Department of Nephrology, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Tokyo 113-8519, Japan.
Biochem Biophys Res Commun. 2012 Aug 24;425(2):456-61. doi: 10.1016/j.bbrc.2012.07.124. Epub 2012 Jul 27.
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.
钠-氯共转运蛋白（NCC）在其氨基末端的磷酸化是基于盐摄入量，受 WNK-OSR1/SPAK 激酶级联的调节。我们观察到，根据 NCC 的磷酸化状态，其在肾脏中的总蛋白丰度及其顶膜表达会发生变化。为了阐明这一机制，我们在低、正常和高盐饮食的小鼠以及 NCC 磷酸化持续升高的假性醛固酮增多症 II 型（PHAII）模型小鼠中检查了 NCC 的泛素化状态。低盐饮食降低了 NCC 的泛素化，而高盐饮食增加了肾脏中 NCC 的泛素化，这与 NCC 的总含量和磷酸化含量呈负相关。在 PHAII 模型中，与野生型同窝仔相比，肾脏中 NCC 的泛素化也较低。为了评估 NCC 磷酸化与泛素化之间的关系，我们在 COS7 细胞中表达了野生型、磷酸化缺陷型和磷酸化模拟型 NCC，并评估了免疫沉淀的总 NCC 和生物素化表面 NCC 的泛素化。在总 NCC 和表面 NCC 中，磷酸化缺陷型 NCC 的 NCC 泛素化增加，磷酸化模拟型 NCC 的 NCC 泛素化减少。因此，我们证明了 NCC 磷酸化降低了 NCC 的泛素化，这可能导致 NCC 丰度的增加，主要是在质膜上。
