CDC20 function, regulation and proteolysis

The destruction of mitotic cyclins and other key regulators uses ubiquitin mediated proteolysis controlled via the activation of the ubiquitin ligase the Anaphase Promoting Complex/Cyclosome (APC/C), and its adaptor proteins Cdc20 and Cdh1. The spindle assembly checkpoint coordinates the APC/C with microtubule attachment and sets the timing from NEBD to anaphase. Cdc20 is inactivated by the spindle assembly checkpoint to prevent premature anaphase onset. Once the spindle assembly checkpoint is satisfied, Cdc20 can be released and activate the APC/C. However, cyclin A is degraded independently of the spindle assembly checkpoint before cyclin B1 and the anaphase inhibitor Securin. How Cdc20 can target different substrates for degradation at different times during mitosis is not yet clear. Using live-cell imaging and RNA interference and immunofluorescence techniques, I have studied the degradation of endogenous and ectopicly expressed APC/C substrates in cells with reduced Cdc20 levels. In this dissertation, I show that cyclin A degradation strongly depends on Cdc20, whereas cyclin B1 and Securin degradation do not. I identified the region of Cdc20 required for cyclin A binding and by mutating this site, I found that Cdc20 was no longer properly localised. I also show that Cdc20 proteolysis in human somatic cells does not require the KEN motif and gone on to find the motif required for Cdc20 degradation. I also show that the function of Cdc20 in the spindle assembly checkpoint can be influenced by the serine/threonine kinase Aurora A. Co-expression of a fluorescently tagged Cdc20 and Aurora A in human somatic cells causes an accelerated progression through mitosis and premature substrate degradation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo