Godfrey, MD; (2015) Cell Cycle Regulation by Phosphatases. Doctoral thesis , UCL (University College London).

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Abstract

In eukaryotes, the timing of cell cycle events and transitions is controlled via substrate phosphorylation and dephosphorylation, by kinases and phosphatases. In budding yeast, the ratio of Cdk kinase/Cdk-opposing phosphatase activity regulates mitotic exit by controlling the timing of substrate dephosphorylation in a quantitative manner. The main mitotic exit phosphatase in budding yeast is Cdc14. In interphase and early mitosis, Cdc14 is sequestered in the nucleolus through inhibitory binding to Net1, from which it is released in early anaphase. At this time, Net1 is phosphorylated by Cdk (thus relieving its inhibition of Cdc14), an event promoted by proteins of the Cdc14 Early Anaphase Release (FEAR) network. Later in anaphase, the Mitotic Exit Network (MEN) signaling cascade maintains Cdc14 release. We have yet to understand how Cdc14 activity can increase in early anaphase, while Cdk activity, that is required for Net1 phosphorylation, decreases and the MEN is not yet active. I show that the nuclear rim protein Nur1 interacts with Net1 and, in its Cdk phosphorylated form, inhibits Cdc14 release. Cdc14 dephosphorylates Nur1 in early anaphase, relieving the inhibition and promoting further Cdc14 release. Nur1 dephosphorylation thus describes a positive feedback loop in Cdc14 phosphatase activation during mitotic exit. The importance of Cdk-opposing phosphatase activity in mitotic exit is established, but it is postulated that similarly, Cdk-opposing phosphatase activity in interphase could be an important element in a quantitative model for cell cycle progression. In budding yeast, PP2A, Cdc14, and PP1 are candidates for such a phosphatase. In the absence of PP2A-Cdc55, global interphase Cdk phosphorylation levels are elevated, independently of PP2A’s role in regulating Cdk tyrosine phosphorylation. Strikingly, absence of PP2A-Cdc55 leads to a specific increase in threonine over serine Cdk phosphorylation. I have also identified specific Cdk targets whose phosphorylation is advanced and increased in this case. This indicates that PP2A-Cdc55 may be directly opposing Cdk phosphorylation events throughout interphase, from G1 until the onset of mitosis, thus forming a part of a mechanism that sets Cdk thresholds for cell cycle progression. The roles of PP1 and Cdc14 in interphase remain as yet undetermined.

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