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Originally published In Press as doi:10.1074/jbc.M100048200 on June 4, 2001

J. Biol. Chem., Vol. 276, Issue 32, 30216-30223, August 10, 2001
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Identification of a Novel A20-binding Inhibitor of Nuclear Factor-kappa B Activation Termed ABIN-2*

Sofie Van Huffel, Filip Delaei, Karen HeyninckDagger, Dirk De Valck§, and Rudi Beyaert

From the Department of Molecular Biology, Unit for Molecular Signal Transduction in Inflammation, Flanders Interuniversity Institute for Biotechnology and Ghent University, 9000 Ghent, Belgium

Received for publication, January 3, 2001, and in revised form, June 1, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The nuclear factor kappa B (NF-kappa B) plays a central role in the regulation of genes implicated in immune responses, inflammatory processes, and apoptotic cell death. The zinc finger protein A20 is a cellular inhibitor of NF-kappa B activation by various stimuli and plays a critical role in terminating NF-kappa B responses. The underlying mechanism for NF-kappa B inhibition by A20 is still unknown. A20 has been shown to interact with several proteins including tumor necrosis factor (TNF) receptor-associated factors 2 and 6, as well as the inhibitory protein of kappa B kinase (IKK) gamma  protein. Here we report the cloning and characterization of ABIN-2, a previously unknown protein that binds to the COOH-terminal zinc finger domain of A20. NF-kappa B activation induced by TNF and interleukin-1 is inhibited by overexpression of ABIN-2. The latter also inhibits NF-kappa B activation induced by overexpression of receptor-interacting protein or TNF receptor-associated factor 2. In contrast, NF-kappa B activation by overexpression of IKKbeta or direct activators of the IKK complex, such as Tax, cannot be inhibited by ABIN-2. These results indicate that ABIN-2 interferes with NF-kappa B activation upstream of the IKK complex and that it might contribute to the NF-kappa B-inhibitory function of A20.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The nuclear factor kappa B (NF-kappa B)1 is activated by stimulation of cells with various stimuli including cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1) and regulates the expression of numerous genes involved in immune responses and inflammatory processes (1). NF-kappa B plays also a role in the inhibition of apoptosis (2-5), either by up-regulation of a number of antiapoptotic genes (6) or by a transcription-independent mechanism (7-9). Aberrant activation of NF-kappa B is observed in a number of diseases such as arthritis, inflammatory bowel disease, and cancer (10).

The activation pathway of NF-kappa B has been largely elucidated. In resting cells, NF-kappa B is sequestered in the cytoplasm as an inactive dimer by the inhibitory protein of kappa B (Ikappa B). After stimulation of cells, Ikappa B is phosphorylated on two serine residues by two Ikappa B kinases (IKKs), namely IKKalpha and IKKbeta (11-13). This targets the Ikappa B protein for ubiquitinylation and subsequent degradation by the 26S proteasome. The active NF-kappa B is then free for translocation to the nucleus, where it can bind specific sequences in the promoters of responsive genes. IKKalpha and IKKbeta are part of a larger IKK complex that also contains an essential regulatory component IKKgamma /NEMO (12, 14, 15), which is believed to link the IKK complex to upstream signaling proteins that might be stimulus-specific. In the case of TNF-induced activation, a number of intracellular proteins involved in NF-kappa B activation are recruited to the intracellular part of the TNF receptor p55 after triggering by TNF. These include the TNF receptor-associated death domain (16), the receptor-interacting protein (RIP) (17), and TNF receptor-associated factor (TRAF) 2 (18). After treatment with TNF, cells lacking RIP or TRAF2 are deficient in or hyporesponsive to NF-kappa B activation, respectively (19-21). Furthermore, dominant negative forms of TRAF2 inhibit NF-kappa B activation (22). Recently, evidence for TNF-dependent recruitment and activation of the IKK complex to the TNF receptor complex has been provided. TRAF2 has been proposed to be required for IKKgamma recruitment to the TNF receptor, whereas RIP mediates IKK activation via IKKgamma -mediated oligomerization (23-27). The proposed involvement of two putative IKK-activating kinases, NF-kappa B-inducing kinase and MEKK1, has been countered recently by knockout studies (28-30).

Besides inhibition by Ikappa B, NF-kappa B-dependent gene expression in response to different stimuli is also inhibited by the zinc finger protein A20 (31, 32). The latter was first cloned as an immediate early response gene up-regulated by TNF in endothelial cells (33). In the meantime, A20 expression has been shown to be strongly increased in several cell types in response to TNF (33-35). The expression of A20 is controlled by NF-kappa B (34), suggesting that A20 is involved in a negative feedback loop for NF-kappa B activation. Recently, it was shown that A20-deficient cells fail to terminate TNF-induced NF-kappa B responses (36). In addition, mice deficient for A20 develop severe inflammation and cachexia and are hyperresponsive to TNF. These results further indicate that A20 is critical for terminating TNF-induced NF-kappa B responses. The underlying mechanism for inhibition of NF-kappa B activation by A20 is still unclear. A number of A20-binding proteins have been described previously (37). A20 can be recruited to TRAF complexes via its NH2-terminal TRAF-binding domain (38, 39), whereas its COOH-terminal domain mediates inhibition of NF-kappa B activation. Binding of this domain to IKKgamma and the NF-kappa B-inhibitory protein ABIN has been proposed to be involved in its NF-kappa B-regulating activity (23, 40).

Here we report the molecular cloning of ABIN-2, a previously unknown protein that binds to the COOH-terminal domain of A20. When expressed ectopically, ABIN-2 inhibits activation of NF-kappa B by TNF, IL-1, or tetradecanoylphorbol acetate (TPA). Moreover, evidence was obtained that ABIN-2 interferes with the NF-kappa B signaling pathway at a level upstream of the IKK complex.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and Reagents-- Human embryonic kidney cells (HEK293T) were a kind gift of Dr. M. Hall (University of Birmingham, Birmingham, United Kingdom) and were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 0.4 mM sodium pyruvate, and antibiotics. HeLa cells were obtained from Dr. P. Agostinis (University of Leuven, Leuven, Belgium) and were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. L929r2 cells (41) were grown in Dulbecco's modified Eagle's medium supplemented with 5% newborn bovine serum 5% fetal bovine serum, and antibiotics. Mf4/4 macrophages (42) were grown in endotoxin-free RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics. Recombinant human TNF and recombinant murine IL-1beta were produced in Escherichia coli in our laboratory and purified to at least 99% homogeneity. TNF had a specific biological activity of 8.8 × 106 IU/mg purified protein, as determined with the international standard code 87/650 (National Institute for Biological Standards and Control, Potters Bar, United Kingdom). IL-1beta had a specific activity of 3.65 × 108 IU/mg purified protein, as determined with the international standard code 93/668.

Expression Plasmids-- Expression vectors contain mutant green fluorescent protein (GFP) S65T, as well as a fusion of the former with murine A20 and deletion mutants thereof (GFP-A20-(1-369) and GFP-A20-(370-775)) (40). Plasmids coding for RIP (40), FLAG-tagged A20 (43), and the yeast plasmid pAS2-A20 (44) were described previously. The HA-tagged ABIN plasmid consists of a fusion of the ABIN cDNA with an NH2-terminal HA tag in the mammalian expression vector pCAGGS. Plasmids encoding TRAF2 and TRAF6 were gifts of Dr. D. V. Goeddel (Tularik, San Francisco, CA). The plasmid coding for Tax was a kind gift of Dr. K.-T. Jeang (National Institutes of Health, Bethesda, MD). The plasmid encoding IKKbeta was a kind gift from Dr. J. Schmidt (Department of Vascular Biology and Thrombosis Research, University of Vienna, Vienna, Austria), and the plasmid coding for IL-1 receptor-associated kinase was a gift from Dr. L. O'Neill (Department of Biochemistry and Biotechnology, Trinity College, Dublin, Ireland). The reporter plasmid pSRE-luc was obtained from Stratagene (La Jolla, CA). The plasmid pNFconluc, encoding the luciferase (Luc) gene driven by a minimal NF-kappa B-responsive promoter, was a gift from Dr. A. Israël (Institut Pasteur, Paris, France). The plasmid pUT651, encoding beta -galactosidase (Gal), was supplied by Eurogentec (Seraing, Belgium).

Cloning of Full-length Murine ABIN-2 cDNA-- RNA was extracted from mouse heart and lung tissue using Trizol reagent (Life Technologies, Inc.). 5'-RACE was performed using a SMART PCR cDNA synthesis kit (CLONTECH). Full-length ABIN-2 cDNA was constructed by assembling the RACE fragment and the expressed sequence tag clone AA105249 by fusion PCR (45). Forward and reverse primers used for amplification of the expressed sequence tag clone were 5'-agagaaggaggtagtcttgcttcg-3' and 5'-gtccttgatgtaccaagtgtgcc-3', respectively. Forward and reverse primers for amplification of the 5'-RACE fragment were 5'-ttggagacgcccaagtccccacgg-3' and 5'-gcaagactacctccttctcttgc-3', respectively. Forward and reverse primers used for fusion PCR of both fragments were 5'-ccgctcgagatgGGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGCGTgaattcgggatccgtatgtcgtctggggacccaagg-3' (uppercase letters represent the sequence for the inserted E tag) and 5'-ggagatcttcattggcagcactcagacag-3', respectively; they were designed to facilitate cloning in the expression vector pCAGGS. Deletion mutants were constructed by PCR. All cDNA inserts were sequenced to verify their correctness.

Yeast Two-hybrid System-- The Matchmaker yeast two-hybrid system was purchased from CLONTECH. L929r2 cDNA library screening was performed as described previously (46). cDNA inserts encoding candidate A20-interacting proteins were sequenced on both strands with a cycle sequencer (Applied Biosystems, Foster City, CA). BLAST searches were performed with the online program offered by the National Center for Biotechnology Information.

Northern Blotting and Reverse Transcription-PCR-- Mouse multiple tissue Northern blot and mouse tissue first-strand cDNA (mouse MTC) were purchased from CLONTECH. Polyadenylated RNA was extracted from 2 × 106 L929r2 or Mf4/4 cells using a Micro-FastTrack Kit (Invitrogen, San Diego, CA). After electrophoresis on a 1% formaldehyde agarose gel, the RNA was transferred by capillary elution to a Hybond-N+ membrane. Hybridization of the Northern blot was performed with partial ABIN-2 cDNA isolated by yeast two-hybrid screening as a probe. The intensity of the ABIN-2 signal, which was determined using a PhosphorImager and an ImageQuant program (Molecular Dynamics, Sunnyvale, CA), is expressed relative to the intensity of the beta -actin signal of the same tissues. PCR on the mouse tissue first-strand cDNA was performed using a touch-down PCR program and primers to ABIN-2 that amplify an internal 400-bp fragment (namely, 5'-tgtcagctcctggtttgcttccgc-3' and 5'-agagaaggaggtagtcttgcttcg-3'). Primers amplifying a 200-bp beta -actin fragment were used as control (5'-ttccgatgccctgaggctct-3' and 5'-caggaggagcaatgatcttg-3').

