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Tohru Ishitani, Giichi Takaesu
1
,
Jun Ninomiya-Tsuji, Hiroshi Shibuya
2
,
Richard B.Gaynor
1
and
Kunihiro Matsumoto
3
Department of Molecular Biology, Graduate School of Science,
Institute for Advanced Research, Nagoya University, and CREST,
Japan Science and Technology Corporation, Chikusa-ku, Nagoya
464-8602,
2
Department of Molecular Cell Biology, Medical Research
Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo
101-0062, Japan and
1
Division of Hematology-Oncology, Department
of Medicine, Harold Simmons Cancer Center, University of Texas,
Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas,
TX 75390-8594, USA
3
Corresponding author
e-mail: g44177a@nucc.cc.nagoya-u.ac.jp
The cytokines IL-1 and TNF induce expression of a
series of genes that regulate in¯ammation through
activation of NF-kB signal transduction pathways.
TAK1, a MAPKKK, is critical for both IL-1- and
TNF-induced activation of the NF-kB pathway. TAB2,
a TAK1-binding protein, is involved in IL-1-induced
NF-kB activation by physically linking TAK1 to
TRAF6. However, IL-1-induced activation of NF-kB
is not impaired in TAB2-de®cient embryonic ®bro-
blasts. Here we report the identi®cation and charac-
terization of a novel protein designated TAB3, a
TAB2-like molecule that associates with TAK1 and
can activate NF-kB similar to TAB2. Endogenous
TAB3 interacts with TRAF6 and TRAF2 in an IL-1-
and a TNF-dependent manner, respectively. Further-
more, IL-1 signaling leads to the ubiquitination of
TAB2 and TAB3 through TRAF6. Cotransfection of
siRNAs directed against both TAB2 and TAB3 inhibit
both IL-1- and TNF-induced activation of TAK1 and
NF-kB. These results suggest that TAB2 and TAB3
function redundantly as mediators of TAK1 activation
in IL-1 and TNF signal transduction.
Keywords: IL-1/NF-kB/TAB2/TAB3/TAK1
Introduction
The pro-in¯ammatory cytokines IL-1 and TNF have
several effects in the in¯ammation process. Stimulation
of cells with IL-1 or TNF initiates a cascade of signaling
events, including activation of NF-kB and mitogen-
activated protein kinases (MAPKs) such as JNK and
p38. These, in turn, upregulate the expression of many pro-
in¯ammatory genes in the nucleus (Dinarello, 1996; Baud
and Karin, 2001). NF-kB is normally sequestered in the
cytoplasm of resting cells by association with inhibitory
IkB proteins. This interaction masks the nuclear localiza-
tion signal of NF-kB, preventing its nuclear translocation
(Ghosh et al., 1998; Karin and Ben-Neriah, 2000; Li and
Verma, 2002). Stimulation by IL-1 or TNF results in the
phosphorylation of the IkB proteins, tagging them for
ubiquitination and subsequent proteosome-mediated deg-
radation. This results in the release of NF-kB, which
translocates to the nucleus where it activates the tran-
scription of speci®c target genes (Karin and Ben-Neriah,
2000; Li and Verma, 2002). Phosphorylation of IkBin
response to extracellular stimuli is carried out by the IkB
kinase (IKK) complex, which is comprised of two catalytic
subunits, IKKaand IKKb, as well as the modulator
NEMO/IKKg(Silverman and Maniatis, 2001; Ghosh and
Karin 2002; Li and Verma, 2002).
Members of the TNF-receptor-associated factor
(TRAF) family of adaptor proteins are involved in
coupling stimulation of the TNF receptors (TNFRs) and
the IL-1 receptor (IL-1R) to NF-kB activation and other
downstream events (Silverman and Maniatis, 2001).
TRAF2 plays a critical role in signal transduction medi-
ated by both TNFR1 and TNFR2, and has been implicated
in TNF-induced activation of NF-kB and MAPKs (Yeh
et al., 1997). Similarly, TRAF6 is important for the
transduction of IL-1-induced signals, including those
resulting in NF-kB and MAPK activation (Cao et al.,
1996; Lomaga et al., 1999; Naito et al., 1999). The TRAF
proteins physically and functionally connect TNFRs and
IL-1R to intracellular protein kinases, thereby linking
these receptors to downstream signaling pathways.
TAK1, a member of the MAPKKK family, also
participates in the IL-1-mediated signaling pathway
(Yamaguchi et al., 1995; Ninomiya-Tsuji et al., 1999).
Following exposure of cells to IL-1, endogenous TAK1 is
recruited to the TRAF6 complex and activated, whereupon
it stimulates NF-kB and MAPK activation. In previous
studies, the yeast two-hybrid system was employed to
isolate TAB1 and TAB2 proteins that interact with TAK1
(Shibuya et al., 1996; Takaesu et al., 2000). TAB1 was
found to augment the kinase activity of TAK1 when
coexpressed (Shibuya et al., 1996), indicating that it
functions as an activator of TAK1. TAB2 was shown to be
an intermediate in the IL-1 signaling pathway (Takaesu
et al., 2000, 2001). TAB2 functions as an adaptor that links
TAK1 and TRAF6 in response to IL-1 and thereby
mediates TAK1 activation. These results suggest that IL-1
activation of the NF-kB and MAPK cascades involves the
formation of a TRAF6±TAB2±TAK1 complex. In add-
ition, a biochemical study has identi®ed TRIKA1 and
TRIKA2 as signaling components that are able to activate
the IKK complex in a TRAF6-dependent manner (Deng
et al., 2000; Wang et al., 2001). TRIKA1 consists of the
ubiquitin-conjugating enzyme Ubc13 and the Ubc-like
protein Uev1A, while TRIKA2 is a ternary complex
composed of TAK1, TAB1 and TAB2. Thus, TRAF6-
mediated ubiquitination appears to play an important role
in TAK1 activation.
Role of the TAB2-related protein TAB3 in IL-1 and
TNF signaling
The EMBO Journal Vol. 22 No. 23 pp.6277±6288, 2003
ãEuropean Molecular Biology Organization 6277
Previously, in order to explore the physiological
importance of TAK1 in activating the NF-kB pathway in
response to IL-1, we utilized small interfering RNA
(siRNA) directed against TAK1 (Takaesu et al., 2003).
Our previous studies con®rmed that TAK1 is critical for
IL-1-induced activation of the NF-kB pathway.
Furthermore, our results indicated that TAK1 is also
important for NF-kB activation in response to TNF via the
inducible association of TAK1 with TRAF2 (Takaesu
et al., 2003). On the other hand, recent studies have
demonstrated that mouse embryo ®broblasts (MEFs)
de®cient in TAB2 exhibit normal IL-1- and TNF-induced
activation of NF-kB (Sanjo et al., 2003). This result
indicates that TAB2 is not essential for IL-1 or TNF
signaling in MEFs. However, there remains the possibility
that another TAB2-like molecule may compensate for the
loss of TAB2 and support TAK1 kinase activity.
