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

该项目将鉴定在自由生活的蛔虫C中发挥作用的基因产物。它们是MPK-1 ERK MAP激酶的磷酸化靶点。 ERK MAP激酶在许多细胞外信号级联的末端起作用，如受体酪氨酸激酶途径，其在动物发育和稳态中具有重要功能。 在许多情况下，当被细胞外信号调节激酶（ERK）磷酸化时被激活或失活的下游靶蛋白是未知的。 在模式实验生物C.在线虫中，存在与ERK类似的单个基因（ERK直系同源物），并且被称为MPK-1。这在十种不同的生殖系过程中起作用，并且被MPK-1 ERK磷酸化以介导这些生殖系过程的靶蛋白是未知的。 在这个项目中，一个三部分的方法将被用来确定MPK-1 ERK磷酸化的目标，在生殖细胞发育的功能。 在第一部分中，对接位点序列将用于计算识别C。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对MPK-1 ERK信号传导敏感的遗传背景中的相应基因的RNA介导的干扰来进行。 在第三部分中，将使用纯化的哺乳动物ERK通过体外磷酸化来确认所选择的体内验证的靶标。 该项目的更广泛影响体现在三个层面：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。 第二，新的基因，介导的生殖细胞发育的10个不同方面，直接调控ERK将被确定。 第三，考虑到ERK信号传导和生殖细胞发育的保守性，在C.线虫也将是其他动物中ERK的靶点。

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.