Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9437-9442, September 1996 Biochemistry TRAF5, a novel tumor necrosis factor receptor-associated factor family protein, mediates CD40 signaling (signal transduction/protein-protein interaction/yeast two-hybrid system) TAKAoMI ISHIDA*, TADASHI ToJo*, TSUTOMU AOKI*, NORIHIKO KOBAYASHI*, TSUKASA OHISHI*, TOSHIKI WATANABEt, TADASHI YAMAMOTO*, AND JUN-ICHIRO INOUE*t Departments of *Oncology and tPathology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan Communicated by David Baltimore, Massachusetts Institute of Technology, Cambridge, MA, May 22, 1996 (received for review March 8, 1996) ABSTRACT Signals emanating from CD40 play crucial called a death domain, suggesting that these receptors could roles in B-cell function. To identify molecules that transduce have either common or similar signaling mechanisms (13). CD40 signalings, we have used the yeast two-hybrid system to Biochemical purification of receptor-associated proteins or the clone cDNAs encoding proteins that bind the cytoplasmic tail recently developed cDNA cloning system that uses yeast of CD40. A cDNA encoding a putative signal transducer genetic selection led to the discovery of two groups of signal protein, designated TRAF5, has been molecularly cloned. transducer molecules. Members of the first group are proteins TRAF5 has a tumor necrosis factor receptor-associated factor with a TRAF domain for TNFR2 and CD40 such as TRAF1, (TRAF) domain in its carboxyl terminus and is most homol- TRAF2 (17), and TRAF3, also known as CD40bp, LAP-1, or ogous to TRAF3, also known as CRAF1, CD40bp, or LAP-1, CRAF1 or CD40 receptor-associated factor (18-20). Those of a previously identified CD40-associated factor. The amino second group are proteins with a death domain for FAS and terminus has a RING finger domain, a cluster of zinc fingers TNFR1 such as FADD (21), also known as MORT1 (22), RIP and a coiled-coil domain, which are also present in other (23), and TRADD (24). members of the TRAF family protein except for TRAF1. In CD40 signalings were reported to include modulation of the vitro binding assays revealed that TRAF5 associates with the activity of nonreceptor-type tyrosine kinases such as Lyn, Fyn, cytoplasmic tail of CD40, but not with the cytoplasmic tail of and Syk, activation of phosphatidylinositol-3-kinase, phos- tumor receptor factor receptor type 2, which associates with phorylation of phospholipase C-y2 (25-27), activation of the TRAF2. Based on analysis of the association between TRAF5 Rel/nuclear factorKB (NFKB) transcription factors (28), and and various CD40 mutants, residues 230-269 of CD40 are induction of the Bcl-XL, Cdk4, and Cdk6 proteins (29). It has required for the association with TRAF5. In contrast to also been shown that tyrosine kinase activity is essential for TRAF3, overexpression of TRAF5 activates transcription factor CD40-mediated rescue of the germinal center B cells from nuclear factor #cB. Furthermore, amino-terminally truncated apoptosis (30). Among these signals, the Rel/NFKB activation forms ofTRAF5 suppress the CD40-mediated induction ofCD23 was shown to be mediated by TRAF2 (31). Although it was expression, as is the case with TRAF. These results suggest that demonstrated that all three known TRAF proteins can be TRAF5 and TRAF3 could be involved in both common and recruited to the cytoplasmic domain of CD40, the precise distinct signaling pathways emanating from CD40. mechanism by which CD40 mediates diverse signals remains to be elucidated. To characterize the initial stage of signaling by CD40, we have used the yeast two-hybrid system to identify CD40 is expressed in late B cells in bone marrow, mature B cDNAs encoding a novel protein containing a TRAF domain, cells, and certain accessory cells, including bone-marrow de- TRAF5, which is closely related to TRAF3 (18-20). We also rived dendritic cells and follicular dendritic cells (1-3), and is show that TRAF5 is involved in CD40 signaling linked to a receptor for CD40 ligand (CD40L) present on activated NFKB activation and CD23 expression. CD4+ T cells (4). Signaling through CD40 rescues B cells from apoptosis induced by crosslinking of the surface immunoglob- MATERIALS AND METHODS ulin M (IgM) complex (5) and also induces B cells to differ- Yeast Two-Hybrid System. DNA encoding the intracellular entiate and to undergo Ig isotype switching (6, 7). CD40L has domain of mouse CD40 (amino acids 216-306) was cloned into been shown to be defective in patients with X-linked hyper IgM the yeast LexA DNA-binding domain vector pBTM116. The syndrome, whose B cells do not form germinal centers and resulting plasmid, pBTM40cyt, was used as bait in two-hybrid produce only IgM and/or IgD (8). In addition, their B cells screens of a murine C57 Black Kaplan cDNA library fused to have the ability to switch from IgM to IgG, IgE, or IgA the