From the Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142
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ABSTRACT |
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We describe here the identification and
characterization of tumor necrosis factor receptor (TNFR)-associated
factor 2A (TRAF2A), a splice variant of the TRAF2 molecule utilized for
signal transduction by members of the TNFR family. TRAF2A and TRAF2
cDNAs are identical in sequence with the exception of an extra 21 base pairs of sequence encoding a 7-amino acid insert within the TRAF2A
RING finger domain. TRAF2A mRNA expression is regulated in a
tissue-specific manner, with relative TRAF2A mRNA levels being
highest in spleen and lowest in brain. TRAF2A protein is capable of
binding to the cytoplasmic domain of TNFR2 (p75) and is detectable in
T-lymphoma cells stably transfected with the TRAF2A cDNA. Unlike
TRAF2, TRAF2A has a short half-life (~100 min) in these cells and is
expressed at only low levels in transiently transfected COS-7 cells.
However, TRAF2A levels in transiently transfected COS-7 cells approach
those of TRAF2 upon coexpression with TRAF1 and/or TRAF2, indicating
that TRAF2A stability is regulated by the binding of other TRAF family proteins. Also in contrast to TRAF2, TRAF2A is unable to stimulate NF-B activity when overexpressed in 293 cells and acts as a dominant inhibitor of TNFR2-dependent NF-
B activation. TRAF2A
thus represents a novel signal transduction protein, the expression of
which can act to inhibit TRAF2-dependent NF-
B
activation.
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INTRODUCTION |
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Type 1 and 2 TNF1
receptors (TNFR1 and TNFR2) are members of a family of structurally
related membrane receptors that includes lymphotoxin receptor, Fas,
WSL-1, DR4, CD40, CD30, CD27, 4-1BB, OX40, and p75 nerve growth factor
receptor (1). Members of the TNFR family can interact through their
cytoplasmic domains with a range of intracellular signal transduction
proteins, most of which fall into one of two distinct homology groups.
The first of these is the death domain-containing proteins, including
TRADD, FADD/MORT1, and RIP, which associate directly with receptors
also containing death domains, such as TNFR1 and Fas (2-4). The second is the TRAF proteins.
TRAF family molecules share a conserved carboxyl-terminal domain, which mediates receptor binding, homooligomerization, and, in the case of TRAF1 and TRAF2, heterooligomerization (5, 6). In the cases of TRAF2-6, homology is also found in the form of an amino-terminal RING finger domain followed by a string of four or five zinc finger motifs (5-12). TRAF1 and TRAF2 were originally identified by their association with the cytoplasmic domain of TNFR2 (5). TRAF2 has subsequently been found to associate with a number of TNFR family molecules including CD40, CD30, and TNFR1 (13-17). In the case of TNFR1, TRAF2 association is indirect, occurring via the TRADD adaptor protein (17). At least four other members of the TRAF homology family have been identified, three of which (TRAF3 (CRAF1/CD40bp/LAP1) (6-8), TRAF5 (10, 11), and TRAF6 (12)) bind directly to members of the TNFR family. Receptor binding activity has not been described for the TRAF4 (CART1) molecule, which, unlike the other TRAF family members, exhibits a nuclear localization (9).
TRAF proteins do not carry motifs characteristic of enzymatic activity
but instead appear to function as adaptor proteins. In the case of
TRAF2, direct binding of at least eight intracellular molecules has
been demonstrated, including TRAF1, c-IAP1, c-IAP2, I-TRAF/TANK, A20,
TRIP, RIP, and NIK (5, 14, 18-23). The best characterized
TRAF-mediated signal transduction pathway is the activation of NF-B
transcription factors, an activity shared by TRAFs 2, 5, and 6 (10-13). In the case of TRAF2, both the RING finger domain and the
first two zinc finger domains are essential for NF-
B
activation (24). TRAF2 mediates NF-
B activation via the
recruitment of the serine/threonine kinase NIK (23), which can in turn
activate CHUK, an I
B-specific kinase that triggers I
B degradation
(25, 26). In addition to recruiting mediators of NF-
B activation,
TRAF2 can bind at least three other molecules (I-TRAF/TANK, A20, TRIP)
that inhibit its ability to activate NF-
B (19-21). The efficiency
of NF-
B activation via TRAF2 in a given cell therefore depends
on the relative levels and activities of a number of different
regulatory molecules.
