Portal de Periódicos da CAPES

Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling

2003; Springer Nature; Volume: 22; Issue: 23 Linguagem: Inglês

10.1093/emboj/cdg605

1460-2075

Tohru Ishitani, Giichi Takaesu, Jun Ninomiya‐Tsuji, Hiroshi Shibuyà, Richard B. Gaynor, Kunihiro Matsumoto,

interferon and immune responses

Article1 December 2003free access Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling Tohru Ishitani Tohru Ishitani Department of Molecular Biology, Graduate School of Science, Institute for Advanced Research, Nagoya University, and CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, 464-8602 Japan Search for more papers by this author Giichi Takaesu Giichi Takaesu Division of Hematology-Oncology, Department of Medicine, Harold Simmons Cancer Center, University of Texas, Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390-8594 USA Search for more papers by this author Jun Ninomiya-Tsuji Jun Ninomiya-Tsuji Department of Molecular Biology, Graduate School of Science, Institute for Advanced Research, Nagoya University, and CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, 464-8602 Japan Search for more papers by this author Hiroshi Shibuya Hiroshi Shibuya Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, 101-0062 Japan Search for more papers by this author Richard B. Gaynor Richard B. Gaynor Division of Hematology-Oncology, Department of Medicine, Harold Simmons Cancer Center, University of Texas, Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390-8594 USA Search for more papers by this author Kunihiro Matsumoto Corresponding Author Kunihiro Matsumoto Department of Molecular Biology, Graduate School of Science, Institute for Advanced Research, Nagoya University, and CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, 464-8602 Japan Search for more papers by this author Tohru Ishitani Tohru Ishitani Department of Molecular Biology, Graduate School of Science, Institute for Advanced Research, Nagoya University, and CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, 464-8602 Japan Search for more papers by this author Giichi Takaesu Giichi Takaesu Division of Hematology-Oncology, Department of Medicine, Harold Simmons Cancer Center, University of Texas, Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390-8594 USA Search for more papers by this author Jun Ninomiya-Tsuji Jun Ninomiya-Tsuji Department of Molecular Biology, Graduate School of Science, Institute for Advanced Research, Nagoya University, and CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, 464-8602 Japan Search for more papers by this author Hiroshi Shibuya Hiroshi Shibuya Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, 101-0062 Japan Search for more papers by this author Richard B. Gaynor Richard B. Gaynor Division of Hematology-Oncology, Department of Medicine, Harold Simmons Cancer Center, University of Texas, Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390-8594 USA Search for more papers by this author Kunihiro Matsumoto Corresponding Author Kunihiro Matsumoto Department of Molecular Biology, Graduate School of Science, Institute for Advanced Research, Nagoya University, and CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, 464-8602 Japan Search for more papers by this author Author Information Tohru Ishitani1, Giichi Takaesu2, Jun Ninomiya-Tsuji1, Hiroshi Shibuya3, Richard B. Gaynor2 and Kunihiro Matsumoto 1 1Department of Molecular Biology, Graduate School of Science, Institute for Advanced Research, Nagoya University, and CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, 464-8602 Japan 2Division of Hematology-Oncology, Department of Medicine, Harold Simmons Cancer Center, University of Texas, Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390-8594 USA 3Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, 101-0062 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:6277-6288https://doi.org/10.1093/emboj/cdg605 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The cytokines IL-1 and TNF induce expression of a series of genes that regulate inflammation through activation of NF-κB signal transduction pathways. TAK1, a MAPKKK, is critical for both IL-1- and TNF-induced