Zeisner, Theresa Ursula; (2024) The role of CDK-opposing phosphatases in cell cycle regulation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Temporal ordering of cell cycle events is ensured by the reversible phosphorylation of hundreds of proteins. This is orchestrated by Cyclin-dependent kinases (CDKs) and counteracting phosphatases. To understand phosphorylation signalling networks, the specific substrates of kinases and phosphatases must be identified. While phosphoproteomic studies have revealed CDK substrates and their phosphorylation timing, the role of CDK-opposing phosphatases in regulating this ordering remains incomplete. Using a phosphoproteomics approach, I identify which CDK sites are dephosphorylated by PP2A-Pab1, PP2A-Par1&2 and Clp1 in fission yeast, by comparing dephosphorylation kinetics of CDK sites in the presence and absence of each of these phosphatases. The majority of CDK sites are dephosphorylated by a specific phosphatase, and only a minority of phosphosites are targeted by multiple phosphatases. The groups of phosphatase substrates differ in their preference for the phosphorylated residue, the surrounding amino acids and short linear interaction motifs. The timing of CDK substrate phosphorylation during a normal cell cycle correlates with which phosphatase targets the site. CDK sites targeted by Clp1 or PP2A-Par1&2 are phosphorylated earlier during the cell cycle, while sites opposed by PP2A-Pab1 are phosphorylated significantly later during the cell cycle. This suggests that PP2A-Pab1 may restrict some CDK substrates from being phosphorylated early during the cell cycle. Using a fluorescent phospho-sensor, based on a PP2A-Pab1 substrate, I show that it is net phosphorylated earlier in the absence of Pab1. This suggests that PP2A-Pab1 actively restricts its substrates from being phosphorylated early during the cell cycle, thereby imposing order on the timing of CDK substrate phosphorylation. Together, these data show that there is a division of labour between different cell cycle phosphatases and highlight the importance of phosphatases in regulating the timing of CDK substrate phosphorylation during the cell cycle.

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