Protein Interactions Regulating Pituitary Signaling

描述(由申请人提供)： 转录活性不仅取决于转录因子与顺式作用元件的结合，还取决于这些因子招募复杂的蛋白质层次结构以稳定基本转录机制的能力。大鼠催乳素(RPRL)启动子的致癌RAS信号依赖于原癌蛋白c-ETS-1与垂体特异性转录因子Pit-1在rPRL启动子的Ets/Pit-1 DNA结合元件(RRE)上的相互作用。这一信号级联利用了由Pit-1和Ets-1与独特的RRE元件结合组成的三方密码，Ets-1 Rill TAD与Pit-1同源结构域的物理相互作用，以及RAS刺激的MAPkinase82在Ets-1中的磷酸化。总而言之，这一密码为转录调控复合体的结合创建了一个结构平台，RAS可以通过该平台介导rPRL启动子的激活。最近的研究表明，Pit-1的磷酸化可以同时调节RAS信号和Ets-1/Pit-1的结合。因此，我假设与RRE结合的Pit-1/Ets-1复合体通过将特定的转录调节蛋白招募到一个独特的相互作用面来介导rPRL启动子的致癌RAS反应。该方案的目的是确定Pit-1的磷酸化抑制rPRL启动子RAS激活的机制，并使用液相色谱/质谱仪方法鉴定RAS信号向Ets-1/Pit-1复合体招募的蛋白质组分。因此，这些研究对我的职业发展很重要，因为它们将使我在蛋白质组学和蛋白质结构-功能关系的尖端领域获得专业知识，然后我可以利用这些专业知识来建立我作为独立研究员的职业生涯。

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.