Evidence for substrate control of Cdk phosphorylation timing during the budding yeast cell cycle 2522

The main intention of this project is to investigate how Cdk substrate phosphorylation is ordered during the cell cycle. We want to understand what makes a ‘good’ Cdk substrate that is phosphorylated early during the cell cycle, e. g. to promote S phase, as compared to a ‘bad’ Cdk substrate that is phosphorylated much later, e. g. when cells enter mitosis. This could be because a late substrate is indeed a poor Cdk substrate, or it could be that the late substrate is a good substrate for Cdk counteracting phosphatases. The two possibilities are not mutually exclusive. We want to compare unperturbed phosphorylation and rephosphorylation from an artificially low phosphorylation state in metaphase arrest. To do that, we arrest cells in metaphase, inhibit Cdk until substrates get dephosphorylated, and reactivate Cdk and record the time and kinetics of the substrate rephosphorylation.