Characterization and Control of the Renal WNK1 Signaling Pathway

肾脏 WNK1 信号通路的表征和控制

基本信息

批准号：
9040930
负责人：
AROHAN R SUBRAMANYA
金额：
$ 33.5万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9040930
关键词：
Address Affect Aldosterone Antibodies Binding Biochemical Blood Pressure Cell Line Cell membrane Cessation of life Comparative Study Congestive Heart Failure Data Disease Distal Employee Strikes End stage renal failure Epithelium Equilibrium Essential Hypertension Event Excretory function Exhibits Family member Health Heart Diseases Hormones Hypertension Kidney Knockout Mice Knowledge Light Lysine Measures Mediating Membrane Protein Traffic Modeling Molecular Mus N-terminal Nephrons Pathogenesis Phosphorylation Phosphotransferases Populations at Risk Protein Isoforms Protein-Serine-Threonine Kinases Proteins Regulation Role SWI1 Signal Pathway Signal Transduction Sodium Sodium Chloride Stimulus Stroke Testing Transcript Ubiquitination Work base blood pressure regulation cardiovascular health clinically relevant gene product improved in vivo insight knock-down novel protein degradation protein expression response thiazide trafficking treatment strategy ubiquitin-protein ligase urinary

项目摘要

DESCRIPTION (provided by applicant): The thiazide-sensitive NaCl cotransporter (NCC) mediates salt reabsorption in the distal nephron (DN) of the kidney and is a key determinant of the blood pressure set point. With-No-Lysine (WNK) kinases regulate NCC membrane trafficking, phosphorylation status, and activity. One member of this family, WNK1, is expressed in the distal nephron as two major classes of isoforms with opposing functions. "Long" isoforms of WNK1 that possess intact serine-threonine kinase activity (L-WNK1) stimulate NCC. In contrast, short "kidney-specific" WNK1 isoforms that lack a kinase domain (KS-WNK1) inhibit NCC by antagonizing L-WNK1. Prior work indicates that the balance of these kinase-active and -defective products controls NCC activity, effectively functioning as an "isoform switch". The upstream mechanisms regulating WNK1 isoform balance, however, remain obscure. Our preliminary data suggest that aldosterone is a physiologically relevant stimulus that signals through the WNK1 switch to activate NCC-mediated salt reabsorption in the kidney. This proposal is driven by three novel observations: (1) First, in DN cell lines, aldosterone increases total WNK1 protein expression, but has a stronger effect on kinase active L-WNK1 than kinase defective KS-WNK1; this triggers downstream signaling events that increase NCC plasma membrane abundance and phosphorylation. (2) Second, WNK1 isoforms enriched at the protein level in the DN contain "PY motifs"- sequences which bind to Nedd4-2, an E3 ubiquitin ligase whose activity is suppressed by aldosterone. (3) Third, although KS-WNK1 transcript levels are high in the DN, it is an inherently unstable protein, and comparative studies with L-WNK1 indicate striking differences in steady state expression and protein turnover. Based on these findings, we hypothesize that aldosterone increases the total protein abundance of WNK1 isoforms in the distal nephron via Nedd4-2 inhibition, adjusting their ratio to favor increased L-WNK1 activity and NCC activation. To critically test this model, we propose to answer three questions about the regulation of WNK1 isoforms that remain incompletely addressed. First, how does the inhibition of Nedd4-2 by aldosterone regulate WNK1 protein expression and NCC activation? Second, why do L-WNK1 and KS-WNK1 exhibit different protein turnover rates? Third, how does aldosterone affect the WNK1 isoform switch in vivo? Answering these questions should provide novel insights into the molecular basis of aldosterone action, NCC regulation, and blood pressure homeostasis. Completion of the proposed aims will therefore improve our understanding of the pathogenesis of essential hypertension and highlight new strategies for its treatment.

描述（由申请人提供）：噻嗪类敏感的氯化钠协同转运蛋白（NCC）介导肾脏远端肾单位（DN）的盐重吸收，是血压设定点的关键决定因素。无赖氨酸 (WNK) 激酶调节 NCC 膜运输、磷酸化状态和活性。该家族的一个成员 WNK1 在远端肾单位中表达为具有相反功能的两大类同种型。具有完整丝氨酸-苏氨酸激酶活性的 WNK1“长”亚型 (L-WNK1) 可刺激 NCC。相比之下，缺乏激酶结构域的短“肾脏特异性”WNK1 亚型 (KS-WNK1) 通过拮抗 L-WNK1 来抑制 NCC。先前的研究表明，这些激酶活性和缺陷产物的平衡控制着 NCC 活性，有效地发挥着“异构体开关”的作用。然而，调节 WNK1 同工型平衡的上游机制仍然不清楚。我们的初步数据表明，醛固酮是一种生理相关的刺激物，通过 WNK1 开关发出信号，激活肾脏中 NCC 介导的盐重吸收。该提议由三个新的观察结果推动：（1）首先，在 DN 细胞系中，醛固酮增加总 WNK1 蛋白表达，但对激酶活性 L-WNK1 的影响比激酶缺陷型 KS-WNK1 更强；这会触发下游信号事件，从而增加 NCC 质膜丰度和磷酸化。 (2) 其次，DN 中蛋白质水平富集的 WNK1 同工型含有“PY 基序”，即与 Nedd4-2 结合的序列，Nedd4-2 是一种 E3 泛素连接酶，其活性被醛固酮抑制。 (3)第三，尽管 KS-WNK1 转录水平在 DN 中很高，但它本质上是一种不稳定的蛋白质，与 L-WNK1 的比较研究表明稳态表达和蛋白质周转存在显着差异。基于这些发现，我们假设醛固酮通过抑制 Nedd4-2 增加远端肾单位中 WNK1 亚型的总蛋白丰度，调整其比例以有利于增加 L-WNK1 活性和 NCC 激活。为了严格测试这个模型，我们建议回答有关 WNK1 同工型监管的三个尚未完全解决的问题。首先，醛固酮抑制Nedd4-2如何调节WNK1蛋白表达和NCC激活？其次，为什么L-WNK1和KS-WNK1表现出不同的蛋白质周转率？第三，醛固酮如何影响体内WNK1亚型转换？回答这些问题将为醛固酮作用、NCC 调节和血压稳态的分子基础提供新的见解。因此，完成所提出的目标将提高我们对原发性高血压发病机制的理解，并突出其治疗的新策略。