Functional Interactions of Transforming Growth Factor β-activated Kinase 1 with IκB Kinases to Stimulate NF-κB Activation*

Several mitogen-activated protein kinase kinase kinases play critical roles in nuclear factor-κB (NF-κB) activation. We recently reported that the overexpression of transforming growth factor-β-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, together with its activator TAK1-binding protein 1 (TAB1) stimulates NF-κB activation. Here we investigated the molecular mechanism of TAK1-induced NF-κB activation. Dominant negative mutants of IκB kinase (IKK) α and IKKβ inhibited TAK1-induced NF-κB activation. TAK1 activated IKKα and IKKβ in the presence of TAB1. IKKα and IKKβ were coimmunoprecipitated with TAK1 in the absence of TAB1. TAB1-induced TAK1 activation promoted the dissociation of active forms of IKKα and IKKβ from active TAK1, whereas the IKK mutants remained to interact with active TAK1. Furthermore, tumor necrosis factor-α activated endogenous TAK1, and the kinase-negative TAK1 acted as a dominant negative inhibitor against tumor necrosis factor-α-induced NF-κB activation. These results demonstrated a novel signaling pathway to NF-κB activation through TAK1 in which TAK1 may act as a regulatory kinase of IKKs.

Several mitogen-activated protein kinase kinase kinases play critical roles in nuclear factor-B (NF-B) activation. We recently reported that the overexpression of transforming growth factor-␤-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, together with its activator TAK1-binding protein 1 (TAB1) stimulates NF-B activation. Here we investigated the molecular mechanism of TAK1-induced NF-B activation. Dominant negative mutants of IB kinase (IKK) ␣ and IKK␤ inhibited TAK1induced NF-B activation. TAK1 activated IKK␣ and IKK␤ in the presence of TAB1. IKK␣ and IKK␤ were coimmunoprecipitated with TAK1 in the absence of TAB1. TAB1-induced TAK1 activation promoted the dissociation of active forms of IKK␣ and IKK␤ from active TAK1, whereas the IKK mutants remained to interact with active TAK1. Furthermore, tumor necrosis factor-␣ activated endogenous TAK1, and the kinase-negative TAK1 acted as a dominant negative inhibitor against tumor necrosis factor-␣-induced NF-B activation. These results demonstrated a novel signaling pathway to NF-B activation through TAK1 in which TAK1 may act as a regulatory kinase of IKKs.
Transcription factor nuclear factor B (NF-B) 1 is composed of homodimers and heterodimers of Rel family proteins and plays a pivotal role in the gene expression involved in inflammatory and immune responses (1)(2)(3). NF-B is sequestered in the cytoplasm by inhibitory proteins such as IB␣, IB␤, and IB⑀, which mask the nuclear localization signal of NF-B (4 -8). The phosphorylation of two Ser residues at an N-terminal regulatory domain of IB proteins triggers polyubiquitination of IB proteins, which targets them for rapid degradation through a proteasome-dependent pathway, thereby releasing NF-B to enter the nucleus (9 -15). Diverse extracellular stimuli such as tumor necrosis factor (TNF)-␣ and interleukin-1␤, phorbol esters, and environmental stresses lead to NF-B activation utilizing the common mechanism for the IB degradation, suggesting the diversity of the upstream signaling pathways for phosphorylation of IB proteins.
Several regulatory kinases involved in the signal-induced phosphorylation of IB proteins have recently been reported. Two closely related kinases designated IB kinase (IKK) ␣ and IKK␤ have been identified as components of the multiprotein IKK complex (500 -900 kDa) that directly phosphorylates the critical Ser residues of IB proteins (16 -20). Together, IKK␣ and IKK␤ form a heterodimer through their C-terminal leucine zipper motifs, and the functional IKK complex contains both IKK subunits. NF-B-inducing kinase (NIK) is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, which was first identified as a TNF-␣ receptor-associated factor (TRAF) 2-interacting protein (21). The ligand-mediated trimerization of the TNF-␣ receptor triggers the recruitment of NIK to TRAF2, and this association results in the activation of NIK, which in turn phosphorylates and activates IKKs. NIK also interacts with TRAF6, another member of the TRAF family, which is required for interleukin-1␤-induced NF-B activation (22). In addition, MAPK/extracellular signalregulated kinase kinase kinase 1 (MEKK1), another member of the MAPKKK family, stimulates NF-B activation by preferentially activating IKK␤ over IKK␣ (23)(24)(25). These findings suggest that several MAPKKKs play a key role in the NF-B activation pathway by regulating the kinase activity of the IKK complex. However, little is known about the regulatory molecular mechanisms of the kinase activity of the IKK complex induced by diverse extracellular stimuli.
Transforming growth factor (TGF) ␤-activated kinase 1 (TAK1) was first identified as a MAPKKK that can be activated by TGF-␤ and bone morphological protein (26). TAK1 activity is regulated by its activator, TAK1-binding protein 1 (TAB1) (27). TAK1 is suggested to act as a MAPKKK in the c-Jun Nterminal kinase (JNK)/stress-activated protein kinase (SAPK) and the p38 MAPK cascades, in which TAK1 phosphorylates MAPK kinase (MKK) 4, MKK3, and MKK6 (28,29). In addition, hematopoietic progenitor kinase 1 induces the activation of the JNK pathway mediated by TAK1 but not MEKK1 and mixed lineage kinase 3 (30). However, the biological role of TAK1 in the intracellular signaling pathways is poorly understood.
We recently reported that the overexpression of TAK1 together with TAB1 stimulates NF-B activation (31). In the present study, we investigated the molecular mechanisms of TAK1-induced NF-B activation. We found functional interactions of TAK1 with IKK␣ and IKK␤. In the activation of TAK1induced IKKs, two Ser residues in the activation loop of the IKKs were critically involved.

MATERIALS AND METHODS
Expression Vectors-In our previous study, three isoforms of human TAK1 cDNA were isolated (31). TAK1a is the most abundantly expressed in HeLa cells and was used in the present study. Full-length * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Cell Cultures and Transfection-HeLa cells were maintained in highglucose Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin at 37°C in 5% CO 2 . Cells were transfected with expression vectors using LipofectAMINE reagents (Life Technologies, Inc.).
Gel Shift Assay and Luciferase Assay-Twenty-four h after transfection, the cells were harvested, and gel shift assays were performed with nuclear extracts as described previously (32). Luciferase reporter gene assay was performed by using pNFB-Luc plasmid (Stratagene). pRSV-␤-gal plasmid was kindly provided by Dr. M. Tsuda (Toyama Medical and Pharmaceutical University).
MAP Kinase Assay-The JNK activity was determined by an in vitro immunocomplex kinase assay. Immunoprecipitation was carried out using an anti-JNK1 (FL) antibody (Santa Cruz Biotechnology), and a kinase assay was performed with GST-c-Jun (1-79) as a substrate using the procedure described above. The GST-c-Jun expression plasmid was kindly provided by Dr. M. Hibi (Osaka University). p38 MAPK activation was monitored by its phosphorylation status at both the Thr 180 and Tyr 182 residues by the immunoblotting of cell lysates with an antiphospho p38 antibody (New England Biolabs).
Phosphatase Treatment-Cell lysates were immunoprecipitated with the anti-Flag antibody. After washes, the beads were incubated with 2 units/l calf intestinal alkaline phosphatase (Takara) at 37°C for 30 min. Where indicated, sodium orthovanadate (1 M) was added in the reaction mixture.

NF-B Activation by TAK1-
We previously demonstrated the ability of TAK1 to activate p50/p65 NF-B in a TAB1-dependent manner (31). To investigate the molecular mechanism of TAK1-induced NF-B activation, N-terminal Flag epitope-tagged wild-type TAK1 or a kinase inactive mutant (TAK1K63W) was transiently expressed in HeLa cells. A gel shift assay showed that wild-type TAK1 together with TAB1 induced the nuclear translocation of NF-B, whereas TAK1K63W could not induce the translocation even when TAB1 was coexpressed (Fig. 1A). In contrast, the Oct-1 DNA binding activity was not affected by the overexpression of TAK1 and TAB1 (Fig. 1A). In addition, two major inhibitory proteins, IB␣ and IB␤, were degraded in cells expressing both wild-type TAK1 and TAB1 (Fig. 1B). The degradation of IB␣ was blocked by a proteasome inhibitor, N-acetyl-leucylleucyl-norleucinal (data not shown), indicating that TAK1 may activate NF-B through the ubiquitination-proteasome pathway.
Mechanism of TAK1 Activation by TAB1-TAB1 was first identified as a TAK1 activator in a yeast two-hybrid system (27). Here, we characterized the molecular mechanism of the TAK1 activation by TAB1 in mammalian cells. Flag-TAK1 and Flag-TAK1K63W were expressed with or without TAB1 in HeLa cells, and anti-Flag immunoprecipitates were analyzed for the coprecipitation of TAB1 by immunoblotting. TAB1 was coimmunoprecipitated with wild-type and kinase inactive TAK1 ( Fig. 2A). TAB1 migrated slowly on a SDS-polyacrylamide gel when coexpressed with TAK1, but not with TAK1K63W ( Fig. 2A). Wild-type TAK1, but not TAK1K63W, also migrated slowly when coexpressed with TAB1 ( Fig. 2A). In addition, TAK1 appeared to be stabilized as a consequence of the association with TAB1 ( Fig. 2A). The reduced mobility of coexpressed TAK1 and TAB1 may reflect the phosphorylation of both proteins induced by their functional interaction, as has been described for several protein kinases including interleukin 1 receptor-associated kinase (33). To investigate this possibility, an in vitro kinase assay was conducted using the anti-Flag immunoprecipitates. The phosphorylation of TAK1 and TAB1 was detected only when wild-type TAK1 and TAB1 were coexpressed (Fig. 2B). Furthermore, treatment of the immunoprecipitated TAK1/TAB1 complex with calf intestinal alkaline phosphatase converted the slower-migrating forms to the faster-migrating forms (Fig. 2C). A phosphatase inhibitor, sodium orthovanadate, blocked this mobility shift of TAK1. The mobility of TAB1 was partially reduced by the inhibitor, suggesting multiple phosphorylation sites in TAB1. These results suggest that the association of TAB1 with TAK1 causes the activation of TAK1, during which TAK1 autophosphorylation and phosphorylation of TAB1 by TAK1 may be occurring.

Involvement of IKKs in TAK1-induced NF-B Activation-
The marked degradation of IB proteins by TAK1 raises the possibility of the involvement of the IKK complex in TAK1induced NF-B activation. To investigate this possibility, the effects of dominant negative mutants of the IKKs were examined. The TAK1-induced nuclear translocation of NF-B was inhibited by the kinase inactive mutants IKK␣ (K44M) and IKK␤ (K44M) (Fig. 3A). In contrast, TAK1-induced JNK and p38 MAPK activation was not inhibited by these IKK mutants (Fig. 3B). These results suggest that TAK1-induced NF-B activation is mediated by the IKK complex, but not through the MAPK signaling cascades.
The TAK1-induced regulation of IKK kinase activity was investigated. First, the endogenous IKK kinase activity was determined by an in vitro anti-IKK␣ immunocomplex kinase assay using bacterially expressed GST-IB␣ (1-54) as a substrate. The kinase activity was significantly increased when wild-type TAK1 and TAB1 were coexpressed, whereas TAK1K63W did not enhance the IKK activity (Fig. 4). The specificity of the IKK activity was confirmed by using a mutant substrate, GST-IB␣ (1-54) (SS32 and 36AA), in which the critical Ser residues for IKKs were replaced with Ala (Fig. 4). The anti-IKK␣ antibody was able to recognize IKK␤ as well as IKK␣, suggesting that both IKK subunits contribute to the IKK activity. Similar results were obtained by an immunocomplex kinase assay using an anti-MAPK phosphatase-1 antibody (data not shown), which has been shown to precipitate the multisubunit IKK complex (17).
To further elucidate the contribution of the two IKK subunits, N-terminal Xpress epitope-tagged IKK␣ and IKK␤ were overexpressed with TAK1 and TAB1, and the kinase activities of the IKKs were measured by an anti-Xpress immunocomplex kinase assay. TAK1, but not TAK1K63W, induced the kinase activity of IKK␣ when coexpressed with TAB1 (Fig. 5A). Similarly, IKK␤ activity was enhanced by TAK1 plus TAB1, whereas IKK␤ alone showed constitutive activity (Fig. 5B). In addition, TAK1K63W slightly inhibited the constitutive IKK␤ activity (Fig. 5B). These results indicate that TAK1 acts as an activator for IKK␣ and IKK␤ in the signaling pathway of FIG. 1. NF-B activation by TAK1. HeLa cells (1 ϫ 10 6 cells/60-mm dish) were transfected with expression vectors for Flag-TAK1 (1 g) or Flag-TAK1K63W (1 g) with or without an expression vector for TAB1 (1 g). The total amount of DNA was adjusted with an empty vector at 2 g. A, 24 h after transfection, nuclear extracts were prepared, and gel shift assays were carried out with oligonucleotide probes containing a B site or an octamer binding site. B, whole cell lysates were prepared, and immunoblotting was carried out with anti-IB␣ and anti-IB␤ antibodies.

TAK1-induced NF-B activation.
Interaction of TAK1 with IKKs-NIK has been shown to directly associate with both IKKs and enhance their kinase activities (19,20). Because TAK1 is also a member of the MAPKKK family, we investigated the interaction of TAK1 with IKKs. HeLa cells were transiently transfected with the expression vectors for Flag-TAK1 or Flag-TAK1K63W with Xpress-IKK␣ and Xpress-IKK␤. Anti-Flag immunoprecipitates were analyzed for the presence of IKKs by immunoblotting with the anti-Xpress antibody. The interaction of wild-type TAK1 with IKK␣ was detected in the absence of TAB1 (Fig. 6A). However, the interaction was not detected when TAK1 was activated by TAB1 (Fig. 6A). Similarly, the interaction of TAK1 with IKK␤ was detected only in the absence of TAB1 (Fig. 6B). In contrast, interactions of NIK with both IKK␣ and IKK␤ were detected through their active forms (data not shown). TAK1K63W interacted weakly with IKK␣ and IKK␤ (Fig. 6, A and B), whereas this molecule had the potential to interact with TAB1 ( Fig. 2A). The immunoblotting of cell lysates with the anti-Xpress antibody showed that IKK␣ and IKK␤ migrated slowly on SDS-PAGE when cotransfected with both TAK1 and TAB1 (Fig. 6). These results indicate that TAK1 interacts with both IKK subunits to induce their kinase activities.
The Significant Role of Ser Residues in the Activation Loop of IKKs-Most of the interactions between activated protein kinases and phosphorylated substrates have been shown to be transient. However, a stable interaction could be detected when the kinase defective mutant or the mutated substrate that lacks the target residues for phosphorylation was used. To examine the features of TAK1-IKKs interactions, Xpresstagged IKK mutants (KM and SSAA) were coexpressed with Flag-TAK1 in HeLa cells. The coimmunoprecipitation assay showed that interactions between TAK1 and all IKK mutants were detectable in both the absence and presence of coexpressed TAB1 (Fig. 7A). These results indicate that the kinase activities of IKKs are necessary for the dissociation of TAK1 from IKKs, in which TAK1 may phosphorylate the Ser residues in the activation loop of the IKKs. In addition, the immunoblotting of cell lysates with the anti-Xpress antibody showed that all IKK mutants did not migrate slowly on SDS-PAGE even when in the presence of active TAK1, suggesting that the reduced mobility of wild-type IKKs reflects autophosphorylation. Furthermore, IKK␣-SSAA and IKK␤-SSAA acted as dominant negative inhibitors in TAK1-induced NF-B activation (Fig. 7B). These results indicate that the activation loop is critically involved in TAK1-induced IKKs activation.
Selective Depletion of TAK1 and TAB1-Interestingly, TAK1 and TAB1 appeared to be selectively depleted in cells cotransfected with IKK␤, whereas the expression of IKK␤ was not affected (Figs. 6B and 7A). Such a depletion was not observed in the presence of IKK␣ (Fig. 6A). The depletion of TAK1 and TAB1 was dependent on the kinase activities of TAK1 and IKK␤, because this was not observed in cells expressing kinasenegative mutants of TAK1 and IKK␤ (Figs. 6B and 7A). This observation may indicate a novel regulatory mechanism of TAK1 kinase activity.
TNF-␣-induced NF-B Activation through TAK1-In A673 human rhabdomyosarcoma cells, endogenous TAK1 is activated by TNF-␣ in which the TAK1 activity was measured for its ability to activate SEK1 (29). Here we investigated the effect of TNF-␣ on TAK1 activation in HeLa cells. The anti-TAK1 immunocomplex in vitro kinase assay using 6xHis-MKK6 as a substrate showed that TNF-␣ activated endogenous TAK1 transiently, and the maximal activation was observed at 2-5 min after stimulation (Fig. 8A). TAK1 activity was also detected together with its autophosphorylation and TAB1 phosphorylation (data not shown), which was similar to the data from the overexpression experiment (Fig. 2B). Interestingly, TAK1 activation was preceded by the activation of endogenous IKK complex, which was detected at 5-10 min after stimulation (Fig. 8A). In contrast, TGF-␤ did not induce TAK1 activation as well as IKK activation (Fig. 8A). These results suggest that TAK1/TAB1 might act as signal transducers of the NF-B activation pathway through the TNF-␣ receptor. To clarify this possibility, we examined the effect of kinase-negative TAK1 on TNF-␣-induced NF-B activation. TAK1K63W inhibited B-dependent luciferase gene expression (Fig. 8B). These results indicate that the TAK1/TAB1 complex plays a role in TNF-␣induced NF-B activation.
Effect of the NIK Mutant on TAK1-induced NF-B Activation-NIK plays a key role in TNF-␣-induced NF-B activation through IKK activation (19 -21). Here we further investigated the effect of the NIK mutant. A truncated mutant NIK (NIK624 -947) acted as a dominant negative inhibitor against the TNF-␣-induced NF-B activation (Fig. 9A). Furthermore, the NIK mutant partially inhibited TAK1-induced NF-B activation (Fig. 9B). DISCUSSION TAK1 was first identified as a MAPKKK that can be activated by TGF-␤ and bone morphological protein (26) and was reported to play a role in bone morphological protein signaling in early Xenopus development (34). cDNA cloning of Xenopus TAK1 revealed that the amino acid sequence of the catalytic domain is highly conserved (98%) between Xenopus TAK1 and human TAK1 (31,34). Recent studies have shown that Smad proteins are critically involved in the signaling pathway from TGF-␤ and bone morphological protein receptors (35,36). The injection of kinase-negative TAK1 mRNA into the Xenopus embryo reverses the Smad1-or Smad5-induced expression of ventral mesoderm markers, suggesting cooperation between TAK1 and Smad proteins (34). Here we demonstrated a novel function of TAK1 as an activator of the IKK complex to stimulate NF-B activation. However, little is known about the functional relationship between TGF-␤ signaling and the NF-B activation pathways. We previously reported that TGF-␤ could not induce the nuclear translocation of NF-B in HeLa cells (31). In addition, we showed that TAK1 was not activated by TGF-␤ in HeLa cells. These results suggest that  (1 g each) for Flag-TAK1, Flag-TAK1K63W, TAB1, Xpress-IKK␣, and Xpress-IKK␤. The total amount of DNA was adjusted with an empty vector at 3 g. The interactions of TAK1 with IKK␣ (A) and IKK␤ (B) were examined by coimmunoprecipitation assays. Twenty-four h after transfection, whole cell lysates were immunoprecipitated with an anti-Flag antibody and analyzed for coprecipitating IKKs by immunoblotting with an anti-Xpress antibody (top panels). The same blots were reprobed with an anti-TAK1 antibody (third panels). Similar results were obtained in the immunoblotting of lysates with