activation of the NF-κB pathway. TAB2, a TAK1-binding protein, is involved in IL-1-induced NF-κB activation by physically linking TAK1 to TRAF6. However, IL-1-induced activation of NF-κB is not impaired in TAB2-deficient embryonic fibroblasts. Here we report the identification and characterization of a novel protein designated TAB3, a TAB2-like molecule that associates with TAK1 and can activate NF-κB similar to TAB2. Endogenous TAB3 interacts with TRAF6 and TRAF2 in an IL-1- and a TNF-dependent manner, respectively. Further more, IL-1 signaling leads to the ubiquitination of TAB2 and TAB3 through TRAF6. Cotransfection of siRNAs directed against both TAB2 and TAB3 inhibit both IL-1- and TNF-induced activation of TAK1 and NF-κB. These results suggest that TAB2 and TAB3 function redundantly as mediators of TAK1 activation in IL-1 and TNF signal transduction. Introduction The pro-inflammatory cytokines IL-1 and TNF have several effects in the inflammation process. Stimulation of cells with IL-1 or TNF initiates a cascade of signaling events, including activation of NF-κB and mitogen-activated protein kinases (MAPKs) such as JNK and p38. These, in turn, upregulate the expression of many pro-inflammatory genes in the nucleus (Dinarello, 1996; Baud and Karin, 2001). NF-κB is normally sequestered in the cytoplasm of resting cells by association with inhibitory IκB proteins. This interaction masks the nuclear localization signal of NF-κB, preventing its nuclear translocation (Ghosh et al., 1998; Karin and Ben-Neriah, 2000; Li and Verma, 2002). Stimulation by IL-1 or TNF results in the phosphorylation of the IκB proteins, tagging them for ubiquitination and subsequent proteosome-mediated degradation. This results in the release of NF-κB, which translocates to the nucleus where it activates the transcription of specific target genes (Karin and Ben-Neriah, 2000; Li and Verma, 2002). Phosphorylation of IκB in response to extracellular stimuli is carried out by the IκB kinase (IKK) complex, which is comprised of two catalytic subunits, IKKα and IKKβ, as well as the modulator NEMO/IKKγ (Silverman and Maniatis, 2001; Ghosh and Karin 2002; Li and Verma, 2002). Members of the TNF-receptor-associated factor (TRAF) family of adaptor proteins are involved in coupling stimulation of the TNF receptors (TNFRs) and the IL-1 receptor (IL-1R) to NF-κB activation and other downstream events (Silverman and Maniatis, 2001). TRAF2 plays a critical role in signal transduction mediated by both TNFR1 and TNFR2, and has been implicated in TNF-induced activation of NF-κB and MAPKs (Yeh et al., 1997). Similarly, TRAF6 is important for the transduction of IL-1-induced signals, including those resulting in NF-κB and MAPK activation (Cao et al., 1996; Lomaga et al., 1999; Naito et al., 1999). The TRAF proteins physically and functionally connect TNFRs and IL-1R to intracellular protein kinases, thereby linking these receptors to downstream signaling pathways. TAK1, a member of the MAPKKK family, also participates in the IL-1-mediated signaling pathway (Yamaguchi et al., 1995; Ninomiya-Tsuji et al., 1999). Following exposure of cells to IL-1, endogenous TAK1 is recruited to the TRAF6 complex and activated, whereupon it stimulates NF-κB and MAPK activation. In previous studies, the yeast two-hybrid system was employed to isolate TAB1 and TAB2 proteins that interact with TAK1 (Shibuya et al., 1996; Takaesu et al., 2000). TAB1 was found to augment the kinase activity of TAK1 when coexpressed (Shibuya et al., 1996), indicating that it functions as an activator of TAK1. TAB2 was shown to be an intermediate in the IL-1 signaling pathway (Takaesu et al., 2000, 2001). TAB2 functions as an adaptor that links TAK1 and TRAF6 in response to IL-1 and thereby mediates TAK1 activation. These results suggest that IL-1 activation of the NF-κB and MAPK cascades involves the formation of a TRAF6–TAB2–TAK1 complex. In addition, a biochemical study has identified TRIKA1 and TRIKA2 as signaling components that are able to activate the IKK complex in a TRAF6-dependent manner (Deng et al., 2000; Wang et al., 2001). TRIKA1 consists of the ubiquitin-conjugating enzyme Ubc13 and the Ubc-like protein Uev1A, while