Identification of ESE1 SAR-Domain Binding Proteins

转录因子，如ETS家族中的转录因子，调节癌基因和肿瘤抑制基因的表达。 包括ESE1在内的几种ETS因子的过度表达与乳腺癌高度相关。vbl.使用 在软琼脂实验中，我们已经证明了在 40aa高酸性结构域的细胞质，称为富含丝氨酸和天冬氨酸(SAR)结构域 ESE1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，不内源性表达ESE1。这些数据表明 ESE1通过胞浆表达在乳腺细胞转化过程中发挥作用。因此，总体上， 这一建议的假设是，ESE1的SAR结构域通过与特定的细胞质结合而起作用 控制转化的乳房细胞表型的调节蛋白。我的具体目标是：1)净化 用GST-SAR亲和结合分析鉴定SAR域的细胞质结合伙伴(S)，2)鉴定 特定目的蛋白(S)的提纯1利用质谱学，3)确定合成孔径雷达的功能相关性 结合蛋白(S)在乳腺细胞功能丧失和功能获得转化中的作用

Transcription factors such as, those in the Ets family, regulate oncogene and tumor suppressor expression. 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.