Karyopherin-alpha2 protein interacts with Chk2 and contributes to its nuclear import.
Chk2 is a nuclear protein kinase involved in the DNA damage-induced ataxia telangiectasia mutated-dependent checkpoint arrest at multiple cell cycle phases. Searching for Chk2-binding proteins by a yeast two-hybrid system, we identified a strong interaction with karyopherin-alpha2 (KPNA-2), a gene product involved in active nuclear import of proteins bearing a nuclear localization signal (NLS). This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. Of the three predicted Chk2 NLSs, located at amino acids 179-182 (NLS-1), 240-256 (NLS-2), and 515-522 (NLS-3), only the latter mediated the interaction with KPNA-2 in the yeast two-hybrid system, and in particular with its C terminus. Unlike mutations in NLS-1 or NLS-2, which left the nuclear localization of Chk2 unaffected, mutations in NLS-3 caused a cytoplasmic relocalization, indicating that the NLS-3 motif acts indeed as NLS for Chk2 in vivo. Finally, co-transfection experiments with green fluorescent protein (GFP)-Chk2 and wild type or mutant KPNA-2 confirmed the role of KPNA-2 in nuclear import of Chk2.
Zannini
Laura
L
Department of Experimental Oncology, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan, Italy.
Lecis
Daniele
D
Lisanti
Sofia
S
Benetti
Roberta
R
Buscemi
Giacomo
G
Schneider
Claudio
C
Delia
Domenico
D
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D064447:Checkpoint Kinase 2; D006801:Humans; D009154:Mutation; D019913:Nuclear Localization Signals / Q000235:genetics; D029543:Nucleocytoplasmic Transport Proteins / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D028901:alpha Karyopherins / Q000378:metabolism*
D019913:Nuclear Localization Signals; D029543:Nucleocytoplasmic Transport Proteins; D028901:alpha Karyopherins; C087978:karyopherin alpha 2; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases