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J Biol Chem. 2016 Jan 29;291(5):2469-84. doi: 10.1074/jbc.M115.691121. Epub 2015 Dec 8.

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

Author information

1
From the Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, 8000 Aarhus, Denmark.
2
the Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
3
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.
4
the Division of Nephrology, Department of Medicine, Stanford University, Palo Alto, California 94305.
5
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.

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.

KEYWORDS:

Vasopressin; aquaporin; intracellular trafficking; kidney; phosphorylation; post-translational modification (PTM); ubiquitin; water channel

PMID:
26645691
PMCID:
PMC4732228
DOI:
10.1074/jbc.M115.691121
[Indexed for MEDLINE]
Free PMC Article

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