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 i s 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 i s 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 of ?-catenin can be uncoupled from its function as a transcriptional coactivator, a question relevant to anti- cancer therapies.
The final aim i s 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.
Retinal development is regulated by a conserved set of signaling pathways, and Drosophila genetics offers a powerful, rapid and economical method to identify novel components required for this process and to understand their molecular mechanisms. We propose to characterize how protein turnover regulates signaling through two pathways that are essential for normal photoreceptor differentiation and the misregulation of which is a major cause of cancer. Evolutionary conservation suggests that our findings will be applicable to higher organisms, including humans, and may lead to tools for the diagnosis or treatment of genetic disorders affecting the development of the eye or other organs, or to new cancer therapies.
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