TRIKA2 is a ternary complex composed of TAK1, TAB1 and TAB2. Thus, TRAF6-mediated ubiquitination appears to play an important role in TAK1 activation. Previously, in order to explore the physiological importance of TAK1 in activating the NF-κB pathway in response to IL-1, we utilized small interfering RNA (siRNA) directed against TAK1 (Takaesu et al., 2003). Our previous studies confirmed that TAK1 is critical for IL-1-induced activation of the NF-κB pathway. Furthermore, our results indicated that TAK1 is also important for NF-κB activation in response to TNF via the inducible association of TAK1 with TRAF2 (Takaesu et al., 2003). On the other hand, recent studies have demonstrated that mouse embryo fibroblasts (MEFs) deficient in TAB2 exhibit normal IL-1- and TNF-induced activation of NF-κB (Sanjo et al., 2003). This result indicates that TAB2 is not essential for IL-1 or TNF signaling in MEFs. However, there remains the possibility that another TAB2-like molecule may compensate for the loss of TAB2 and support TAK1 kinase activity. Here we describe the identification and characterization of TAB3, a novel TAK1-binding protein that is closely related to TAB2 in structure. TAB3 activates TAK1 and mediates its interaction with TRAF2 and TRAF6. TAB3 rapidly and transiently associates with TRAF6 and TRAF2 in an IL-1- and a TNF-dependent manner, respectively. IL-1 stimulation and TRAF6 overexpression induce ubiquitination of the TAB2 and TAB3 proteins. In this study, we utilized siRNA directed against TAB2 and TAB3 to further explore their roles in activation of the NF-κB and MAPK pathways following treatment of cells with IL-1 and TNF. These studies demonstrate that TAB2 and TAB3 are important for NF-κB and MAPK activation in response to IL-1 and TNF, and suggest that TAB2 and TAB3 have redundant functions as mediators of TAK1 activation in IL-1 and TNF signal transduction. Results Isolation of TAB3 In an attempt to identify new signal transducers involved in TAK1 activation in the IL-1 and TNF signaling pathways, we searched EST databases for sequences similar to TAB2. Multiple human and murine EST sequences were found which encode polypeptides sharing significant homology with TAB2. We cloned one full-length human TAB2 homolog by PCR from a human kidney cDNA library. This human TAB2-like gene, termed TAB3, was predicted to encode a 712 amino acid polypeptide (Figure 1A). Similar to TAB2, TAB3 contains a ubiquitin-binding motif (Shih et al., 2003) near its N-terminus and is predicted to encode an α-helical coiled-coil region in its C-terminus. Thus TAB3 is structurally related to TAB2. We also found two highly conserved homologs of TAB3 in Xenopus laevis. While this manuscript was in preparation, one of the homologs was reported by Munoz-Sanjuan and coworkers (Munoz-Sanjuan et al., 2002). Both of the Xenopus cDNAs resemble TAB3 more closely than TAB2. These workers (Munoz-Sanjuan et al., 2002) also described mouse and human TAB3. Furthermore, Drosophila contains a TAB2/3-like protein carrying the ubiquitin-binding motif and the α-helical coiled-coil region in its N- and C-termini, respectively (Figure 1A). Figure 1.Structure of TAB3. (A) Comparison of amino acid sequences among hTAB3 (human), hTAB2 (human) and DTAB2 (Drosophila). They share the CUE domain (bold underline) and coiled-coil structure (box). Identical and conserved amino acids are indicated by black and gray boxes, respectively. DDBJ/EMBL/GenBank accession No. for hTAB3 is AY437560. (B) Schematic representation of various TAB3 constructs. Gray and black boxes indicate the CUE domain and coiled-coil structure, respectively. Download figure Download PowerPoint TAB3 associates with TAK1 We have shown previously that TAB2 interacts with TAK1 through the C-terminal region of TAB2, and that overexpression of TAB2 can induce TAK1 kinase activity (Takaesu et al., 2000). To examine whether TAB3 functions similarly, the interaction of TAB3 with TAK1 was investigated in mammalian cells by in vivo coprecipitation. Human 293 embryonic kidney cells were cotransfected with T7-TAB3 and HA-TAK1. Cell extracts were immunoprecipitated with anti-T7 antibody, and coprecipitated HA-TAK1 was