Phosphorylation of claudin-4 is required for tight junction formation in a human keratinocyte cell line

Exp Cell Res. 2008 Nov 1;314(18):3326-39. doi: 10.1016/j.yexcr.2008.08.012. Epub 2008 Aug 28.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae / genetics
  • Amino Acid Sequence
  • Animals
  • Anthracenes / pharmacology
  • Base Sequence
  • Cell Line
  • Claudin-4
  • Conserved Sequence
  • Dogs
  • Enzyme Inhibitors / pharmacology
  • Fluorescent Antibody Technique
  • Genetic Vectors / genetics
  • Humans
  • Immunoblotting
  • JNK Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • Keratinocytes / cytology
  • Keratinocytes / metabolism*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • Molecular Sequence Data
  • Mutation
  • Pan troglodytes
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein Kinase C / metabolism
  • Rats
  • Sequence Alignment
  • Tight Junctions / drug effects
  • Tight Junctions / metabolism*
  • Zonula Occludens-1 Protein


  • Anthracenes
  • CLDN4 protein, human
  • Claudin-4
  • Cldn4 protein, mouse
  • Enzyme Inhibitors
  • Membrane Proteins
  • Phosphoproteins
  • TJP1 protein, human
  • Tjp1 protein, mouse
  • Tjp1 protein, rat
  • Zonula Occludens-1 Protein
  • pyrazolanthrone
  • PKC-3 protein
  • Protein Kinase C
  • JNK Mitogen-Activated Protein Kinases