Transfection, Coimmunoprecipitation, and Western Blotting-- For immunoprecipitation, 106 HEK293T cells were seeded in 10-cm Petri dishes and transfected using the DNA calcium phosphate coprecipitation method (47). For immunoprecipitation with anti-GFP antibody, cells were lysed in 500 µl of lysis buffer (20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 150 mM NaCl, and 1 mM EDTA) supplemented with protease inhibitors (10 µg/ml leupeptin, 200 units/ml aprotinin, and 1 mM Pefablock) and phosphatase inhibitors (10 mM NaF, 1 mM sodium vanadate, and 5.5 mg/ml beta -glycerophosphate). Immunoprecipitation was performed with a polyclonal anti-GFP antibody (CLONTECH) and binding to protein A-Trisacryl beads (Pierce). Beads were washed four times with lysis buffer. For immunoprecipitation with anti-HA antibody (Babco, Richmond, CA), cells were lysed in lysis buffer E1A (50 mM Hepes, pH 7.6, 250 mM NaCl, 5 mM EDTA, and 0.1% Nonidet P-40) supplemented with protease and phosphatase inhibitors. Protein A-Trisacryl-bound immunocomplexes were washed twice with buffer E1A, twice with the same buffer containing 1 M NaCl, and twice more with buffer E1A. Binding proteins were eluted with Laemmli buffer and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. Detection of coprecipitating proteins was achieved with a monoclonal anti-E tag antibody (Amersham Pharmacia Biotech) or a monoclonal anti-FLAG tag antibody (Sigma Chemical Co.), both of which were coupled to horseradish peroxidase. Immunoreactivity was revealed with a Renaissance enhanced chemiluminescence system (PerkinElmer Life Sciences).

For detection of endogenous ABIN-2 protein expression, L929r2 or Mf4/4 cells were left untreated or treated with TNF for 24 h. Cells were lysed in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromphenol blue. Lysates were incubated at 95 °C for 10 min, run on a 15% SDS-polyacrylamide gel electrophoresis gel, and transferred to a nitrocellulose membrane. The membrane was probed with a rabbit polyclonal antibody raised against a mixture of three ABIN-2-specific peptides (NH2-lnakwqrydasrdeyc-COOH, NH2-ggwrpessrsqqmepsc-COOH, and NH2-myqvsqrqdsrepgpc-COOH) coupled with keyhole limpet hemocyanin and with a secondary donkey anti-rabbit immunoglobulin antibody coupled to horseradish peroxidase (Amersham Pharmacia Biotech). Signals were detected with a Renaissance enhanced luminescence system.

NF-kappa B-dependent Gene Expression Assays-- To determine NF-kappa B activation by TNF, IL-1, or TPA stimulation, HEK293T cells were grown in 6-well plates and transiently transfected by DNA calcium phosphate coprecipitation (47) with a total of 1 µg of DNA. The DNA mixture comprised 100 ng of pUT651, 100 ng of pNFconluc, and 800 ng of specific expression plasmids. 24 h after transfection, cells were seeded in 24-well plates at a density of 4 × 104 cells/well. 24 h later, cells were left untreated or stimulated with TNF (1000 IU/ml), IL-1 (40 ng/ml), or TPA (200 ng/ml) for 6 h; they were subsequently lysed in 200 µl of lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-cyclohexanediaminetetraacetic acid, 10% glycerol, and 1% Triton X-100). Luc and Gal activities were analyzed as described previously (46). Luc values were normalized for Gal values to correct differences in transfection efficiency (plotted as Luc/Gal).

To determine NF-kappa B activation induced by overexpression of intracellular signaling proteins, cells were seeded directly in 24-well plates and transiently transfected with 20 ng of pUT651, 20 ng of pNFconluc, and a total of 160 ng of specific expression plasmids. 24 h after transfection, cells were lysed and analyzed as described above.

Immunolocalization-- HeLa cells were grown in 6-well plates and transiently transfected with 1 µg of total DNA using LipofectAMINE Plus reagent (Life Technologies, Inc.). 24 h after transfection, cells were seeded on coverslips; 24 h later, cells were fixed with 3% paraformaldehyde, permeabilized with 1% Triton X-100, and sequentially incubated for 2 h with a monoclonal E tag antibody (Amersham Pharmacia Biotech), followed by incubation for 2 h with a biotinylated anti-mouse immunoglobulin antibody and incubation for 30 min with avidin-Texas red (Amersham Pharmacia Biotech). Nuclei were stained for 10 min with 40 ng/ml 4,6-diamidino-2-phenylindole. Fluorescence was analyzed by fluorescence microscopy (Axiophot) at an excitation wavelength of 485 nm (emission, 510 nm) for GFP and 543 nm (emission, 600 nm) for Texas red.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of ABIN-2 as an A20-interacting Protein-- Thus far, the mechanism by which A20 inhibits NF-kappa B activation remains unclear. Because no enzymatic activity of the A20 protein has been described, we attempted to identify A20-interacting proteins that might play a role in the inhibition of NF-kappa B activation. A20 was used as a bait in a yeast two-hybrid screening of a murine fibrosarcoma L929r2 cDNA library. Ten clones expressed A20-interacting proteins, including A20 (44), 14-3-3 proteins (46), and ABIN (40). One clone contained the 3' end (832-1967 bp) of a cDNA encoding a previously unknown protein that we termed ABIN-2 (A20-binding inhibitor of NF-kappa B activation-2). BLAST searches with this partial cDNA revealed homology to a mouse expressed sequence tag with GenBankTM accession number AA105249. Full-length ABIN-2 (Fig. 1) was subsequently isolated by 5'-RACE on RNA samples obtained from mouse lung and heart tissues. Because two independent RNA sources and two independent RACE experiments resulted in the same 5' nucleotide, we concluded that ABIN-2 cDNA was complete. Full-length murine ABIN-2 cDNA (1967 bp) has a 5'-untranslated region of 82 nucleotides, an open reading frame of 1290 nucleotides, and a 3'-untranslated region of 594 nucleotides. The corresponding full-length human ABIN-2 cDNA could be reconstituted from the expressed sequence tag clones with GenBankTM accession numbers AA081482 and AW170250. Murine and human ABIN-2 showed 76% identity at the amino acid level. To examine the tissue distribution of ABIN-2 mRNA, we used the fragment isolated in the yeast two-hybrid screening as a probe in a multiple tissue Northern blot. ABIN-2 mRNA was detected in all tissues examined (Fig. 2A). Analysis of ABIN-2 expression relative to beta -actin expression in the same tissue showed that ABIN-2 was maximally expressed in kidney and only weakly expressed in skeletal muscle. An ABIN-2-specific fragment could also be PCR-amplified from first-strand cDNA prepared from all tissues examined (Fig. 2B), further demonstrating the general expression of ABIN-2. ABIN-2 is expressed in all tissues as an mRNA species with an estimated length of 2000 nucleotides, which is in agreement with the length of the cloned ABIN-2 cDNA. A similar ABIN-2 mRNA was also detectable in several cell lines, including murine L929r2 fibroblasts and Mf4/4 macrophages. ABIN-2 mRNA expression levels were not modulated after treatment with TNF, lipopolysaccharide, or interferon-gamma , which is in contrast with the inducible expression of A20 (Fig. 2C). Western blotting of L929r2 and Mf4/4 cell extracts revealed that endogenous ABIN-2 is expressed as a 50-kDa protein, which corresponds to the molecular mass predicted from the amino acid sequence. Scanning of the Prosite data base with the predicted amino acid sequence did not reveal any known protein motifs.