detected by immunoblotting with anti-HA antibody. TAB3 was found to associate with TAK1 (Figure 2A, lane 2). To verify that TAK1 associates with the C-terminal region of TAB3 in mammalian cells, we used two truncated proteins: T7-TAB3N, consisting of the N-terminus of TAB3 (amino acids 1–392), and T7-TAB3C, consisting of the C-terminus (amino acids 393–712) (Figure 1B). Immune complex assays showed that TAK1 coimmunoprecipitated with T7-TAB3C (Figure 2A, lane 4), but not with T7-TAB3N (lane 3). The C-terminal domain of TAB3 contains a coiled-coil structure (Figure 1A). To examine whether this coiled-coil region is involved in the interaction with TAK1, we constructed the mutant protein TAB3Δcc, which lacks this domain (amino acids 478–661) (Figure 1B). Coimmunoprecipitation analysis from cells coexpressing T7-TAB3Δcc and HA-TAK1 demonstrated that the TAB3Δcc protein failed to interact with TAK1 (Figure 2A, lane 5). These results confirm that the C-terminal coiled-coil region of TAB3 is responsible for its association with TAK1. Figure 2.TAB3 interacts with and activates TAK1. (A) Interaction of TAB3 with TAK1. The 293 cells were transfected with plasmids encoding T7-TAB3 full-length (F), T7-TAB3N (N), T7-TAB3C (C), T7-TAB3Δcc (Δcc) and HA-TAK1 as indicated. Complexes immunoprecipitated with anti-T7 antibody were immunoblotted with anti-HA or anti-T7 antibodies. Whole-cell extracts were immunoblotted with anti-HA antibody. (B and C) Activation of TAK1 and JNK by TAB3. The 293 cells were transfected with plasmids encoding HA-TAK1, HA-JNK, T7-TAB2 (2) and T7-TAB3 (3) as indicated. HA-TAK1 or HA-JNK was immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to an in vitro phosphorylation assay using bacterially expressed MKK6 (B) or GST-c-Jun (C) as an exogenous substrate. The immunoprecipitates were analyzed by immunoblotting with anti-HA antibody. (D) Effect of TAB2 on the interaction between TAK1 and TAB3. The 293 cells were transfected with plasmids encoding HA-TAB3, T7-TAB2 and Flag–TAK1 as indicated. Complexes immunoprecipitated with anti-HA antibody were immunoblotted with anti-Flag, anti-T7 or anti-HA antibodies. Whole-cell extracts were immunoblotted with anti-Flag or anti-T7 antibodies. (E) Interaction among TAK1-binding proteins. The 293 cells were transfected with plasmids encoding HA-TAB2 (2), HA-TAB3 (3), T7-TAB1 (1), T7-TAB2 (2) and T7-TAB3 (3) as indicated. Complexes immunoprecipitated with anti-T7 antibody were immunoblotted with anti-HA or anti-T7 antibodies. Whole-cell extracts were immunoblotted with anti-HA antibody. Download figure Download PowerPoint To examine whether TAB3 can induce TAK1 activation, we transfected 293 cells with HA-TAK1 in the presence or absence of the TAB3 expression vector. Transiently expressed TAK1 was immunoprecipitated using anti-HA antibody, and kinase activity was measured by in vitro kinase assay using MKK6 as a substrate. When expressed alone, TAK1 exhibited low basal kinase activity (Figure 2B, lane 1). However, coexpression of TAB3 led to a marked enhancement in TAK1 catalytic activity (lane 3), although the degree of activation was weaker than that induced by TAB2 (lane 2). Since activation of TAK1 induces JNK activation (Shirakabe et al., 1997), we analyzed the effect of ectopically expressed TAB3 on JNK activity. First, 293 cells were cotransfected with HA-JNK and TAB3 or TAB2. Then, JNK activity was determined by immunoprecipitation of JNK followed by in vitro kinase assay using GST-c-Jun protein as a substrate. We observed that TAB2 and TAB3 significantly induced activation of JNK (Figure 2C, lanes 2 and 3). Taken together, these results suggest that the function of TAB3 is similar to that of TAB2. Since TAB2 and TAB3 each interact with TAK1 via their respective C-terminal coiled-coil domain, we examined whether the bindings of TAB2 and TAB3 to TAK1 are mutually exclusive. We cotransfected 293 cells with Flag-TAK1 and HA-TAB3 in the presence or absence of T7-TAB2. We confirmed that TAK1 and TAB3 could interact in the absence of TAB2 coexpression (Figure 2D, top panel, lane 3). Coexpression of T7-TAB2 did not block the interaction of TAB3 with TAK1 (lane 4). Furthermore, T7-TAB2 