Characterisation of WEE1 regulation at mitosis
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Embargo End Date
ICR Authors
Authors
Johnson, V
Document Type
Thesis or Dissertation
Date
2024-08-22
Date Accepted
Abstract
Wee1 is a tyrosine kinase that plays a major role in regulating mitotic entry. Throughout G2 phase, it phosphorylates and inhibits Cdk1, the master mitotic regulator, thereby preventing mitotic entry. At the end of G2 phase Wee1 is inactivated, which causes the rapid activation of Cdk1 and irreversible entry into mitosis. It is important, therefore, that Wee1 activity is tightly regulated, to avoid premature mitotic entry in the face of incomplete DNA replication or cell stress including DNA damage. However, the regulation of Wee1 in human cells is poorly understood. My thesis aims to address this gap in knowledge by characterising Wee1 behaviour in RPE-1 cells. I use CRISPR-Cas9 technology to generate cell lines in which endogenous Wee1 is tagged with a fluorescent protein. Using live cell fluorescence microscopy, I show that Wee1 is neither degraded nor exported from the nucleus prior to mitosis, in contrast to previous reports. I also characterise the changes in phosphorylation of Wee1 across the cell cycle using mass spectrometry and identify a single phosphorylation site (S312) that, when mutated, causes a striking delay in G2/M progression. I use both live cell microscopy and immunofluorescence to show that expression of this Wee1 mutant causes defects in chromosome segregation and in the degradation of Cyclin A2 and B1 by APC/C during late mitosis. Finally, I carry out in vitro kinase assays to characterise the effect of this phosphosite mutation on the kinase activity of Wee1.
Citation
2024
DOI
Source Title
Publisher
Institute of Cancer Research (University Of London)
ISSN
eISSN
Collections
Research Team
Cell Division