Identification of TRAF6, a novel tumor necrosis factor receptor-associated factor protein that mediates signaling from an amino-terminal domain of the CD40 cytoplasmic region.

CD40 signalings play crucial roles in B-cell function. To identify molecules which transduce CD40 signalings, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer, designated TRAF6, has been molecularly cloned. TRAF6 has a tumor necrosis factor receptor (TNFR)-associated factor (TRAF) domain in its carboxyl terminus and has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other TRAF family proteins. TRAF6 does not associate with the cytoplasmic tails of TNFR2, CD30, lymphotoxin-β receptor, and LMP1 of Epstein-Barr virus. Deletion analysis showed that residues 246-269 of CD40 which are required for its association with TRAF2, TRAF3, and TRAF5 are dispensable for its interaction with TRAF6, whereas residues 230-245 were required. Overexpression of TRAF6 activates transcription factor NFκB, and its TRAF-C domain suppresses NFκB activation triggered by CD40 lacking residues 246-277. These results suggest that TRAF6 could mediate the CD40 signal that is transduced by the amino-terminal domain (230-245) of the CD40 cytoplasmic region and appears to be independent of other known TRAF family proteins.


CD40 signalings play crucial roles in B-cell function.
To identify molecules which transduce CD40 signalings, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer, designated TRAF6, has been molecularly cloned. TRAF6 has a tumor necrosis factor receptor (TNFR)-associated factor (TRAF) domain in its carboxyl terminus and has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other TRAF family proteins. TRAF6 does not associate with the cytoplasmic tails of TNFR2, CD30, lymphotoxin-␤ receptor, and LMP1 of Epstein-Barr virus. Deletion analysis showed that residues 246 -269 of CD40 which are required for its association with TRAF2, TRAF3, and TRAF5 are dispensable for its interaction with TRAF6, whereas residues 230 -245 were required. Overexpression of TRAF6 activates transcription factor NFB, and its TRAF-C domain suppresses NFB activation triggered by CD40 lacking residues 246 -277. These results suggest that TRAF6 could mediate the CD40 signal that is transduced by the amino-terminal domain (230 -245) of the CD40 cytoplasmic region and appears to be independent of other known TRAF family proteins.
CD40 is a member of the tumor necrosis factor receptor (TNFR) 1 superfamily, which includes TNFR1 and -2 (1, 2), Fas (3), lymphotoxin-␤ receptor (4), CD30 (5), OX40 (6), and the low affinity nerve growth factor receptor (7), all of which share a ligand-binding domain composed of tandemly repeated cysteine-rich modules. CD40 is expressed in late B cells in bone marrow, mature B cells, and certain accessory cells, including bone marrow-derived dendritic cells and follicular dendritic cells (8 -10), and is a receptor for CD40 ligand (CD40L) present on activated CD4 ϩ T cells (11). Signals through CD40 rescue B cells from apoptosis induced by cross-linking of the surface immunoglobulin M complex (12) and also induce B cells to differentiate and to undergo Ig isotype switching (13,14).
CD40 signaling events were reported to include modulation of the activity of non-receptor-type tyrosine kinases such as Lyn, Fyn, and Syk, activation of phosphatidylinositol-3-kinase, phosphorylation of phospholipase C␥2 (15)(16)(17), activation of the Rel/NFB transcription factors (18), and induction of the Bclx L , Cdk4, and Cdk6 proteins (19). However, the mechanisms of signal transduction from CD40 are uncertain. Biochemical purification of receptor-associated proteins or the recently developed cDNA cloning system that uses yeast genetic selection led to the discovery of two groups of signal transducer molecules that are utilized by members of the TNFR superfamily. Members of the first group are proteins with a highly conserved domain known as the TRAF domain and include TRAF1, TRAF2 (20), and TRAF3, also known as CD40bp, LAP-1, or CRAF1 (21)(22)(23). TRAF proteins have been implicated in signal transduction from TNFR2 and CD40. We (24) and Nakano et al. (25) have recently cloned a cDNA encoding TRAF5 which mediates signals emanating from CD40 and the lymphotoxin-␤ receptor. The second group includes proteins with a death domain involved in Fas and TNFR1 signaling such as FADD (26) also known as MORT1 (27) or RIP (28), and TRADD (29). Among various CD40 signaling events, the Rel/NFB activation was demonstrated to be mediated by TRAF2 (30), TRAF5 (24,25), and RIP (31). To further characterize the initial stage of signaling by CD40, we have used the yeast two-hybrid system to identify cDNAs encoding proteins that interact with the cytoplasmic tail of CD40. Here we report the identification of a novel member of the TRAF family, designated TRAF6, that binds to the amino-terminal region of the CD40 cytoplasmic tail, which is distinct from the binding domain for TRAF2, TRAF3, and TRAF5.

