Phosphorylation and ubiquitylation are opposing players in regulating endocytosis of the water channel Aquaporin-2

Abstract

The post-translational modifications (PTMs) phosphorylation and ubiquitylation regulate plasma membrane protein function. Here, we examine the interplay between phosphorylation and ubiquitylation of the membrane protein aquaporin-2 (AQP2) and demonstrate that phosphorylation can override the previously suggested dominant endocytic signal of K63-linked polyubiquitylation. In polarized epithelial cells, although S256 is an important phosphorylation site for AQP2 membrane localization, the rate of AQP2 endocytosis was reduced by prolonging phosphorylation specifically at S269. Despite their close proximity, AQP2 phosphorylation at S269 and ubiquitylation at K270 can occur in parallel, with increased S269 phosphorylation and decreased AQP2 endocytosis occurring when K270 polyubiquitylation levels are maximal. In vivo studies support this data, with maximal levels of AQP2 ubiquitylation occurring in parallel to maximal S269 phosphorylation and enhanced AQP2 plasma membrane localization. In conclusion, we demonstrate for the first time that although K63-linked polyubiquitylation marks AQP2 for endocytosis, site-specific phosphorylation can counteract polyubiquitylation to determine its final localization. Similar mechanisms might exist for other plasma membrane proteins.

Regulation of the Water Channel Aquaporin-2 via 14-3-3θ and -ζ

“…Quantification data from 3 individual immunoprecipitations is shown in Fig. 5, B and C. AVP Washout in the Presence of Phosphatase Inhibitors Preserves the AQP2 and 14-3-3 Interaction-We previously determined that several AQP2 protein/protein interactions are dependent on the phosphorylation status of AQP2 at Ser-256 and Ser-269 (29) and that the phosphorylation of AQP2 (particularly at Ser-269) can be enhanced and prolonged during AVP washout by inhibition of specific protein phosphatases using OA and CA (23). To examine a potential role of phosphoryla- tion in mediating the interaction between AQP2 and 14-3-3, mpkCCD 14 cells were acutely stimulated with dDAVP followed by a washout period in the presence or absence of OA and CA.…”

“…Immunoprecipitation (IP) Using mpkCCD 14 Cells-Immunoprecipitation was performed as previously described (23).…”

“…14-3-3 Far Western Blotting (Overlay Assay)-mpkCCD 14 cells were grown in dDAVP for 4 days, treated acutely with dDAVP where indicated (description above), and AQP2 was immunoprecipitated as previously described (23). Some samples were solubilized with 1% SDS for 10 min with heating at 60°C, after which the SDS was diluted to 0.1% using lysis buffer before IP.…”

“…These phosphorylation sites play alternative roles in the subcellular distribution and function of AQP2, e.g. the apical plasma membrane accumulation of AQP2 in response to AVP treatment is modulated by Ser-256 and Ser-269 phosphorylation (23)(24)(25)(26)(27)(28). Although the underlying mechanisms for AQP2 regulation via phosphorylation are not completely clear, phosphorylation-dependent protein interactions appear to play a crucial role (29).…”

“…In addition, the carboxylterminal tail of AQP2 is further modified by ubiquitylation (30). A complex interplay between AQP2 phosphorylation and ubiquitylation is responsible for modulating the abundance of AQP2 on the plasma membrane (23).…”