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Title: Quantitative analysis of regulated proteolysis and protein dephosphorylation during mitotic exit
Author: Holder, James
ISNI:       0000 0004 7966 1265
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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The critical function of the cell cycle is to enable the faithful inheritance of genetic material. Correct attachment of chromosomes triggers degradation of cyclin B and entry into anaphase, the cell subsequently undergoing a series of rapidly occurring changes. Precise regulation of these events is necessary to ensure that they take place in the correct order and is crucial for the success of mitotic exit. This regulation has been attributed to the activity of both phosphatases and the anaphase-promoting complex/cyclosome (APC/C), but the relative contribution of each process remains unclear. Using a quantitative, high-resolution mass spectrometric analysis, this work shows that the order of events in anaphase is controlled by a series of graded dephosphorylations whose rate is encoded into the system at the level of each individual phospho-site, based on the site's sequence characteristics. Phospho-serines surrounded by acidic residues are seen to be dephosphorylated more slowly than more basic phospho-threonine sites. This preference also applies to PP1 dependent dephosphorylations and is consistent with the previously identified substrate specificity of PP2A-B55. This indicates a general rate-determining principle of phospho-protein phosphatase-mediated dephosphorylation. In addition, the results presented in this thesis indicate that PP1 activates itself following CDK1 inactivation, promoting the dephosphorylation of over 1000 phospho-sites. Finally, in the absence of CDK activity, APC/C-dependent proteolysis is not necessary for ordered dephosphorylation. A single proteolytic event, the destruction of cyclin B, is therefore sufficient for orderly mitotic exit through relieving inhibition of phosphatase activity.
Supervisor: Barr, Francis Sponsor: Wellcome Trust ; Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry ; Biology ; Mitosis ; Cell cycle ; Mass spectrometry