The nuclear localization sequences of the BRCA1 protein interact with the importin-alpha subunit of the nuclear transport signal receptor
The nuclear localization sequences of the BRCA1 protein interact with the importin-alpha subunit of the nuclear transport signal receptor
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DOI：
10.1074/jbc.271.51.32863
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发表时间：
1996-12-20
影响因子：
4.8
通讯作者：
Lee, WH
中科院分区：
文献类型：
--
作者：
Chen, CF;Li, S;Lee, WH
BRCA1基因产物是一种核磷蛋白，异常定位于大多数乳腺癌细胞的细胞质中。为了阐明BRCA1蛋白核转运的可能机制，(503)KRKRRP(508)，(606)PKKNRLRRKS(615)和(KKKKYN656)-K-651三个高电荷碱性残基区域被确定为潜在的核定位信号(NLSS)。这三个区域随后分别突变为(503)KLP(508)、(607)KLS(615)和(651)KLN(656)。用FLAG表位标记野生型和突变型蛋白，在人DU145细胞中表达，并用M2单抗进行检测。在DU145细胞中，KLP突变体完全不能定位在细胞核内，而KLS突变体主要是胞质内的，偶尔有核定位。KLN蛋白定位于细胞核内。在以BRCA1为诱饵的酵母双杂交筛选中，一致地鉴定出NLS受体复合体的一种成分hSRP1α(importin-α)。BRCA1与Importin-α相互作用的特异性进一步证明，(503)KRKRRP(508)和(606)PKKNRLRRKS(615)区域对这种相互作用是关键的，而不是(KKKKYN656)-K-651。为了确定内源性BRCA1在乳腺癌细胞中的胞质错位是否是由于细胞的缺陷，我们在6个乳腺癌细胞系中异位表达了标记有FLAG表位的野生型BRCA1蛋白。分析表明，在所有六个细胞中，该蛋白定位于这些细胞的细胞质中。相反，该构建体在四个非乳腺癌细胞系中的表达导致了核定位。这些数据支持这样一种可能性，即BRCA1蛋白在乳腺癌细胞中的错误定位可能是由于NLS受体介导的核输入途径中涉及的细胞学机制的缺陷。
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, (503)KRKRRP(508), (606)PKKNRLRRKS(615), and (KKKKYN656)-K-651, were identified as potential nuclear localization signals (NLSs). These three regions were subsequently mutated to (503)KLP(508), (607)KLS(615), and (651)KLN(656), 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, hSRP1 alpha (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 (503)KRKRRP(508) and (606)PKKNRLRRKS(615) regions, but not (KKKKYN656)-K-651, 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.