CEBPALPHA, Pit1and CBP interactons during GH regulation

描述：(摘自申请者的摘要)生长激素是一种 线性生长和代谢稳态的关键调节器 在垂体前叶有选择地合成生长激素。抄写 生长激素基因之间的相互作用受到严格调控。 有限的转录调控因子，包括Pit-1，C/EBPalpha 以及共同激活剂CBP。本文件中概述的研究的总体目标 应用是定义这些蛋白质之间相互作用的时间和地点 发生在脑下垂体生长激素中。关于C/EBPA的许多工作都集中在 它在脂肪细胞终末分化中的作用；然而，人们对此知之甚少 关于其在调节脑垂体细胞分化中的作用。我们会 用永生化小鼠垂体细胞系GHFT1-5建立 C/EBPA的不同结构域在其激活生长激素能力中的重要性 并控制其与Pit-1和CBP的相互作用。这种文化 模型将允许在多个层次上分析蛋白质的相互作用。这个 第一个目标是确定C/EBPA蛋白结构域在指导 与其他蛋白质和生长激素启动子的相互作用，以及检查 PKA和PKC信号在这些相互作用中的作用。第二个目标是 确定C/EBPA的蛋白结构域如何影响其亚核分布 在活细胞中以及随后与Pit-1和CBP的相互作用中。这将是 通过使用标记有水母绿颜色变体的蛋白质来完成 荧光蛋白。此外，我将检查PKA和PKC信号 影响活细胞中的这些相互作用。这些研究将定义 C/EBPA与Pit-1和CBP相互作用的关键蛋白结构域 获得有关控制生长激素基因的分子机制的基本信息 表情。预计这些研究将产生深远的影响 关于转录因子如何影响核的暗示 组织。

DESCRIPTION: (Scanned from the applicant's abstract) Growth hormone (GH) is a critical regulator of linear growth and metabolic homeostatis that is selectively synthesized in anterior pituitary somatotropes. The transcription of the GH gene is tightly regulated through the cooperative interactions of a limited repertoire of transcriptional regulators that include Pit-1, C/EBPalpha and the co-activator CBP. The broad objective of the studies outlined in this application is to define when and where the interactions between these proteins occur in the pituitary somatotropes. Much work concerning C/EBPa has focused on its role during adipocyte terminal differentiation; however, little is known about its function in the regulating pituitary cell differentiation. We will use the immortalized murine pituitary cell line GHFT1-5 to establish the importance of the various domains of C/EBPa in its ability to activate the GH promoter as well as control its interactions with Pit-1 and CBP. This culture model will allow the analysis of protein interactions at several levels. The first aim will establish the role of C/EBPa protein domains in directing interactions with other proteins and the GH promoter, as well as examine the effects of PKA and PKC signaling on these interactions. The second aim will determine how the protein domains of C/EBPa affect its subnuclear distribution in living cells and subsequent interactions with Pit-1 and CBP. This will be accomplished by use of proteins tagged with color variants of jellyfish green fluorescent protein. Additionally, I will examine if PKA and PKC signaling affects these interactions in the living cell. These studies will define the key protein domains of C/EBPa that direct interactions with Pit-1 and CBP and yield essential information about the molecular mechanisms that control GH gene expression. It is expected that these studies will have far-reaching implications regarding how transcription factors may influence nuclear organization.