A20-binding Inhibitor of Nuclear Factor- B (NF- B)-2 (ABIN-2) Is an ...

A20-binding Inhibitor of Nuclear Factor-�B (NF-�B)-2(ABIN-2) Is an Activator of Inhibitor of NF-�B (I�B) Kinase �(IKK�)-mediated NF-�B Transcriptional Activity*

Received for publication, March 14, 2011, and in revised form, July 13, 2011 Published, JBC Papers in Press, July 22, 2011, DOI 10.1074/jbc.M111.236448

Laurent Leotoing‡§¶1, Fanny Chereau‡§¶1, Silvere Baron�**2, Florent Hube‡‡2, Hugo J. Valencia‡§¶,Didier Bordereaux‡§¶, Jeroen A. Demmers§§, John Strouboulis¶¶3, and Veronique Baud‡§¶4

From ‡INSERM, U1016, Institut Cochin, 75014 Paris, France, §CNRS, UMR 8104, Paris, France, ¶Universite Paris Descartes, SorbonneParis Cite, Paris, France, �Clermont Universite, Universite Blaise Pascal, Genetique Reproduction et Developpement, BP 10448,F-63000 Clermont-Ferrand, France, **CNRS, UMR 6247, GReD, F-63177 Aubiere, France, ‡‡Universite Paris Diderot Paris 7,CNRS, UMR 7216, Epigenetics and Cell Fate, Paris, France, and the §§Department of Biochemistry and Proteomics Center and¶¶Department of Cell Biology, Erasmus Medical Center, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands

NF-�B transcription factors are pivotal players in controllinginflammatory and immune responses, as well as cell prolifera-tion and apoptosis. Aberrant regulation of NF-�B and the sig-naling pathways that regulate its activity have been involved invarious pathologies, particularly cancers, as well as inflamma-tory and autoimmune diseases. NF-�B activation is tightly reg-ulated by the I�B kinase (IKK) complex, which is composed oftwo catalytic subunits IKK� and IKK�, and a regulatory subunitIKK�/��. Although IKK� and IKK� share structural simi-larities, IKK� has been shown to have distinct biological func-tions. However, themolecularmechanisms thatmodulate IKK�activity have not yet been fully elucidated. To understand betterthe regulation of IKK� activity, we purified IKK�-associatedproteins and identified ABIN-2. Here, we demonstrate thatIKK� and IKK� both interact with ABIN-2 and impair its con-stitutive degradation by the proteasome. Nonetheless, ABIN-2enhances IKK�- but not IKK�-mediated NF-�B activation byspecifically inducing IKK� autophosphorylation and kinaseactivity. Furthermore, we found that ABIN-2 serine 146 is crit-ical for the ABIN-2-dependent IKK� transcriptional up-regula-tion of specific NF-�B target genes. These results imply thatABIN-2 acts as a positive regulator of NF-�B-dependent tran-scription by activating IKK�.

The NF-�B family of transcription factors regulates theexpression of a wide range of genes implicated in immune andinflammatory responses, cell proliferation, apoptosis, and

oncogenesis (1–9). Although activation of NF-�B occurs inresponse to a remarkable diversity of stimuli including cyto-kines (e.g. TNF�, IL1�, lymphotoxin �), bacterial lipopolysac-charide (LPS), stress, and viral proteins, most of the respectivesignaling pathways converge at the level of the IKK5 complex(10–12). Once activated, IKK phosphorylates the I�B inhibi-tory proteins on two specific serine residues (e.g. serines 32 and36 of I�B�), leading to their ubiquitination by the multisubunitSCF�-TrCP E3 ligase and subsequent proteosomal degradation,which in turn allows NF-�B dimers to translocate to thenucleus and activate the transcription of their specific targetgenes (13–16).IKK is composed of two catalytic subunits, IKK� and IKK�,

and a regulatory subunit IKK�/NEMO (10, 17, 18). AlthoughIKK� and IKK� share a high degree of sequence similarity, theyhave largely distinct functions, due to their different substratespecificities andmodes of regulation. IKK� (and IKK�) is essen-tial for rapid and transient NF-�B induction by proinflamma-tory signaling cascades, such as those triggered by TNF� orLPS, via the “classical NF-�B pathway” that mainly relies onI�B� degradation (19–21). In contrast, IKK� is required for thelate and sustained activation of NF-�B in response to a specificsubset of TNF family members (e.g. lymphotoxin �, B cell acti-vating factor (BAFF), CD40 ligand) via the “alternative NF-�Bpathway” that relies on the inducible proteolysis of p100 (22,23). Nonetheless, there is evidence indicating that IKK� con-tributes to cytokine-induced I�B� phosphorylation and subse-quent degradation, thus participating in the classical NF-�Bactivation pathway as well. For instance, IKK� is essential forefficient TNF�-induced I�B� phosphorylation in HeLa cells(24), and it has been reported that IKK� is also required forreceptor activator of nuclear factor �-B ligand (RANKL) medi-ated classical NF-�B activation in mammary epithelial cells(25). Furthermore, IKK� exerts nuclear NF-�B transcriptionalactivating functions through the control of histone phosphory-lation (26, 27) and can also act in specific cell types as a negativemediator of NF-�B activation by attenuating IKK�-drivenNF-�B activation ormodulating RelA Ser-536 phosphorylation

* This work was supported by grants from the Agence Nationale pour laRecherche, Association pour la Recherche sur le Cancer, Belgian InterUni-versity Attraction Pole, Canceropole Ile-de-France, Institut National duCancer, and Universite Paris Descartes (to V. B.), postdoctoral funding fromthe Agence Nationale de la Recherche (to L. L.), grants from the LigueNationale contre le Cancer (to S. B. and F. H.), and a doctoral fellowshipfrom the Ministere de la Recherche et des Technologies (to F. C.).

