A20-binding Inhibitor Of NF-κB Research Articles

ABIN1 is a signal-induced autophagy receptor that attenuates NF-κB activation by recognizing linear ubiquitin chains.

Linear ubiquitin chains play pivotal roles in immune signaling by augmenting NF-κB activation and suppressing programmed cell death induced by various stimuli. A20-binding inhibitor of NF-κB 1 (ABIN1) binds to linear ubiquitin chains and attenuates NF-κB activation and cell death induction. Although interactions with linear ubiquitin chains are thought to play a role in ABIN1-mediated suppression of NF-κB and cell death, the underlying molecular mechanisms remain unclear. Here, we show that upon stimulation by Toll-like receptor (TLR) ligands, ABIN1 is phosphorylated on Ser 83 and functions as a selective autophagy receptor. ABIN1 recognizes components of the MyD88 signaling complex via interaction with linear ubiquitin chains conjugated to components of the complex in TLR signaling, which leads to autophagic degradation of signaling proteins and attenuated NF-κB signaling. Our current findings indicate that phosphorylation and linear ubiquitination also play a role in downregulation of signaling via selective induction of autophagy.

Structural and Biochemical Basis for Higher-Order Assembly between A20-Binding Inhibitor of NF-κB 1 (ABIN1) and M1-Linked Ubiquitins

Polyubiquitination is important in controlling NF-κB signaling. Excessive NF-κB activity has been linked to inflammatory disorders and autoimmune diseases, while ABIN1 could attenuate NF-κB activation to maintain immune homeostasis by utilizing UBAN to recognize linear (M1)-linked polyubiquitinated NF-κB activation mediators, including NEMO, IRAK1 and RIP1. PolyUb-mediated UBAN recruitment remains undetermined, since the recognition studies focused mostly on di-ubiquitin (diUb). Here we report three crystal structures of human ABIN1 UBAN (hABIN1UBAN) in complex with M1-linked diUb, triUb, and tetraUb, respectively. Notably, the hABIN1UBAN:diUb structure reveals that a diUb randomly binds one of the Ub-binding sites of the hABIN1UBAN dimer and leaves the other site vacant. Together with the ITC and gel-filtration analyses, we found that M1-triUb and M1-tetraUb adopt two unique conformations, instead of an elongated one, and they preferentially use the N-terminal two-Ub unit to bind the primary Ub-binding site of a hABIN1UBAN dimer and the C-terminal two-Ub unit to bind the secondary Ub-binding site of another hABIN1UBAN dimer. Especially, our results suggest that two ABIN1UBAN dimers cooperatively bind two UBAN-binding units of a tetraUb or vice versa. Since the UBAN family members share a conserved diUb-binding mode, our results suggest that M1-polyUb modification allows multiple copies of the two-tandem Ub unit to simultaneously coordinate multiple and/or different binding partners to increase their local concentrations and to facilitate the formation of a large signaling complex. Our study provides a structural-functional glimpse of M1-polyUb as a multiple-molecule binding platform to exert its intrinsic structural plasticity in mediating cellular signaling.

A20-Binding Inhibitor of NF-κB 1 Ameliorates Neuroinflammation and Mediates Antineuroinflammatory Effect of Electroacupuncture in Cerebral Ischemia/Reperfusion Rats.

A20-binding inhibitor of NF-κB 1 (ABIN1) is an inhibitor of NF-κB and exerts anti-inflammatory effect. Electroacupuncture (EA) is considered as a neuroprotective strategy by inhibiting neuroinflammatory damage after cerebral ischemia. This study was performed to explore the role of ABIN1 and investigate whether the ABIN1 is involved in the mechanism of EA in cerebral ischemia/reperfusion (I/R) rats. Male Sprague-Dawley (SD) rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and received EA after reperfusion once a day. Lentivirus-mediated ABIN1 gene knockdown was used to detect the role of ABIN1 in neuroinflammation after I/R. ABIN1 expression, proinflammatory cytokine levels, microglial activation, neurological function, infarct volumes, and NF-κB activation were assessed. ABIN1 expression was elevated in the peri-infarct cortex and was further upregulated by EA. ABIN1 knockdown increased the levels of proinflammatory cytokines and activation of microglia, worsened neurological deficits, and enlarged the infarct volume. Moreover, ABIN1 was blocked to partially reverse the neuroprotective effect of EA, and this treatment weakened the ability of EA to suppress NF-κB activity. Based on these findings, ABIN1 is a potential suppressor of neuroinflammation and ABIN1 mediates the antineuroinflammatory effect of EA in cerebral I/R rats.

