PKCι interacts with Rab14 and modulates epithelial barrier function through regulation of claudin-2 levels.
PKCι interacts with Rab14 and modulates epithelial barrier function through regulation of claudin-2 levels.
复制标题
PKCι与RAB14相互作用，并通过调节Claudin-2水平来调节上皮屏障功能。
DOI：
10.1091/mbc.e14-12-1613
复制
发表时间：
2015-04-15
影响因子：
3.3
通讯作者：
Wilson JM
中科院分区：
文献类型：
--
作者：
Lu R;Dalgalan D;Mandell EK;Parker SS;Ghosh S;Wilson JM
PKCι在上皮极性中具有重要作用。其敲除通过控制密蛋白-2的运输来增加上皮屏障。PKCι直接与小的GTdR ab 14相互作用，并且这些蛋白质在细胞中共定位。有人提出，这些蛋白质共同作用，以调节claudin-2的运输。PKCι对于上皮极性的建立和紧密连接的正常组装是必不可少的。我们发现，PKCι敲低不会损害大多数紧密连接蛋白的稳态分布，但会导致跨上皮阻力（TER）增加和细胞旁通透性降低。对紧密连接组分水平的分析表明紧密连接蛋白-2蛋白水平降低。然而，其他紧密连接蛋白，如claudin-1，ZO-1和occludin，是不变的。用aPKC假底物孵育重现了PKC i敲低的表型，包括TER增加和紧密连接蛋白-2水平降低。此外，PKC 1的过表达导致密蛋白-2水平增加。ELISA和免疫共沉淀显示TGN/内体小GTdR ab 14和PKCι直接相互作用。免疫标记显示，PKC 1和Rab 14共定位于细胞内斑点和质膜上，并且Rab 14表达是3D培养中胞囊中正常PKC 1分布所需的。我们先前表明，Rab 14的敲低导致TER增加和claudin-2减少。我们的研究结果表明，Rab 14和aPKC相互作用，调节运输的claudin-2的溶酶体导向的途径。
PKCι has an essential role in epithelial polarity. Its knockdown increases epithelial barrier through control of the trafficking of claudin-2. PKCι interacts directly with the small GTPase Rab14, and these proteins colocalize in cells. It is proposed that these proteins act together to regulate claudin-2 trafficking. PKCι is essential for the establishment of epithelial polarity and the normal assembly of tight junctions. We find that PKCι knockdown does not compromise the steady-state distribution of most tight junction proteins but results in increased transepithelial resistance (TER) and decreased paracellular permeability. Analysis of the levels of tight junction components demonstrates that claudin-2 protein levels are decreased. However, other tight junction proteins, such as claudin-1, ZO-1, and occludin, are unchanged. Incubation with an aPKC pseudosubstrate recapitulates the phenotype of PKCι knockdown, including increased TER and decreased levels of claudin-2. In addition, overexpression of PKCι results in increased claudin-2 levels. ELISA and coimmunoprecipitation show that the TGN/endosomal small GTPase Rab14 and PKCι interact directly. Immunolabeling shows that PKCι and Rab14 colocalize in both intracellular puncta and at the plasma membrane and that Rab14 expression is required for normal PKCι distribution in cysts in 3D culture. We showed previously that knockdown of Rab14 results in increased TER and decreased claudin-2. Our results suggest that Rab14 and aPKC interact to regulate trafficking of claudin-2 out of the lysosome-directed pathway.