Regulation of the water channel Aquaporin-2 by Cullin E3 ubiquitin ligases

Aquaporin 2 (AQP2) is a vasopressin (VP) regulated water channel in the renal collecting duct. Phosphorylation and ubiquitylation of AQP2 play essential roles in controlling the cellular abundance of AQP2 and its accumulation on the plasma membrane in response to VP. Cullin-RING ubiquitin ligases (CRLs) are multi-subunit E3 ligases involved in ubiquitylation and degradation of their target proteins, eight of which are expressed in the collecting duct. Here, we utilized an established cell model of the collecting duct (mpkCCD14 cells) to study the role of Cullins in modulating AQP2. Western blotting identified Cul-1 to -5 in mpkCCD14 cells. Treatment of cells for 4 h with a pan-cullin inhibitor (MLN4924) decreased AQP2 abundance, prevented a VP-induced reduction in AQP2 Ser261 phosphorylation and attenuated VP-induced plasma membrane accumulation of AQP2 relative to vehicle. AQP2 ubiquitylation levels were significantly higher after MLN4924 treatment compared to controls, and they remained higher despite VP treatment. Cullin inhibition increased ERK1/2 activity, a kinase that regulates AQP2 Ser261 phosphorylation, and VP-induced reductions in ERK1/2 phosphorylation were absent during MLN4924 treatment. Furthermore, the greater Ser261 phosphorylation and reduction in AQP2 abundance during MLN4924 treatment were attenuated during ERK1/2 inhibition. MLN4924 increased intracellular calcium levels via calcium-release-activated calcium channels, inhibition of which abolished MLN4924 effects on Ser261 phosphorylation and AQP2 abundance. In conclusion, CRLs play a vital role in mediating some of the effects of VP to increase AQP2 plasma membrane accumulation and AQP2 abundance. Whether modulation of cullin activity can contribute to body water homeostasis requires further studies.