title: Intact structure of EGAM1 homeoproteins and basic amino acid residues in the common homeodomain of EGAM1 and EGAM1C contribute to their nuclear localization in mouse embryonic stem cells.
abstract: Recently, we identified the structurally related homeoproteins EGAM1, EGAM1N, and EGAM1C in both preimplantation mouse embryos and mouse embryonic stem (ES) cells. These EGAM1 homeoproteins act as positive or negative regulators of differentiation and cell growth in mouse ES cells, such that these proteins are considered transcriptional regulators. In this study, we investigated their nuclear localization and identified the amino acid residues crucial for the nuclear translocation of EGAM1 and EGAM1C. When expressed exogenously in pluripotent ES cells and somatic NIH3T3 cells, all EGAM1 homeoproteins localized to the nucleus. Analysis using the web-based tool PSORTII predicted a potential nuclear localization signal (NLS) motif, RKDLIRSWFITQRHR, in the homeodomain shared by EGAM1 and EGAM1C. The introduction of mutations, such as mutations from K or R, both basic amino acid residues, to A, in this potential NLS resulted in significant impairment of the nuclear localization of both EGAM1 and EGAM1C. In contrast, GFP fusion proteins of all the full-length EGAM1 homeoproteins failed to localize to the nucleus. These results, when taken together, suggest that basic amino acid residues in the common homeodomain of EGAM1 and EGAM1C and the intact structures of the EGAM1 homeoproteins contribute, at least in part, to the nuclear localization of these proteins in mouse ES cells.
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan.
authors.forename: Sho
authors.initials: S
authors.lastname: Sato
authors.forename: Sanae
authors.initials: S
authors.lastname: Morita
authors.forename: Momoe
authors.initials: M
authors.lastname: Iha
authors.forename: Yuki
authors.initials: Y
authors.lastname: Mori
authors.forename: Saiko
authors.initials: S
authors.lastname: Sugawara
authors.forename: Kano
authors.initials: K
authors.lastname: Kasuga
authors.forename: Ikuo
authors.initials: I
authors.lastname: Kojima
authors.forename: Noriaki
authors.initials: N
authors.lastname: Ozaki
authors.forename: Hajime
authors.initials: H
authors.lastname: Muraguchi
authors.forename: Keiju
authors.initials: K
authors.lastname: Okano
authors.forename: Jun
authors.initials: J
authors.lastname: Iwashita
authors.forename: Jun
authors.initials: J
authors.lastname: Murata
authors.forename: Masahiro
authors.initials: M
authors.lastname: Hosaka
authors.forename: Masayuki
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D000595:Amino Acid Sequence; D024361:Amino Acids, Basic / Q000737:chemistry*; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D019913:Nuclear Localization Signals; D017434:Protein Structure, Tertiary
chemical_list: D024361:Amino Acids, Basic; C558397:EGAM1C protein, mouse; D018398:Homeodomain Proteins; D019913:Nuclear Localization Signals; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins