title: Identification of functional nuclear export sequences in human topoisomerase IIalpha and beta.
abstract: Nuclear localization of topoisomerase IIalpha and beta is important for normal cell function as well as being a determinant of tumour cell sensitivity to topoisomerase II-targeting chemotherapeutic agents. However, topoisomerase II is cytoplasmic under certain circumstances, indicating that it may undergo active nuclear export. We have examined the ability of Leu-rich potential nuclear export signal (NES) sequences present in human topoisomerase IIalpha and beta to direct the export of a green fluorescent protein-glutathione-S-transferase fusion protein following microinjection into HeLa cell nuclei. Of 12 sequences tested, only one potential NES sequence from the comparable location in each isoform (alphaNES(1018-1028) and betaNES(1034-1044)) was active. Mutation of hydrophobic residues in alphaNES(1018-1028) and betaNES(1034-1044) substantially reduced their nuclear export activity as did leptomycin B treatment of microinjected cells. Our results provide the first evidence of active nuclear export of topoisomerase II and suggest it is mediated by a CRM1-dependent pathway.
authors.affiliation: Cancer Research Laboratories, Queen's University, Ont, Kingston, Canada K7L 3N6. selm@post.queensu.ca
authors.forename: Shelagh E L
authors.initials: SE
authors.lastname: Mirski
authors.forename: Juliane C
authors.initials: JC
authors.lastname: Bielawski
authors.forename: Susan P C
authors.initials: SP
authors.lastname: Cole
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000951:Antigens, Neoplasm; D002467:Cell Nucleus / Q000378:metabolism*; D002645:Chickens; D004252:DNA Mutational Analysis; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D000097606:Exportin 1 Protein
chemical_list: D000903:Antibiotics, Antineoplastic; D000951:Antigens, Neoplasm; D004250:DNA Topoisomerases, Type II; D004268:DNA-Binding Proteins; D005231:Fatty Acids, Unsaturated; D005982:Glutathione Transferase; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D008164:Luminescent Proteins; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D000097606:Exportin 1 Protein; C038753:leptomycin B