Identification and characterization of two putative nuclear localization signals (NLS) in the DNA-binding protein NUCKS.
Immunofluorescence analyses show that the vertebrate specific and DNA-binding protein NUCKS is distributed throughout the cytoplasm in mitotic cells and targeted to the reforming nuclei in late telophase of the cell cycle. Computer analysis of the primary structure of NUCKS revealed the presence of two regions of highly charged, basic residues, which were identified as potential nuclear localization signals (NLSs). One of these signals (NLS1) is highly conserved between the species investigated, and fits to the description of being a classical bipartite NLS. The other amino acid motif (NLS2) is less conserved and does not constitute a classical bipartite NLS consensus sequence. We have shown that each of the two putative NLSs is capable of translocating green fluorescent protein (GFP) into the nucleus. The highly conserved NLS1 is monopartite, resembling the signals of c-Myc and RanBP3. Surprisingly, a natural occurring splice variant of NUCKS lacking 40 amino acids including NLS1, is not capable of translocating a corresponding NUCKS-GFP fusion protein into the nucleus, indicating that NLS1 is the main nuclear localization signal in NUCKS. This is also confirmed by site-directed mutagenesis of the full-length protein. By GFP-immunoprecipitation and GST-pull down experiments, we show that NUCKS binds to importin alpha3 and importin alpha5 in vitro, suggesting that the nuclear targeting of NUCKS follows a receptor-mediated and energy-dependent import mechanism.
Grundt
Kirsten
K
Department of Biochemistry, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1112, Blindern, 0317 Oslo, Norway.
Haga
Ingvild V
IV
Huitfeldt
Henrik S
HS
Ostvold
Anne Carine
AC
D021581:Active Transport, Cell Nucleus / Q000235:genetics / Q000502:physiology; D000595:Amino Acid Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000737:chemistry*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013692:Telophase; D028901:alpha Karyopherins / Q000378:metabolism
D004268:DNA-Binding Proteins; C423142:KPNA1 protein, human; C106259:KPNA4 protein, human; C426654:NUCKS1 protein, human; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; D049452:Green Fluorescent Proteins; D008156:Luciferases