The spatiotemporal control of CDK activity
File(s)
Author(s)
Roberts, Emma
Type
Thesis
Abstract
The oscillating activity of cyclin-dependent kinases (CDKs) in complex with cyclins drives progression through the eukaryotic cell cycle. It has long been appreciated that the temporal control of cyclin-CDK activity is critical to cell cycle progression, but there is increasing evidence that the spatial control of cyclin-CDK activity is also important in cell cycle regulation. The centrosome (metazoa) and spindle pole body (SPB, yeast) act as key signalling hubs in cell cycle transitions including mitotic entry. Understanding CDK localisation and regulation at the centrosome and SPB will inform our understanding of the importance of CDK spatial regulation and how it impacts cell cycle progression.
I have studied the localisation of the mitotic cyclin Cdc13 in the fission yeast S. pombe, identifying that the hydrophobic patch region of Cdc13 is responsible for Cdc13 localisation to the SPB in early G2. A hydrophobic patch mutant (HPM) of Cdc13 does not localise to the SPB, and does not enter mitosis. Restoring the SPB localisation of Cdc13HPM-Cdc2 promotes a wave of mitotic entry, leading to the hypothesis that cyclin-CDK activity at the SPB is essential for entry into mitosis. Unlike human Cyclin B1, Cyclin B1HPM is not detected at the centrosome, suggesting that the involvement of the Cyclin B hydrophobic patch in SPB and centrosomal localisation is similar between fission yeast and human cells.
I also show that there is a hydrophobic patch-independent mechanism of Cdc13 localisation to the SPB after Polo kinase localisation to the SPB in late G2/mitosis, which operates when there is a wild-type copy of Cdc13 present in cells. Finally, I present initial work developing a system to compare CDK substrate phosphorylation across different subcellular locations. Overall, this work supports the body of work indicating that the SPB and centrosome play an important role in mitotic entry.
I have studied the localisation of the mitotic cyclin Cdc13 in the fission yeast S. pombe, identifying that the hydrophobic patch region of Cdc13 is responsible for Cdc13 localisation to the SPB in early G2. A hydrophobic patch mutant (HPM) of Cdc13 does not localise to the SPB, and does not enter mitosis. Restoring the SPB localisation of Cdc13HPM-Cdc2 promotes a wave of mitotic entry, leading to the hypothesis that cyclin-CDK activity at the SPB is essential for entry into mitosis. Unlike human Cyclin B1, Cyclin B1HPM is not detected at the centrosome, suggesting that the involvement of the Cyclin B hydrophobic patch in SPB and centrosomal localisation is similar between fission yeast and human cells.
I also show that there is a hydrophobic patch-independent mechanism of Cdc13 localisation to the SPB after Polo kinase localisation to the SPB in late G2/mitosis, which operates when there is a wild-type copy of Cdc13 present in cells. Finally, I present initial work developing a system to compare CDK substrate phosphorylation across different subcellular locations. Overall, this work supports the body of work indicating that the SPB and centrosome play an important role in mitotic entry.
Version
Open Access
Date Issued
2022-02
Date Awarded
2022-08
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Nurse, Paul
Aragon Alcaide, Luis
Sponsor
Cancer Research UK
Wellcome Trust (London, England)
Medical Research Council (Great Britain)
The Lord Leonard and Lady Estelle Wolfson Foundation
Grant Number
FC01121
214183
Publisher Department
Institute of Clinical Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)