In this study, we describe TRAF2A, a splice variant of TRAF2 that
contains a 7-amino acid insertion within the RING finger domain. TRAF2A
is incapable of mediating the activation of NF-B and can act as a
dominant inhibitor of TNFR2-mediated NF-
B activation. Thus,
alternative splicing of primary transcripts from the TRAF2/TRAF2A gene
to produce TRAF2A as opposed to TRAF2 mRNAs may represent a novel
mechanism by which cells can regulate NF-
B activation through TNF
family receptors.
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EXPERIMENTAL PROCEDURES |
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cDNAs-- Full-length TRAF1 and TRAF2 cDNAs were obtained by RT-PCR from spleen and kidney RNA, respectively, from a female C57BL/6 mouse. Sequence-specific RT was performed using TACTCTAGACCCCATCCCTAAGCACTA (TRAF1) and TATTCTAGACTGGCCTAATGTGACAGC (TRAF2). These oligonucleotides were utilized as lower primers for PCR along with the upper primers AAGAATGCGGCCGCACCTGAAACCCCAAGAT (TRAF1) and AAGAATGCGGCCGCGTGTGGGGGTTGTAACTCA (TRAF2). PCR was performed using cloned Pfu DNA polymerase (Stratagene). PCR products of the correct size were digested with NotI and XbaI and cloned into pRC-CMV (Invitrogen). The same approach was taken to prepare TNFR2 cDNA from spleen RNA except that RT was with GGGTCCCTTTGCAGGGTG, and PCR was with AATGCGGCCGCCACCGCTGCCCCTATG and TACTCTAGACAGGGGTCAGGCCACTTT. cDNA clones were verified by restriction enzyme mapping and DNA sequencing. Constructs encoding HA-tagged TRAF2 and TRAF2A were generated by PCR-mediated insertion of DNA sequence encoding the peptide AYPYDVPDYAEF immediately following the methionine initiation codon.
RT-PCR Detection of TRAF2 and TRAF2A mRNA-- Cytoplasmic poly(A)+ mRNA was prepared from cultured cells using an Oligotex kit and total RNA from tissues using the RNeasy kit (Qiagen). TRAF2 sequence-specific RT was performed as above, and PCR was performed using upper (GACCAGGTTAGAAGCCAAGTA) and lower (TAGACACAGGCAGCACAGTTC) primers corresponding to TRAF2 sequences flanking the site of the TRAF2A insert. 147- and 168-bp fragments derived from TRAF2 and TRAF2A mRNAs, respectively, were separated by electrophoresis on 2-4% agarose gels. These primers were also used to amplify a 2.0-kbp fragment from C57BL/6 embryonic stem cell genomic DNA. This fragment was cloned and sequenced to reveal the genomic location of the TRAF2A insert.
GST and Maltose-binding Protein (MBP) Fusion Proteins-- Fusion proteins of Escherichia coli glutathione S-transferase (GST) and MBP were produced using the vectors pGEX-4T-1 (Pharmacia Biotech Inc.) and pMAL-c2 (New England Biolabs), respectively. DNA fragments coding for the relevant protein domains were PCR-amplified from cDNAs with Pfu DNA polymerase using upper primers flanked by an EcoRI site and lower primers that included an in frame stop codon followed by a SalI site. PCR fragments were digested with EcoRI and SalI and subcloned into the corresponding vector, and inserts were confirmed by partial DNA sequencing. Expression and purification of GST fusion proteins was performed as described previously (27) and that of maltose-binding protein fusion proteins according to the manufacturer's instructions. Except where indicated, TRAF2/TRAF2A fusion proteins were grown in the presence of 1 mM ZnCl2.
Antibodies-- Rabbit anti-TRAF2 antiserum was raised by HRP Inc. (Denver, PA) using as the immunogen a fusion protein between GST and amino acids 2-178 of TRAF2 (GST-TRAF2-(2-178)). To obtain affinity purified anti-TRAF2 antiserum, MBP-TRAF2-(2-178) fusion protein was covalently coupled to Affi-Gel 10/15 (1:1 mixture) (Bio-Rad), and material from immune serum that bound to the resulting affinity resin eluted with 100 mM glycine/HCl, pH 2.5. Mouse monoclonal antibody against the hemagglutinin epitope YPYDVPDYA (clone 12CA5) was from Boehringer Mannheim.
