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. J. Cell. Physiol. 214:221–229, 2008. © 2007 Wiley‐Liss, Inc.