The water channel aquaporin 2 (AQP2) has four phosphorylation sites at Ser256, Ser261, Ser264, and Ser269 in the C-terminus and these sites are important for AQP2 bioactivity. However, the exact role of each phosphorylation site still remains unclear. In this study, we generated unique AQP2 mutants in which we eliminated three phosphorylation sites but maintained only one site at the C-terminal end. The AQP2 phosphorylation of each single site by protein kinase A (PKA) was examined by in vitro translation and 32P incorporation. The ability of AQP2 trafficking to the cell membrane was evaluated by cell surface biotinylation. Among the four phosphorylation sites, AQP2 mutant with only S256 preserved the most ability of AQP2 to cell membrane expression. The AQP2 water permeability was measured in oocyte. Ser256 is the most important site for AQP2 function. Interestingly, Ser261 and Ser264 significantly inhibit AQP2 activity. Ser269 slightly but not statistically reduced AQP2 activity. Our data suggest that the four phosphorylation sites execute differential roles in concert in AQP2 functional regulation. AQP2 activity regulated by phosphorylation at Ser256 can be counterbalanced by phosphorylation at Ser261 and Ser264. (C) 2020 Elsevier Inc. All rights reserved.