Nonreceptor tyrosine kinase c-Yes interacts with occludin during tight junction formation in canine kidney epithelial cells
Nonreceptor tyrosine kinase c-Yes interacts with occludin during tight junction formation in canine kidney epithelial cells
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DOI：
10.1091/mbc.01-08-0423
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发表时间：
2002-04-01
影响因子：
3.3
通讯作者：
Jeansonne, B
中科院分区：
文献类型：
--
作者：
Chen, YH;Lu, Q;Jeansonne, B
Occludin是一种完整的膜蛋白，当定位在紧密连接时酪氨酸磷酸化。当Ca2+从培养基中消失时，Madin-Darby犬肾上皮细胞的occludin酪氨酸磷酸化在2分钟内减少。这种去磷酸化与经上皮电阻（TER）的显著降低相关，表明紧密连接屏障功能的全局丧失。Ca2+的重构导致occludin的酪氨酸再磷酸化，这与TER的增加暂时相关。此外，我们在这项研究中证明，occludin与非受体酪氨酸激酶c-Yes在细胞连接区域共定位，并在体内与c-Yes形成免疫沉淀复合物。当细胞在没有Ca2+的培养基中孵育或用c-Yes抑制剂CGP77675处理时，该复合物会分离。Ca2+补充后，CGP77675存在时，occludin酪氨酸磷酸化被完全消除，紧密连接的形成和TER的增加都受到抑制。因此，我们的研究提供了强有力的证据，表明occludin酪氨酸磷酸化与上皮细胞中紧密连接的形成密切相关，并且非受体酪氨酸激酶c-Yes参与了这一过程的调节。
Occludin is an integral membrane protein that is tyrosine phosphorylated when localized at tight junctions. When Ca2+ was depleted from the culture medium, occludin tyrosine phosphorylation was diminished from Madin-Darby canine kidney epithelial cells in 2 min. This dephosphorylation was correlated with a significant reduction in transepithelial electrical resistance (TER), indicating a global loss of the tight junction barrier function. Reconstitution of Ca2+ resulted in a robust tyrosine rephosphorylation of occludin that was temporally associated with an increase in TER. Moreover, we demonstrate in this study that occludin was colocalized with the nonreceptor tyrosine kinase c-Yes at cell junction areas and formed an immunoprecipitable complex with c-Yes in vivo. This complex dissociated when the cells were incubated in medium without Ca2+ or treated with a c-Yes inhibitor, CGP77675. In the presence of CGP77675 after Ca2+ repletion, occludin tyrosine phosphorylation was completely abolished and both tight junction formation and the increase of the TER were inhibited. Our study thus provides strong evidence that occludin tyrosine phosphorylation is tightly linked to tight junction formation in epithelial cells, and that the nonreceptor tyrosine kinase c-Yes is involved in the regulation of this process.