Extracellular Mg(2+) regulates the tight junctional localization of claudin-16 mediated by ERK-dependent phosphorylation.
Claudin-16 is involved in the paracellular reabsorption of Mg(2+) in the thick ascending limb of Henle. Little is known about the mechanism regulating the tight junctional localization of claudin-16. Here, we examined the effect of Mg(2+) deprivation on the distribution and function of claudin-16 using Madin-Darby canine kidney (MDCK) cells expressing FLAG-tagged claudin-16. Mg(2+) deprivation inhibited the localization of claudin-16 at tight junctions, but did not affect the localization of other claudins. Re-addition of Mg(2+) induced the tight junctional localization of claudin-16, which was inhibited by U0126, a MEK inhibitor. Transepithelial permeability to Mg(2+) was also inhibited by U0126. The phosphorylation of ERK was reduced by Mg(2+) deprivation, and recovered by re-addition of Mg(2+). These results suggest that the MEK/ERK-dependent phosphorylation of claudin-16 affects the tight junctional localization and function of claudin-16. Mg(2+) deprivation decreased the phosphothreonine levels of claudin-16. The phosphothreonine levels of T225A and T233A claudin-16 were decreased in the presence of Mg(2+) and these mutants were widely distributed in the plasma membrane. Furthermore, TER and transepithelial Mg(2+) permeability were decreased in the mutants. We suggest that the tight junctional localization of claudin-16 requires a physiological Mg(2+) concentration and the phosphorylation of threonine residues via a MEK/ERK-dependent pathway.
Ikari
Akira
A
Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. ikari@u-shizuoka-ken.ac.jp
Kinjo
Keishi
K
Atomi
Kosuke
K
Sasaki
Yohei
Y
Yamazaki
Yasuhiro
Y
Sugatani
Junko
J
D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002451:Cell Compartmentation / Q000187:drug effects; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D057167:Claudins; D004285:Dogs; D017097:Electric Impedance; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D005110:Extracellular Space / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009570:Nitriles / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D019108:Tight Junctions / Q000187:drug effects* / Q000201:enzymology*; D062826:Zonula Occludens-1 Protein
D002070:Butadienes; D057167:Claudins; D008565:Membrane Proteins; D009570:Nitriles; D010750:Phosphoproteins; C569044:Tjp1 protein, rat; C113580:U 0126; D062826:Zonula Occludens-1 Protein; C502010:claudin 16; D010769:Phosphothreonine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D008274:Magnesium