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J. Biol. Chem., Vol. 280, Issue 27, 25901-25906, July 8, 2005

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Glutathionylation of Two Cysteine Residues in Paired Domain Regulates DNA Binding Activity of Pax-8^*

Xia Cao, Fukushi Kambe, Xiuli Lu, Natsuko Kobayashi, Sachiko Ohmori, and Hisao Seo

From the Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan

We reported that the first two cysteine residues out of three present in paired domain (PD), a DNA-binding domain, are responsible for redox regulation of Pax-8 DNA binding activity. We show that glutathionylation of these cysteines has a regulatory role in PD binding. Wild-type PD and its mutants with substitution of cysteine to serine were synthesized and named CCC, CSS, SCS, SSC, and SSS according to the positions of substituted cysteines. They were incubated in a buffer containing various ratios of GSH/GSSG and subjected to gel shift assay. Binding of CCC, CSS, and SCS was impaired with decreasing GSH/GSSG ratio, whereas that of SSC and SSS was not affected. Because [³H]glutathione was incorporated into CCC, CSS, and SCS, but not into SSC and SSS, the binding impairment was ascribed to glutathionylation of the redox-reactive cysteines. This oxidative inactivation of PD binding was reversed by a reductant dithiothreitol and by redox factor (Ref)-1 in vitro. To explore the glutathionylation in cells, Chinese hamster ovary cells overexpressing CSS and SCS were labeled with [³⁵S]cysteine in the presence of cycloheximide. Immunoprecipitation with an antibody against PD revealed that treatment of the cells with an oxidant diamide induced the ³⁵S incorporation into both mutants, suggesting the PD glutathionylation in cells. Since the two cysteine residues in PD are conserved in all Pax members, this novel posttranslational modification of PD would provide a new insight into molecular basis for modulation of Pax function.

Received for publication, October 7, 2004 , and in revised form, March 30, 2005.

^* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-52-789-3866; Fax: 81-52-789-3891; E-mail: kambe{at}riem.nagoya-u.ac.jp.

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