Arthur Julian, Huang Jianmin, Nomura Naohiro, Jin William W, Li Wei, Cheng Xiang, Brown Dennis, Lu Hua Jenny
Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Am J Physiol Renal Physiol. 2015 Oct 15;309(8):F673-9. doi: 10.1152/ajprenal.00152.2015. Epub 2015 Aug 19.
Vasopressin (VP) stimulates a signaling cascade that results in phosphorylation and apical membrane accumulation of aquaporin-2 (AQP2), leading to water reabsorption by kidney collecting ducts. However, the roles of most C-terminal phosphorylation events in stimulated and constitutive AQP2 recycling are incompletely understood. Here, we generated LLC-PK1 cells containing point mutations of all potential phosphorylation sites in the AQP2 C terminus: S226, S229, T244, S256, S261, S264, and S269, to determine their impact on AQP2 trafficking. We produced an All Null AQP2 construct in which these serine (S) or threonine (T) residues were mutated to alanine (A) or glycine (G), and we then reintroduced the phosphorylation mimic aspartic acid (D) individually to each site in the All Null mutant. As expected, the All Null mutant does not accumulate at the plasma membrane in response to VP but still undergoes constitutive recycling, as shown by its membrane accumulation when endocytosis is blocked by methyl-β-cyclodextrin (MβCD), and accumulation in a perinuclear patch at low temperature (20°C). Single phosphorylation mimics S226D, S229D, T244D, S261D, S264D, and S269D were insufficient to cause membrane accumulation of AQP2 alone or after VP treatment. However, AQP2 S256 reintroduced into the All Null mutant maintains its trafficking response to VP. We conclude that 1) constitutive recycling of AQP2 does not require phosphorylation at any C-terminal sites; 2) forced "phosphorylation" of sites in the AQP2 C terminus is insufficient to stimulate membrane accumulation in the absence of S256 phosphorylation; and 3) phosphorylation of S256 alone is necessary and sufficient to cause membrane accumulation of AQP2.
血管加压素（VP）刺激信号级联反应，导致水通道蛋白2（AQP2）磷酸化并在顶端膜积聚，从而使肾集合管重吸收水分。然而，大多数C末端磷酸化事件在刺激型和组成型AQP2循环中的作用尚未完全明确。在此，我们构建了LLC-PK1细胞，使其含有AQP2 C末端所有潜在磷酸化位点（S226、S229、T244、S256、S261、S264和S269）的点突变，以确定它们对AQP2转运的影响。我们制备了一种全无效AQP2构建体，其中这些丝氨酸（S）或苏氨酸（T）残基被突变为丙氨酸（A）或甘氨酸（G），然后将磷酸化模拟物天冬氨酸（D）分别重新引入全无效突变体的每个位点。正如预期的那样，全无效突变体在VP刺激下不会在质膜积聚，但仍会进行组成型循环，这表现为当内吞作用被甲基-β-环糊精（MβCD）阻断时其在膜上的积聚，以及在低温（20°C）下在核周斑块中的积聚。单个磷酸化模拟物S226D、S229D、T244D、S261D、S264D和S269D单独或在VP处理后均不足以导致AQP2在膜上积聚。然而，重新引入全无效突变体的AQP2 S256保留了其对VP的转运反应。我们得出以下结论：1）AQP2的组成型循环不需要任何C末端位点的磷酸化；2）在没有S256磷酸化的情况下，AQP2 C末端位点的强制“磷酸化”不足以刺激膜积聚；3）单独S256的磷酸化是导致AQP2膜积聚的必要且充分条件。
