Chen C F, Li S, Chen Y, Chen P L, Sharp Z D, Lee W H
Department of Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245-3207, USA.
J Biol Chem. 1996 Dec 20;271(51):32863-8. doi: 10.1074/jbc.271.51.32863.
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, hSRP1alpha (importin-alpha), 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-alpha 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.
BRCA1基因产物是一种核磷蛋白，在大多数乳腺癌细胞的细胞质中定位异常。为了阐明BRCA1蛋白核转运的潜在机制，三个富含电荷的碱性残基区域，即503KRKRRP508、606PKKNRLRRKS615和651KKKKYN656，被确定为潜在的核定位信号（NLS）。随后，这三个区域分别突变为503KLP508、607KLS615和651KLN656。野生型和突变型蛋白用flag表位标记，在人DU145细胞中表达，并用M2单克隆抗体检测。在DU145细胞中，KLP突变体完全不能定位于细胞核，而KLS突变体大多位于细胞质中，偶尔有核定位。KLN蛋白始终位于细胞核中。同样，在以BRCA1为诱饵的酵母双杂交筛选中，鉴定出NLS受体复合物的一个组分hSRP1α（输入蛋白α）。通过显示503KRKRRP508和606PKKNRLRRKS615区域而非651KKKKYN656区域对这种相互作用至关重要，进一步证明了BRCA1与输入蛋白α之间相互作用的特异性。为了确定乳腺癌细胞中内源性BRCA1的细胞质错位是否是由于细胞缺陷所致，用flag表位标记的野生型BRCA1蛋白在六种乳腺癌细胞系中异位表达。分析表明，在所有六种细胞系中，这种蛋白都定位于这些细胞的细胞质中。相反，在四种非乳腺癌细胞系中表达该构建体导致核定位。这些数据支持这样一种可能性，即乳腺癌细胞中BRCA1蛋白的错位可能是由于参与NLS受体介导的核输入途径的细胞机制存在缺陷。
