Identification of ESE1 SAR-Domain Binding Proteins

描述(申请人提供)：转录因子，如ETS家族中的转录因子，调节细胞存活、增殖和肿瘤发生。包括ESE1在内的几种ETS因子的过度表达与乳腺癌高度相关。在软琼脂实验中使用锚定非依赖性集落形成，我们已经证明了ESE1的一个40aa的高酸性结构域，即富含丝氨酸和天冬氨酸(SAR)结构域的细胞质中稳定表达是MCF12-A乳腺细胞转化的必要条件和充分条件。MCF-12A细胞系永生化，但未完全转化，不表达内源性ESE1。这些数据表明，ESE1通过胞浆表达在乳腺细胞转化中发挥作用。因此，这一建议的总体假设是，ESE1的SAR结构域通过与控制转化的乳腺细胞表型的特定细胞质调节蛋白结合来发挥作用。我的具体目标是：1)通过GST-SAR亲和结合分析纯化SAR域的细胞质结合伙伴(S)；2)鉴定针对特定目的纯化的蛋白(S)；3)通过功能丧失和功能获得的分析，确定SAR结合蛋白(S)(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.