Here we describe the identi®cation and characterization
of TAB3, a novel TAK1-binding protein that is closely
related to TAB2 in structure. TAB3 activates TAK1 and
mediates its interaction with TRAF2 and TRAF6. TAB3
rapidly and transiently associates with TRAF6 and TRAF2
in an IL-1- and a TNF-dependent manner, respectively.
IL-1 stimulation and TRAF6 overexpression induce
ubiquitination of the TAB2 and TAB3 proteins. In this
study, we utilized siRNA directed against TAB2 and
TAB3 to further explore their roles in activation of the
NF-kB and MAPK pathways following treatment of cells
with IL-1 and TNF. These studies demonstrate that TAB2
and TAB3 are important for NF-kB and MAPK activation
in response to IL-1 and TNF, and suggest that TAB2 and
TAB3 have redundant functions as mediators of TAK1
activation in IL-1 and TNF signal transduction.
Results
Isolation of TAB3
In an attempt to identify new signal transducers involved
in TAK1 activation in the IL-1 and TNF signaling
pathways, we searched EST databases for sequences
similar to TAB2. Multiple human and murine EST
sequences were found which encode polypeptides sharing
signi®cant homology with TAB2. We cloned one full-
length human TAB2 homolog by PCR from a human
kidney cDNA library. This human TAB2-like gene,
termed TAB3, was predicted to encode a 712 amino acid
polypeptide (Figure 1A). Similar to TAB2, TAB3 contains
a ubiquitin-binding motif (Shih et al., 2003) near its
N-terminus and is predicted to encode an a-helical coiled-
coil region in its C-terminus. Thus TAB3 is structurally
related to TAB2. We also found two highly conserved
homologs of TAB3 in Xenopus laevis. While this manu-
script was in preparation, one of the homologs was
reported by Munoz-Sanjuan and coworkers (Munoz-
Sanjuan et al., 2002). Both of the Xenopus cDNAs
resemble TAB3 more closely than TAB2. These workers
(Munoz-Sanjuan et al., 2002) also described mouse and
human TAB3. Furthermore, Drosophila contains a TAB2/
3-like protein carrying the ubiquitin-binding motif and the
a-helical coiled-coil region in its N- and C-termini,
respectively (Figure 1A).
TAB3 associates with TAK1
We have shown previously that TAB2 interacts with
TAK1 through the C-terminal region of TAB2, and that
overexpression of TAB2 can induce TAK1 kinase activity
(Takaesu et al., 2000). To examine whether TAB3
functions similarly, the interaction of TAB3 with TAK1
was investigated in mammalian cells by in vivo copreci-
pitation. Human 293 embryonic kidney cells were
cotransfected with T7-TAB3 and HA-TAK1. Cell extracts
were immunoprecipitated with anti-T7 antibody, and
coprecipitated HA-TAK1 was detected by immunoblot-
ting with anti-HA antibody. TAB3 was found to associate
with TAK1 (Figure 2A, lane 2). To verify that TAK1
associates with the C-terminal region of TAB3 in mam-
malian cells, we used two truncated proteins: T7-TAB3N,
consisting of the N-terminus of TAB3 (amino acids 1±
392), and T7-TAB3C, consisting of the C-terminus (amino
acids 393±712) (Figure 1B). Immune complex assays
showed that TAK1 coimmunoprecipitated with
T7-TAB3C (Figure 2A, lane 4), but not with T7-TAB3N
(lane 3). The C-terminal domain of TAB3 contains a
coiled-coil structure (Figure 1A). To examine whether this
coiled-coil region is involved in the interaction with
TAK1, we constructed the mutant protein TAB3Dcc,
which lacks this domain (amino acids 478±661)
(Figure 1B). Coimmunoprecipitation analysis from cells
coexpressing T7-TAB3Dcc and HA-TAK1 demonstrated
that the TAB3Dcc protein failed to interact with TAK1
(Figure 2A, lane 5). These results con®rm that the
C-terminal coiled-coil region of TAB3 is responsible for
its association with TAK1.
To examine whether TAB3 can induce TAK1 acti-
vation, we transfected 293 cells with HA-TAK1 in the
presence or absence of the TAB3 expression vector.
Transiently expressed TAK1 was immunoprecipitated
using anti-HA antibody, and kinase activity was measured
by in vitro kinase assay using MKK6 as a substrate. When
expressed alone, TAK1 exhibited low basal kinase activity
(Figure 2B, lane 1). However, coexpression of TAB3 led
to a marked enhancement in TAK1 catalytic activity
(lane 3), although the degree of activation was weaker than
that induced by TAB2 (lane 2). Since activation of TAK1
induces JNK activation (Shirakabe et al., 1997), we
analyzed the effect of ectopically expressed TAB3 on JNK
activity. First, 293 cells were cotransfected with HA-JNK
and TAB3 or TAB2. Then, JNK activity was determined
by immunoprecipitation of JNK followed by in vitro
kinase assay using GST-c-Jun protein as a substrate. We
observed that TAB2 and TAB3 signi®cantly induced
activation of JNK (Figure 2C, lanes 2 and 3). Taken
together, these results suggest that the function of TAB3 is
similar to that of TAB2.
Since TAB2 and TAB3 each interact with TAK1 via
their respective C-terminal coiled-coil domain, we exam-
Fig. 1. Structure of TAB3. (A) Comparison of amino acid sequences among hTAB3 (human), hTAB2 (human) and DTAB2 (Drosophila). They share
the CUE domain (bold underline) and coiled-coil structure (box). Identical and conserved amino acids are indicated by black and gray boxes, respect-
ively. DDBJ/EMBL/GenBank accession No. for hTAB3 is AY437560. (B) Schematic representation of various TAB3 constructs. Gray and black
boxes indicate the CUE domain and coiled-coil structure, respectively.
T.Ishitani et al.
6278
Role of TAB3 in IL-1 and TNF signaling pathways
6279
ined whether the bindings of TAB2 and TAB3 to TAK1
are mutually exclusive. We cotransfected 293 cells with
Flag-TAK1 and HA-TAB3 in the presence or absence of
T7-TAB2. We con®rmed that TAK1 and TAB3 could
interact in the absence of TAB2 coexpression (Figure 2D,
top panel, lane 3). Coexpression of T7-TAB2 did not block
the interaction of TAB3 with TAK1 (lane 4). Furthermore,
T7-TAB2 coimmunoprecipitated with HA-TAB3 (second
panel, lane 4), suggesting that TAB2 and TAB3 form a
complex. To con®rm this possibility, T7-TAB2 and
HA-TAB3 were coexpressed in 293 cells and coimmuno-
precipitation analysis was performed. We found that
TAB2 associated with TAB3 (Figure 2E, lane 3).
Furthermore, when 293 cells were cotransfected with the
combination of HA-TAB3 and T7-TAB3 or HA-TAB2
and T7-TAB2, we found that TAB3 and TAB2 each self-
associated (lanes 2 and 6). These results suggest that
homo- and hetero-oligomers of TAB2 and TAB3 form a
complex with TAK1. We failed to detect any association
of TAB1 with TAB3 or TAB2 when overexpressed (lanes 4
and 7).
TAB3 is involved in NF-
k
B activation
Given the sequence homology of TAB3 to TAB2, a known
activator of NF-kB (Takaesu et al., 2000), we investigated
whether TAB3 might play a role in NF-kB activation.
When TAB3 and an NF-kB-dependent luciferase reporter
were cotransfected into 293 cells, TAB3 was found to
activate the reporter gene in a dose-dependent manner
(Figure 3A). Intact TAB3 was required for this activity, as
truncated derivatives of TAB3 failed to induce NF-kB
activity. Wild-type and mutant proteins were expressed in
comparable amounts, as shown by western blot analysis
(data not shown).
We next tested whether TAB3 is involved in the IL-1
or TNF signaling pathway that leads to NF-kB
activation. Since mutants of TAB2 lacking the
C-terminus function as dominant negatives (Takaesu
Fig. 2. TAB3 interacts with and activates TAK1. (A) Interaction of TAB3 with TAK1. The 293 cells were transfected with plasmids encoding
T7-TAB3 full-length (F), T7-TAB3N (N), T7-TAB3C (C), T7-TAB3Dcc (Dcc) and HA-TAK1 as indicated. Complexes immunoprecipitated with anti-
T7 antibody were immunoblotted with anti-HA or anti-T7 antibodies. Whole-cell extracts were immunoblotted with anti-HA antibody. (Band
C) Activation of TAK1 and JNK by TAB3. The 293 cells were transfected with plasmids encoding HA-TAK1, HA-JNK, T7-TAB2 (2) and
T7-TAB3 (3) as indicated. HA-TAK1 or HA-JNK was immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to an
in vitro phosphorylation assay using bacterially expressed MKK6 (B) or GST-c-Jun (C) as an exogenous substrate. The immunoprecipitates were ana-
lyzed by immunoblotting with anti-HA antibody. (D) Effect of TAB2 on the interaction between TAK1 and TAB3. The 293 cells were transfected
with plasmids encoding HA-TAB3, T7-TAB2 and Flag±TAK1 as indicated. Complexes immunoprecipitated with anti-HA antibody were immuno-
blotted with anti-Flag, anti-T7 or anti-HA antibodies. Whole-cell extracts were immunoblotted with anti-Flag or anti-T7 antibodies. (E) Interaction
among TAK1-binding proteins. The 293 cells were transfected with plasmids encoding HA-TAB2 (2), HA-TAB3 (3), T7-TAB1 (1), T7-TAB2 (2) and
T7-TAB3 (3) as indicated. Complexes immunoprecipitated with anti-T7 antibody were immunoblotted with anti-HA or anti-T7 antibodies. Whole-cell
extracts were immunoblotted with anti-HA antibody.
T.Ishitani et al.
6280
et al., 2000), we made a homologous truncated deriva-
tive of TAB3 (TAB3C) (Figure 1B). First, 293 cells
were transfected with an NF-kB-dependent luciferase
reporter and increasing concentrations of TAB3C. Later,
cells were treated with IL-1 or TNF and luciferase
activity was determined. Figure 3B shows that increas-
ing concentrations of TAB3C potently inhibited induc-
tion of NF-kB by IL-1 and TNF. These results suggest
that TAB3 may be a common downstream mediator of
NF-kB activation by IL-1 and TNF.
TAB3 associates with TRAF6 and TRAF2
IL-1 and TNF activate their signal pathways via distinct
families of cell-surface receptors. However, both path-
ways utilize members of the TRAF family of adaptor
proteins as signal transducers (Silverman and Maniatis,
2001). The TRAF proteins share homology at their
C-terminal domains, but their binding properties and
activities differ. For example, whereas TRAF6 is
essential for IL-1 signaling (Lomaga et al., 1999;
Naito et al., 1999), TRAF2 is involved in TNF signaling
(Yeh et al., 1997). Recently, it was shown that TAB2
interacts speci®cally with TRAF6 (Takaesu et al., 2000).
Therefore we tested the ability of TAB3 to bind to
TRAF6 and TRAF2. T7-TAB2 or T7-TAB3 was
expressed together with Flag-TRAF6 or Flag-TRAF2
in 293 cells and immunoprecipitated with anti-T7
antibody. The immune complexes were subjected to
immunoblotting with anti-Flag antibody. As observed
previously, TAB2 was found to interact with TRAF6
(Figure 4A, lane 2), but not with TRAF2 (Figure 4B,
lane 2). In contrast, TAB3 was found to coprecipitate
both TRAF6 (Figure 4A, lane 3) and TRAF2 (Figure 4B,
lane 3) ef®ciently. This is consistent with the notion that
TAB3 is involved in both the IL-1 and TNF signaling
pathways. To determine whether TAB2 and TAB3
competitively bind to TRAF6, we analyzed the inter-
action between TAB3 and TRAF6 in the presence or
absence of TAB2. When Flag-TRAF6 and HA-TAB3
were expressed together with T7-TAB2 in 293 cells,
TAB2 formed a complex with TAB3 (Figure 4C, second
panel, lane 4) and did not interfere with the interaction
between TAB3 and TRAF6 (top panel, lane 4). These
results suggest that TAB2 and TAB3 bind cooperatively,
but not competitively, to TRAF6.
To determine the regions within TAB3 responsible for
its interaction with TRAF6 and TRAF2, we performed
immunoprecipitation assays using deletion mutants of
TAB3. We found that TRAF6 and TRAF2 coprecipitated
with TAB3C (Figure 4A and B, lane 5), but not with
TAB3N (lane 4). Thus the C-terminal domain of TAB3 is
required for binding to TRAF6 and TRAF2. This is similar
to TAB2, which also requires its C-terminal domain for
binding to TRAF6. We next tested whether the C-terminal
coiled-coil region of TAB3 is essential for its interaction
with TRAF6 and TRAF2. We found that the TAB3Dcc
mutant (Figure 1B), which lacks the coiled-coil motif, was
still able to associate with TRAF6 (see Figure 6A below)
and TRAF2 (data not shown). Thus, the coiled-coil motif
of TAB3 is required for its interaction with TAK1, but not
with TRAF6 or TRAF2.
We have previously shown that TAB2 functions as an
adaptor protein mediating the association of TAK1 with
TRAF6 (Takaesu et al., 2000). The ability of TAB3 to
interact with both TAK1 and TRAF6 led us to hypothesize
that TAB3 similarly acts as a link between TRAF6 and
TAK1. To test this possibility, we analyzed the interaction
between TAK1 and TRAF6 in both the presence and
absence of TAB3. Although a small amount of TAK1 was
found to associate with TRAF6 in the absence of
Fig. 3. TAB3 is involved in NF-kB activation pathway. (A) Effects of TAB3 on NF-kB-dependent reporter gene activity. The 293 cells were trans-
fected with luciferase reporter plasmid (0.1 mg) and the indicated amounts of plasmids encoding TAB3 full-length (F), TAB3N (N) and TAB3C (C).
After 24 h incubation, cells were harvested and luciferase activity measured. The values shown are the average of one representative experiment in
which each transfection was performed in duplicate. (B) Effects of TAB3 on IL-1- and TNFa-induced NF-kB-activation. 293IL-1RI cells were trans-
fected with luciferase reporter plasmid (0.1 mg) and the indicated amounts of plasmids encoding TAB3N (N) and TAB3C (C). IL-1b(5 ng/ml) or
TNFa(10 ng/ml) was added to each plate 3 h after transfection. Cells were harvested 24 h after transfection and luciferase activity was measured. The
values shown are the average of one representative experiment in which each transfection was performed in duplicate.
Role of TAB3 in IL-1 and TNF signaling pathways
6281
exogenous TAB3 (Figure 4D, lane 5), overexpression of
TAB3 strongly enhanced the association between TRAF6
and TAK1 (lane 6). This indicates that TAB3, at least
when overexpressed, can link TRAF6 to TAK1. We next
examined the effect of the TAB3Dcc mutant, lacking the
coiled-coil motif, on TRAF6±TAK1 complex formation.
As described above, we had observed that TAB3Dcc
interacted with TRAF6 but not with TAK1. In the present
experiment, we observed that overexpression of TAB3Dcc
did not enhance the association between TRAF6 and
TAK1 (lane 7). These results support the idea that TAB3 is
an intermediate signaling molecule linking TAK1 and
TRAF6.
We were interested to determine whether the presence
of TAB3 could also affect the ability of TAK1 to interact
with TRAF2. To address this point, Flag-TRAF2 and
HA-TAK1 were cotransfected into 293 cells with or
without T7-TAB3. In the absence of TAB3 expression, no
association of TAK1 with TRAF2 could be determined
(Figure 4D, lane 2). However, upon coexpression of
TAB3, we were able to detect the association between
TAK1 and TRAF2 (lane 3). Overexpression of TAB3Dcc
failed to enhance the interaction of TAK1 with TRAF2
(lane 4). Taken together, these results suggest that TAB3
functions as an adaptor for the association of TAK1 with
both TRAF6 and TRAF2.
Ligand-dependent endogenous interaction of TAB3
with TRAF6 and TRAF2
To evaluate the interaction of TAB3 with TRAF6 and
TRAF2 under more physiological conditions, we exam-
ined the association of endogenous TAB3 with TRAF6
and TRAF2 in 293IL-1RI cells. A rabbit anti-TAB3
polyclonal antibody was generated to identify endogenous
TAB3 protein. When lysates prepared from 293IL-1RI
cells were subjected to immunoprecipitation followed by
western blotting with anti-TAB3 antibody, one band at
approximately 90 kDa was observed (Figure 5A, bottom
panel, lane 10). This band was not observed when control
IgG was used for immunoprecipitation (lane 9), indicating
that it represents endogenous TAB3. Lysates from IL-1-
treated cells were immunoprecipitated with anti-TAB3
antibody and then analyzed by immunoblotting with anti-
TRAF6 antibody. We found that TRAF6 rapidly associ-
Fig. 4. TAB3 mediates the interaction of TAK1 with TRAF6 and TRAF2. (Aand B) Interaction of TAB3 with TRAF6 and TRAF2. The 293 cells
were transfected with plasmids encoding T7-TAB2 full-length (2F), T7-TAB3 full-length (3F), T7-TAB3N (3N), T7-TAB3C (3C), Flag-TRAF6 and
Flag-TRAF2 as indicated. Complexes immunoprecipitated with anti-T7 antibody were immunoblotted with anti-Flag or anti-T7 antibodies. Whole-cell
extracts were immunoblotted with anti-Flag antibody. (C) Effect of TAB2 on the interaction between TRAF6 and TAB3. The 293 cells were trans-
fected with plasmids encoding HA-TAB3, T7-TAB2 and Flag-TRAF6 as indicated. Complexes immunoprecipitated with anti-HA antibody were im-
munoblotted with anti-Flag, anti-T7 or anti-HA antibodies. Whole-cell extracts were immunoblotted with anti-Flag or anti-T7 antibodies. (D) Effect of
TAB3 on the interaction of TAK1 with TRAF2 and TRAF6. The 293 cells were transfected with plasmids encoding HA-TAK1, T7-TAB3 full-
length (F), T7-TAB3Dcc (Dcc), Flag-TRAF2 (2) and Flag-TRAF6 (6) as indicated. Complexes immunoprecipitated with anti-Flag antibody were im-
munoblotted with anti-HA or anti-Flag antibodies. Whole-cell extracts were immunoblotted with anti-HA antibody.
T.Ishitani et al.
6282
ated with TAB3 in an IL-1-dependent manner (top panel,
lanes 10±12). This interaction was observed within 3 min
after IL-1 treatment, and decreased thereafter. Thus the
interaction between TAB3 and TRAF6 is physiologically
induced by IL-1. The kinetics of IL-1-induced TRAF6±
TAB3 association were similar to those for TRAF6±TAK1
(lanes 2±4) and TRAF6±TAB2 (lanes 6±8) association.
TNF did not induce the interaction of TRAF6 with TAB2
or TAB3 (see Figure 6B). Thus the interaction of TRAF6
with TAB2 and TAB3 is signaled speci®cally and
physiologically by IL-1. When TAK1 was immunopreci-
pitated with anti-TAK1 antibody, the TAK1 immunocom-
plexes were found to contain TAB2 and TAB3 even in the
absence of IL-1 stimulation (Figure 5A, third and bottom
panels, lane 2). In addition, we found that TAB2 and
TAB3 associated constitutively (bottom panel, lane 6, and
third panel, lane 10). Thus TAK1, TAB2 and TAB3 form a
complex in the absence of stimulation. This is consistent
with recent observations that the TRIKA2 complex
containing TAK1, TAB1 and TAB2 is formed before
IL-1 stimulation (Wang et al., 2001) and that TAK1,
TAB1 and TAB2 are pre-associated on the membrane
before stimulation (Jiang et al., 2002). Western blot
analysis revealed that TAB3 proteins migrated more
slowly on SDS±PAGE in cells treated with IL-1 (bottom
panel, lanes 4, 8 and 12). These slowly migrating bands
were eliminated by phosphatase treatment (data not
shown), indicating that they may represent phosphorylated
TAB3.
