title: Coactivation of the N-terminal transactivation of mineralocorticoid receptor by Ubc9.
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 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.
authors.affiliation: Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Yokota
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Shibata
authors.forename: Isao
authors.initials: I
authors.lastname: Kurihara
authors.forename: Sakiko
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Noriko
authors.initials: N
authors.lastname: Suda
authors.forename: Ayano
authors.initials: A
authors.lastname: Murai-Takeda
authors.forename: Ikuo
authors.initials: I
authors.lastname: Saito
authors.forename: Hirochika
authors.initials: H
authors.lastname: Kitagawa
authors.forename: Shigeaki
authors.initials: S
authors.lastname: Kato
authors.forename: Takao
authors.initials: T
authors.lastname: Saruta
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Itoh
mesh_terms: D000450:Aldosterone / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018161:Receptors, Mineralocorticoid / Q000378:metabolism*; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000502:physiology*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D018161:Receptors, Mineralocorticoid; D000450:Aldosterone; D044763:Ubiquitin-Conjugating Enzymes; D005982:Glutathione Transferase; D000098843:Ubiquitin-Conjugating Enzyme UBC9