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

描述（申请人提供）：无赖氨酸蛋白激酶（K）4（WNK 4）突变与II型假性醛固酮减少症（PHAII）相关，PHAII是一种遗传性高血压。WNK 4是参与血压调节的肾电解质转运蛋白的整合调节剂。越来越多的证据表明，WNK 4是磷酸化级联反应的关键组成部分，该级联反应将肾脏中的肾素-血管紧张素-醛固酮系统的激活与电解质转运联系起来。然而，WNK 4中的血管紧张素II/醛固酮反应元件和WNK 4稳定性的醛固酮反应调节仍不清楚。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稳态紊乱来了解肾脏电解质转运生理学，以便为罕见和常见原因的相关疾病制定治疗策略。本申请的目的是鉴定血管紧张素II和/或醛固酮激活WNK 4激酶和调节WNK 4稳定性的机制。初步研究表明，WNK 4的调节结构域含有钙调蛋白结合和磷酸化位点。该结构域抑制WNK 4激酶活性，并且该作用需要钙调蛋白结合位点。在该结构域中模拟磷酸化消除了抑制作用。此外，WNK 4蛋白的稳定性受到Kelch样3（KLHL 3）的强烈调节，Kelch样3是在PHAII中突变的泛素E3连接酶组分。该提议的中心假设是WNK 4和KLHL 3中的PHAII突变导致WNK 4活性升高。WNK 4中的突变提高了特异性激酶活性/蛋白丰度，KLHL 3中的突变提高了WNK 4蛋白丰度。该假设将在两个特定目的中进行测试：1）确定WNK 4的调节结构域对WNK 4激酶活性的调节;和2）确定含有KLHL 3的泛素E3连接酶对WNK 4蛋白稳定性的调节。在目的1中，将使用体外和体内测定来评估通过钙调蛋白和通过磷酸化对WNK 4激酶活性的调节，并且将使用核磁共振（NMR）光谱来确定调节结构域与钙调蛋白和激酶结构域的相互作用表面。在目的2中，将确定WNK 4 C-末端区域的KLHL 3识别基序。此外，将在生物化学和敲入小鼠模型中评估KLHL 3中PHAII突变的影响。将在动物中测定KLHL 3对醛固酮的反应性。WNK 4激酶激活和蛋白稳定性调节的机制是重要的，因为它们是WNK 4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。调控结构域的相互作用表面信息对于开发WNK 4的小分子抑制剂作为研究工具和潜在的新的抗高血压药物是至关重要的。

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.