Janus kinase 1/2 inhibition for the treatment of autoinflammation associated with heterozygous TNFAIP3 mutation

Janus kinase 1/2 inhibition for the treatment of autoinflammation associated with heterozygous TNFAIP3 mutation

ABIN2 Function Is Required To Suppress DSS-Induced Colitis by a Tpl2-Independent Mechanism.

The A20-binding inhibitor of NF-κB 2 (ABIN2) interacts with Met1-linked ubiquitin chains and is an integral component of the tumor progression locus 2 (Tpl2) kinase complex. We generated a knock-in mouse expressing the ubiquitin-binding-defective mutant ABIN2[D310N]. The expression of Tpl2 and its activation by TLR agonists in macrophages or by IL-1β in fibroblasts from these mice was unimpaired, indicating that the interaction of ABIN2 with ubiquitin oligomers is not required for the stability or activation of Tpl2. The ABIN2[D310N] mice displayed intestinal inflammation and hypersensitivity to dextran sodium sulfate-induced colitis, an effect that was mediated by radiation-resistant cells rather than by hematopioetic cells. The IL-1β-dependent induction of cyclooxygenase 2 (COX2) and the secretion of PGE2 was reduced in mouse embryonic fibroblasts and intestinal myofibroblasts (IMFs) from ABIN2[D310N] mice. These observations are similar to those reported for the Tpl2 knockout (KO) mice (Roulis et al. 2014. Proc. Natl. Acad. Sci. USA 111: E4658-E4667), but the IL-1β-dependent production of COX2 and PGE2 in mouse embryonic fibroblasts or IMFs was unaffected by pharmacological inhibition of Tpl2 in wild-type mice. The expression of ABIN2 is decreased drastically in Tpl2 KO mice. These and other lines of evidence suggest that the hypersensitivity of Tpl2 KO mice to dextran sodium sulfate-induced colitis is not caused by the loss of Tpl2 catalytic activity but by the loss of ABIN2, which impairs COX2 and PGE2 production in IMFs by a Tpl2 kinase-independent pathway.

A20-binding inhibitor of NF-κB (ABIN) 2 negatively regulates allergic airway inflammation.

TPL-2 MAP 3-kinase promotes inflammation in numerous mouse disease models and is an attractive anti-inflammatory drug target. However, TPL-2-deficient (Map3k8 -/-) mice develop exacerbated allergic airway inflammation to house dust mite (HDM) compared with wild type controls. Here, we show that Map3k8D270A/D270A mice expressing kinase dead TPL-2 had an unaltered response to HDM, indicating that the severe airway inflammation observed in Map3k8 -/- mice is not due to blockade of TPL-2 signaling and rather reflects a TPL-2 adaptor function. Severe allergic inflammation in TPL-2-deficient mice was likely due to reduced levels of ABIN-2 (TNIP2), whose stability depends on TPL-2 expression. Tnip2E256K knock-in mutation, which reduced ABIN-2 binding to A20, augmented the HDM-induced airway inflammation, but did not affect TPL-2 expression or signaling. These results identify ABIN-2 as a novel negative regulator of allergic airway responses and importantly indicate that TPL-2 inhibitors would not have unwanted allergic comorbidities.

Association of Tumor Necrosis Factor Alpha-Induced Protein 3 Interacting Protein 1 (TNIP1) Gene Polymorphism (rs7708392) with Lupus Nephritis in Egyptian Patients.

Lupus nephritis (LN) is a major cause of morbidity and mortality in systemic lupus erythematosus (SLE). Previous studies suggest that mutant A20 binding inhibitor of NF-κB 1 (ABIN1) protein encoded by tumor necrosis factor alpha-induced protein 3 interacting protein 1 (TNIP1) gene is associated with LN via NF-κB dysregulation. The aim of the current study was to evaluate the association of TNIP1 gene SNP rs7708392 with SLE and LN in Egyptian patients. 5' nuclease Allelic discrimination was used to evaluate the frequency of TNIP1 SNP rs7708392 in 53 patients with LN, 57 SLE patients without nephritis and 85 healthy controls. The genotyping analysis revealed that the CC genotype was more frequent in controls than SLE patients, while GC and GG genotypes were more common in SLE patients. Moreover, the GG genotype and the G allele were significantly more prevalent among LN patient than non-LN patients (P < 0.001). In LN patients, the most common genotype was GG (56.6%), while among the non-LN patients; the CG genotype was the most common (59.6%). Regression analysis demonstrated that SLE patients carrying only one G allele had a 3.4 folds increased risk for LN. Our results suggested that TNIP1 SNP (rs7708392) might be associated with the LN in Egyptian SLE patients. TNIP1 SNP (rs7708392) might be used to identify patients at risk of developing LN, which could help in early detection and treatment before progression to end-stage renal disease, improving patients' outcome and quality of life.

