The Zinc Finger Protein A20 Inhibits TNF-induced NF-κB–dependent Gene Expression by Interfering with an RIP- or TRAF2-mediated Transactivation Signal and Directly Binds to a Novel NF-κB–inhibiting Protein ABIN

Abstract

The zinc finger protein A20 is a tumor necrosis factor (TNF)– and interleukin 1 (IL-1)-inducible protein that negatively regulates nuclear factor-kappa B (NF-κB)–dependent gene expression. However, the molecular mechanism by which A20 exerts this effect is still unclear. We show that A20 does not inhibit TNF- induced nuclear translocation and DNA binding of NF-κB, although it completely prevents the TNF- induced activation of an NF-κB–dependent reporter gene, as well as TNF-induced IL-6 and granulocyte macrophage–colony stimulating factor gene expression. Moreover, NF-κB activation induced by overexpression of the TNF receptor–associated proteins TNF receptor–associated death domain protein (TRADD), receptor interacting protein (RIP), and TNF recep- tor–associated factor 2 (TRAF2) was also inhibited by expression of A20, whereas NF-κB activation induced by overexpression of NF-κB–inducing kinase (NIK) or the human T cell leukemia virus type 1 (HTLV-1) Tax was unaffected. These results demonstrate that A20 inhibits NF-κB–dependent gene expression by interfering with a novel TNF-induced and RIP- or TRAF2-mediated pathway that is different from the NIK–IκB kinase pathway and that is specifically involved in the transactivation of NF-κB. Via yeast two-hybrid screening, we found that A20 binds to a novel protein, ABIN, which mimics the NF-κB inhibiting effects of A20 upon overexpression, suggesting that the effect of A20 is mediated by its interaction with this NF-κB inhibiting protein, ABIN.

Ubiquitin binding mediates the NF-κB inhibitory potential of ABIN proteins

“…We have already pointed out that ABIN proteins interfere with NF-kB signaling downstream of TRAF2/6, but upstream of IKKb (Heyninck et al, 1999;Van Huffel et al, 2001;Wullaert et al, 2007), and others showed that ABIN proteins can bind NEMO (Liu et al, 2004;Mauro et al, 2006;Weaver et al, 2007). Moreover, we here showed that both the UBAN domain and the NBD of ABIN-1 are involved in its NF-kB inhibitory function ( Figure 3c).…”

“…The ABIN proteins were originally identified as three different intracellular NF-kB inhibitory proteins that bind the deubiquitinating protein A20 (Heyninck et al, 1999;Van Huffel et al, 2001;Wullaert et al, 2007). ABINs show partial sequence homology in a number of short regions, designated ABIN homology domains (AHDs).…”

ABINs: A20 binding inhibitors of NF-κB and apoptosis signaling

“…Two different splice variants of approximately 2800 and 2600 nucleotides long were found, containing open reading frames of respectively 1941 and 1792 nucleotides and encoding proteins of 72 and 68 kDa, respectively [9]. In contrast to the human ABIN-1 isoforms, the two murine splice variants differ at their N-terminus due to the initiation from two different methionines.…”

“…Yeast two-hybrid screening of a mouse fibroblast L929r2 cDNA library with A20 as bait originally led to the identification of A20-binding inhibitor of NF-B (ABIN)-1 and ABIN-2 [9]. A TBLASTN search in the non-redundant and expressed sequence tag database identified ABIN-3 as another related protein [10].…”

“…In addition, ABIN-1 contains a NEMO binding domain (NBD) in its C-terminus [27], a leucine zipper (LZ) structure, and four coiled-coil structures [9,16]. Furthermore, ABIN-1 also has four putative leucine-rich nuclear 8 export signals distributed throughout its sequence, as well as a nuclear localization signal in its C-terminal part.…”