Protein phosphatases 2A and 1 interact with occludin and negatively regulate the assembly of tight junctions in the CACO-2 cell monolayer
Protein phosphatases 2A and 1 interact with occludin and negatively regulate the assembly of tight junctions in the CACO-2 cell monolayer
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DOI：
10.1074/jbc.m610597200
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发表时间：
2007-04-13
影响因子：
4.8
通讯作者：
Rao, Radhakrishna
中科院分区：
文献类型：
--
作者：
Seth, Ankur;Sheth, Parimal;Rao, Radhakrishna
在完整的上皮紧密连接（TJ）中，Occludin在Ser和Thr残基上过度磷酸化;然而，这种磷酸化在TJ组装中的作用尚不清楚。在Caco-2细胞中评估了蛋白磷酸酶PP 2A和PP 1对TJ组装和occludin磷酸化的影响。蛋白磷酸酶抑制剂和PP 2A-C α和PP 1 α的表达减少加速了钙诱导的跨上皮电阻和菊粉渗透性屏障的增加，并增强了闭合蛋白和ZO-1在TJ组装过程中的连接组织。闭合蛋白的磷酸化的Thr残基，但不是在Ser残基，大大减少在拆卸TJ和重组过程中逐渐增加。PP 2A和PP 1与occludin共免疫沉淀，这种关联在TJ组装过程中减少。利用重组GST-occludin的GST-转移酶（GST）pull-down分析表明，细胞PP 2A和PP 1与occludin的C-末端尾部结合，并且这些相互作用在TJ组装过程中也被减少。使用GST- occludin和纯化的PP 2A和PP 1的成对结合测定表明PP 2A和PP 1直接与occludin的C-末端尾部相互作用。PP 2A和PP 1与occludin的体外孵育表明PP 2A使occludin的磷酸化Thr残基去磷酸化，而PP 1使occludin的磷酸化Ser残基去磷酸化，表明PP 2A和PP 1直接与occludin相互作用，通过调节occludin的磷酸化状态负调控TJ的组装.
Occludin is hyperphosphorylated on Ser and Thr residues in intact epithelial tight junction ( TJ); however, the role of this phosphorylation in the assembly of TJ is unclear. The influence of protein phosphatases PP2A and PP1 on the assembly of TJ and phosphorylation of occludin was evaluated in Caco-2 cells. Protein phosphatase inhibitors and reduced expression of PP2A-C alpha and PP1 alpha accelerated the calcium-induced increase in transepithelial electrical resistance and barrier to inulin permeability and also enhanced the junctional organization of occludin and ZO-1 during TJ assembly. Phosphorylation of occludin on Thr residues, but not on Ser residues, was dramatically reduced during the disassembly of TJ and was gradually increased during the reassembly. PP2A and PP1 co-immunoprecipitate with occludin, and this association was reduced during the assembly of TJ. Glutathione S-transferase ( GST) pull-down assay using recombinant GST-occludin demonstrated that cellular PP2A and PP1 bind to the C-terminal tail of occludin, and these interactions were also reduced during the assembly of TJ. Apairwise binding assay using GST- occludin and purified PP2A and PP1 demonstrates that PP2A and PP1 directly interacts with the C-terminal tail of occludin. In vitro incubation of phospho- occludin with PP2A or PP1 indicated that PP2A dephosphorylates occludin on phospho- Thr residues, whereas PP1 dephosphorylates it on phospho- Ser. This study shows that PP2A and PP1 directly interact with occludin and negatively regulate the assembly of TJ by modulating the phosphorylation status of occludin.