Structural Insights into Linear Tri-ubiquitin Recognition by A20-Binding Inhibitor of NF-κB, ABIN-2

Recognition of linear polyubiquitin by specific ubiquitin-binding proteins plays an important role in mediating nuclear factor-κB (NF-κB) signaling. A20 binding proteins, ABINs, recognize linear polyubiquitin and A20 through UBAN and AHD1, respectively, for the inhibition of NF-κB activation. Here we report the crystal structure of the AHD1-UBAN fragment of ABIN2 in complex with linear tri-ubiquitin, which reveals a 2:1 stoichiometry of the complex. Structural analyses together with mutagenesis, pull-down, and isothermal titration calorimetry assays show that the hABIN2:tri-ubiquitin interaction is mainly through the primary ubiquitin-binding site, and also through the secondary ubiquitin-binding site under a high local protein concentration. Surprisingly, three ubiquitin units could form a right-handed helical trimer to bridge two ABIN2 dimers. The residues around the M1-linkage are crucial for ABIN2 to recognize tri-ubiquitin. The tri-ubiquitin bridging two ABIN2 dimers model suggests a possible higher-ordersignaling complex assembled between M1-linked polyubiquitinated proteins, ubiquitin-binding proteins, and effector signaling proteins in signal transduction.

Ebola virus VP35 induces high-level production of recombinant TPL-2–ABIN-2–NF-κB1 p105 complex in co-transfected HEK-293 cells

Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)–ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]–NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells.

TNIP1, a Retinoic Acid Receptor Corepressor and A20-binding Inhibitor of NF-κB, Distributes to Both Nuclear and Cytoplasmic Locations

An increasingly wide range of functions, from repression of NF-κB signaling to protection from apoptosis, is being recognized for tumor necrosis factor α-induced protein 3-interacting protein 1 (TNIP1). The authors recently demonstrated TNIP1 interaction with and repression of liganded retinoic acid receptors, distinguishing it from the more typical NCoR and SMRT corepressors, which function only in the absence of ligand. To improve their understanding of TNIP1's roles in physiologic and pathologic events, the authors examined its distribution in normal and malignant human tissues and cultured cells. They found cytoplasmic and nuclear TNIP1 in normal skin keratinocytes as it colocalized with retinoic acid receptor α, one of the nuclear receptors it corepresses. Nuclear and cytoplasmic TNIP1 was also found in the malignant keratinocytes of squamous cell carcinomas. Compared to adjacent normal tissues of other organs, TNIP1 staining and distribution varied with increased levels in esophageal cancer and marked decreases in prostate cancer. The varying levels and distribution of TNIP1 in normal and disease state tissues could be expected to affect processes in which TNIP1 is involved, such as NF-κB and nuclear receptor signaling, possibly contributing to the disease course or response to therapies targeting these key players of cell growth and differentiation.

A20-binding inhibitor of NF-κB (ABIN1) controls Toll-like receptor-mediated CCAAT/enhancer-binding protein β activation and protects from inflammatory disease

Toll-like receptors (TLRs) are expressed on innate immune cells and trigger inflammation upon detection of pathogens and host tissue injury. TLR-mediated proinflammatory-signaling pathways are counteracted by partially characterized anti-inflammatory mechanisms that prevent exaggerated inflammation and host tissue damage as manifested in inflammatory diseases. We biochemically identified a component of TLR-signaling pathways, A20-binding inhibitor of NF-κB (ABIN1), which recently has been linked by genome-wide association studies to the inflammatory diseases systemic lupus erythematosus and psoriasis. We generated ABIN1-deficient mice to study the function of ABIN1 in vivo and during TLR activation. Here we show that ABIN1-deficient mice develop a progressive, lupus-like inflammatory disease characterized by expansion of myeloid cells, leukocyte infiltrations in different parenchymatous organs, activated T and B lymphocytes, elevated serum Ig levels, and the appearance of autoreactive antibodies. Kidneys develop glomerulonephritis and proteinuria, reflecting tissue injury. Surprisingly, ABIN1-deficient macrophages exhibit normal regulation of major proinflammatory signaling pathways and mediators but show selective deregulation of the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) and its target genes, such as colony-stimulating factor 3 (Csf3), nitric oxide synthase, inducible (Nos2), and S100 calcium-binding protein A8 (S100a8). Their gene products, which are intimately linked to innate immune cell expansion (granulocyte colony-stimulating factor), cytotoxicity (inducible nitric oxide synthase), and host factor-derived inflammation (S100A8), may explain, at least in part, the inflammatory phenotype observed. Together, our data reveal ABIN1 as an essential anti-inflammatory component of TLR-signaling pathways that controls C/EBPβ activity.

ABIN1 Protein Cooperates with TAX1BP1 and A20 Proteins to Inhibit Antiviral Signaling

Upon virus infection, the innate immune response provides the first line of protection and rapidly induces type I interferons (IFNα/β), which mediate potent antiviral effects. To maintain homeostasis and prevent autoimmunity, IFN production is tightly regulated; however, the mechanisms of negative regulation are poorly understood. Herein, we demonstrate that the A20 binding inhibitor of NF-κB 1 (ABIN1) is a novel negative regulator of antiviral signaling. Overexpression of ABIN1 inhibited IFN-β promoter activation in response to virus infection or poly(I:C) transfection, whereas siRNA-mediated knockdown of ABIN1 enhanced IFN-β production upon virus infection. ABIN1 interacted with the A20 regulatory molecule TAX1BP1 and was essential for the recruitment of TAX1BP1 and A20 to the noncanonical IκB kinases TBK1 and IKKi in response to poly(I:C) transfection. ABIN1 and TAX1BP1 together disrupted the interactions between the E3 ubiquitin ligase TRAF3 and TBK1/IKKi to attenuate lysine 63-linked polyubiquitination of TBK1/IKKi. Finally, an intact ubiquitin binding domain of ABIN1 was essential for ABIN1 to interact with TBK1/IKKi and inhibit IFN-β production upon poly(I:C) transfection or virus infection. Together, these results suggest that ABIN1 requires its ubiquitin binding domain and cooperates with TAX1BP1 and A20 to restrict antiviral signaling.

A20, ABIN-1/2, and CARD11 Mutations and Their Prognostic Value in Gastrointestinal Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of aggressive lymphomas with the activated B-cell-like subtype characterized by constitutive NF-κB activation. Activating mutations of CARD11 and inactivating mutations of A20 are frequent events in DLBCL. However, the full extent of genetic alterations in the NF-κB pathway regulators and their potential prognostic value in DLBCL remain to be investigated. We investigated the genetic abnormalities of CARD11, A20, and ABIN-1/2/3 (the A20 binding inhibitor of NF-κB) and their clinicopathologic correlation in gastrointestinal DLBCL. The somatic mutation and copy number changes of CARD11, A20, and ABIN-1/2/3 were investigated in 71 gastrointestinal DLBCLs by PCR/sequencing, and interphase FISH/array comparative genomic hybridization, respectively. The mutations identified were functionally characterized by NF-κB reporter assays and immunoprecipitation experiments. Recurrent somatic mutations were found in CARD11 (10%), A20 (17%), ABIN-1 (4%), and ABIN-2 (3%), but not in ABIN-3. In comparison with the wild-type, all CARD11 mutants were potent NF-κB activators in vitro. On the basis of the destructive nature of the observed mutations, and the findings by reporter assays and immunoprecipitation studies, most if not all of the somatic mutations that were seen in A20, ABIN-1, and ABIN-2 could impair their normal functions. Among these genetic abnormalities, A20 somatic mutation was significantly associated with both poor overall survival and event-free survival. We show further evidence of NF-κB pathway genetic abnormalities in DLBCL, which are potentially valuable in the prognosis and design of future therapeutic strategies.

