Characterization of a novel substrate of ERK MAP kinase that functions during vul

描述（由申请人提供）：在许多多细胞生物的发育过程中，Ras/MAP激酶信号通路促进细胞分裂。研究这一信号通路具有重要意义，因为ras基因的突变是人类癌变肿瘤发生的重要步骤。许多蛋白质参与Ras/MAP激酶信号的调控和传递，我们已经开始研究秀丽隐杆线虫发育过程中这一信号通路的新靶点。线虫预测的开放阅读框T08D10.1编码dna结合蛋白CCAAT-box家族成员，并具有多个潜在的细胞外调节激酶（ERK） MAP激酶磷酸化位点和对接位点。初步数据表明，T08D10.1作为ERK MAP激酶的高亲和力底物，在秀丽隐杆线虫外阴发育过程中具有抑制Ras信号的功能。我们建议通过生化和遗传方法来表征Ras信号通路中这一新成分的功能和表达。T08D10.1作为ERK MAP激酶底物的能力以及推测的对接位点序列的作用将通过生化激酶试验进行研究。T08D10.1的突变表型将在秀丽隐杆线虫在野生型和敏化背景下的发育过程中进行表征。通过转基因动物的产生，研究T08D10.1的时空表达模式。最后，T08D10.1作为转录因子的功能将通过体外电泳迁移转移试验和在培养细胞中进行的转激活电位试验进行测试。研究T08D10.1作为Ras通路下游介质的作用，不仅有助于理解秀丽隐杆线虫发育过程中特定细胞命运指定所需的机制，而且有助于我们理解正常和异常后生动物发育过程中一般上游信号事件导致适当细胞命运决定的一般机制。Ras信号通路成分的突变可导致人类癌症的发展。该途径的许多核心成分在多细胞生物中是保守的，包括模式生物秀丽隐杆线虫。该项目旨在描述秀丽隐杆线虫Ras信号通路的一种新的调节因子，该调节因子可能具有抑制细胞分裂的功能。

DESCRIPTION (provided by applicant): The Ras/MAP kinase signaling pathway promotes cell divisions during the development of many multicellular organisms. Study of this signaling pathway is significant since mutation of the ras gene in humans is an important step in the generation of cancerous tumors. Many proteins are involved in the regulation and transmission of the Ras/MAP kinase signal, and we have initiated a study of a novel target of this signaling pathway during Caenorhabditis elegans development. The C. elegans predicted open-reading-frame, T08D10.1, codes for member of the CCAAT-box family of DNA-binding proteins and possesses multiple potential phosphorylation sites and docking sites for extracellular regulated kinase (ERK) MAP kinase. Preliminary data indicate that T08D10.1 acts as a high affinity substrate for ERK MAP kinase and functions to inhibit Ras signaling during C. elegans vulval development. We propose to characterize the function and expression of this novel component of the Ras signaling pathway through biochemical and genetic methods. The ability of T08D10.1 to function as a substrate for ERK MAP kinase and the role of putative docking site sequences will be investigated using biochemical kinase assays. The mutant phenotype of T08D10.1 will be characterized during C. elegans development in wild-type and sensitizes backgrounds. The temporal and spatial expression pattern of T08D10.1 will be investigated through the generation of transgenic animals. Finally, the function of T08D10.1 as a transcription factor will be tested using in vitro electrophoretic mobility shift assays and transactivation potential assays conducted in cultured cells. Investigation of the role of T08D10.1 as a downstream mediator of the Ras pathway will not only contribute to the understanding of the mechanisms required for the specification of specific cell fates during the development of C. elegans, but will also contribute to our understanding of the general mechanisms by which general uptstream signaling events lead to appropriate cell fate decisions during normal and abnormal metazoan development. Mutation of components of the Ras signaling pathway can lead to the development of cancer in humans. Many of the core components of this pathway are conserved in multicellular organisms including the model organism Caenorhabditis elegans. This project seeks to characterize a novel regulator of the Ras signaling pathway in C. elegans that may function to inhibit cell divisions.