is not necessary for the IL-1-induced activation of JNK. Supporting this conclusion, we have recently found (15) that the IL-1induced phosphorylation of IRAK correlates strongly with the activation of NFB but is not necessary for the activation of JNK. Therefore, the IL-1 signaling pathways leading to NFB and JNK activation bifurcate at or upstream of IRAK and upstream of TAK1, TAB1, and TAB2. Our results suggest that the IRAK-mediated activation of TAK1, TAB1, and TAB2 is required for the IL-1-induced activation of NFB but not of JNK. Analysis of tak1, tab1, and tab2 gene knock-out mice and cell lines derived from them will further elucidate the roles of these proteins in the IL-1 signaling pathway.