Inhibition of NF-kappaB by ZAS3, a zinc-finger protein that also binds to the kappaB motif

Abstract

The ZAS proteins are large zinc-finger transcriptional proteins implicated in growth, signal transduction, and lymphoid development. Recombinant ZAS fusion proteins containing one of the two DNA-binding domains have been shown to bind specifically to the kappaB motif, but the endogenous ZAS proteins or their physiological functions are largely unknown. The kappaB motif, GGGACTTTCC, is a gene regulatory element found in promoters and enhancers of genes involved in immunity, inflammation, and growth. The Rel family of NF-kappaB, predominantly p65.p50 and p50.p50, are transcription factors well known for inducing gene expression by means of interaction with the kappaB motif during acute-phase responses. A functional link between ZAS and NF-kappaB, two distinct families of kappaB-binding proteins, stems from our previous in vitro studies that show that a representative member, ZAS3, associates with TRAF2, an adaptor molecule in tumor necrosis factor signaling, to inhibit NF-kappaB activation. Biochemical and genetic evidence presented herein shows that ZAS3 encodes major kappaB-binding proteins in B lymphocytes, and that NF-kappaB is constitutively activated in ZAS3-deficient B cells. The data suggest that ZAS3 plays crucial functions in maintaining cellular homeostasis, at least in part by inhibiting NF-kappaB by means of three mechanisms: inhibition of nuclear translocation of p65, competition for kappaB gene regulatory elements, and repression of target gene transcription.

Fig. 1.

NF-κB and ZAS3 are the major κB DNA-binding proteins in B lymphocytes. (A) EMSA of ³²P-κB oligonucleotide and 38B9 nuclear extracts. Antibodies or unlabeled DNA (20 ng; 100-fold excess) supplemented to binding reactions are indicated at the top of each lane. The major DNA–protein complexes were designated as C1, -2, and -3. (B) EMSA of ³²P-κB oligonucleotide and nuclear extracts. Lane 1, 38B9; lanes 2 and 3, representative ZAS3-deficient cells ZAS3^–/–B1 and ZAS3^–/–B2. (C) Gel supershift assays show the presence of p65 and p50 in the predominant κB–DNA protein complex derived from ZAS3^–/–B1 nuclear extracts. Triangles indicate antibody supershifted complexes.

Fig. 2.

Constitutive activation of NF-κB in ZAS3^–/– cells. (A) EMSA of ³²P-κB oligonucleotide, nuclear extracts (N), or cytoplasmic extracts (C), and with (+) or without (–) LPS (10 μg/ml for 4 h). (B) Immunoblot analysis of nuclear extracts. (C) Reporter gene assays. Indicated cells were cotransfected with p7×κB (0.25 μg) and pCH110 (0.1 μg). Thirty-six hours posttransfection, duplicated cell samples were incubated with LPS (10 μg/ml, 4 h). Luciferase activity, normalized to β-galactosidase activity, from unstimulated 38B9 cells was assigned as +1. (D and E) Immunoblot analyses of cytoplasmic extracts.

Fig. 3.

ZAS3 represses NF-κB-mediated transcription. (A) Expression of recombinant FLAG-ZAS3 proteins in HEK 293 cells. (Upper) Structural domains of recombinant FLAG-ZAS3 protein. NLS, nuclear localization signal. (Lower) Immunoblot analyses of HEK 293 cells transiently transfection with FLAG-ZAS3 expression plasmid or parental plasmid, pCMV-Tag2 (FLAG). N, nuclear extracts; C, cytoplasmic extracts. (B) Reporter gene assays. Plasmids 3×κB-Luc (10 ng) and pCH110 (10 ng) were cotransfected with the control parental vector (pCMV-Tag2; white bar) or indicated amount of FLAG-ZAS3 expression plasmid (black bars) in HEK 293 cells. The normalized luciferase activity of cells cotransfected with the control vector was assigned to 100%. (C) ZAS3 repressed NF-κB-activated transcription. Transient transfection experiments were performed with (+) or without (–) p65 and p50 expression plasmids (10 ng each) in HEK 293 cells. The normalized luciferase activity of cells transfected with p65 and p50 expression plasmids was assigned as 100. (D) ZAS3 repressed a κB-reporter gene in ZAS3^–/– cells. Normalized luciferase activity of transfection in 38B9 cells with the reporter gene only was assigned as 1.

Fig. 4.

ZAS3 inhibits nuclear localization of p65. Immunoblot analysis of 38B9 and ZAS3^–/– cells after transient transfection with FLAG-ZAS3 (+) or parental FLAG (–) expression plasmids showed that p65 was localized mainly in the cytoplasmic extracts (CE) of 38B9, but in the nuclear extracts (NE) of ZAS3^–/– cells. ZAS3 expression in both cells reduced the amount of nuclear p65 with a concomitant increase of cytoplasmic p65. The second p65 was an overexposure of the first to highlight the reduction of nuclear p65 by ZAS3 expression in 38B9 cells. As controls for protein loading and cell fractionation, immunoblot analyses were also performed with a nuclear protein control, histone H1, and a cytosolic protein control, heat-shock protein 90 (hsp90).

Fig. 5.

ZAS3 also represses transcription independent of p65. (A) Reporter gene assays. Transient transfection was performed in p65^–/– MEFs (black bars) or p65^+/+ control MEFs (white bars). Normalized activity of the κB-reporter in p65^+/+ MEF transfected with the empty vector is taken as 100. A representative experiment of three is shown here. (B) ZASC inhibits transcription activation of VP16. Cos-7 cells were transiently transfected with pCMV110 (5 μg), and pSG-VP16, pSG-ZASC, or pSG424 expression plasmids, as indicated. The relative chloramphenicol acetyl transferase activity of duplicates is illustrated for each experiment. The amounts of plasmid DNA used are: +,10 μg; ++,15 μg; +++, 20 μg; and –, 0 μg. Activity from cells transfected with pCMV110 and PSG-VP16 was assigned as 100.

Fig. 6.

A model of ZAS3 in inhibiting NF-κB. In the nucleus, ZAS3 interferes with NF-κB-mediated transcription by competing for κB gene regulatory elements and by repressing transcription. In the cytoplasm, ZAS3, most likely a protein isoform, inhibits the nuclear translocation of p65 by association with TRAF2, which blocks the formation of the IKK complex. TRADD, TNF receptor (TNFR)-associated death domain; RIP, receptor-interacting protein; TRAF2, TNFR-associated factor 2; FADD, Fas-associated death domain; IKK, IκB kinase; and NEMO, NF-κB essential modulator.