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  		Fig. 1.   cDNA sequence and predicted amino acid sequence of murine ABIN-2. The region of homology with ABIN is underlined.


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  		Fig. 2.   Expression pattern of ABIN-2. A, mouse multiple tissue Northern blot for ABIN-2. The blot was hybridized with a radiolabeled ABIN-2 cDNA fragment isolated in a yeast two-hybrid screen and analyzed by autoradiography. The same blot was reprobed with beta -actin cDNA as a control for sample loading. B, mouse multiple tissue reverse transcription-PCR. ABIN-2-specific PCR amplification on first-strand cDNA from each tissue yields a 400-bp product. Amplification of beta -actin was used as a control. C, mRNA expression in L929r2 and Mf4/4 cells. Cells were stimulated for 1 h with 1000 IU/ml TNF, 4 h with 10 µg/ml cycloheximide (CHX), 1 h with 1 µg/ml lipopolysaccharide (LPS), 1 h with interferon-gamma (IFN-gamma ), or combinations thereof. ABIN-2 mRNA expression was analyzed by Northern blotting. D, protein expression of ABIN-2. Lysates of L929r2 and Mf4/4 cells, which were either left untreated or treated with 1000 IU/ml TNF for 24 h, were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with a polyclonal ABIN-2-specific antibody.

The ABIN-2 fragment isolated by yeast two-hybrid screening from the cDNA library corresponds to its COOH terminus (amino acids 250-430). In addition, the full-length protein ABIN-2 also bound A20 in a yeast two-hybrid assay. To confirm the interaction in mammalian cells, the coding region of ABIN-2 was NH2-terminally fused to the E tag and cloned into the eukaryotic expression plasmid pCAGGS. Transient transfection of this vector into HEK293T cells and immunoblotting with an anti-E tag antibody revealed a protein of 50 kDa (data not shown), which corresponds to the predicted molecular mass. Full-length ABIN-2 was also found to interact in mammalian cells with A20 because both proteins were able to coimmunoprecipitate in HEK293T cells transiently transfected with an expression plasmid for E-tagged ABIN-2 and chimeric GFP-A20 protein (Fig. 3A). No interaction was observed between ABIN-2 and GFP as a negative control. The interaction between A20 and ABIN-2 was not influenced by stimulation of cells with TNF (data not shown). Partial deletion mutagenesis of A20 indicated that ABIN-2 specifically interacts with the zinc finger-containing COOH-terminal half of A20 (amino acids 370-775), which was previously shown to mediate the NF-kappa B-inhibitory activity of A20. Characterization of the subcellular distribution of ectopically expressed E-tagged ABIN-2 and GFP-A20 in HeLa cells by fluorescence microscopy showed that ABIN-2 colocalizes with GFP-A20 in the cytoplasm (Fig. 3B).


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  		Fig. 3.   A, coimmunoprecipitation of ABIN-2 and A20. 2 × 106 HEK293T cells were transfected with 2.5 µg of expression vector for E-tagged ABIN-2 combined with 2.5 µg of expression vector for GFP-A20, GFP, GFP-A20-(370-775), or GFP-A20-(1-369). A20 complexes were isolated by immunoprecipitation with anti-GFP polyclonal antibodies. Associated ABIN-2 was determined by immunoblotting with anti-E tag antibodies (top panel). Aliquots of total cell extracts were immunoblotted with anti-E tag antibody (middle panel) and anti-GFP antibody (bottom panel). B, colocalization of ABIN-2 and A20 in the cytoplasm. HeLa cells were transiently transfected with E-tagged ABIN-2 together with GFP or a GFP-A20 fusion protein. The overlay shows combined images of E-tagged ABIN-2, GFP, and DNA stained with 4,6-diamidino-2-phenylindole.

ABIN-2 Inhibits TNF-induced and IL-1-induced NF-kappa B Activation-- Because A20 is known to inhibit NF-kappa B activation by different stimuli, we examined the influence of ectopically expressed ABIN-2 on NF-kappa B-dependent expression of a Luc reporter gene in transiently transfected HEK293T cells. Ectopically expressed GFP and GFP-A20 were used as negative and positive controls, respectively. Like GFP-A20, ABIN-2 was able to inhibit NF-kappa B-dependent Luc expression in response to TNF, IL-1, and TPA (Fig. 4A and B). To exclude that ABIN-2 is acting as a general inhibitor of inducible genes, we also tested its effect on TPA-induced activation of SRE-dependent Luc expression. However, ectopic expression of ABIN-2 did not inhibit SRE-dependent gene expression in response to TPA (Fig. 4C). In contrast, ABIN-2 expression induced a 3-fold increase in basal SRE-dependent gene expression.


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  		Fig. 4.   Effect of ABIN-2 on NF-kappa B-dependent (A and B) and SRE-dependent (C) gene expression. HEK293T cells were transiently transfected with 300 ng of expression plasmid for GFP, ABIN-2, GFP-A20, or an empty vector (EV), each time with 100 ng of pUT651 and 100 ng of pNFconluc (A and B) or with 100 ng of pSRE-luc (C). Cells were left untreated () or stimulated with 1000 IU/ml TNF (black-square), 40 ng/ml IL-1 (), or 200 ng/ml TPA () for 6 h. Cell lysates were assayed for Luc and Gal activity. Results are representative of at least three independent experiments. Each bar represents the mean (± S.D. <10%) of three samples.

