PDF(Pubmed)
Abstract：
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, in vitro 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.
摘要：
蛋白磷酸酶2A（PP2A）是一种必需的丝氨酸/苏氨酸蛋白磷酸酶，它的功能障碍与癌症和神经退行性疾病的发生有关。PP2A充当三聚体全酶，其组成受PP2A催化亚基（PP2Ac）的甲酯化（甲基化）调节。蛋白磷酸酶甲基酯酶-1（PME-1）是唯一的PP2Ac甲基酯酶，在各种癌症和神经退行性疾病中观察到较高的PME-1表达。除了作为甲基酯酶，PME-1作为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轴来阻碍药物开发。本研究应用NanoBiT系统，基于生物发光的蛋白质相互作用测定，阐明调节未知PME-1/PP2Ac蛋白-蛋白相互作用（PPI）的分子机制。化合物筛选鉴定CHK1抑制剂抑制PME-1/PP2Ac缔合而不影响PP2Ac甲基化水平。CHK1直接磷酸化PP2Ac以促进PME-1缔合。磷酸质谱鉴定了PP2Ac上的多个磷酸位点，包括影响PME-1相互作用的Thr219。产生了抗磷酸-Thr219PP2Ac抗体，并显示CHK1调节细胞中该位点的磷酸化水平。相反,体外磷酸酶测定显示CHK1是PP2A的底物,和PME-1阻碍PP2A介导的CHK1去磷酸化。我们的数据为控制PME-1/PP2AcPPI的分子机制和PP2A之间的三联体关系提供了新的见解。PME-1和CHK1。