1 Both authors contributed equally to this work.2 Both authors contributed equally to this work.3 Present address: Institute of Molecular Oncology, Biomedical Sciences

Research Center “Alexander Flemming,” 34 Fleming St., Vari, Greece.4 To whom correspondence should be addressed: Departement

d’Immunologie-Hematologie, Institut Cochin, INSERM U1016, MaternitePort-Royal, 123 Boulevard de Port-Royal, 75014 Paris, France. Tel.:33153104370; Fax: 33143251167; E-mail: [email protected].

5 The abbreviations used are: IKK, I�B kinase; ABIN-2, A20-binding inhibitor ofNF-�B; KD, kinase-inactive; MEF, mouse embryonic fibroblast; Ub, ubiqui-tin; RANTES, regulated on activation T cell expressed and secreted.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 37, pp. 32277–32288, September 16, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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(28, 29). IKK� is also involved in epidermal differentiation, butindependently of its kinase activity (30–32).IKK� and IKK� activation is dependent on phosphorylation

of specific serine residues located within the activation loop (Tloop), either by upstream kinases or by trans-autophosphoryla-tion (33–36). In addition, conformational changes by proteininteractionsmay also be amechanism to stimulate IKK activity.For example, Cdc37 andHsp90 forma chaperone complexwithIKK�/� that is required for IKK activation in response to TNF�and DNA damage (37, 38), and NIK and IKK� binding protein(NIBP) is an enhancer of TNF�-induced NF-�B activity byinteracting with IKK� and increasing IKK kinase activity (39).Because IKK� is involved in many ways in the regulation of

NF-�B activity, the possibility that IKK�-interacting proteinsmight specifically regulate its activity is a crucial issue with pro-found implications toward specific NF-�B response. We havepurified IKK�-associated proteins in mouse embryonic fibro-blasts (MEFs), andmass spectrometry analysis of the copurifiedproteins revealed the presence of A20-binding inhibitor ofNF-�B (ABIN-2) (40, 41). In the study presented here, we showthat IKK� and IKK� both interact with ABIN-2 and impair itsconstitutive degradation by the proteasome. Nonetheless,ABIN-2 increases IKK� but not IKK�-mediated NF-�B activa-tion by specifically increasing IKK� autophosphorylation andkinase activity. In addition, we found that ABIN-2 serine 146 iscritical for ABIN-2-dependent IKK� transcriptional up-regula-tion of specific NF-�B target genes.

MATERIALS AND METHODS

Antibodies and Reagent—The antibodies were purchasedfrom Abcam (E tag), Roche Diagnostic (HA), Sigma (FLAG,�-actin), Cell Signaling (IKK�, phospho-I�B� serine 32–36,phospho-IKK�/� serine 176/180), BDBiosciences Pharmingen(IKK�), Santa Cruz Biotechnology (RelA, RelB, cRel, I�B�,p105/p50, p100/p52, ABIN-2), Southern Biotech (R-phyco-erythrin conjugated anti-rat), and Molecular Probes (AlexaFluor 488-conjugated anti-mouse).MG132 andmurine recom-binant TNF� were purchased from Calbiochem and Sigma,respectively.Plasmid Constructs—Expression vectors for E tag-ABIN-1

and -ABIN-2 were obtained fromR. Beyaert (Ghent University,Zwijnaarde, Belgium). FLAG-ABIN-2 and GST-ABIN-2 fromS. C. Ley (National Institute for Medical Research, MillHill,UK).GST-I�B�(1–54), wild-typeHA-IKK� andHA-IKK�, andkinase-inactive K44M mutants (KD) of HA-IKK� andHA-IKK� were from M. Karin (University of California, SanDiego, CA). FLAG-IKK� and FLAG-IKK� were from M. Hu(University of Texas, Houston, TX). HA-Ub WT and mutantswere fromC. Pique (Institut Cochin, Paris, France), and 3�NF-�B-luciferase reporter was from D. Baltimore (California Insti-tute of Technology, Pasadena, CA). pTrip-ABIN-2 was gener-ated by subcloning human full-length ABIN-2 cDNA intopTRIP-�U3-EF1�-IRES GFP lentiviral vector (42). GST-ABIN-2 deletionmutants were obtained by subcloning ABIN-2coding sequences from amino acids 1–97 (�1), 94–184 (�2),180–279 (�3), 275–345 (�4), and 348–429 (�5) followingstandard recombinant DNA procedures; and details are avail-able upon request. GST-ABIN-2 point mutants were generated

by substituting serine 2, 7, 62, 113, 129, 140, and 146 to alanineusing site-directed mutagenesis (Stratagene) and confirmed bysequencing.Cell Culture and Transient Transfections—HEK293, 293T,

HeLa andMEF cells were cultured in DMEM (Invitrogen) sup-plemented with 10% heat-inactivated FBS and 100 units/mlpenicillin-streptomycin. For transfections, 106 HEK293 cells in35-mm dishes were transfected with 0.8–1 �g of DNA usingLipofectamine (Invitrogen) in Opti-MEM (Invitrogen) asdescribed (43). Cells were harvested 24 h after transfection.Lentiviral Production and Transduction—Production of in-

fectious recombinant lentiviruses was performed by transienttransfection of 293T cells as described in Ref. 42. For infections,105 cells in 35-mmdisheswere transducedwith 5000 ng/ml p24(HIV-1 capsid protein). 48 h later, cells were washed, and freshmedium was added. The culture was then continued asdescribed above.In Vivo Biotinylation Approach—The approach was adapted

from the method described by de Boer et al. based on efficientbiotinylation in vivo and single-step purification of tagged tran-scription factors in mammalian cells (44). Briefly, we first gen-erated a lentiviral vector named pTRIP-BirA by subcloning thebacterial protein-biotin ligase BirA cDNA into the lentiviralvector pTRIP-�U3-EF1�-IRES GFP (42) and then subclonedthe cDNA encoding full-length IKK� tagged at the N terminusby a peptide of 23 amino acids that can be biotinylated by BirAin vivo (IKK�-BP). IKK�-deficient MEFs were then stablytransduced with either pTRIP-BirA empty vector or pTRIP-BirA expressing IKK�-BP. Following large scale cell culture,cytoplasmic and nuclear extracts were incubated with strepta-vidin beads. Eluted proteins were separated by SDS-PAGE andthen stained with Colloidal blue. Each entire lane was dividedinto 2–3-mm gel slices (at least 20 gel plugs/lane) that wereeach treated by an in-gel-digestedmethod usingmodified tryp-sin and further analyzed by nanoflow liquid chromatography-tandem coupled to a Q-TOF system. Data base searches wereperformed using MASCOT and Profound.Indirect Immunofluorescence Microscopy—Cells seeded on

coverslips were transfected using Lipofectamine. 24 h later,cells were fixed in 4% (w/v) paraformaldehyde in PBS for 25min, washed twice in PBS, and permeabilized for 15 min in0.25%TritonX-100 in PBS at room temperature.After blockingwith 10%FBS in PBS for 10min at room temperature, cells werethen incubated for 1 h at room temperature with the primaryantibody in 3% FBS in PBS, washed twice in PBS, and furtherincubated with the fluorophore-conjugated secondary anti-body for 1 h. After two washes in PBS, the coverslips weremounted with Vectashield� mounting medium containing 1.5�g/ml DAPI (Vector Laboratories). Fluorescence imaging wasperformed using a Leica DMI6000 inverted microscope and aMicroMAX-1300Y/HS (Princeton Instruments) camera usingthe Metamorph v.7 software (Molecular Devices).Coimmunoprecipitation and Immunoblotting—Coimmuno-

precipitation and immunoblotting were performed as reportedin Ref. 45.EMSAs—EMSAs were realized as described in Ref. 45. For

supershift assays, nuclear extracts were incubated with specific

Activation of IKK�-dependent NF-�B Activity by ABIN-2

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antibodies for 30 min on ice before incubation with the labeledprobe.Kinase Assays—For IKK immunocomplex kinase assays,

transfected cells were collected in whole cell lysis buffer (300mMNaCl, 25 mMHepes, pH 7.7, 1.5 mMMgCl2, 0.2 mM EDTA,0.5% Triton X-100 supplemented with 10 mM p-nitrophenylphosphate disodium salt, 20 mM �-glycerol phosphate, 100 �M

Na3VO4, 1mMPMSF, 1�Complete protease inhibitormixture(Roche Diagnostics)). HA-IKK� or HA-IKK� was immunopre-cipitated from 100�g of cell lysates withHA antibody for 2 h orovernight at 4 °C, after which protein A/G-agarose beads wereadded and incubation continued for 90min at 4 °C. The immu-noprecipitateswere collected, washed three times in lysis bufferand once in kinase buffer (20mMHepes, pH 7.6, 10mMMgCl2),and then incubated at 30 °C for 25 min in 30 �l of kinase reac-tion mixture containing 1 �Ci of [�-32P]ATP, 1.5 �g of eitherbacterial expressed GST-I�B�(1–54) or GST-ABIN-2 full-length and mutant forms as substrate. The reaction wasstopped by addition of an equal volume of Laemmli buffer andheat denaturation for 5 min at 90 °C. Proteins were separatedon 10% SDS-polyacrylamide gel and transferred to nitrocellu-lose membranes, stained with Ponceau S, subjected to autora-diography to visualize phosphorylated GST fusion proteins,and finally analyzed by immunoblotting for normalization onamount of immunoprecipitated IKKs.Ubiquitination Assays—Cells were lysed for 20 min on ice in

ubiquitination assay buffer (150mMNaCl, 50mMTris-HCl, pH7.5, 1% deoxycholic acid, 1% Nonidet P-40, 50 �M MgCl2, 0.1%SDS, supplemented with 10 mM N-ethylmaleimide, 50 �M

MG132, 10 mM p-nitrophenyl phosphate disodium salt, 20 mM

�-glycerol phosphate, 100 �M Na3VO4, 1 mM PMSF, and 1 �Complete protease inhibitor mixture), after which the SDSconcentration was adjusted to 1%, and whole cell extractsboiled for 10 min, diluted 10-fold with ubiquitination assaybuffer without SDS, and subjected to immunoprecipitation andimmunoblotting.Calf Intestinal Phosphatase Assays—Whole cells extracts

prepared in ubiquitination assay buffer as described abovewereincubated for 30min at 37 °C with 1 unit of calf intestinal phos-phatase (New England Biolabs) either untreated or heat-inacti-vated for 10 min at 95 °C and analyzed by immunoblotting.Luciferase Reporter Assays—Firefly and Renilla luciferase

activities were determined using theDual Luciferase Assay Sys-tem (Promega) according to the manufacturer’s instructions.Experiments were conducted three times in triplicate.RT-qPCR—Total RNA extraction and RTwere performed as

described previously (45). Real-time PCR analysis was carriedout with LightCycler FastStart DNAMaster plus SYBRGreen Ion a Light Cycler 1.5 (Roche Applied Science). All values werenormalized to the level of hypoxanthine-guanine phosphoribo-syltransferase (HPRT) mRNA. Primer sequences are availableupon request.

RESULTS

ABIN-2 Interacts with IKK� and IKK�—Because IKK� playspivotal and complex roles in the regulation ofNF-�B activation,we undertook to identify new IKK�-interacting partners thatcould participate in the activation of NF-�B transcriptional

activity. We applied a biochemical approach based on specificbiotinylation in vivo using IKK� as a bait (see “Materials andMethods”), taking advantage of the very high affinity of avidin/streptavidin for biotinylated templates and efficient single-steppurification and identified ABIN-2 as a new IKK�-interactingpartner candidate.To examine further the significance of this ABIN-2-IKK�

interaction, we first examined the subcellular localization ofABIN-2 when coexpressed with IKK�/� in HeLa cells. Asshown in Fig. 1A, when expressed alone, ABIN-2 formedpunctate structures of various sizes in the cytoplasm. How-ever, when coexpressed with IKK� or IKK�, ABIN-2 relocal-izes uniformly all over the cytoplasm, indicating that IKK�/�have an effect on the subcellular localization of ABIN-2. Wenext performed coimmunoprecipitation experiments usingwhole cell extracts from HEK293 cells cotransfected withE tag-ABIN-2 along with either FLAG-IKK� or FLAG-IKK�.As shown in Fig. 1B, reciprocal experiments with anti-FLAG- and anti-E tag specific antibodies showed thatABIN-2 coimmunoprecipitates with both IKK� and IKK� atsimilar levels. Finally, we confirmed the association betweenABIN-2 and IKK�/� on endogenous proteins in HEK293 andHeLa cells (Fig. 1C).IKK� and IKK� Impair ABIN-2 Constitutive Degradation by

the Proteasome through Deubiquitination of Lys-48-linkedPolyubiquitin Chains—In the course of our experiments, wenoted that when expressed alone in HEK293 cells, ABIN-2 pro-tein was hardly detectable (Fig. 1B), although the total mRNAlevel of ABIN-2mRNApresented a 500-fold increase over basallevel of untransfected cells (Fig. 2B). Remarkably, ABIN-2 pro-tein expression is also extremely low in wild-type MEFs stablytransduced with a lentivirus expressing E tag-ABIN-2 (Fig. 2A,left, lane 1). These observations suggest that ABIN-2 might beconstitutively targeted for degradation. Treatment of E tag-ABIN-2 expressingMEFs (Fig. 2A, left), as well as HEK293 cells(Fig. 2A, right), with the proteasome inhibitor MG132 led to astrong increase in ABIN-2 protein levels, indicating thatABIN-2 is constitutively degraded in a proteasome-dependentmanner. Coexpression of IKK�/�withABIN-2 inHEK293 cellsresulted in a strong increase of ABIN-2 protein expression lev-els in an IKK kinase activity-dependent manner (Fig. 2C),whereas it had only a slight effect on ABIN-2 mRNA level (Fig.2B). Taken together, these observations led us to speculate thatIKK�/� might induce a reduction in the extent of conjugationof ABIN-2 to the Lys-48-linked polyubiquitin chains whichprovide a tag for recognition by the 26 S proteasome (46). Totest this hypothesis, we first examined whether ABIN-2 can beubiquitinated via a Lys-48 linkage by cotransfection of HEK293cells of E tag-ABIN-2 with an HA-tagged ubiquitin Lys-48mutant containing only one lysine at position 48 (Lys-48-Ub).After immunoprecipitation ofABIN-2 (E tag antibody), Lys-48-linked polyubiquitination was analyzed by immunoblottingwith an HA antibody. As shown in Fig. 2D, left, ABIN-2 is effi-ciently ubiquitinated with ubiquitin conjugated through Lys-48. Most importantly, coexpression of either HA-IKK� orHA-IKK� led to a marked reduction in Lys-48-linked ubiquiti-nation of ABIN-2. Significantly, coexpression of IKK�/� withwild-type ubiquitin also decreased ABIN-2 polyubiquitination




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(Fig. 2D, center), whereas it does not affect Lys-63-linked ubiq-uitination (Fig. 2D, right). Together, these results indicate thatABIN-2 is constitutively degraded by the proteasome and that

IKK�/� can increase ABIN-2 stability, possibly via the induc-tion of deubiquitination of Lys-48-linked polyubiquitin chainsof ABIN-2.

FIGURE 1. ABIN-2 interacts with IKK� and IKK�. A, subcellular relocalization of ABIN-2 upon IKK�/� ectopic expression. HeLa cells were transfectedeither with E tag-ABIN-2, HA-IKK�, or HA-IKK� alone, or with E tag-ABIN-2 along with either HA-IKK� or HA-IKK�. Localization of transiently expressedproteins was evaluated by indirect immunofluorescence microscopy using anti-E tag or anti-HA antibody, followed by immunofluorescent staining withAlexa Fluor 488-conjugated anti-mouse or phycoerythrin-conjugated anti-rat, respectively. Nuclei were stained with DAPI. The merged images areshown on the right. B, interaction of ABIN-2 with IKK� and IKK� within transfected HEK293 cells. HEK293 cells were transiently transfected withequivalent amounts of the indicated expression plasmids and whole cell extracts were subjected to immunoprecipitation (IP) with anti-FLAG (upperpanel) or anti-E tag (central panel) and analyzed by immunoblotting (IB) for the indicated proteins. C, ABIN-2 constitutively interacts with IKK� and IKK�in vivo. Whole cell extracts from HEK293 (left) and HeLa (right) cells were subjected to immunoprecipitation with anti-ABIN-2 or nonimmune controlantibody and analyzed by immunoblotting for the indicated proteins. Of note, immunoprecipitated endogenous ABIN-2 comigrates with the IgG heavychains.




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ABIN-2 Enhances IKK�-mediated NF-�B Activation—Giventhe ability of ABIN-2 to interact with and to be stabilized byIKK� and IKK�, we wanted to see whether ABIN-2 could affectIKK�/�-mediated NF-�B activation. To our surprise, theexpression of ABIN-2 led to a strong increase in IKK�-medi-ated NF-�B DNA binding activity in an ABIN-2 dose-depen-dent manner (Fig. 3A). In contrast, although the basal level ofNF-�BDNAbinding complexes was slightly higher when IKK�was expressed alone compared with that seen with IKK� alone(lane 3 versus lane 8), ABIN-2 expression had little effect onIKK�-mediated NF-�B DNA binding activity (Fig. 3A). Wenext examined the subunit composition of the NF-�B DNA-

binding complexes. Supershift analysis of nuclear extracts fromHEK293 cells transfected with HA-IKK� and E tag-ABIN-2(extracts from Fig. 3A, lane 7) revealed that �B-binding com-plexesweremainly composed of RelA-p50 and cRel-p50 dimers(Fig. 3B). Not surprisingly, expression of ABIN-2 had no effecton catalytically inactive K44M IKK� and IKK� mutants (KD)(Fig. 3C). Most importantly, coexpression of IKK� withABIN-1, another ABIN family member (41), had no effect onthe overall NF-�B DNA binding activity, indicating thatABIN-2 exerts specific function in enhancing IKK�-mediatedNF-�B activation (Fig. 3D). To address whether ABIN-2-acti-vating function could also be seen on IKK�-dependent NF-�B

FIGURE 2. IKK� and IKK� impair constitutive degradation of ABIN-2 by the proteasome through deubiquitination of Lys-48-linked polyubiquitinchains. A, ABIN-2 is constitutively degraded by the ubiquitin-proteasome pathway. ABIN-2-deficient MEFs stably transduced with a lentivirus encodingE tag-ABIN-2 (left) or HEK293 cells (right) were treated with MG132 (10 �M) for the indicated periods of time, and whole cell extracts were analyzed byimmunoblotting for the indicated proteins. B, ABIN-2 mRNA level upon ABIN-2 overexpression. Total RNA was isolated from HEK293 cells transfected witheither empty vector or E tag-ABIN-2 either alone or along with HA-IKK� or HA-IKK� expression plasmids. ABIN-2 mRNA expression was quantified by real-timePCR analysis. Data are normalized to hypoxanthine-guanine phosphoribosyltransferase mRNA expression and shown as mean � S.D. (error bars; n � 3– 4). C,IKK�/� kinase activity is required for ABIN-2 stabilization. Whole cell extracts from HEK293 cells transfected with E tag-ABIN-2 along with either wild-type or KDmutants of IKK�/� were analyzed by immunoblotting for the indicated proteins. D, Lys-48-linked poly(Ub) chains of ABIN-2 deubiquinated by IKK� and IKK�.HEK293 cells were transiently transfected with E tag-ABIN-2 and either HA-WT Ub or HA-Ub Lys-48 or HA-Ub Lys-63 along with an empty vector or FLAG-IKK�/�.Whole cell extracts prepared as described under the section on ubiquitination assays (“Materials and Methods”) were subjected to immunoprecipitation (IP)with anti-E tag and analyzed by immunoblotting for the indicated proteins.




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FIGURE 3. ABIN-2 enhances IKK�-mediated NF-�B activation. A, ABIN-2 enhances IKK�- but not IKK�-mediated NF-�B DNA binding activity. Nuclear extractsfrom HEK293 cells transfected with either HA-IKK� or HA-IKK� alone, or along with increasing amounts of E tag-ABIN-2 were analyzed by EMSA using a32P-labeled HIV-LTR tandem �B oligonucleotide as a probe (upper panel). Protein expression levels were analyzed by immunoblotting for the indicated proteins(lower panels). Arrows indicate various migrating forms of ABIN-2. B, for supershift analysis, nuclear extracts from HEK293 cells cotransfected with HA-IKK� andE tag-ABIN-2 (conditions of panel A, lane 7) were incubated with the indicated antibodies before incubation with the labeled probe. Complex I, RelA/p50;complex II, cRel/p50. C, IKK� kinase activity is required for the ABIN-2-dependent IKK�-mediated NF-�B DNA binding activity. Nuclear extracts from HEK293cells transfected with E tag-ABIN-2 along with either WT IKK�/� or KD mutants were analyzed for NF-�B activity by EMSA. Protein expression levels wereanalyzed by immunoblotting for the indicated proteins. D, ABIN-2 but not ABIN-1 synergizes with IKK� to induce NF-�B DNA binding activity. Nuclear extractsfrom HEK293 cells transfected with either E tag-ABIN-1 or E tag-ABIN-2 alone or with either wild-type or KD mutant of HA-IKK� were analyzed for NF-�B activityby EMSA. Protein expression levels were analyzed by immunoblotting for the indicated proteins. E, ABIN-2 enhances IKK�-mediated NF-�B transcriptionalactivity. HEK293 cells were transfected with 3�NF-�B-luciferase reporter along with the indicated expression plasmids, and luciferase activity was determined.F, ABIN-2 increases IKK� activity in vitro. Whole cell extracts from HEK293 cells transiently transfected with HA-IKK� alone or with E tag-ABIN-2 were subjectedto immunoprecipitation with anti-HA, and kinase assays were performed using either GST alone or GST-I�B�(1–54) as substrates. Immunoprecipitated HA-IKK�protein was detected by immunoblotting with anti-HA. Ponceau S staining ensures equal amounts of substrates. Arrows indicate phosphorylated substrateand IKK� autophosphorylation. G, ABIN-2 increases IKK� activity in vivo. Whole cell extracts from HEK293 cells transiently transfected with equivalent amountsof the indicated expression plasmids were analyzed by immunoblotting for the indicated proteins. H, ABIN-2 controls NF-�B DNA binding activity upon prolongedstimulation by TNF� in primary MEFs. Nuclear extracts from WT and ABIN-2-deficient MEFs treated with TNF� for the indicated periods of time were analyzed for NF-�Bactivity by EMSA. I, ABIN-2 controls IKK� activity upon prolonged stimulation by TNF� in primary MEFs. Upper, whole cell extracts from WT and ABIN-2-deficient MEFstreated with TNF� for the indicated periods of time were analyzed for IKK� kinase activity and expression as described above. Lower, whole cell extracts from WT andABIN-2-deficient MEFs were analyzed by immunoblotting for the indicated proteins. * indicates nonspecific band.