ABIN-2 Inhibits NF-kappa B Activation Upstream of the IKK Complex-- TNF-induced NF-kappa B activation involves recruitment and clustering of a number of adaptor proteins to the p55 TNF receptor, which is the main signaling receptor for TNF. These include TNF receptor-associated death domain, which binds directly to the TNF receptor death domain, as well as RIP and TRAF2, which bind to TNF receptor-associated death domain. Multiple evidence points to a role for both proteins in the activation of NF-kappa B by the p55 TNF receptor (19-22). Both TRAF2 and RIP then activate the IKK complex by a mechanism that is still largely unknown but that seems to involve recruitment to the p55 TNF receptor and IKK oligomerization. Also, a role for IKK phosphorylation by specific mitogen-activated protein kinase kinase kinase-related kinases, such as NF-kappa B-inducing kinase and MEKK1, has been proposed (48, 49), but this has been countered recently by the generation of specific NF-kappa B-inducing kinase or MEKK1 knockout mice in which TNF-induced NF-kappa B activation was not altered (28-30, 50). To investigate the place in the TNF-induced signaling pathway where ABIN-2 interferes with NF-kappa B activation, we relied on the fact that signaling initiated by TNF-induced clustering of signaling proteins is mimicked by their overexpression. As shown in Fig. 5A, coexpression of ABIN-2 can inhibit NF-kappa B activation in response to overexpression of RIP or TRAF2. In contrast, ABIN-2 had no effect on NF-kappa B activation in response to overexpression of IKKbeta , as well as in response to human T-cell lymphotrophic virus type I-Tax, which is a direct activator of the IKK complex (51) (Fig. 5B). Also, no effect was observed on NF-kappa B activation in response to NF-kappa B-inducing kinase overexpression (data not shown). These results suggest that ABIN-2 inhibits NF-kappa B activation by TNF upstream of the IKK complex, but downstream of TRAF2 and RIP. Similar observations were made with A20, confirming previous findings (40). A20 and ABIN-2 also inhibited NF-kappa B activation in response to overexpression of IL-1 receptor-associated kinase or TRAF6, which are upstream activators of the IKK complex in the IL-1 signaling pathway (Fig. 5C).


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  		Fig. 5.   Effect of A20 and ABIN-2 on NF-kappa B activation in response to overexpression of signaling proteins involved in NF-kappa B activation. 4 × 104 HEK293T cells were transiently transfected with 20 ng of pUT651, 20 ng of pNFconluc, and 40 ng of an expression plasmid for a specific NF-kappa B-activating protein. In each case, cells were also transfected with 60 ng of an empty vector () or with an expression plasmid for GFP (), GFP-A20 (), or ABIN-2 (black-square). As a control, cells that were not transfected with NF-kappa B-inducing proteins were left untreated or treated with 1000 IU/ml TNF or 40 ng/ml IL-1 for 6 h. All cells were lysed 24 h after transfection, and Luc and Gal activities were determined. Results are representative of at least three independent experiments. Each bar represents the mean (± S.D. <10%) of three samples.

ABIN-2 Is Partially Related to ABIN-- We previously identified ABIN as another A20-binding protein with NF-kappa B-inhibiting activity (40). ABIN and ABIN-2 bind to the COOH-terminal zinc finger-containing region of A20 (Fig. 3A) (46). To determine any simultaneous interaction of ABIN and ABIN-2 with A20, we analyzed whether coexpression of ABIN-2 in HEK293T cells affects the interaction of ABIN with A20 and whether ABIN and ABIN-2 can be coprecipitated with A20 in a single complex. Fig. 6 shows that coexpression of ABIN-2 significantly reduces the amount of A20 coprecipitated with ABIN, suggesting that ABIN and ABIN-2 compete for A20 binding. Moreover, ABIN-2 did not coprecipitate with ABIN, even in the absence or presence of A20. This shows that ABIN and ABIN-2 cannot bind to a single A20 molecule and do not form ABIN/ABIN-2 heterodimers. BLAST searches with full-length ABIN-2 did not reveal overall homology to any known protein but revealed a region of ABIN-2 (amino acids 256-321) with significant homology to amino acids 423-496 of ABIN (Figs. 1 and 7A). To study the functional role of this region of homology between ABIN and ABIN-2 in A20 binding, we constructed a number of deletion mutants (Fig. 7B) and assayed them for their ability to associate with A20 in a yeast two-hybrid test. These studies demonstrated an essential role for the region of homology between ABIN and ABIN-2 in the binding of A20 (Fig. 7B). Similar results were obtained in coimmunoprecipitation experiments (data not shown). To investigate whether the NF-kappa B-inhibiting potential of ABIN-2 also resides in the homologous region, we tested the ABIN-2 mutants for their ability to inhibit NF-kappa B-dependent gene expression in a reporter gene assay (Fig. 7C). None of the mutants missing the region of homology was able to inhibit TNF-induced NF-kappa B activation, indicating an important role for amino acids 256-321 in NF-kappa B inhibition. These results also suggest a correlation between A20 binding and NF-kappa B inhibition. However, a mutant that still contained the region of homology but lacked the 90 last amino acids, viz. ABIN-2 (1), was unable to inhibit NF-kappa B activation despite its binding to A20. These results demonstrate that binding of ABIN-2 with A20 is not sufficient for NF-kappa B inhibition and also indicate a role for the COOH-terminal 90 amino acids of ABIN-2. We also tested whether coexpression of the ABIN-2 (1) mutant had a dominant negative effect on NF-kappa B inhibition by A20 or full-length ABIN-2. However, the activity of A20 or ABIN-2 was not changed in the presence of ABIN-2 (1) (data not shown).


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  		Fig. 6.   ABIN and ABIN-2 are not part of a single complex. 2 × 106 HEK293T cells were transiently transfected with 1 µg of FLAG-tagged A20, 1 µg of HA-tagged ABIN, 3 µg of E-tagged ABIN-2, or combinations thereof. ABIN complexes were isolated by immunoprecipitation with anti-HA tag antibody; coprecipitating A20 and ABIN-2 proteins were revealed by immunoblotting with anti-FLAG tag and anti-E tag antibodies. Aliquots of total lysates were analyzed to confirm expression of transfected proteins, using anti-HA tag, anti-FLAG tag, and anti-E tag antibodies as indicated.