activation domain of Gal4 in the pACT plasmid (CLON- production in vitro by the stimulation of CD40 (9). This genetic TECH). Seventy-two of the 2 x 106 transformants screened evidence strongly supports the idea that the intercellular grew in the absence of histidine and had detectable f3-galac- communication through CD40-CD40L is essential for germi- tosidase staining within 20 min of incubation. To remove nal center formation and Ig class switching. clones containing either TRAF3 or TRAF2, plasmids ex- CD40 is a member of the tumor necrosis factor receptor tracted from each yeast colony were subjected to Southern (TNFR) superfamily, which includes TNFR1 and TNFR2 (10, blotting probed with TRAF3 and TRAF2 cDNA. Ten clones, 11), lymphotoxin 13 receptor (12), Fas antigen (13), OX40 (14), which were not hybridized with either of two probes, were used CD30 (15), and the low-affinity nerve growth factor receptor (16), all of which share a ligand-binding domain composed of Abbreviations: TRAF, tumor necrosis factor receptor-associated fac- tandemly repeated cysteine-rich modules. Among these, Fas tor; NFKB, nuclear factor KB; CD40L, CD40 ligand; IgM, immuno- antigen and TNFR1 have significant similarity in their cyto- globulin M; TNFR, tumor necrosis factor receptor; GST, glutathione plasmic domain over 46 amino acids, a part of the domain S-transferase; CAT, chloramphenicol acetyltransferase. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. D83528). The publication costs of this article were defrayed in part by page charge ITo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" in Oncology, The Institute of Medical Science, The University ofTokyo, accordance with 18 U.S.C. §1734 solely to indicate this fact. 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan. 9437 9438 Biochemistry: Ishida et al. Proc. Natl. Acad. Sci. USA 93 (1996) in a cotransformation assay with pBTM40cyt or control pBT- CD23 Expression Assay. Mouse WEHI-231 B-cell clones MLamin bait to reconfirm the specificity. Then these clones expressing the FLAG-C40-3 protein were generated by intro- were subjected to nucleotide sequencing. ducing pME-FLAG-C40-3 together with an expression vector cDNA Cloning and Northern Blotting. The cDNA insert of for the puromycin resistant gene (pApuro) followed by selec- approximately 1 kbp from two-hybrid clone C40-3 was used as tion in the presence of 0.5 ,ug/ml of puromycin (38). For a probe to screen mouse testis cDNA library in AZAPII controls, cells were transfected with pApuro alone and sub- (Stratagene, provided by M. Ohsugi) by standard methods. jected to selection. More than 10 clones were isolated and Two independent positive clones were obtained and subjected expression of FLAG-C40-3 was checked by Western blotting. to nucleotide sequencing by the BcaBest sequence system Every clone was confirmed to express normal levels of mouse (Takara Shuzo, Shiga). The total RNA from various tissue was CD40. WEHI-231 cell clones with or without expression of prepared as described (32). The purification of poly(A)+ RNA FLAG-C40-3 were stimulated with mouse CD40L-CD8 chi- was performed using oligo(dT) latex (Takara Shuzo). Seven meric protein (39) for 48 hr. For controls, medium was added micrograms of poly(A)+ RNA was separated by 1% formal- instead of stimulator. After stimulation, cells were stained with dehyde denaturing agarose gel and transferred to nylon mem- fluorescein isothiocyanate-conjugated anti-CD23 antibody brane (Hybond N, Amersham). The filter was incubated with followed by FACScan analysis using the Lysis II program. Side 32P-labeled C40-3 cDNA probe in hybridization buffer [0.2 M scatter values were also measured to confirm that the charac- NaHPO4, pH 7.2/1 mM EDTA/1% (wt/vol) BSA/7% (wt/vol) terization of cells was not dramatically changed during the SDS] at 65°C. The filter was finally washed with 0.5 x drug selection process. SSC/0.2% (wt/vol) SDS at 65°C for 30 min. Glutathione S-Transferase (GST) Fusion Protein and the RESULTS Binding Assay. Plasmids encoding CD40 mutants with Thr-254 Structure and Expression of TRAF5. From 2 x 106 clones to Ala-254 substitutions, carboxyl-terminal truncation, or in- of a murine C57 Black Kaplan T-cell lymphoma cDNA library, ternal deletion were generated by the method of Kunkel (33). 10 independent clones were isolated that met all specific DNA fragments encoding cytoplasmic tail of CD40 and its criteria for binding to the cytoplasmic tail of CD40 in yeast. mutants were subcloned into the GST fusion protein vector Nucleotide sequencing of all cDNA fragments revealed that pGEX2T (Pharmacia LKB) and transformed into the Esche- three (clones C40-3, C40-6, and C40-72) encoded portions of richia coli strain BL21. GST and GST fusion proteins were the same protein, which has a TRAF domain in its carboxyl prepared by standard methods (34), and the recombinant terminal region (Fig.