Cell Lines and Transfections-- COS-7, LBRM-33-1A5, and 293 cells were from the ATCC. COS-7 and 293 cells were maintained in Dulbecco's modified Eagle's medium plus 10% fetal calf serum (Sigma), and LBRM cells were maintained in RPMI, 10% FCS, 25 µM 2-mercaptoethanol. LBRM clones stably expressing TRAF cDNAs were obtained by electroporation (0.29 kV, 960 microfarads) with PvuI-linearized pRC-CMV expression plasmids, selection in medium containing 0.4 mg/ml geneticin (Sigma), and propagation of resistant colonies. Transient transfections of COS-7 and 293 cells were achieved using the DEAE-dextran and calcium phosphate (28) methods, respectively.
Immunoprecipitations and Precipitations with GST Fusion Proteins-- Cells were washed in ice-cold PBS and then lysed for 20 min at 4 °C in 0.5% Nonidet P-40 lysis buffer also containing 25 mM HEPES, pH 7.4, 250 mM NaCl, 2 mM EDTA, 10 µg/ml leupeptin, and 0.1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochlorine. Nontransfected and stably transfected LBRM cells were lysed at a concentration of 2-5 × 107 cells/ml, and transiently transfected COS-7 cells were lysed at 1-5 × 106 cells/ml. Metabolic labeling was with [35S]methionine/cysteine (100 µCi/ml). Cellular debris was removed from lysates by centrifugation at 14,000 × g for 10 min at 4 °C. For immunoprecipitations, lysates were precleared with 25-50 µl of protein A-Sepharose (Pharmacia) for 1 h at 4 °C. Antiserum (1 µg of purified antibody) was mixed with 25 µl of protein A-Sepharose beads for 1 h and mixed with precleared lysates for 4-16 h, and beads were washed extensively in lysis buffer. For coprecipitation with GST fusion proteins, lysates were precleared with 25-50 µl of glutathione-agarose beads (Sigma) for 1 h at 4 °C. 10 µl of beads containing 0.5-2.0 µg of GST fusion protein were then added to precleared lysates, mixed for 12-16 h, and washed extensively in lysis buffer. For secondary immunoprecipitations with anti-TRAF2, beads were boiled for 10 min in 30 µl of 1% SDS and diluted with 400 µl of lysis buffer, and the supernatant was subjected to immunoprecipitation for 3-4 h. Precipitated proteins were removed from the beads by boiling in reducing sample buffer for 2 min and then fractionated by SDS-PAGE. To detect 35S-labeled proteins, dried gels were exposed directly to Biomax film (Eastman Kodak Co.). For Western blots, fractionated proteins were transferred to a nitrocellulose membrane and incubated with primary antibody followed by horseradish peroxidase conjugated to either anti-rabbit or anti-mouse IgG (Amersham Corp.). Positive bands were detected by electrochemiluminscence.
Measurement of NF-B Activation--
Activation of NF-
B by
TNFR2 and TRAF molecules was measured following transient
cotransfection of 293 cells with the pRC-CMV expression plasmids and a
luciferase-based NF-
B reporter construct. Cells seeded 18-24 h
beforehand in six-well dishes (2 × 105 cells/well)
were cotransfected with 20 ng of NF-
B-luciferase reporter construct
(14), 500 ng of pCH110 (expresses lacZ from SV40 early
promoter) (Pharmacia), 2 µg of pRC-TRAF expression plasmid, and 1 µg of either empty pRC-CMV vector or pRC-TNFR2. Luciferase and
-galactosidase activity was measured in cell lysates after 48 h
as described (14) except that chlorophenol
red-
-D-galactopyranoside (10 mg/ml stock) (Sigma) was
used as the
-galactosidase substrate, and activity was measured as
increased absorbance at 570 nm. This readout exhibited a linear
relationship to
-galactosidase concentration over the experimental
range and served as the internal control for standardization of
readings of luciferase activity.
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RESULTS |
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TRAF2A mRNA Encodes TRAF2 with an Extended RING Finger Domain and Is Produced by Alternate Splicing of a Common Primary Transcript-- Using an RT-PCR strategy based on the published sequence of murine TRAF2 cDNA (5), full-length TRAF2 cDNAs were obtained from mouse kidney RNA. Restriction enzyme mapping and DNA sequencing revealed that several clones (3 of 21) carried an additional 21 bp of sequence encoding a 7-amino acid insert within the TRAF2 RING finger domain (Fig. 1A). This sequence was identical in all three clones carrying the insert and in two further clones obtained from mouse spleen RNA. Sequencing of full-length cDNAs revealed that, with the exception of the 21-bp insert, the two classes of TRAF2 clones were identical both to each other and to the published TRAF2 sequence. The newly identified TRAF2 variant was designated TRAF2A.
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Relative Levels of TRAF2A mRNA Varies between Different Tissues-- Northern blot analysis has revealed ubiquitous expression of mRNA species hybridizing to a TRAF2 probe (5). To determine what proportion of these molecules encode TRAF2A versus TRAF2 in different tissues, we employed an RT-PCR assay using primers flanking the TRAF2A insertion site. While coexpression of TRAF2 and TRAF2A transcripts was found in all tissues examined, the ratio of TRAF2A to TRAF2 mRNAs varied from very low in brain, lung, and heart to almost 1:1 in spleen (Fig. 2). Thus, the balance between TRAF2 and TRAF2A mRNAs is differentially regulated in different tissues, suggesting that TRAF2A may have tissue- or cell-type specific functions.
TRAF2 and TRAF2A RING Fingers Can Form Zinc-stabilized Structures-- Although RING finger domains exhibit a high conservation of spacing between most of the eight zinc-coordinating residues, the gap between the sixth and seventh of these can vary from 4 to 48 amino acids in length (29) (Fig. 1). The TRAF2A insert is situated in this heterogeneous region of the RING finger (Fig. 1A), suggesting that the resulting increase in the number of residues may still permit formation of a zinc-stabilized RING finger fold. To test this, fusion proteins were expressed in bacteria that consisted of GST linked to either wild type TRAF2 or TRAF2A RING finger domains or RING fingers in which the first two zinc-coordinating cysteine residues were mutated to alanine. Efficient purification from bacteria of intact GST-RING finger fusion protein, as opposed to the GST core from which the RING finger structure had been degraded, was reliant on the presence of added Zn2+ in the culture medium and on the first two zinc-coordinating cysteine residues but was independent of the TRAF2A insert (Fig. 3). Thus TRAF2A as well as TRAF2 RING finger domains can form zinc-stabilized structures that presumably conform to the RING finger fold (30, 31).
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Expression of TRAF2A Protein-- Since TRAF2A mRNA expression is high in spleen, we examined a lymphoid cell line (LBRM-33-1A5 T-lymphoma) in an attempt to detect TRAF2A protein. As controls, LBRM cells stably transfected with the TRAF2 cDNA (LBRM/TRAF2) or TRAF2A cDNA (LBRM/TRAF2A) were used. RT-PCR analysis revealed that in untransfected and vector-transfected cells, the ratio of TRAF2A to TRAF2 mRNA was approximately 1:3 and that this ratio was appropriately shifted down and up in LBRM/TRAF2 and LBRM/TRAF2A cells, respectively (Fig. 4A).
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TRAF2A Has a Significantly Shorter Half-life than TRAF2-- The fact that TRAF2A protein could only be detected easily in LBRM/TRAF2A cells expressing relatively high levels of TRAF2A mRNA suggested that it may possess a shorter half-life than TRAF2. To verify this, a pulse-chase experiment was performed using LBRM/TRAF2A cells. TRAF2/TRAF2A proteins were prepared following a 40-min pulse with [35S]methionine/cysteine and at various times following the reintroduction of the pulsed cells into culture medium containing nonradioactive amino acids. While little change was observed in the amount of 35S-labeled TRAF2 present in the cells over the 3-h chase period, 35S-labeled TRAF2A levels dropped rapidly, the half-life of TRAF2A molecules labeled during the pulse being about 100 min (Fig. 5).
