Phosphorylation of claudin-4 is required for tight junction formation in a human keratinocyte cell line.
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.
Aono
Shinya
S
Department of Morphoregulation, Institute for Frontier Medical Sciences, Kyoto University, Shogoinkawahara-cho 53, Sakyo-Ku, Kyoto 606-8507, Japan. midnight@frontier.kyoto-u.ac.jp
Hirai
Yohei
Y
D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D062506:Claudin-4; D017124:Conserved Sequence; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D002679:Pan troglodytes; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein
D000873:Anthracenes; C568157:CLDN4 protein, human; D062506:Claudin-4; C568158:Cldn4 protein, mouse; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; C432165:pyrazolanthrone; C115015:PKC-3 protein; D011493:Protein Kinase C; D048031:JNK Mitogen-Activated Protein Kinases