The Deubiquitylase USP4 Interacts with the Water Channel AQP2 to Modulate Its Apical Membrane Accumulation and Cellular Abundance

Sathish Murali,Robert Fenton,Takwa Aroankins,Hanne Moeller

doi:10.3390/cells8030265

Abstract

Aquaporin 2 (AQP2) mediates the osmotic water permeability of the kidney collecting duct in response to arginine vasopressin (VP) and is essential for body water homeostasis. VP effects on AQP2 occur via long-term alterations in AQP2 abundance and short-term changes in AQP2 localization. Several of the effects of VP on AQP2 are dependent on AQP2 phosphorylation and ubiquitylation; post-translational modifications (PTM) that modulate AQP2 subcellular distribution and function. Although several protein kinases, phosphatases, and ubiquitin E3 ligases have been implicated in AQP2 PTM, how AQP2 is deubiquitylated or the role of deubiquitylases (DUBS) in AQP2 function is unknown. Here, we report a novel role of the ubiquitin-specific protease USP4 in modulating AQP2 function. USP4 co-localized with AQP2 in the mouse kidney, and in mpkCCD14 cells USP4 and AQP2 abundance are increased by VP. AQP2 and USP4 co-immunoprecipitated from mpkCCD14 cells and mouse kidney, and in vitro, USP4 can deubiquitylate AQP2. In mpkCCD14 cells, shRNA mediated knockdown of USP4 decreased AQP2 protein abundance, whereas no changes in AQP2 mRNA levels or VP-induced cAMP production were detected. VP-induced AQP2 membrane accumulation in knockdown cells was significantly reduced, which was associated with higher levels of ubiquitylated AQP2. AQP2 protein half-life was also significantly reduced in USP4 knockdown cells. Taken together, the data suggest that USP4 is a key regulator of AQP2 deubiquitylation and that loss of USP4 leads to increased AQP2 ubiquitylation, decreased AQP2 levels, and decreased cell surface AQP2 accumulation upon VP treatment. These studies have implications for understanding body water homeostasis.

Highlights

Aquaporin 2 (AQP2) is the arginine vasopressin (VP) regulated water channel of the kidney collecting duct (CD)
The data suggest that ubiquitin-specific protease 4 (USP4) is a key regulator of AQP2 deubiquitylation and that loss of USP4 leads to increased AQP2 ubiquitylation, decreased AQP2 levels, and decreased cell surface AQP2 accumulation upon VP treatment
We demonstrate that USP4 is abundantly expressed in mouse kidney and mpkCCD14 cells, where it co-localizes and interacts with AQP2

Summary

Introduction

Aquaporin 2 (AQP2) is the arginine vasopressin (VP) regulated water channel of the kidney collecting duct (CD). A major role of VP in regulating body water homeostasis involves binding of VP to the vasopressin type 2 receptor (V2R) in collecting duct principal cells, which initiates a complex intracellular signaling response leading to apical plasma membrane targeting of AQP2 [1,2]. Various post-translational modifications of AQP2 in its intracellular carboxyl-terminal tail play a vital role in determining its subcellular distribution and function. AQP2 membrane accumulation, rate of degradation, or AQP2 recycling pathways [3,4]. AQP2 is polyubiquitylated with K63 linked chains at Lys270, which increases AQP2 endocytosis and lysosomal degradation [5]. AQP2 can be phosphorylated and ubiquitylated simultaneously, phosphorylation of AQP2 is able to override the internalization signal of ubiquitin [3]

Methods

Results

Conclusion