Phosphatase specificity influences phosphorylation timing of CDK substrates during the cell cycle

Cell cycle events are ordered by cyclin-dependent kinases (CDKs), which phosphorylate hundreds of substrates. Multiple phosphatases oppose CDK substrate phosphorylation, yet a systematic understanding of how these phosphatases collectively influence phosphorylation timing is lacking. Here, we show that phosphatases influence the timing of CDK substrate phosphorylation during G2 and mitosis in fission yeast. We identify substrates of four phosphatases (PP2A-B55, PP2A-B56, CDC14, and PP1), showing that each phosphatase targets a distinct subset of CDK substrate sites. On average, sites dephosphorylated by CDC14 and PP2A-B56 are phosphorylated earlier during G2, followed by sites dephosphorylated by PP1 and then PP2A-B55. This suggests that the identity of the phosphatase impacts the timing of CDK substrate phosphorylation, establishing different phosphorylation thresholds at the G2/M transition. Consistent with this, depletion of PP2A-B55 and CDC14 advances mitotic onset independently of CDK activity regulation, likely due to the earlier phosphorylation of their respective CDK substrates.