Raymond J. Deshaies
Division of Biology, 156-29
California Institute of Technology
1200 East California Blvd.
Pasadena, CA 91125
1995 Searle Scholar
Regulation of cyclin/CDK activity in yeast
Progression through the cell division cycle in eukaryotes is driven by the sequential activation/inactivation of a set of cyclin/cyclin-dependent kinase (CDK) complexes. Several transcriptional and posttranslational mechanisms collaborate to choreograph these successive waves of cyclin/CDK activity. My lab is particularly interested in how cyclin/CDK complexes and cell cycle progression are regulated by the action of the ubiquitin-dependent proteolytic pathway. Ubiquitin-dependent proteolysis of specific polypeptides is required for the execution of at least two steps in the cell division program. First, the transition from G1 to S phase requires the destruction of p40SIC1, which inhibits the activity of a cyclin B/CDK complex that is absolutely required for DNA synthesis. Mutants that are unable to destroy p40SIC1 accumulate inactive cyclin B/CDK/p40SIC1 complexes and fail to initiate DNA synthesis. Second, for cells to transit from metaphase to G1 phase, at least two proteins must be degraded; chromosome segregation is initiated by the destruction of an unknown protein that regulates the association of sister chromatids, and the inactivation of the mitotic state is triggered by the destruction of cyclin_B. Both of these proteolytic events depend upon a multisubunit complex that has ubiquitin conjugating activity.
My lab is currently addressing several questions about these two critical cell cycle-specific destruction pathways. What are the components that make up these pathways, what are their mechanisms of action, and how are they regulated? How are the activities of these pathways influenced by checkpoints that restrain DNA replication and chromosome segregation in the presence of damaged DNA and defective microtubule spindles, respectively? Our basic approach is to recapitulate in crude yeast extracts the ubiquitination reactions that target specific proteins for degradation at the G1/S and metaphase/anaphase transitions. These biochemical assays will subsequently be exploited to identify the components involved in these reactions, and investigate how their activities are turned on and off during cell cycle progression. Ultimately, we hope to provide a molecular description of the proteolytic events that govern the G1 to S transition and the exit from mitosis.
My poster will summarize progress we've made in unraveling the mechanism of p40SIC1 destruction at the G1/S transition. Assembly of multiubiquitin chains on p40SIC1 triggers its rapid proteolysis. Multiubiquitination of p40SIC1 in vitro requires the activities of Cdc34p, Cdc4p, and G1 cyclin/CDK complexes. Whereas Cdc34p is a ubiquitin conjugating enzyme, the function of Cdc4p is unknown. The requirement for cyclin/CDK activity suggests that the ubiquitination of p40SIC1 is triggered by protein phosphorylation. Our results to date suggest the following model. In early G1-phase cells p40SIC1 is very stable despite the presence of an active Cdc34p pathway. Late in G1 phase, G1 cyclin/CDK complexes are assembled, and these complexes phosphorylate p40SIC1 on multiple sites. Phosphorylation of p40SIC1 by cyclin/CDK complexes is both necessary and sufficient to target its ubiquitination by the Cdc34p pathway. In agreement with our in vitro data, mutation of a set of cyclin/CDK phosphorylation sites in p40SIC1 severely reduces ubiquitination of p40SIC1 in vitro, and stabilizes p40SIC1 in vivo.. Our current goals are to determine how phosphorylation of p40SIC1 directs its ubiquitination by the Cdc34p pathway, and to identify a set of components that is sufficient to catalyze the Cdc34p-dependent ubiquitination of p40SIC1.
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