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Title: Studies of the E3 ubiquitin ligase Sina-Homologue
Author: Cooper, S. E.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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I have identified Sina-Homologue (SinaH) as a novel Drosophila protein that is homologous to the E3 ubiquitin ligase Sina, but has different expression patterns throughout development. In this thesis I investigate the biochemical mechanisms of SinaH directed degradation in cells, and the physiological role of SinaH in Drosophila. I show that SinaH can direct the degradation of the transcriptional repressor Tramtrack using two different mechanisms. One is similar to Sina and requires the adaptor Phyllopod (Phyl), and the other is a novel mechanism of recognition. This novel mode of targeting for degradation is specific for the Tramtrack isoform, Ttk69. Ttk69 contains a region that is required for binding of SinaH and for SinaH directed degradation. This region contains an AxVxP motif, which is the consensus sequence found in substrates of the mammalian Sina like proteins. These results suggest that degradation directed by SinaH is more similar to that found in higher eukaryotes. In order to identify novel SinaH substrates and potential adaptor proteins, a yeast 2-hybrid screen was carried out. As well as others, this identified Numb as a potential substrate, and the protein Bruce as an E2 ligase and adaptor molecule. GST pulldown assays and coimmunoprecipitation experiments were used to verify some of these interactors, and Numb was found to be directed for degradation in cells. Flies that were deficient in SinaH and in Sina and SinaH were created using homogolous recombination. The genetic data, cell degradation assays and expression profiles together suggest that Sina and SinaH have distinct functions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available