From the Division of Cellular and Molecular Biology,
Department of Cancer Biology, The Institute of Medical Science, The
University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan,
§ Graduate School of Science and Technology, Keio
University, Yokohama, Kanagawa 223-8522, Japan, and ¶ Graduate
School of Pharmaceutical Sciences, Kumamoto University, Oe-honmachi,
Kumamoto 862-0973, Japan
Received for publication, January 22, 2003
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ABSTRACT |
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Tumor necrosis factor receptor-associated factor
6 (TRAF6) transduces signals from members of the Toll/interleukin-1
(IL-1) receptor family by interacting with IL-1 receptor-associated
kinase-1 (IRAK-1) after IRAK-1 is released from the receptor-MyD88
complex upon IL-1 stimulation. However, the molecular mechanisms
underlying regulation of the IRAK-1/TRAF6 interaction are largely
unknown. We have identified TIFA, a TRAF-interacting protein with a
forkhead-associated (FHA) domain. The FHA domain is a motif known to
bind directly to phosphothreonine and phosphoserine. In transient
transfection assays, TIFA activates NF Interleukin-1 (IL-1)1 is
a principal cytokine responsible for the induction of mediators that
orchestrate immune and inflammatory responses by activating
transcription factors NF IL-1 signaling is initiated by ligand-induced formation of a receptor
complex that consists of the IL-1 receptor and IL-1 receptor accessory
protein. Then, the cytosolic protein MyD88 (4, 5) and Tollip (6) are
recruited to this complex. MyD88 in turn recruits members of the IRAK
family including IRAK-1, IRAK-2, IRAK-M (4, 7, 8), and IRAK-4 (9) via
interaction between their death domains. IRAK-1 is activated presumably
via phosphorylation by IRAK-4, leaving the receptor complex to interact with TRAF6. Subsequently, TRAF6 activates transforming growth factor
Yeast Two-hybrid Screening and Northern
Blotting--
Full-length mouse TRAF6 was subcloned into pBD-GAL4-Cam
(Stratagene, La Jolla, CA) and used as bait in a two-hybrid screening of an ICR (8-week-old female) bone marrow cDNA library fused to the
activation domain of Gal4 in pAD-GAL4-2.1 (Stratagene). The cDNA
encoding human TIFA was identified from a human B-cell cDNA library. Sequence data for mouse, and human TIFA have been submitted to
DDBJ/EMBL/GenBankTM databases under accession number
AB062111 and AB062110, respectively. A mouse multiple tissue RNA blot
(Clontech, Palo Alto, CA) was incubated with
32P-labeled full-length mouse TIFA cDNA and Plasmids and Antibodies--
pME-FLAG-TRAF6 and
pME-FLAG-TRAF6-(275-530) were described previously (15).
pME-FLAG-TIFA and pME-Myc-TIFA were constructed by insertion of a DNA
fragment encoding FLAG- or Myc-tagged TIFA, respectively, into pME18S
(16). For expression of GST fusion proteins, a DNA fragment encoding
GST-tagged TIFA or a mutant was inserted into pME18S. A cDNA
encoding IRAK-1-(1-211) was cloned by PCR and inserted into pME18S.
pEF-MyD88 and pEF-MyD88-(152-296) were kindly provided by S. Akira
(Osaka University); pEF-TLR4 and pEF-MD-2 were provided by K. Miyake
(University of Tokyo); pEF-TAK1, pEF-TAK1(K63W), and pEF-TAB1 were
provided by K. Matsumoto (Nagoya University); pEF-IRAK-1 was provided
by T. Naka (Osaka University); 3x GST Pull-down Assay, Western Blotting, and
Immunoprecipitation--
For the GST pull-down assay, HEK293T cells
were cotransfected with pME-GST-TIFA and either pME-FLAG-TRAF6,
pME-FLAG-TIFA, or pEF-IRAK-1. Thirty-six hours after transfection,
cells were lysed in 500 µl of TNE buffer (50 mM Tris-HCl,
pH 8.0, 1% Nonidet P-40, 1 mM EDTA, 150 mM
NaCl, 0.5 mM dithiothreitol) and centrifuged to remove
cellular debris. The resulting supernatant was used as a cell lysate.
Cell lysates were incubated with glutathione-Sepharose (Amersham
Biosciences), and the GST fusion protein complexes were separated on
8.5% polyacrylamide/SDS gels. For analysis of the IRAK-1·TIFA·TRAF6 complex, HEK293T cells were cotransfected with the indicated combinations of pEF-IRAK-1, pME-Myc-TIFA, and
pME-FLAG-TRAF6. Cell lysates were subjected to immunoprecipitation by
the addition of anti-FLAG antibody and protein G-Sepharose (Amersham
Biosciences). The resulting immunoprecipitates were separated on 10%
or 12.5% polyacrylamide/SDS gels. Cell lysates were also used to
analyze the expression level of each protein. Establishment of mouse
embryonic fibroblasts (MEFs) and TRAF6-deficient MEFs and introduction
of FLAG-TIFA into MEFs by retrovirus vector were performed as described (17). Mouse 70Z and MEF cells were either untreated or treated with
IL-1 (20 ng/ml) for 5 min. Cell lysates were subjected to immunoprecipitation with 1 µg of the appropriate antibody and 20 µl
of protein G-Sepharose (Amersham Biosciences). Immunoprecipitates or
whole-cell lysates were separated by SDS-PAGE and transferred to
polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes
were immunoblotted with specific antibodies and visualized with
appropriate horseradish peroxidase-conjugated second antibody and the
ECL Western blotting system (Amersham Biosciences).
