New insights into the functions and regulation of the APC/C ubiquitin ligase

The Anaphase-Promoting Complex/Cyclosome (APC/C) is a macromolecular E3 ubiquitin ligase that, through targeting protein substrates for polyubiquitylation and 26S proteasome-mediated degradation coordinates the progression of cells through mitosis and the successive G1 phase of the cell-cycle. APC/C E3 ligase activity is stimulated, in the presence of the E2-ubiquitin conjugating enzymes UbcH10 and Ube2S, by the temporally coordinated recruitment of one of two related activator proteins, Cdc20 or Cdh1, to tetratricopeptide repeat (TPR)-containing APC/C subunits such as APC3; Cdc20 and Cdh1 also serve in conjunction with particular APC/C subunits to bind substrates. Work from our laboratory has determined previously that the ubiquitin ligase activity of APC/C-Cdc20 and APC/C-Cdh1 is also regulated by the transcriptional co-activators CBP and p300, which bind specifically to APC/C subunits APC5 and APC7, through interaction domains conserved in adenovirus E1A. We have also determined that the DNA damage response, and scaffold protein, MDC1 regulates APC/C-Cdc20 activity during mitosis by promoting Cdc20 association with the APC/C, whilst the transcriptional repressor and tumour suppressor, TIF1y, also regulates APC/C-Cdc20 activity in mitosis. The current project will use mass spectrometry to identify novel APC/C substrates and APC/C regulators, and define the role of phosphorylation in substrate recognition and regulator function. We will also use conventional molecular and cell biology techniques, well established in our laboratory, to study the cell cycle phase-specific degradation of new APC/C substrates and the regulation of APC/C function by novel APC/C-interacting proteins.