Abstract：
Aquaporin 2 (AQP2) is a vasopressin (VP)-regulated water channel in the renal collecting duct. Phosphorylation and ubiquitylation of AQP2 play an essential role in controlling the cellular abundance of AQP2 and its accumulation on the plasma membrane in response to VP. Cullin-RING ubiquitin ligases (CRLs) are multisubunit E3 ligases involved in ubiquitylation and degradation of their target proteins, eight of which are expressed in the collecting duct. Here, we used an established cell model of the collecting duct (mpkCCD14 cells) to study the role of cullins in modulating AQP2. Western blotting identified Cul-1 to Cul-5 in mpkCCD14 cells. Treatment of cells for 4 h with a pan-cullin inhibitor (MLN4924) decreased AQP2 abundance, prevented a VP-induced reduction in AQP2 Ser261 phosphorylation, and attenuated VP-induced plasma membrane accumulation of AQP2 relative to the vehicle. AQP2 ubiquitylation levels were significantly higher after MLN4924 treatment compared with controls, and they remained higher despite VP treatment. Cullin inhibition increased ERK1/2 activity, a kinase that regulates AQP2 Ser261 phosphorylation, and VP-induced reductions in ERK1/2 phosphorylation were absent during MLN4924 treatment. Furthermore, the greater Ser261 phosphorylation and reduction in AQP2 abundance during MLN4924 treatment were attenuated during ERK1/2 inhibition. MLN4924 increased intracellular calcium levels via calcium release-activated calcium channels, inhibition of which abolished MLN4924 effects on Ser261 phosphorylation and AQP2 abundance. In conclusion, CRLs play a vital role in mediating some of the effects of VP to increase AQP2 plasma membrane accumulation and AQP2 abundance. Whether modulation of cullin activity can contribute to body water homeostasis requires further studies.NEW & NOTEWORTHY Aquaporin 2 (AQP2) is essential for body water homeostasis and is regulated by the antidiuretic hormone vasopressin. The posttranslational modification ubiquitylation is a key regulator of AQP2 abundance and plasma membrane localization. Here we demonstrate that cullin-RING E3 ligases play a vital role in mediating some of the effects of vasopressin to increase AQP2 abundance and plasma membrane accumulation. The results suggest that manipulating cullin activity could be a novel strategy to alter kidney water handling.
摘要：
水通道蛋白2(AQP2)是一种血管加压素(VP)调控肾集合管的水通道。AQP2的磷酸化和泛素化在控制AQP2的细胞丰度及其响应VP在质膜上的积累中起着至关重要的作用。Cullin-RING泛素连接酶（CRL）是多亚基E3连接酶，参与其靶蛋白的泛素化和降解，其中八个在收集管中表示。这里,我们利用已建立的收集导管细胞模型(mpkCCD14细胞)来研究Cullins在调节AQP2中的作用.Western印迹鉴定了mpkCCD14细胞中的Cul-1至-5。用pan-cullin抑制剂（MLN4924）处理细胞4小时降低了AQP2的丰度，防止了VP诱导的AQP2Ser261磷酸化减少，并减弱了VP诱导的AQP2质膜相对于载体的积累。与对照组相比，MLN4924治疗后AQP2的泛素化水平显着升高，尽管接受了VP治疗，但它们仍然较高。Cullin抑制增加ERK1/2活性，一种调节AQP2Ser261磷酸化的激酶，和VP诱导的ERK1/2磷酸化减少在MLN4924治疗期间不存在。此外，在ERK1/2抑制期间,MLN4924治疗期间Ser261磷酸化和AQP2丰度的降低减弱.MLN4924通过钙释放激活的钙通道增加细胞内钙水平，抑制其消除了MLN4924对Ser261磷酸化和AQP2丰度的影响。总之,CRL在介导VP的某些作用以增加AQP2质膜积累和AQP2丰度中起着至关重要的作用。cullin活性的调节是否可以促进体内水稳态需要进一步研究。
