Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
Cells. 2020 Mar 10;9(3):673. doi: 10.3390/cells9030673.
Arginine-vasopressin (AVP) facilitates water reabsorption in renal collecting duct principal cells through regulation of the water channel aquaporin-2 (AQP2). The hormone binds to vasopressin V2 receptors (V2R) on the surface of the cells and stimulates cAMP synthesis. The cAMP activates protein kinase A (PKA), which initiates signaling that causes an accumulation of AQP2 in the plasma membrane of the cells facilitating water reabsorption from primary urine and fine-tuning of body water homeostasis. AVP-mediated PKA activation also causes an increase in the AQP2 protein abundance through a mechanism that involves dephosphorylation of AQP2 at serine 261 and a decrease in its poly-ubiquitination. However, the signaling downstream of PKA that controls the localization and abundance of AQP2 is incompletely understood. We carried out an siRNA screen targeting 719 kinase-related genes, representing the majority of the kinases of the human genome and analyzed the effect of the knockdown on AQP2 by high-content imaging and biochemical approaches. The screening identified 13 hits whose knockdown inhibited the AQP2 accumulation in the plasma membrane. Amongst the candidates was the so far hardly characterized cyclin-dependent kinase 18 (CDK18). Our further analysis revealed a hitherto unrecognized signalosome comprising CDK18, an E3 ubiquitin ligase, STUB1 (CHIP), PKA and AQP2 that controls the localization and abundance of AQP2. CDK18 controls AQP2 through phosphorylation at serine 261 and STUB1-mediated ubiquitination. STUB1 functions as an A-kinase anchoring protein (AKAP) tethering PKA to the protein complex and bridging AQP2 and CDK18. The modulation of the protein complex may lead to novel concepts for the treatment of disorders which are caused or are associated with dysregulated AQP2 and for which a satisfactory treatment is not available, e.g., hyponatremia, liver cirrhosis, diabetes insipidus, ADPKD or heart failure.
精氨酸加压素（AVP）通过调节水通道蛋白-2（AQP2）促进肾集合管主细胞的水重吸收。该激素与细胞表面的血管加压素 V2 受体（V2R）结合并刺激 cAMP 合成。cAMP 激活蛋白激酶 A（PKA），启动信号通路导致 AQP2 在细胞的质膜中积累，促进原尿中的水重吸收，并精细调节体内水的平衡。AVP 介导的 PKA 激活还通过涉及丝氨酸 261 去磷酸化和 AQP2 多聚泛素化减少的机制，增加 AQP2 蛋白的丰度。然而，PKA 下游控制 AQP2 定位和丰度的信号通路尚不完全清楚。我们针对 719 个激酶相关基因进行了 siRNA 筛选，这些基因代表了人类基因组中大多数激酶，并通过高内涵成像和生化方法分析了敲低对 AQP2 的影响。筛选鉴定了 13 个靶点，其敲低抑制了 AQP2 在质膜中的积累。候选物之一是迄今为止几乎没有被描述的周期蛋白依赖性激酶 18（CDK18）。我们的进一步分析揭示了一个前所未有的信号复合物，包括 CDK18、一种 E3 泛素连接酶、STUB1（CHIP）、PKA 和 AQP2，它控制 AQP2 的定位和丰度。CDK18 通过丝氨酸 261 的磷酸化和 STUB1 介导的泛素化来控制 AQP2。STUB1 作为 A 激酶锚定蛋白（AKAP）将 PKA 锚定在蛋白质复合物上，并连接 AQP2 和 CDK18。该蛋白质复合物的调节可能为治疗由 AQP2 失调引起或与之相关的疾病提供新的概念，这些疾病目前尚无满意的治疗方法，例如低钠血症、肝硬化、尿崩症、多囊肾病或心力衰竭。
