Redox Regulation of the DNA Binding Activity in Transcription Factor PEBP2
THE ROLES OF TWO CONSERVED CYSTEINE RESIDUES*
- Yoshiko Akamatsu‡§,
- Tetsuya Ohno§¶,
- Kiichi Hirota¶,
- Hiroshi Kagoshima‖,
- Junji Yodoi¶ and
- Katsuya Shigesada**
- From the Laboratory of Biochemistry, Department of Genetics and Molecular Biology and ¶Laboratory of Infection and Prevention, Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto 606-01,-Japan
Abstract
Transcription factor PEBP2/CBF consists of a DNA binding subunit, α, and a regulatory subunit, β. The α subunit has an evolutionarily conserved 128-amino acid region termed “Runt domain” that is responsible for both DNA binding and heterodimerization with the β subunit. The Runt domain in all mammalian submembers of the α 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 PEBP2αA 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 β 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 β subunit as key modulators.
Footnotes
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↵* This work was supported in part by Research Grant RG-357/94 (to K. S.) from the Human Frontier Science Program Organization (Strasbourg) and by Research Project 08NP0601 (to J. Y.) under the “New Program System” of the Ministry of Education, Science, Culture and Sports of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Recipient of a research fellowship from the Japan Society for the Promotion of Science for Young Scientists. Present address: Dept. of Molecular Biology, Massachusetts General Hospital, 50 Blossom St., Boston, MA 02114.
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↵§ The first two authors contributed to this work equally.
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↵‖ Present address: Biozentrum, der Universität Basel, Abteilung Zellbiologie, Klingelbergstr. 70, CH-4056 Basel, Switzerland.
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↵** To whom correspondence should be addressed. Tel.: 81-75-751-4019; Fax: 81-75-751-3992; E-mail: kshigesa{at}virus.kyoto-u.ac.jp.
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↵1 The abbreviations used are: PEBP, polyoma virus enhancer-binding protein; Trx, thioredoxin; TrxR, thioredoxin reductase; EMSA, electrophoretic mobility shift assay; DTT, dithiothreitol.
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↵2 H. Kagoshima and T. Bürglin, personal communication.
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- Received March 19, 1997.
- Revision received March 31, 1997.
