Functional Genomic Approach to Identify and Characterize MPK-1 ERK Phosphorylation Targets in Caenorhabditis Elegans Germline Development

该项目将确定在自由生活的线虫胚系发育中发挥作用的基因产物，这些基因产物是MPK-1 ERK MAP Kinase的磷酸化靶标。ERK-MAP在许多细胞外信号通路的末端发挥作用，如受体酪氨酸激酶通路，在动物发育和动态平衡中具有重要的功能。在许多情况下，被细胞外信号调节激酶(ERK)磷酸化时激活或失活的下游靶蛋白是未知的。在模式实验生物秀丽线虫中，有一个与ERK(ERK直系同源)相似的基因，被称为MPK-1。它在10个不同的生殖系过程中发挥作用，而被MPK-1 ERK磷酸化以介导这些生殖系过程的靶蛋白尚不清楚。在这个项目中，将使用一个三部分的方法来确定在生殖系发育中起作用的MPK-1 ERK磷酸化靶点。在第一部分，将使用对接位点序列来计算识别线虫蛋白，这些蛋白是MPK-1 ERK磷酸化的潜在靶标。在第二部分中，对预测的靶基因产物的体内验证将通过RNA介导的干扰对MPK-1 ERK信号敏感的遗传背景中的相应基因进行。在第三部分中，选定的体内有效靶点将通过使用纯化的哺乳动物ERK进行体外磷酸化来确认为靶点。该项目的广泛影响有三个层面：首先，该项目将为一名博士后研究员和一些本科生提供遗传学、基因组学和发育生物学方面的研究培训。其次，将识别直接受ERK调控的调节生殖系发育的十个不同方面的新基因。第三，考虑到ERK信号和生殖系发育的保守性，在线虫中确定的ERK靶标很可能也将成为其他动物的ERK靶标。

This project will identify gene products that function in the free-living roundworm C. elegans germline development which are phosphorylation targets of MPK-1 ERK MAP Kinase. ERK MAP kinases act at the end of numerous extracellular signaling cascades, such as receptor tyrosine kinase pathways, which have essential functions in animal development and homeostasis. In many cases, the downstream target proteins that are activated or inactivated when phosphorylated by extracellular signal-regulated kinase (ERK) are unknown. In the model experimental organism C. elegans, there is a single gene similar to ERK (ERK ortholog) and is known as MPK-1. This functions in ten different germline processes and the target proteins that are phosphorylated by MPK-1 ERK to mediate these germline processes are unknown. In this project, a three-part approach will be used to identify MPK-1 ERK phosphorylation targets that function in germline development. In the first part, docking site sequences will be used to computationally identify C. elegans proteins that are potential targets of MPK-1 ERK phosphorylation. In the second part, in vivo validation of the predicted target gene products will be performed by RNA mediated interference of the corresponding genes in genetic backgrounds that are sensitized for MPK-1 ERK signaling. In the third part, selected in vivo validated targets will be confirmed as targets by in vitro phosphorylation using purified mammalian ERK. The Broader Impact of this project is at three levels: First, the project will provide research training in genetics, genomics and developmental biology for a postdoctoral fellow and a number of undergraduate students. Second, novel genes that mediate ten different aspects of germline development that are directly regulated by ERK will be identified. Third, given the conservation of both ERK signaling and germline development, it is very likely that the ERK targets identified in C. elegans will also be ERK targets in other animals.