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transcriptional activity, we compared the ability of ABIN-2 topotentiate IKK� and IKK� activation of an NF-�B-dependentreporter gene in transiently transfectedHEK293 cells.WhereasIKK� and IKK� alone led to similar activation of the NF-�Breporter, overexpression of ABIN-2 greatly enhanced IKK�-inducedNF-�B activation in a dose-dependentmanner but hadlittle or no effect on IKK�-mediated activation (Fig. 3E).Because ABIN-2 interacts with IKK� and specifically acti-

vates IKK�-dependent NF-�B activation, we wonderedwhether ABIN-2 might increase IKK� activity. We first mea-sured IKK� activity in an in vitro kinase assay. Whereas in theabsence of ABIN-2, only a weak IKK� kinase activity towardI�B� could be detected (Fig. 3F), this activity was enhancedmarkedly when cells were cotransfected with E tag-ABIN-2

(Fig. 3F, lane 2 versus lane 3). Interestingly, ABIN-2 also greatlyenhanced IKK� autophosphorylation.Wenext examined IKK�by immunoblotting (Fig. 3G) and similarly observed thatABIN-2 also markedly increased I�B� phosphorylation on ser-ine 32 and serine 36 in vivo as well as IKK� phosphorylation.

Further in vivo evidence for a role for ABIN-2 in regulatingIKK� activity was obtained by comparing IKK/NF-�B activ-ity in WT and ABIN-2-deficient primary MEFs upon pro-longed stimulation with TNF�. As shown in Fig. 3H, TNF�treatment of WT MEFs resulted in the characteristic bipha-sic time course of NF-�B DNA binding activity. In contrast,the late phase of NF-�B activity that occurs at 4 h and per-sisted for at least 8 h in WT MEFs was strongly decreased inABIN-2-deficient MEFs.

FIGURE 3—continued




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Because temporal control of IKK activity has been reportedpreviously to be crucial for dynamic of NF-�B activation (47),we thus compared the IKK� activation profile in WT andABIN-2-deficientMEFs at late time points of TNF� treatment.Examining IKK� kinase activity in WT MEFs revealed thatIKK� was highly active at early time points, dropped to lowlevels after about 45 min, and was reactivated at 4 h of TNF�stimulation and persisted over 6 h inWTMEFs (Fig. 3I, upper).In contrast, the late phase of TNF�-induced IKK� activity wasalmost completely abolished in absence of ABIN-2. Similarly,IKK� phosphorylation induced by prolonged stimulation byTNF� was markedly reduced in ABIN-2-deficient MEFs com-pared with that seen in WT MEFs (Fig. 3I, lower). Together,these results indicate that ABIN-2 induces IKK� autophos-phorylation and kinase activity in vivo, thus resulting inincreased IKK�-mediated NF-�B transcriptional activity.IKK� Induces the Phosphorylation of ABIN-2—During the

course of ABIN-2 immunoblotting assays, we noticed thatmore slowly migrating ABIN-2 species could be detected whenABIN-2 was coexpressed with HA-IKK� or HA-IKK� (Fig. 1B,Inputs, and Fig. 2C). Importantly, highly resolutive SDS-poly-acrylamide gels indicated that coexpression of ABIN-2 withIKK� resulted in the appearance of an even more shifted bandcompared with the one observed upon coexpression of ABIN-2with IKK� (Fig. 2C, lane 2 versus lane 4). Because coexpressionof ABIN-2 with IKK� led to a strong increase in IKK� kinaseactivity and subsequent IKK�-mediated NF-�B activation,these results raise the intriguing possibility that IKK� may alsodirectly phosphorylateABIN-2. Thus, we decided to investigatewhether ABIN-2 might be phosphorylated by IKK� andwhether such a phosphorylation might be, at least in part,responsible for the enhancement of IKK�-dependent NF-�Btarget gene expression.Interestingly, the slower migrating form of ABIN-2 was con-

verted to its basal form upon treatment with calf intestinalphosphatase, revealing phosphorylation as the nature ofABIN-2 modification (Fig. 4A). In a second step, to determinewhether IKK� directly phosphorylates ABIN-2, we generated aGST-ABIN-2 full-length fusion protein and performed in vitroIKK� immunocomplex kinase assays using IKK� that wasimmunoprecipitated from HEK293 cells overexpressing HA-IKK�, and the GST-ABIN-2 protein as a substrate. The GST-ABIN-2 protein was significantly phosphorylated by IKK�, therelative level of phosphorylation being slightly lower than thatof IKK� autophosphorylation (Fig. 4B, right). Moreover, theABIN-2 phosphorylation was specific, because IKK� did notphosphorylate the GST protein alone (Fig. 4B, right). To local-ize the region(s) of ABIN-2 that is (are) phosphorylated byIKK�, five GST-ABIN-2 deletion mutants, each containing asubdomain of theABIN-2 coding region, as described in Fig. 4C(�1–�5 mutants), were used as substrates in IKK� kinaseassays. As shown in Fig. 4D, only mutants �1 and �2 werephosphorylated efficiently by IKK�. Note that the bandobserved with mutant �3 is nonspecific, because its size doesnot correspond to that of the substrate detected by Ponceau Sstaining (Fig. 4D). Mutant �1 and mutant �2 contained threeand four serines, respectively. Because IKK� is known to be aserine/threonine kinase (10), one or more of these serines are

likely involved in ABIN-2 phosphorylation. We have mutatedeach of the serine residues to alanine into the �1 and �2 GST-ABIN-2 deletion mutants, and in vitro kinase assays revealedthat a point mutation at serine 62 and serine 146 abolishedphosphorylation of the�1 and the�2 ABIN-2 deletionmutant,respectively (Fig. 4E). Together, these results suggest thatABIN-2 serine 62 and/or serine 146 phosphorylation by IKK�might be required for enhancement of IKK�-mediated NF-�Bactivity.ABIN-2 Serine 146 Is Critical to Enhance IKK�-dependent

