title: Regulation of the Water Channel Aquaporin-2 via 14-3-3θ and -ζ.
abstract: The 14-3-3 family of proteins are multifunctional proteins that interact with many of their cellular targets in a phosphorylation-dependent manner. Here, we determined that 14-3-3 proteins interact with phosphorylated forms of the water channel aquaporin-2 (AQP2) and modulate its function. With the exception of σ, all 14-3-3 isoforms were abundantly expressed in mouse kidney and mouse kidney collecting duct cells (mpkCCD14). Long-term treatment of mpkCCD14 cells with the type 2 vasopressin receptor agonist dDAVP increased mRNA and protein levels of AQP2 alongside 14-3-3β and -ζ, whereas levels of 14-3-3η and -θ were decreased. Co-immunoprecipitation (co-IP) studies in mpkCCD14 cells uncovered an AQP2/14-3-3 interaction that was modulated by acute dDAVP treatment. Additional co-IP studies in HEK293 cells determined that AQP2 interacts selectively with 14-3-3ζ and -θ. Use of phosphatase inhibitors in mpkCCD14 cells, co-IP with phosphorylation deficient forms of AQP2 expressed in HEK293 cells, or surface plasmon resonance studies determined that the AQP2/14-3-3 interaction was modulated by phosphorylation of AQP2 at various sites in its carboxyl terminus, with Ser-256 phosphorylation critical for the interactions. shRNA-mediated knockdown of 14-3-3ζ in mpkCCD14 cells resulted in increased AQP2 ubiquitylation, decreased AQP2 protein half-life, and reduced AQP2 levels. In contrast, knockdown of 14-3-3θ resulted in increased AQP2 half-life and increased AQP2 levels. In conclusion, this study demonstrates phosphorylation-dependent interactions of AQP2 with 14-3-3θ and -ζ. These interactions play divergent roles in modulating AQP2 trafficking, phosphorylation, ubiquitylation, and degradation.
authors.affiliation: From the Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, 8000 Aarhus, Denmark.
authors.forename: Hanne B
authors.initials: HB
authors.lastname: Moeller
authors.affiliation: From the Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, 8000 Aarhus, Denmark.
authors.forename: Joachim
authors.initials: J
authors.lastname: Slengerik-Hansen
authors.affiliation: From the Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, 8000 Aarhus, Denmark.
authors.forename: Takwa
authors.initials: T
authors.lastname: Aroankins
authors.affiliation: the Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Mette
authors.initials: M
authors.lastname: Assentoft
authors.affiliation: the Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Nanna
authors.initials: N
authors.lastname: MacAulay
authors.affiliation: From the Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, 8000 Aarhus, Denmark, the Institute of Molecular Medicine, University of Southern Denmark, 5000 Odense, Denmark, and.
authors.forename: Soeren K
authors.initials: SK
authors.lastname: Moestrup
authors.affiliation: the Division of Nephrology, Department of Medicine, Stanford University, Palo Alto, California 94305.
authors.forename: Vivek
authors.initials: V
authors.lastname: Bhalla
authors.affiliation: From the Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, 8000 Aarhus, Denmark, robert.a.fenton@biomed.au.dk.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fenton
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D000818:Animals; D051399:Aquaporin 2 / Q000378:metabolism*; D019921:Biotinylation; D003894:Deamino Arginine Vasopressin / Q000737:chemistry; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism; D007684:Kidney Tubules / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020349:Surface Plasmon Resonance; D025801:Ubiquitin / Q000378:metabolism; D014667:Vasopressins / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; C496024:Aqp2 protein, mouse; D051399:Aquaporin 2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D025801:Ubiquitin; D014667:Vasopressins; D005982:Glutathione Transferase; D003894:Deamino Arginine Vasopressin
keywords: Vasopressin; aquaporin; intracellular trafficking; kidney; phosphorylation; post-translational modification (PTM); ubiquitin; water channel