title: Assembly of epithelial tight junctions is negatively regulated by Par6.
abstract: Epithelial cells display apical-basal polarity, and the apical surface is segregated from the basolateral membranes by a barrier called the tight junction (TJ). TJs are constructed from transmembrane proteins that form cell-cell contacts-claudins, occludin, and junctional adhesion molecule (JAM)-plus peripheral proteins such as ZO-1. The Par proteins (partitioning-defective) Par3 and Par6, plus atypical protein kinase C (aPKC) function in the formation or maintenance of TJs and more generally in metazoan cell polarity establishment. Par6 contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain and binds the small GTPase Cdc42. Here, we show that Par6 inhibits TJ assembly in MDCK II epithelial cells after their disruption by Ca(2+) depletion but does not inhibit adherens junction (AJ) formation. Transepithelial resistance and paracellular diffusion assays confirmed that assembly of functional TJs is delayed by Par6 overexpression. Strikingly, the isolated, N-terminal fragment of PKCzeta, which binds Par6, also inhibits TJ assembly. Activated Cdc42 can disrupt TJs, but neither a dominant-negative Cdc42 mutant nor the CRIB domain of gammaPAK (p21-activated kinase), which inhibits Cdc42 function, observably inhibit TJ formation. These results suggest that Cdc42 and Par6 negatively regulate TJ assembly in mammalian epithelial cells.
authors.affiliation: Center for Cell Signaling and Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA 22908-0577, USA.
authors.forename: Lin
authors.initials: L
authors.lastname: Gao
authors.forename: Gérard
authors.initials: G
authors.lastname: Joberty
authors.forename: Ian G
authors.initials: IG
authors.lastname: Macara
mesh_terms: D022005:Adherens Junctions / Q000378:metabolism / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004058:Diffusion; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D019108:Tight Junctions / Q000378:metabolism*; D013997:Time Factors; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D000099221:Protein Kinase C zeta
chemical_list: D002118:Calcium; D020033:Protein Isoforms; D011493:Protein Kinase C; D011506:Proteins; D020764:cdc42 GTP-Binding Protein; D000099221:Protein Kinase C zeta