title: Claudin-16 is directly phosphorylated by protein kinase A independently of a vasodilator-stimulated phosphoprotein-mediated pathway.
abstract: Claudin-16 (CLDN-16) is involved in the paracellular reabsorption of Mg(2+) in the thick ascending limb of Henle. The tight junctional localization and Mg(2+) transport of CLDN-16 are regulated by cAMP/PKA-dependent phosphorylation. Here, we examined whether PKA phosphorylates CLDN-16 in a direct or indirect manner. CLDN-16 was stably expressed in Madin-Darby canine kidney (MDCK) cells using a Tet-OFF system. The phosphorylation of CLDN-16 is upregulated by fetal calf serum (FCS). This phosphorylation was completely inhibited by a PKA inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride. Without FCS, dibutyryl cAMP (DBcAMP) increased the phosphoserine level of CLDN-16 in a concentration-dependent manner. The phosphorylated CLDN-16 elicited increases of transepithelial electrical resistance (TER) and transepithelial transport of Mg(2+). Vasodilator-stimulated phosphoprotein (VASP) was also phosphorylated in the presence of FCS or DBcAMP. In the glutathione-S-transferase (GST) pull down assay, a cytosolic carboxyl domain of CLDN-16 was associated with PKA, but not with VASP. Furthermore, PKA was immunoprecipitated with CLDN-16 in MDCK cells, but VASP was not. In cells expressing a dephosphorylated mutant (Ser160Ala) of VASP, CLDN-16 was phosphorylated by DBcAMP and was associated with ZO-1, a tight junctional-scaffolding protein, without integral cell-cell junctions. We suggest that PKA directly phosphorylates CLDN-16, resulting in the localization to tight junctions (TJs) and the maintenance of Mg(2+) reabsorption.
authors.affiliation: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Japan. ikari@u-shizuoka-ken.ac.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Ikari
authors.forename: Midori
authors.initials: M
authors.lastname: Ito
authors.forename: Chiaki
authors.initials: C
authors.lastname: Okude
authors.forename: Hayato
authors.initials: H
authors.lastname: Sawada
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Harada
authors.forename: Masakuni
authors.initials: M
authors.lastname: Degawa
authors.forename: Hideki
authors.initials: H
authors.lastname: Sakai
authors.forename: Tadanobu
authors.initials: T
authors.lastname: Takahashi
authors.forename: Junko
authors.initials: J
authors.lastname: Sugatani
authors.forename: Masao
authors.initials: M
authors.lastname: Miwa
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000502:physiology; D003994:Bucladesine / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D057167:Claudins; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D018076:DNA, Complementary; D004285:Dogs; D017097:Electric Impedance; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D007365:Intercellular Junctions / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism; D014162:Transfection; D062826:Zonula Occludens-1 Protein; D000098662:Vasodilator-Stimulated Phosphoprotein
chemical_list: D015815:Cell Adhesion Molecules; D057167:Claudins; D018076:DNA, Complementary; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009842:Oligopeptides; D010455:Peptides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; C502010:claudin 16; D000098662:Vasodilator-Stimulated Phosphoprotein; D003994:Bucladesine; C085706:FLAG peptide; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D008274:Magnesium