title: Aquaporin-2 Ser-261 phosphorylation is regulated in combination with Ser-256 and Ser-269 phosphorylation.
abstract: Aquaporin-2 (AQP2) is a water channel in collecting duct principal cells in the kidney. Vasopressin catalyzes AQP2 phosphorylation at several serine sites in its C-terminus: Ser-256, Ser-261, and Ser-269. Upon stimulation by vasopressin, Ser-269 phosphorylation increases and Ser-261 phosphorylation decreases. Ser-256 phosphorylation is relatively constant. However, whether these types of phospho-regulation occur independently in distinct AQP2 populations or sequentially in the same AQP2 population is unclear. Especially, the manner of vasopressin-mediated Ser-261 phospho-regulation has been in controversy. In this study, we established phospho-specific AQP2 immunoprecipitation assays and investigated how pS256-positive AQP2 and pS269-positive AQP2 are catalyzed by forskolin or vasopressin, focusing on their Ser-261 phosphorylation status in polarized Madin-Darby canine kidney (MDCK) cells and in mice. In forskolin-treated MDCK cells, Ser-269 phosphorylation preceded Ser-261 dephosphorylation and Ser-256 phosphorylation was constant. In both MDCK cells and mouse kidney, phospho-specific immunoprecipitation revealed that the regulated Ser-269 phosphorylation occurred in the pS256-positive AQP2 population. Importantly, basal-state Ser-261 phosphorylation and its regulated dephosphorylation occurred in the pS256- and pS269-positive AQP2 population. These results provide the direct evidence that the Ser-261 dephosphorylation is involved in the pS256- and pS269-related AQP2 regulation.
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Tokyo, Japan. Electronic address: nyui.kid@tmd.ac.jp.
authors.forename: Naofumi
authors.initials: N
authors.lastname: Yui
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Sei
authors.initials: S
authors.lastname: Sasaki
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Uchida
mesh_terms: D000818:Animals; D051399:Aquaporin 2 / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000378:metabolism*; D004285:Dogs; D007668:Kidney / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation; D051381:Rats; D012694:Serine / Q000032:analysis / Q000378:metabolism; D014667:Vasopressins / Q000378:metabolism*
chemical_list: D051399:Aquaporin 2; D014667:Vasopressins; D005576:Colforsin; D012694:Serine
keywords: Aquaporin-2; Dephosphorylation; Forskolin; MDCK; Phosphorylation; Vasopressin