Claudin-16 is directly phosphorylated by protein kinase A independently of a vasodilator-stimulated phosphoprotein-mediated pathway

J Cell Physiol. 2008 Jan;214(1):221-9. doi: 10.1002/jcp.21178.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Transport / physiology
  • Bucladesine / pharmacology
  • Cell Adhesion Molecules / genetics
  • Cell Adhesion Molecules / metabolism*
  • Cell Line
  • Cell Membrane Permeability
  • Claudins
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • DNA, Complementary
  • Dogs
  • Electric Impedance
  • Escherichia coli / genetics
  • Glutathione Transferase / metabolism
  • Intercellular Junctions / metabolism
  • Kidney / cytology
  • Magnesium / metabolism
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism*
  • Mutation
  • Oligopeptides
  • Peptides / chemistry
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Plasmids
  • Protein Binding
  • Protein Structure, Tertiary
  • Rats
  • Recombinant Fusion Proteins / metabolism
  • Tight Junctions / metabolism
  • Transfection
  • Zonula Occludens-1 Protein

Substances

  • Cell Adhesion Molecules
  • Claudins
  • DNA, Complementary
  • Membrane Proteins
  • Microfilament Proteins
  • Oligopeptides
  • Peptides
  • Phosphoproteins
  • Recombinant Fusion Proteins
  • Tjp1 protein, rat
  • Zonula Occludens-1 Protein
  • claudin 16
  • vasodilator-stimulated phosphoprotein
  • Bucladesine
  • FLAG peptide
  • Glutathione Transferase
  • Cyclic AMP-Dependent Protein Kinases
  • Magnesium