title: JNK1/2-dependent phosphorylation of angulin-1/LSR is required for the exclusive localization of angulin-1/LSR and tricellulin at tricellular contacts in EpH4 epithelial sheet.
abstract: Tricellular tight junctions (tTJs) are specialized structural variants of tight junctions within tricellular contacts of an epithelial sheet and comprise several transmembrane proteins including lipolysis-stimulated lipoprotein receptor (angulin-1/LSR) and tricellulin. To elucidate the mechanism of its formation, we carried out stepwise screening of kinase inhibitors followed by RNAi screening to identify kinases that regulate intracellular localization of angulin-1/LSR to the tTJs using a fluorescence image-based screen. We found that the activity of JNK1 and JNK2, but not JNK3, was required for the exclusive localization of angulin-1/LSR at the tTJs. Based on a bioinformatics approach, we estimated the potential phosphorylation site of angulin-1/LSR by JNK1 to be serine 288 and experimentally confirmed that JNK1 directly phosphorylates angulin-1/LSR at this site. We found that JNK2 was also involved in the phosphorylation of angulin-1/LSR. Furthermore, GFP-tagged angulin-1/LSR(S288A), in which serine 288 was substituted by alanine, was observed to be dispersed to bicellular junctions, indicating that phosphorylation of Ser288 is crucial for the exclusive localization of angulin-1/LSR and tricellulin at tTJs. Our fluorescence image-based screening for kinases inhibitor or siRNAs combined with the phosphorylation site prediction could become a versatile and useful tool to elucidate the mechanisms underlying the maintenance of tTJs regulated by kinase networks.
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo, 153-8902, Japan.
authors.forename: Daiki
authors.initials: D
authors.lastname: Nakatsu
authors.forename: Fumi
authors.initials: F
authors.lastname: Kano
authors.forename: Yuki
authors.initials: Y
authors.lastname: Taguchi
authors.forename: Taichi
authors.initials: T
authors.lastname: Sugawara
authors.forename: Takashi
authors.initials: T
authors.lastname: Nishizono
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Nishikawa
authors.forename: Yukako
authors.initials: Y
authors.lastname: Oda
authors.forename: Mikio
authors.initials: M
authors.lastname: Furuse
authors.forename: Masayuki
authors.initials: M
authors.lastname: Murata
mesh_terms: D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D047310:Apigenin / Q000494:pharmacology; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism*; D062794:MARVEL Domain Containing 2 Protein / Q000378:metabolism*; D051379:Mice; D048055:Mitogen-Activated Protein Kinase 8 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048056:Mitogen-Activated Protein Kinase 9 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009570:Nitriles; D010766:Phosphorylation; D018110:Receptors, Lipoprotein / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism*; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: D000873:Anthracenes; D047310:Apigenin; D062794:MARVEL Domain Containing 2 Protein; D048055:Mitogen-Activated Protein Kinase 8; D048056:Mitogen-Activated Protein Kinase 9; D009570:Nitriles; D018110:Receptors, Lipoprotein; D012694:Serine; D020032:Tyrphostins; C508309:Marveld2 protein, mouse; C000592022:LSR protein, mouse; C432165:pyrazolanthrone; C001784:SF 6847