We next examined the effect of TNF on the interaction
of endogenous TAB3 with TRAF2. 293IL-1RI cells were
either left untreated or were stimulated with TNF and the
interaction of TRAF2 with TAK1, TAB2 and TAB3 was
examined by immunoprecipitation. Stimulation with TNF
resulted in a markedly greater interaction between TRAF2
and TAB3, whereas no signi®cant interaction was
observed in the absence of TNF (Figure 5B, top panel,
lanes 10±12). In addition, TNF stimulation substantially
increased the interaction of TRAF2 with TAK1 (lanes 2±
4) and TAB2 (lanes 6±8). The kinetics of TNF-induced
TRAF2±TAB3 association were similar to those for
TRAF2±TAK1 and TRAF2±TAB2 association. The asso-
ciation was speci®c for TNF stimulation, because IL-1
treatment did not induce the interaction of TRAF2 with
TAB2 or TAB3 (Figure 6B). Taken together, these results
suggest that IL-1 and TNF stimulation transiently induces
the formation of complexes composed of TRAF6±TAB2±
TAB3±TAK1 and TRAF2±TAB2±TAB3±TAK1, respect-
ively.
TAB2 and TAB3 are ubiquitinated in response to
IL-1 stimulation
IL-1 signaling leads to TRAF6 polyubiquitination, thereby
triggering activation of TAK1 (Deng et al., 2000; Wang
et al., 2001). TRAF6 functions as an E3 ligase, facilitating
the synthesis of polyubiquitination chains. Thus one of the
targets of IL-1-induced ubiquitination is TRAF6 itself.
The interaction of TRAF6 with TAB2 and TAB3
suggested that TAB2 and TAB3 might be substrates for
TRAF6 ligase activity. To test this possibility, we
transfected 293 cells with either T7-TAB2 or T7-TAB3
in the presence or absence of Flag-TRAF6. Cell extracts
were immunoprecipitated with anti-T7 antibody and then
immunoblotted for the presence of ubiquitin (Ub). TRAF6
association with TAB2 and TAB3 was detected in cells
cotransfected with TRAF6 (Figure 6A, top panel, lanes 3
and 6). Immunoblot detection of Ub revealed a smear of
material containing TAB2 and TAB3 between 100 kDa
and 220 kDa (second panel, lanes 3 and 6). These results
indicate that overexpression of TRAF6 can induce Ub
modi®cation in TAB2 and TAB3. When T7-TAB3Dcc,
lacking the coiled-coil domain, was coexpressed with
Flag-TRAF6, it was found to continue to associate with
TRAF6 (top panel, lane 8) but was ubiquitinated very
poorly (second panel, lane 8). Thus the coiled-coil domain
of TAB3 is required for stimulation of TAB3 ubiquitina-
tion by TRAF6. TRAF2 has also been shown to act as an
E3 ubiquitin ligase and to ubiquitinate itself (Shi et al.,
2003). Coexpression of TRAF2 with TAB2 or TAB3 did
not induce Ub modi®cation of the TAB2 or TAB3 proteins
(second panel, lanes 2 and 5).
We next tested whether IL-1 stimulation could trigger
ubiquitination of TAB2 and TAB3. Endogenous TAB2 or
TAB3 proteins were immunoprecipitated with anti-TAB2
or anti-TAB3 antibody, respectively, from 293IL-1RI
cells, which had been treated with IL-1 or were untreated.
Immunoprecipitates were immunoblotted with anti-
TRAF6, anti-TRAF2 or anti-Ub antibody. We again
observed that IL-1 stimulation induced association of
TRAF6 with TAB2 and TAB3 (Figure 6B, top panel,
lanes 3 and 9). Following IL-1 treatment, Ub modi®cation
of TAB2 or TAB3 was observed (third panel, lanes 3, 4, 9
and 10). Similarly, we examined the effect of TNF
stimulation on Ub modi®cation of TAB2 and TAB3
Fig. 5. Ligand-dependent association of TRAF6 and TRAF2 with
TAK1, TAB2 and TAB3. 293IL-1RI cells were treated with (A) IL-1
(10 ng/ml) or (B) TNFa(10 ng/ml) for the indicated time periods.
Endogenous TAK1, TAB2 and TAB3 were immunoprecipitated with
anti-TAK1 (T1), anti-TAB2 (T2) and anti-TAB3 (T3) antibodies,
respectively. Complexes immunoprecipitated with control IgG (C) or
each antibody was immunoblotted with anti-TRAF6, anti-TRAF2, anti-
TAK1, anti-TAB2 or anti-TAB3 antibodies. Whole-cell extracts were
immunoblotted with anti-TRAF6 or anti-TRAF2 antibodies.
Role of TAB3 in IL-1 and TNF signaling pathways
6283
proteins. We observed that TNF stimulation induced
association of TRAF2 with TAB2 and TAB3 (second
panel, lanes 5 and 11). However, TNF induced weak
ubiquitination of TAB3 (third panel, lane 12) but not
TAB2 (lanes 5 and 6). Next, we investigated whether
TRAF6 was required for the IL-1-induced Ub modi®cation
of TAB2. Since the E3 ligase activity of TRAF6 is
dependent on its N-terminal RING ®nger motif (Deng
et al., 2000; Wang et al., 2001), we examined the activity
of a mutant TARF6 lacking the RING ®nger domain,
TRAF6DN, on TAB2 modi®cation in response to IL-1. We
found that overexpression of TRAF6DN prevented IL-1-
induced ubiquitination of TAB2 (Figure 6C, lane 4).
Taken together, these results suggest that IL-1 signaling
leads to ubiquitination of TAB2 and TAB3 through the
action of TRAF6.
