From the Institute of Pharmacology, Medical School
Hannover, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany and the
§ Department of Molecular Biology, Graduate School of
Science, Nagoya University, Nagoya 464-8602, Japan
Received for publication, May 22, 2000, and in revised form, October 23, 2000
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ABSTRACT |
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Mechanisms of fulminant gene induction during an
inflammatory response were investigated using expression of the
chemoattractant cytokine interleukin-8 (IL-8) as a model. Recently we
found that coordinate activation of NF- The proinflammatory cytokine
IL-11 mediates its effects
primarily by reprogramming gene expression in inflamed tissues. It is
therefore likely that clarifying the molecular mechanisms of IL-1-induced gene expression will yield novel molecular targets for
anti-inflammatory therapy (1). Among the many genes induced by IL-1 are
those of chemokines, such as IL-8 (2). Blockade of
IL-1 receptors by application of the IL-1 receptor antagonist consistently reduces tissue-neutrophilia in a variety of disease models
presumably by preventing IL-1-induced synthesis of IL-8 and related
chemokines (reviewed in Ref. 1), thus illustrating the
pathophysiological importance of IL-1-induced expression of these genes.
IL-1 receptors have been molecularly cloned and are ubiquitously
expressed. Binding of IL-1 to the cell surface results in formation of
an IL-1 receptor complex consisting of the type I IL-1 receptor, the
IL-1 receptor accessory protein, MyD88, TRAF6, and the protein kinase
IRAK. TRAF6 couples the receptor complex to the I- MAPKKs require phosphorylation in the conserved kinase domain VIII for
activation. This is achieved by MAP kinase kinase kinases (MAPKKK). To
date, a remarkably large group of distinct enzymes has been shown to
fulfill this function, comprising Raf-1, A-Raf, B-Raf, Mos, NIK,
MEKK1-4, MLK2, MLK3, DLK/MUK, ASK1, Tpl-2/Cot, and TAK1 (8). The JNK
and p38 MAPK signaling pathways can be activated by ectopically
expressed MEKK1-4, MLK2, MLK3, DLK/MUK, ASK1, Tpl-2/Cot, and TAK1,
albeit to varying degrees (7, 8). Moreover, it is becoming increasingly
evident that most of these MAPKKKs also directly activate the I- It is unclear which MAPKKKs are utilized by the activated IL-1 receptor
complex. It is also unclear if different stimuli use the same MAPKKK
for a particular biological response. IL-1 shares many biological
responses with the other major proinflammatory cytokine TNF (1).
Inactive mutants of NIK, MEKK1, ASK, and TAK1 suppressed
TNF-dependent NF- TAK1 binds to novel TAK binding proteins (TAB) 1 and 2. Its interaction
with TAB1 is crucial for activation (33). Recently, it was shown that
TAK1 and TAB1 associate with the adapter molecule TRAF6 of the IL-1
receptor complex in an IL-1-dependent fashion (13).
IL-8 induction represents an important (patho)physiological response to
IL-1. IL-8 gene regulation is under complex
transcriptional and post-transcriptional control, involving AP-1,
CAAT/enhancer-binding protein, and NF- Based on the above evidence for a role of TAK1 in IL-1 signaling we set
up experiments to investigate the role of TAK1 in IL-1-induced
IL-8 gene expression.
Cells and Materials--
HeLa cells stably expressing the
tet transactivator protein (35, kindly provided by H. Bujard) were cultured in Dulbecco's modified Eagle's medium
complemented with 10% fetal calf serum. [ Plasmids--
The expression plasmid for GST-JUN (amino acids
1-135) was kindly provided by J. R. Woodgett. GST fusion proteins
were expressed and purified from Escherichia coli by
standard methods. pCDNA3MYC-MK2 was a kind gift of M. Gaestel.
pCMV-TAB11-418 encoding C-terminally truncated TAB1,
pEF-TAB1, pcDLR Transfections and Reporter Assays for Transcription and mRNA
Stability--
Transient transfections by the calcium phosphate method
and determination of luciferase reporter gene activity were performed as described (17). Equal amounts of plasmid DNA within each experiment
were obtained by adding empty vector. For determination of promoter
activity cells (seeded at 5 × 105 per well of 6-well
plates) were transfected with 0.25 µg of the IL-8-promoter or the
5xNF-
For measurements of IL-8 secretion 2 × 105 cells per
well were seeded in 6-well plates and transfected with 4 µl of
LipofectAMINE (Life Technologies) and 2 µg of DNA according to the
manufacturer's instructions. 24 h later the medium was changed
and cells were stimulated with IL-1 Preparation of Cell Extracts--
Cells were harvested and
washed in ice-cold phosphate-buffered saline. For preparation of
whole-cell extracts, cells were lysed in 10 mM Tris, pH
7.05, 30 mM NaPPi, 50 mM NaCl, 1%
Triton X-100, 2 mM Na3VO4, 50 mM NaF, 20 mM Immune Complex Protein Kinase Assays--
Whole-cell extract
(100-250 µg of protein) was diluted in 500 µl of ice-cold
immunoprecipitation (IP) buffer A (20 mM Tris, pH 7.4, 154 mM NaCl, 50 mM NaF, 1 mM
Na3VO4, 1% Triton X-100). Samples were
incubated for 3 h with 2 µl of antiserum SAK9 to immunoprecipitate endogenous JNK, 1 µg of 9E10 (anti-MYC) or 1 µg of 12CA5 (anti-HA) antibodies to immunoprecipitate epitope-tagged MYC-MK2 or HA-JNK2, respectively, followed by the addition of 20 µl
of protein A-Sepharose beads (SAK9) or protein G-Sepharose beads (9E10,
12CA5) for 1-2 h at 4 °C. Beads were spun down, washed 3× in 1 ml
of IP buffer A, and resuspended in 10 µl of the same buffer. Then 1 µg of recombinant protein substrates (GST-JUN or HSP27,
respectively) in 10 µl of H2O and 10 µl of kinase
buffer (150 mM Tris, pH 7.4, 30 mM
MgCl2, 60 µM ATP, 4 µCi of
[ Western Blotting--
Cell extract proteins were separated on
10% SDS-PAGE and electrophoretically transferred to polyvinylidene
difluoride membranes (Immobilon, Millipore). After blocking with 5%
dried milk in Tris-buffered saline overnight, membranes were incubated
for 4-24 h with primary antibodies, washed in Tris-buffered saline,
and incubated for 2-4 h with the peroxidase-coupled secondary
antibody. Proteins were detected by using the Amersham Pharmacia
Biotech enhanced chemiluminescence system.
