Pattern Formation in the Drosophila Eye Disc

描述（由申请人提供）：Wnt和受体酪氨酸激酶信号通路在调节脊椎动物和无脊椎动物系统的眼睛发育中发挥重要作用。通过这些途径的信号转导依赖于蛋白质稳定性的变化，蛋白质降解也是抵抗衰老和疾病的重要防御手段。蛋白质被标记为通过泛素化降解，泛素连接酶本身受到类化修饰的调节，这种修饰增加了它们的活性，但也促进了它们的周转。本课题将探讨泛素连接酶复合物在Wnt和表皮生长因子受体（EGFR）信号通路及果蝇眼发育其他方面的功能和调控以及蛋白稳定性。对正常光感受器分化所需基因的遗传筛选发现，编码COP9信号体亚基的CSN1b突变导致Wnt信号传导减少和EGFR信号传导增加。COP9最著名的功能是使Cullins（泛素连接酶复合物的支架亚基）去基化；然而，CSN1b表型不能用Cullin功能的丧失来解释。第一个目的是测试CSN1b的作用是否由于COP9信号体的去醛化失败，并了解CSN1b如何通过Wnt和EGFR途径改变信号传导。遗传学和蛋白质组学将用于确定每条通路中受影响的组分，并测试它们是否为COP9泛素化、类化修饰或其他活性的底物。第二个目标是了解在同一筛选中发现的另一个基因，解除武装（dsm），如何通过阻止-catenin积累而促进其转录活性来调节Wnt信号。由于dsm已被定位到基因组的一个小区域，因此有可能快速克隆该基因，确定其在Wnt通路中的作用位点，并研究其作用机制。这一结果将有助于深入了解如何将-连环蛋白的稳定性与其作为转录辅助激活因子的功能解耦，这是一个与抗癌治疗相关的问题。最终目的是寻找泛素连接酶复合物在眼睛发育中的新功能和底物。转基因RNAi策略将用于筛选cullin基泛素连接酶复合物的底物结合亚基在眼睛发育中的功能。具有有趣敲低表型的保守亚基的底物将通过表型和生化分析的结合来确定。该屏幕将揭示在眼睛发育过程中蛋白质稳定性受到调节的程度，并将有助于我们了解蛋白质在发育和疾病中的作用和机制。

DESCRIPTION (provided by applicant): The Wnt and receptor tyrosine kinase signaling pathways play important roles in regulating eye development in both vertebrate and invertebrate systems. Signal transduction through these pathways depends on changes in protein stability, and protein degradation is also an important defense against aging and disease. Proteins are marked for degradation by ubiquitination, and ubiquitin ligases themselves are regulated by neddylation, a modification that increases their activity but also promotes their turnover. This proposal will investigate the functions and regulation of ubiquitin ligase complexes and protein stability in Wnt and Epidermal Growth Factor Receptor (EGFR) signaling and in other aspects of Drosophila eye development. A genetic screen for genes required for normal photoreceptor differentiation found that mutations in CSN1b, which encodes a subunit of the COP9 signalosome, result in reduced Wnt signaling and increased EGFR signaling. The best-known function of COP9 is to deneddylate Cullins, the scaffolding subunits of ubiquitin ligase complexes; however, CSN1b phenotypes cannot be explained by loss of Cullin function. The first aim is to test whether the effects of CSN1b are due to failure of deneddylation by the COP9 signalosome, and to understand how CSN1b alters signaling through the Wnt and EGFR pathways. Genetics and proteomics will be used to determine the components of each pathway that are affected, and test whether they are substrates for ubiquitination, neddylation, or other activities of COP9. The second aim is to understand how another gene identified in the same screen, disarmed (dsm), regulates Wnt signaling by preventing �-catenin accumulation but promoting its transcriptional activity. As dsm has been mapped to a small region of the genome, it should be possible to rapidly clone the gene, pinpoint its sites of action in the Wnt pathway, and investigate its mechanism of action. The results will lead to insights into how the stability o �-catenin can be uncoupled from its function as a transcriptional coactivator, a question relevant to anti- cancer therapies. The final aim is to search for novel functions and substrates for ubiquitin ligase complexes in eye development. A transgenic RNAi strategy will be used to screen the substrate-binding subunits of Cullin-based ubiquitin ligase complexes for functions in eye development. Substrates for conserved subunits with interesting knockdown phenotypes will be identified through a combination of phenotypic and biochemical analysis. The screen will reveal the extent to which protein stability is regulated during eye development, and will contribute to our understanding of the roles and mechanisms of protein turnover in development and disease.