Separation of Recombinant TIFA Protein by Gel Filtration Column
Chromatography--
MBP-tagged TIFA protein was expressed in
Escherichia coli BL21 and affinity purified with amylose
resin (New England BioLabs, Beverly, MA). MBP-TIFA (24 µg) was
cleaved with 2.4 units of PreScission Protease (Amersham Biosciences)
at 4 °C for 4 h. Fifty microliters of the reaction mixture was
applied to a Superdex 75 column (0.32 × 30 cm), and fractions (20 µl) were collected. The fractions were analyzed on 10%
polyacrylamide-SDS gel, and proteins were visualized by Coomassie
Brilliant Blue Staining. Molecular weight marker proteins were analyzed
under the column conditions described above.
Luciferase Reporter Assay and in Vitro Kinase Assay--
HEK293T
cells were transfected with 1 ng of 3x Identification of TIFA as a TRAF6-interacting Protein--
To
identify the protein(s) that regulate association of IRAK-1 with TRAF6,
we performed yeast two-hybrid screening of a mouse bone marrow cDNA
library using murine TRAF6 as bait. We identified a cDNA encoding a
184-amino acid protein, identical to T2BP previously identified as a
TRAF2-binding protein, that activates NF
To determine whether interaction of TIFA with TRAF6 is required for
TIFA-induced NF Possible Implication of TIFA Self-association in TRAF6
Activation--
TRAF6 transduces signals when oligomerized (13,
14). Because a part of the expressed TIFA is phosphorylated (data not shown), homo-oligomerization of TIFA via the FHA domain/phosphopeptide interaction may facilitate or induce oligomerization of TRAF6. To
address whether TIFA forms a homo-oligomer, the binding of FLAG-TIFA to
various GST-TIFA mutants was analyzed (Fig.
3A). GST-TIFA, but not GST,
associated with FLAG-TIFA, indicating that TIFA forms a homo-oligomer.
Both the FHA domain and the C domain were required, whereas the N
domain was not necessary for the TIFA self-association. The structural
requirement for the self-association is similar to that for NF TIFA Enhances the Association of TRAF6 and IRAK-1--
We next
addressed whether TIFA interacts with IRAK-1. Coexpression of GST-TIFA
with IRAK-1 followed by GST pull-down assay revealed that TIFA
associates with IRAK-1 (Fig.
4A). Both GST-G50ES66A and
GST-E178A bind to IRAK-1 as efficiently as wild-type TIFA, indicating
that the IRAK-1/TIFA interaction is not mediated by recognition of the
phosphopeptide by the FHA domain. Because TIFA binds both TRAF6 and
IRAK-1, we hypothesized that TIFA may affect the IRAK-1/TRAF6
interaction. To test this, the interaction between TRAF6 and IRAK-1 was
analyzed in both the absence and presence of increasing expression of
TIFA. Transfection experiments were carried out using 0.2 µg of TRAF6
and IRAK-1 expression vectors to minimize the IRAK-1/TRAF6 interaction
in the absence of TIFA expression. The amount of IRAK-1 coprecipitated
with TRAF6 was increased dramatically at the optimal expression of TIFA
(Fig. 4B), indicating that TIFA, when overexpressed, can
link TRAF6 to IRAK-1.
Interaction of TIFA with TRAF6 and IRAK-1 in Vivo--
For
investigation of the physiological significance of the
IRAK-1·TIFA·TRAF6 complex, the interactions of endogenous proteins were analyzed. For identification of endogenous TIFA protein, rabbit
anti-TIFA polyclonal antibody was generated. When lysates prepared from
70Z mouse pre-B cells were subjected to immunoprecipitation followed by
Western blotting with anti-TIFA antibody, two bands at ~24 kDa that
comigrated with protein expressed from TIFA cDNA in HEK293T cells
were identified (Fig. 5A,
left four lanes). These two bands were not observed when
control IgG was used for immunoprecipitation, indicating that they
represent endogenous TIFA. We next addressed whether TIFA interacts
with TRAF6. Endogenous TRAF6 was immunoprecipitated from either
unstimulated 70Z cells or from cells stimulated with IL-1 for 5 min,
and coprecipitated TIFA was analyzed by Western blotting with anti-TIFA
antibody. Similar amounts of TIFA were detected irrespective of IL-1
stimulation (Fig. 5A, right two lanes),
indicating that TIFA interacts with TRAF6 constitutively in the 70Z
pre-B cell line. To determine whether TIFA interacts with TRAF6 in a
different cell type, wild-type MEF and TRAF6-deficient MEF (17) were
analyzed. Because the reactivity of anti-TIFA antibody was relatively
weak, wild-type MEF and TRAF6
We next investigated the interaction of TIFA and IRAK-1 in
vivo. When FLAG-TIFA was immunoprecipitated with anti-FLAG
antibody from wild-type MEF in the absence or presence of IL-1
stimulation for 5 min followed by Western blotting with anti-IRAK-1
antibody, phosphorylated IRAK-1 was coprecipitated only in the presence of IL-1 stimulation (Fig. 5C). Because activated
phospho-IRAK-1 is reported to be released from the receptor-MyD88
complex (5), it is possible that IRAK-1 associated with TIFA after
release from the receptor complex, consistent with the previous finding that only activated phospho-IRAK-1 is coprecipitated with TRAF6 upon
IL-1 stimulation (30). IRAK-1 was not precipitated from MEFs infected
with control virus. These results clearly indicate that TIFA interacts
with IRAK-1 in an IL-1 signal-dependent manner in
vivo and suggest that TIFA links TRAF6 to IRAK-1 upon IL-1 stimulation.
TIFA Mediates IL-1-induced NF
TIFA-induced NF We report the identification of TIFA as a TRAF6-binding protein
that can mediate IL-1 signaling. In transient transfection assays, TIFA
binds both TRAF6 and IRAK-1. Furthermore, IRAK-1 is efficiently
coprecipitated with TRAF6 under conditions of optimal TIFA expression.
Thus, TIFA, when overexpressed, can link TRAF6 to IRAK-1 in the absence
of IL-1 stimulation. By analyzing the interaction of endogenous TIFA
with TRAF6 in two different types of cells, we showed that TIFA can
bind TRAF6 irrespective of IL-1 stimulation. In contrast, interaction
of TIFA and IRAK-1 is dependent on IL-1 stimulation in vivo.
These results strongly suggest that TIFA links TRAF6 to IRAK-1 in an
IL-1 stimulation-dependent manner in vivo. TIFA
may play a similar role in the signaling from members of the TLR
family, because the TLR signal transduction pathways are also mediated
by IRAK-1 and TRAF6 (3, 32). In fact, TLR4-mediated NF The association of signal transducers including kinases and adapter
proteins is largely regulated through protein phosphorylation, allowing
dissociation as the balance shifts from kinase to phosphatase activity.
Phosphorylation of proteins on serine and threonine residues has
traditionally been recognized as a way to regulate enzyme conformation.
Recent studies have revealed that serine/threonine phosphorylation can
play critical roles in the formation of multimolecular signaling
complexes through specific interaction of phosphorylated peptides and
Ser(P)/Thr(P) binding modules including FHA domains, 14-3-3, WW
domains, and WD40 and leucine-rich repeat domains (25). Among the
identified FHA domain-containing proteins, a specific target protein
has been determined for some. In each case,
phosphorylation-dependent interaction of the FHA domain
with the target protein is thought to be responsible for the
physiologically significant regulation of cell function (20). The FHA2
domain of Rad53p, a protein kinase involved in the DNA damage response
and in cell cycle arrest in Saccharomyces cerevisiae, binds
Rad9p when Rad9p is phosphorylated by DNA damage signals. Mutation of
the FHA2 domain of Rad53p abolishes DNA damage-induced G2/M
cell cycle arrest, indicating the biological relevance of the
Rad53p-Rad9p interaction (21). In addition, the FHA domain of
Arabidopsis kinase-associated protein phosphatase interacts
with a plasma membrane-integrated receptor, leucine-rich repeat
receptor-like protein kinase, when receptor-like protein kinase is
autophosphorylated upon ligand binding (22). Receptor-like protein
kinase is a product of the CLAVATA1 (CLV1) gene,
mutations that result in plants with enlarged shoots and floral
meristems (33). These observations and the fact that the G50ES66A
mutant of TIFA is not able to activate NF Whether TIFA is essential for the IRAK-1/TRAF6 interaction or whether
it stabilizes the interaction is not clear. One interesting possibility
is that TIFA may augment the signal strength. IRAK-1 contains three
consensus TRAF6 binding sites (26), and TIFA contains a single
consensus TRAF6 binding site. Because TIFA and IRAK-1 interacts upon
IL-1 stimulation, the number of TRAF6 molecules involved in signaling
in cells abundantly expressing TIFA could be higher than that in cells
with less TIFA expression. Furthermore, IRAK-1/TIFA interaction may
induce their structural changes and increase their affinity for TRAF6.
It is possible that the FHA domain of TIFA may bind to the substrate of
IRAK-1 or IRAK-4, which leads to the stabilization of
IRAK-1·TIFA·TRAF6 complex. It has been reported that kinase
activity of IRAK-1 is not required for IL-1 signaling in a human
embryonic kidney 293 cell line (34). Perhaps it can affect signal
strength in other specific cell types. In this sense, it is interesting
that TIFA is highly expressed in spleen but scarcely expressed in
skeletal muscle. During the preparation of our manuscript, Pellino 1 was reported to be associated with IRAK-1·IRAK-4·TRAF6 complex in
an IL-1 signal-dependent manner (35). Thus, Pellino 1 and
TIFA may have similar roles. However, they have distinct tissue
specificities; Pellino 1 is weakly expressed in spleen but
significantly expressed in skeletal muscle.
TRAF6 is also involved in mediating signals from members of the TNF
receptor superfamily, including CD40, RANK, X-linked ectodysplatin-A2 receptor (XEDAR), and p75 neutrophin nerve growth factor receptor (11,
12, 36, 37). Although a recent study showed that the p75 nerve growth
factor receptor recruits IRAK-1 upon ligand stimulation (38), other
receptors are thought to form trimers and do not require IRAK-1 (39).
In our preliminary experiments, TIFA did not bind the cytoplasmic tail
of CD40 or RANK, suggesting that TIFA may not be required for
oligomerization of TRAF6 to transduce signals from members of the TNF
receptor superfamily with the exception of the p75 nerve growth factor
receptor. TIFA may regulate signals that are mediated by both TRAF6 and
IRAK-1. To elucidate the molecular mechanism underlying TIFA signaling, especially to identify a role of the FHA domain, crystallographic studies of TIFA and identification of a target protein of the FHA
domain are required. Further studies of TIFA may reveal a novel
regulation of inflammation and innate immunity.
B and c-Jun amino-terminal
kinase. However, TIFA carrying a mutation that abolishes TRAF6 binding
or mutations in the FHA domain that are known to abolish FHA domain
binding to phosphopeptide fails to activate NF
B and c-Jun
amino-terminal kinase. TIFA, when overexpressed, binds both TRAF6 and
IRAK-1 and significantly enhances the IRAK-1/TRAF6 interaction.
Furthermore, analysis of endogenous proteins indicates that TIFA
associates with TRAF6 constitutively, whereas it associates with IRAK-1
in an IL-1 stimulation-dependent manner in
vivo. Thus, TIFA is likely to mediate IRAK-1/TRAF6 interaction
upon IL-1 stimulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and AP-1 (1). The Toll-like
receptors (TLRs) have also recently emerged as critical molecules in
the establishment of innate immune and inflammatory responses due to
their ability to recognize various pathogen-associated molecules (2).
Thus, the Toll/IL-1 receptor family members play a critical role in the
switch from innate to adaptive immunity. The signal transduction
pathways of the Toll/IL-1 receptor family members consist of common
intermediates including MyD88, members of IL-1 receptor-associated
kinase (IRAK) family, and tumor necrosis factor receptor-associated
factor 6 (TRAF6), whereas TLR3 and TLR4 have MyD88-independent pathways
(3). Furthermore, the signaling cascade is evolutionarily conserved
with that initiated by the Drosophila Toll receptor (2).
Therefore, the MyD88/IRAK/TRAF6 pathway is an essential protein linkage
required for the immune and inflammatory systems.
-activated kinase 1 (TAK1), which activates a number of downstream
signaling cascades, including those of I
B kinase, p38, and c-Jun
amino-terminal kinase (JNK), leading to the activation of transcription
factors such as NF
B and AP-1 (10). All members of the IRAK
family activate NF
B when overexpressed in cells. However, only
IRAK-4 requires kinase activity to activate NF
B, and IRAK-4
phosphorylates IRAK-1 in vitro (9). Furthermore, introduction of IRAK-1, IRAK-2, or IRAK-M can restore IL-1-induced NF
B activation in IRAK-1-deficient cells, whereas IRAK-4 cannot compensate for the loss of IRAK-1. Thus, IRAK-4 may act upstream of
IRAK-1 and phosphorylate IRAK-1 in a stimulation-dependent manner. TRAF6-deficient mice are defective in IL-1 signaling (11, 12).
Furthermore, TRAF6 activates TAK1 when TRAF6 was artificially oligomerized without IL-1 stimulation (13, 14). Although these recent
data indicate that IRAK-1 and TRAF6 play pivotal roles in the Toll/IL-1
receptor signaling, the molecular mechanisms underlying the interaction
of TRAF6 with IRAK-1 released from the receptor complex and how the
IRAK-1·TRAF6 complex activates downstream signals remain to be
elucidated. In this study, we identified an adapter protein that is
likely to link IRAK-1 to TRAF6 and activates TRAF6 upon IL-1 stimulation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin
cDNA at 65 °C. The filter was washed with 0.5× SSC (1×
SSC = 0.15 M NaCl and 0.015 M sodium
citrate), 0.2 (w/v)% SDS at 65 °C for 30 min.
B-luc and 3xM
B-luc were provided
by S. Miyamoto (University of Wisconsin); pEF-T7-JNK was provided by S. Ohno (Yokohama City University); pEF-Ubc13(C87A) was provided by
Z. J. Chen (University of Texas). Anti-TRAF6, anti-IRAK-1, and
anti-Myc antibodies (Santa Cruz Biotechnology, Santa Cruz, CA),
anti-FLAG M2 antibody (Sigma) and anti-T7 antibody (Novagen, Madison,
WI) were purchased. Anti-GST, anti-TIFA, and anti-maltose-binding protein (MBP) antibodies were generated by injection of recombinant GST
protein or MBP-TIFA fusion protein into rabbits.
B-luc or 3xM
B-luc (mutant
B sites), 10 ng of
-actin-
-galactosidase and the indicated
amounts of various expression plasmids. Thirty-six hours after
transfection, luciferase activity was measured with the PicaGene
luciferase assay system (TOYO INK, Tokyo), and
-galactosidase activity was used to standardize transfection efficiency. For the
in vitro JNK kinase assay, HEK293T cells were transfected with 10 ng of pEF-T7-JNK and the indicated amounts of pME-FLAG-TIFA. Thirty-six hours after transfection, His-JNK was immunoprecipitated with anti-T7 antibody. Immunoprecipitates were incubated with 2 µg of
GST-c-JUN (1-89) fusion protein (New England BioLabs) in 20 µl of
kinase buffer (20 mM HEPES, pH 7.5, 20 mM
MgCl2, 5 µCi of [
-32P]ATP (3000 Ci/mmol)) at 30 °C for 30 min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and JNK in transient
transfection assays (Ref. 18 and data not shown). Because the present
study showed that T2BP associates with TRAF6 and a forkhead-associated
(FHA) domain of the T2BP (Fig.
1A) is likely to play a
critical role in the signaling (see "Discussion"), we
designated T2BP as a TRAF-interacting
protein with an FHA domain, TIFA.
TIFA mRNA was expressed highly in spleen and moderately in other
tissues, whereas it was not detectable in skeletal muscle (Fig.
1B). FHA domains are conserved sequences of 60-100 amino acids found mainly in eukaryotic nuclear proteins (19) that participate
in establishing or maintaining cell-cycle checkpoints, DNA repair, or
transcriptional regulation (20). More importantly, some of them were
shown to bind directly to phosphoserine/phosphothreonine (Ser(P)/Thr(P)) residues (21-24) in the same way that SH2 domains interact with phosphotyrosine residues (20, 25).
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Fig. 1.
Identification of TIFA as a TRAF6-interacting
protein. A, diagram of the overall structure of TIFA
and alignment of the FHA domain of TIFA with that of other proteins.
Amino acids identical or similar in least six proteins are indicated by
gray boxes. Asterisks indicate amino acids
selected for site-directed mutagenesis. h, human;
m, murine. B, multiple-tissue Northern blot. A
mouse multiple-tissue Northern blot (Clontech) was
probed with a cDNA fragment of mouse TIFA (upper) or
-actin (lower). C, structure of TIFA and its
mutants and alignment of TRAF6 binding sites. D,
identification of the TRAF6 binding site in TIFA. HEK293T cells were
cotransfected with pME-GST-TIFA or pME-GST-TIFA mutant and
pME- FLAG-TRAF6. Thirty-six hours after transfection, cell lysates
were subjected to GST pull-down assay. WB, Western
blot.
B and JNK activation, we identified the TRAF6
binding site in TIFA. Wild-type TIFA and various mutants were expressed
as GST fusion proteins in HEK293T cells (Fig. 1C) in
conjunction with FLAG-TRAF6. Subsequent GST pull-down assays revealed
that the carboxyl-terminal portion of TIFA (C domain) is sufficient for
TRAF6 binding and that the amino-terminal portion (N domain) and the
FHA domain are not necessary (Fig. 1D). Recent analysis of
the crystal structure of the TRAF-C domain of TRAF6 in complex with
TRAF6-binding peptides from CD40 and RANK revealed that eight amino
acid residues of the TRAF6-binding peptide contact the TRAF-C domain of
TRAF6 and identified
XXPXEXX-(aromatic/acidic) as a
consensus sequence for TRAF6 binding (26) (Fig. 1C).
Substitution of Ala for Glu at the fifth amino acid position of the
TRAF6 binding site in CD40 abolished binding to TRAF6 (27, 28). A
consensus TRAF6 binding motif is present in the C domain of both human
and mouse TIFA (Fig. 1C). Substitution of Ala for Glu-178
(E178A) abolished binding of TIFA to TRAF6 (Fig. 1D), which
is consistent with the notion that the TRAF domain of TRAF6 is
responsible for binding to both CD40 (15) and TIFA (data not shown). We
then analyzed the ability of various TIFA mutants to activate NF
B and JNK. Both the FHA and the C domain were required, whereas the N
domain was not necessary for NF
B activation (Fig.
