title: Phosphorylation of Na-Cl cotransporter by OSR1 and SPAK kinases regulates its ubiquitination.
abstract: 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.
authors: Department of Nephrology, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Tokyo 113-8519, Japan.
authors: Muhammad Zakir
authors: MZ
authors: Hossain Khan
authors: Eisei
authors: E
authors: Sohara
authors: Akihito
authors: A
authors: Ohta
authors: Motoko
authors: M
authors: Chiga
authors: Yuichi
authors: Y
authors: Inoue
authors: Kiyoshi
authors: K
authors: Isobe
authors: Mai
authors: M
authors: Wakabayashi
authors: Katsuyuki
authors: K
authors: Oi
authors: Tatemitsu
authors: T
authors: Rai
authors: Sei
authors: S
authors: Sasaki
authors: Shinichi
authors: S
authors: Uchida
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004039:Diet, Sodium-Restricted; D004195:Disease Models, Animal; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011546:Pseudohypoaldosteronism / Q000378:metabolism*; D011955:Receptors, Drug / Q000378:metabolism*; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D011955:Receptors, Drug; C578875:Slc12a3 protein, mouse; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters; C486407:Stk39 protein, mouse; C534421:OXSR1 protein, mouse; D017346:Protein Serine-Threonine Kinases