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New insights into the function of the Anaphase Promoting Complex ubiquitin ligase in cell cycle control

Principal Supervisor: Dr Andy Turnell - Institute of Cancer and Genomic Sciences

Co-supervisor: Dr Aga Gambus

PhD project title: New insights into the function of the Anaphase Promoting Complex ubiquitin ligase in cell cycle control

University of Registration: Birmingham

Project outline:

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 (1). 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 (1). Work from our laboratory has previously determined 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 (2). 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 (3), whilst the transcriptional repressor and tumour suppressor, TIF1, also regulates APC/C-Cdc20 activity in mitosis (4).

The principal aim of this studentship is to further our understanding of the molecular function of the APC/C during the cell cycle. To do this, we will explore the temporal nature of protein-protein interactions during the cell cycle, by analysing the interactomes of the both the APC/C and proteins known to regulate APC/C activity. In this regard, we will also analyse the post-translational modification status (e.g. phosphorylation, acetylation, ubiquitylation and sumoylation) of the APC/C and associated proteins. We will employ SILAC to discern quantitative changes in APC/C composition, and PTM status. Furthermore, we will also use SILAC to detect and quantify the levels, and PTM status of over 3,000 cellular proteins during cell cycle progression. We will also employ multiple reaction monitoring (MRM) mass spectrometry to quantify the dynamic changes in the levels of candidate proteins during the cell cycle. Where appropriate, we will adopt conventional protein biochemistry techniques to validate protein-protein interactions and PTM status. We will also use biochemical assays to establish whether proteins found to be at reduced levels during mitosis or G1, or, found associated with APC/C regulators, are novel substrates for APC/C activity. Taken together, these data will help further our understanding of APC/C, and the dynamic nature of the cellular proteome.

References:

  1. Sivakumar S, Gorbsky GJ. (2015) Spatiotemporal regulation of the anaphase-promoting complex in mitosis. Nat. Rev. Mol. Cell. Biol. 16:82-94.
  2. Turnell AS, Stewart GS, Grand RJA., Rookes SM, Martin A, Yamano H, Elledge SJ, Gallimore PH. (2005) The APC/C and CBP/p300 cooperate to regulate transcription and cell-cycle progression. Nature 438:690-695.
  3. Townsend K, Mason H, Blackford A N, Miller ES, Chapman JR, Sedgwick GG, Barone G, Turnell AS, Stewart GS (2009) MDC1 regulates mitotic progression. J. Biol. Chem. 284:33939-33948.
  4. Sedgwick GG, Townsend K, Martin A, Shimwell NJ, Grand RJA, Stewart GS, Nilsson J, Turnell AS. (2013) Transcriptional Intermediary Factor 1y binds to the Anaphase-Promoting Complex/Cyclosome and promotes mitosis. Oncogene 32:4622-4633.

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

  • Protein Biochemistry: PAGE, IP/GST pulldowns, Western Blots; bacterial protein expression and purification; biochemical assays – Ubiquitination assays, kinase assays; mass spectrometry-interactomics and post-translational modifications.
  • Molecular Biology: cloning; sequencing; transformation; DNA and RNA purification; qPCR; mutagenesis.
  • Cell Biology: Tissue culture; Transfection- DNA and siRNA; generation of cell lines- FlpIn and plasmid; confocal microscopy; flow cytometry; use of UV irradiation and ionizing radiation.
  • Bioinformatics: Handling large data-sets generated from mass spectrometry; bioinformatics tools for sequences analysis – DNA and Proteins.

Contact: Dr Andy Turnell, University of Birmingham