an anti-Flag antibody. To monitor the expression of IKKs and TAB1, lysates were immunoblotted with anti-Xpress (second panels) or anti-TAB1 antibodies (bottom panels), respectively. TAK1 is involved in the NF-B activation pathway induced by extracellular stimuli other than TGF-␤. In this study, we demonstrated that TNF-␣ activated TAK1 to stimulate NF-B activation. It has been shown that NIK plays a significant role in TNF-␣-induced NF-B activation. Our previous study showed that NIK624 -947 did not inhibit the TAK1-induced nuclear translocation of NF-B (31). In contrast, the truncated NIK mutant inhibits TAK1-induced NF-B-dependent luciferase gene expression in human embryonal kidney 293 cells. 2 We also observed the partial dominant negative effect of the NIK mutant in HeLa cells. These results suggest that TAK1 might be a regulatory kinase of NIK. Otherwise, TAK1 may regulate IKKs directly, when the NIK mutant could interact with and inactivate endogenous IKKs. Understanding the precise functional relationship between TAK1 and NIK in TNF-␣-induced NF-B activation requires further investigation.
Hematopoietic progenitor kinase 1 is a serine/threonine kinase with restricted expression in hematopoietic tissues (37,38). It has been shown that hematopoietic progenitor kinase 1 activates the JNK pathway mediated by TAK1 (30). It is interesting to evaluate the ability of hematopoietic progenitor kinase 1 to stimulate NF-B activation through TAK1, which may present a physiological function of TAK1-induced NF-B activation in hematopoietic differentiation.
In the present study, we demonstrated that the recruitment of TAB1 to TAK1 may trigger both TAK1 autophosphorylation and phosphorylation of TAB1. The C-terminal 68 amino acids of TAB1 were shown to be sufficient for binding and activating TAK1 (27). In contrast, the N-terminal domain lacking the TAK1 binding domain acts as a dominant negative inhibitor in TGF-␤ signaling (27). In addition, the deletion of 20 amino acids from the N terminus of TAK1 renders the protein kinase constitutively active (26). These findings strongly suggest that TAK1 phosphorylates the Cterminal domain of TAB1 and the N-terminal domain of TAK1. In fact, these domains contain a Ser/Thr-rich sequence (26,27). The identification of the phosphorylation sites of TAK1 will provide more information regarding the molecular mechanism of TAK1 activation by TAB1.
The functional implications of MAPK cascades in the signaling pathways to NF-B activation have been characterized. The 90-kDa ribosomal S6 kinase (pp90 rsk ) that lies downstream of the Raf-MAPK/extracellular signal-regulated kinase pathway is involved in phorbol ester-induced NF-B activation by phosphorylating Ser 32 but not Ser 36 of IB␣ (39). MAPK cascades that are sensitive to the MAPK/extracellular signal-regulated kinase inhibitor PD098059 and the p38 MAPK inhibitor SB203580 were shown to enhance the TNF-␣-induced transactivation of the p65 NF-B subunit (40). Several MAPKKKs including NIK (21), MEKK1 (41,42), and MEKK3 (43) were recently shown to have the potential to activate NF-B. NIK and MEKK1 preferentially activate IKK␣ and IKK␤, respectively (23,44). Here we demonstrated that TAK1 is a new member of the MAPKKK family that activates IKKs. TAK1 as well as NIK interacts with both IKK␣ and IKK␤. In contrast, the interaction of MEKK1 with IKKs has not yet been demonstrated, although a MEKK1 catalytic subunit was copurified with the TNF-␣-induced multiprotein IKK complex (17). A recent study attempting to isolate rat MEKK1 cDNA clarified that MEKK1 is a 195-kDa protein with a large N-terminal regulatory domain (45), raising the possibility that the regulatory domain may play a role in the interaction with IKKs. In fact, the human T cell leukemia virus type I Tax protein binds to the regulatory domain of MEKK1 to stimulate IKK kinase activity (24). Thus, these observations indicate that MAPKKKs stimulate NF-B activation through direct interactions with IKKs, but not through the MAPKK-MAPK signaling pathways.