2A). GST-E178A did not
activate NF
B or JNK (Fig. 2, A and B),
indicating that interaction of TIFA with TRAF6 is essential for
TIFA-mediated NF
B and JNK activation. To clarify the role of the
FHA domain of TIFA in NF
B and JNK activation, Gly-50 and Ser-66
were replaced with Glu and Ala, respectively (G50ES66A, Fig. 1,
A and C), since identical mutations in the FHA
domain of the Arabidopsis kinase-associated protein
phosphatase abolished its binding to the phosphorylated receptor-like
protein kinase (22). Interestingly, GST-G50ES66A, which binds to TRAF6
(Fig. 1D), did not activate NF
B or JNK (Fig. 2,
A and B), indicating that interaction of TIFA
with TRAF6 is necessary but not sufficient for activation. Thus, the
FHA domain of TIFA plays a critical role in the NF
B and JNK
activation.
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Fig. 2.
Both the TRAF6 binding site and the intact
FHA domain are required for TIFA-induced activation of
NF B and JNK. A, activation of
NF
B by TIFA mutants. HEK293T cell were transfected with 1 ng of
3x
B-luc or 3xM
B-luc (mutant
B sites), 10 ng of
-actin-
-galactosidase, and 5 µg of expression plasmid for TIFA
or its mutants. Thirty-six hours after transfection, luciferase assays
were performed. Results shown are the mean ± S.D. of triplicate
experiments and are representative of two independent experiments.
B, activation of JNK by TIFA mutants. HEK293T cells were
transfected with 10 ng of pEF-T7-JNK and the indicated amounts of
expression plasmid for TIFA or its mutants. Thirty-six hours after
transfection, His-JNK was immunoprecipitated with anti-T7 antibody.
Immunoprecipitates were subjected to in vitro kinase assay
using GST-Jun as a substrate. The results shown are representative of
duplicate experiments. wt, wild type.
B and
JNK activation, suggesting that the self-association of TIFA may be a
prerequisite for activation of NF
B and JNK. However, GST-G50ES66A
associated with FLAG-G50ES66A, suggesting that the FHA
domain/phosphopeptide interaction is not likely to be involved in the
self-association. More importantly, because the G50ES66A mutant does
not activate downstream signals, homo-oligomer of wild-type TIFA and
that of the G50ES66A mutant could have different physical properties.
To address this question, recombinant TIFA was generated as an
MBP-tagged protein with a cleavage site for human rhinovirus type-14 3C
protease (29) at the junction (MBP-TIFA). Purified MBP-TIFA was
digested almost completely by a fusion protein of GST with the 3C
protease (GST-3C), and the reaction mixture was analyzed by Superdex 75 gel filtration chromatography (Fig. 3B, upper).
According to the elution profile of the marker proteins, MBP (43 kDa)
was present as a monomer, and GST-3C (46 kDa) formed a dimer. TIFA (24 kDa) was eluted at around fraction 14, which corresponds to TIFA
trimer. However, when recombinant TIFA-G50ES66A protein was subjected
to a similar experiment, the mutant protein was eluted at around
fraction 7, which corresponds to TIFA pentamer or hexamer (Fig
3B, lower). These results indicate that
introduction of the G50ES66A mutation result in loss of the ability to
form a trimer, presumably by altering the conformation of the FHA and
the C domains, which are responsible for the self-association. Taken
together, the self-association of TIFA may be required for activation
of downstream signals, but the size or the conformation of
homo-oligomer could be important.
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Fig. 3.
Self-association of TIFA. A,
identification of the domain required for TIFA self-association.
GST-TIFA or its mutants were coexpressed with FLAG-TIFA or
FLAG-G50ES66A in HEK293T cells. GST pull-down assays were performed,
and coprecipitated FLAG-TIFA was analyzed by Western blotting
(WB) with anti-FLAG antibody. B, oligomer
formation of recombinant TIFA protein and that of mutant TIFA protein.
MBP-TIFA (24 µg, upper) or MBP-TIFA-G50ES66A
(lower) was cleaved with 2.4 units of PreScission Protease
(GST-3C) at 4 °C for 4 h. The reaction mixture was applied to a
Superdex 75 column, and fractions (20 µl) were collected. Fractions
were analyzed on 10% polyacrylamide-SDS gel and proteins were
visualized by Coomassie Brilliant Blue staining. Arrows
indicate the elution profile of the molecular mass marker proteins (158 kDa, -globulin; 44 kDa, ovalbumin; 17 kDa, myoglobin) and the
position of the void volume.
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Fig. 4.
TIFA enhances the association of TRAF6 and
IRAK-1. A, interaction of TIFA with IRAK-1. HEK293T
cells were cotransfected with pME-GST-TIFA, pME-GST-TIFA mutant, or
pME-GST and pEF-IRAK-1. Thirty-six hours after transfection, cell
lysates were subjected to GST pull-down assay. WB, Western
blot. B, TIFA links TRAF6 to IRAK-1. HEK293T cells were
cotransfected with 0.2 µg of pME-FLAG-TRAF6 and/or pEF-IRAK-1 and
increasing amounts of pME-Myc-TIFA. FLAG-TRAF6 was immunoprecipitated
with anti-FLAG antibody.
