Aim: 

Vasopressin regulates water reabsorption in renal collecting duct activating a cAMP-dependent pathway, causing the phosphorylation of AQP2 at S256 which is essential for its translocation to the apical membrane. However, AQP2 can also be phosphorylated at S261. Activation of the PKC pathway induces AQP2 ubiquitination at K270 and its internalization. The relationship between S256 and S261 phosphorylation and ubiquitination is investigated here.

Methods: 

MDCK cells were unstimulated, stimulated with 10^-5M forskolin for 45 min, or incubated with 10^-7M TPA and forskolin for 30 min following forskolin stimulation. Subsequently, cells were analyzed by immunofluorescence or immunoblotting.

Results: 

Forskolin stimulation increased pS256, decreased pS261 and redistributed AQP2 to the apical membrane. These responses were reversed with forskolin/TPA treatment. For the treatments above AQP2-S256A always localized to intracellular vesicles, AQP2-S256D localized in the apical membrane with/without forskolin and was internalized with forskolin/TPA, and AQP2-S261A and AQP2-S261D always localized to vesicles. For these AQP2 mutants, however, the changes in pS261 and pS256 were similar to wt-AQP2, indicating that the changes in pS261 and pS256 occur independent from the localization of AQP2. Interestingly, AQP2-S256D-Ub always localized to vesicles and was constitutively phosphorylated at S261. Indeed, AQP2-S261D-K270R, which cannot undergo ubiquitination, was always localized in the apical membrane.

Conclusions: 

Activation of the PKA and PKC pathway induce a reciprocal change in S256 and S261 phosphorylation of AQP2 independently of the AQP2 localization. Phosphorylation of the pS256 is needed and sufficient for AQP2 translocation to the apical membrane. Phosphorylation of S261 seems to induce AQP2 internalization and involves AQP2 ubiquitination.