MAPKKKs activate MAPKKs by phosphorylating Ser residues in the activation loop (S-X-X-X-S) located between kinase subdomains VII and VIII (46). These Ser residues are conserved in both IKK␣ and IKK␤. The Ser residues in IKK␤ were shown to be essential for NF-B activation. In the signaling pathway to NF-B activation, IKK␤ mutants in which Ser 177 and Ser 181 are replaced with Ala or Glu act as a dominant negative inhibitor and a constitutively active mutant, respectively (17). In addition, NIK activates IKK␣ by phosphorylating Ser 176 in the activation loop (44). In the present study, we demonstrated the functional significance of the Ser residues in the activation loop of both IKK subunits in TAK1-induced IKK activation. Collectively, these findings indicate that the molecular mechanism of the regulation of IKKs by TAK1 may be as follows. TAK1 interacts with IKKs in unstimulated cells. The recruitment of TAB1 to TAK1 activates the kinase activity of TAK1, where TAB1 phosphorylation by TAK1 and the autophosphorylation of TAK1 may be occurring. The active TAK1 then phosphorylates the Ser residues in the activation loop of IKKs, resulting in the dissociation of TAK1 from IKKs, depending on the kinase activity of IKKs. Recently, the subunits of the multiprotein IKK complex NEMO (IKK␥) and IKAP were isolated (47)(48)(49). The characterization of these subunits and the identification of other subunits of the IKK complex will provide more information on the regulatory mechanisms of IKK activation by TAK1.
A selective depletion of TAK1 and TAB1 was detected in the presence of IKK␤. IKK␤ activation leads to the phosphorylation of IB proteins in the NF-B/IB complexes, which triggers the degradation of IB proteins through the ubiquitination-proteasome pathway. These results suggest a novel regulatory mechanism of TAK1 kinase activity in which selective protein degradation through a proteasome pathway might be involved. The IKK complex was first isolated from unstimulated HeLa cells as a ubiquitination-dependent kinase complex (50). In addition, Seeger et al. (51) recently reported that a novel 450-kDa protein complex possessing similarities to 26 S proteasome subunits was involved in the phosphorylation of IB␣. Interestingly, the subunit similar to 26 S proteasome, signalsome (sgn) 6, and other components, sgn1 and sgn7, contain the Ser-X-X-X-Ser MAPKK activation loop motif, raising the possibility that MAPKKKs regulate proteasome-like activity by phosphorylating the Ser residues in the activation loop of these subunits. Future studies of the regulatory mechanisms of depletion of the TAK1/TAB1 complex will shed light on the role of protein degradation in the signaling pathways to NF-B activation.
In summary, we demonstrated that TAK1 is a new regulatory kinase of IKKs that stimulates NF-B activation. Our findings, together with previous observations, indicate that the multiprotein complexes composed of core IKK subunits and regulatory kinases such as TAK1, NIK, and MEKK1 may be involved in the signaling pathways to NF-B activation by diverse extracellular stimuli. Selective intervention of the activation and the function of TAK1 is likely to have therapeutic value in treating inflammatory diseases, in which NF-B may play significant pathogenic roles.