The Spindle Assembly Checkpoint (SAC) delays the onset of anaphase in response to unattached kinetochores. The mechanism by which the SAC works is by inhibiting the activity of the Anaphase-promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase that targets several anaphase inhibitors for proteasome-mediated degradation, including securin and cyclin B. When the SAC is satisfied, the APC/C becomes active and this allows progression through the cell cycle. Work from the last decade identified the mitotic checkpoint complex (MCC) as the main transducer of the SAC. The MCC is composed of BubR1, Bub3, Mad2 and Cdc20 and it is a very potent inhibitor of the APC/C. When the SAC is active, the MCC binds the APC/C and it inhibits its activity. Once the SAC is satisfied, the MCC becomes disassembled, which allows APC/C activation and mitotic progression. However, the mechanisms that dictate MCC assembly and how it inhibits the APC/C remain to be understood. Here, I used a combination of cell biology and in vitro biochemistry to investigate the mechanism by which the MCC component BubR1 participates in the SAC. My data shows that through its interaction with Bub3, BubR1 localises to kinetochores and this event greatly facilitates its assembly onto the MCC and its SAC function. On the other hand, MCC formation and APC/C binding were only dependent on BubR1's N-terminus, therefore questioning the existence of a second Cdc20 binding site. Within this region, TPR domains and an N-terminal motif known as the KEN box (KEN1) mediates these interactions. By contrast, BubR1's second KEN box (KEN2) does not participate in MCC assembly or APC/C binding. However, both in cells and in vitro, the KEN2 box is required for APC/C inhibition. Indeed, I show that this second KEN box promotes SAC function by blocking the interaction of the APC/C with its substrates. Thus, both KEN boxes in BubR1 participate differentially in the SAC, the first to promote MCC assembly and the second one to block substrate recruitment to the APC/C.In addition, I investigated the mechanisms that mediate MCC inactivation, following SAC silencing. I observed that p31comet and APC/C activity cooperate to promote MCC turnover. The implication of these observations in our understanding of the SAC is discussed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764271 |
| Date | January 2013 |
| Creators | Lara González, Pablo |
| Contributors | Taylor, Stephen |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/investigating-how-the-spindle-assembly-checkpoint-inhibits-the-onset-of-anaphase(91d10d3a-9fc9-4395-9a18-d29969d604f8).html |
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