MATERIALS AND METHODS
Yeast Two-hybrid System-A DNA fragment encoding the cytoplasmic tail of mouse CD40 (amino acids 216 -306) was cloned into the yeast LexA DNA-binding domain vector pBTM116. The resulting plasmid, pBTM40cyt, was used as bait in a two-hybrid screening of a murine C57 Black Kaplan cDNA library fused to the activation domain of Gal4 in the pACT plasmid (Clontech). Seventy-two out of the 2 ϫ 10 6 transformants screened grew in the absence of histidine and had detectable * This work was supported by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports and Culture of Japan, a grant-in-aid from the research fellowships of the Japan Society for the Promotion of Science for Young Scientists, and Grand-in-aid CA47006 from the National Cancer Institute (to E. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This 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 GenBank TM /EBI Data Bank with accession number(s) D84655.
␤-galactosidase staining within 20 min of incubation. Ten clones were used in a cotransformation assay with pBTM40cyt or control pBTM-Lamin bait to reconfirm the specificity.
cDNA Cloning and Northern Blotting-A fragment of approximately 400 bp derived from the 5Ј-region of the C40 -65 cDNA (probe D, see Fig. 2B) was used as a probe to screen a murine T cell leukemia line EL-4 cDNA library in ZAPII. Two independent positive clones were obtained and subjected to nucleotide sequencing. A mouse multiple tissue RNA blot (Clontech) was incubated with 32 P-labeled 65-69 cDNA ( Fig. 2B) and ␤-actin cDNA at 65°C as described (19). The filter was finally washed with 0.5 ϫ SSC, 0.2% (w/v) SDS at 65°C for 30 min. Total RNA from WEHI-231 and EL-4 cell lines was prepared, and the purification of poly(A) ϩ RNA was performed using oligo(dT) latex (Takara). Five micrograms of the poly(A) ϩ RNA was separated by 1% formaldehyde denaturing agarose gel and transferred to nylon membrane (Hybond N, Amersham). The filter was then incubated with 32 P-labeled probe A or B (Fig. 2B) and then washed as described above. Reverse Transcription-PCR and Southern Blotting-Mouse spleen total RNA was converted to cDNA with random hexanucleotide primers (pd(N) 6 , Pharmacia Biotech Inc.). The PCR primers were designed based on the C40 -65 cDNA sequence (see Fig. 2B). A pair of primers (primer a, 5Ј-CAAGCAGTGGGAGAGGTG-3Ј; primer b, 5Ј-GTTGCT-GTCATCATCCACGAG-3Ј) were used to amplify a 782-bp fragment. The PCR products were separated on a 1.5% agarose gel and transferred to nylon membrane (Hybond N, Amersham). The filter was incubated with a 32 P-labeled oligonucleotide (probe c, 5Ј-ATCACCACT-GCCTGTGCTGGTGCC-3Ј) at 30°C and then washed with 1 ϫ SSC, 0.2%(w/v) SDS at 25°C for 30 min.
In Vivo and in Vitro Binding Assay-For in vivo binding assay, human 293T kidney cells were cotransfected with FLAG-tagged TRAF6 or TRAF2 and GST-tagged cytoplasmic tail of CD40 or its mutants (24). Thirty-six hours after transfection, 1 ϫ 10 7 transfected cells were harvested and lysed with TNE buffer (24) followed by centrifugation. The supernatant was incubated with glutathione-agarose beads (GSHbeads) for 2 h at 4°C. After washing the beads, the GST fusion protein complexes were separated on a 8.5% polyacrylamide-SDS gel, and the FLAG-TRAF6 or FLAG-TRAF2 protein was detected by Western blotting using anti-FLAG antibody M2 and alkaline phosphatase-conjugated anti-mouse IgG. For in vitro binding assays, purified GST and various GST fusion proteins expressed in Escherichia coli (BL-21) were immobilized onto GSH-beads. Extracts were prepared from COS-7 cells transfected with FLAG-tagged TRAF6 and mixed with GST or GST fusion proteins immobilized on beads. After washing, FLAG-TRAF6 bound to GST fusion proteins was detected as described above.
Cell Culture, Plasmid, and Transient Transfection Assays-Human Jurkat T cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were transfected with 0.5 g of the reporter plasmid, [B] 6 TK-CAT or [BM] 6 TK-CAT (32), 0.5 g of ␤-galactosidase expression vector driven by ␤-actin promoter (␤-actin-␤-gal), and indicated amounts of various expression plasmids by the DEAEdextran method. The amount of DNA transfected was always adjusted to 5 g with a control expression vector, pME18S-FLAG. Forty hours after transfection, cell extracts were prepared by freeze-thawing followed by centrifugation. ␤-Galactosidase activity was used to standardize transfection efficiency, and CAT assays were performed for 1 h at 37°C as described (32).

RESULTS AND DISCUSSION
From 2 ϫ 10 6 clones of a murine C57 Black Kaplan T cell lymphoma cDNA library, 10 independent clones were isolated that met all specific criteria for binding to the cytoplasmic tail of mouse CD40 in yeast. Nucleotide sequencing of all cDNA fragments revealed that one of them (clone C40 -65) encoded a peptide which has a TRAF domain in its carboxyl-terminal region (Fig. 1A). Since it is the sixth member of the TRAF family of proteins, we termed this protein "TRAF6." Using the C40 -65 cDNA as a probe, a cDNA encompassing the entire coding region was obtained from a cDNA library prepared from the murine EL-4 T cell line. The longest cDNA clone (clone 65-69) contained 2312 base pairs (bp) including a potential poly(A) tail (Fig. 2B). An in-frame stop codon 69 nucleotides upstream from the first methionine indicates that the clone 65-69 encodes the full-length TRAF6 protein. However, Northern blotting of poly(A) ϩ RNA from various murine tissues and cell lines revealed that the TRAF6 mRNA is 5.5 kb (Fig. 2, A  and C). The C40 -65 cDNA starts at nucleotide 829 of the 65-69 cDNA and has an extra 2986 bp of 3Ј-untranslated region (Fig. 2B). To further investigate the relation between the 65-69 and the C40 -65 cDNAs, Northern blotting was performed using the 5Ј-region of the 65-69 cDNA (probe A) and the 3Ј-region of the C40 -65 (probe B) as probes (Fig. 2B). Both probes hybridized to a 5.5-kb mRNA (Fig. 2C). Furthermore, the C40 -65 cDNA was not artificially generated by ligating two independent cDNAs during the preparation of the cDNA library, because a TRAF6 cDNA obtained from spleen mRNA contains an extra 3Ј-untranslated region identical with that of the C40 -65 cDNA (Fig. 2D). Thus, these short cDNAs could be generated due to the minor poly(A) addition site at 2291 or misannealing of oligo(dT) primer for cDNA synthesis. The length of combined TRAF6 cDNAs is 5277 bp, which is consistent with the size of the TRAF6 mRNA ( Fig. 2A).
The TRAF6 cDNA can encode a protein of 530 amino acids (Fig. 1B) with a calculated molecular weight of 60,083. The carboxyl terminus of the encoded protein is homologous to that of TRAF family protein for approximately 150 amino acids, called TRAF-C domain (20). Although the TRAF-C domain of TRAF6 is most homologous to that of TRAF2 (35.9% identity), identities among other TRAF family proteins range from 41.7% to 66.2%. In addition to the TRAF-C domain, TRAF6 has three potential domains including a coiled-coil domain, a cluster of five zinc fingers, and a RING finger, which are also present in other members of the TRAF family of proteins. The TRAF6 mRNA is expressed in all tissues examined, especially high in brain, lung, liver, skeletal muscle, and kidney ( Fig. 2A). Low levels of the transcripts were detected in heart, spleen, and testis.
