Spatial redox regulation of a critical cysteine residue of NF-kappa B in vivo.
Reduction-oxidation (redox) regulation has been implicated in the activation of the transcription factor NF-kappaB. However, the significance and mechanism of the redox regulation remain elusive, mainly due to the technical limitations caused by rapid proton transfer in redox reactions and by the presence of many redox molecules within cells. Here we establish versatile methods for measuring redox states of proteins and their individual cysteine residues in vitro and in vivo, involving thiol-modifying reagents and LC-MS analysis. Using these methods, we demonstrate that the redox state of NF-kappaB is spatially regulated by its subcellular localization. While the p65 subunit and most cysteine residues of the p50 subunit are reduced similarly in the cytoplasm and in the nucleus, Cys-62 of p50 is highly oxidized in the cytoplasm and strongly reduced in the nucleus. The reduced form of Cys-62 is essential for the DNA binding activity of NF-kappaB. Several lines of evidence suggest that the redox factor Ref-1 is involved in Cys-62 reduction in the nucleus. We propose that the Ref-1-dependent reduction of p50 in the nucleus is a necessary step for NF-kappaB activation. This study also provides the first example of a drug that inhibits the redox reaction between two specific proteins.
Nishi
Takeyuki
T
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Shimizu
Noriaki
N
Hiramoto
Masaki
M
Sato
Iwao
I
Yamaguchi
Yuki
Y
Hasegawa
Makoto
M
Aizawa
Shin
S
Tanaka
Hirotoshi
H
Kataoka
Kohsuke
K
Watanabe
Hajime
H
Handa
Hiroshi
H
D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D005786:Gene Expression Regulation; D006801:Humans; D019169:Jurkat Cells; D008301:Maleimides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013438:Sulfhydryl Compounds; D013997:Time Factors; D051996:Transcription Factor RelA; D014158:Transcription, Genetic
D008301:Maleimides; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D051996:Transcription Factor RelA; C043592:maleimide; D004247:DNA; D003545:Cysteine