Modulation of transcription factor NF-cB binding activity by oxidation-reduction in vitro

NF-icB is a widely used regulator of inducible and tissue-specific gene control. In the cytosol, when complexed to an inhibitory molecule, IKB, NF-#cB is in an inactive form and cannot bind DNA. Activation of cells with appropriate stimuli results in the dissociation of NF-mcB from IjcB and its translocation to the nucleus as an active binding protein. We now demonstrate that NF-ucB binding in vitro can be inhibited by agents that modify free sulfhydryls. Binding is eliminated after treatment with N-ethylmaleimide, an alkylating agent, and diamide, an oxidizing agent. The diamide effect can be reversed by 2-mercaptoethanol. Further, 2-mercaptoethanol acts synergistically with deoxycholate plus Nonidet P-40 in converting inactive cytosolic NF-KB to an active DNA-binding form. It is therefore possible that modulation of the redox state of NF-KB could represent a post-translational control mechanism for this factor. Regulatory proteins that influence gene expression can be controlled both transcriptionally and post-transcriptionally. For example, the T-cell-specific DNA-binding protein NFAT-1, which is critical for interleukin 2 gene expression, is regulated principally at the level of transcription (1), whereas the binding of CREB (2) is influenced by its phosphorylation state, and in vitro the iron response element RNA-binding protein (IRE-BP) is influenced by the redox state of the protein (3). NF-KB is an inducible DNA-binding protein detected in multiple cell types and has been implicated in the transcriptional induction of many genes (4). NF-KB is at least in part controlled by its cellular localization (5, 6). It consists of a DNA-binding subunit of 50 kDa (7, 8) that associates with a second protein of 65 kDa (9). In the cytosol of uninduced non-B cells, NF-KB exists in an inactive form complexed to an inhibitory molecule, IKB (5, 6). Activation of cells by appropriate stimuli results in the dissociation ofNF-KB from IKB and its translocation to the nucleus as a complex containing both p50 and p65 (5, 6). NF-KB therefore represents a paradigm for the study of cytoplasmic-to-nuclear signaling and induced gene expression (4). We now demonstrate that NF-KB binding in vitro can be modulated by variations in the redox state of the protein. We discuss the potential implications of this phenomenon. MATERIALS AND METHODS Oligodeoxynucleotides. Complementary oligonucleotides were prepared on an Applied Biosystems DNA synthesizer (model 381A), annealed, purified on native polyacrylamide gels, and used as unlabeled competitors or as probes after end-labeling with [a-32P]dCTP and the Klenow fragment'of DNA polymerase I. Radiolabeled probes were separated from free nucleotides by using push columns (Stratagene). Cells and Preparation of Nuclear and Cytoplasmic Extracts. Jurkat (acute lymphocytic T-cell leukemia), MT-2 (human T-cell lymphotropic virus I-infected T cell line), and HeLa (cervical carcinoma-derived cell line) were grown in RPMI 1640 medium supplemented with 10%6 (vol/vol) fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml), and streptomycin (100 rAg/ml). Where indicated, cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) 10-14 hr before harvesting. Nuclear extracts were prepared per Dignam et al. (10) with the omission of dithiothreitol (DTT) in each buffer. The post-nuclear supernatant was centrifuged at 50,000 X g and the supernatant was concentrated using a Centricon-10, dialyzed against buffer D (10) not containing DTT [buffer D(-)], and saved as cytosolic extracts. Protein concentrations were determined by the bicinchoninic acid (BCA) method. Diazinedicarboxylic acid bis(N,N-dimethylamide) (diamide, Sigma) was constituted as a 1 M solution in water. N-Ethylmaleimide (NEM, Calbiochem-Behring) was constituted as a 250mM solution in water. Dialysis of nuclear extracts, after treatment with diamide or NEM, was performed using 1500 vol of buffer D(-) for . 4 hr. In Vitro Transcription and Translation. pBluescript vector containing a truncated (Rsa I digestion at nucleotide 1372, amino acids 1-399) human NF-KB/KBF-1 (p50) cDNA insert (11) was linearized with Xba I and transcribed into mRNA by using T7 RNA polymerase with the cap analog to produce capped transcripts according to the manufacturer's recommendations (Pharmacia). After purification on a push column, 1 ,ug of mRNA was used in rabbit reticulocyte lysate and wheat germ extract cell-free translation systems, according to the manufacturer's directions (Promega). Electrophoretic Mobility-Shift Assays (EMSAs). DNAbinding reaction mixtures (20 ,ul) contained 10 mM Tris'HCl (pH 7.6), 50mM NaCl, 1 mM EDTA, 1 ,ug ofpoly(dI-dC), and 1.5-2.5 x 104 cpm of the probe. Depending on the experiment, 5 jug of nuclear extracts, 10 pg of cytosolic extracts, 2 ,ul of rabbit reticulocyte lysates, or 4 ILI of wheat germ extracts were added prior to the probe. Diamide, NEM, 2-mercaptoethanol (2-ME), sodium deoxycholate (DOC), and Nonidet P-40 (NP40) were added to the binding reaction mixtures at 20°C, for approximately 10 min before addition of the probe. Binding reactions and subsequent analyses on native 4% polyacrylamide gels were performed as described (12). Autoradiograms were analyzed using an UltraScan XL densitometer (LKB).