Protein phosphatase 2A (PP2A) is an essential serine/threonine protein phosphatase, and its dysfunction is involved in the onset of cancer and neurodegenerative disorders. PP2A functions as a trimeric holoenzyme whose composition is regulated by the methyl-esterification (methylation) of the PP2A catalytic subunit (PP2Ac). Protein phosphatase methylesterase-1 (PME-1) is the sole PP2Ac methylesterase, and the higher PME-1 expression is observed in various cancer and neurodegenerative diseases. Apart from serving as a methylesterase, PME-1 acts as a PP2A inhibitory protein, binding directly to PP2Ac and suppressing its activity. The intricate function of PME-1 hinders drug development by targeting the PME-1/PP2Ac axis. This study applied the NanoBiT system, a bioluminescence-based protein interaction assay, to elucidate the molecular mechanism that modulates unknown PME-1/PP2Ac protein–protein interaction (PPI). Compound screening identified that the CHK1 inhibitors inhibited PME-1/PP2Ac association without affecting PP2Ac methylation levels. CHK1 directly phosphorylates PP2Ac to promote PME-1 association. Phospho-mass spectrometry identified multiple phospho-sites on PP2Ac, including the Thr219, that affect PME-1 interaction. An anti-phospho-Thr219 PP2Ac antibody was generated and showed that CHK1 regulates the phosphorylation levels of this site in cells. On the contrary, phosphatase assay showed that CHK1 is the substrate of PP2A, and PME-1 hindered PP2A-mediated dephosphorylation of CHK1. Our data provides novel insights into the molecular mechanisms governing the PME-1/PP2Ac PPI and the triad relationship between PP2A, PME-1, and CHK1.

中文翻译：

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基于荧光素酶的体内蛋白质-蛋白质相互作用测定表明 CHK1 促进 PP2A 和 PME-1 相互作用


蛋白磷酸酶 2A (PP2A) 是一种必需的丝氨酸/苏氨酸蛋白磷酸酶，其功能障碍与癌症和神经退行性疾病的发病有关。 PP2A 作为三聚体全酶发挥作用，其组成由 PP2A 催化亚基 (PP2Ac) 的甲基酯化（甲基化）调节。蛋白磷酸酶甲酯酶-1 (PME-1) 是唯一的 PP2Ac 甲酯酶，在各种癌症和神经退行性疾病中观察到较高的 PME-1 表达。除了充当甲酯酶外，PME-1 还充当 PP2A 抑制蛋白，直接与 PP2Ac 结合并抑制其活性。 PME-1 的复杂功能通过靶向 PME-1/PP2Ac 轴阻碍药物开发。本研究应用 NanoBiT 系统（一种基于生物发光的蛋白质相互作用测定法）来阐明调节未知 PME-1/PP2Ac 蛋白质-蛋白质相互作用 (PPI) 的分子机制。化合物筛选发现 CHK1 抑制剂抑制 PME-1/PP2Ac 关联而不影响 PP2Ac 甲基化水平。 CHK1 直接磷酸化 PP2Ac 以促进 PME-1 结合。磷质谱鉴定出 PP2Ac 上的多个磷酸位点，包括影响 PME-1 相互作用的 Thr219。生成了抗磷酸化 Thr219 PP2Ac 抗体，并表明 CHK1 调节细胞中该位点的磷酸化水平。相反，磷酸酶检测显示CHK1是PP2A的底物，PME-1阻碍PP2A介导的CHK1去磷酸化。我们的数据为控制 PME-1/PP2Ac PPI 的分子机制以及 PP2A、PME-1 和 CHK1 之间的三联关系提供了新的见解。