Medical Scientist Training Program, University of Colorado Denver, Aurora, CO 80045, USA.
Mol Cancer. 2011 Aug 28;10:103. doi: 10.1186/1476-4598-10-103.
The ETS family transcription factor ESE-1 is often overexpressed in human breast cancer. ESE-1 initiates transformation of MCF-12A cells via a non-transcriptional, cytoplasmic process that is mediated by a unique 40-amino acid serine and aspartic acid rich (SAR) subdomain, whereas, ESE-1's nuclear transcriptional property is required to maintain the transformed phenotype of MCF7, ZR-75-1 and T47D breast cancer cells.
To map the minimal functional nuclear localization (NLS) and nuclear export (NES) signals, we fused in-frame putative NLS and NES motifs between GFP and the SAR domain. Using these GFP constructs as reporters of subcellular localization, we mapped a single NLS to six basic amino acids (242 HGKRRR 247) in the AT-hook and two CRM1-dependent NES motifs, one to the pointed domain (NES1: 102 LCNCALEELRL 112) and another to the DNA binding domain (DBD), (NES2: 275 LWEFIRDILI 284). Moreover, analysis of a putative NLS located in the DBD (316 GQKKKNSN 323) by a similar GFP-SAR reporter or by internal deletion of the DBD, revealed this sequence to lack NLS activity. To assess the role of NES2 in regulating ESE-1 subcellular localization and subsequent transformation potency, we site-specifically mutagenized NES2, within full-length GFP-ESE-1 and GFP-NES2-SAR reporter constructs. These studies show that site-specific mutation of NES2 completely abrogates ESE-1 transforming activity. Furthermore, we show that exclusive cytoplasmic targeting of the SAR domain is sufficient to initiate transformation, and we report that an intact SAR domain is required, since block mutagenesis reveals that an intact SAR domain is necessary to maintain its full transforming potency. Finally, using a monoclonal antibody targeting the SAR domain, we demonstrate that the SAR domain contains a region accessible for protein - protein interactions.
These data highlight that ESE-1 contains NLS and NES signals that play a critical role in regulating its subcellular localization and function, and that an intact SAR domain mediates MEC transformation exclusively in the cytoplasm, via a novel nontranscriptional mechanism, whereby the SAR motif is accessible for ligand and/or protein interactions. These findings are significant, since they provide novel molecular insights into the functions of ETS transcription factors in mammary cell transformation.
ETS 家族转录因子 ESE-1 在人乳腺癌中常过表达。ESE-1 通过非转录的细胞质过程启动 MCF-12A 细胞的转化，该过程由独特的 40 个氨基酸丝氨酸和天冬氨酸丰富（SAR）亚结构域介导，而 ESE-1 的核转录特性是维持 MCF7、ZR-75-1 和 T47D 乳腺癌细胞转化表型所必需的。
为了绘制最小的功能性核定位（NLS）和核输出（NES）信号，我们将推定的 NLS 和 NES 基序在 GFP 和 SAR 结构域之间进行框内融合。使用这些 GFP 构建体作为亚细胞定位的报告物，我们将一个 NLS 映射到 AT 钩中的六个碱性氨基酸（242 HGKRRR 247）和两个 CRM1 依赖性 NES 基序之一，指向结构域（NES1：102 LCNCALEELRL 112）和另一个到 DNA 结合域（DBD）（NES2：275 LWEFIRDILI 284）。此外，通过类似的 GFP-SAR 报告物或通过 DBD 内部缺失分析位于 DBD 中的推定 NLS（316 GQKKKNSN 323），揭示该序列缺乏 NLS 活性。为了评估 NES2 在调节 ESE-1 亚细胞定位和随后的转化效力中的作用，我们在全长 GFP-ESE-1 和 GFP-NES2-SAR 报告构建体中对 NES2 进行了定点突变。这些研究表明，NES2 的定点突变完全消除了 ESE-1 的转化活性。此外，我们表明 SAR 结构域的专有细胞质靶向足以启动转化，并且我们报告说完整的 SAR 结构域是必需的，因为阻断突变揭示完整的 SAR 结构域是维持其全部转化效力所必需的。最后，使用针对 SAR 结构域的单克隆抗体，我们证明 SAR 结构域包含一个可用于蛋白质-蛋白质相互作用的区域。
这些数据强调 ESE-1 包含 NLS 和 NES 信号，这些信号在调节其亚细胞定位和功能中起着关键作用，并且完整的 SAR 结构域通过一种新型的非转录机制仅在细胞质中介导 MEC 转化，其中 SAR 基序可用于配体和/或蛋白质相互作用。这些发现意义重大，因为它们为 ETS 转录因子在乳腺细胞转化中的功能提供了新的分子见解。
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