TAB2 and TAB3 have redundant functions in
mediating IL-1 and TNF signaling
To investigate the functional roles of TAB2 and TAB3 in
IL-1- and TNF-mediated signaling, we used siRNAs
directed against their respective mRNAs. Western blot
analysis showed that TAB2 siRNA transfection of HeLa
cells caused a reduction in TAB2 protein levels (Figure 7A
and B, fourth panels, lanes 4±6), but had little effect on
TAB3 levels (®fth panels, lanes 4±6). Conversely, cells
Fig. 6. Ubiquitination of TAB2 and TAB3. (A) Effect of TRAF2 and TRAF6 on ubiquitination of TAB2 and TAB3. 293IL-1RI cells were transfected
with plasmids encoding T7-TAB2 (2), T7-TAB3 (3), T7-TAB3Dcc (Dcc), Flag-TRAF2 (2) and Flag-TRAF6 (6) as indicated. Complexes immunopreci-
pitated with anti-T7 antibody were immunoblotted with anti-Flag, anti-ubiquitin (anti-Ub) or anti-T7 antibodies. Whole-cell extracts were immuno-
blotted with anti-Flag antibody. (B) Effect of IL-1 and TNFastimulation on ubiquitination of TAB2 and TAB3. 293IL-1RI cells were treated with
IL-1 (10 ng/ml) or TNFa(20 ng/ml) for the indicated time periods. Cell extracts were subjected to immunoprecipitation with control IgG (C), anti-
TAB2 (T2) or anti-TAB3 (T3) antibodies. Immunoprecipitated complexes were immunoblotted with anti-TRAF6, anti-TRAF2, anti-Ub, anti-TAB2 or
anti-TAB3 antibodies. Whole-cell extracts were immunoblotted with anti-TRAF6 or anti-TRAF2 antibodies. (C) Effect of TRAF6DN on IL-1-induced
ubiquitination of TAB2. 293IL-1RI cells were transfected with a plasmid encoding TRAF6DN. At 48 h post-transfection, cells were treated with IL-1
(10 ng/ml) for 10 min. Cell extracts were subjected to the immunoprecipitation with anti-TAB2 antibody. Immunoprecipitated complexes were immu-
noblotted with anti-Ub or anti-TAB2 antibodies.
T.Ishitani et al.
6284
treated with TAB3 siRNA showed reduced levels of TAB3
protein (®fth panels, lanes 7±9) but had no effect on TAB2
(fourth panels, lanes 7±9). Furthermore, when cells were
cotransfected with TAB2 and TAB3 siRNAs, levels of
both proteins decreased (fourth and ®fth panels, lanes 10±
12). These results demonstrated that TAB2 and TAB3
siRNAs speci®cally blocked expression of their corres-
ponding proteins. In the presence of both TAB2 and TAB3
siRNAs, TAK1 protein levels did not change (top panels,
lanes 10±12), but TAB1 protein levels were slightly
decreased (Figure 7B, bottom panel, lanes 10±12),
suggesting that a decrease of TAB2/TAB3 protein levels
may affect the stability of TAB1 protein.
Previous studies have shown that activation of TAK1
coincides with TAK1 autophosphorylation, as evidenced
by a shift in the mobility of TAK1 in SDS±PAGE
(Kishimoto et al., 2000). To test the effect of TAB2 and
TAB3 siRNAs on IL-1- and TNF-induced activation of
TAK1, cells were transfected with the various siRNAs and
treated with either IL-1 or TNF. Lysates were prepared and
assayed for TAK1 activation. A shift in TAK1 mobility
was observed in cells transfected with control GFP siRNA
(Figure 7A and B, top panels, lane 3), or single transfection
of TAB2 siRNA (lane 6) or TAB3 siRNA (lane 9).
However, mobility shift following stimulation with IL-1
and TNF was signi®cantly inhibited by the cotransfection
of both TAB2 and TAB3 siRNAs (lane 12). These results
suggest that TAB2 and TAB3 play a redundant but critical
role in the IL-1- and TNF-induced activation of TAK1.
We next examined the ability of siRNAs directed
against TAB2 and TAB3 to prevent IL-1- and TNF-
induced NF-kB activation. Western blot analysis demon-
strated increases in both phospho-IkBaand IkBadegrad-
ation in control cells following treatment with either IL-1
or TNF (Figure 7A and B, second and third panels, lanes 2
and 3). Transfection with either TAB2 or TAB3 siRNA
alone had little effect on IkBaphosphorylation or
degradation in response to IL-1 or TNF (lanes 5, 6, 8
and 9). However, when cells were transfected with TAB2
and TAB3 siRNAs together and then treated with either
IL-1 or TNF, the amount of phospho-IkBadecreased and
IkBadegradation was slightly inhibited (lanes 11 and 12).
Fig. 7. Effects of TAB2 and TAB3 siRNAs on IL-1 and TNF signaling pathways. HeLa cells were transfected with annealed sense and antisense 21-
mer siRNA oligonucleotides directed against Jelly®sh GFP (GFP; as a control siRNA), TAB2 or TAB3 using Oligofectamine: 400 nM GFP siRNA;
200 nM TAB2 siRNA + 200 nM GFP siRNA; 200 nM TAB3 siRNA + 200 nM GFP siRNA; 200 nM TAB2 siRNA + 200 nM TAB3 siRNA. At 72 h
post-transfection, the cells were treated with IL-1 (10 ng/ml) (Aand C) or TNFa(10 ng/ml) (Band D) for the indicated times (A and B) or for
10 min (C and D). Western blot analysis was performed on extracts prepared from these cells using antibodies directed against TAK1, phospho-
IkBa(IkB-P), IkBa, TAB2, TAB3, TAB1, phospho-p38, p38 or JNK1. Western blot analysis for b-catenin indicates that an equivalent amount of
protein was present in each lane.
Role of TAB3 in IL-1 and TNF signaling pathways
6285
These ®ndings were consistent with those obtained by gel
retardation analysis, which demonstrated that IL-1 or TNF
stimulation induced smaller increases in NF-kB binding in
extracts prepared from cells transfected with both TAB2
and TAB3 siRNAs compared with control cells or cells
transfected with each individual siRNA (Supplementary
®gure S1 available at The EMBO Journal Online). These
data suggest that TAB2 and TAB3 have redundant
functions in activating the IL-1- and TNF-induced
NF-kB pathway.
In addition to NF-kB activation, IL-1 and TNF induce
activation of p38 and JNK MAPKs (Baud and Karin,
2001). Similar experiments were performed to determine
the effect of siRNAs directed against both TAB2 and
TAB3 on IL-1- and TNF-induced activation of p38 and
JNK. We observed no effect of transfecting individual
TAB2 or TAB3 siRNA on activation of p38 and JNK
induced by IL-1 or TNF (Figure 7C and D, top and third
panels, lanes 4 and 6). However, the combination of TAB2
and TAB3 siRNAs reduced both IL-1- and TNF-induced
activation of p38 and JNK (lane 8). Thus, TAB2 and
TAB3 are important for activation of p38 and JNK in
response to IL-1 and TNF. Taken together, these results
suggest that TAB2 and TAB3 have redundant functions in
mediating both IL-1 and TNF signal transduction path-
ways.
Discussion
Previous studies have demonstrated that the TAK1-
associating protein TAB2 interacts with TRAF6 in an
IL-1-dependent manner, resulting in the formation of a
TRAF6±TAB2±TAK1 complex (Takaesu et al., 2000).
