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秀丽隐杆线虫种系发育中起作用的基因产物，这些基因产物是MPK-1 ERK MAP激酶的磷酸化靶点。ERK MAP激酶作用于许多细胞外信号级联的末端，如受体酪氨酸激酶途径，在动物发育和体内平衡中具有重要功能。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。在模型实验生物秀丽隐杆线虫中，存在一个与ERK （ERK同源物）相似的基因，称为MPK-1。这在十种不同的种系过程中起作用，而MPK-1 ERK磷酸化介导这些种系过程的靶蛋白尚不清楚。在本项目中，将采用三部分方法来鉴定MPK-1 ERK磷酸化靶点，这些靶点在种系发育中起作用。在第一部分中，对接位点序列将用于计算鉴定秀丽隐杆线虫蛋白，这些蛋白是MPK-1 ERK磷酸化的潜在靶点。在第二部分中，预测的靶基因产物的体内验证将通过RNA介导的遗传背景中对MPK-1 ERK信号敏感的相应基因的干扰来进行。在第三部分中，选定的体内验证靶点将通过纯化的哺乳动物ERK进行体外磷酸化来确认为靶点。本项目的广泛影响有三个层面：第一，本项目将为一名博士后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，将确定介导由ERK直接调节的种系发育的十个不同方面的新基因。第三，考虑到ERK信号和种系发育的保守性，在秀丽隐杆线虫中发现的ERK靶点很可能也是其他动物的ERK靶点。

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.