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

WNK4（一种在遗传性高血压中发生突变的蛋白激酶）的调节

基本信息

批准号：
9248355
负责人：
JI-BIN PENG
金额：
$ 22.05万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-03-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9248355
关键词：
Affect Aldosterone Angiotensin II Animal Model Animals Antihypertensive Agents Binding Binding Sites Biochemical Biological Assay Blood Pressure C-terminal Ca(2+)-Calmodulin Dependent Protein Kinase Calmodulin Chronic Kidney Failure Complex Disease Electrolyte Disorder Electrolytes Elements Essential Hypertension Ethnic group Excision Foundations Functional disorder Genetic Polymorphism Goals Homeostasis Hypertension In Vitro Inherited Intervention Kidney Knock-in Link Lysine Mediating Mutate Mutation Mutation Analysis Myocardial Infarction NMR Spectroscopy Nuclear Magnetic Resonance Phosphorylation Phosphorylation Site Phosphotransferases Physiological Physiology Protein Kinase Proteins Public Health Regulation Renin-Angiotensin-Aldosterone System Reporting Research Risk Factors Signal Transduction Stroke Surface Syndrome Testing Therapeutic Type II Pseudohypoaldosteronism Xenopus oocyte base blood pressure regulation cullin-3 design in vivo inhibitor/antagonist mouse model new therapeutic target novel therapeutics public health relevance response small molecule small molecule inhibitor tool ubiquitin-protein ligase

项目摘要

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.

描述（由申请人提供）：无赖氨酸（K）4（WNK4）蛋白激酶的突变与II型假性醛固酮增多症（PHAII）（一种遗传性高血压）相关。 WNK4 是参与血压调节的肾电解质转运蛋白的综合调节剂。越来越多的证据表明，WNK4 是磷酸化级联的关键组成部分，该级联将肾素-血管紧张素-醛固酮系统的激活与肾脏中的电解质转运联系起来。然而，WNK4 中的血管紧张素 II/醛固酮响应元件以及 WNK4 稳定性的醛固酮响应调节仍不清楚。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。本申请的目的是确定 WNK4 激酶的激活机制以及血管紧张素 II 和/或醛固酮调节 WNK4 稳定性的机制。初步研究表明 WNK4 的调节域包含钙调蛋白结合和磷酸化位点。该结构域抑制 WNK4 激酶活性，并且该作用需要钙调蛋白结合位点。模仿该结构域的磷酸化会消除抑制作用。此外，WNK4 蛋白的稳定性受到 Kelch-like 3 (KLHL3) 的强有力调节，Kelch-like 3 是 PHAII 中突变的泛素 E3 连接酶成分。该提议的中心假设是 WNK4 和 KLHL3 中的 PHAII 突变导致 WNK4 活性升高。 WNK4 的突变提高了特定激酶活性/蛋白质丰度，而 KLHL3 的突变则提高了 WNK4 蛋白质丰度。该假设将在两个具体目标中得到检验：1）确定WNK4调节域对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，将使用体外和体内测定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) 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光谱学。在目标 2 中，将确定 WNK4 C 末端区域的 KLHL3 识别基序。此外，还将通过生化和敲入小鼠模型评估 KLHL3 中 PHAII 突变的影响。 KLHL3 对醛固酮的反应性将在动物中测定。 WNK4 激酶激活和蛋白质稳定性调节的机制非常重要，因为它们对于 WNK4 响应生理信号至关重要。这些机制的功能障碍会导致 PHAII。阐明这些机制为高血压的新干预措施铺平了道路。调节域的相互作用表面信息对于开发 WNK4 小分子抑制剂作为研究工具和潜在的新型抗高血压药物至关重要。