Coactivation of the N-terminal transactivation of mineralocorticoid receptor by Ubc9

Kenichi Yokota, Hirotaka Shibata, Isao Kurihara, Sakiko Kobayashi, Noriko Suda, Ayano Takeda, Ikuo Saito, Hirochika Kitagawa, Shigeaki Kato, Takao Saruta, Hiroshi Itoh

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Abstract

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 sumoylationinactive 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.

Original languageEnglish
Pages (from-to)1998-2010
Number of pages13
JournalJournal of Biological Chemistry
Volume282
Issue number3
DOIs
Publication statusPublished - 2007 Jan 19

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Mineralocorticoid Receptors
Transcriptional Activation
Nuclear Receptor Coactivator 1
Assays
Sumoylation
Ubiquitin
Aldosterone
Transfection
Genes
Chromatin Immunoprecipitation
COS Cells
Enzymes
Gene expression

ASJC Scopus subject areas

  • Biochemistry

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Coactivation of the N-terminal transactivation of mineralocorticoid receptor by Ubc9. / Yokota, Kenichi; Shibata, Hirotaka; Kurihara, Isao; Kobayashi, Sakiko; Suda, Noriko; Takeda, Ayano; Saito, Ikuo; Kitagawa, Hirochika; Kato, Shigeaki; Saruta, Takao; Itoh, Hiroshi.

In: Journal of Biological Chemistry, Vol. 282, No. 3, 19.01.2007, p. 1998-2010.

Research output: Contribution to journalArticle

Yokota, Kenichi ; Shibata, Hirotaka ; Kurihara, Isao ; Kobayashi, Sakiko ; Suda, Noriko ; Takeda, Ayano ; Saito, Ikuo ; Kitagawa, Hirochika ; Kato, Shigeaki ; Saruta, Takao ; Itoh, Hiroshi. / Coactivation of the N-terminal transactivation of mineralocorticoid receptor by Ubc9. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 3. pp. 1998-2010.
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abstract = "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 sumoylationinactive 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.",
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AU - Suda, Noriko

AU - Takeda, Ayano

AU - Saito, Ikuo

AU - Kitagawa, Hirochika

AU - Kato, Shigeaki

AU - Saruta, Takao

AU - Itoh, Hiroshi

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