Interferon regulatory factor 2 binding protein 2 is a new NFAT1 partner and represses its transcriptional activity

Abstract

The nuclear factor of activated T cells (NFAT) family of transcription factors is expressed in a wide range of cell types and regulates genes involved in cell cycle, differentiation, and apoptosis. NFAT proteins share two well-conserved regions, the regulatory domain and the DNA binding domain. The N- and C-terminal ends are transactivation sites and show less sequence similarity, whereas their molecular functions remain poorly understood. Here, we identified a transcriptional repressor, interferon regulatory factor 2 binding protein 2 (IRF-2BP2), which specifically interacts with the C-terminal domain of NFAT1 among the NFAT family members. IRF-2BP2 was described as a corepressor by inhibiting both enhancer-activated and basal transcription. Gene reporter assays demonstrated that IRF-2BP2 represses the NFAT1-dependent transactivation of NFAT-responsive promoters. The ectopic expression of IRF-2BP2 in CD4 T cells resulted in decreased interleukin-2 (IL-2) and IL-4 production, supporting a repressive function of IRF-2BP2 for NFAT target genes. Furthermore, NFAT1 and IRF-2BP2 colocalized in the nucleus in activated cells, and the mutation of a newly identified nuclear localization signal in the IRF-2BP2 rendered it cytoplasmic, abolishing its repressive effect on NFAT1 activity. Collectively, our data demonstrate that IRF-2BP2 is a negative regulator of the NFAT1 transcription factor and suggest that NFAT1 repression occurs at the transcriptional level.

Fig. 1.

IRF-2BP2 interacts with NFAT1 TAD-C in a yeast two-hybrid assay. (A) Schematic representation of the NFAT1 transcriptional factor with its conserved domains: the NFAT homology region (NHR), the DNA binding domain (DBD), and the transactivation domains (TAD-N and TAD-C). The boundary of each region is labeled above the sequence; numbering refers to the amino acid position of the protein. The NFAT1 C-terminal end (amino acids 727 to 925) used in the yeast two-hybrid system also is represented. (B) Yeast two-hybrid system interaction assays on plates containing synthetic minimal medium and 0, 5, or 15 mM 3-AT. Region 1, positive control (pTL-Nip7p + pACT-Nop8p); region 2, test (pTL-CT-NFAT1 + pACT-IRF-2BP2); regions 3, 4, and 5, negative controls (pTL-CT-NFAT + pGAD424, pBTM-Nip7 + pGAD-IRF-2BP2, pBTM-Nip7 + pGAD-424, respectively). (C) β-Galactosidase filter assay. The L40 strain, which contains the HIS3 and lacZ reporter genes, was cotransformed with vectors containing the LexA DBD fusion proteins and Gal4p activation domain fusion proteins. All results are representative of at least three independent experiments.

Fig. 2.

IRF-2BP2 interacts with NFAT1, and these proteins colocalize in the nucleus. (A) In vitro binding assay. Glutathione-Sepharose beads were loaded with GST or GST-IRF-2BP2-RING proteins, washed, and incubated with Jurkat extracts. The proteins then were eluted as described in Materials and Methods. Samples from the extracts and eluted from the resin were fractionated by SDS-PAGE and subjected to immunoblot analysis using anti-GST and anti-NFAT1 antibodies. (B) NFAT1 coimmunoprecipitates (IP) with IRF-2BP2. Nuclear lysates of HEK293T cells cotransfected with pcDNA4-IRF-2BP2 and pLIRES-EGFP-CA-NFAT1 were immunoprecipitated with anti-c-myc antibody. Bound proteins then were analyzed by SDS-PAGE followed by Western blotting using anti-tag antibody (c-myc) for IRF-2BP2 or anti-NFAT1 antibody. (C) Immunofluorescence analysis of IRF-2BP2 and NFAT1. HEK293T cells cotransfected with GFP-NFAT1 and IRF-2BP2, grown on glass coverslips, and treated with ionomycin (Iono) (5 μM), CsA (2 μM), or both for 15 min. Untreated cells were used as a control. NFAT1 was fused to EGFP, and IRF-2BP2 was detected with an anti-tag primary antibody (c-myc) followed by a rhodamine-conjugated antibody. DAPI staining shows the position of the nucleus. All results are representative of at least three independent experiments. Unst, unstimulated.