Electrophoretic Mobility Shift Assay--
Double-stranded
oligonucleotides corresponding to the IL-8 promoter sequence
5'-ccatgggtggaatttcctctgacatg-3' (NF- ELISA--
IL-8 protein concentrations in the cell culture
medium were determined using the human IL-8 duo set kit (R&D Systems)
exactly following the manufacturer's instructions.
The NF-
Initially we confirmed that TAK1 activates all three signal
transduction pathways within the same cell (Fig.
1). Simultaneous overexpression of TAK1
and its coactivator TAB1 in HeLa cells activated endogenous NF- Overexpression of TAK1 + TAB1 Is Sufficient to Activate
Transcription from a Minimal IL-8 Promoter in a
JNK-dependent Manner--
The effect of TAK1 activation on
IL-8 gene transcription was determined using a reporter
construct in which a minimal IL-8 promoter encompassing the AP-1,
NF-IL-6, and NF-
To compare the requirements for transcriptional activation by TAK1 + TAB1 and IL-1, the promoter was mutated at its NF- Overexpression of TAK1 + TAB1 Is Sufficient to Induce Stabilization
of a Mutants of TAK1 and TAB1 Interfere with IL-1-induced Transcription
and mRNA Stabilization--
Taken together, the data so far
indicate that TAB1-TAK1 activates mechanisms of transcription and
mRNA stabilization in a way similar to IL-1. The involvement of
TAK1 in IL-1-induced activation of both modes of gene regulation was
directly addressed using the kinase-inactive mutant
TAK1K63W. Its coexpression largely abrogated the activation
of NF-
In theory, TAK1K63W could exert its inhibitory function by
binding to and masking downstream targets, thereby preventing their phosphorylation and activation by other more relevant MAPKKKs. Therefore, it was necessary to construct mutants that would act more
selectively. Two such mutants were applied based on the following: first, TAK1 requires the interaction with TAB1 to function.
TAB11-418, a C-terminally truncated form of TAB1, which
lacks the TAK1 binding domain, acts as a dominant negative inhibitor in
transforming growth factor-
We initially confirmed that the TAK11-77 peptide and
the TAB11-418 deletion mutant alone were functionally
inactive in HeLa cells. Replacement of TAK1 by TAK11-77 or
replacement of TAB1 by TAB11-418 resulted in loss of
promoter activation by TAB1-TAK1 (Fig.
7A). Importantly, as shown in
Fig. 7B, expressing either of the two mutants strongly and
dose-dependently impaired promoter activation in response
to IL-1. The extent of inhibition was comparable with that achieved
with the full-length TAK1K63W mutant in parallel cell
cultures (Fig. 7B). In contrast, wild type TAK1 or
wild type TAB1 did not inhibit IL-1-induced IL-8 transcription,
indicating a specific dominant negative effect of TAK11-77
and TAB11-418 (Fig. 7C). Additional experiments showed that TAK11-77 did not affect activation of the IL-8 promoter by constitutively active MEK1, a specific activator of the ERK
pathway, thus ruling out a general suppressive effect of the TAK1 N
terminus (data not shown).
Finally we assessed the relevance of TAK1 for inducible formation of
IL-8 protein. Coexpression of TAK1 and TAB1, but not of each of them
alone, resulted in a strong increase in IL-8 secretion (Fig.
8A), showing that
TAB1-activated TAK1 is sufficient to activate the endogenous
IL-8 gene. To analyze the effect of kinase-inactive TAK1 on
IL-1-induced IL-8 formation, a liposome-based transfection procedure
was employed that yielded higher rates of transfected cells. For these
experiments the TAK1 cDNAs were fused to that of enhanced green
fluorescent protein (GFP). The GFP-tagged wild type TAK1 and
TAK1K63W behaved functionally similar to the respective HA-tagged forms in reporter gene assays (data not shown). Reproducibly more than 50% of cells expressed the GFP-TAK1 fusion proteins as
estimated by fluorescence-activated cell sorting analysis (data not
shown). Expression of GFP-TAK1 and GFP-TAK1K63W was also
monitored by Western blot (Fig. 8C). In these cell cultures
IL-1 induced a 10-fold increase in IL-8 secretion, which was hardly
affected by expressing GFP-TAK1 (Fig. 8B). In contrast,
expression of the kinase-inactive GFP-TAK1K63W resulted in
marked inhibition of IL-1-induced IL-8 secretion (by 52-67% in three
individual experiments). Taking into account the observed transfection
efficiency, this result suggests that in cells expressing
GFP-TAKK63W, IL-1-induced IL-8 secretion is almost
completely inhibited.
The data presented in Figs. 5-8 show that IL-1-induced
IL-8 gene expression is sensitive to disruption of IL-1
signaling by dominant negative mutants of both TAK1 and TAB1. In
conclusion, our data therefore suggest that the MAPKKK TAK1, together
with its coactivator TAB1, links the major mechanisms contributing to
IL-8 gene expression to the IL-1 receptor.
Studying induction of IL-8 as an example for highly inducible
gene expression, we have recently developed a model in which formation of large amounts of IL-8 requires the coordinated activation of at least three signaling pathways. First, activation of NF-B and c-Jun N-terminal
protein kinase (JNK) is required for strong IL-8 transcription, whereas
the p38 MAP kinase (MAPK) pathway stabilizes the IL-8 mRNA. It is
unclear how these pathways are coupled to the receptor for IL-1, an
important physiological inducer of IL-8. Expression of the MAP kinase
kinase kinase (MAPKKK) TAK1 together with its coactivator TAB1 in HeLa cells activated all three pathways and was sufficient to induce IL-8
formation, NF-
B + JNK2-mediated transcription from a minimal IL-8
promoter, and p38 MAPK-mediated stabilization of a reporter mRNA
containing IL-8-derived regulatory mRNA sequences. Expression of a
kinase-inactive mutant of TAK1 largely blocked IL-1-induced transcription and mRNA stabilization, as well as formation of endogenous IL-8. Truncated TAB1, lacking the TAK1 binding domain, or a
TAK1-derived peptide containing a TAK1 autoinhibitory domain were also
efficient in inhibition. These data indicate that the previously
described three-pathway model of IL-8 induction is operative in
response to a physiological stimulus, IL-1, and that the MAPKKK TAK1
couples the IL-1 receptor to both transcriptional and RNA-targeted
mechanisms mediated by the three pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B kinase pathway,
resulting in the release of I-
B from NF-
B and nuclear
translocation of the latter (3, 4). Furthermore, IL-1 activates the
c-Jun N-terminal kinase (JNK) and p38 MAP kinase (MAPK) pathways (5,
6). These MAP kinases are activated by dual-specificity MAP kinase
kinases (MAPKKs). JNKs are activated by MKK7, whereas p38 MAPKs are
activated by MKK3 and -6. Additionally, MKK4 activates both JNK and p38
MAPK (7, 8).
B
kinase/NF-
B signaling pathway in parallel, as demonstrated for
MEKK1-3 (9-12), TAK1 (13-15), and Tpl-2/Cot (16). On the other hand,
MAPKKKs exist that, like NIK, only activate NF-
B, but not JNK and
p38 MAPK (17-19).
B, JNK, and p38 MAPK activation, suggesting a role for these MAPKKKs in biological responses to TNF (9,
14, 19-24). However, for IL-1, similar information is limited to NIK
and TAK1 mutants (18, 13). An important question, therefore, that is
yet to be answered is which MAPKKKs are physiologically involved in
IL-1-induced gene expression. Changes in enzymatic activity of
endogenous MAPKKKs in immunoprecipitation experiments have proven
difficult to detect (25). Recently, however, endogenous TAK1 was shown
to be activated by IL-1 as well as by TNF (13, 26). In that context it
is interesting to note that TAK1 was originally found to function in
signaling of transforming growth factor-
(15, 27-30), a cytokine
which has been described to have effects distinct from and in part
opposing those of IL-1 and TNF (31, 32).
B binding sites and AU-rich
mRNA regions as cis-elements, respectively (17, 34). We
have recently shown by expressing active forms of NIK, MKK7, and MKK6
that IL-8 transcription is controlled by the coordinated action of
NF-
B and JNK pathways, whereas IL-8 mRNA degradation is
regulated by the p38 MAPK pathway (17, 34).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was
purchased from Hartmann Analytics. Rabbit antiserum SAK9 to the C
terminus of JNK2 (36) was a kind gift of Jeremy Saklatvala. Polyclonal
rabbit antibodies against TAK1 have been described (13) or were
obtained from Santa Cruz (SC-M-579). Antibodies 12CA5 against
hemagglutinin (HA) and 9E10 against C-MYC epitopes were from Roche
Molecular Biochemicals. Horseradish peroxidase-coupled secondary
antibodies against mouse, rabbit, and rat IgG were from Sigma. Protein
A-, protein G-, and glutathione-Sepharose were from Amersham Pharmacia
Biotech. Human recombinant IL-1-
was produced as described
previously (36). Recombinant bacterially expressed HSP27 was a kind
gift of M. Gaestel.
HA-TAK1, and pcDLR
HA-TAK1K63W have
been described elsewhere (13, 26, 33). pcDLR
HA-TAK11-77 was constructed by excising a SacI/SacI fragment
encoding the C-terminal 520 amino acids of TAK1 from the
pcDLR
HA-TAK1 plasmid. To generate expression vectors for GFP-TAK1
fusion proteins, the cDNAs (nucleotides 159-1896) of TAK1 or
TAK1K63W were amplified by polymerase chain reaction using
the oligonucleotides 5'-cgcggaattcgtcgacagcctccgccg-3' (sense) and
5'-cgcggaattctcatgaagtgccttgtcgtttc-3' (antisense) and subcloned into
the EcoRI site of peGFPC1 (CLONTECH).
peVHA-JNK2, peVHA-JNK2K55R, and pCS3FLAG-p38AGF
have been described previously (17, 34). The NF-
B reporter plasmid
pNF-
B-luc was obtained from Stratagene. pSV-
-gal coding for SV40
promoter driven
-galactosidase was from Promega. The IL-8
promoter-luciferase reporter plasmid pUHC13-3-IL-8pr (nucleotides
1348-1527 of the IL-8 gene) and the corresponding mutants
in the NF-
B and AP-1 cis elements have been described in
detail (17). The
-globin-IL-8 hybrid mRNA, containing a fragment
of the 3'-untranslated region of the IL-8 mRNA (nucleotides
972-1310), which includes the AU-rich regulatory region, was expressed
under the control of the tetracycline-regulated promoter, using the
ptetBBB-IL-8972-1310 plasmid (34).
B luciferase reporter plasmid and 0.5 µg of pSV-
-gal
(Promega).
-Galactosidase activity was determined (using reagents
from CLONTECH) to allow normalization of luciferase activity in different transfections. RNA degradation kinetics were
measured using the tet-off system as described (34).
Briefly, cells (5 × 106 seeded per 9-cm-diameter
dish) were transfected with the ptetBBB-IL-8972-1310 plasmid (3 µg). For each kinetics, cells from one transfection were
distributed into parallel cultures. The next day transcription of the
-globin-IL-8 DNA was stopped by addition of the tetracycline analog
doxycycline (3 µg/ml). At the indicated times thereafter, total RNA
was isolated, and Northern blot analysis was performed using a
-globin antisense RNA probe labeled with digoxygenin (Roche
Molecular Biochemicals). The RNA half-life was quantified as in
a previous study (34) using a video-imaging system and the Molecular
Analyst program (Bio-Rad).
(10 ng/ml) or left untreated.
After 17 h, culture media were collected for IL-8 ELISA. Cell
pellets were lysed, and transfected proteins were analyzed by Western blotting.
-glycerophosphate, and freshly added 0.5 mM PMSF, 0.5 µg/ml leupeptin, 0.5 µg/ml
pepstatin, 10 mM para-nitrophenyl phosphate, 400 nM okadaic acid, 2 mM DTT (whole cell lysis
buffer) at 4 °C. After 10 min on ice, lysates were cleared by
centrifugation at 10,000 × g for 15 min at 4 °C.
