PUBLICATION
Identification and characterization of two putative nuclear localization signals (NLS) in the DNA-binding protein NUCKS
- Authors
- Grundt, K., Haga, I.V., Huitfeldt, H.S., and Ostvold, A.C.
- ID
- ZDB-PUB-080428-2
- Date
- 2007
- Source
- Biochimica et biophysica acta. Molecular cell research 1773(9): 1398-1406 (Journal)
- Registered Authors
- Keywords
- NUCKS, Nuclear transport, NLS, Importin, Cell cycle
- MeSH Terms
-
- DNA-Binding Proteins/chemistry*
- DNA-Binding Proteins/genetics
- DNA-Binding Proteins/metabolism
- Precipitin Tests
- Phosphoproteins/chemistry*
- Phosphoproteins/genetics
- Phosphoproteins/metabolism
- Fluorescent Antibody Technique, Indirect
- Sequence Homology, Amino Acid
- Humans
- Cell Nucleus/metabolism*
- Active Transport, Cell Nucleus/genetics
- Active Transport, Cell Nucleus/physiology
- Protein Binding
- Genes, Reporter
- Telophase
- Binding Sites
- Amino Acid Sequence
- Nuclear Proteins/chemistry*
- Nuclear Proteins/genetics
- Nuclear Proteins/metabolism
- Recombinant Fusion Proteins/chemistry
- Recombinant Fusion Proteins/metabolism
- Mutagenesis, Site-Directed
- Luciferases/metabolism
- HeLa Cells
- Signal Transduction
- Green Fluorescent Proteins/metabolism
- Protein Structure, Tertiary
- alpha Karyopherins/metabolism
- Molecular Sequence Data
- Nuclear Localization Signals/chemistry*
- PubMed
- 17604136 Full text @ BBA Molecular Cell Research
Citation
Grundt, K., Haga, I.V., Huitfeldt, H.S., and Ostvold, A.C. (2007) Identification and characterization of two putative nuclear localization signals (NLS) in the DNA-binding protein NUCKS. Biochimica et biophysica acta. Molecular cell research. 1773(9):1398-1406.
Abstract
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping