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Investigating 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 Clare Davies, Institute of Cancer and Genomic Sciences

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

University of Registration: University of 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 (DDR), 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). More recently we have established that the DDR protein, 53BP1, is both an inhibitor, and substrate of the APC/C, and regulates entry into, and progression through, mitosis (5).

The main objective of this studentship is to gain further insight into the function and regulation of the APC/C. Specifically, this study aims to identify novel APC/C substrates and characterize the function of individual APC/C subunits in APC/C E3 ubiquitin ligase control. To identify novel APC/C substrates we will characterize the interactomes of Cdc20 and Cdh1 and determine the post-translational status of interacting proteins, by mass spectrometry. We will then investigate the role of the APC/C in regulating the protein levels of Cdc20- and Cdh1- interacting proteins during the cell cycle and in response to cell cycle checkpoint activation. To explore in more detail the role of APC/C subunits in APC/C function we will first investigate the effects of APC/C post-translational modification, or depletion upon cell cycle progression and cell cycle checkpoint activation. We will then characterize the interactomes of individual APC/C subunits, and determine, using the techniques outlined above, whether APC/C- interacting proteins serve to regulate the APC/C, or are substrates for the APC/C.

References

  • Sivakumar S, Gorbsky GJ. (2015) Spatiotemporal regulation of the anaphase-promoting complex in mitosis. Nat. Rev. Mol. Cell. Biol. 16:82-94.
  • 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.
  • 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.
  • Sedgwick GG, Townsend K, Martin A, Shimwell NJ, Grand RJA, Stewart GS, Nilsson J, Turnell AS. (2013) Transcriptional Intermediary Factor 1 binds to the Anaphase-Promoting Complex/Cyclosome and promotes mitosis. Oncogene 32:4622-4633.
  • Kucharski TJ, Minshall PE, Moustafa-Kamal M, Turnell AS, Teodoro JG. (2017) Reciprocal regulation between 53BP1 and the Anaphase-Promoting Complex/Cyclosome is required for genomic stability in response to mitotic stress. Cell Rep. 18(8):1982-1995.

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 Tet-inducible cell lines- FlpIn system, retroviral transduction; confocal microscopy; flow cytometry; Live-cell imaging; use of UV irradiation and ionizing radiation.
  • Bioinformatics: Handling large data-sets generated from mass spectrometry; bioinformatics tools for sequences analysis.

Contact: Dr Andy Turnell, Institute of Cancer and Genomic Sciences