Fig. 3.

IRF-2BP2 represses NFAT1-mediated transactivation and cytokine expression in primary lymphocytes. (A and B) Jurkat cells were electroporated with empty vectors (8 μg each), the NFAT1 vector (8 μg), the IRF-2BP2 vector (0.5 to 8 μg), and luciferase reporter vectors (1 μg). After 24 h, cells were stimulated for 6 h with PMA (10 nM) plus ionomycin (1 μM) for 3× NFAT vector-transfected cells (left) or PMA (10 nM) alone for 6× NF-κB vector-transfected cells (right). (A) Luciferase assays using reporter plasmids containing the NFAT binding site (3×NFAT-Luc) or NF-κB binding site (6×NFκB). Unst, unstimulated. (B) Luciferase assays using reporter plasmids containing the κ3 element of the TNF-α promoter or IL-2 or IL-4 promoter. For all experiments, the firefly luciferase reporter gene was normalized with a renilla vector (0.1 μg pRL-TK). Results represent the means from three independent experiments ± standard deviations. (C) Primary CD4 T cells from lymph nodes of C57/BL6 mice were stimulated with anti-CD3 and anti-CD28 for 48 h and then transduced with pIRES-EGFP or pILRES-EGFP-IRF-2BP2 vector. Twenty hours after transduction, cells were restimulated with PMA (10 nM) plus ionomycin (1 μM) for 6 h, and the intracellular cytokines IL-2 and IL-4 were stained and analyzed by flow cytometry.

Fig. 4.

IRF-2BP2 interacts with the C-terminal end of NFAT1, and this region is essential for the repressing phenotype mediated by IRF-2BP2. (A) GST and GST–IRF-2BP2 proteins were immobilized on glutathione-Sepharose beads and further incubated with E. coli extracts expressing each NFAT1 domain independently (N terminus, DBD, or C terminus) as indicated. The beads were washed and then proteins were eluted. Samples from the extracts (inputs) and eluted from the resin (pull down) were fractionated by SDS-PAGE, followed by immunoblot analysis using anti-GST and anti-NFAT1 antibodies. (B) Extracts prepared from HEK293T cells transfected with pcDNA5-NFAT1 or pCDNA5-NFAT1ΔC were incubated with the recombinant proteins GST and GST–IRF-2BP2, which previously were immobilized on glutathione-Sepharose beads. After incubation, the beads were washed, proteins were eluted, and samples from the extracts (inputs) and eluted from the resin (pulldown) were fractionated by SDS-PAGE, followed by immunoblot analysis using anti-GST and anti-NFAT1 antibodies. (C) Jurkat cells were electroporated with empty vectors (8 μg each), NFAT1 vector (8 μg), NFAT1ΔC vector (8 μg), IRF-2BP2 vector (8 μg), and the luciferase reporter vector 3× NFAT (1 μg), as indicated. After 24 h, cells were stimulated for 6 h with PMA (10 nM) plus ionomycin (1 μM). The firefly luciferase reporter gene was normalized with a renilla vector (0.1 μg pRL-TK). Results represent the means from three independent experiments ± standard deviations. (D) The interaction sites of CT-NFAT1 and IRF-2BP2 were mapped by the yeast two-hybrid system in the L40 strain cotransformed with CT-NFAT1 and IRF-2BP2 constructs. A β-galactosidase filter assay was performed to test the lacZ reporter gene. Different deletions of CT-NFAT1 were constructed as shown. The boundary of each region is labeled above the sequence; numbers refer to the amino acid position of the protein. All results are representative of at least three independent experiments.

Fig. 5.