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  		Fig. 7.   A, amino acid alignment of the region of homology between ABIN and ABIN-2. Identical amino acids are underlined. B, schematic overview of deletion mutants of ABIN-2, with an indication of their potential to interact with A20 in a yeast two-hybrid assay (+, interaction; -, no interaction) and to inhibit TNF-induced NF-kappa B activation. C, effect of ABIN-2 deletion mutants on NF-kappa B-dependent reporter gene expression. HEK293T cells were transiently transfected with 100 ng of pUT651, 100 ng of pNFconluc, and 300 ng of an expression plasmid for GFP-A20, ABIN-2, or ABIN-2 mutants. Cells were left untreated () or stimulated with 1000 IU/ml TNF (black-square) for 6 h. Cell lysates were assayed for Luc and Gal activity. Results are representative of at least three independent experiments. Each bar represents the mean (± S.D. <10%) of three samples.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because of the potential role of NF-kappa B in several diseases, NF-kappa B and its regulators have recently drawn much attention as therapeutic targets. One of the cellular regulators of NF-kappa B activation is the zinc finger protein A20. The latter is encoded by an immediate early response gene, which is induced by different stimuli including TNF, IL-1, CD40, and phorbol esters, but also by overexpression of the viral proteins Tax and LMP1 (33, 52-54). Because A20 expression is NF-kappa B-dependent, A20 is likely to be involved in a negative regulatory loop for NF-kappa B activation. The role of A20 in termination of TNF-induced NF-kappa B activation has recently been confirmed with mice deficient for A20 (36). These mice develop severe inflammation and cachexia and are hypersensitive to both TNF and lipopolysaccharide. Although it is known that the NF-kappa B-inhibiting potential of A20 resides in its COOH-terminal zinc finger-containing domain (38, 39, 55), the underlying mechanism is still unclear. Recently, a number of A20-interacting proteins have been described (37), some of which might be involved in regulating or mediating the activity of A20. Here we describe the isolation and initial characterization of ABIN-2 as a novel A20-interacting protein. ABIN-2 was isolated in a yeast two-hybrid screening of a L929r2 fibrosarcoma cDNA library with A20 as bait. The interaction was confirmed in human cells by coimmunoprecipitation and shown to map to the COOH-terminal zinc finger-containing domain of A20. In contrast to A20, which is only expressed after stimulation of cells with NF-kappa B-activating stimuli, ABIN-2 is constitutively expressed in different cell types. Overexpression of ABIN-2 was shown to considerably reduce NF-kappa B-dependent expression of a reporter gene in response to TNF or IL-1 as well as to overexpression of signaling proteins involved in activation of the IKK complex by TNF or IL-1 (RIP or TRAF2 in the case of TNF; IL-1 receptor-associated kinase or TRAF6 in the case of IL-1). However, ABIN-2 had no effect on NF-kappa B activation induced by overexpression of IKKbeta or by overexpression of human T-cell lymphotrophic virus type I-Tax, which has been shown to directly activate the IKK complex by direct binding to IKK-gamma (51). Similar observations were made when A20 was coexpressed instead of ABIN-2. This indicates that A20 and ABIN-2 act at a level upstream of the IKK kinases. The recent observation that the IKK complex can be recruited to the TNF receptor complex via direct interaction between TRAF2 and IKKalpha /IKKbeta (27) and between RIP and IKKgamma (23), together with the finding that A20 can bind directly to TRAF-2 and IKKgamma (23, 38), points to a complex role for A20 and ABIN-2 in the regulation of IKK activation. Moreover, A20 can also bind to another protein (ABIN) that is able to inhibit TNF-induced NF-kappa B activation upon overexpression. The interaction of A20 with ABIN and ABIN-2 is mediated in both cases by the COOH-terminal region of A20 containing seven zinc finger structures. We recently showed that a minimum of four zinc fingers is sufficient for binding to ABIN and ABIN-2 as well as for inhibiting NF-kappa B-dependent gene expression (43). In addition, the zinc fingers of A20 were found to function in a redundant manner because either the first four or the last four zinc fingers could mediate ABIN or ABIN-2 binding as well as NF-kappa B inhibition. This suggests that a single A20 molecule might bind ABIN and ABIN-2 together. However, we demonstrate here that both interactions are competitive, suggesting that at least two different A20 complexes can be formed in a cell. Cell type-specific complexes are rather unlikely because ABIN and ABIN-2 were found to be expressed together in multiple tissues and cell lines. The existence of multiple A20 complexes might allow a stimulus-specific regulation of NF-kappa B-dependent gene expression. In this context, it should be mentioned that endogenous ABIN-2 expression was only detectable when cells were lysed directly under denaturing conditions, indicating that ABIN-2 is degraded very rapidly. Thus far, the functional link between A20 and ABIN-2 is still unclear. Although A20 can bind to TRAF2 and IKKgamma , previous studies have shown that these interactions are not sufficient for NF-kappa B inhibition by A20 (43, 56), suggesting the involvement of other A20-binding proteins, among which ABIN-2 is a good candidate. A20 and ABIN-2 are both localized in the cytoplasm, but A20 is only expressed upon NF-kappa B activation by certain stimuli. Previously, we demonstrated that A20 mutants that had lost their ability to bind ABIN and ABIN-2 were also unable to inhibit NF-kappa B-dependent gene expression (43). In the present study, we demonstrate that ABIN-2 mutants that have lost their A20 binding potential also no longer inhibit NF-kappa B activation upon overexpression, further indicating that binding to A20 and NF-kappa B inhibition are correlated. However, the discovery of an ABIN-2 mutant that still binds A20 but does not inhibit NF-kappa B activation also demonstrates that binding of ABIN-2 to A20 is not sufficient for NF-kappa B inhibition, suggesting an active role for ABIN-2. Surprisingly, the latter mutant did not behave as a dominant negative when coexpressed with A20 or ABIN-2. It might be that full-length ABIN-2 has a higher affinity for A20 or other proteins involved in the NF-kappa B-inhibiting effect of ABIN-2.

Particularly interesting is the observation that a short region of ABIN-2 that is critical for NF-kappa B inhibition shows significant homology to a similar region in ABIN. Moreover, the COOH-terminal part of this region is also present in IKKgamma (amino acids 294-314). Although the role of this region in IKKgamma is still unknown, a possible mechanism by which ABIN-2 (and also ABIN) might interfere with NF-kappa B activation is by competing with IKKgamma for the binding of an upstream activator of IKKgamma . Binding of A20 to TRAF2 or IKKgamma might facilitate the effect of ABIN-2 on the activation of the IKK complex. Final proof for the role of ABIN-2 and its interaction with A20 in the regulation of NF-kappa B-dependent gene expression will require the generation of ABIN-2-deficient cells, as well as analysis of the effect of ABIN-2 in A20-deficient cells.

Proteins of the NF-kappa B activation pathway are interesting targets for the development of novel therapeutic strategies to treat inflammatory diseases and cancer. Therefore, the identification of protein-protein interactions, such as A20-ABIN-2, might offer interesting targets for the development of drugs that can modulate NF-kappa B-dependent gene expression. Moreover, overexpression of ABIN-2 or other NF-kappa B inhibitors by gene therapy might provide additional tools to challenge different diseases in the future.

    ACKNOWLEDGEMENTS

We thank Drs. D. V. Goeddel, J. Schmidt, K. T. Jeang, L. O'Neill, M. Klinkenberg, and A. Israël for providing plasmids, Dr. J. Vandekerckhove for peptide synthesis, and A. Meeus for technical assistance.

    FOOTNOTES

* This work was supported in part by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, the Interuniversitaire Attractiepolen, the Sportvereniging tegen Kanker, the Belgische Federatie tegen Kanker, and EC-TMR Grant ERBFMRXCT970153.The costs of publication of this article were defrayed in part by the payment of page charges. The 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 GenBankTM/EMBL Data Bank with accession number(s) AJ304865 and AJ304866.

Dagger Postdoctoral research assistant with the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen.

§ Present address: Catholic University of Leuven, Faculty of Medicine, Department of Pathophysiology, Rheumatology Section, Herestraat 49, B-3000 Leuven, Belgium.

To whom correspondence should be addressed: Dept. of Molecular Biology, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium. Tel.: 32-9-264-51-31; Fax: 32-9-264-53-48; E-mail: rudi.beyaert@dmb.rug.ac.be.

