The tumour suppressor p53 is a key regulatory protein that prevents proliferation of damaged cells. Under unperturbed conditions, the ubiquitin ligase murine double minute 2 (MDM2) mediates p53 ubiquitination and further degradation by the proteasome. In consequence p53 is present at low levels, but becomes rapidly stabilised and activated in response to a variety of stimuli, such as DNA damage or virus infection. P53 responds to these diverse stresses to regulate the expression of many target genes that induce cell cycle arrest, DNA repair, or apoptosis. The attenuation of p53 interaction with MDM2 is maintained by enzymes catalysing p53 post-translational modifications such as phosphorylation. Casein kinase 1 α (CK1α) is one such enzyme; it stimulates p53 after DNA virus infection. Surprisingly depletion of CK1α using small interfering RNA or inhibition using a CK1 kinase inhibitor activated the transcription factor p53, indicating that p53 steady-state level is controlled by CK1α. Disrupting MDM2-p53 interaction using small molecule Nutlin-3 displayed similar pharmacological properties to the CK1 inhibitor on p53, indicating that the MDM2-CK1α complex co-regulates p53 stability. Indeed co-immunoprecipitation of endogenous CK1α with MDM2 occurred in undamaged cells. CK1α was shown in vitro to directly bind to and phosphorylate MDM2. Therefore it appears that CK1α must be recruited into specific complexes under different conditions, which can influence its substrate selectivity and explain its dual role on the p53 pathway. Apart from CK1, there are few other kinases whose action can directly contribute to the inhibition of p53. A novel pyrazolo-pyridine analogue showing dual activity against CK1 and Checkpoint kinase 1 led to increased p53 activation. These data highlighted the potential value of dual kinase inhibitors as therapeutics in cancer. The dominant protein-protein interface that stabilises the MDM2-CK1α complex was mapped using a peptide-based approach. One CK1α peptide bound strongly to MDM2, it specifically disrupted the protein-protein interaction, and its transfection was able to reduce cancer cell growth. A peptide phage display approach was finally combined with Next-Generation Sequencing to define the change in MDM2 binding motifs when the CK1α peptide or Nutlin-3 is bound, compared to ligand-free MDM2, and thus will help to understand protein-protein interaction network re-wirings which led to cell growth inhibition.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:699955 |
| Date | January 2014 |
| Creators | Huart, Anne-Sophie |
| Contributors | Hupp, Ted |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/17282 |
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