To characterize the CD40-TRAF6 interaction, we carried out in vivo binding assays using GST pull down experiments. Cell extracts were prepared from 293T cells cotransfected with FLAG-tagged TRAF6 or TRAF2, and GST-tagged CD40 or its mutants. Whereas the interaction of TRAF2 (middle), TRAF3 (34), or TRAF5 (24) with CD40 is mediated by residues 246 -269 of CD40, the interaction of TRAF6 with CD40 does not fully require residues 245-269 and is mediated by 230 -245 (lanes  2-5, top). The interaction of TRAF6 with CD40 was little affected when Thr 254 of CD40 was converted to Ala (TA mutant, our amino acid numbering includes the signal peptide) (lane 6, top), an alteration known to impair CD40 signaling linked to growth inhibition (33), whereas this mutation abolished the interaction of CD40 with TRAF2 (lane 6, middle), TRAF3 (21), and TRAF5 (24). However, we could not rule out the possibility that TRAF6 might also recognize residues 245-269, because the amount of TRAF6 bound to GST⌬246 or GST-TA was about 65% of that bound to GST⌬270 or GST-WT. These results suggest that TRAF6 could mediate CD40 signaling events that are distinct from those mediated by TRAF2, TRAF3, or TRAF5. Since it is possible that the tertiary structure of CD40 cytoplasmic region in GST fusion protein is different from that in natural membrane-bound CD40, our conclusion described above should be interpreted in consideration of this point.
We next analyzed the interaction of TRAF6 with other members of the TNF receptor superfamily by in vitro binding assay. TRAF6 did not bind to any other receptor examined including TNF receptor type 2, CD30, and lymphotoxin-␤ receptor (Fig.  3C, lanes 3-5), and to LMP-1 of Epstein-Barr virus (lane 6) to which TRAF3 binds (23).
One of the signals emanating from CD40 is the activation of the transcription factor NFB. It has been demonstrated that TRAF2 and TRAF5, but not TRAF3 (24,25,30), mediate signals linked to NFB activation (26). To examine the possible role of TRAF6 in CD40-mediated NFB activation, transient transfection experiments were performed to determine whether TRAF6 expression might lead to activation of transcription from a B-site-dependent reporter gene. The B-sitedependent reporter construct ([B] 6 TK-CAT) (32) was cotransfected with a TRAF6 expression vector (pME-FLAG-TRAF6) into human Jurkat T cells. To confirm the specificity of transcription, the same reporter construct carrying mutant B sites ([BM] 6 TK-CAT) was transfected. In Jurkat cells, TRAF6 as FIG. 3. Mapping of the TRAF6-binding domain of the CD40 cytoplasmic tail. A, structure of CD40 mutants. The amino acid sequence of the cytoplasmic tail of CD40 is shown on the top. B, association of TRAF6 or TRAF2 with the cytoplasmic tail of CD40. Cell extracts from 293T cells cotransfected with FLAG-tagged TRAF6 or TRAF2 and GST-tagged cytoplasmic tail of CD40 or its mutants were prepared. The GST pull-down assays were performed, and the proteins were separated on a 8.5% polyacrylamide-SDS gel. The Western blot was probed with anti-FLAG antibody M2 to detect coprecipitated FLAG-TRAF6 (top) or FLAG-TRAF2 (middle), or with anti-GST antibody to detect GST fusion proteins (bottom). Comparable expression of TRAF6 and TRAF2 in each transfection was confirmed (data not shown). C, association of TRAF6 with various receptors. The FLAG-tagged TRAF6 was transiently expressed in COS-7 cells. The GST pull-down assays were performed, and TRAF6 bound to beads was then analyzed by Western blotting probed with anti-FLAG antibody M2. An asterisk indicates the position of immunoglobulin heavy chain used for immunoprecipitation (upper panel). Two microliters of GST and various GST fusion proteins attached to agarose beads were separated on 12.5% polyacrylamide-SDS gel and visualized by Coomassie Brilliant Blue R-250 staining (lower panel).  