Nuclear and cytosolic extracts were prepared as described previously
(17). Briefly, cells were suspended and pelleted in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM
MgCl2, 0.3 mM Na3VO4,
20 mM
-glycerophosphate and freshly added 2.5 µg/ml
leupeptin, 10 µM E-64, 300 µM PMSF, 1.0 µg/ml pepstatin, 5 mM DTT, 400 nM okadaic
acid). The pellet was resuspended in buffer A + 0.1% Nonidet P-40,
left on ice for 10 min, and then vortexed. After centrifugation at
10,000 × g for 5 min at 4 °C, supernatants were
taken as cytosolic extracts. Pellets were resuspended in buffer B (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25%
glycerol, 0.3 mM Na3VO4, 20 mM
-glycerophosphate, 2.5 µg/ml leupeptin, 10 µM E-64, 300 µM PMSF, 1.0 µg/ml
pepstatin, 5 mM DTT, 400 nM okadaic acid). After 1 h on ice, samples were vortexed and cleared at 10,000 × g for 5 min at 4 °C. Supernatants were collected as
nuclear extracts. Protein concentration of extracts was determined by the method of Bradford and samples stored at
80 °C.
-32P]ATP) were added. After 30 min at room
temperature, SDS-PAGE sample buffer was added and proteins were eluted
from the beads by boiling for 5 min. After centrifugation at
10,000 × g for 5 min, supernatants were separated on
10% SDS-PAGE. Phosphorylated proteins were visualized by autoradiography.
B site underlined) were end-labeled using
[
-32P]ATP and T4 polynucleotide kinase and purified by
gel filtration on S-200 spin columns (Amersham Pharmacia Biotech).
Protein-DNA binding reactions were performed with 5-20 µg of nuclear
extract protein, labeled oligonucleotide (1 × 105
cpm, 0.2-0.5 pmol), 1 µg of poly(dI-dC) in 10 mM Tris,
pH 7.5, 10 mM EDTA, 0.05% (w/v) dried low-fat milk, 50 mM NaCl, 10 mM DTT, and 10% glycerol in a
total volume of 10 µl. After incubation at room temperature for 30 min, protein-DNA complexes were resolved on 5% PAGE and visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, JNK, and p38 MAPK Pathways Are Activated by
Overexpressed TAK1 + TAB1--
IL-1 is a strong inducer of IL-8
synthesis. We previously suggested that the NF-
B and JNK pathways
cooperate to activate transcription, whereas the p38 MAPK pathway
stabilizes IL-8 mRNA (17, 34). How these downstream effects are
coupled to the IL-1 receptor complex remained unclear. IL-1 has been
shown to activate the MAPKKK TAK1 (13). Because it has been reported previously that wild-type TAK1 in a TAB1-dependent manner
activates NF-
B, JNK, and also p38 MAPK in a variety of cell systems
(13-15, 26, 30, 37-39), TAK1 is a candidate for a common upstream
activator of signaling leading to transcriptional as well as
post-transcriptional mechanisms involved in IL-8 induction.
B,
determined in EMSA with a radiolabeled probe corresponding to the
NF-
B binding site of the IL-8 promoter sequence (17), JNK as
determined by in vitro phosphorylation of its substrate
GST-JUN, and p38 MAPK as determined by in vitro phosphorylation of HSP27 as substrate for the p38 MAPK-activated kinase
MK2 (Fig. 1). Overexpression of a kinase-inactive mutant of TAK1,
TAK1K63W (13), with TAB1 (Fig. 1) as well as overexpression of TAK1 without TAB1 (not shown) had no effect.
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Fig. 1.
Coexpression of TAK1 and its activator TAB1
activates endogenous NF- B, JNK, and p38 MAPK
signaling pathways. A and B, cells were
transfected with empty vector or with pEF-TAB1 together with
pcDLR
HA-TAK1 or pcDLR
HA-TAK1K63W (2.5 µg each).
24 h later cytosolic, and nuclear extracts were prepared.
A, TAK1-dependent nuclear translocation and DNA
binding of NF-
B to the IL-8 promoter was measured by EMSA using 20 µg of nuclear extracts. B, endogenous JNK was
immunoprecipitated from cytosolic extracts with rabbit polyclonal
anti-JNK antibodies, and its activity was measured in vitro
using GST-JUN and [
-32P]ATP as substrates.
C, cells were transfected as in A and 2.5 µg of
pCDNA3MYC-MK2 in addition as indicated. Total amount of DNA was
kept constant in all transfections by adding empty vector. 24 h
later, cells were lysed in whole cell lysis buffer, and
TAK1-dependent activation of the p38 MAPK pathway was
measured by immune complex protein kinase assay of transfected
epitope-tagged MK2 using HSP27 and [
-32P]ATP as
substrates.
B binding sites was placed upstream of the
luciferase cDNA (17). Expression of TAK1 was controlled by Western
blot using anti-TAK1 antibodies (Fig.
2A). Cotransfection of
cDNAs for TAK1 and TAB1 resulted in strong activation of luciferase
activity (Fig. 2B). Consistent with earlier reports on the
mode of action of TAK1, transfection of TAK1 or TAB1 alone had no
significant effect. Transcriptional activation of the IL-8 promoter has
been suggested by us to involve signaling through both NF-
B and JNK
(17). To evaluate the contribution of the JNK pathway in
TAB1-TAK1-induced transcription, a kinase-inactive mutant of JNK2 in
which the ATP-binding lysine was changed to arginine,
JNK2K55R, was cotransfected. This resulted in a strong and
dose-dependent impairment of TAB1-TAK1-induced
transcription (Fig. 2, C and D). On the other
hand, a nonactivatable mutant of p38 MAPK in which the
phosphorylatable threonine and tyrosine residues were changed to
alanine and phenylalanine, p38AGF, had no significant
effect (Fig. 2D). Thus activation of the JNK pathway appears
crucial for TAB1-TAK1-induced transcriptional activation of the minimal
IL-8 promoter.
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Fig. 2.
TAK1 + TAB1-induced IL-8 transcription
requires the JNK but not the p38 MAPK pathway. HeLa cells were
cotransfected with a minimal IL-8 promoter-luciferase cDNA
construct (0.25 µg) (17), with 0.5 µg of pSV- -gal and with empty
vector or (A, B) expression vectors for TAB1 and
wild type or mutant TAK1 as described in the legend of Fig. 1, or
(C, D) expression vectors for TAK1 + TAB1 and
dominant negative JNK2 (JNK2K55R) or dominant negative p38
MAPK (p38AGF) (2.5 µg or indicated amounts of DNA).
A, TAK1 overexpression was confirmed by Western blotting
using polyclonal anti-TAK1 antibodies. B-D, luciferase
activity was determined in cell lysates. Results normalized for
transfection efficiency with
-gal (for details see "Experimental
Procedures") are expressed in relative light units (RLU), mean ± S.E. for triplicate determinations of one representative of two
experiments for C, mean ± S.E. from three independent
experiments performed in triplicates for B and
D.