IRF-2BP2 interacts specifically with NFAT1. (A) Amino acid sequence alignment comparing the C-terminal end of NFAT1-4 family members. (B) In vitro binding assays. The recombinant proteins GST and GST–IRF-2BP2 expressed in E. coli and immobilized on glutathione-Sepharose beads were incubated with extracts prepared from HEK 293T cells transfected with pcDNA5-NFAT1, pcDNA3-NFAT2, pcDNA5-NFAT3, or pcDNA4-NFAT4. After incubation, the beads were washed, proteins were eluted, and samples from the extracts (inputs) and eluted from the resin (pulldown) were fractionated by SDS-PAGE, followed by immunoblot analysis using anti-GST and anti-NFAT1 antibodies. (C) Jurkat cells were electroporated with empty vectors (8 μg each), NFAT1 vector (8 μg), NFAT2 vector (8 μg), IRF-2BP2 vector (8 μg) as indicated, and the luciferase reporter vector 3× NFAT (1 μg). After 24 h, cells were stimulated for 6 h with PMA (10 nM) plus ionomycin (1 μM). The firefly luciferase reporter gene was normalized with a renilla vector (0.1 μg pRL-TK). Results represent the means from three independent experiments ± standard deviations.

Fig. 6.

RING domain and zinc finger are necessary for the repressive function of IRF-2BP2. (A) Mapping the IRF-2BP2 interaction sites with NFAT1 via the yeast two-hybrid system (β-galactosidase reporter assay). The L40 strain was cotransformed with the following CT-NFAT1 and IRF-2BP2 constructs: full-length IRF-2BP2, IRF-2BP2Δring, or IRF-2BP2Δzinc. A schematic diagram of the IRF-2BP2 constructs is shown on the left. The boundary of each region is labeled above the sequence; numbers refer to the amino acid position of the protein. Luciferase reporter assays testing the truncated proteins IRF-2BP2Δring (B) and IRF-2BP2Δzinc (C) are shown. Jurkat cells were electroporated with 3× NFAT-Luc (1 μg), empty vectors (8 μg), the NFAT1 vector (8 μg), and increasing amounts (1 to 8 μg) of IRF-2BP2 constructs (full length, Δring, or Δzinc). After 24 h, cells were stimulated for 6 h with PMA (10 nM) plus ionomycin (1 μM). For all experiments, the firefly luciferase reporter gene was normalized with a renilla vector (0.1 μg pRL-TK). Results represent the means from three independent experiments ± standard deviations.

Fig. 7.

NFAT1 protein stability is not affected by IRF-2BP2. (A) HEK293T cells were transfected with pLIRES-EGFP-CA-NFAT1 (2 μg) and increasing amounts of pcDNA4-IRF-2BP2 (0.25 to 2 μg). After 48 h, the indicated proteins were detected by Western blotting with specific antibodies. Results are representative of three independent experiments. (B) Jurkat cells were electroporated with the luciferase reporter vector 3× NFAT-Luc (1 μg), empty vectors (8 μg), the pcDNA5-NFAT1 vector (8 μg), and increasing amounts of the pcDNA4-IRF-2BP2 vector (1 to 8 μg). After 24 h, cells were left untreated or were treated with MG132 (20 μM) and stimulated for 6 h with PMA (10 nM) plus ionomycin (1 μM). The firefly luciferase reporter gene was normalized with a renilla vector (0.1 μg pRL-TK). Results represent means from three independent experiments ± standard deviations.

Fig. 8.

IRF-2BP2 repression phenotype depends on its nuclear localization. (A) Schematic representation of IRF-2BP2 and its nuclear localization signal (NLS). The RKRK amino acids from the wild-type protein (WT) were changed to alanine to generate a mutated protein (Mut: AAAA). The boundary of each region is labeled above the sequence; numbers refer to the amino acid position of the protein. (B) Immunofluorescence analysis of HEK293T cells transfected with vectors encoding wild-type IRF-2BP2 (RKRK) or the AAAA-mutated protein. Proteins were labeled with a conjugated rhodamine antibody. DAPI staining shows the position of the nucleus. Results are representative of three independent experiments. (C) Luciferase reporter assay comparing IRF-2BP2 (wild type) and IRF-2BP2MutNLS (AAAA mutation) using the reporter 3× NFAT-Luc. Jurkat cells were electroporated with 3× NFAT-Luc (1 μg), empty vectors (8 μg), the NFAT1 vector (8 μg), and increasing amounts (1 to 8 μg) of IRF-2BP2 constructs. After 24 h, cells were stimulated for 6 h with PMA (10 nM) plus ionomycin (1 μM). The firefly luciferase reporter gene was normalized with a renilla vector (0.1 μg pRL-TK). Results represent means from three independent experiments ± standard deviations.