Detection of a nuclear protein which binds specifically to the antioxidant responsive element (ARE) of the human NAD(P) H:quinone oxidoreductase gene.
We have detected a protein or complex of proteins with a native molecular mass of 160 kDa from the nuclear extract of HeLa cells, which binds specifically to the human antioxidant responsive element (ARE) in the 5'-flanking region of the NAD(P)H:quinone oxidoreductase gene. Binding of the 160 kDa protein to oligonucleotides containing the ARE in gel mobility shift assays is diminished or abolished by increasing concentrations of the reducing agent dithiothreitol, but not by anti-Jun or anti-Fos antibodies. The effect of dithiothreitol is opposite to that observed for the Ref-1-mediated binding of Fos/Jun to the ARE or to the related 12-O-tetradecanoyl phorbol-13-acetate responsive element (TRE). Competition assays indicated that the binding of the 160 kDa protein requires the ARE sequence, TGACNNNGCA, with T as the most important base, and that the TRE sequence (TGACTCA) is not sufficient. F9 cells, which contain no AP-1 protein, were able to form a complex with the same mobility as the 160 kDa protein in gel mobility shift assays. We conclude that a 160 kDa protein or complex of proteins binds specifically to the human ARE sequence but not to the TRE. The 160 kDa protein does not contain Fos or Jun proteins, and its binding is abolished by the reducing agent, dithiothreitol.
Wang
B
B
Food Molecular Biochemistry Department, Norwich Laboratory, Colney, UK.
Williamson
G
G
D000975:Antioxidants / Q000378:metabolism*; D001483:Base Sequence; D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D005982:Glutathione Transferase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000032:analysis; D016755:Proto-Oncogene Proteins c-jun / Q000032:analysis; D011808:Quinone Reductases / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D000975:Antioxidants; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases; D005982:Glutathione Transferase