B site or its AP-1
site or at both sites. Either mutation strongly impaired induction by
TAB1-TAK1 as well as by IL-1, and mutation of both sites abolished
inducibility by both stimuli (Fig. 3, A and B). These results show a similarity in the
effect of TAB1-TAK1 and IL-1 on transcription and thus are in line with
(but do not prove) involvement of the former in IL-1-induced
transcription.
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Fig. 3.
Activation of the minimal IL-8 promoter by
TAK1 + TAB1 or by IL-1 involves the AP-1 and
NF- B binding sites. HeLa cells were
transfected with reporter plasmids containing the minimal IL-8 promoter
(wt) (see legend to Fig. 2) or derivatives thereof with
mutations in the AP-1 site (AP-1), NF-
B site
(NF
B), or both sites
(AP-1/NF
B) and cultured for 2 days.
A, cells were cotransfected with empty vector (open
bars) or TAK1 and TAB1 expression vectors (filled
bars). B, cells were kept without (open
bars) or with IL-1
(10 ng/ml, filled bars) for the
final 5 h of the cultivation period. Luciferase activity was
determined as for Fig. 2 (mean RLU ± S.E. from three independent
experiments performed in triplicates).
-Globin-IL-8 Reporter mRNA via the p38 MAPK
Pathway--
Triggering of the p38 MAPK pathway can induce
stabilization of IL-8 mRNA as measured with a
tetracycline-controlled expression system, an effect that requires
regulatory sequences in the 3'-untranslated region of the IL-8
transcript and occurs via MK2 (34). The degradation of a
-globin
mRNA, which carries the regulatory region of the IL-8 mRNA in
its 3'-untranslated region, was monitored by sequential Northern blot
analyses following stop of its transcription by adding the tetracycline
analog doxycycline. The mRNA shows rapid basal degradation and
IL-1-induced, p38 MAPK-dependent stabilization (34). As
shown in Fig. 4 coexpression of TAK1 + TAB1 also induced marked stabilization of the mRNA. That
stabilization was largely reversed by a dominant negative mutant of p38
MAPK, whereas the kinase-negative JNKK55R mutant, which
strongly suppressed transcription (see Fig. 3), did not interfere with
mRNA stabilization (Fig. 4). This indicates that activation of
TAK1, just as exposure to IL-1 (17, 34), can induce mRNA
stabilization via the p38 MAPK pathway.
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Fig. 4.
Ectopic expression of TAK1 + TAB1 induces
stabilization of a -globin-IL-8 hybrid
mRNA through the p38 MAPK pathway. HeLa cells constitutively
expressing the tet transactivator protein were transfected
with ptetBBB-IL-8972-1310 encoding a
-globin mRNA
with the regulatory region of the IL-8 mRNA inserted
(BBB-IL-8) and cotransfected with empty vector and
expression vectors for TAK1 + TAB1, inactive JNK2 (JNK2K55R)
and inactive p38 MAPK (p38AGF) as indicated. The following day
doxycycline (3 µg/ml) was added, and total RNA was isolated at the
indicated times thereafter. Northern blot analysis was performed with a
-globin antisense RNA probe. Ethidium bromide staining of 28 S rRNA
(EtBR) is shown to allow comparison of RNA amounts loaded.
mRNA half-life was determined as described under "Experimental
Procedures."
B, JNK, and p38 MAPK in response to IL-1 (Fig.
5) (increased MK2 expression with
increasing doses of TAK1 expression plasmid was not reproduced in other
experiments). Furthermore, expression of TAK1K63W
dose-dependently inhibited IL-1-induced activation of the
minimal IL-8 promoter, with complete inhibition at 5 µg of
transfected DNA (Fig. 6A).
Likewise, IL-1-induced stabilization of the
-globin-IL8 hybrid
mRNA was impaired by the kinase-inactive mutant of TAK1 (Fig.
6B). These results provide strong evidence for a crucial
role of TAB1-TAK1 in both IL-1-induced transcription and mRNA
stabilization.
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Fig. 5.
A kinase-inactive mutant of TAK1,
TAK1K63W, inhibits IL-1-induced activation of
NF- B, JNK, and p38 MAPK. A,
cells were transfected with an NF-
B-luciferase reporter plasmid
(0.25 µg), pSV-
-gal (0.5 µg), and
pcDLR
HA-TAK1K63W or empty vector (5 µg each). 43 h after transfection, cells were stimulated for 5 h with 10 ng/ml
IL-1
or left untreated. Then cells were lysed and analyzed for
luciferase activity. Shown are the mean RLU ± S.E. from two
independent experiments performed in triplicates. B and
C, cells were transfected with empty vector, 0.3-6 µg of
pcDLR
HA-TAK1K63W and 2.5 µg of peVHA-JNK2
(B) or pCDNA3MYC-MK2 (C). 24 h later,
cells were stimulated with 10 ng/ml IL-1
for 30 min or left
untreated. Then cells were lysed in whole cell lysis buffer and
ectopically expressed HA-JNK2 or MYC-MK2 immunoprecipitated by
anti-epitope tag antibodies. Kinase activity of JNK (B) and
MK2 (C) was measured in vitro with
[
-32P]ATP and GST-JUN or HSP27, respectively, as
substrates. 100 µg of extract proteins were separated on 10%
SDS-PAGE and analyzed for expression of TAK1K63W, HA-JNK2,
or MYC-MK2 with polyclonal rabbit anti-TAK1, anti-HA, or anti-MYC
antibodies, respectively.
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Fig. 6.
IL-1-induced transcription and mRNA
stabilization is blocked by the inactive TAK1K63W
mutant. A, cells transfected with the minimal IL-8
promoter-luciferase plasmid, 0.5 µg of pSV- -gal, and the indicated
amounts of pcDLR
HA-TAK1K63W were incubated for 48 h
without (open bars) or with (filled bars) 10 ng/ml IL-1
added for the last 5 h. Then cells were lysed and
luciferase activity was determined (mean RLU ± S.E. from three
independent experiments performed in triplicates). B, cells
transfected with ptetBBB-IL-8972-1310 and
pcDLR
HA-TAK1K63W where indicated received doxycycline (3 µg/ml) alone or together with IL-1
(10 ng/ml). At the indicated
times thereafter
-globin-IL-8 hybrid mRNA (BBB-IL-8)
was detected by Northern blotting as for Fig. 4 (EtBr,
ethidium bromide staining of 28 S rRNA). mRNA half-life was
determined as described under "Experimental Procedures."
signaling (33). Second, the TAK1
N-terminal 22 amino acids have an inhibitory function, TAK1 lacking
this region is constitutively active without requiring TAB1 (27, 29,
33). TAK11-77, an N-terminal peptide containing this
inhibitory region should block TAK1 activity by masking a putative TAK1
activation domain, which interacts with the TAB1 C terminus (29,
33).
