title: Phosphorylation of claudin-2 on serine 208 promotes membrane retention and reduces trafficking to lysosomes.
abstract: Claudins are critical components of epithelial and endothelial tight junction seals, but their post-transcriptional regulation remains poorly understood. Several studies have implicated phosphorylation in control of claudin localisation and/or function, but these have focused on single sites or pathways with differing results, so that it has been difficult to draw general functional conclusions. In this study, we used mass spectrometry (MS) analysis of purified claudin-2 from MDCK II cells and found that the cytoplasmic tail is multiply phosphorylated on serines, a threonine and tyrosines. Phos-tag SDS PAGE revealed that one site, S208, is heavily constitutively phosphorylated in MDCK II cells and in mouse kidney; this site was targeted for further study. Mutational analysis revealed that the phosphomimetic mutant of claudin-2, S208E, was preferentially localised to the plasma membrane while claudin-2 S208A, which could not be phosphorylated at this site, both immunolocalized and co-fractionated with lysosomal markers. Mutations at sites that were previously reported to interfere with plasma membrane targeting of claudin-2 reduced phosphorylation at S208, suggesting that membrane localisation is required for phosphorylation; however phosphorylation at S208 did not affect binding to ZO-1 or ZO-2 Administration of forskolin or PGE2 resulted in dephosphorylation at S208 and transient small increases in transepithelial electrical resistance (TER). Together these data are consistent with phosphorylation at S208 playing a major role in the retention of claudin-2 at the plasma membrane.
authors: National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Christina.vanitallie@nih.gov
authors: Christina M
authors: CM
authors: Van Itallie
authors: Amber Jean
authors: AJ
authors: Tietgens
authors: Kirsten
authors: K
authors: LoGrande
authors: Angel
authors: A
authors: Aponte
authors: Marjan
authors: M
authors: Gucek
authors: James M
authors: JM
authors: Anderson
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D062446:Claudin-2 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005576:Colforsin / Q000008:administration & dosage; D004252:DNA Mutational Analysis; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D009154:Mutation; D054731:PDZ Domains; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport; D012694:Serine / Q000235:genetics* / Q000378:metabolism; D019108:Tight Junctions / Q000235:genetics / Q000378:metabolism
chemical_list: D062446:Claudin-2; D005576:Colforsin; D012694:Serine