Aquaporin-2 Ser-261 phosphorylation is regulated in combination with Ser-256 and Ser-269 phosphorylation

• Published in: Biochemical and biophysical research communications Vol. 482; no. 4; pp. 524 - 529
• Main Authors: Yui, Naofumi, Sasaki, Sei, Uchida, Shinichi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22.01.2017
• Subjects: Animals; Aquaporin 2 - chemistry; Aquaporin 2 - metabolism; Aquaporin-2; Cell Line; Colforsin - metabolism; Dephosphorylation; Dogs; Forskolin; Kidney - chemistry; Kidney - metabolism; MDCK; Mice; Mice, Inbred C57BL; Phosphorylation; Rats; Serine - analysis; Serine - metabolism; Vasopressin; Vasopressins - metabolism; PP; Phosphorylation; FK; IP; Dephosphorylation; AQP2; Vasopressin; Aquaporin-2; PMP; NDI; λPP; ANOVA; Forskolin; VP; MDCK
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2016.11.118

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. •A phospho-specific AQP2 isolation technique was developed.•Ser-269 phosphorylation precedes Ser-261 dephosphorylation.•Regulated Ser-269 phosphorylation occurs in pS256-positive AQP2.•Regulated Ser-261 dephosphorylation occurs in pS256-positive AQP2.•Regulated Ser-261 dephosphorylation occurs in pS269-positive AQP2.