Protein Interactions Regulating Pituitary Signaling

描述（由申请人提供）： 转录活性不仅依赖于转录因子与顺式作用元件的结合，而且依赖于这些因子募集复杂的蛋白质层级以稳定基础转录机制的能力。致癌Ras信号传导至大鼠催乳素（rPRL）启动子依赖于原癌蛋白c-Ets-1（Ets家族转录因子的成员）与垂体特异性转录因子Pit-1在rPRL启动子中的复合Ets/Pit-1 DNA结合元件（RRE）处的相互作用。该信号级联利用由Pit-1和Ets-1结合至独特的RRE元件、Ets-1 RIII酶与Pit-1同源结构域的物理相互作用以及Ras刺激的Ets-1中苏氨酸82的MAP激酶磷酸化组成的三重密码。该编码共同创建了一个用于结合转录调控复合物的结构平台，通过该平台Ras可以介导rPRL启动子的激活。最近的研究表明，Pit-1的磷酸化可以调节Ras信号和Ets-1/Pit-1结合。因此，我假设Pit-1/Ets-1复合物结合到RRE介导的致癌Ras反应的rPRL启动子通过招募特定的转录调控蛋白到一个独特的相互作用面。本提案的目标是确定Pit-1的磷酸化抑制Ras激活rPRL启动子的机制，并使用液相色谱/质谱方法来鉴定由Ras信号转导至Ets-1/Pit-1复合物的蛋白质组分。因此，这些研究对我的职业发展很重要，因为它们将使我获得蛋白质组学和蛋白质结构-功能关系的前沿领域的专业知识，然后我可以利用这些知识来建立我作为独立研究者的职业生涯。

DESCRIPTION (provided by applicant): Transcriptional activity is dependent not only on the binding of transcription factors to cis-acting elements, but also on the ability of those factors to recruit a complex hierarchy of proteins to stabilize the basal transcriptional machinery. Oncogenic Ras signaling to the rat prolactin (rPRL) promoter is dependent on the interaction of the proto-oncoprotein c-Ets-1, a member of the Ets family of transcription factors, with the pituitary-specific transcription factor, Pit-1, at a composite Ets/Pit-1 DNA binding element (RRE) in the rPRL promoter. This signaling cascade utilizes a tripartite code comprised of Pit-1 and Ets-1 binding to the unique RRE element, the physical interaction of the Ets-1 Rill TAD with the Pit-1 homeodomain, and Ras-stimulated MAPkinase phosphorylation of threonine 82 in Ets-1. Together this code creates a structural platform for the binding of a transcriptional regulatory complex through which Ras can mediate activation of the rPRL promoter. Recent studies suggest that the phosphorylation of Pit-1 can modulate both Ras signaling and Ets-1/Pit-1 binding. Thus, I hypothesize that the Pit-1/Ets-1 complex binding to the RRE mediates the oncogenic Ras response of the rPRL promoter by recruiting specific transcription regulatory proteins to a unique interaction face. The goals of this proposal are to determine the mechanism by which phosphorylation of Pit-1 inhibits Ras-activation of the rPRL promoter, and to use liquid chromatography/mass spectrometry methods to identify the protein components that are recruited by Ras signaling to the Ets-1/Pit-1 complex. Therefore, these studies are important to my career development as they will allow me to gain expertise in the cutting edge field of proteomics and protein structure-function relationships, which I can then utilize to establish my career as an independent investigator.