coimmunoprecipitated with HA-TAB3 (second panel, lane 4), suggesting that TAB2 and TAB3 form a complex. To confirm this possibility, T7-TAB2 and HA-TAB3 were coexpressed in 293 cells and coimmunoprecipitation analysis was performed. We found that TAB2 associated with TAB3 (Figure 2E, lane 3). Furthermore, when 293 cells were cotransfected with the combination of HA-TAB3 and T7-TAB3 or HA-TAB2 and T7-TAB2, we found that TAB3 and TAB2 each self-associated (lanes 2 and 6). These results suggest that homo- and hetero-oligomers of TAB2 and TAB3 form a complex with TAK1. We failed to detect any association of TAB1 with TAB3 or TAB2 when overexpressed (lanes 4 and 7). TAB3 is involved in NF-κB activation Given the sequence homology of TAB3 to TAB2, a known activator of NF-κB (Takaesu et al., 2000), we investigated whether TAB3 might play a role in NF-κB activation. When TAB3 and an NF-κB-dependent luciferase reporter were cotransfected into 293 cells, TAB3 was found to activate the reporter gene in a dose-dependent manner (Figure 3A). Intact TAB3 was required for this activity, as truncated derivatives of TAB3 failed to induce NF-κB activity. Wild-type and mutant proteins were expressed in comparable amounts, as shown by western blot analysis (data not shown). Figure 3.TAB3 is involved in NF-κB activation pathway. (A) Effects of TAB3 on NF-κB-dependent reporter gene activity. The 293 cells were transfected with luciferase reporter plasmid (0.1 μg) and the indicated amounts of plasmids encoding TAB3 full-length (F), TAB3N (N) and TAB3C (C). After 24 h incubation, cells were harvested and luciferase activity measured. The values shown are the average of one representative experiment in which each transfection was performed in duplicate. (B) Effects of TAB3 on IL-1- and TNFα-induced NF-κB-activation. 293IL-1RI cells were transfected with luciferase reporter plasmid (0.1 μg) and the indicated amounts of plasmids encoding TAB3N (N) and TAB3C (C). IL-1β (5 ng/ml) or TNFα (10 ng/ml) was added to each plate 3 h after transfection. Cells were harvested 24 h after transfection and luciferase activity was measured. The values shown are the average of one representative experiment in which each transfection was performed in duplicate. Download figure Download PowerPoint We next tested whether TAB3 is involved in the IL-1 or TNF signaling pathway that leads to NF-κB activation. Since mutants of TAB2 lacking the C-terminus function as dominant negatives (Takaesu et al., 2000), we made a homologous truncated derivative of TAB3 (TAB3C) (Figure 1B). First, 293 cells were transfected with an NF-κB-dependent luciferase reporter and increasing concentrations of TAB3C. Later, cells were treated with IL-1 or TNF and luciferase activity was determined. Figure 3B shows that increasing concentrations of TAB3C potently inhibited induction of NF-κB by IL-1 and TNF. These results suggest that TAB3 may be a common downstream mediator of NF-κB activation by IL-1 and TNF. TAB3 associates with TRAF6 and TRAF2 IL-1 and TNF activate their signal pathways via distinct families of cell-surface receptors. However, both pathways utilize members of the TRAF family of adaptor proteins as signal transducers (Silverman and Maniatis, 2001). The TRAF proteins share homology at their C-terminal domains, but their binding properties and activities differ. For example, whereas TRAF6 is essential for IL-1 signaling (Lomaga et al., 1999; Naito et al., 1999), TRAF2 is involved in TNF signaling (Yeh et al., 1997). Recently, it was shown that TAB2 interacts specifically with TRAF6 (Takaesu et al., 2000). Therefore we tested the ability of TAB3 to bind to TRAF6 and TRAF2. T7-TAB2 or T7-TAB3 was expressed together with Flag-TRAF6 or Flag-TRAF2 in 293 cells and immunoprecipitated with anti-T7 antibody. The immune complexes were subjected to immunoblotting with anti-Flag antibody. As observed previously, TAB2 was found to interact with TRAF6 (Figure 4A, lane 2), but not with TRAF2 (Figure 4B, lane 2). In contrast, TAB3 was found to coprecipitate both TRAF6 (Figure 4A, lane 3) and TRAF2 (Figure 4B, lane 3) efficiently. This is consistent with the notion that TAB3 is involved in both the IL-1 and TNF signaling