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Fig. 7.
Expression of TAB1 lacking the TAK1-binding
domain or of the putative autoinhibitory domain of TAK1 disrupts
signaling from the IL-1 receptor to the IL-8 promoter. Cells were
transfected with the IL-8 promoter-luciferase construct (0.25 µg),
pSV- -gal (0.5 µg), and empty vector. pcDLR
HA-TAK1
(TAK1wt), pcDLR
HA-TAK1K63W
(TAK1K63W), pEF-TAB1 (TAB1wt),
pCMV-TAB11-418 (TAB11-418), or
pcDLR
HA-TAK11-77 (TAK11-77) were
cotransfected at 2.5 µg in the indicated combinations in
A, 0.5, 2.5 and 5 µg in B and 5 µg in C. Cells were
cultured for 48 h without further additions (A) or with
IL-1
(10 ng/ml) for the final 5 h as indicated
(B, C). Thereafter, cells were lysed and
luciferase activity was measured (mean RLU ± S.E. from three
(A, C), and five (B) independent
experiments performed in triplicates).
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Fig. 8.
Formation of IL-8 protein is affected by
TAK1. A, cells were transfected using LipofectAMINE
with pcDLR HA-TAK1 and pEF-TAB1 alone or in combination (1 µg each)
and pCS3MT (vector), which was also used to adjust total
amount of DNA to 2 µg per transfection. B, cells were
transfected using LipofectAMINE with peGFPC1, peGFPC1-TAK1, or
peGFPC1-TAK1K63W (2 µg each) as indicated. 24 h
after transfection, medium was changed and cells were cultivated
further in the presence or absence of 10 ng/ml IL-1
as indicated.
Thereafter, IL-8 secreted into the cell culture supernatant was
quantified by specific ELISA (see "Experimental Procedures" section
for details). Results represent means ± S.E. from two
(A) or three (B) independent experiments.
C, equal expression of transfected GFP-TAK1 fusion proteins
in the experiments summarized in B was verified by Western
blotting of lysates using anti-TAK1 (M-579) antibodies. One Western
blot representative for three is shown (end.,
endogenous).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B alone was able to induce IL-8 transcription and secretion, but only to
very low levels. Second, selective activation of the JNK pathway by
expression of active mutants of MKK7 also induced low levels of IL-8
secretion and transcription (17). Activation of the JNK pathway is
necessary for triggering IL-8 synthesis by its physiological inducer
IL-1, because inhibition of that pathway by dominant negative JNK or
its antisense mRNA strongly suppressed IL-1-induced IL-8 mRNA
and protein expression (40). The NF-
B and JNK pathways markedly
synergized on the transcriptional level and required intact AP-1 and
NF-
B binding sites in the IL-8 promoter (17). Third, selective
triggering of the p38 MAPK pathway by MKK6 stabilized IL-8 mRNA,
and IL-1 induced mRNA stabilization in a p38
MAPK-dependent manner (17, 34). We now confirm and extend
this model by demonstrating that IL-1 induces both transcription and
mRNA stabilization through the MAPKKK TAK1 (Fig.
9).
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Fig. 9.
Scheme of signaling pathways cooperating in
IL-1-induced IL-8 formation. For details see text.
TAK1, together with its coactivator TAB1, activated in parallel the
NF-B, JNK, and p38 MAPK pathways in HeLa cells and induced transcription from a minimal IL-8 promoter. The latter effect was
inhibited by dominant negative JNK2 and required intact NF-
B and
AP-1 sites. Thus TAB1-TAK1 activates transcription in a way suggested
by us previously, namely through the coordinate function of both the
NF-
B and JNK pathways. TAB1-TAK1 overexpression also induced
mRNA stabilization. Although this effect was unaffected by dominant
negative JNK2, it was largely abrogated by a dominant negative mutant
of p38 MAPK.
Although the p38 MAPK pathway is capable of activating transcription by phosphorylation of several transcription factors (8), the dominant negative p38 MAPK mutant was devoid of any effect on TAK1-induced IL-8 transcription. The striking selectivity of effects of TAK1-activated JNK on IL-8 transcription and TAK1-activated p38 MAPK on IL-8 mRNA stabilization may be achieved by binding of the two MAPKs to specific scaffold proteins. Recently, three different scaffold protein families involved in JNK activation have been identified in mammalian cells, i.e. JIP1-3, JSAP1, and JNKBP1 (41-45). In yeast, scaffold proteins have been described also for the p38 MAPK homolog HOG1 (44). It appears that the FUS3 and HOG1 MAPK modules in yeast are activated by the same MAPKKK, Ste11, but are routed to distinct functions by different adapter proteins (8, 44). Our observations may therefore be explained by the formation of specific signaling complexes, which direct the cellular function of JNK and p38 MAPK in the IL-1-TAK1 signaling pathway toward transcriptional activation and mRNA stabilization, respectively.
IL-1 is an important physiological inducer of IL-8 formation. It
simultaneously activates the NF-B, JNK, and p38 MAPK signaling pathways. A number of different MAPKKKs have emerged as candidate molecules that serve as upstream activators of the JNK, p38, and NF-
B pathways. Very little information exists, however, that makes
it possible to distinguish between the following hypothetical situations: 1) in response to a given stimulus several different MAPKKKs are activated and function in a promiscuous manner; 2) different MAPKKKs in a given setting activate distinct pathways selectively, implying a signal bifurcation point upstream of them; or
3) one MAPKKK species is activated and in turn activates the different
downstream pathways, thus itself representing the bifurcation point.