Regulation and function of TPL-2, an IκB kinase-regulated MAP kinase kinase kinase

The IκB kinase (IKK) complex plays a well-documented role in innate and adaptive immunity. This function has been widely attributed to its role as the central activator of the NF-κB family of transcription factors. However, another important consequence of IKK activation is the regulation of TPL-2, a MEK kinase that is required for activation of ERK-1/2 MAP kinases in myeloid cells following Toll-like receptor and TNF receptor stimulation. In unstimulated cells, TPL-2 is stoichiometrically complexed with the NF-κB inhibitory protein NF-κB1 p105, which blocks TPL-2 access to its substrate MEK, and the ubiquitin-binding protein ABIN-2 (A20-binding inhibitor of NF-κB 2), both of which are required to maintain TPL-2 protein stability. Following agonist stimulation, the IKK complex phosphorylates p105, triggering its K48-linked ubiquitination and degradation by the proteasome. This releases TPL-2 from p105-mediated inhibition, facilitating activation of MEK, in addition to modulating NF-κB activation by liberating associated Rel subunits for translocation into the nucleus. IKK-induced proteolysis of p105, therefore, can directly regulate both NF-κB and ERK MAP kinase activation via NF-κB1 p105. TPL-2 is critical for production of the proinflammatory cytokine TNF during inflammatory responses. Consequently, there has been considerable interest in the pharmaceutical industry to develop selective TPL-2 inhibitors as drugs for the treatment of TNF-dependent inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. This review summarizes our current understanding of the regulation of TPL-2 signaling function, and also the complex positive and negative roles of TPL-2 in immune and inflammatory responses.

Epstein-Barr latent membrane protein 1 transformation site 2 activates NF-κB in the absence of NF-κB essential modifier residues 133–224 or 373–419

Epstein Barr virus latent membrane protein 1 (LMP1) induces NF-κB activation through transformation effector sites (TES) 1 and 2, both of which are critical for B-lymphocyte transformation. TES2 principally activates canonical NF-κB, which we confirm is NF-κB essential modifier (NEMO)-dependent and requires an intact ubiquitin binding in A20 binding inhibitor of NF-κB and NEMO (UBAN) domain. LMP1 TES2 activated NF-κB in Jurkat cell lines harboring NEMO truncated at 372 (A45) or NEMO with an in-frame deletion of 133-224 (2C), whereas TNFα, 12-O-Tetradecanoylphorbol-13-acetate, human T-cell leukemia virus 1 Tax, and CD40 did not. In both A45 and 2C Jurkat cell lines, LMP1 TES2-mediated NF-κB activation was blocked by siRNAs to TNFα receptor-associated factor 6 and NEMO, by IκB kinase inhibitors, and by the IκBα superrepressor, indicating that the NEMO mutants function to support canonical NF-κB activation. Expression of A45 or 2C mutants in NEMO-deficient murine embryonic fibroblasts reproduced the Jurkat phenotypes: LMP1 TES2 activated NF-κB in fibroblasts lacking NEMO amino acids 133-224 or 373-419, but TNFα and Tax did not. Further analysis indicated that TES2 did not activate NF-κB in cells expressing the double deletion mutant Δ133-224/Δ372-419. These data provide further evidence of the essential role for NEMO in LMP1 TES2 NF-κB activation and highlight the importance of unique domains within NEMO for sensing distinct NF-κB stimuli.

IRAK1-independent pathways required for the interleukin-1-stimulated activation of the Tpl2 catalytic subunit and its dissociation from ABIN2

The protein kinase Tpl2 (tumour progression locus 2) is activated by LPS (lipopolysaccharide), TNFalpha (tumour necrosis factor alpha) and IL (interleukin)-1. Activation of the native Tpl2 complex by these agonists requires the IKKbeta {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase beta}-catalysed phosphorylation of the p105/NF-kappaB1 subunit and is accompanied by the release of the catalytic subunit from both p105/NF-kappaB1 and another subunit ABIN2 (A20-binding inhibitor of NF-kappaB 2). In the present study we report that IL-1 activates the transfected Tpl2 catalytic subunit in an HEK (human embryonic kidney)-293 cell line that stably expresses the IL-1R (IL-1 receptor), but does not express the protein kinase IRAK1 (IL-1R-associated kinase). In these cells IL-1 does not activate IKKbeta or induce the phosphorylation of p105/NF-kappaB1, and nor does the IKKbeta inhibitor PS1145 prevent the IL-1-induced activation of transfected Tpl2. However, the IL-1-stimulated activation of transfected Tpl2 in IRAK1-null cells or activation of the endogenous Tpl2 complex in IRAK1-expressing cells is suppressed by the protein kinase inhibitor PP2 by a mechanism that does not involve inhibition of Src family protein tyrosine kinases. The IL-1-stimulated activation of transfected Tpl2 is accompanied by its phosphorylation at Thr290 and Ser400 and by enhanced phosphorylation of Ser62, which we demonstrate are autophosphorylation events catalysed by Tpl2 itself. We further show that IL-1 triggers the dissociation of Tpl2 from co-transfected ABIN2 in IRAK1-null IL-1R cells, which is not suppressed by PP2 or by the inhibition of Tpl2 or IKKbeta. These studies identify two new signalling events involved in activation of the native Tpl2 complex by IL-1. First, the IRAK1-, IKKbeta- and PP2-independent dissociation of Tpl2 from ABIN2; secondly, the IRAK1- and IKKbeta-independent, but PP2-sensitive, activation of the Tpl2 catalytic subunit.

