A CRM1-mediated nuclear export signal governs cytoplasmic localization of BRCA2 and is essential for centrosomal localization of BRCA2.
Germ-line mutations of the BRCA2 gene cause inherited susceptibility to breast and ovarian cancers. BRCA2 contains two nuclear localization signals, predominantly localizes in the nucleus and plays significant roles in DNA double-strand break repair. Recently, we reported that BRCA2 localizes to the centrosomes during the S and early M phases of the cell cycle. In this study, for the first time, we identified a functional nuclear export sequence (NES1; (1383)DLSDLTFLEVA(1393)) in BRCA2. The green fluorescent protein (GFP)-NES1 fusion protein was localized in the cytoplasm and could be blocked by the chromosomal region maintenance 1-specific export inhibitor leptomycin B. Mutation of a leucine residue in the NES1 motif to alanine (L1384A) resulted in both cytoplasmic and nuclear localization of the GFP-NES1 fusion protein and a nuclear accumulation of ectopic full-length BRCA2-FLAG. Moreover, treatment of cells with leptomycin B decreased centrosomal localization of BRCA2. Finally, by microinjection of an anti-BRCA2 antibody into the cytoplasm of HeLa S3 cells, we found that depletion of normal BRCA2 proteins in the cytoplasm leads to centrosome amplification and binucleated cells. Our results suggest that disruption of the NES function by genetic changes results in deregulation of BRCA2 export, which ultimately leads to centrosome disorder.
Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.
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X
Han
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Saito
Y
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Miki
A
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Nakanishi
D000595:Amino Acid Sequence; D000818:Animals; D024682:BRCA2 Protein / Q000378:metabolism*; D001483:Base Sequence; D019556:COS Cells; D018385:Centrosome / Q000187:drug effects / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D049790:Nuclear Export Signals / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D000097606:Exportin 1 Protein
D024682:BRCA2 Protein; D017931:DNA Primers; D005231:Fatty Acids, Unsaturated; D028884:Karyopherins; D049790:Nuclear Export Signals; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein; C038753:leptomycin B