Identification of Genes Regulated by Proteolysis
Final rept. 1 Jul 2001-30 Jun 2005
HARVARD MEDICAL SCHOOL BOSTON MA
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Substrate selection in ubiquitination reactions is achieved by ubiquitin ligases, which simultaneously bind both the target protein and a ubiquitin conjugating enzyme. We have developed a phosphopeptide based approach to facilitate the identification of ubiquitin ligases e.g. F-box proteins that recognize regulatory proteins in a phosphorylation-dependent manner. Thus far, we have used this approach to identify substrates of two F-box proteins, Fbw7 and beta-TRCP. Cyclin E associates with Fbw7 through a phosphodegron near its C-terminus. Experiments in vitro and in vivo have identified critical phosphorylation sites in this degron that are required for interaction with Fbw7. Interestingly, this motif is found in a number of other unstable oncogenic proteins, including c-myc, c-jun, and SREBP. In a second series of studies, we have identified the cell cycle regulatory protein Cdc25A as a target of the beta-TRCP protein. We have found that Chk1 phosphorylates Cdc25A in response to DNA damage to generate a priming event that facilitates phosphorylation of Cdc25A on a motif that then binds to beta-TRCP. Using biochemical and genetic techniques, we demonstrate that this interaction is required for regulated proteolysis of Cdc25A.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research