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J. Biol. Chem., Vol. 282, Issue 3, 1998-2010, January 19, 2007

This Article Full Text Full Text (PDF) All Versions of this Article: 282/3/1998    most recent M607741200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yokota, K. Articles by Itoh, H. Search for Related Content PubMed PubMed Citation Articles by Yokota, K. Articles by Itoh, H. Social Bookmarking                      What's this?

Coactivation of the N-terminal Transactivation of Mineralocorticoid Receptor by Ubc9^*

Kenichi Yokota, Hirotaka Shibata¹, Isao Kurihara, Sakiko Kobayashi, Noriko Suda, Ayano Murai-Takeda, Ikuo Saito, Hirochika Kitagawa^¶, Shigeaki Kato^¶, Takao Saruta, and Hiroshi Itoh

From the Department of Internal Medicine, School of Medicine, and the Health Center, Keio University, Tokyo 160-8582 and the ^¶Institute of Molecular and Cellular Bioscience, University of Tokyo, Tokyo 113-0032, Japan

Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood. Various transcription factors are post-translationally modified by small ubiquitin-related modifier-1 (SUMO-1). We investigated the role of the SUMO-1-conjugating enzyme Ubc9 in MR transactivation. Yeast two-hybrid, GST-pulldown, and coimmunoprecipitation assays showed that Ubc9 interacted with N-terminal MR-(1-670). Endogenous Ubc9 is associated with stably expressing MR in 293-MR cells. Transient transfection assays in COS-1 cells showed that Ubc9 increased MR transactivation of reporter constructs containing MRE, ENaC, or MMTV promoter in a hormone-sensitive manner. Moreover, reduction of Ubc9 protein levels by small interfering RNA attenuated hormonal activation of a reporter construct as well as an endogenous target gene by MR. A sumoylation-inactive mutant Ubc9(C93S) similarly interacted with MR and potentiated aldosterone-dependent MR transactivation. An MR mutant in which four lysine residues within sumoylation motifs were mutated into arginine (K89R/K399R/K494R/K953R) failed to be sumoylated, but Ubc9 similarly enhanced transactivation by the mutant MR, indicating that sumoylation activity is dispensable for coactivation capacity of Ubc9. Coexpression of Ubc9 and steroid receptor coactivator-1 (SRC-1) synergistically enhanced MR-mediated transactivation in transient transfection assays. Indeed, chromatin immunoprecipitation assays demonstrated that endogenous MR, Ubc9, and SRC-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner. Coimmunoprecipitation assays showed a complex of MR, Ubc9, and SRC-1 in mammalian cells, and the endogenous proteins were colocalized in the nuclei of the mouse collecting duct cells. These findings support a physiological role of Ubc9 as a transcriptional MR coactivator, beyond the known SUMO E2-conjugating enzyme.

Received for publication, August 14, 2006 , and in revised form, October 23, 2006.

^* This work was supported by a Grant-in-aid for Scientific Research (C) 17590966, 2005-2006 (to H. S.), from the Japan Society for the Promotion of Science, a grant from Yamaguchi Endocrine Research Association (to H. S.), a grant from the Smoking Research Foundation (to H. I.), an independent research grant from Pfizer Pharmaceuticals Inc. (to H. S.), and a Health Labor Science Research Grant for Disorders of Adrenocortical Hormone Production from the Ministry of Health, Labor, and Welfare, Japan (to H. S.). 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.

¹ To whom correspondence should be addressed. Tel.: 81-3-3353-1211 (ext. 62312); Fax: 81-3-5363-3635; E-mail: hiro-405{at}cb3.so-net.ne.jp.

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