Phosphorylation of claudin-4 is required for tight junction formation in a human keratinocyte cell line |
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Authors: | Aono Shinya Hirai Yohei |
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Institution: | Department of Morphoregulation, Institute for Frontier Medical Sciences, Kyoto University, Shogoinkawahara-cho 53, Sakyo-Ku, Kyoto 606-8507, Japan |
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Abstract: | Extensive studies have identified a large number of the molecular components of cellular tight junctions (TJ), including the claudins, occludin and ZO-1/2, and also many of the physical interactions between these molecules. However, the regulatory mechanisms of TJ formation are as yet poorly understood. In HaCaT, a human epidermal keratinocyte cell line, TJ were newly formed when cells were cultured in the presence of SP600125, a JNK inhibitor. Moreover, claudin-4 was newly phosphorylated during this process. We found that claudin-4 contains a sequence which is phosphorylated by atypical PKC (aPKC). Kinase assay demonstrated that the 195th serine (serine195) of mouse claudin-4 was phosphorylated by aPKC in vitro. The 194th serine (serine194) of human claudin-4 corresponding to serine195 of mouse claudin-4 was phosphorylated in HaCaT cells when TJ were formed, and the phosphorylated claudin-4 co-localized with ZO-1 at TJ. aPKC activity was required for both the claudin-4 phosphorylation and TJ formation in HaCaT. Furthermore, overexpression of mutant claudin-4 protein S195A, which was not phosphorylated by aPKC, perturbed the TJ formation mediated by SP600125. These findings suggest that aPKC regulates TJ formation through the phosphorylation of claudin-4. |
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Keywords: | TJ tight junctions aPKC atypical protein kinase C C-DMEM DMEM containing 9 8  mM of calcium SPC-DMEM C-DMEM containing 40  μM of SP600125 AJ adherens junctions NHEK normal human epidermal keratinocyte TER transepithelial resistance |
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