/
MEF expressing FLAG-TIFA
were generated by retrovirus vector-mediated gene transfer. To avoid
overexpression of FLAG-TIFA, infection with retrovirus carrying
FLAG-TIFA and a puromycin resistance gene was performed with a low
titer of virus followed by puromycin selection. The amount of FLAG-TIFA
expressed in wild-type MEF and TRAF6
/
MEF was ~3
times that of endogenous TIFA protein in MEF and, because of this low
expression of FLAG-TIFA, the basal activity of NF
B and JNK in MEF
was not affected (data not shown). Thus, we may characterize the
FLAG-TIFA as endogenous TIFA. When lysates from wild-type or
TRAF6
/
MEF were subjected to immunoprecipitation with
anti-TRAF6 antibody followed by Western blotting with anti-FLAG
antibody, TIFA was precipitated only in the presence of TRAF6 (Fig.
5B). Therefore, anti-TRAF6 antibody did not
immunoprecipitate TIFA directly due to its cross-reactivity. From these
data, we conclude that TIFA binds TRAF6 constitutively both in 70Z
pre-B cells and in MEF in vivo.
View larger version (23K):
[in a new window]
Fig. 5.
Interaction of TIFA with TRAF6 and IRAK-1
in vivo. A, interaction of TRAF6 with
TIFA in 70Z pre-B cells. Lysates prepared from 70Z cells with (+) or
without ( ) IL-1 stimulation (20 ng/ml, 5 min) were immunoprecipitated
(IP) with either anti-TIFA, control rabbit IgG, or
anti-TRAF6 antibody. Immunoprecipitates were analyzed by Western
blotting (WB) with anti-TIFA, anti-IRAK-1, and anti-TRAF6
antibodies. Lysates prepared from HEK293T cells transfected with
pME-TIFA or control vector were analyzed on identical gels.
Open and solid arrowheads indicate the Ig light
chain and nonspecific bands, respectively. B, interaction of
TRAF6 with TIFA in MEF cells. Both wild-type MEF (+/+) and
TRAF6-deficient MEF (
/
) were infected with retrovirus expressing
FLAG-TIFA. Anti-TRAF6 antibody immunoprecipitates were analyzed by
Western blotting with anti-FLAG antibody. Open and
solid arrowheads indicate the Ig heavy chain and Ig light
chain, respectively. Lysates were also subjected to immunoblotting
directly to analyze FLAG-TIFA expression. C, IL-1
stimulation-dependent interaction of TIFA and IRAK-1 in MEF
cells. Wild-type MEFs were infected with retrovirus expressing
FLAG-TIFA or control virus. Both types of MEFs were unstimulated (
) or stimulated (+) with
IL-1 (20 ng/ml, 5 min). Anti-FLAG antibody immunoprecipitates were
analyzed by Western blotting with anti-IRAK-1 antibody. The solid
arrowhead indicates Ig light chain. Lysates were also subjected to
immunoblotting directly to analyze IRAK-1 expression.
B Activation via IRAK-1 and
TRAF6--
To further investigate the notion that TIFA
functions by interacting with IRAK-1 and TRAF6,
the effect of a dominant-negative mutant (DN) of TIFA and DN of
previously identified proteins involved in IL-1 signaling on
NF
B-mediated transcription was analyzed. GST-G50ES66A suppressed
IL-1- and TLR4-induced NF
B activation but not TNF
-induced
activation (Fig. 6A),
suggesting that TIFA functions in TRAF6-mediated signaling. MyD88- and
IRAK-1-induced NF
B activation was also suppressed by GST-G50ES66A,
whereas TAK1/TAB1-induced activation (10) was not affected (Fig.
6B, left, luciferase activity without
GST-G50ES66A was set to 100). Unexpectedly, TRAF6-induced NF
B
activation was further augmented by GST-G50ES66A (Fig. 6B, right, luciferase activity with reporter alone was set to
1), which binds to TRAF6 but does not activate NF
B without
expressing exogenous TRAF6. Transfection of a suboptimal dose of TRAF6
leads to the activation of NF
B to some extent, suggesting that
exogenously expressed TRAF6 can be partially activated presumably by
its conformational change or by its oligomerization under this
transient transfection condition. GST-G50ES66A may be able to activate
partially activated exogenous TRAF6.
View larger version (20K):
[in a new window]
Fig. 6.