Published, JBC Papers in Press, June 4, 2001, DOI 10.1074/jbc.M100048200

    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor kappa B; Gal, beta -galactosidase; GFP, green fluorescent protein; Ikappa B, inhibitory protein of kappa B; IKK, inhibitory protein of kappa B kinase; IL-1, interleukin-1; Luc, luciferase; RIP, receptor-interacting protein; SRE, serum-responsive element; TNF, tumor necrosis factor; TPA, tetradecanoylphorbol acetate; TRAF, tumor necrosis factor receptor-associated factor; MEKK, mitogen-activated protein kinase kinase/extracellular signal-regulated kinase kinase kinase; HA, hemagglutinin; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; bp, base pair(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Pahl, H. L. (1999) Oncogene 18, 6853-6866
2. Beg, A., Sha, W. C., Bronson, R. T., Ghosh, G., and Baltimore, D. (1995) Nature 276, 167-170
3. Beg, A. A., and Baltimore, D. (1996) Science 274, 782-784
4. Wang, C. Y., Mayo, M. W., and Baldwin, A. S. J. (1996) Science 274, 784-787
5. Van Antwerp, D. J., Martin, S. J., Kafri, T., Green, D. R., and Verma, I. M. (1996) Science 274, 787-789
6. Wang, C. Y., Mayo, M. W., Korneluk, R. G., Goeddel, D. V., and Baldwin, A. S. J. (1998) Science 281, 1680-1683
7. Kajino, S., Suganuma, M., Teranishi, F., Takahashi, N., Tetsuka, T., Ohara, H., Itoh, M., and Okamoto, T. (2000) Oncogene 19, 2233-2239
8. Mori, T., Okamoto, H., Takahashi, N., Ueda, R., and Okamoto, T. (2000) FEBS Lett. 465, 124-128
9. Yang, J. P., Hori, M., Takahashi, N., Kawabe, T., Kato, H., and Okamoto, T. (1999) Oncogene 18, 5177-5186
10. Chen, F., Castranova, V., Shi, X., and Demers, L. M. (1999) Clin. Chem. 45, 7-17
11. Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997) Cell 91, 243-252
12. Regnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z., and Rothe, M. (1997) Cell 90, 373-383
13. Karin, M. (1999) Oncogene 18, 6867-6874
14. Yamaoka, S., Courtois, G., Bessia, C., Whiteside, S. T., Weil, R., Agou, F., Kirk, H. E., Kay, R. J., and Israel, A. (1998) Cell 93, 1231-1240
15. Rothwarf, D. M., Zandi, E., Natoli, G., and Karin, M. (1998) Nature 395, 297-300
16. Hsu, H., Xiong, J., and Goeddel, D. V. (1995) Cell 81, 495-504
17. Hsu, H., Huang, J., Shu, H. B., Baichwal, V., and Goeddel, D. V. (1996) Immunity 4, 387-396
18. Shu, H. B., Takeuchi, M., and Goeddel, D. V. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13973-13978
19. Kelliher, M. A., Grimm, S., Ishida, Y., Kuo, F., Stanger, B. Z., and Leder, P. (1998) Immunity 8, 297-303
20. Ting, A. T., Pimentel-Muinos, F. X., and Seed, B. (1996) EMBO J. 15, 6189-6196
21. Yeh, W. C., Shahinian, A., Speiser, D., Kraunus, J., Billia, F., Wakeham, A., de la Pompa, J. L., Ferrick, D., Hum, B., Iscove, N., Ohashi, P., Rothe, M., Goeddel, D. V., and Mak, T. W. (1997) Immunity 7, 715-725
22. Rothe, M., Sarma, V., Dixit, V. M., and Goeddel, D. V. (1995) Science 269, 1424-1427
23. Zhang, S. Q., Kovalenko, A., Cantarella, G., and Wallach, D. (2000) Immunity 12, 301-311
24. Inohara, N., Koseki, T., Lin, J., del Peso, L., Lucas, P. C., Chen, F. F., Ogura, Y., and Nunez, G. (2000) J. Biol. Chem. 275, 27823-27831
25. Devin, A., Cook, A., Lin, Y., Rodriguez, Y., Kelliher, M., and Liu, Z. (2000) Immunity 12, 419-429
26. Poyet, J.-L., Srinivasula, S. M., Lin, J., Fernandes-Alnemri, T., Yamaoka, S., Tsichlis, P. N., and Alnemri, E. S. (2000) J. Biol. Chem. 275, 37966-37977
27. Devin, A., Lin, Y., Yamaoka, S., Li, Z., Karin, M., and Liu, Z. G. (2001) Mol. Cell. Biol. 21, 3986-3994
28. Matsushima, A., Kaisho, T., Rennert, P. D., Nakano, H., Kurasawa, K., Uchida, D., Takeda, K., Akira, S., and Matsumoto, M. (2001) J. Exp. Med. 193, 631-636
29. Yujiri, T., Ware, M., Wildmann, C., Oyer, R., Russell, D., Chan, E., Zaitsu, Y., Clarke, P., Tyler, K., Oka, Y., Fanger, G., Henson, P., and Johnson, G. (2001) Proc. Natl. Acad. Sci. U. S. A. 97, 7272-7277
30. Xia, Y., Makris, C., Su, B., Li, E., Yang, J., Nemerow, G. R., and Karin, M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5243-5248
31. Jäättelä, M., Mouritzen, H., Elling, F., and Bastholm, L. (1996) J. Immunol. 156, 1166-1173
32. Cooper, J. T., Stroka, D. M., Brostjan, C., Palmetshofer, A., Bach, F. H., and Ferran, C. (1996) J. Biol. Chem. 271, 18068-18073
33. Dixit, V. M., Green, S., Sarma, V., Holzman, L. B., Wolf, F. W., O'Rourke, K., Ward, P. A., Prochownik, E. V., and Marks, R. M. (1990) J. Biol. Chem. 235, 2973-2978
34. Krikos, A., Laherty, C. D., and Dixit, V. M. (1992) J. Biol. Chem. 267, 17971-17976
35. Tewari, M., Wolf, F. W., Seldin, M. F., O'Shea, K. S., Dixit, V. M., and Turka, L. A. (1995) J. Immunol. 154, 1699-1706
36. Lee, E. G., Boone, D. L., Chai, S., Libby, S. L., Chien, M., Lodolce, J. P., and Ma, A. (2000) Science 289, 2350-2354
37. Beyaert, R., Heyninck, K., and Van Huffel, S. (2000) Biochem. Pharmacol. 60, 1143-1151
38. Song, H. Y., Rothe, M., and Goeddel, D. V. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6721-6725
39. Heyninck, K., and Beyaert, R. (1999) FEBS Lett. 442, 147-150
40. Heyninck, K., De Valck, D., Vanden Berghe, W., Van Criekinge, W., Contreras, R., Fiers, W., Haegeman, G., and Beyaert, R. (1999) J. Cell Biol. 145, 1471-1482
41. Vanhaesebroeck, B., Van Bladel, S., Lenaerts, A., Suffys, P., Beyaert, R., Lucas, R., Van Roy, F., and Fiers, W. (1991) Cancer Res. 51, 2469-2477
42. Desmedt, M., Rottiers, P., Dooms, H., Fiers, W., and Grooten, J. (1998) J. Immunol. 160, 5300-5308
43. Klinkenberg, M., Van Huffel, S., Heyninck, K., and Beyaert, R. (2001) FEBS Lett. 498, 93-97
44. De Valck, D., Heyninck, K., Van Criekinge, W., Contreras, R., Beyaert, R., and Fiers, W. (1996) FEBS Lett. 384, 61-64
45. Olsen, O. (1992) Methods Mol. Cell. Biol. 3, 159-160
46. De Valck, D., Heyninck, K., Van Criekinge, W., Vandenabeele, P., Fiers, W., and Beyaert, R. (1997) Biochem. Biophys. Res. Commun. 238, 590-594
47. O'Mahoney, J. V., and Adams, T. E. (1994) DNA Cell Biol. 13, 5043-5049
48. Lee, F. S., Peters, R. T., Dang, L. C., and Maniatis, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9319-9324
49. Ling, L., Cao, Z., and Goeddel, D. V. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3792-3797
50. Yin, L., Wu, L., Wesche, H., Arthur, C. D., White, J. M., Goeddel, D. V., and Schreiber, R. D. (2001) Science 291, 2161-2165
51. Jin, D. Y., Giordano, V., Kibler, K. V., Nakano, H., and Jeang, K. T. (1999) J. Biol. Chem. 274, 17402-17405
52. Sarma, V., Lin, Z., Clark, L., Rust, B. M., Tewari, M., Noelle, R. J., and Dixit, V. M. (1995) J. Biol. Chem. 270, 12343-12346
53. Laherty, C. D., Hu, H. M., Opipari, A. W., Wang, F., and Dixit, V. M. (1992) J. Biol. Chem. 267, 24157-24160
54. Laherty, C. D., Perkins, N. D., and Dixit, V. M. (1993) J. Biol. Chem. 268, 5032-5039
55. Natoli, G., Costanzo, A., Guido, F., Moretti, F., Bernardo, A., Burgio, V. L., Agresti, C., and Levrero, M. (1998) J. Biol. Chem. 273, 31262-31272
56. Lademann, U., Kallunki, T., and Jäättelä, M. (2001) Cell Death Differ. 8, 265-272

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