Identification of ESE1 SAR-Domain Binding Proteins

描述（由申请人提供）：转录因子，如Ets家族的转录因子，调节细胞存活、增殖和肿瘤发生。包括ESE1在内的几个Ets因子的过度表达与乳腺癌高度相关。通过在软琼脂实验中进行锚定独立集落形成，我们发现在细胞质中稳定表达一个40AA高酸性结构域，即ESE1的Serine and Aspartic Rich （SAR）结构域，是MCF12-A乳腺细胞转化的必要和充分条件。MCF-12A细胞系是永生化的，但没有完全转化，也不内源性表达ESE1。这些数据表明，ESE1通过其细胞质表达在乳腺细胞转化中发挥作用。因此，该建议的总体假设是ESE1的SAR结构域通过与控制转化乳腺细胞表型的特定细胞质调节蛋白结合而起作用。我的具体目标是：1)使用GST-SAR亲和结合试验纯化SAR结构域的细胞质结合伙伴，2)使用质谱法鉴定在特定目标1中纯化的蛋白质，3)通过功能丧失和功能获得试验确定SAR结合蛋白（SBP）在乳腺细胞转化中的功能相关性。

DESCRIPTION (provided by applicant): Transcription factors, such as those in the Ets family, regulate cellular survival, proliferation and tumor genesis. The over-expression of several Ets factors, including ESE1, is highly associated with breast cancer. Using anchorage independent colony formation in soft agar assays, we have shown that stable expression in the cytoplasm of a 40AA highly acidic domain, referred to as the Serine and Aspartic Rich (SAR) domain of ESE1, is necessary and sufficient for MCF12-A mammary cell transformation. The MCF-12A cell-line is immortalized but not fully transformed and does not express ESE1 endogenously. These data suggest ESE1 plays a role in mammary cell transformation due to its cytoplasmic expression. Thus, the overall hypothesis of this proposal is that the SAR domain of ESE1 acts by binding to specific cytoplasmic regulatory proteins that govern the transformed breast cell phenotype. My specific aims are: 1) Purify cytoplasmic binding partner(s) of the SAR domain using GST-SAR affinity binding assays, 2) Identify the protein(s) purified in specific aim 1 using mass spectrometry, 3) Determine functional relevance of SAR binding protein(s) (SBP) in mammary cell transformation by loss of function and gain of function assays.