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The Tumor Necrosis Factor-Inducible Zinc Finger Protein A20 Interacts

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The Tumor Necrosis Factor-Inducible Zinc Finger Protein A20 Interacts Proc. Natl. Acad. Sci. USA Vol. 93, pp. 6721-6725, June 1996 Biochemistry The tumor necrosis factor-inducible zinc finger protein A20 interacts with TRAF1/TRAF2 and inhibits NF-cB activation (tumor necrosis factor receptors/CD40/signal transduction) Ho YEONG SONG, MiKE ROTHE, AND DAVID V. GOEDDEL* Tularik, Inc., 270 East Grand Avenue, South San Francisco, CA 94080 Contributed by David V Goeddel, March 13, 1996 ABSTRACT TRAF1 and TRAF2 form an oligomeric com- this family, TRAF3 and TRAF4 (CART1), have been iden- plex that associates with the cytoplasmic domains of various tified. TRAF3, which is also called CD40bp (11), CRAF-1 members of the tumor necrosis factor (TNF) receptor super- (12), LAP1 (13), and CAP1 (14), is structurally similar to family. TRAF2 action is required for activation of the tran- TRAF2. TRAF3 interacts with the intracellular domain of scription factor NF-KB triggered by TNF and the CD40 ligand. CD40 and may be important for certain CD40-mediated signal Here we show that TRAF1 and TRAF2 interact with A20, a transduction pathways (12). TRAF3 also interacts with the zinc finger protein, whose expression is induced by agents that Epstein-Barr virus-encoded protein LMP1, which is crucial for activate NF-#cB. Mutational analysis revealed that the N- Epstein-Barr virus-induced B cell transformation (13). terminal half of A20 interacts with the conserved C-terminal TRAF4 (CART1) is the most recently cloned TRAF family TRAF domain of TRAF1 and TRAF2. In cotransfection ex- member (15). In contrast to TRAF2 and TRAF3, which are periments, A20 blocked TRAF2-mediated NF-KB activation. ubiquitously expressed and localized in the cytoplasm (5, 13), A20 also inhibited TNF and IL-i-induced NF-cB activation, TRAF4 is a nuclear protein that was found to be predomi- suggesting that it may inhibit NF-KcB activation signaled by nantly expressed in breast carcinoma (15). This raises the diverse stimuli. The ability of A20 to block NF-cB activation possibility that TRAF4 may belong to a subfamily of TRAFs was mapped to its C-terminal zinc finger domain. Thus, A20 that function as nuclear-signaling or transcription factors. is composed of two functionally distinct domains, an N- TRAF2 can interact with both TNFR2 and CD40, and it acts terminal TRAF binding domain that recruits A20 to the as a common mediator for NF-KB activation through these two TRAF2-TRAF1 complex and a C-terminal domain that me- receptors (16). TRAF2 can also be recruited to TNFR1 diates inhibition of NF-cB activation. Our findings suggest a through its interaction with TRADD (10). A dominant neg- possible molecular mechanism that could explain A20's abil- ative TRAF2 blocked TNFR1-mediated NF-KB activation in ity to negatively regulate its own TNF-inducible expression. 293 cells, suggesting that TRAF2 is also involved in the TNFR1-induced NF-KB activation pathway. In contrast to Tumor necrosis factor (TNF) is a cytokine that is produced by TRAF2, neither TRAF1 nor TRAF3 can activate NF-KB in activated macrophages and plays an important role in the cotransfection experiments (16). regulation of immune and inflammatory responses (1). TNF TRAF2 consists of at least three domains (5, 17). The signaling is mediated by cell surface receptors of 55 kDa N-terminal RING finger motif (18) is required for NF-KB (TNFR1) and 75 kDa (TNFR2) that are present on most cell activation by TRAF2 (16). The central cysteine-rich domain types (2, 3). The intracellular domains of TNFR1 and TNFR2 consists of five zinc finger-like structures. The C-terminal are unrelated in primary amino acid sequence, suggesting they TRAF domain (residues 272-501) can be further divided into use distinct signaling mechanisms (2, 3). TNFR1 has been two subdomains. The TRAF-N subdomain (residues 272-355) regarded as the major signal transducer for many TNF func- comprises a predicted coil-coil structure, which may be in- tions, including cytotoxicity, whereas TNFR2 signals a more volved in oligomerization of TRAFs (5, 16). This domain is limited number of responses (2, 3). However, both receptors also responsible for interaction with cellular inhibitors of are capable of independently activating the transcription factor apoptosis proteins that associate with the TNFR2-TRAF NF-KB (4, 5). Searches for TNFR-associated factors have complex (19). The TRAF-C subdomain (residues 356-501) is resulted in cloning of TRADD (6), which interacts with required for binding to TNFR2 (19). TNFR1, and TRAFM and TRAF2, which associate with In this study we used the yeast two-hybrid system to search TNFR2 (5). for TRAF2-interacting proteins that might participate in TNFR1 contains a so-called "death domain" of "80 resi- NF-KB activation signaled by TRAF2. We identified A20, a dues near its C terminus, which is required for TNF-induced TNF-inducible zinc finger protein, that also interacts with cytotoxicity (7) and receptor oligomerization (8, 9). TRADD TRAF1. Overexpression of A20 blocks NF-KB activation by contains a region at its C terminus that shares homology with several stimuli, including TRAF2. This negative feedback the death domain of TNFR1 (6). The interaction of the death regulation of TNF-induced NF-KB activation by A20 is prob- domains of TRADD and TNFR1 is TNF-dependent, suggest- ably mediated by A20's association with the TRAF2-TRAF1 ing TNFR1 oligomerization is a prerequisite for the recruit- complex. ment of TRADD to the TNFR1 complex (10). When overex- pressed, TRADD induces apoptosis and activates NF-KB (6), MATERIALS AND METHODS which are hallmark activities signaled by TNFR1. TRAF proteins are a family of signal transducers having a Cell Culture and Reagents. The human embryonic kidney C-terminal homology region termed the "TRAF domain" (5). 293 cell line and the murine interleukin 2-dependent CT6 cell In addition to TRAF1 and TRAF2, which interact with each line were maintained as described (5). TNF and interleukin 1 other and form a complex with TNFR2, two other members of (IL-1) were provided by Genentech. The monoclonal anti-Flag antibody (M2) was obtained from IBI, and the monoclonal The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: IL-1, interleukin 1; TNF, tumor necrosis factor. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 6721 Downloaded by guest on September 23, 2021 6722 Biochemistry: Song et al. Proc. Natl. Acad. Sci. USA 93 (1996) anti-human A20 antibody was provided by V. Dixit (University murine A20 protein (22) lacking only eight amino acids from of Michigan). The polyclonal anti-mouse TRAF1 antibody was its N terminus. raised using glutathione S-transferase-TRAF1 as antigen. The A20 was originally identified as a TNF-inducible zinc finger rabbit anti-murine TNFR2 antibody was described (5). For protein of 80 kDa (23). It has been reported that A20 can Western blot analysis, anti-mouse IgG antibody conjugated protect several cell lines from TNF-induced cytotoxicity (24) with horseradish peroxidase and enhanced chemilumines- and can induce antiapoptotic activity in B cells following CD40 cence (ECL) kit (Amersham) were used. activation (25). A20 was 'also shown to be constitutively Expression Vectors. Mammalian expression vectors for expressed at high level in lymphoid tissues, suggesting that A20 TRADD, TRAF1, TRAF2, TNFR2, and CD40 have been may have an important function in lymphoid systems (22). described (6, 16). Flag epitope-tagged TRAF1 and TRAF2 Retransformation assays performed with the A20 clone S-21 expression vectors have been described (19). The human A20 confirmed that the interaction between A20 and TRAF2 was expression plasmid was provided by V. Dixit. Truncated A20 specific. We then performed two-hybrid deletion mapping expression vectors for the N-terminal half (amino acids 1-386) analysis on TRAF2 to delineate the domains required for and the C-terminal half (amino acids 387-790) of A20 were interaction with A20. The N-terminal RING and central zinc prepared by PCR. The NF-KB reporter plasmid, pELAM-luc, finger domains of TRAF2 were not required for binding to and the f-galactosidase control plasmid, pCMV-,BGal, were A20, because TRAF2(875ol.) and TRAF2(2645o1) mutants in- described (10). teracted strongly with A20, whereas a TRAF2(1l86) mutant Yeast Two-Hybrid System. Vectors and procedures for the failed to interact (Table 1). TRAF2(1358), which lacks the two-hybrid library screening were as described (5). The mouse TRAF-C domain, also interacts with A20. This demonstrates peripheral lymph node two-hybrid cDNA library was supplied that the TRAF-N domain is probably important for interaction by L. Lasky (Genentech). Deletion constructs for two-hybrid with A20. mapping were made by PCR. Yeast strain Y190 was used for We next tested whether TRAFM and TRAF3 could also library screening and retransformation experiments. We fol- interact with A20 in the two-hybrid system. The interaction lowed the Matchmaker Two Hybrid System protocol (Clon- between TRAF1 and A20 was as strong as that observed tech) for transformation and plasmid DNA rescue from yeast. between TRAF2 and A20. In contrast, TRAF3 did not interact Transfection and Coimmunoprecipitation. The calcium with A20 in the two-hybrid system (Table 1). This result is phosphate precipitation method was used for transfections consistent with A20 interacting with the TRAF-N domain (20). For coimmunoprecipitations, 2 x 106 cells per 100-mm of TRAF1 and TRAF2, because this domain is highly con- plate were transfected with 5 gg of appropriate expression served between TRAF1 and TRAF2 but poorly conserved vectors. Twenty-four hours after transfection, the cells were in TRAF3. washed once with cold PBS, lysed in 500 ,sl of lysis buffer Sequence analysis of A20 had previously revealed two containing 50 mM Hepes (pH 7.6), 250 mM NaCl, 0.1% distinct structural domains (22, 23).
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