title: Nuclear import of insulin-like growth factor-binding protein-3 and -5 is mediated by the importin beta subunit.
abstract: Although insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-5 are known to modulate cell growth by reversibly sequestering extracellular insulin-like growth factors, several reports have suggested that IGFBP-3, and possibly also IGFBP-5, have important insulin-like growth factor-independent effects on cell growth. These effects may be related to the putative nuclear actions of IGFBP-3 and IGFBP-5, which we have recently shown are transported to the nuclei of T47D breast cancer cells. We now describe the mechanism for nuclear import of IGFBP-3 and IGFBP-5. In digitonin-permeabilized cells, where the nuclear envelope remained intact, nuclear translocation of wild-type IGFBP-3 appears to occur by a nuclear localization sequence (NLS)-dependent pathway mediated principally by the importin beta nuclear transport factor and requiring both ATP and GTP hydrolysis. Under identical conditions, an NLS mutant form of IGFBP-3, IGFBP-3[(228)KGRKR --> MDGEA], was unable to translocate to the nucleus. In cells where both the plasma membrane and nuclear envelope were permeabilized, wild-type IGFBP-3, but not the mutant form, accumulated in the nucleus, implying that the NLS was also involved in mediating binding to nuclear components. By fusing wild-type and mutant forms of NLS sequences (IGFBP-3 [215-232] and IGFBP-5 [201-218]) to the green fluorescent protein, we identified the critical residues of the NLS necessary and sufficient for nuclear accumulation. Using a Western ligand binding assay, wild-type IGFBP-3 and IGFBP-5, but not an NLS mutant form of IGFBP-3, were shown to be recognized by importin beta and the alpha/beta heterodimer but only poorly by importin alpha. Together these results suggest that the NLSs within the C-terminal domain of IGFBP-3 and IGFBP-5 are required for importin-beta-dependent nuclear uptake and probably also accumulation through mediating binding to nuclear components.
authors.affiliation: Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, Sydney, New South Wales 2065, Australia. lyns@med.usyd.edu.au
authors.forename: L J
authors.initials: LJ
authors.lastname: Schedlich
authors.forename: S L
authors.initials: SL
authors.lastname: Le Page
authors.forename: S M
authors.initials: SM
authors.lastname: Firth
authors.forename: L J
authors.initials: LJ
authors.lastname: Briggs
authors.forename: D A
authors.initials: DA
authors.lastname: Jans
authors.forename: R C
authors.initials: RC
authors.lastname: Baxter
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D017931:DNA Primers; D019281:Dimerization; D004734:Energy Metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics / Q000378:metabolism*; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics / Q000378:metabolism*; D028884:Karyopherins; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D017931:DNA Primers; D018972:Insulin-Like Growth Factor Binding Protein 3; D018975:Insulin-Like Growth Factor Binding Protein 5; D028884:Karyopherins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate