Regulation of WNK4, a protein kinase mutated in a hereditary form of hypertension

描述(申请人提供)：蛋白激酶-非赖氨酸(K)4(WNK4)突变与假性低醛固酮增多症II型(PHAII)有关，PHAII是一种遗传性高血压。WNK4是参与血压调节的肾脏电解质转运体的综合调节器。越来越多的证据表明，WNK4是将肾素-血管紧张素-醛固酮系统激活与肾脏电解质转运联系起来的磷酸化级联反应的关键组成部分。然而，WNK4中的血管紧张素II/醛固酮反应元件以及醛固酮反应对WNK4稳定性的调节仍不清楚。长期目标是通过分析电解质转运蛋白及其调节因子的突变如何导致电解质平衡紊乱来了解肾脏电解质转运生理学，从而为罕见和常见原因的相关疾病制定治疗策略。本应用的目的是确定血管紧张素II和/或醛固酮激活WNK4激酶和调节WNK4稳定性的机制。初步研究表明，WNK4的一个调控结构域含有钙调蛋白结合和磷酸化位点。该结构域抑制WNK4的活性，钙调素结合部位是这一作用所必需的。在这个结构域中模拟磷酸化可以消除这种抑制作用。此外，WNK4蛋白的稳定性受到Kelch-like 3(KLHL3)的强烈调控，KLHL3是在PHAII中突变的泛素E3连接酶组分。这一建议的中心假设是WNK4和KLHL3中的PHAII突变导致WNK4活性升高。WNK4基因突变可提高特定的激酶活性/蛋白丰度，KLHL3基因突变可提高WNK4蛋白丰度。这一假说将在两个特定的目标下得到验证：1)确定WNK4调节结构域对WNK4激酶活性的调节；2)确定含有KLHL3的泛素E3连接酶对WNK4蛋白稳定性的调节。在目标1中，将使用体外和体内试验来评估钙调蛋白和磷酸化对WNK4激酶活性的调节，并将使用核磁共振(NMR)光谱来确定调节结构域与钙调蛋白和激活域的相互作用表面。在目标2中，将确定WNK4 C-末端区域的KLHL3识别基序。此外，KLHL3中PHAII突变的影响将在生物化学和基因敲除小鼠模型中进行评估。KLHL3对醛固酮的反应将在动物身上确定。WNK4激活和蛋白质稳定性调节的机制是重要的，因为它们是WNK4响应生理信号所必需的。这些机制的功能障碍导致PHAII。阐明这些机制为高血压的新干预措施铺平了道路。调节结构域的相互作用表面信息对于开发WNK4的小分子抑制剂作为研究工具和潜在的降压新药至关重要。

DESCRIPTION (provided by applicant): Mutations in protein kinase with-no-lysine (K) 4 (WNK4) are associated with pseudohypoaldosteronism type II (PHAII), a hereditary form of hypertension. WNK4 is an integrative regulator of renal electrolyte transporters that are involved in blood pressure regulation. Accumulating evidence indicates that WNK4 is a key component of a phosphorylation cascade that links the activation of renin-angiotensin-aldosterone system to electrolyte transport in the kidney. However, the angiotensin II/aldosterone-responsive elements in WNK4 and the aldosterone-responsive regulation of WNK4 stability remain unclear. The long-term goal is to understand renal electrolyte transport physiology via analyzing how mutations in electrolyte transporters and their regulators cause disordered electrolyte homeostasis, so that therapeutic strategies could be developed for relevant disorders of both rare and common causes. The objective in this application is to identify the mechanisms for the activation of WNK4 kinase and for the regulation of WNK4 stability by angiotensin II and/or aldosterone. The preliminary studies indicate that a regulatory domain in WNK4 harbors calmodulin binding and phosphorylation sites. This domain inhibits WNK4 kinase activity and the calmodulin binding site is required for this action. Mimicking phosphorylation in this domain abolishes the inhibitory effect. Furthermore, the stability of WNK4 protein is robustly regulated by Kelch-like 3 (KLHL3), an ubiquitin E3 ligase component mutated in PHAII. The central hypothesis of this proposal is that PHAII mutations in both WNK4 and KLHL3 result in elevated WNK4 activity. Mutations in WNK4 elevate specific kinase activity/protein abundance and those in KLHL3 raise WNK4 protein abundance. This hypothesis will be tested in two specific aims: 1) Determine the regulation of WNK4 kinase activity by the regulatory domain of WNK4; and 2) Determine the regulation of WNK4 protein stability by the ubiquitin E3 ligase containing KLHL3. In Aim 1, the regulation of WNK4 kinase activity by calmodulin and by phosphorylation will be assessed using in vitro and in vivo assays, and the interaction surfaces of the regulatory domain with calmodulin and the kinase domain will be determined using nuclear magnetic resonance (NMR) spectroscopy. In Aim 2, the KLHL3-recognition motif at WNK4 C-terminal region will be determined. In addition, the effects of PHAII mutations in KLHL3 will be assessed biochemically and in knock-in mouse model. The responsiveness of KLHL3 to aldosterone will be determined in animals. The mechanisms for WNK4 kinase activation and protein stability regulation are significant, because they are essential for WNK4 to respond to physiological signals. Dysfunction of these mechanisms results in PHAII. Elucidating these mechanisms paves the way to new interventions for hypertension. The interaction surface information of the regulatory domain is crucial for developing small molecule inhibitors of WNK4 as research tools and potentially as new antihypertensive drugs.