Cyclins on the move : a time and a place for cyclin A2 and cyclin B1 in the human cell cycle

Abstract

The ultimate aim of the cell cycle is to create an identical daughter cell. Therefore, correct progression through the different phases of the cell cycle is crucial to ensure faithful cell division. Successful execution of the different processes in the cell cycle is achieved by the coordinated action of a complex network of protein kinases and phosphatases at the centre of which stand Cyclin-Cdk complexes. Human cells possess a variety of cyclins and Cdks, which form complexes that regulate cell cycle transitions.

In an unperturbed cell cycle, preparing a cell for mitosis requires faithful DNA replication and reorganisation of the cell’s structures and organelles. In this scenario, cells initiate successive waves of Cdk activity that orchestrate the timely and spatially controlled phosphorylation of a multitude of targets. In contrast, upon DNA damage cells must halt cell cycle progression in order to prevent mitotic entry of damaged cells and subsequently avoid potential propagation of mutations.

Strict control of Cyclin-Cdk complexes is, therefore, essential both for correct cell division and to maintain genome integrity. However, the exact mechanisms underlying the activation of Cyclin- Cdk complexes in these different scenarios remain largely unknown. In this thesis, I have investigated several aspects of the regulation of Cdk activity both in the unperturbed cell cycle and during a DNA damage response.

To address Cdk activity in the unperturbed cell cycle we established a novel quantitative immunofluorescence method and assessed the dynamics of cyclin accumulation and Cdk target phosphorylation in the unperturbed cell cycle. We found that the mitotic entry network first becomes activated at the S/G2 transition. This finding shifts the classical view of an abrupt Cdk activation at mitotic entry to an earlier and more gradual activation. Furthermore, it provides a potential link between S phase and mitosis, suggesting the existence of a mechanism that maintains pro-mitotic activities under a certain threshold until DNA replication is completed (Paper I).

Interestingly, in parallel to an increase of pro-mitotic activities at the S/G2 transition, we observed a change in the localisation of Cyclin A2. Using genome-edited cell lines that express endogenous Cyclin A2-eYFP we were able to determine the cell cycle-dependent localisation of Cyclin A2 to the cytoplasm. Interestingly, despite coinciding with an increase of Cdk activity in the cell cycle we found that the cytoplasmic accumulation Cyclin A2 is modulated by p21 and the presence rather than activity of Cdk1. These findings suggest that complex formation and interaction with Cdk inhibitor proteins (CKI) might regulate Cyclin A2 localisation throughout the cell cycle (Paper IV). Despite not having uncovered a role for cytoplasmic Cyclin A2, we hypothesise that the cell cycle-dependent localisation of cyclins may be an important step to regulate Cdk activity.

In order to understand how cells modulate Cdk activity upon DNA damage we made use of endogenously tagged cell lines expressing Cyclin B1-eYFP. We found that upon DNA damage cells continue to accumulate Cyclin B1 until reaching levels that are normally present in G2 phase. At this point, cells translocate Cyclin B1 to the nucleus in a p21 and p53- dependent manner where it is degraded by APC/CCdh1. We identified nuclear translocation and degradation of Cyclin B1 as a restriction point in the cell cycle when cells irreversibly exit the cell cycle and become senescent (Paper II).

Senescence is regarded as an early barrier for tumorigenesis as it prevents the propagation of cells with damaged DNA. Our findings in Paper II suggested a link between mitotic inducers and the induction of senescence; therefore we decided to investigate the role of Cdk activity in terminal cell cycle exit. We found that upon DNA damage cells preserve low levels of Cdk activity to ensure that damaged cells continue to progress through the cell cycle until they reach a point where they can be forced into senescence. In this context, we found that Cdk activity induces p21 expression in a p53-independent manner to promote nuclear translocation and degradation of Cyclin B1 and other mitotic inducers (Paper III).

Altogether, the data presented in this thesis points towards the existence of a link between the mitotic entry network and the DNA damage response to modulate the activity of Cyclin-Cdk complexes in time and space to trigger ensure correct progression to mitosis or, when needed, to trigger senescence.