6 TK-CAT was little affected by the expression of any TRAF proteins. Each set of experiments was done at least three times. B, involvement of TRAF6 on CD40-mediated NFB activation. pME-FLAG-TRAF6-C and pME-FLAG-TRAF2-C directs the synthesis of the TRAF-C domain of TRAF6 and TRAF2, respectively. Jurkat cells (2 ϫ 10 6 ) were transfected with 0.5 g of [B] 6 TK-CAT, 0.5 g of ␤-actin-␤-galactosidase, 2 or 6 g of pME-FLAG-TRAF6-C or pME-FLAG-TRAF2-C, various combinations of 0.01 g of pMECD40⌬246 and 0.03 g of CD40L expression vector, and enough pME18FLAG control plasmid to give 5 g of total DNA by the DEAE-dextran method. Values correspond to means Ϯ S.E. of at least three independent experiments. well as TRAF2 activates B-site-dependent transcription in a dose-dependent manner (Fig. 4A). Cheng and Baltimore (34) demonstrated the existence of two non-overlapping regions in the cytoplasmic tail of CD40, each of which is sufficient for NFB activation. One region, called TIMct, consists of 17 amino acids (residues 250 -266) and is required and sufficient for the association of CD40 with TRAF2 and TRAF3. The other is a more amino-terminal region which partly overlaps with the TRAF6 binding region. We have also shown here that residues 230 -245 of CD40 required for NFB activation (19) coincide with the region required for the binding of TRAF6, suggesting that activation of NFB by CD40 signaling could be partly mediated by TRAF6. To further elucidate the role of TRAF6 in CD40-mediated NFB activation, we asked whether the TRAF-C domain of TRAF6 acts as a transdominant negative mutant to suppress NFB activation induced by CD40⌬246 mutant in which the binding site for TRAF2 was removed. Expression of the TRAF-C domain of TRAF6 resulted in the suppression of NFB activity, while expression of the TRAF-C domain of TRAF2 had little effect (Fig. 4B). Thus, it is possible that NFB activation through TIMct could be mediated by either TRAF2 or TRAF5 or both, whereas NFB activation from a more amino-terminal region is mediated by TRAF6.
The TNF receptor family of proteins, including Fas, CD40, CD30, and the lymphotoxin-␤ receptor, have been shown to associate with downstream signal transducer molecules carrying either a TRAF or a death domain (20 -29). TRAF2 can interact with the death domain containing molecule TRADD (35), indicating that cross-talk between TRAF proteins and death domain proteins could mediate diverse signaling processes emanating from the TNF receptor family members. Accumulating evidence indicated that three members of the TRAF family proteins including TRAF2, TRAF3, and TRAF5 can directly associate with CD40 (21)(22)(23)(24)30). We have now identified and characterized a new member of CD40-associated factors, designated as TRAF6. We have previously shown that CD40 residues 246 -266, which contains the TIMct domain (34), were required for blocking apoptosis of WEHI 231 cells induced by surface IgM signaling (19). Furthermore, TRAF5 also requires this region to associate with CD40 (24). In contrast, TRAF6 can recognize a more amino-terminal region of CD40 (230 -245) and does not fully require TIMct to associate with CD40 (Fig. 3A). Whether the TRAF6-binding region in CD40 is required for other biological signaling events in addition to NFB activation is unclear; however, it is likely that two distinct signals could emanate from different regions of the CD40 cytoplasmic tail and those signals could cooperate to induce biological phenomena such as blocking of apoptosis or immunoglobulin class switch.
Since TRAF1 associates with TRAF2, CD40 could potentially recruit five members of the TRAF family members to transduce signals. Extensive studies to define complex formation of CD40 with the TRAF family of proteins and the discovery of the downstream signal transducers will clarify further the mechanisms of CD40 signal transduction.