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


CONCLUSIONS
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.
Haenni
Sandra S
SS
Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. haennis@vetbio.uzh.ch
Altmeyer
Matthias
M
Hassa
Paul O
PO
Valovka
Taras
T
Fey
Monika
M
Hottiger
Michael O
MO
D000595:Amino Acid Sequence; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism*
D000903:Antibiotics, Antineoplastic; D005231:Fatty Acids, Unsaturated; D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; C483041:PARP2 protein, human; D011065:Poly(ADP-ribose) Polymerases; D008239:Lysine; C038753:leptomycin B