Schuhmacher Diana, Sontag Jean-Marie, Sontag Estelle
School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW, Australia.
Front Cell Dev Biol. 2022 Jul 13;10:911279. doi: 10.3389/fcell.2022.911279. eCollection 2022.
Tight junctions (TJs) are multiprotein complexes essential for cell polarity and the barrier function of epithelia. The major signaling molecule, protein serine/threonine phosphatase 2A (PP2A), interacts with the TJ and modulates the phosphorylation state of TJ proteins. An important PP2A regulatory mechanism involves leucine carboxyl methyltransferase-1 (LCMT1)-dependent methylation and protein phosphatase methylesterase-1 (PME1)-mediated demethylation of its catalytic subunit on Leu309. Here, using MDCK cells, we show that overexpression of LCMT1, which enhances cellular PP2A methylation, inhibits TJ formation, induces TJ ruffling, and decreases TJ barrier function. Conversely, overexpression of PME1 accelerates TJ assembly and enhances TJ barrier function. PME1-dependent PP2A demethylation increases during early Ca-dependent junctional assembly. Inhibition of endogenous PME1 delays the initial Ca-mediated redistribution of TJ proteins to cell-cell contacts and affects TJ morphology and barrier function. Manipulating one-carbon metabolism modulates TJ assembly, at least in part by affecting PP2A methylation state. The integrity of PP2A methylation is critical for proper targeting of PP2A to the TJ. It is necessary for PP2A complex formation with the TJ proteins, occludin and ZO-1, and proteins of the PAR complex, Par3 and atypical protein kinase C ζ (aPKCζ), which play a key role in development of cell polarity. Expression of a methylation incompetent PP2A mutant induces defects in TJ assembly and barrier function. aPKCζ-mediated Par3 phosphorylation is also required for targeting of the PP2A ABαC holoenzyme to the TJ. Our findings provide the first evidence for a role of LCMT1, PME1 and PP2A methylation/demethylation processes in modulating TJ assembly and functional integrity. They also position PP2A at the interface of one-carbon metabolism and the regulation of key TJ and polarity proteins that become deregulated in many human diseases.
紧密连接（TJs）是多蛋白复合物，对于细胞极性和上皮细胞的屏障功能至关重要。主要信号分子蛋白丝氨酸/苏氨酸磷酸酶2A（PP2A）与紧密连接相互作用，并调节紧密连接蛋白的磷酸化状态。一种重要的PP2A调节机制涉及亮氨酸羧基甲基转移酶-1（LCMT1）依赖性甲基化以及蛋白磷酸酶甲基酯酶-1（PME1）介导的其催化亚基在Leu309位点的去甲基化。在此，我们利用MDCK细胞表明，增强细胞PP2A甲基化的LCMT1过表达会抑制紧密连接形成、诱导紧密连接褶皱，并降低紧密连接屏障功能。相反，PME1过表达会加速紧密连接组装并增强紧密连接屏障功能。在早期钙依赖性连接组装过程中，PME1依赖性PP2A去甲基化增加。抑制内源性PME1会延迟紧密连接蛋白最初由钙介导的向细胞-细胞接触部位的重新分布，并影响紧密连接形态和屏障功能。操纵一碳代谢至少部分通过影响PP2A甲基化状态来调节紧密连接组装。PP2A甲基化的完整性对于PP2A正确靶向紧密连接至关重要。这对于PP2A与紧密连接蛋白闭合蛋白和ZO-1以及PAR复合物蛋白Par3和非典型蛋白激酶Cζ（aPKCζ）形成复合物是必要的，这些蛋白在细胞极性发展中起关键作用。甲基化无活性的PP2A突变体的表达会诱导紧密连接组装和屏障功能缺陷。aPKCζ介导的Par3磷酸化对于PP2A ABαC全酶靶向紧密连接也是必需的。我们的研究结果首次证明了LCMT1、PME1和PP2A甲基化/去甲基化过程在调节紧密连接组装和功能完整性中的作用。它们还将PP2A定位在一碳代谢与关键紧密连接和极性蛋白调节的界面，而这些蛋白在许多人类疾病中会失调。
