Redox regulation of the DNA binding activity in transcription factor PEBP2. The roles of two conserved cysteine residues.
Transcription factor PEBP2/CBF consists of a DNA binding subunit, alpha, and a regulatory subunit, beta. The alpha subunit has an evolutionarily conserved 128-amino acid region termed "Runt domain" that is responsible for both DNA binding and heterodimerization with the beta subunit. The Runt domain in all mammalian submembers of the alpha subunit contains two conserved cysteine residues, and its DNA binding activity undergoes redox regulation. To investigate the mechanism of this redox regulation, we performed site-directed mutagenesis of the two conserved cysteines in the Runt domain of the mouse PEBP2alphaA homolog. Substitution of Cys-115 to serine resulted in a partially impaired DNA binding, which remained highly sensitive to a thiol-oxidizing reagent, diamide. Conversely, the corresponding substitution of Cys-124 caused an increased DNA binding concomitant with an increased resistance to diamide. In contrast, substitution of either cysteine to aspartate was destructive to DNA binding to marked extents. These results have revealed that both Cys-115 and Cys-124 are responsible for the redox regulation in their own ways with low and high oxidizabilities, respectively. We have also found that two cellular thiol-reactive proteins, thioredoxin and Ref-1, work effectively and synergistically for activation of the Runt domain. Interestingly, the beta subunit further enhanced the activation by these proteins and reciprocally prevented the oxidative inactivation by diamide. These findings collectively suggest the possibility that the Runt domain's function in vivo could be dynamically regulated by the redox mechanism with Trx, Ref-1, and the beta subunit as key modulators.
Akamatsu
Y
Y
Laboratory of Biochemistry, Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Kyoto 606-01,-Japan.
Ohno
T
T
Hirota
K
K
Kagoshima
H
H
Yodoi
J
J
Shigesada
K
K
D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D003545:Cysteine; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D003958:Diamide / Q000494:pharmacology; D019281:Dimerization; D019143:Evolution, Molecular; D007700:Kinetics; D046911:Macromolecular Substances; D008322:Mammals; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016324:Sequence Tagged Sites; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D003958:Diamide; D004247:DNA; D003545:Cysteine