ABIN-1 Binds to NEMO/IKKγ and Co-operates with A20 in Inhibiting NF-κB

Nuclear factor kappaB (NF-kappaB) plays a pivotal role in inflammation, immunity, stress responses, and protection from apoptosis. Canonical activation of NF-kappaB is dependent on the phosphorylation of the inhibitory subunit IkappaBalpha that is mediated by a multimeric, high molecular weight complex, called IkappaB kinase (IKK) complex. This is composed of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, NEMO/IKKgamma. The latter protein is essential for the activation of IKKs and NF-kappaB, but its mechanism of action is not well understood. Here we identified ABIN-1 (A20 binding inhibitor of NF-kappaB) as a NEMO/IKKgamma-interacting protein. ABIN-1 has been previously identified as an A20-binding protein and it has been proposed to mediate the NF-kappaB inhibiting effects of A20. We find that both ABIN-1 and A20 inhibit NF-kappaB at the level of the IKK complex and that A20 inhibits activation of NF-kappaB by de-ubiquitination of NEMO/IKKgamma. Importantly, small interfering RNA targeting ABIN-1 abrogates A20-dependent de-ubiquitination of NEMO/IKKgamma and RNA interference of A20 impairs the ability of ABIN-1 to inhibit NF-kappaB activation. Altogether our data indicate that ABIN-1 physically links A20 to NEMO/IKKgamma and facilitates A20-mediated de-ubiquitination of NEMO/IKKgamma, thus resulting in inhibition of NF-kappaB.

The inhibitor ABIN-2 disrupts the interaction of receptor-interacting protein with the kinase subunit IKKgamma to block activation of the transcription factor NF-kappaB and potentiate apoptosis.

NF-kappaB (nuclear factor kappaB) proteins are key transcription factors that regulate gene expression in response to various extracellular stimuli. The pathway leading to the activation of NF-kappaB involves a complicated network that includes a number of signalling molecules. The recent identification of a wide range of negative regulators of NF-kappaB has given another layer of complexity in NF-kappaB activation. We and others have previously identified the protein ABIN-2 (A20 binding inhibitor of NF-kappaB 2) as an inhibitor of NF-kappaB activation. In the present paper, we demonstrate that ABIN-2 exerts its inhibitory function by blocking the interaction of RIP (receptor-interacting protein) with the downstream effector IKKgamma, a non-kinase component of the IkappaB (inhibitory kappaB) kinase complex. When overexpressed in cells, ABIN-2 bound to IKKgamma and prevented the association of IKKgamma with RIP. By a deletion mapping, a stretch of 50 amino acids on ABIN-2 is found to be essential for its interaction with IKKgamma. The ABIN-2 mutant that lacked these 50 amino acids did not interact with IKKgamma and, consequently, failed to inhibit NF-kappaB activation. Strikingly, a portion of RIP, which is similar to this 50-residue domain of ABIN-2, is also essential for RIP interaction with IKKgamma. The RIP mutant with deletion of this similar region did not associate with IKKgamma and had substantial reduction of its ability to mediate NF-kappaB activation. Taken together, these conserved 50 residues of ABIN-2 and RIP define a novel structural domain in mediating a key step in the NF-kappaB signalling pathway through the interaction with IKKgamma. Finally, the signalling pathway of NF-kappaB activation is known to promote survival in many cellular events. The mechanism for decision between cell death and survival is under fine regulation. In the present paper, we demonstrated further that the expression of ABIN-2 could promote the RIP-mediated apoptosis by presumably suppressing the anti-apoptotic effect of NF-kappaB.