Evidence supporting the latter model was obtained by investigating the
involvement of TAK1 in IL-1-induced expression of IL-8. First we found
that IL-1-induced activation of the NF-B and JNK pathways was
blocked by a kinase-inactive mutant of TAK1, thus confirming previous
results (13). Furthermore, the mutant also blocked IL-1-induced
activation of p38 MAPK. So far, no function has been demonstrated
for TAK1-activated NF-
B, JNK, or p38 MAPK signaling pathways in
IL-1-dependent cellular responses. As shown here,
IL-1-induced transcriptional activation was blocked by the kinase-inactive TAK1 mutant, strongly suggesting a sequence of events involving the IL-1 receptor complex, TAK1 and JNK + NF-
B activation. The kinase-inactive form of TAK1 also inhibited
IL-1-induced, p38 MAPK-dependent mRNA stabilization. Thus
for both kinds of effects on gene expression, IL-1 appears to utilize
the TAK1 MAPKKK.
Several other MAPKKKs that activate NF-B, JNK, and p38 MAPK may be
involved in IL-1 or TNF signaling (9, 18-24). Because the inactive
TAK1 mutant could possibly interfere with signaling in a nonspecific
way, i.e. by competing with those MAPKKKs through its
interaction with common downstream targets, we sought to apply more
specific inhibitors. TAK1 activation requires a protein-protein interaction with the TAB1 molecule (33). Available knowledge indicates
that, within the group of MAPKKK enzymes, this mode of activation is
unique for TAK1. In support of this, TAB1 has not been reported to bind
to and activate other MAPKKKs. Mapping of the regions in TAK1 and TAB1
that are required for this interaction showed that a region within the
first 303 amino acids of TAK1 binds to the C terminus of TAB1, which
contains a region homologous to the serine-rich N terminus of TAK1
(33). Because deletion of the N-terminal 22 amino acids yields an
activated form of TAK1 (29), it can be speculated that the N terminus
exerts an autoinhibitory interaction with the same region in the TAK1
molecule that also binds TAB1. Binding of the N terminus would render
the wild type TAK1 kinase inactive. TAB1 would activate TAK1 by
dislodging the N terminus (and possibly exerting a stimulatory effect).
The two mutants expected to act in a dominant negative fashion
accordingly, the N-terminal TAK1 peptide and the C-terminally truncated
TAB1, both effectively inhibited IL-1-induced IL-8 transcription.
Therefore the results are indicative of a specific recruitment of TAK1
by the IL-1 receptor complex and suggest that IL-1-induced downstream effects are dependent on TAB1-mediated TAK1 activation.
Taken together the evidence presented in this study assigns a central
role for IL-1-induced IL-8 formation in HeLa cells to the TAB1 and TAK1
molecules. Further proof will have to await the results of chemokine
expression in mice deficient of TAB1 and TAK1. It will also be
important to find out if this model holds true for other human cell
types, stimuli, and induced genes. That information will shed light on
the suitability of TAK1 and its coactivator TAB1 as targets for
interference with gene induction by inflammatory stimuli.
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ACKNOWLEDGEMENTS |
---|
We thank Hermann Bujard for providing us with plasmids pUHD10-3 and pUHC13-3 and HeLa cells expressing the tet transactivator protein, James R. Woodgett for his gift of expression plasmid for GST-JUN, Jeremy Saklatvala for providing us with antiserum SAK9, Matthias Gaestel for MK2 plasmids and recombinant HSP27, and Eisuke Nishida for TAK1 and TAB1 expression plasmids.
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FOOTNOTES |
---|
* This work was supported by grants (Kr1143/2-3, SFB 244/B15, SFB 244/B18, Ho1116/2-1, III GK-GRK 99/2-98 (P4)) from the Deutsche Forschungsgemeinschaft (to H. H. and M. K.).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.
¶ To whom correspondence should be addressed. Tel.: 49-511-532-2800; Fax: 49-511-532-4081; E-mail: Kracht.Michael@MH-Hannover.de.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M004376200
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ABBREVIATIONS |
---|
The abbreviations used are: IL-1, interleukin-1; EMSA, electrophoretic mobility shift assay; HA, hemagglutinin; IP, immunoprecipitation; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; RLU, relative light units; TNF, tumor necrosis factor; TAB, TAK binding protein; GST, glutathione S-transferase; ELISA, enzyme-linked immunosorbent assay; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; GFP, green fluorescent protein.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Dinarello, C.
(1996)
Blood
87,
2095-2147 |
2. | Baggiolini, M., and Clark-Lewis, I. (1992) FEBS Lett. 307, 97-101[CrossRef][Medline] [Order article via Infotrieve] |
3. | Martin, M. U., and Falk, W. (1997) Eur. Cytokine Netw. 8, 5-17[Medline] [Order article via Infotrieve] |
4. | O'Neill, L. A., and Greene, C. (1998) J. Leukoc. Biol. 63, 650-657[Abstract] |
5. | Kracht, M., Truong, O., Totty, N. F., Shiroo, M., and Saklatvala, J. (1994) J. Exp. Med. 180, 2017-2025[Abstract] |
6. | Freshney, N. W., Rawlinson, L., Guesdon, F., Jones, E., Cowley, S., Hsuan, J., and Saklatvala, J. (1994) Cell 78, 1039-1049[Medline] [Order article via Infotrieve] |
7. | Davis, R. J. (1999) Biochem. Soc. Symp. 64, 1-12[Medline] [Order article via Infotrieve] |
8. | Garrington, T. P., and Johnson, G. L. (1999) Curr. Opin. Cell Biol. 11, 211-218[CrossRef][Medline] [Order article via Infotrieve] |
9. | Lee, F. S., Hagler, J., Chen, Z. J., and Maniatis, T. (1997) Cell 88, 213-222[Medline] [Order article via Infotrieve] |
10. |
Meyer, C. F.,
Wang, X.,
Chang, C.,
Templeton, D.,
and Tan, T. H.
(1996)
J. Biol. Chem.
271,
8971-8976 |
11. |
Zandi, E.,
and Karin, M.
(1999)
Mol. Cell. Biol.
19,
4547-4551 |
12. |
Zhao, Q.,
and Lee, F. S.
(1999)
J. Biol. Chem.
274,
8355-8358 |
13. | Ninomiya-Tsuji, J., Kishimoto, K., Hiyama, A., Inoue, J., Cao, Z., and Matsumoto, K. (1999) Nature 398, 252-256[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Sakurai, H.,
Miyoshi, H.,
Toriumi, W.,
and Sugita, T.
