Phosphorylation of paracellin-1 at Ser217 by protein kinase A is essential for localization in tight junctions.
Although paracellin-1 (PCLN-1) is known to have a crucial role in the control of Mg2+ reabsorption in the kidney, the molecular pathways involved in the regulation of PCLN-1 have not been clarified. We used FLAG-tagged PCLN-1 to investigate these pathways further, and found that PCLN-1 is phosphorylated at Ser217 by protein kinase A (PKA) under physiological conditions in Madin-Darby canine kidney (MDCK) cells. PCLN-1 expression decreased Na+ permeability, resulting in a decrease in the transepithelial electrical resistance (TER). By contrast, PCLN-1 enhanced transepithelial Mg2+ transport. PKA inhibitors, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and myristoylated protein kinase A inhibitor 14-22 amide PKI, and an adenylate cyclase inhibitor, 2',5'-dideoxy adenosine (DDA), reduced the phosphoserine level of PCLN-1. The inhibitory effect of DDA was rescued by 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP). PKA and adenylate cyclase inhibitors decreased transepithelial Mg2+ transport and TER. Dephosphorylated PCLN-1 moved from detergent-insoluble to soluble fractions and was dissociated from ZO-1. A fusion protein of PCLN-1 with glutathione-S-transferase revealed that Ser217 was phosphorylated by PKA. Phosphorylated PCLN-1 was localized in the tight junction (TJ) along with ZO-1, whereas dephosphorylated PCLN-1 and the S217A mutant were translocated into the lysosome. The degradation of dephosphorylated PCLN-1 and S217A mutant was inhibited by chloroquine, a specific lysosome inhibitor. Thus, the PKA-dependent phosphorylation of Ser217 in PCLN-1 is essential for its localization in the TJ and transepithelial Mg2+ transport.
Ikari
Akira
A
Department of Environmental Biochemistry and Toxicology, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan. ikari@u-shizuoka-ken.ac.jp
Matsumoto
Satomi
S
Harada
Hitoshi
H
Takagi
Kuniaki
K
Hayashi
Hisayoshi
H
Suzuki
Yuichi
Y
Degawa
Masakuni
M
Miwa
Masao
M
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D057167:Claudins; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016048:Dideoxyadenosine / Q000031:analogs & derivatives / Q000378:metabolism; D004285:Dogs; D017097:Electric Impedance; D004594:Electrophysiology; D004847:Epithelial Cells / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010539:Permeability; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein
D057167:Claudins; D008565:Membrane Proteins; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D062826:Zonula Occludens-1 Protein; C502010:claudin 16; D012694:Serine; D016048:Dideoxyadenosine; C021008:2',5'-dideoxyadenosine; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D008274:Magnesium