title: Inhibition of DNA binding activity of cAMP response element-binding protein by 1,2-naphthoquinone through chemical modification of Cys-286.
abstract: 1,2-Naphthoquinone (1,2-NQ) is an atmospheric electrophile that reacts covalently with protein thiols. Our previous study revealed that exposure of bovine aortic endothelial cells to 1,2-NQ causes covalent modification of cAMP response element-binding protein (CREB), thereby inhibiting its DNA binding activity and substantial gene expression of B-cell lymphoma-2 (Bcl-2) that is regulated by this transcription factor. In this study, we identified the modification sites of CREB that are associated with the decreased transcriptional activity. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis indicated that three amino acids (Cys-286, Lys-290, and Lys-319) were irreversibly modified by 1,2-NQ. Mutational analysis revealed that electrophilic modification of Cys-286, but not the other two amino acids, at the DNA binding domain is essential for the reduced CREB activity. Substitution of Cys-286 with tryptophan (C286W), which mimics CREB modification by 1,2-NQ, supported this notion. These results suggest that the covalent interaction of CREB with 1,2-NQ through Cys-286 blocks the DNA binding activity of CREB, resulting in the repression of CREB-regulated genes.
authors.affiliation: Doctoral Programs in Medical Sciences, Graduate School of Comprehensive Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Endo
authors.forename: Daigo
authors.initials: D
authors.lastname: Sumi
authors.forename: Noriko
authors.initials: N
authors.lastname: Iwamoto
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Kumagai
mesh_terms: D045744:Cell Line, Tumor; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004785:Environmental Pollutants / Q000633:toxicity*; D015870:Gene Expression / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D009153:Mutagens / Q000378:metabolism; D009285:Naphthoquinones / Q000633:toxicity*
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D004785:Environmental Pollutants; D009153:Mutagens; D009285:Naphthoquinones; C040756:1,2-naphthoquinone; D003545:Cysteine