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TRAF2A Is Stabilized by TRAF1 and/or TRAF2-- As described above, the intrinsic instability of TRAF2A results in it being expressed at low levels in COS-7 cells transfected with the TRAF2A cDNA alone (Fig. 4B). Interestingly, however, cotransfection of COS-7 cells with the TRAF2 and TRAF2A cDNAs results in greatly increased levels of TRAF2A protein (Fig. 4B). Since TRAF2 binds both to itself and to TRAF1 independently of the RING finger domain (5), we hypothesized that TRAF2A may be stabilized by binding to TRAF2 and potentially to TRAF1. To test this, cDNAs encoding HA-tagged TRAF2 or TRAF2A were cotransfected into COS-7 cells with combinations of untagged TRAF1, TRAF2, and TRAF2A cDNAs. Expression of the tagged molecules was assessed by Western blotting with anti-HA antibody of material precipitated from Nonidet P-40 lysates with full-length GST-TNFR2ic. Coexpression with the various untagged TRAF molecules did not markedly alter the relatively high levels of HA-TRAF2 detected in the cell lysates (Fig. 6A). In contrast, HA-TRAF2A levels were significantly increased by coexpression with either TRAF1 or TRAF2 alone and by expression with TRAF1 plus TRAF2. However, coexpression of HA-TRAF2A with untagged TRAF2A did not detectably increase HA-TRAF2A levels (Fig. 6A). Cotransfection of HA-TRAF2A cDNA with increasing amounts of untagged TRAF1 and TRAF2 but not TRAF2A cDNAs also resulted in increased HA-TRAF2A levels (Fig. 6B). In each of these cases, the amount of HA-TRAF2A bound to GST-TNFR2ic reflected the levels present in the cell lysates (data not shown). Thus, despite the lower innate stability of TRAF2A, stabilization by coexpressed TRAF1 and/or TRAF2 facilitated its expression at levels comparable with those exhibited by TRAF2.
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TRAF2A Does Not Activate NF-B and Inhibits Its Activation by
TNFR2--
TRAF2 mediates the activation of NF-
B transcription
factors through TNFR1, TNFR2, and CD40, an activity for which the RING finger domain is essential (13, 24). We therefore asked whether TRAF2A,
with its altered RING finger domain, also had this activity. To do
this, we took advantage of the fact that overexpression and presumably
autoaggregation of TRAF2 in transiently transfected 293 cells induces
ligand-independent activation of NF-
B (13). A luciferase reporter
construct containing three
B recognition sites was cotransfected
with various TRAF constructs to measure induced NF-
B activity.
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DISCUSSION |
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We describe here the identification and primary characterization of TRAF2A, a previously unknown splice variant of the gene encoding TRAF2. TRAF2A mRNA is produced by alternate usage of splice donor sites present at the 3'-end of (presumably) exon 1 and differs from TRAF2 mRNA in encoding an additional 7 amino acid residues within the amino-terminal RING finger domain (Fig. 1). The production of TRAF2A and TRAF2 mRNAs is regulated in a tissue-specific manner, the ratio of TRAF2A to TRAF2 mRNAs varying significantly between the different tissues examined (Fig. 2). Given the functional and biochemical differences identified here between the TRAF2 and TRAF2A proteins, alternate splicing of the TRAF2/TRAF2A gene may represent an important mechanism for regulating cellular responses to signaling through members of the TNFR family.
Studies utilizing GST fusion proteins of TRAF2 and TRAF2A RING finger domains suggested that the extra 7 amino acids within the TRAF2A RING finger do not significantly disrupt the zinc-stabilized RING finger fold (Fig. 3). This is consistent with the apparent permissiveness of the of the spacing of the sixth and seventh zinc-coordinating residues, which ranges from 4 to 48 residues in different RING finger proteins (29). Given the high conservation of spacing between nearly all of the other zinc-coordinating residues (29), alternative splicing of small amounts of sequence into this region of the RING finger may have evolved to allow specific physical and presumably functional alterations to the domain without disrupting the overall RING finger fold. The fact that a splice site is positioned in the same region of TRAF4 (CART1) and TRAF3 (9, 32) indicates that such a mechanism could be utilized in the expression of other RING finger proteins.
The apparent stability of the TRAF2A RING finger domain in bacteria stands in contrast to the short half-life of TRAF2A compared with TRAF2 in LBRM T-lymphoma cells (Fig. 5) and in COS-7 cells (Fig. 4B). This observation may be explained by the presence in mammalian but not bacterial cells of proteolytic systems that target the TRAF2A but not the TRAF2 RING finger for degradation. One possible candidate for this is the ubiquitin/proteosome pathway, a common regulator of proteins in signal transduction pathways (33). Whatever the mechanism responsible for the short intrinsic half-life of TRAF2A, it is greatly inhibited by the coexpression of TRAF2A with and presumably binding to TRAF1 and/or TRAF2 (Fig. 5). Given that production of high levels of TRAF2A mRNA in normal cells is probably accompanied by down-regulation of TRAF2 mRNA, TRAF1 seems more likely to stabilize TRAF2A under physiological conditions. In this respect, it is interesting to note that TRAF1, which exhibits tissue-specific and inducible expression (5), is expressed at high levels in spleen, the tissue found here to express the highest levels of TRAF2A mRNA (Fig. 2).
The precise role of TRAF2A remains to be identified. It is possible
that TRAF2A, by virtue of its unique RING finger structure, may
initiate a unique signaling cascade in response to ligation of TNF
family receptors. Nevertheless, the demonstration here that TRAF2A
cannot activate NF-B and has a dominant inhibitory effect on this
function suggests that one consequence of TRAF2A expression may be to
down-regulate the TRAF2-dependent NF-
B activation pathway. While the ability of TRAF2A to act as dominant inhibitor may
depend on it being stabilized in some way (e.g. by TRAF1), a
shift toward exclusive production of TRAF2A mRNA would be expected to inhibit TRAF2-dependent NF-
B activation by simply
down-regulating TRAF2 levels. Such a mechanism may act in addition to
or complement the actions of other inhibitors of
TRAF-dependent NF-
B activation. For example, unlike the
actions of I-TRAF/TANK and A20 (19, 20), splicing to TRAF2A is likely
to specifically inhibit TRAF2-mediated NF-
B activation and not
activation of NF-
B through TRAF6. Also, the specific conditions
under which alternate splicing to TRAF2A occurs may differ from those
required to activate other inhibitors of TRAF2-mediated NF-
B
activation.
The presence of multiple mechanisms for down-regulating NF-B
responses indicates the importance of this form of signal regulation. Evidence for the role such mechanisms may play in regulating cellular responses comes from experiments demonstrating that inhibition of
TNFR1-mediated NF-
B activation can enhance the apoptotic signal transduced through this receptor (34-37). Since TRAF2 mediates the
activation of NF-
B by TNFR1, switching to the production of TRAF2A
by cells may mark them for killing by TNF or related cytokines. To
clarify this and other potential functions of the alternate splicing of
the TRAF2/TRAF2A gene, it will be important to identify the cells
(e.g. splenic subpopulation) that produce TRAF2A mRNA as
the major splice product. This search is currently under way.
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ACKNOWLEDGEMENTS |
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We thank Drs U. Klingmüller, X. Liu, and K. Luo for valuable discussions and advice. We also thank R. Quinn and Dr N. Pearce for practical assistance and Prof. A. Basten for support.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant R01 CA-63260 (to H. F. L.) and by a Leo & Jenny Leukaemia & Cancer Foundation of Australia grant (to R. B.).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/EMBL Data Bank with accession number(s) AF027570.
A C. J. Martin Fellow of the National Health and Medical
Research Council of Australia. To whom correspondence should be
addressed: Centenary Institute of Cancer Medicine and Cell Biology,
Locked Bag No. 6, Newtown NSW 2042, Australia. Tel.: 61-2-9565-6136; Fax: 61-2-9565-6105; E-mail: R.Brink{at}centenary.usyd.edu.au.
1 The abbreviations used are: TNF, tumor necrosis factor; TNFR, TNF receptor; TRAF, TNFR-associated factor; bp, base pair(s); kbp, kilobase pair(s); GST, glutathione S-transferase; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; RT, reverse transcription; PCR, polymerase chain reaction; MBP, maltose-binding protein.
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REFERENCES |
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