The global dynamics of CDK substrate phosphorylation in a simplified CDK network

Cyclin-CDK complexes initiate S-phase and mitosis, but how these events are temporally separated is not fully understood. In S. pombe a single Cyclin-CDK is sufficient to order the cell cycle. Understanding how and when CDK substrates are phosphorylated, during the cell cycle, will inform how CDK orders cell cycle events. To this end, I have combined in vivo chemical inhibition of CDK and phosphoproteomics to study the behaviour of CDK-mediated phosphorylation. Hundreds of CDK-dependent phosphorylation events have been identified and their cell cycle phosphorylation dynamics quantified. CDK substrates falls into three categories: i) the vast majority become phosphorylated during mitosis, ii) S-phase substrates, which are first phosphorylated at G1/S and are stable until mitosis, and iii) biphasic substrates that increases in phosphorylation at both transitions but peak at G2/M. All three classes are dephosphorylated simultaneously at mitotic exit. S-phase substrates are more sensitive to CDK activity in vivo than mitotic substrates. The causal relationship between CDK activity, substrate phosphorylation, and cell cycle fate is corroborated by the fact that when the timing of substrate phosphorylation is reordered, the order of cell cycle events is correspondingly reorganised. These data argue that rising CDK activity orders the cell cycle via the attainment of substrate-specific thresholds. Low activity results in S-phase substrate phosphorylation, and the initiation of DNA synthesis, whilst higher CDK activity results in mitotic substrate phosphorylation and chromosome segregation. Furthermore, the timing of mitotic substrate phosphorylation during G2/M correlates with sensitivity to CDK activity, indicating that passage through sequential CDK activity thresholds orders substrate phosphorylation, and likely mitotic events, during the entry into mitosis. Phosphoproteomic analysis also indicates that G1/S cyclins contribute mainly to the accumulation of CDK activity, and that phosphorylation by other mitotic kinases is organised into sequential waves, allowing the amplification and diversification of CDK signalling