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.

[1]  K. Loveland,et al.  Importin α mRNAs have distinct expression profiles during spermatogenesis , 2006 .

[2]  I. Macara,et al.  RanBP3 Contains an Unusual Nuclear Localization Signal That Is Imported Preferentially by Importin-α3 , 1999, Molecular and Cellular Biology.

[3]  C. Christophe-Hobertus,et al.  Nuclear targeting of proteins: how many different signals? , 2000, Cellular signalling.

[4]  M. Mumby,et al.  Identification of Phosphoproteins and Their Phosphorylation Sites in the WEHI-231 B Lymphoma Cell Line* , 2004, Molecular & Cellular Proteomics.

[5]  S. Laland,et al.  The phosphate groups of the high mobility group like protein P1 strengthens its affinity for DNA. , 1992, Biochemical and biophysical research communications.

[6]  U. Kutay,et al.  Transport between the cell nucleus and the cytoplasm. , 1999, Annual review of cell and developmental biology.

[7]  H. Huitfeldt,et al.  A putative DNA-binding domain in the NUCKS protein. , 2002, Archives of biochemistry and biophysics.

[8]  S. Quake,et al.  Identification and confirmation of a module of coexpressed genes. , 2002, Genome research.

[9]  T. Misteli,et al.  Mitotic Phosphorylation Prevents the Binding of HMGN Proteins to Chromatin , 2001, Molecular and Cellular Biology.

[10]  R. Laskey,et al.  Nuclear targeting sequences--a consensus? , 1991, Trends in biochemical sciences.

[11]  Steven P Gygi,et al.  Large-scale characterization of HeLa cell nuclear phosphoproteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[12]  D. Jans,et al.  Regulation of protein transport to the nucleus: central role of phosphorylation. , 1996, Physiological reviews.

[13]  U. Kutay,et al.  Nucleocytoplasmic transport: taking an inventory , 2003, Cellular and Molecular Life Sciences CMLS.

[14]  R. Laskey,et al.  Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids , 1996, Current Biology.

[15]  Joel Greshock,et al.  High resolution genomic analysis of sporadic breast cancer using array-based comparative genomic hybridization , 2005, Breast Cancer Research.

[16]  G. Pan,et al.  Nuclear localization of the phosphatidylserine receptor protein via multiple nuclear localization signals. , 2004, Experimental cell research.

[17]  S. Chatterjee,et al.  Monitoring nuclear transport in HeLa cells using the green fluorescent protein. , 1996, BioTechniques.

[18]  C. Ball,et al.  Identification of genes periodically expressed in the human cell cycle and their expression in tumors. , 2002, Molecular biology of the cell.

[19]  P. Volpe,et al.  A method for measuring cell cycle phases in suspension cultures. , 1973, Methods in cell biology.

[20]  W. Richardson,et al.  The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen , 1988, The Journal of cell biology.

[21]  J. Norum,et al.  Molecular cloning of a mammalian nuclear phosphoprotein NUCKS, which serves as a substrate for Cdk1 in vivo. , 2001, European journal of biochemistry.

[22]  E. Hartmann,et al.  Cloning of two novel human importin‐α subunits and analysis of the expression pattern of the importin‐α protein family , 1997 .

[23]  S. Nadler,et al.  Differential Expression and Sequence-specific Interaction of Karyopherin α with Nuclear Localization Sequences* , 1997, The Journal of Biological Chemistry.

[24]  D. Chelsky,et al.  Sequence requirements for synthetic peptide-mediated translocation to the nucleus , 1989, Molecular and cellular biology.

[25]  F. Huang,et al.  Phosphorylation of HMG‐I by Protein Kinase C Attenuates Its Binding Affinity to the Promoter Regions of Protein Kinase C γ and Neurogranin/RC3 Genes , 2000, Journal of neurochemistry.

[26]  D. Goldfarb,et al.  Facilitated nuclear transport of histone H1 and other small nucleophilic proteins , 1990, Cell.

[27]  K. Grundt,et al.  Characterisation of the NUCKS gene on human chromosome 1q32.1 and the presence of a homologous gene in different species. , 2004, Biochemical and biophysical research communications.

[28]  K. Yamamoto,et al.  Two signals mediate hormone‐dependent nuclear localization of the glucocorticoid receptor. , 1987, The EMBO journal.

[29]  C. Xiao,et al.  Nuclear targeting signal recognition: a key control point in nuclear transport? , 2000, BioEssays : news and reviews in molecular, cellular and developmental biology.