Formation of this complex appears to be required for IL-1-
mediated activation of NF-kB. TAB2 acts as an adaptor
that links TAK1 and TRAF6, and thereby mediates the
activation of TAK1 in the IL-1 signaling pathway. By this
model, TAK1 and TAB2 are important components in the
IL-1 pathway, but their physiological functions in this
signal cascade have yet to be fully clari®ed. Recent studies
using siRNA have demonstrated that TAK1 is critical not
only for IL-1-mediated activation of the NF-kB pathway
but also for TNF activation of this pathway (Takaesu et al.,
2003). In addition, Vidal and coworkers have reported that
dTAK1, a Drosophila melanogaster homolog of TAK1,
controls the activity of the Rel/NF-kB-like transactivator
Relish in the Imd pathway (Vidal et al., 2001). Taken
together, these ®ndings support the idea that TAK1 plays
an essential role in the NF-kB activation pathway. Mice
de®cient for TAB2 exhibit embryonic lethal due to liver
degeneration and apoptosis, yet embryonic ®broblasts
de®cient for TAB2 show no impairment in IL-1- or TNF-
induced activation of NF-kB (Sanjo et al., 2003). This is in
contrast with cells transfected with TAK1 siRNA, which
do exhibit impaired IL-1 and TNF signaling. These
®ndings demonstrate that the embryonic lethality caused
by TAB2 deletion is not due to defective IL-1- or TNF-
mediated signaling itself, but to some other defect leading
to liver apoptosis. We and other workers (Munoz-Sanjuan
et al., 2002) have now identi®ed a novel TAK1-binding
protein, termed TAB3, which is closely related to TAB2 in
structure, in mouse, human and Xenopus laevis. In this
study, we demonstrate that TAB2 and TAB3 have
redundant functions in activating both IL-1 and TNF
signaling pathways, and that TAB3 may compensate for
the loss of TAB2.
We present several lines of evidence supporting the role
of TAB3 as a signal transducer in both IL-1 and TNF
signaling pathways. First, ectopic expression of TAB3
results in the activation of both NF-kB and JNK, and
therefore mimics these two IL-1- and TNF-induced
cellular responses. Secondly, the C-terminal half of
TAB3 behaves as a dominant negative mutant that blocks
both IL-1- and TNF-induced activation of NF-kB. Thirdly,
endogenous TAB3 associates with TRAF6 and TRAF2 in
an IL-1- and TNF-dependent manner, respectively.
Fourthly, TAB3 interacts with TAK1 and thereby medi-
ates its association with TRAF6 and TRAF2. Finally, IL-1
and TNF stimulation induces ubiquitination of TAB3.
Collectively, these data are consistent with the idea that
TAB3 participates in both IL-1 and TNF signaling by
linking TAK1 with TRAF6 and TRAF2, which are
upstream components of each pathway.
As compared with transgenic knockout mice, siRNA
does not totally prevent the translation of its target mRNA.
However, siRNA has the advantage that it can be
introduced either individually or in combination into the
same cells to decrease the level of speci®c proteins. Here,
siRNA has been utilized to speci®cally decrease the
expression of TAB2 and TAB3 in order to better de®ne
their respective roles in regulating the IL-1 and TNF
signaling pathways. Neither TAB2 nor TAB3 siRNA
alone had any signi®cant effect on IL-1- or TNF-induced
activation of TAK1 and NF-kB, even though each of these
individual siRNAs speci®cally decreased expression of
their respective target protein. However, cotransfection of
the TAB2 and TAB3 siRNAs resulted in inhibition of both
IL-1- and TNF-induced activation of TAK1 and NF-kB.
Thus, at least in HeLa cells, it seems likely that TAB2 and
TAB3 have redundant roles in IL-1- and TNF-mediated
signaling. Although our previous studies demonstrated
that TAB2 functions mainly in the IL-1 signaling pathway
(Takaesu et al., 2000, 2001), it appears that TAB2 is able
to compensate for the loss of TAB3 in the TNF signaling
pathway. The generation and analysis of TAB3-de®cient
mice will reveal whether mammalian TAB3 is in fact
involved in IL-1- and TNF-mediated activation of NF-kB.
Furthermore, it would be interesting to determine whether
aDrosophila TAB2/3 homolog would be involved in
dTAK1-mediated Imd pathway.
Our previous and present studies suggest a model in
which IL-1 and TNF stimulation facilitates the formation
of TRAF6±TAB2/TAB3±TAK1 and TRAF2±TAB2/
TAB3±TAK1 complexes, respectively, leading to the
activation of TAK1. Our result demonstrating that
TAB2, TAB3 and TAK1 preform a complex in the
absence of stimulation is consistent with a recent obser-
vation that the TRIKA2 complex containing TAK1, TAB1
and TAB2 is formed constitutively (Wang et al., 2001),
and further suggests that TAB3 may be a component of the
TRIKA2 complex. In the case of the IL-1 signaling
pathway, TRAF6 acts as a mediator between the IL-1
receptor complex and TAK1, and TAB2/TAB3 function as
adaptors that link TAK1 and TRAF6. In addition, these
authors have demonstrated that ubiquitination of TRAF6 is
involved in TAK1 activation by IL-1, leading to phos-
T.Ishitani et al.
6286
phorylation and activation of the IKK complex (Wang
et al., 2001). How TRAF6 activates TAK1 remains
unknown, although it apparently depends upon the E3
ligase activity of TRAF6, which promotes or perhaps
stabilizes the oligomerization of TRAF6 (Deng et al.,
2000; Wang et al., 2001). Interestingly, TAB2 and TAB3
contain a ubiquitin-binding domain, raising the possibility
that TAB2 and TAB3 mediate TAK1 activation through a
ubiquitin-mediated step. Consistent with this possibility,
we found that TAB2 and TAB3 were ubiquitinated by
either IL-1 stimulation or TRAF6 overexpression. These
results suggest that stimulation of cells with IL-1 leads to
the ubiquitination of TAB2 and TAB3 by stimulating the
E3 ligase activity of TRAF6. Other studies have shown
that TAK1 is activated by autophosphorylation
(Kishimoto et al., 2000). Based on these results, we
speculate that ubiquitination of the TAB2±TAB3 complex
may alter the conformation of TAK1, which induces its
kinase activity leading to autophosphorylation. Although
TRAF2 is another E3 that plays an important role in TNF
signaling (Yeh et al., 1997; Shi and Kehrl, 2003), it
remains to be determined whether TRAF2 mediates
ubiquitination of TAB2 and TAB3. Further study is
required to clarify the exact mechanisms by which
ubiquitination of TAB2 and TAB3 activates TAK1.
Materials and methods
Cloning of human TAB3
Full-length human TAB3 gene was ampli®ed by PCR from a human
kidney cDNA library (Clontech) with the following primers: 5¢-GCC-
GGTTAACATCCATTTCC-3¢(5¢-primer), 5¢-TTTCACTTTCAACC-
TGGCGC-3¢(3¢-primer) for the ®rst reaction, and 5¢-ATTTGCTCTGG-
CATGGCGCAAAG-3¢(5¢-primer), 5¢-ATTCAGGTGTACCGTGGCA-
TCTC-3¢(3¢-primer) for the second reaction. The ampli®ed cDNAs were
subcloned into pCR2.1-TOPO (Invitrogen) and completely sequenced.
Expression vectors and antibodies
To overexpress T7-TAB3, we constructed pCMV-T7-TAB3, a vector
expressing T7-TAB3 under the control of the cytomegalovirus (CMV)
promoter. Mammalian expression vectors encoding TAK1, TAB2,
TRAF6, TRAF2 and JNK have been described (Ninomiya-Tsuji et al.,
1999; Takaesu et al., 2000). The TAB3 mutants, TAB3N, TAB3C and
TAB3Dcc, were generated by PCR. Polyclonal rabbit antibody against
TAB3 was produced using peptides corresponding to amino acids 635±
648 of TAB3. This antibody reacted with TAB3 but not TAB2.
Polyclonal rabbit antibodies against TAK1 and TRAF6 have been
described (Ninomiya-Tsuji et al., 1999; Takaesu et al., 2000). Anti-
TRAF2 and anti-b-catenin were from Pharmingen. Anti-ubiquitin, anti-
p65 and anti-JNK were from Santa Cruz. Anti-phospho-IkBa, anti-IkBa,
anti-phospho-p38 and anti-p38 were from Cell Signaling. Anti-T7 was
from Novagen. Anti-HA (HA.11) was from Babco. Anti-Flag (M2) and
control immunoglobulin G (IgG) were from Sigma.
Cell culture and transfection
The 293IL-1RI cells were described previously (Ninomiya-Tsuji et al.,
1999). The HEK293, 293IL-1RI and HeLa cells were grown in
Dulbecco's modi®ed Eagle's medium (DMEM) supplemented with
10% fetal bovine serum. The 293 cells in 100 mm plates were transfected
with the expression plasmids (10 mg) by calcium phosphate precipitation.
Reporter gene assays
The 293 and 293IL-1RI cells (1.6 3105cells/well) were seeded into six-
well (35 mm) plates. Cells were transfected by the calcium phosphate
precipitate method 24 h after seeding with the NF-kB-Luc reporter gene
plasmid, along with each expression vector as indicated. The total DNA
concentration (1.7 mg) was kept constant by supplementing with empty
vector DNAs. IL-1b(5 ng/ml) or TNFa(10 ng/ml) was added to each
plate 3 h after transfection. At 24 h after transfection, luciferase activity
was determined with the Luciferase Assay System (Promega). b-Gal
vector (0.1 mg), under the control of the b-actin promoter, was used for
normalizing transfection ef®ciencies. The values shown are the averages
of one representative experiment in which each transfection was
performed in duplicate.
Immunoprecipitation and immunoblotting
Cells were either left untreated or treated with IL-1b(5 ng/ml) or TNFa
(10 ng/ml) for the indicated times. Cells were washed once with ice-cold
phosphate-buffered saline and lysed in 0.3 ml 0.5% Triton X-100 lysis
buffer containing 20 mM HEPES pH 7.4, 150 mM NaCl, 12.5 mM
glycerophosphate, 1.5 mM MgCl
2
, 2 mM EGTA, 10 mM NaF, 2 mM
dithiothreitol (DTT), 1 mM sodium orthovanadate, 1 mM phenylmethyl-
sulfonyl ¯uoride (PMSF) and 20 mM aprotinin. Proteins from cell extracts
were immunoprecipitated with various antibodies and protein G-
Sepharose (Pharmacia). For immunoblotting, immunoprecipitates or
whole-cell extracts were resolved by SDS±PAGE and transferred to
Hybond-P membranes (Amersham). The membranes were immuno-
blotted with various antibodies, and the bound antibodies were visualized
with horseradish-peroxidase-conjugated antibodies against rabbit or
mouse IgG by using the Enhanced Chemiluminescence (ECL) Western
Blotting System (Amersham).
In vitro kinase assays
Bacterially expressed GST-c-Jun or MKK6 were described previously
(Shirakabe et al., 1997). Aliquots of immunoprecipitates were incubated
with substrates (1 mg) in 10 ml kinase buffer containing 10 mM HEPES
pH 7.4, 1 mM DTT, 5 mM MgCl
2
and 5 mCi [g-32P]ATP at 25°C for 2 min.
Samples were resolved by SDS±PAGE and phosphorylated proteins were
visualized by autoradiography.
RNA oligonucleotides
siRNAs with two thymidine residues (dTdT) at the 3¢end of the sequence
were designed against the TAB3 (sense 5¢-CCACCUCAACAGCCA-
UCUU-3¢) and TAB2 (sense 5¢-CCUCCAGCACUUCCUCUUC-3¢)
mRNAs along with their corresponding antisense RNA oligonucleotides
(Japan Bio Service). These RNAs were dissolved in DEPC-treated water
to 50 mM, heated to 90°C in buffer (30 mM HEPES±KOH pH 7.4,
100 mM potassium acetate, 2 mM magnesium acetate) and annealed at
37°C. As a control, we used siRNA directed against Jelly®sh GFP
(Nippon Gene).
Transfection of RNA oligonucleotides
HeLa cells (1 3105cells/well) were seeded into six-well (35 mm) plates
to 20±30% con¯uency, and transfection of the RNA oligonucleotides was
performed using Oligofectamine (Invitrogen) to a ®nal RNA concentra-
tion of 400 nM. The cells were harvested at different time points post-
treatment with IL-1b(10 ng/ml) or TNFa(10 ng/ml) and lysed for use in
western blot analysis.
Gel retardation assays
HeLa cells were harvested 30 min after treatment with IL-1b(10 ng/ml)
or TNFa(10 ng/ml) and lysed for use in gel retardation assays. NF-kB
oligonucleotides were from Promega. Binding reactions were performed
at room temperature for 10 min by incubating 7 ml of total lysates and
0.035 pmol of labeled oligonucleotides in 15 ml of binding buffer (10 mM
Tris pH 7.5, 50 mM NaCl, 1 mM MgCl
2
, 0.5 mM EDTA, 0.5 mM DTT,
4% glycerol, 0.75 mg poly(dI-dC), 1.5 mg salmon sperm DNA, 1.5 mg
BSA).
Supplementary data
Supplementary data are available at The EMBO Journal Online.
Acknowledgements
We thank M.Lamphier for critical reading of the manuscript. This work
was supported by special grants from CREST, Advanced Research on
Cancer from the Ministry of Education, Culture and Science of Japan and
Yamanouchi Foundation for Research on Metabolic Disorders (K.M.).
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Received June 25, 2003; revised September 15, 2003;
accepted October 14, 2003
T.Ishitani et al.
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