NF-�B Target Gene Expression—We therefore analyzed thefunctional consequence of ABIN-2 serine 62 or serine 146mutations on endogenousNF-�B target gene expression. Usingreal-time PCR, we compared the expression of 15 knownNF-�B target genes, including inflammatory chemokines andcytokines as well as antiapoptotic genes, in HEK293 cells trans-fected with HA-IKK� along with either wild-type ABIN-2 orS62A or S146A mutants. Remarkably, whereas the expressionof RANTES and monocyte chemoattractant protein-1 wasstrongly increased in HEK293 cells transfected with IKK� andeither wild-type ABIN-2 or the S62A ABIN-2 mutant (Fig. 5A,upper panels), coexpression of IKK� with the S146A mutantresulted in a marked reduction in RANTES andmonocyte che-moattractant protein-1 mRNA levels. The decrease is specific,because it was not observed for TNF� and cIAP2, two otherNF-�B target genes for which the expression level is alsostrongly induced in cells coexpressing IKK� and wild-typeABIN-2 (Fig. 5A, middle panels). Moreover, analysis of wild-type and mutant ABIN-2 expression showed that their mRNAlevels are similar when coexpressed with IKK� (Fig. 5A, lowerleft panel). Similarly, IKK� expression is not affected by coex-pressionwith either formofABIN-2 (Fig. 5A, lower right panel).Taken together, our results show that ABIN-2 exerts a serine146-dependent selective activating function serving to enhanceIKK�-mediated NF-�B activity.

DISCUSSION

In this report, we reveal an unexpected and intimate relation-ship between IKK� and ABIN-2. We show that ABIN-2 inter-acts with and is stabilized by IKK�. Importantly, ABIN-2 acti-vates IKK� kinase activity, thus enhancing IKK�-mediatedNF-�B transcriptional activity. Furthermore, ABIN-2 is phos-phorylated by IKK� at serine 146, a serine that appears to becritical for ABIN-2-dependent IKK� transcriptional up-regula-tion of specific NF-�B target genes. We propose that ABIN-2serves as an adaptor protein to activate IKK� which in turn canalso phosphorylate ABIN-2. This cross-regulation betweenABIN-2 and IKK� seems to be essential for activation of selec-tive NF-�B target gene expression (Fig. 5B). However, it is notyet clear whether ABIN-2 stabilization is absolutely requiredfor an ABIN-2-dependent increase in IKK� activity.

In contrast to our findings that ABIN-2 interacts with IKK�and IKK� both in overexpressing conditions and when theinteractions of the endogenous proteins were examined, a pre-vious report showed that ABIN-2 binds to IKK�/NEMO, butnot to IKK� and IKK� in transfected HEK293 cells (48). Thereasons for these discrepant results are not clear but could bedue to differences in conditions employed for the coimmuno-




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precipitation experiments. Nevertheless, because all three IKKsubunits are known to form a complex in vivo (49), it is likelythat ABIN-2 could be part of the classical tripartite IKK com-plex (Fig. 5B). Nevertheless it cannot be excluded that ABIN-2might also bind to the alternative IKK�-containing complexindependently of IKK� and IKK� (50).

Based on its ability to bindA20 and block inflammatory cyto-kine-induced NF-�B activation upon overexpression, ABIN-2was originally described as a negative regulator of NF-�B-de-pendent gene expression (40). Such an inhibition was alsoobserved in response to TPA and EGF, thus reinforcing the

concept that ABIN-2 overexpression leads to inhibition ofNF-�B (40, 51). Interestingly, ABIN-2 interferes with NF-�Bactivation induced by RIP1, TRAF2, TRAF6, and IRAK1, butnot IKK�, suggesting that the inhibitory effect of ABIN-2occurs upstream of IKK� (40). In the present report, we dem-onstrate that ABIN-2 can act as an activator of NF-�B throughits interaction with IKK� and subsequent enhancement ofIKK�-mediated NF-�B transcriptional activity. It thus appearsthat ABIN-2 may exert dual functions depending on its targetswithin the NF-�B activation pathways. Interestingly, IKK� wasshown to associate with RelA in the nucleus and activate the

FIGURE 4. IKK� induces the phosphorylation of ABIN-2. A, differential migration of ABIN-2 upon ectopic expression of IKK� is due to phosphorylation.HEK293 cells were transfected with the indicated expression plasmids. Whole cell extracts prepared as in Fig. 2D were incubated with calf intestinalphosphatase, either untreated (CIP) or heat-inactivated (CIP*), and analyzed by immunoblotting for the indicated proteins. B, IKK� phosphorylatesABIN-2 in vitro. Whole cell extracts from HEK293 cells transiently transfected with HA-IKK� were subjected to immunoprecipitation with anti-HA, andkinase assays were performed using either GST alone or GST-ABIN-2 full-length as substrates. Immunoprecipitated HA-IKK� protein was detected byimmunoblotting with anti-HA. Arrows indicate phosphorylated substrate and IKK� autophosphorylation, and * indicates nonspecific bands. C, sche-matic representation of GST-ABIN-2 deletion mutants. Numbers refer to amino acid positions within human ABIN-2. �1, 1–97; �2, 94 –184; �3, 180 –279;�4, 275–345; and �5, 348 – 429. Structural subdomains are indicated. AHD1/2/4, ABIN homology domains 1, 2, and 4; UBAN, ubiquitin binding domain;ZF, zinc finger. D, IKK� phosphorylation of N-terminal domain of ABIN-2. Kinase assays were performed as in B using either GST alone or GST-ABIN-2deletion mutants as substrates. Immunoprecipitated HA-IKK� protein was detected by immunoblotting. * indicates nonspecific band. E, IKK� phos-phorylates ABIN-2 on serine 62 and serine 146 in vitro. Kinase assays were performed as in B using as substrates various GST-ABIN-2 point mutants inwhich serine (S) was individually replaced by alanine (A).




