PHOSPHORYLATION IN A DEVELOPMENT SIGNALING PATHWAY

描述（由申请人提供）：本研究的长期目标是了解通过控制调控蛋白的亚细胞定位来调节基因表达程序的信号通路。在果蝇中，细胞表面受体Toll的信号转导促进胚胎中控制背腹轴形成和成人先天免疫的转录因子的核易位。先天免疫应答中的通路功能广泛保守，在植物和哺乳动物中都有同源通路诱导抗微生物防御。在果蝇中，Toll信号的靶点是Dorsal和Dif，一对NF-KappaB相关的转录因子，以及抑制蛋白Cactus。从Toll到Cactus和Dorsal/Dif的信号转导需要两个适配蛋白MyD88和Tube，以及一个蛋白激酶Pelle。事实上，这些途径成分已经在遗传和分子水平上被表征，并且可以在胚胎、全蝇和细胞培养系统中进行检测，这使得该途径特别适合实验研究。因此，现在有可能解决有关信号转导机制的基本问题。拟议研究的重点将从一系列蛋白质相互作用的静态图像转移到对整个途径结构背景下信号转导的动态理解。我们将开发用于快速和同步启动信号的试剂，然后利用这些工具来探索途径成分如何寡聚，结合和解离，以及如何被修饰，重新定位和降解，以影响信号转导。在进行这些研究时，我们将利用诱导二聚化系统来触发途径激活，RNAi技术用于大规模反向遗传筛选，定位修饰小颗粒蛋白激酶以确定催化的直接目标，以及荧光共振能量转移（FRET）来监测蛋白质复合物的组装。同时，我们将开展靶基因的顺式调控序列研究，重点研究Toll通路信号在整体先天免疫应答中的整合。鉴于信号通路的保守性，拟议的研究结果应该对广泛生物体的信号转导和先天免疫具有重大意义。

DESCRIPTION (provided by applicant): The long term goal of this research is to understand a signaling pathway that regulates programs of gene expression by controlling the subcellular localization of regulatory proteins. In the fruit fly Drosophila, signal transduction by the cell surface receptor Toll promotes nuclear translocation of transcription factors governing dorsoventral axis formation in the embryo and innate immunity in the adult. Pathway function in innate immune responses has been widely conserved, with homologous pathways inducing antimicrobial defenses in both plants and mammals. In flies, the targets of Toll signaling are Dorsal and Dif, a pair of NF-KappaB related transcription factors, and the inhibitory protein Cactus. Signal transduction from Toll to Cactus and Dorsal/Dif requires two adaptor proteins, MyD88 and Tube, as well as a protein kinase, Pelle. The fact that these pathway components have been characterized at both the genetic and molecular level and can be assayed in embryo, whole fly, and cell culture systems makes this pathway particularly amenable to experimental investigation. It is now possible, therefore, to address fundamental questions about the mechanism for signal transduction. The focus of the proposed research will be to move from a static picture of a series of protein interactions to a dynamic understanding of signal transduction in the context of overall pathway architecture. We will develop reagents for the rapid and synchronous initiation of signaling, then exploit these tools to probe how pathway components oligomerize, associate and dissociate, and are modified, relocalized and degraded, to effect signal transduction. In carrying out these studies, we will exploit inducible dimerization systems for triggering pathway activation, RNAi technology for large-scale reverse genetic screens, site directed modifications of the Pelle protein kinase to identify direct targets of catalysis, and fluorescence resonance energy transfer (FRET) to monitor the assembly of protein complexes. At the same time, we will carry out studies of cis-regulatory sequences in target genes, focusing on integration of Toll pathway signals into the overall innate immune response. Given the conserved nature of the signaling pathway, the results of the proposed research should be of substantial interest with regard to signal transduction and innate immunity in a broad range of organisms.