The reciprocal regulation of phosphoprotein phosphatases (PPPs) by protein kinases is essential to cell cycle progression and control, particularly during mitosis for which the role of kinases has been extensively studied. PPPs perform much of the serine/threonine dephosphorylation in eukaryotic cells and achieve substrate selectivity and specificity through the interaction of distinct regulatory subunits with conserved catalytic subunits in holoenzyme complexes. Using a mass spectrometry–based chemical proteomics approach to enrich, identify, and quantify endogenous PPP holoenzyme complexes combined with kinase profiling, we investigated the phosphorylation-dependent regulation of PPP holoenzymes in mitotic cells. We found that cyclin-dependent kinase 1 (CDK1) phosphorylated a threonine residue on the catalytic subunit of the phosphatase PP2A, which disrupted its holoenzyme formation with the regulatory subunit B55. The consequent decrease in the dephosphorylation of PP2A-B55 substrates promoted mitotic entry. This direct phosphorylation by CDK1 was in addition to a previously reported indirect mechanism, thus adding a layer to the interaction between CDK1 and PP2A in regulating mitotic entry.

蛋白激酶对磷蛋白磷酸酶 (PPP) 的相互调节对于细胞周期进程和控制至关重要，特别是在有丝分裂期间，激酶的作用已得到广泛研究。PPP 在真核细胞中执行大部分丝氨酸/苏氨酸去磷酸化，并通过不同调节亚基与全酶复合物中保守催化亚基的相互作用实现底物选择性和特异性。使用基于质谱的化学蛋白质组学方法来富集、鉴定和量化内源性 PPP 全酶复合物，并结合激酶分析，我们研究了有丝分裂细胞中 PPP 全酶的磷酸化依赖性调节。我们发现细胞周期蛋白依赖性激酶 1 (CDK1) 磷酸化磷酸酶 PP2A 催化亚基上的苏氨酸残基，它用调节亚基 B55 破坏了其全酶的形成。PP2A-B55 底物去磷酸化的随之减少促进了有丝分裂的进入。CDK1 的这种直接磷酸化是先前报道的间接机制的补充，从而为 CDK1 和 PP2A 之间调节有丝分裂进入的相互作用增加了一层。