WNK3 Prevents the Nedd4‐2 Inhibition of the Renal Na‐Cl Cotransporter (NCC)

Abstract

NCC and ENaC are inhibited by the E3 ubiquitin ligase Nedd4‐2 (JASN, 2011) and elimination of two canonical SGK1 phosphorylation sites on Nedd4‐2 (S222A and S328A), renders the Nedd4‐2 mediated inhibition of NCC and ENaC irrecoverable by SGK1. In search of new regulatory proteins, we found that the NH2 terminal domain of WNK3 interacts with Nedd4‐2. In Xenopus laevis oocytes inhibition of NCC by Nedd4‐2 is completely reversed in the presence of WNK3. In oocytes injected with NCC alone, WNK3 increases NCC activity by 264 ± 22 %, while in the presence of NCC and Nedd4‐2, the WNK3‐induced increase was of 353 ± 44 %. Additionally, in contrast to SGK1, WNK3 further activates NCC in the presence of double mutant Nedd4‐2 S222A/S328A to 501 ± 48 %. Thus, in the presence of wild type or mutant Nedd4‐2, WNK3 is a more potent stimulator of NCC. In the absence or presence of Nedd4‐2, NCC activation by WNK3 is prevented by elimination of WNK3 histidine 1497 and glycine 1508 that have been shown to be critical for interaction between WNKs (TBiochem J, 2011). In order to test for non‐specific WNK interactions we tested WNK4 and saw no difference in the Nedd4‐2 mediated inhibition of NCC. Thus, we propose that WNK3 is a regulator of the Nedd4‐2‐NCC pathway.