Background:The sodium chloride cotransporter NCC mediates NaCl reabsorption in the kidney distal convoluted tubule. Results: NCC internalization from the plasma membrane is clathrin-mediated and regulated by NCC phosphorylation and ubiquitylation. Conclusion: Phosphorylation of NCC can regulate NCC internalization and ubiquitylation. Significance: Impaired NCC endocytosis could be implicated in salt-sensitive hypertension in vivo.

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.

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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.

Protein post-translational modification by the small-ubiquitin-related-modifier (SUMO) is a mechanism that allows a diverse response of cells to stress. Five SUMO family members, SUMO1-5, are expressed in mammals. We hypothesized that because kidney epithelial cells are often subject to stresses arising from various physiological conditions, multiple proteins in the kidney will be SUMOylated. Here we profiled SUMO1 and SUMO2 modified proteins in a polarized epithelial cell model of the renal cortical collecting duct (mpkCCD14 cells). Modified forms of SUMO1 or SUMO2, with a histidine tag and a Thr to Lys mutation preceding the carboxyl-terminal di-gly motif were expressed in mpkCCD14 cells allowing SUMO-conjugated proteins to be purified and identified. Protein mass spectrometry identified 1428 SUMO1 and 1957 SUMO2 sites, corresponding to 741 SUMO1 and 971 SUMO2 proteins. Gene ontology indicated that the function of the majority of SUMOylated proteins in mpkCCD14 cells was related to gene transcription. After treatment of the mpkCCD14 cells for 24 h with aldosterone, the levels of SUMOylation at a specific site on the proton and oligopeptide/antibiotic cotransporter protein Pept2 were greatly increased. In conclusion, the SUMOylation landscape of mpkCCD14 cells suggests that protein modification by SUMOylation is a mechanism within renal epithelial cells to modulate gene transcription under various physiological conditions.