TIFA activates NF B
via TRAF6 and IRAK-1. A, HEK293T cells or HepG2 cells
were transfected with 3x
B-luc and increasing amounts (0, 3, or 10 µg) of pME-GST-G50ES66A. Thirty hours after transfection, cells were
either stimulated with TNF
(10 ng/ml, HEK293T) or IL-1 (20 ng/ml,
HepG2) for 6 h. HEK293T cells were transfected with pEF-TLR4 and
pEF-MD-2 together with 3x
B-luc and increasing amounts of
pME-GST-G50ES66A. B, HEK293T cells were transfected with an
expression plasmid encoding one of the NF
B activators including
MyD88, IRAK-1, TAK1/TAB1, or TRAF6 in the presence of increasing
amounts (0, 3, or 10 µg) of pME-GST-G50ES66A. C, HEK293T
cells were transfected with 0.1 µg of pME-TIFA in the absence or
presence of increasing amounts (3 or 10 µg) of expression vector
encoding MyD88-(152-296), IRAK-1-(1-211), TRAF6-(275-530),
TAK1(K63W), or Ubc13(C87A). Thirty-six hours after transfection,
luciferase activity was measured. Relative values in which the fold
activation in the absence of each dominant-negative mutant was set to
100 (A, B except TRAF6 columns, C) or
in which the luciferase activity with reporter alone was set to 1 (TRAF6 columns in B) are shown. Results shown are
the mean ± S.D. of triplicate experiments and are representative
of two independent experiments.
B activation was blocked by TRAF6-DN and TAK1-DN
(Fig. 6C). An inactive form of Ubc13 (Ubc13(C87A)) also
suppressed TIFA-induced activation (Fig. 6C), suggesting
that the TIFA signal leads to Lys-63-linked polyubiquitination of
TRAF6, which is required for TAK1-mediated activation of I
B kinase
and MKK6 (13, 31). TIFA-induced activation was not affected by
MyD88-DN, but it was blocked by IRAK-1-DN (Fig. 6C). Because
TIFA binds IRAK-1 when overexpressed (Fig. 4A), it is
possible that IRAK-1-DN inhibited TIFA-mediated NF
B activation by
interfering with the activation process of TIFA even if IRAK-1
functions upstream of TIFA in vivo. Taken together, these
findings support the notion that TIFA links TRAF6 to IRAK-1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activation
was inhibited by expression of the DN of TIFA. Thus, TIFA may function
in innate immunity. Although T2BP, identical to TIFA, was shown to
associate with TRAF2, when they overexpressed, the T2BP/TRAF2
interaction in vivo has never been demonstrated (18).
Nevertheless, TIFA may regulate both TRAF2 and TRAF6 in vivo.
B or JNK suggest that a
specific target protein for the FHA domain of TIFA may exist and play a role in the regulation of TIFA-mediated signaling. In this study, we
identified three TIFA-binding proteins including TRAF6, IRAK-1, and
TIFA itself. However, they were able to bind the G50ES66A TIFA mutant
as well as wild-type TIFA, which suggests that none of them is a
candidate protein that binds to the FHA domain in a
phosphorylation-dependent manner. Because TRAF6 transduces
signals when oligomerized (13, 14) and recombinant TIFA can form
trimers, one may postulate that TIFA is involved in the oligomerization of TRAF6 in concert with IRAK-1 to activate NF
B and JNK.
Interestingly, recombinant TIFA G50ES66A mutant formed a pentamer or
hexamer, not a trimer. Moreover, the G50ES66A mutant is able to
activate NF
B in the presence of the suboptimal dose of TRAF6 but not
in concert with endogenous TRAF6 in the absence of exogenous TRAF6. Given that transient transfection of the suboptimal dose of TRAF6 results in the partial activation of TRAF6, the intact structure of the
FHA domain of TIFA may be required for the initial step of the TRAF6
activation that may require specific oligomer formation of TRAF6.
Further studies are required to clarify exact roles of the FHA domain
of TIFA; that is, binding to the putative phosphoprotein, holding the
TIFA protein in an active conformation, or some other functions.
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ACKNOWLEDGEMENTS |
---|
We thank Z. J. Chen, S. Miyamoto, K. Miyake, S. Akira, K. Matsumoto, T. Naka, and S. Ohno for providing reagents. We thank H. Hayashi, R. Ajima, and T. Yamamoto for technical support and valuable discussions. We are grateful to Z. J. Chen and S. Miyamoto for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by a grant-in-aid for Scientific Research on Priority Areas and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese government.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB062111 and AB062110.
To whom correspondence should be addressed. Tel.:
813-5449-5275; Fax: 813-5449-5421; E-mail:
jun-i@ims.u-tokyo.ac.jp.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M300720200
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ABBREVIATIONS |
---|
The abbreviations used are:
IL-1, interleukin-1;
TLR, Toll-like receptor;
IRAK-1, IL-1 receptor-associated kinase-1;
TRAF6, tumor necrosis factor receptor-associated factor 6;
TAK1, transforming growth factor -activated kinase 1;
JNK, c-Jun
amino-terminal kinase;
GST, glutathione S-transferase;
MBP, maltose-binding protein;
MEF, mouse embryonic fibroblast;
FHA domain, forkhead-associated domain;
DN, dominant negative;
MKK, mitogen-activated kinase kinase II.
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