(1999)
J. Biol. Chem.
274,
10641-10648 |
15. | Sakurai, H., Shigemori, N., Hasegawa, K., and Sugita, T. (1998) Biochem. Biophys. Res. Commun. 243, 545-549[CrossRef][Medline] [Order article via Infotrieve] |
16. | Lin, X., Cunningham, E. T., Jr., Mu, Y., Geleziunas, R., and Greene, W. C. (1999) Immunity 10, 271-280[Medline] [Order article via Infotrieve] |
17. |
Holtmann, H.,
Winzen, R.,
Holland, P.,
Eickemeier, S.,
Hoffmann, E.,
Wallach, D.,
Malinin, N. L.,
Cooper, J. A.,
Resch, K.,
and Kracht, M.
(1999)
Mol. Cell. Biol.
19,
6742-6753 |
18. | Malinin, N. L., Boldin, M. P., Kovalenko, A. V., and Wallach, D. (1997) Nature 385, 540-544[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Song, H. Y.,
Regnier, C. H.,
Kirschning, C. J.,
Goeddel, D. V.,
and Rothe, M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
9792-9796 |
20. |
Baud, V.,
Liu, Z. G.,
Bennett, B.,
Suzuki, N.,
Xia, Y.,
and Karin, M.
(1999)
Genes Dev.
13,
1297-1308 |
21. |
Ichijo, H.,
Nishida, E.,
Irie, K.,
ten Dijke, P.,
Saitoh, M.,
Moriguchi, T.,
Takagi, M.,
Matsumoto, K.,
Miyazono, K.,
and Gotoh, Y.
(1997)
Science
275,
90-94 |
22. |
Lin, X.,
Mu, Y.,
Cunningham, E. T., Jr.,
Marcu, K. B.,
Geleziunas, R.,
and Greene, W. C.
(1998)
Mol. Cell. Biol.
18,
5899-5907 |
23. |
Nemoto, S.,
DiDonato, J. A.,
and Lin, A.
(1998)
Mol. Cell. Biol.
18,
7336-7343 |
24. |
Xia, Y.,
Wu, Z.,
Su, B.,
Murray, B.,
and Karin, M.
(1998)
Genes Dev.
12,
3369-3381 |
25. |
Karin, M.,
and Delhase, M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9067-9069 |
26. |
Shirakabe, K.,
Yamaguchi, K.,
Shibuya, H.,
Irie, K.,
Matsuda, S.,
Moriguchi, T.,
Gotoh, Y.,
Matsumoto, K.,
and Nishida, E.
(1997)
J. Biol. Chem.
272,
8141-8144 |
27. |
Sano, Y.,
Harada, J.,
Tashiro, S.,
Gotoh-Mandeville, R.,
Maekawa, T.,
and Ishii, S.
(1999)
J. Biol. Chem.
274,
8949-8957 |
28. |
Shibuya, H.,
Iwata, H.,
Masuyama, N.,
Gotoh, Y.,
Yamaguchi, K.,
Irie, K.,
Matsumoto, K.,
Nishida, E.,
and Ueno, N.
(1998)
EMBO J.
17,
1019-1028 |
29. | Yamaguchi, K., Shirakabe, K., Shibuya, H., Irie, K., Oishi, I., Ueno, N., Taniguchi, T., Nishida, E., and Matsumoto, K. (1995) Science 270, 2008-2011[Abstract] |
30. |
Zhou, G.,
Lee, S. C.,
Yao, Z.,
and Tan, T. H.
(1999)
J. Biol. Chem.
274,
13133-13138 |
31. | Letterio, J. J., and Roberts, A. B. (1998) Annu. Rev. Immunol. 16, 137-161[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Song, X. Y.,
Gu, M.,
Jin, W. W.,
Klinman, D. M.,
and Wahl, S. M.
(1998)
J. Clin. Invest.
101,
2615-2621 |
33. | Shibuya, H., Yamaguchi, K., Shirakabe, K., Tonegawa, A., Gotoh, Y., Ueno, N., Irie, K., Nishida, E., and Matsumoto, K. (1996) Science 272, 1179-1182[Abstract] |
34. |
Winzen, R.,
Kracht, M.,
Ritter, B.,
Wilhelm, A.,
Chen, C.-Y. A.,
Shyu, A.-B.,
Müller, M.,
Gaestel, M.,
Resch, K.,
and Holtmann, H.
(1999)
EMBO J.
18,
4969-4980 |
35. | Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5547-5551[Abstract] |
36. | Finch, A., Holland, P., Cooper, J. A., Saklatvala, J., and Kracht, M. (1997) FEBS Lett. 418, 144-148[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Hanafusa, H.,
Ninomiya-Tsuji, J.,
Masuyama, N.,
Nishita, M.,
Fujisawa, J.,
Shibuya, H.,
Matsumoto, K.,
and Nishida, E.
(1999)
J. Biol. Chem.
274,
27161-27167 |
38. |
Moriguchi, T.,
Kuroyanagi, N.,
Yamaguchi, K.,
Gotoh, Y.,
Irie, K.,
Kano, T.,
Shirakabe, K.,
Muro, Y.,
Shibuya, H.,
Matsumoto, K.,
Nishida, E.,
and Hagiwara, M.
(1996)
J. Biol. Chem.
271,
13675-13679 |
39. |
Wang, W.,
Zhou, G.,
Hu, M. C.-T.,
Yao, Z.,
and Tan, T.-H.
(1997)
J. Biol. Chem.
272,
22771-22775 |
40. |
Krause, A.,
Holtmann, H.,
Eickemeier, S.,
Winzen, R.,
Szamel, M.,
Resch, K.,
Saklatvala, J.,
and Kracht, M.
(1998)
J. Biol. Chem.
273,
23681-23689 |
41. |
Ito, M.,
Yoshioka, K.,
Akechi, M.,
Yamashita, S.,
Takamatsu, N.,
Sugiyama, K.,
Hibi, M.,
Nakabeppu, Y.,
Shiba, T.,
and Yamamoto, K. I.
(1999)
Mol. Cell. Biol.
19,
7539-7548 |
42. | Koyano, S., Ito, M., Takamatsu, N., Shiba, T., Yamamoto, K., and Yoshioka, K. (1999) FEBS Lett. 457, 385-388[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Whitmarsh, A. J.,
Cavanagh, J.,
Tournier, C.,
Yasuda, J.,
and Davis, R. J.
(1998)
Science
281,
1671-1674 |
44. | Whitmarsh, A. J., and Davis, R. J. (1998) Trends Biochem. Sci. 23, 481-485[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Yasuda, J.,
Whitmarsh, A. J.,
Cavanagh, J.,
Sharma, M.,
and Davis, R. J.
(1999)
Mol. Cell. Biol.
19,
7245-7254 |