Phosphatase specificity influences the 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 these CDK substrates, yet their collective role in regulating phosphorylation timing in vivo remains unclear. Here, we show that four phosphatases (PP2A-B55, PP2A-B56, CDC14, and PP1) each target distinct subsets of CDK substrate sites in vivo in fission yeast, influencing when phosphorylation occurs during G2 and mitosis. On average, sites dephosphorylated by CDC14 and PP2A-B56 are phosphorylated earlier during G2, followed by sites dephosphorylated by PP1 and PP2A-B55. This suggests that these phosphatases set different phosphorylation thresholds at the G2/M transition. Consistent with this, depleting PP2A-B55 or CDC14 accelerates mitotic onset, likely by advancing phosphorylation of their respective CDK substrates, suggesting these phosphorylation thresholds are important for regulating mitotic onset. Our findings establish in vivo phosphatase substrate specificity as a key factor regulating the timing of CDK substrate phosphorylation throughout the cell cycle.

细胞周期事件的时序由细胞周期蛋白依赖性激酶（cyclin-dependent kinases, CDKs）调控，这类激酶可对数百种底物进行磷酸化修饰。尽管存在多种磷酸酶可拮抗CDK介导的底物磷酸化，但这类磷酸酶在体内对磷酸化时序的集体调控作用仍未明确。本研究证实，在裂殖酵母体内，四种磷酸酶（PP2A-B55、PP2A-B56、CDC14与PP1）各自靶向CDK底物位点的不同子集，并能影响G2期与有丝分裂期的磷酸化发生时机。平均而言，被CDC14与PP2A-B56去磷酸化的位点在G2期更早发生磷酸化，随后是被PP1与PP2A-B55去磷酸化的位点。这表明这些磷酸酶在G2/M转换期设定了不同的磷酸化阈值。与此结论一致，敲低PP2A-B55或CDC14可加速有丝分裂起始，这一效应可能通过提前其对应CDK底物的磷酸化水平实现，提示这类磷酸化阈值对调控有丝分裂起始具有重要意义。本研究结果证实，体内磷酸酶的底物特异性是调控整个细胞周期中CDK底物磷酸化时序的关键因素。