title: Transcription factor single-minded 2 (SIM2) is ubiquitinated by the RING-IBR-RING-type E3 ubiquitin ligases.
abstract: Human single-minded 2 (SIM2) is a member of the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors and is associated with the etiology of Down syndrome phenotype. Here, we examined a possibility of the post-translational modification of SIM2 protein by transfecting various expression constructs followed by the analysis with immunoprecipitation and Western blotting. In fact, transient expression of SIM2 cDNA in HEK293 cells revealed poly-ubiquitination of SIM2 protein. In the stable transfectants, a proteasome inhibitor MG132 protected the poly-ubiquitinated SIM2 protein from degradation. Furthermore, in the cells co-transfected with SIM2 and each of four different E3 ubiquitin ligases, SIM2 was immunoprecipitated with the RING-IBR-RING-type E3 ubiquitin ligases, Parkin and HHARI, but it was not immunoprecipitated with other E3 ligases, such as one RING-type Siah-1 and the PHD type AIRE. A series of deletion constructs revealed that Parkin actually binds to SIM2 with the IBR (294-377)-RING2 (378-465) domains and that the sites for poly-ubiquitination of SIM2 reside within the PAS1-PAS2 region (aa 141-289). We postulated that transcription factor SIM2 and E3 ubiquitin ligase Parkin may interact each other to play an important physiological role in the brain development which is controlled by ubiquitination.
authors.affiliation: Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
authors.forename: Michiyo
authors.initials: M
authors.lastname: Okui
authors.forename: Akiko
authors.initials: A
authors.lastname: Yamaki
authors.forename: Atsushi
authors.initials: A
authors.lastname: Takayanagi
authors.forename: Jun
authors.initials: J
authors.lastname: Kudoh
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Shimizu
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Shimizu
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D019556:COS Cells; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D006801:Humans; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D014452:Ubiquitins
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins; C417032:SIM2 protein, human