ABIN-2 interacts with IKKα and IKKβ. A, subcellular relocalization of ABIN-2 upon IKKα/β ectopic expression. HeLa cells were transfected either with E tag-ABIN-2, HA-IKKα, or HA-IKKβ alone, or with E tag-ABIN-2 along with either HA-IKKα or HA-IKKβ. Localization of transiently expressed proteins was evaluated by indirect immunofluorescence microscopy using anti-E tag or anti-HA antibody, followed by immunofluorescent staining with Alexa Fluor 488-conjugated anti-mouse or phycoerythrin-conjugated anti-rat, respectively. Nuclei were stained with DAPI. The merged images are shown on the right. B, interaction of ABIN-2 with IKKα and IKKβ within transfected HEK293 cells. HEK293 cells were transiently transfected with equivalent amounts of the indicated expression plasmids and whole cell extracts were subjected to immunoprecipitation (IP) with anti-FLAG (upper panel) or anti-E tag (central panel) and analyzed by immunoblotting (IB) for the indicated proteins. C, ABIN-2 constitutively interacts with IKKα and IKKβ in vivo. Whole cell extracts from HEK293 (left) and HeLa (right) cells were subjected to immunoprecipitation with anti-ABIN-2 or nonimmune control antibody and analyzed by immunoblotting for the indicated proteins. Of note, immunoprecipitated endogenous ABIN-2 comigrates with the IgG heavy chains.

ABIN-2 enhances IKKα-mediated NF-κB activation. A, ABIN-2 enhances IKKα- but not IKKβ-mediated NF-κB DNA binding activity. Nuclear extracts from HEK293 cells transfected with either HA-IKKα or HA-IKKβ alone, or along with increasing amounts of E tag-ABIN-2 were analyzed by EMSA using a ³²P-labeled HIV-LTR tandem κB 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-IKKα and E 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, IKKα kinase activity is required for the ABIN-2-dependent IKKα-mediated NF-κB DNA binding activity. Nuclear extracts from HEK293 cells transfected with E tag-ABIN-2 along with either WT IKKα/β or KD mutants were analyzed for NF-κB activity by EMSA. Protein expression levels were analyzed by immunoblotting for the indicated proteins. D, ABIN-2 but not ABIN-1 synergizes with IKKα to induce NF-κB DNA binding activity. Nuclear extracts from 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-IKKα were analyzed for NF-κB activity by EMSA. Protein expression levels were analyzed by immunoblotting for the indicated proteins. E, ABIN-2 enhances IKKα-mediated NF-κB transcriptional activity. HEK293 cells were transfected with 3×NF-κB-luciferase reporter along with the indicated expression plasmids, and luciferase activity was determined. F, ABIN-2 increases IKKα activity in vitro. Whole cell extracts from HEK293 cells transiently transfected with HA-IKKα alone or with E tag-ABIN-2 were subjected to immunoprecipitation with anti-HA, and kinase assays were performed using either GST alone or GST-IκBα(1–54) as substrates. Immunoprecipitated HA-IKKα protein was detected by immunoblotting with anti-HA. Ponceau S staining ensures equal amounts of substrates. Arrows indicate phosphorylated substrate and IKKα autophosphorylation. G, ABIN-2 increases IKKα activity in vivo. Whole cell extracts from HEK293 cells transiently transfected with equivalent amounts of the indicated expression plasmids were analyzed by immunoblotting for the indicated proteins. H, ABIN-2 controls NF-κB DNA binding activity upon prolonged stimulation by TNFα in primary MEFs. Nuclear extracts from WT and ABIN-2-deficient MEFs treated with TNFα for the indicated periods of time were analyzed for NF-κB activity by EMSA. I, ABIN-2 controls IKKα activity upon prolonged stimulation by TNFα in primary MEFs. Upper, whole cell extracts from WT and ABIN-2-deficient MEFs treated with TNFα for the indicated periods of time were analyzed for IKKα kinase activity and expression as described above. Lower, whole cell extracts from WT and ABIN-2-deficient MEFs were analyzed by immunoblotting for the indicated proteins. * indicates nonspecific band.

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

IKKα and IKKβ impair constitutive degradation of ABIN-2 by the proteasome through deubiquitination of Lys-48-linked polyubiquitin chains. A, ABIN-2 is constitutively degraded by the ubiquitin-proteasome pathway. ABIN-2-deficient MEFs stably transduced with a lentivirus encoding E tag-ABIN-2 (left) or HEK293 cells (right) were treated with MG132 (10 μm) for the indicated periods of time, and whole cell extracts were analyzed by immunoblotting for the indicated proteins. B, ABIN-2 mRNA level upon ABIN-2 overexpression. Total RNA was isolated from HEK293 cells transfected with either empty vector or E tag-ABIN-2 either alone or along with HA-IKKα or HA-IKKβ expression plasmids. ABIN-2 mRNA expression was quantified by real-time PCR analysis. Data are normalized to hypoxanthine-guanine phosphoribosyltransferase mRNA expression and shown as mean ± S.D. (error bars; n = 3–4). C, IKKα/β 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 KD mutants of IKKα/β were analyzed by immunoblotting for the indicated proteins. D, Lys-48-linked poly(Ub) chains of ABIN-2 deubiquinated by IKKα and IKKβ. 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-IKKα/β. 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.

IKKα induces the phosphorylation of ABIN-2. A, differential migration of ABIN-2 upon ectopic expression of IKKα 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 intestinal phosphatase, either untreated (CIP) or heat-inactivated (CIP*), and analyzed by immunoblotting for the indicated proteins. B, IKKα phosphorylates ABIN-2 in vitro. Whole cell extracts from HEK293 cells transiently transfected with HA-IKKα were subjected to immunoprecipitation with anti-HA, and kinase assays were performed using either GST alone or GST-ABIN-2 full-length as substrates. Immunoprecipitated HA-IKKα protein was detected by immunoblotting with anti-HA. Arrows indicate phosphorylated substrate and IKKα autophosphorylation, and * indicates nonspecific bands. C, schematic representation of GST-ABIN-2 deletion mutants. Numbers refer to amino acid positions within human ABIN-2. Δ1, 1–97; Δ2, 94–184; Δ3, 180–279; Δ4, 275–345; and Δ5, 348–429. Structural subdomains are indicated. AHD1/2/4, ABIN homology domains 1, 2, and 4; UBAN, ubiquitin binding domain; ZF, zinc finger. D, IKKα phosphorylation of N-terminal domain of ABIN-2. Kinase assays were performed as in B using either GST alone or GST-ABIN-2 deletion mutants as substrates. Immunoprecipitated HA-IKKα protein was detected by immunoblotting. * indicates nonspecific band. E, IKKα phosphorylates 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 in which serine (S) was individually replaced by alanine (A).