title: Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells.
abstract: Vasopressin-dependent translocation of aquaporin-2 (AQP2) between intracellular vesicles and the plasma membrane has been demonstrated in vivo and in vitro. Furthermore, the vasopressin-induced increase in apical membrane water permeability of renal principal cells is dependent on a rise in intracellular adenosine 3',5'-cyclic monophosphate and activation of protein kinase A (PKA). To determine whether trafficking of AQP2 is dependent on PKA phosphorylation, we first examined the effect of the PKA-inhibitor N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89) on AQP2 translocation in transfected LLC-PK1 cells. Vasopressin-induced membrane insertion of AQP2 was completely inhibited by pretreatment of the cells for 60 min with H-89. This reagent also caused a dense accumulation of AQP2 in the Golgi region. Next, LLC-PK1 cells were stably transfected with AQP2 cDNA in which the PKA phosphorylation site, Ser256, was replaced with alanine (S256A). S256A-AQP2 was not phosphorylated in vitro by PKA, and S256A-AQP2 was mainly localized to intracellular vesicles in the basal condition, similar to wild-type AQP2. However, after stimulation with vasopressin or forskolin, the cellular distribution of S256A-AQP2 remained unchanged. In addition, the usual vasopressin-induced increase in endocytosis seen in AQP2-transfected cells was not observed in S256A-AQP2-transfected cells. These results demonstrate that the Ser256 PKA phosphorylation site is possibly involved in the vasopressin-induced trafficking of AQP2 from intracellular vesicles to the plasma membrane and in the subsequent stimulation of endocytosis.
authors.affiliation: Renal Unit, Massachusetts General Hospital, Boston, USA.
authors.forename: T
authors.initials: T
authors.lastname: Katsura
authors.forename: C E
authors.initials: CE
authors.lastname: Gustafson
authors.forename: D A
authors.initials: DA
authors.lastname: Ausiello
authors.forename: D
authors.initials: D
authors.lastname: Brown
mesh_terms: D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018374:LLC-PK1 Cells; D009154:Mutation; D010766:Phosphorylation; D013449:Sulfonamides; D013552:Swine; D014162:Transfection; D014667:Vasopressins / Q000494:pharmacology
chemical_list: D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D004791:Enzyme Inhibitors; D007473:Ion Channels; D007546:Isoquinolines; D013449:Sulfonamides; D014667:Vasopressins; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide