title: PKCι interacts with Rab14 and modulates epithelial barrier function through regulation of claudin-2 levels.
abstract: 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.
authors.affiliation: Department of Cellular and Molecular Medicine, University of Arizona College of Medicine, Tucson, AZ 85724.
authors.forename: Ruifeng
authors.initials: R
authors.lastname: Lu
authors.affiliation: Department of Cellular and Molecular Medicine, University of Arizona College of Medicine, Tucson, AZ 85724.
authors.forename: Dogukan
authors.initials: D
authors.lastname: Dalgalan
authors.affiliation: Department of Neurology, Yale University School of Medicine, New Haven, CT 06511.
authors.forename: Edward K
authors.initials: EK
authors.lastname: Mandell
authors.affiliation: Department of Cellular and Molecular Medicine, University of Arizona College of Medicine, Tucson, AZ 85724.
authors.forename: Sara S
authors.initials: SS
authors.lastname: Parker
authors.affiliation: Department of Neurology, Yale University School of Medicine, New Haven, CT 06511.
authors.forename: Sourav
authors.initials: S
authors.lastname: Ghosh
authors.affiliation: Department of Cellular and Molecular Medicine, University of Arizona College of Medicine, Tucson, AZ 85724 jeanw@email.arizona.edu.
authors.forename: Jean M
authors.initials: JM
authors.lastname: Wilson
mesh_terms: D000818:Animals; D062446:Claudin-2 / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D010539:Permeability; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D019108:Tight Junctions / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000099293:Protein Kinase C-lambda
chemical_list: D062446:Claudin-2; D007527:Isoenzymes; D011493:Protein Kinase C; D020691:rab GTP-Binding Proteins; D000099293:Protein Kinase C-lambda; C553411:Rab14 protein, human