title: Identification of nuclear import and export signals within Fli-1: roles of the nuclear import signals in Fli-1-dependent activation of megakaryocyte-specific promoters.
abstract: The Ets factor Friend leukemia integration 1 (Fli-1) is an important regulator of megakaryocytic (Mk) differentiation. Here, we demonstrate two novel nuclear localization signals (NLSs) within Fli-1: one (NLS1) is located at the N terminus, and another (NLS2) is within the Ets domain. Nuclear accumulation of Fli-1 reflected the combined functional effects of the two discrete NLSs. Each NLS can independently direct nuclear transport of a carrier protein, with mutations within the NLSs affecting nuclear accumulation. NLS1 has a bipartite motif, whereas the NLS2 region contains a nonclassical NLS. Both NLSs bind importin alpha (IMPalpha) and IMPbeta, with NLS1 and NLS2 being predominantly recognized by IMPalpha and IMPbeta, respectively. Fli-1 also contains one nuclear export signal. Leptomycin B abolished its cytoplasmic accumulation, showing CRM1 dependency. We demonstrate that Ets domain binding to specific target DNA effectively blocks IMP binding, indicating that the targeted DNA binding plays a role in localizing Fli-1 to its destination and releasing IMPs for recycling back to the cytoplasm. Finally, by analyzing full-length Fli-1 carrying NLS1, NLS2, and combined NLS1-NLS2 mutations, we conclude that two functional NLSs exist in Fli-1 and that each NLS is sufficient to target Fli-1 to the nucleus for activation of Mk-specific genes.
authors.affiliation: Centre for Thrombosis and Vascular Research, Department of Medicine, SXt. George Clinical School, University of New South Wales, Sydney, New South Wales 2217, Australia.
authors.forename: Wei
authors.initials: W
authors.lastname: Hu
authors.forename: Alana S
authors.initials: AS
authors.lastname: Philips
authors.forename: Juliana C
authors.initials: JC
authors.lastname: Kwok
authors.forename: Michael
authors.initials: M
authors.lastname: Eisbacher
authors.forename: Beng H
authors.initials: BH
authors.lastname: Chong
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics / Q000502:physiology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008533:Megakaryocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D017434:Protein Structure, Tertiary; D051789:Proto-Oncogene Protein c-fli-1; D051381:Rats; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D015533:Transcriptional Activation; D028901:alpha Karyopherins / Q000378:metabolism; D028961:beta Karyopherins / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C078433:FLI1 protein, human; D019913:Nuclear Localization Signals; D051789:Proto-Oncogene Protein c-fli-1; D015534:Trans-Activators; D028901:alpha Karyopherins; D028961:beta Karyopherins