PROTEIN-KINASE-A PHOSPHORYLATION IS INVOLVED IN REGULATED EXOCYTOSIS OF AQUAPORIN-2 IN TRANSFECTED LLC-PK1-CELLS

Vasopressin-dependent translocation of aquaporin-2 (AQP2) between intr acellular vesicles and the plasma membrane has been demonstrated in vi vo and in vitro. Furthermore, the vasopressin-induced increase in apic al membrane water permeability of renal principal cells is dependent o n a rise in intracellular adenosine 3',5'-cyclic monophosphate and act ivation of protein kinase A (PKA). To determine whether trafficking of AQP2 is dependent on PKA phosphorylation, we first examined the effec t of the PKA-inhibitor N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethy l)- 5-isoquinolinesulfonamide (H-89) on AQP2 translocation in transfec ted 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 r egion. Next, LLC-PK1 cells were stably transfected with AQP2 cDNA in w hich the PKA phosphorylation site, Ser256, was replaced with alanine ( S256A). S256A-AQP2 was not phosphorylated in vitro by PKA, and S256A-A QP2 was mainly localized to intracellular vesicles in the basal condit ion, similar to wildtype AQP2. However, after stimulation with vasopre ssin or forskolin, the cellular distribution of S256A-AQP2 remained un changed. In addition, the usual vasopressin-induced increase in endocy tosis seen in AQP2-transfected cells was not observed in S256A-AQP2-tr ansfected cells. These results demonstrate that the Ser256 PKA phospho rylation site is possibly involved in the vasopressin-induced traffick ing of AQP2 from intracellular vesicles to the plasma membrane and in the subsequent stimulation of endocytosis.