Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS

BMC Cell Biol. 2008 Jul 21;9:39. doi: 10.1186/1471-2121-9-39.

Abstract

Background: The enzymes responsible for the synthesis of poly-ADP-ribose are named poly-ADP-ribose polymerases (PARP). PARP-2 is a nuclear protein, which regulates a variety of cellular functions that are mainly controlled by protein-protein interactions. A previously described non-conventional bipartite nuclear localization sequence (NLS) lies in the amino-terminal DNA binding domain of PARP-2 between amino acids 1-69; however, this targeting sequence has not been experimentally examined or validated.

Results: Using a site-directed mutagenesis approach, we found that lysines 19 and 20, located within a previously described bipartite NLS, are not required for nuclear localization of PARP-2. In contrast, lysine 36, which is located within a predicted classical monopartite NLS, was required for PARP-2 nuclear localization. While wild type PARP-2 interacted with importin alpha3 and to a very weak extent with importin alpha1 and importin alpha5, the mutant PARP-2 (K36R) did not interact with importin alpha3, providing a molecular explanation why PARP-2 (K36R) is not targeted to the nucleus.

Conclusion: Our results provide strong evidence that lysine 36 of PARP-2 is a critical residue for proper nuclear targeting of PARP-2 and consequently for the execution of its biological functions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibiotics, Antineoplastic / metabolism
  • Cell Line
  • Cell Nucleus / metabolism*
  • Fatty Acids, Unsaturated / metabolism
  • Humans
  • Lysine / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nuclear Localization Signals* / genetics
  • Nuclear Localization Signals* / metabolism
  • Poly(ADP-ribose) Polymerases* / genetics
  • Poly(ADP-ribose) Polymerases* / metabolism
  • Protein Binding
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Alignment
  • alpha Karyopherins / genetics
  • alpha Karyopherins / metabolism*

Substances

  • Antibiotics, Antineoplastic
  • Fatty Acids, Unsaturated
  • Nuclear Localization Signals
  • Protein Isoforms
  • Recombinant Fusion Proteins
  • alpha Karyopherins
  • PARP2 protein, human
  • Poly(ADP-ribose) Polymerases
  • Lysine
  • leptomycin B