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INTRODUCTION |
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 (1-3). NF-
B is sequestered in the cytoplasm by inhibitory
proteins such as I
B
, I
B
, and I
B
, which mask the
nuclear localization signal of NF-
B (4-8). The phosphorylation of
two Ser residues at an N-terminal regulatory domain of I
B proteins
triggers polyubiquitination of I
B 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 I
B
degradation, suggesting the diversity of the upstream signaling
pathways for phosphorylation of I
B proteins.
Several regulatory kinases involved in the signal-induced
phosphorylation of I
B proteins have recently been reported. Two closely related kinases designated I
B 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 I
B 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 signal-regulated kinase kinase kinase 1 (MEKK1), another member of the MAPKKK family, stimulates NF-
B
activation by preferentially activating IKK
over IKK
(23-25).
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 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-
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 TAK1-induced IKKs, two Ser residues in the
activation loop of the IKKs were critically involved.
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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
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). IKK
and IKK
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 IKK
,
5'-GACGTATGGGATATCTTCAGAAA-3' and 5'-CGAGTCTAGAGTCATTCTGTTAACCAACTCC-3'
for the C terminus of IKK
, 5'-TGACGGTACCAATGAGCTGGTCACCTTCCCTG-3'
and 5'-GGAAGCCATGGAATTCTTCATTTTGG-3' for the N terminus of IKK
, and
5'-CCAAAATGAAGAATTCCATGGCTTCC-3' and
5'-TGACTCTAGATCATGAGGCCTGCTCCAGGC-3' for the C terminus of IKK
. Full-length IKK
and IKK
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), IKK
(SS176, 180AA), IKK
(K44M), IKK
(SS177,
181AA), and IKK
(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 pNF
B-Luc
plasmid (Stratagene). pRSV-
-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
-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
-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-IKK
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
-glycerophosphate, 20 mM disodium
p-nitrophenylphosphate, 0.1 mM sodium
orthovanadate, and 3 µCi of [
-32P]ATP) at 30 °C
for 30 min. For the IKK kinase assay, 2.5 µg of bacterially expressed
GST-I
B
(1-54) or GST-I
B
(1-54) (SS32, 36AA) were added as
a substrate. An expression vector for GST-I
B
(1-54) was
generated by inserting human I
B
cDNA encoding amino acids
1-54 into the BamHI-EcoRI site of pGEX-2T
(Pharmacia). An expression vector for GST-I
B
(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.
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RESULTS |
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,
I
B
and I
B
, were degraded in cells expressing both wild-type
TAK1 and TAB1 (Fig. 1B). The degradation of I
B
was
blocked by a proteasome inhibitor,
N-acetyl-leucyl-leucyl-norleucinal (data not shown),
indicating that TAK1 may activate NF-
B through the
ubiquitination-proteasome pathway.

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Fig. 1.
NF- 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 B site or an octamer binding site. B,
whole cell lysates were prepared, and immunoblotting was carried out
with anti-I B and anti-I B antibodies.
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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 [ -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.
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Involvement of IKKs in TAK1-induced NF-
B Activation--
The
marked degradation of I
B proteins by TAK1 raises the possibility of
the involvement of the IKK complex in TAK1-induced 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.

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Fig. 3.
Inhibition of TAK1-induced NF- 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 IKK (K44M) or IKK (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.
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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-I
B
(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-I
B
(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).

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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-IKK antibody. The IKK kinase activity was measured by an
in vitro immunocomplex kinase assay with GST-I B
(1-54) or GST-I B (1-54) (SS32 and 36AA) as a substrate.
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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 TAK1-induced NF-
B
activation.

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Fig. 5.
Activation of IKK and IKK by TAK1.
HeLa cells were transfected with expression vectors (1 µg 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 µg.
Twenty-four h after transfection, whole cell lysates were
immunoprecipitated with an anti-Xpress antibody. Kinase activities of
IKK (A) and IKK (B) were measured by
in vitro immunocomplex kinase assays with GST-I B
(1-54) or GST-I B (1-54) (SS32 and 36AA) as a substrate. The
expression level of IKKs is shown in Fig. 6.
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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.

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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-IKK , and Xpress-IKK . The
total amount of DNA was adjusted with an empty vector at 3 µg. 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.
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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, 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.

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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, IKK
(K44M), IKK (SS176, 180AA), IKK (K44M), and IKK (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 IKK -SSAA
and IKK -SSAA on TAK1-induced nuclear translocation of NF- B were
examined by gel shift assays. The shifted bands are shown.
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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 kinase-negative 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.

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Fig. 8.
TNF- -induced NF- B activation through
TAK1. A, HeLa cells were stimulated with human recombinant
TNF- (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 ( B)4-luciferase reporter plasmid
(0.25 µg), and pRSV- -gal (2 µg). Twenty-four h after
transfection, cells were stimulated with or without TNF- for 5 h. Luciferase activity was determined and normalized on the basis of
-galactosidase activity. Data are the average of duplicate
determinations from a representative experiment. Similar results were
obtained in the three independent experiments.
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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).

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Fig. 9.
Effect of NIK mutant on TAK1-induced NF- B
activation. A, HeLa cells were transfected with expression
vector for NIK624-947 (0.03, 0.1, or 0.3 µg), a
( B)4-luciferase reporter plasmid (0.25 µg), and
pRSV- -gal (2 µg). Twenty-four h after transfection, cells were
stimulated with or without TNF- 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 -galactosidase activity. Data are the
average of duplicate determinations from a representative experiment.
Similar results were obtained in the two independent experiments.
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|
 |
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 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 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-
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-
B activation by phosphorylating
Ser32 but not Ser36 of I
B
(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 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 IKK
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 (IKK
) 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
IKK
. IKK
activation leads to the phosphorylation of I
B
proteins in the NF-
B/I
B complexes, which triggers the degradation
of I
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 I
B
.
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.