Regulation of WNK signaling by potassium and Mo25: structure, function and physiology

人WNK（无赖氨酸）激酶的突变与高钾血症、高血压和慢性 肾病然而，尽管广泛表征了WNK在肾脏中的（病理）生理作用， 和肾外组织，令人惊讶的是很少了解WNK本身是如何调节的。WNKs 调节哺乳动物肾脏中的上皮离子转运。申请人的长期目标是实现 对与人类肾功能相关的上皮离子转运机制的机械理解。氯化 已知离子调节WNK。本申请的总体目标是定义和理解新的 WNK调节机制。这一更新应用程序建立在三个重大进展，从目前的 资助的赠款。首先，钾通过非氯化物依赖性抑制果蝇和哺乳动物的WNK。 机制等第二，支架蛋白Mo 25（小鼠蛋白25/Cab 39）是WNK的重要调节剂 信号，并激活WNK独立于其对SPAK（Ste 20相关的脯氨酸丙氨酸丰富的已知效应） 激酶）和OSR 1（氧化应激反应）激酶。第三，钾和钼25具有不同的影响， 肾脏表达的哺乳动物WNK 1、3和4。中心假设是钾和Mo 25直接 调节WNK激酶活性，对哺乳动物WNK亚型具有不同的作用。以强有力的初步指导 数据，中心假设将通过追求三个具体目标进行测试：1）确定机制， 钾调节WNK的生理后果; 2）阐明Mo 25调节的新机制 3）确定钾离子对WNK亚型差异调节的分子基础 Mo25该方法是创新的，利用生物物理和结构研究的见解来确定 上皮细胞离子转运调节的分子机制，使用一个独特的平台，果蝇Malpighian 小管，具有强大的分子遗传工具和易处理的生理读数。分析已经 建立，并证明可行的研究人员的手中，以：1）确定WNK钾结合位点， 产生钾不敏感的WNK突变体，并测试它们对跨上皮离子转运的影响; 2）确定 Mo 25如何在体外和小管中调节WNK活性，并探索Mo 25，钾离子和蛋白质之间的相互作用。 和氯化物在WNK调节中的作用;和3）测试WNK 1、3和4的钾和Mo 25调节的差异。 成功完成拟议的研究将阐明最全面的机制理解 迄今为止，WNK监管取得的进展。这是重要的，因为这些WNK的重要性的描述 调节机制在各种（病理）生理背景下，连同分子的见解，从 这里提出的研究，将允许开发靶向方法来治疗调节WNK 发信号。这在广泛的条件下具有转化影响，包括治疗糖尿病， 盐敏感性高血压、心脑血管疾病和肾脏疾病。

Mutations in human WNK (With No Lysine) kinases are associated with hyperkalemia, hypertension and chronic kidney disease. However, despite extensive characterization of (patho)physiological roles of WNKs in the kidney and extrarenal tissues, there is surprisingly little understanding of how WNKs themselves are regulated. WNKs regulate epithelial ion transport in the mammalian kidney. The applicants’ long-term goal is to achieve mechanistic understanding of epithelial ion transport mechanisms relevant to human kidney function. Chloride ion is known to regulate WNKs. The overall objective of this application is to define and understand new mechanisms of WNK regulation. This renewal application builds on three significant advances from the currently funded grant. First, potassium inhibits Drosophila and mammalian WNKs through chloride-independent mechanisms. Second, the scaffold protein Mo25 (Mouse protein 25/Cab39) is an important regulator of WNK signaling, and activates WNKs independent of its known effects on SPAK (Ste20-related proline alanine rich kinase) and OSR1 (oxidative stress response) kinases. Third, potassium and Mo25 have differential effects on the kidney-expressed mammalian WNKs 1, 3 and 4. The central hypothesis is that potassium and Mo25 directly regulate WNK kinase activity, with differential effects on mammalian WNK isoforms. Guided by strong preliminary data, the central hypothesis will be tested by pursuing three specific aims: 1) Determine the mechanism and physiological consequences of WNK regulation by potassium; 2) Elucidate novel mechanisms of Mo25 regulation of WNK signaling; and 3) Determine the molecular basis for differential WNK isoform regulation by potassium and Mo25. The approach is innovative by leveraging insights from biophysical and structural studies to determine molecular mechanisms of epithelial ion transport regulation, using a unique platform, the Drosophila Malpighian tubule, that has powerful molecular genetic tools and tractable physiologic readouts. Assays have been established, and demonstrated feasible in the investigators’ hands, to: 1) identify WNK potassium binding sites, generate potassium-insensitive WNK mutants, and test their effects on transepithelial ion transport; 2) determine how Mo25 regulates WNK activity in vitro and in the tubule, and probe the interactions between Mo25, potassium and chloride in WNK regulation; and 3) test differences in potassium and Mo25 regulation of WNKs 1, 3 and 4. Successful completion of the proposed studies will elucidate the most comprehensive mechanistic understanding of WNK regulation achieved to date. This is significant, because delineation of the importance of these WNK regulatory mechanisms in various (patho)physiological contexts, together with the molecular insights gained from the studies proposed here, will allow the development of targeted approaches to therapeutically modulate WNK signaling. This has translational impact in a broad range of conditions, including the treatment of dyskalemias, salt-sensitive hypertension, cardio- and cerebrovascular disease, and kidney disease.