Controlling kinase activity during the cell cycle: from the DNA damage response to mitosis

Abstract

The cell cycle is the process through which cells execute cell division. This is essential for many basal processes such as organismal development, tissue maintenance and reproduction. Disruption of the cell cycle, for example by damaged DNA, can have vast consequences and can lead to many diseases such as cancer. Therefore, it is of the utmost importance that the state of the cell is continuously monitored during the cell cycle to ensure a correct preparation and execution of cell division. In this thesis, we have studied the role of the kinases Plk1 and Tlk2 during the cell cycle. This thesis contains a comprehensive introduction to the cell cycle with a focus on the role of Plk1 therein. Plk1 is a kinase that is active from the end of S-phase, where DNA replication takes place, until cells have completed cell division. During this period of the cell cycle, Plk1 controls all kinds of essential processes such as centrosome maturation, mitotic entry, checkpoint recovery, spindle assembly, sister chromatid separation, and cytokinesis. During the G2-phase of the cell cycle Plk1 is activated by the kinase Aurora A, an event that depends on binding of Plk1 to the co-activator Bora. We find that during mitosis, levels of Bora are substantially reduced, albeit not complete. However, using a specific FRET-based biosensor for monitoring Plk1 activity in real time we were able to show that the small residual pool of Bora that remains is sufficient to effectively mediate Aurora A-dependent maintenance of Plk1 activity. Furthermore we studied Plk1 activity during the DNA damage response in the G2-phase of the cell cycle, where Plk1 and Aurora A activity are repressed. Surprisingly, the co-activator Bora continues to bind to Plk1 in the early stages of the DNA damage response. Expression of a constitutive active Aurora A was not sufficient to restore Plk1 activity, suggesting that inactivation of Plk1 activity is mediated through recruitment of Aurora A to the Plk1/Bora complex. In addition, we were able to show that Plk1 localizes at the nuclear envelope at the brink of mitosis. Using a selective siRNA-based screening approach for nuclear envelope proteins we were able to identify the nuclear pore protein NUP155 as the recruiting factor of Plk1 to the nuclear envelope. Finally we used an unbiased siRNA-based screening approach to identify new kinases that play a role in recovery from a DNA damage-induced cell cycle arrest. Using this approach we were able to identify the kinase Tlk2. We were able to show that Tlk2 is required for the maintenance of G2-specific proteins during the arrest. Loss of Tlk2 resulted in reduced levels of Cyclin B1 and Plk1 and this resulted in an inability to resume the cell cycle even though the DNA damage foci were resolved. In this context Tlk2 most likely act through the histone chaperone Asf1A, which is required for inserting nucleosomes into newly repaired DNA.