title: Phosphorylation and ubiquitylation are opposing processes that regulate 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.
authors.affiliation: Department of Biomedicine, Center for Interactions of Proteins in Epithelial Transport (InterPrET), Aarhus University, Aarhus DK-8000, Denmark hbmo@ana.au.dk rofe@ana.au.dk.
authors.forename: Hanne B
authors.initials: HB
authors.lastname: Moeller
authors.affiliation: Department of Biomedicine, Center for Interactions of Proteins in Epithelial Transport (InterPrET), Aarhus University, Aarhus DK-8000, Denmark.
authors.forename: Takwa Shaiman
authors.initials: TS
authors.lastname: Aroankins
authors.affiliation: Department of Biomedicine, Center for Interactions of Proteins in Epithelial Transport (InterPrET), Aarhus University, Aarhus DK-8000, Denmark.
authors.forename: Joachim
authors.initials: J
authors.lastname: Slengerik-Hansen
authors.affiliation: Department of Biomedicine, Center for Interactions of Proteins in Epithelial Transport (InterPrET), Aarhus University, Aarhus DK-8000, Denmark Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Trairak
authors.initials: T
authors.lastname: Pisitkun
authors.affiliation: Department of Biomedicine, Center for Interactions of Proteins in Epithelial Transport (InterPrET), Aarhus University, Aarhus DK-8000, Denmark hbmo@ana.au.dk rofe@ana.au.dk.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fenton
mesh_terms: D000818:Animals; D051399:Aquaporin 2 / Q000378:metabolism*; D004285:Dogs; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D051379:Mice; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D017208:Rats, Wistar; D054875:Ubiquitination
chemical_list: D051399:Aquaporin 2
keywords: AQP2; Aquaporin; Phosphatase; Phosphorylation; Ubiquitylation; Vasopressin