The nuclear localization sequences of the BRCA1 protein interact with the importin-α subunit of the nuclear transport signal receptor

The BRCA1 gene product is a nuclear phosphoprotein that is aberrantly localized in the cytoplasm of most breast cancer cells. In an attempt to elucidate the potential mechanism for the nuclear transport of BRCA1 protein, three regions of highly charged, basic residues, ⁵⁰³KRKRRP⁵⁰⁸, ⁶⁰⁶PKKNRLRRKS⁶¹⁵, and ⁶⁵¹KKKKYN⁶⁵⁶, were identified as potential nuclear localization signals (NLSs). These three regions were subsequently mutated to ⁵⁰³KLP⁵⁰⁸, ⁶⁰⁷KLS⁶¹⁵, and ⁶⁵¹KLN⁶⁵⁶, respectively. Wild-type and mutated proteins were tagged with the flag epitope, expressed in human DU145 cells, and detected with the M2 monoclonal antibody. In DU145 cells, the KLP mutant completely fails to localize in nuclei, whereas the KLS mutant is mostly cytoplasmic with occasional nuclear localization. The KLN protein is always located in nuclei. Consistently, hSRP1α (importin-α), a component of the NLS receptor complex, was identified in a yeast two-hybrid screen using BRCA1 as the bait. The specificity of the interaction between BRCA1 and importin-α was further demonstrated by showing that the ⁵⁰³KRKRRP⁵⁰⁸ and ⁶⁰⁶PKKNRLRRKS⁶¹⁵ regions, but not ⁶⁵¹KKKKYN⁶⁵⁶, are critical for this interaction. To determine if the cytoplasmic mislocation of endogenous BRCA1 in breast cancer cells is due to a deficiency of the cells, wild-type BRCA1 protein tagged with the flag epitope was ectopically expressed in six breast cancer cell lines. The analysis demonstrated that, in all six, this protein localized in the cytoplasm of these cells. In contrast, expression of the construct in four non-breast cancer cell lines resulted in nuclear localization. These data support the possibility that the mislocation of the BRCA1 protein in breast cancer cells may be due to a defect in the cellular machinery involved in the NLS receptor-mediated pathway of nuclear import.