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 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 Ser-256 is an important phosphorylation site for AQP2 membrane localization, the rate of AQP2 endocytosis was reduced by prolonging phosphorylation specifically at Ser-269. Despite close association, AQP2 phosphorylation at Ser-269 and ubiquitylation at Lys-270 can occur in parallel, with increased Ser-269 phosphorylation and decreased AQP2 endocytosis occurring when Lys-270 polyubiquitylation levels are maximal. In vivo studies support this data, with maximal levels of AQP2 ubiquitylation occuring in parallel to maximal Ser-269 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 may exist for other plasma membrane proteins.