pathways. To determine whether TAB2 and TAB3 competitively bind to TRAF6, we analyzed the interaction between TAB3 and TRAF6 in the presence or absence of TAB2. When Flag-TRAF6 and HA-TAB3 were expressed together with T7-TAB2 in 293 cells, TAB2 formed a complex with TAB3 (Figure 4C, second panel, lane 4) and did not interfere with the interaction between TAB3 and TRAF6 (top panel, lane 4). These results suggest that TAB2 and TAB3 bind cooperatively, but not competitively, to TRAF6. Figure 4.TAB3 mediates the interaction of TAK1 with TRAF6 and TRAF2. (A and B) Interaction of TAB3 with TRAF6 and TRAF2. The 293 cells were transfected with plasmids encoding T7-TAB2 full-length (2F), T7-TAB3 full-length (3F), T7-TAB3N (3N), T7-TAB3C (3C), Flag-TRAF6 and Flag-TRAF2 as indicated. Complexes immunoprecipitated with anti-T7 antibody were immunoblotted with anti-Flag or anti-T7 antibodies. Whole-cell extracts were immunoblotted with anti-Flag antibody. (C) Effect of TAB2 on the interaction between TRAF6 and TAB3. The 293 cells were transfected with plasmids encoding HA-TAB3, T7-TAB2 and Flag-TRAF6 as indicated. Complexes immunoprecipitated with anti-HA antibody were immunoblotted with anti-Flag, anti-T7 or anti-HA antibodies. Whole-cell extracts were immunoblotted with anti-Flag or anti-T7 antibodies. (D) Effect of TAB3 on the interaction of TAK1 with TRAF2 and TRAF6. The 293 cells were transfected with plasmids encoding HA-TAK1, T7-TAB3 full-length (F), T7-TAB3Δcc (Δcc), Flag-TRAF2 (2) and Flag-TRAF6 (6) as indicated. Complexes immunoprecipitated with anti-Flag antibody were immunoblotted with anti-HA or anti-Flag antibodies. Whole-cell extracts were immunoblotted with anti-HA antibody. Download figure Download PowerPoint To determine the regions within TAB3 responsible for its interaction with TRAF6 and TRAF2, we performed immunoprecipitation assays using deletion mutants of TAB3. We found that TRAF6 and TRAF2 coprecipitated with TAB3C (Figure 4A and B, lane 5), but not with TAB3N (lane 4). Thus the C-terminal domain of TAB3 is required for binding to TRAF6 and TRAF2. This is similar to TAB2, which also requires its C-terminal domain for binding to TRAF6. We next tested whether the C-terminal coiled-coil region of TAB3 is essential for its interaction with TRAF6 and TRAF2. We found that the TAB3Δcc mutant (Figure 1B), which lacks the coiled-coil motif, was still able to associate with TRAF6 (see Figure 6A below) and TRAF2 (data not shown). Thus, the coiled-coil motif of TAB3 is required for its interaction with TAK1, but not with TRAF6 or TRAF2. We have previously shown that TAB2 functions as an adaptor protein mediating the association of TAK1 with TRAF6 (Takaesu et al., 2000). The ability of TAB3 to interact with both TAK1 and TRAF6 led us to hypothesize that TAB3 similarly acts as a link between TRAF6 and TAK1. To test this possibility, we analyzed the interaction between TAK1 and TRAF6 in both the presence and absence of TAB3. Although a small amount of TAK1 was found to associate with TRAF6 in the absence of exogenous TAB3 (Figure 4D, lane 5), overexpression of TAB3 strongly enhanced the association between TRAF6 and TAK1 (lane 6). This indicates that TAB3, at least when overexpressed, can link TRAF6 to TAK1. We next examined the effect of the TAB3Δcc mutant, lacking the coiled-coil motif, on TRAF6–TAK1 complex formation. As described above, we had observed that TAB3Δcc interacted with TRAF6 but not with TAK1. In the present experiment, we observed that overexpression of TAB3Δcc did not enhance the association between TRAF6 and TAK1 (lane 7). These results support the idea that TAB3 is an intermediate signaling molecule linking TAK1 and TRAF6. We were interested to determine whether the presence of TAB3 could also affect the ability of TAK1 to interact with TRAF2. To address this point, Flag-TRAF2 and HA-TAK1 were cotransfected into 293 cells with or without T7-TAB3. In the absence of TAB3 expression, no association of TAK1 with TRAF2 could be determined (Figure 4D, lane 2). However, upon coexpression of TAB3, we were able to detect the association between TAK1 and TRAF2 (lane 3). Overexpression of TAB3Δcc failed to enhance the interaction of TAK1 with TRAF2 (lane 4). Taken together, these results suggest that TAB3 functions as an adaptor for the association of TAK1 with both TRAF6 and TRAF2. Ligand-dependent endogenous interaction of TAB3 with TRAF6 and TRAF2 To evaluate the interaction of TAB3 with TRAF6 and TRAF2 under more physiological conditions, we examined the association of endogenous TAB3 with TRAF6 and TRAF2 in 293IL-1RI cells. A rabbit anti-TAB3 polyclonal antibody was generated to identify endogenous TAB3 protein. When lysates prepared from 293IL-1RI cells were subjected to immunoprecipitation followed by western blotting with anti-TAB3 antibody, one band at approximately 90 kDa was observed (Figure 5A, bottom panel, lane 10). This band was not observed when control IgG was used for immunoprecipitation (lane 9), indicating that it represents endogenous TAB3. Lysates from IL-1-treated cells were immunoprecipitated with anti-TAB3 antibody and then analyzed by immunoblotting with anti-TRAF6 antibody. We found that TRAF6 rapidly associated with TAB3 in an IL-1-dependent manner (top panel, lanes 10–12). This interaction was observed within 3 min after IL-1 treatment, and decreased thereafter. Thus the interaction between TAB3 and TRAF6 is physiologically induced by IL-1. The kinetics of IL-1-induced TRAF6–TAB3 association were similar to those for TRAF6–TAK1 (lanes 2–4) and TRAF6–TAB2 (lanes 6–8) association. TNF did not induce the interaction of TRAF6 with TAB2 or TAB3 (see Figure 6B). Thus the interaction of TRAF6 with TAB2 and TAB3 is signaled specifically and physiologically by IL-1. When TAK1 was immunoprecipitated with anti-TAK1 antibody, the TAK1 immunocomplexes were found to contain TAB2 and TAB3 even in the absence of IL-1 stimulation (Figure 5A, third and bottom panels, lane 2). In addition, we found that TAB2 and TAB3 associated constitutively (bottom panel, lane 6, and third panel, lane 10). Thus TAK1, TAB2 and TAB3 form a complex in the absence of stimulation. This is consistent with recent observations that the TRIKA2 complex containing TAK1, TAB1 and TAB2 is formed before IL-1 stimulation (Wang et al., 2001) and that TAK1, TAB1 and TAB2 are pre-associated on the membrane before stimulation (Jiang et al., 2002). Western blot analysis revealed that TAB3 proteins migrated more slowly on SDS–PAGE in cells treated with IL-1 (bottom panel, lanes 4, 8 and 12). These slowly migrating bands were eliminated by phosphatase treatment (data not shown), indicating that they may represent phosphorylated TAB3. Figure 5.Ligand-dependent association of TRAF6 and TRAF2 with TAK1, TAB2 and TAB3. 293IL-1RI cells were treated with (A) IL-1 (10 ng/ml) or (B) TNFα (10 ng/ml) for the indicated time periods. Endogenous TAK1, TAB2 and TAB3 were immunoprecipitated with anti-TAK1 (T1), anti-TAB2 (T2) and anti-TAB3 (T3) antibodies, respectively. Complexes immunoprecipitated with control IgG (C) or each antibody was immunoblotted with anti-TRAF6, anti-TRAF2, anti-TAK1, anti-TAB2 or anti-TAB3 antibodies. Whole-cell extracts were immunoblotted with anti-TRAF6 or anti-TRAF2 antibodies. Download figure Download PowerPoint Figure 6.Ubiquitination of TAB2 and TAB3. (A) Effect of TRAF2 and TRAF6 on ubiquitination of TAB2 and TAB3. 293IL-1RI cells were transfected with plasmids encoding T7-TAB2 (2), T7-TAB3 (3), T7-TAB3Δcc (Δcc), Flag-TRAF2 (2) and Flag-TRAF6 (6) as indicated. Complexes immunoprecipitated with anti-T7 antibody were immunoblotted with anti-Flag, anti-ubiquitin (anti-Ub) or anti-T7 antibodies. Whole-cell extracts were immunoblotted with anti-Flag antibody. (B) Effect of IL-1 and TNFα stimulation on ubiquitination of TAB2 and TAB3. 293IL-1RI cells were treated with IL-1 (10 ng/ml) or TNFα (20 ng/ml) for the indicated time periods. Cell extracts were subjected to immunoprecipitation with control IgG (C), anti-TAB2 (T2) or anti-TAB3 (T3) antibodies. Immunoprecipitated complexes were immunoblotted with anti-TRAF6, anti-TRAF2, anti-Ub, anti-TAB2 or anti-TAB3 antibodies. Whole-cell extracts were immunoblotted with anti-TRAF6 or anti-TRAF2 antibodies. (C) Effect of TRAF6ΔN on IL-1-induced ubiquitination of TAB2. 293IL-1RI cells were transfected with a plasmid encoding TRAF6ΔN. At