Yui Naofumi, Sasaki Sei, Uchida Shinichi
Department of Nephrology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Tokyo, Japan.
Department of Nephrology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Tokyo, Japan.
Biochem Biophys Res Commun. 2017 Jan 22;482(4):524-529. doi: 10.1016/j.bbrc.2016.11.118. Epub 2016 Nov 23.
Aquaporin-2 (AQP2) is a water channel in collecting duct principal cells in the kidney. Vasopressin catalyzes AQP2 phosphorylation at several serine sites in its C-terminus: Ser-256, Ser-261, and Ser-269. Upon stimulation by vasopressin, Ser-269 phosphorylation increases and Ser-261 phosphorylation decreases. Ser-256 phosphorylation is relatively constant. However, whether these types of phospho-regulation occur independently in distinct AQP2 populations or sequentially in the same AQP2 population is unclear. Especially, the manner of vasopressin-mediated Ser-261 phospho-regulation has been in controversy. In this study, we established phospho-specific AQP2 immunoprecipitation assays and investigated how pS256-positive AQP2 and pS269-positive AQP2 are catalyzed by forskolin or vasopressin, focusing on their Ser-261 phosphorylation status in polarized Madin-Darby canine kidney (MDCK) cells and in mice. In forskolin-treated MDCK cells, Ser-269 phosphorylation preceded Ser-261 dephosphorylation and Ser-256 phosphorylation was constant. In both MDCK cells and mouse kidney, phospho-specific immunoprecipitation revealed that the regulated Ser-269 phosphorylation occurred in the pS256-positive AQP2 population. Importantly, basal-state Ser-261 phosphorylation and its regulated dephosphorylation occurred in the pS256- and pS269-positive AQP2 population. These results provide the direct evidence that the Ser-261 dephosphorylation is involved in the pS256- and pS269-related AQP2 regulation.
水通道蛋白-2（AQP2）是肾脏集合管主细胞中的一种水通道。抗利尿激素催化AQP2在其C末端的几个丝氨酸位点发生磷酸化，即丝氨酸-256、丝氨酸-261和丝氨酸-269。在抗利尿激素的刺激下，丝氨酸-269磷酸化增加，丝氨酸-261磷酸化减少。丝氨酸-256磷酸化相对恒定。然而，尚不清楚这些类型的磷酸化调节是在不同的AQP2群体中独立发生，还是在同一AQP2群体中依次发生。特别是，抗利尿激素介导的丝氨酸-261磷酸化调节方式一直存在争议。在本研究中，我们建立了磷酸化特异性AQP2免疫沉淀分析方法，并研究了毛喉素或抗利尿激素如何催化pS256阳性AQP2和pS269阳性AQP2，重点关注它们在极化的Madin-Darby犬肾（MDCK）细胞和小鼠中的丝氨酸-261磷酸化状态。在毛喉素处理的MDCK细胞中，丝氨酸-269磷酸化先于丝氨酸-261去磷酸化，丝氨酸-256磷酸化保持恒定。在MDCK细胞和小鼠肾脏中，磷酸化特异性免疫沉淀均显示，受调节的丝氨酸-269磷酸化发生在pS256阳性AQP2群体中。重要的是，基础状态下的丝氨酸-261磷酸化及其受调节的去磷酸化发生在pS256和pS269阳性AQP2群体中。这些结果提供了直接证据，表明丝氨酸-261去磷酸化参与了与pS256和pS269相关的AQP2调节。
