Functional Interactions of Transforming Growth Factor beta -activated Kinase 1 with Ikappa B Kinases to Stimulate NF-kappa B Activation*

Hiroaki Sakurai, Hidetaka Miyoshi, Wataru Toriumi, and Takahisa SugitaDagger

From the Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532-8505, Japan

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several mitogen-activated protein kinase kinase kinases play critical roles in nuclear factor-kappa B (NF-kappa B) activation. We recently reported that the overexpression of transforming growth factor-beta -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-kappa B activation. Here we investigated the molecular mechanism of TAK1-induced NF-kappa B activation. Dominant negative mutants of Ikappa B kinase (IKK) alpha  and IKKbeta inhibited TAK1-induced NF-kappa B activation. TAK1 activated IKKalpha and IKKbeta in the presence of TAB1. IKKalpha and IKKbeta were coimmunoprecipitated with TAK1 in the absence of TAB1. TAB1-induced TAK1 activation promoted the dissociation of active forms of IKKalpha and IKKbeta from active TAK1, whereas the IKK mutants remained to interact with active TAK1. Furthermore, tumor necrosis factor-alpha activated endogenous TAK1, and the kinase-negative TAK1 acted as a dominant negative inhibitor against tumor necrosis factor-alpha -induced NF-kappa B activation. These results demonstrated a novel signaling pathway to NF-kappa B activation through TAK1 in which TAK1 may act as a regulatory kinase of IKKs.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transcription factor nuclear factor kappa B (NF-kappa 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 (1-3). NF-kappa B is sequestered in the cytoplasm by inhibitory proteins such as Ikappa Balpha , Ikappa Bbeta , and Ikappa Bepsilon , which mask the nuclear localization signal of NF-kappa B (4-8). The phosphorylation of two Ser residues at an N-terminal regulatory domain of Ikappa B proteins triggers polyubiquitination of Ikappa B proteins, which targets them for rapid degradation through a proteasome-dependent pathway, thereby releasing NF-kappa B to enter the nucleus (9-15). Diverse extracellular stimuli such as tumor necrosis factor (TNF)-alpha and interleukin-1beta , phorbol esters, and environmental stresses lead to NF-kappa B activation utilizing the common mechanism for the Ikappa B degradation, suggesting the diversity of the upstream signaling pathways for phosphorylation of Ikappa B proteins.

Several regulatory kinases involved in the signal-induced phosphorylation of Ikappa B proteins have recently been reported. Two closely related kinases designated Ikappa B kinase (IKK) alpha  and IKKbeta have been identified as components of the multiprotein IKK complex (500-900 kDa) that directly phosphorylates the critical Ser residues of Ikappa B proteins (16-20). Together, IKKalpha and IKKbeta form a heterodimer through their C-terminal leucine zipper motifs, and the functional IKK complex contains both IKK subunits. NF-kappa B-inducing kinase (NIK) is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, which was first identified as a TNF-alpha receptor-associated factor (TRAF) 2-interacting protein (21). The ligand-mediated trimerization of the TNF-alpha 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-1beta -induced NF-kappa B activation (22). In addition, MAPK/extracellular signal-regulated kinase kinase kinase 1 (MEKK1), another member of the MAPKKK family, stimulates NF-kappa B activation by preferentially activating IKKbeta over IKKalpha (23-25). These findings suggest that several MAPKKKs play a key role in the NF-kappa 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) beta -activated kinase 1 (TAK1) was first identified as a MAPKKK that can be activated by TGF-beta 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 N-terminal 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-kappa B activation (31). In the present study, we investigated the molecular mechanisms of TAK1-induced NF-kappa B activation. We found functional interactions of TAK1 with IKKalpha and IKKbeta . In the activation of TAK1-induced IKKs, two Ser residues in the activation loop of the IKKs were critically involved.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 TAK1 cDNA was subcloned into the EcoRI-XbaI site in pFLAG-CMV2 mammalian expression vector (Kodak) and expressed as a Flag epitope-tagged protein. The expression vectors for TAB1 and NIK624-947 were described previously (31). IKKalpha and IKKbeta cDNAs were obtained from human monocytic THP-1 cells by reverse transcription-polymerase chain reaction. The primers used were as follows: 5'-GGCCGCTTGAATTCCCGCCCCATGGA-3' and 5'-TTTCTGAAGATATCCCATACG-3' for the N terminus of IKKalpha , 5'-GACGTATGGGATATCTTCAGAAA-3' and 5'-CGAGTCTAGAGTCATTCTGTTAACCAACTCC-3' for the C terminus of IKKalpha , 5'-TGACGGTACCAATGAGCTGGTCACCTTCCCTG-3' and 5'-GGAAGCCATGGAATTCTTCATTTTGG-3' for the N terminus of IKKbeta , and 5'-CCAAAATGAAGAATTCCATGGCTTCC-3' and 5'-TGACTCTAGATCATGAGGCCTGCTCCAGGC-3' for the C terminus of IKKbeta . Full-length IKKalpha and IKKbeta cDNAs were subcloned into the EcoRI-NotI and KpnI-NotI sites of pcDNA3.1(+) and pcDNA3.1(+) HisB (Invitrogen), respectively. Expression vectors encoding the dominant negative mutants (TAK1 (K63W), IKKalpha (SS176, 180AA), IKKalpha (K44M), IKKbeta (SS177, 181AA), and IKKbeta (K44M)) were constructed using a QuikChange site-directed mutagenesis kit (Stratagene). All of the mutations were verified by DNA sequencing analysis.

Cell Cultures and Transfection-- HeLa cells were maintained in high-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 5% CO2. 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 pNFkappa B-Luc plasmid (Stratagene). pRSV-beta -gal plasmid was kindly provided by Dr. M. Tsuda (Toyama Medical and Pharmaceutical University).

Coimmunoprecipitation Assay-- Twenty-four h after transfection, whole cell lysates were prepared with lysis buffer (25 mM HEPES (pH 7.7), 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 20 mM beta -glycerophosphate, 0.1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Cell lysates were diluted with 3 volumes of dilution buffer (20 mM HEPES (pH 7.7), 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100, 20 mM beta -glycerophosphate, 0.1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and incubated on ice for 10 min. After centrifugation, lysates were incubated with a M5 anti-Flag antibody (Kodak) on ice for 1.5 h and rotated with protein G-conjugated Sepharose (Pharmacia) at 4 °C for 1.5 h. The beads were washed five times with washing buffer (20 mM HEPES (pH 7.7), 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, and 0.05% Triton X-100), and the immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblotting was performed as described previously (31). The antibodies used were anti-Xpress (Omni probe; M-21), anti-TAK1 (M-17), and anti-TAB1 (N-19) (Santa Cruz Biotechnology).

Immunocomplex Kinase Assay-- Cell lysates were immunoprecipitated with the anti-Flag antibody, the anti-Xpress antibody, or an anti-IKKalpha antibody (H-744; Santa Cruz Biotechnology) by the procedure described above. After washes, the beads were incubated with 30 µl of kinase buffer (20 mM HEPES (pH 7.6), 20 mM MgCl2, 2 mM dithiothreitol, 20 µM ATP, 20 mM beta -glycerophosphate, 20 mM disodium p-nitrophenylphosphate, 0.1 mM sodium orthovanadate, and 3 µCi of [gamma -32P]ATP) at 30 °C for 30 min. For the IKK kinase assay, 2.5 µg of bacterially expressed GST-Ikappa Balpha (1-54) or GST-Ikappa Balpha (1-54) (SS32, 36AA) were added as a substrate. An expression vector for GST-Ikappa Balpha (1-54) was generated by inserting human Ikappa Balpha cDNA encoding amino acids 1-54 into the BamHI-EcoRI site of pGEX-2T (Pharmacia). An expression vector for GST-Ikappa Balpha (1-54) (SS32, 36AA) was constructed by using the mutagenesis kit mentioned above. The reaction mixtures were resolved by SDS-PAGE, followed by autoradiography.

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 Thr180 and Tyr182 residues by the immunoblotting of cell lysates with an anti-phospho 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/µl calf intestinal alkaline phosphatase (Takara) at 37 °C for 30 min. Where indicated, sodium orthovanadate (1 µM) was added in the reaction mixture.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NF-kappa B Activation by TAK1-- We previously demonstrated the ability of TAK1 to activate p50/p65 NF-kappa B in a TAB1-dependent manner (31). To investigate the molecular mechanism of TAK1-induced NF-kappa 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-kappa 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, Ikappa Balpha and Ikappa Bbeta , were degraded in cells expressing both wild-type TAK1 and TAB1 (Fig. 1B). The degradation of Ikappa Balpha was blocked by a proteasome inhibitor, N-acetyl-leucyl-leucyl-norleucinal (data not shown), indicating that TAK1 may activate NF-kappa B through the ubiquitination-proteasome pathway.


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Fig. 1.   NF-kappa B activation by TAK1. HeLa cells (1 × 106 cells/60-mm dish) were transfected with expression vectors for Flag-TAK1 (1 µg) or Flag-TAK1K63W (1 µg) with or without an expression vector for TAB1 (1 µg). The total amount of DNA was adjusted with an empty vector at 2 µg. A, 24 h after transfection, nuclear extracts were prepared, and gel shift assays were carried out with oligonucleotide probes containing a kappa B site or an octamer binding site. B, whole cell lysates were prepared, and immunoblotting was carried out with anti-Ikappa Balpha and anti-Ikappa Bbeta antibodies.

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.


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Fig. 2.   TAB1-mediated activation of TAK1. HeLa cells were transfected with expression vectors for Flag-TAK1, Flag-TAK1K63W, and TAB1 as described in the Fig. 1 legend. Whole cell lysates were prepared 24 h after transfection. A, lysates were immunoprecipitated with an anti-Flag antibody and analyzed for coprecipitating TAB1 by immunoblotting with an anti-TAB1 antibody (top panel). The same blots were reprobed with an anti-TAK1 antibody (bottom panel). Similar results were obtained in the immunoblotting of lysates with an anti-Flag antibody. To monitor the expression of TAB1, lysates were immunoblotted with an anti-TAB1 antibody (middle panel). B, lysates were immunoprecipitated with an anti-Flag antibody and incubated with kinase buffer containing [gamma -32P]ATP. The reaction mixtures were resolved by 7.5% SDS-PAGE, followed by autoradiography. C, the TAK1/TAB1 complex immunoprecipitated with anti-Flag antibody was treated with calf intestinal alkaline phosphatase (CIP). Mobility was analyzed by immunoblotting with the anti-TAK1 and the anti-TAB1 antibodies. Sodium orthovanadate (1 µM) was added in the reaction mixture, where indicated. P-TAK1 and P-TAB1 indicate their phosphorylated forms.

Involvement of IKKs in TAK1-induced NF-kappa B Activation-- The marked degradation of Ikappa B proteins by TAK1 raises the possibility of the involvement of the IKK complex in TAK1-induced NF-kappa B activation. To investigate this possibility, the effects of dominant negative mutants of the IKKs were examined. The TAK1-induced nuclear translocation of NF-kappa B was inhibited by the kinase inactive mutants IKKalpha (K44M) and IKKbeta (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-kappa B activation is mediated by the IKK complex, but not through the MAPK signaling cascades.


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Fig. 3.   Inhibition of TAK1-induced NF-kappa B activation by IKK mutants. HeLa cells were transfected with expression vectors for Flag-TAK1 (1 µg) and TAB1 (1 µg) with or without expression vectors (1 µg each) for IKKalpha (K44M) or IKKbeta (K44M). The total amount of DNA was adjusted with an empty vector at 3 µg. A, 24 h after transfection, gel shift assays were carried out with nuclear extracts. The shifted bands are shown. B, 24 h after transfection, whole cell lysates were analyzed for JNK and p38 MAPK activation. JNK activity was determined by an in vitro immunocomplex kinase assay with GST-c-Jun (1-79) as a substrate. The phosphorylation of p38 MAPK was examined by immunoblotting with an anti-phospho p38 antibody.

The TAK1-induced regulation of IKK kinase activity was investigated. First, the endogenous IKK kinase activity was determined by an in vitro anti-IKKalpha immunocomplex kinase assay using bacterially expressed GST-Ikappa Balpha (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-Ikappa Balpha (1-54) (SS32 and 36AA), in which the critical Ser residues for IKKs were replaced with Ala (Fig. 4). The anti-IKKalpha antibody was able to recognize IKKbeta as well as IKKalpha , 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).


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Fig. 4.   Activation of endogenous IKK complex by TAK1. HeLa cells were transfected with expression vectors (1 µg each) for Flag-TAK1, Flag-TAK1K63W, and TAB1. The total amount of DNA was adjusted with an empty vector at 2 µg. Twenty-four h after transfection, whole cell lysates were immunoprecipitated with an anti-IKKalpha antibody. The IKK kinase activity was measured by an in vitro immunocomplex kinase assay with GST-Ikappa Balpha (1-54) or GST-Ikappa Balpha (1-54) (SS32 and 36AA) as a substrate.

To further elucidate the contribution of the two IKK subunits, N-terminal Xpress epitope-tagged IKKalpha and IKKbeta 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 IKKalpha when coexpressed with TAB1 (Fig. 5A). Similarly, IKKbeta activity was enhanced by TAK1 plus TAB1, whereas IKKbeta alone showed constitutive activity (Fig. 5B). In addition, TAK1K63W slightly inhibited the constitutive IKKbeta activity (Fig. 5B). These results indicate that TAK1 acts as an activator for IKKalpha and IKKbeta in the signaling pathway of TAK1-induced NF-kappa B activation.


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Fig. 5.   Activation of IKKalpha and IKKbeta by TAK1. HeLa cells were transfected with expression vectors (1 µg each) for Flag-TAK1, Flag-TAK1K63W, TAB1, Xpress-IKKalpha , and Xpress-IKKbeta . The total amount of DNA was adjusted with an empty vector at 3 µg. Twenty-four h after transfection, whole cell lysates were immunoprecipitated with an anti-Xpress antibody. Kinase activities of IKKalpha (A) and IKKbeta (B) were measured by in vitro immunocomplex kinase assays with GST-Ikappa Balpha (1-54) or GST-Ikappa Balpha (1-54) (SS32 and 36AA) as a substrate. The expression level of IKKs is shown in Fig. 6.

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-IKKalpha and Xpress-IKKbeta . Anti-Flag immunoprecipitates were analyzed for the presence of IKKs by immunoblotting with the anti-Xpress antibody. The interaction of wild-type TAK1 with IKKalpha 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 IKKbeta was detected only in the absence of TAB1 (Fig. 6B). In contrast, interactions of NIK with both IKKalpha and IKKbeta were detected through their active forms (data not shown). TAK1K63W interacted weakly with IKKalpha and IKKbeta (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 IKKalpha and IKKbeta 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.


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Fig. 6.   Interaction of TAK1 with IKKs. HeLa cells were transfected with expression vectors (1 µg each) for Flag-TAK1, Flag-TAK1K63W, TAB1, Xpress-IKKalpha , and Xpress-IKKbeta . The total amount of DNA was adjusted with an empty vector at 3 µg. The interactions of TAK1 with IKKalpha (A) and IKKbeta (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.

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, Xpress-tagged 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, IKKalpha -SSAA and IKKbeta -SSAA acted as dominant negative inhibitors in TAK1-induced NF-kappa B activation (Fig. 7B). These results indicate that the activation loop is critically involved in TAK1-induced IKKs activation.


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Fig. 7.   Functional significance of Ser residues in the activation loop of IKKs. A, HeLa cells were transfected with expression vectors (1 µg each) for Flag-TAK1, TAB1, IKKalpha (K44M), IKKalpha (SS176, 180AA), IKKbeta (K44M), and IKKbeta (SS177, 181AA). The total amount of DNA was adjusted with an empty vector at 3 µg. Interactions of TAK1 with IKKs were examined by the procedure described in the Fig. 6 legend. B, effects of IKKalpha -SSAA and IKKbeta -SSAA on TAK1-induced nuclear translocation of NF-kappa B were examined by gel shift assays. The shifted bands are shown.

Selective Depletion of TAK1 and TAB1-- Interestingly, TAK1 and TAB1 appeared to be selectively depleted in cells cotransfected with IKKbeta , whereas the expression of IKKbeta was not affected (Figs. 6B and 7A). Such a depletion was not observed in the presence of IKKalpha (Fig. 6A). The depletion of TAK1 and TAB1 was dependent on the kinase activities of TAK1 and IKKbeta , because this was not observed in cells expressing kinase-negative mutants of TAK1 and IKKbeta (Figs. 6B and 7A). This observation may indicate a novel regulatory mechanism of TAK1 kinase activity.

TNF-alpha -induced NF-kappa B Activation through TAK1-- In A673 human rhabdomyosarcoma cells, endogenous TAK1 is activated by TNF-alpha in which the TAK1 activity was measured for its ability to activate SEK1 (29). Here we investigated the effect of TNF-alpha on TAK1 activation in HeLa cells. The anti-TAK1 immunocomplex in vitro kinase assay using 6xHis-MKK6 as a substrate showed that TNF-alpha 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-beta 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-kappa B activation pathway through the TNF-alpha receptor. To clarify this possibility, we examined the effect of kinase-negative TAK1 on TNF-alpha -induced NF-kappa B activation. TAK1K63W inhibited kappa B-dependent luciferase gene expression (Fig. 8B). These results indicate that the TAK1/TAB1 complex plays a role in TNF-alpha -induced NF-kappa B activation.


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Fig. 8.   TNF-alpha -induced NF-kappa B activation through TAK1. A, HeLa cells were stimulated with human recombinant TNF-alpha (20 ng/ml) for the indicated time period. The endogenous TAK1 activity was determined by an immunocomplex kinase assay using the anti-TAK1 antibody. The endogenous IKK complex activity was determined by the procedure described in the Fig. 4 legend. B, HeLa cells were transfected with an expression vector for Flag-TAK1K63W (0.03 or 0.1 µg), a (kappa B)4-luciferase reporter plasmid (0.25 µg), and pRSV-beta -gal (2 µg). Twenty-four h after transfection, cells were stimulated with or without TNF-alpha for 5 h. Luciferase activity was determined and normalized on the basis of beta -galactosidase activity. Data are the average of duplicate determinations from a representative experiment. Similar results were obtained in the three independent experiments.

Effect of the NIK Mutant on TAK1-induced NF-kappa B Activation-- NIK plays a key role in TNF-alpha -induced NF-kappa 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-alpha -induced NF-kappa B activation (Fig. 9A). Furthermore, the NIK mutant partially inhibited TAK1-induced NF-kappa B activation (Fig. 9B).


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Fig. 9.   Effect of NIK mutant on TAK1-induced NF-kappa B activation. A, HeLa cells were transfected with expression vector for NIK624-947 (0.03, 0.1, or 0.3 µg), a (kappa B)4-luciferase reporter plasmid (0.25 µg), and pRSV-beta -gal (2 µg). Twenty-four h after transfection, cells were stimulated with or without TNF-alpha for 5 h. B, HeLa cells were transfected with expression vectors for Flag-TAK1 (0.5 µg) and TAB1 (0.5 µg) with or without the expression vector for NIK624-947 (0.3 µg). Luciferase activity was determined and normalized on the basis of beta -galactosidase activity. Data are the average of duplicate determinations from a representative experiment. Similar results were obtained in the two independent experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TAK1 was first identified as a MAPKKK that can be activated by TGF-beta 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-beta 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-kappa B activation. However, little is known about the functional relationship between TGF-beta signaling and the NF-kappa B activation pathways. We previously reported that TGF-beta could not induce the nuclear translocation of NF-kappa B in HeLa cells (31). In addition, we showed that TAK1 was not activated by TGF-beta in HeLa cells. These results suggest that TAK1 is involved in the NF-kappa B activation pathway induced by extracellular stimuli other than TGF-beta . In this study, we demonstrated that TNF-alpha activated TAK1 to stimulate NF-kappa B activation. It has been shown that NIK plays a significant role in TNF-alpha -induced NF-kappa B activation. Our previous study showed that NIK624-947 did not inhibit the TAK1-induced nuclear translocation of NF-kappa B (31). In contrast, the truncated NIK mutant inhibits TAK1-induced NF-kappa 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-alpha -induced NF-kappa 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-kappa B activation through TAK1, which may present a physiological function of TAK1-induced NF-kappa 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-beta 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 C-terminal 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-kappa B activation have been characterized. The 90-kDa ribosomal S6 kinase (pp90rsk) that lies downstream of the Raf-MAPK/extracellular signal-regulated kinase pathway is involved in phorbol ester-induced NF-kappa B activation by phosphorylating Ser32 but not Ser36 of Ikappa Balpha (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-alpha -induced transactivation of the p65 NF-kappa B subunit (40). Several MAPKKKs including NIK (21), MEKK1 (41, 42), and MEKK3 (43) were recently shown to have the potential to activate NF-kappa B. NIK and MEKK1 preferentially activate IKKalpha and IKKbeta , 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 IKKalpha and IKKbeta . In contrast, the interaction of MEKK1 with IKKs has not yet been demonstrated, although a MEKK1 catalytic subunit was copurified with the TNF-alpha -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-kappa 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 IKKalpha and IKKbeta . The Ser residues in IKKbeta were shown to be essential for NF-kappa B activation. In the signaling pathway to NF-kappa B activation, IKKbeta mutants in which Ser177 and Ser181 are replaced with Ala or Glu act as a dominant negative inhibitor and a constitutively active mutant, respectively (17). In addition, NIK activates IKKalpha by phosphorylating Ser176 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 (IKKgamma ) and IKAP were isolated (47-49). 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 IKKbeta . IKKbeta activation leads to the phosphorylation of Ikappa B proteins in the NF-kappa B/Ikappa B complexes, which triggers the degradation of Ikappa B 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 Ikappa Balpha . 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-kappa B activation.

In summary, we demonstrated that TAK1 is a new regulatory kinase of IKKs that stimulates NF-kappa 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-kappa 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-kappa B may play significant pathogenic roles.

    ACKNOWLEDGEMENTS

We are grateful to Dr. K. Matsumoto for helpful discussions and advice on the manuscript. We are also grateful to Drs. M. Tsuda and M. Hibi for the generous gift of pRSV-beta -gal and an expression plasmid for GST-c-Jun (1-79), respectively. We thank N. Shigemori-Kageyama, Dr. N. Yanaka, Dr. Y. Suzuki, T. Murakami, and K. Kyono for technical support and discussions and Y. Kawashima for DNA sequencing.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-6-6300-2571; Fax.: 81-6-6300-2593; E-mail: t-sugita{at}tanabe.co.jp.

2 Tsuji, N. J., Kishimoto, K., Hiyama, A., Inoue, J., Cao, Z., Matsumoto, K. (1999) Nature, in press.

    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor kappa B; MAPK, mitogen-activated protein kinase; MAPKKK, MAPK kinase kinase; TGF, transforming growth factor; TAK1, transforming growth factor beta -activated kinase 1; IKK, Ikappa B kinase; NIK, NF-kappa B-inducing kinase; TNF, tumor necrosis factor; TAB1, TAK1-binding protein 1; MEKK1, MAPK/extracellular signal-regulated kinase kinase kinase 1; JNK, c-Jun N-terminal kinase; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; GST, glutathione S-transferase; TRAF, TNF-alpha receptor-associated factor; MKK, MAPK kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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