Phosphorylation of Na-Cl cotransporter by OSR1 and SPAK kinases regulates its ubiquitination

Na-Cl cotransporter (NCC) is phosphorylated in its amino terminus based on salt intake under the regulation of the WNK-OSR1/SPAK kinase cascade. We have observed that total protein abundance of NCC and its apical membrane expression varies in the kidney based on the phosphorylation status. To clarify the mechanism, we examined NCC ubiquitination status in mice fed low, normal and high salt diets, as well as in a model mouse of pseudohypoaldosteronism type II (PHAII) where NCC phosphorylation is constitutively elevated. Low-salt diet decreased NCC ubiquitination, while high-salt diet increased NCC ubiquitination in the kidney, and this was inversely correlated with total and phosphorylated NCC abundance. In the PHAII model, the ubiquitination of NCC in kidney was also lower when compared to that in wild-type littermates. To evaluate the relationship between phosphorylation and ubiquitination of NCC, we expressed wild-type, phospho-deficient and -mimicking NCC in COS7 cells, and the ubiquitination of immunoprecipitated total and biotinylated surface NCC was evaluated. NCC ubiquitination was increased in the phospho-deficient NCC and decreased in phospho-mimicking NCC in both total and surface NCC. Thus, we demonstrated that NCC phosphorylation decreased NCC ubiquitination, which may contribute to the increase of NCC abundance mostly on plasma membranes.