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FIGURE 5. ABIN-2 serine 146 is critical to enhance IKK�-dependent NF-�B target gene expression. A, total RNA was isolated from HEK293 cells transfectedwith the indicated expression plasmids, and gene expression was quantified by real-time PCR analysis. Data are normalized to hypoxanthine-guanine phos-phoribosyltransferase mRNA expression and shown as mean � S.D. (error bars; n � 3– 4). B, model of NF-�B activation by ABIN-2. Upon recruitment of ABIN-2to the IKK complex, ABIN-2 is stabilized through deubiquitination of Lys-48-linked poly(Ub) chains; ABIN-2 is phosphorylated on serine 146; and IKK� becomesactivated, thus leading to the phosphorylation, ubiquitination, and degradation of I�B�, and subsequent activation of NF-�B target gene expression.




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expression of NF-�B-responsive genes through direct modula-tion of histone function (26, 27). Although we were unable todetect nuclear ABIN-2 by immunofluorescence microscopyexperiments, there is evidence to suggest that ABIN-2 mayfunction as a transcriptional coactivator. First, tandem affinitypurification indicated that ABIN-2 forms a constitutive com-plex with several NF-�B subunits (RelA, p50, p52) (52); second,a fusion protein of ABIN-2 and the Gal4-DNA binding domainleads to the induction of a Gal4-dependent reporter gene (53);and third, the C-terminal domain of ABIN-2 can enter thenucleus and exert transactivating activity (53). In the nearfuture, chromatin immunoprecipitation experiments designedto determine whether ABIN-2, potentially in conjunction withIKK� and/or RelA, is recruited to the promoter regions ofNF-�B-responsive genes will provide a direct test of thishypothesis.Analysis of ABIN-2-deficient mice revealed that ABIN-2 is

required for ERK activation in response to TNF� and LPS inmacrophages as well as upon CD40 ligation in B cells by stabi-lization of the upstream kinase TPL-2 (54). In contrast, LPS-and TNF�-induced activation of ERK was unaffected byABIN-2 deficiency in B cells and dendritic cells, respectively(54). Therefore, it is likely that the ABIN-2-activating functionin the ERK signaling pathway is both stimulus- and cell type-specific, reflecting the alternate use of TPL-2 and other MEKkinases to trigger ERKactivation.Nodefect inNF-�Bactivationtriggered by LPS was observed in primary B cells isolated fromABIN-2-deficient mice (54). Similarly, activation of NF-�Binduced by TNF� and LPS was unaffected in ABIN-2-deficientmacrophages (54). However, because overexpression experi-ments point to a role of ABIN-2 in the control of NF-�B tran-scriptional activity, it is tempting to hypothesize that ABIN-2,just as it does for the ERKpathway, plays a role in the regulationof NF-�B activation in vivo in a cell type- and stimulus-specificmanner. In support of this idea, a recent report demonstratesthe existence of recurrent ABIN-2 somatic mutations in diffuselarge B cell lymphoma that altered the capacity of ABIN-2 toinhibit NF-�B (55).

In an effort to address the physiological relevance of the acti-vation of NF-�B activity by ABIN-2, we have performed real-time PCR on several known NF-�B target genes with roles ininflammation and cell survival. We observed that coexpressionof ABIN-2 and IKK� leads to a strong induction of severalinflammatory cytokines and chemokines compared with whatwas seen upon expression of either IKK� or ABIN-2 alone, sug-gesting a proinflammatory role for ABIN-2, most likely due toits NF-�B-activating properties described in this report. Inter-estingly, serine 146, located within the N-terminal domain ofABIN-2, seems to be a critical phosphorylation site for ABIN-2-dependent IKK� transcriptional up-regulation of specificNF-�B target genes. Because ABIN-2 serine 146 is phosphory-lated by IKK� in vitro, our data suggest an intimate interplaybetweenABIN-2 and IKK� that is crucial for activation of selec-tive NF-�B target gene expression. However, further experi-ments are required to determine whether IKK� is responsiblefor ABIN-2 phosphorylation in vivo and whether other kinasesmight be involved.

In conclusion, our study has highlighted a new and unex-pected NF-�B-activating function for ABIN-2 and defined apreviously unrecognized mechanism of cross-regulationbetween ABIN-2 and IKK� that seems to be an essential regu-lator of NF-�B-dependent gene expression.

Acknowledgments—We thank S. Ley, R. Beyaert, P. Cohen, D. Balti-more, G. Courtois, Robert Weil, V. M. Dixit, M. Hu, M. Karin, M.Korner, A.Ma, T. Tchenio, C. Pique, I. Verma, C.Ware, and J. Brown-ing for valuable material; N. Cagnard for assistance in bioinformaticanalysis; S. Spaggiari for technical assistance; L. L. Pritchard for crit-ically reading the manuscript; and E. Derudder for fruitful discussionand constant support.

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Didier Bordereaux, Jeroen A. Demmers, John Strouboulis and Véronique BaudLaurent Leotoing, Fanny Chereau, Silvère Baron, Florent Hube, Hugo J. Valencia,

ActivityB Transcriptionalκ)-mediated NF-α (IKKαB) Kinase κB (IκInhibitor of NF-

B)-2 (ABIN-2) Is an Activator ofκB (NF-κA20-binding Inhibitor of Nuclear Factor-

doi: 10.1074/jbc.M111.236448 originally published online July 22, 20112011, 286:32277-32288.J. Biol. Chem.

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