Mechanisms of Kinase-dependent Regulation of the V-ATPase in Kidney

肾脏中 V-ATP 酶的激酶依赖性调节机制

基本信息

批准号：
7698955
负责人：
NURIA M. PASTOR-SOLER
金额：
$ 30.3万
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：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7698955
关键词：
5&apos -AMP-activated protein kinase 6-carboxyfluorescein ATP phosphohydrolase Abbreviations Acids Address Adenylate Cyclase Apical Bicarbonates Biological Assay Cell Culture Techniques Cell Line Cell membrane Cells Chronic Clear Cell Coupled Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cystic Fibrosis Transmembrane Conductance Regulator Disease Distal renal tubular acidosis Type 1 Duct (organ) structure Embryo Environment Enzymes Epithelial Epithelial Cells Esters Excretory function Figs - dietary Functional disorder Generations Genitourinary system Horseradish Peroxidase Human In Vitro Individual Intercalated Cell Kidney Lead Maintenance Male Infertility Maps Mass Spectrum Analysis Mediating Metabolic Metabolic Activation Metabolic acidosis Metabolism Organ Oryctolagus cuniculus Patients Peanut Agglutinin Phospholipase C Phosphorylation Phosphorylation Site Phosphotransferases Physiological Physiology Protein Kinase Inhibitors Protons Pump Recovery Regulation Role Site-Directed Mutagenesis Sperm Maturation Stimulus Tight Junctions Vas deferens structure Whole Organism apical membrane base bone carbonate dehydratase collecting tubule structure complement C2b epithelial Na+ channel extracellular in vivo inhibitor/antagonist insight interest male pH Homeostasis prevent protein complex protein kinase inhibitor public health relevance reproductive sensor sperm cell vacuolar H+-ATPase water channel

项目摘要

DESCRIPTION (provided by applicant): V-ATPase function in kidney is important for excretion of non-volatile acids generated by metabolism. Individuals with defective V-ATPase activity in kidney intercalated cells (ICs) may suffer from severe chronic metabolic acidosis with potential severe sequelae in bone, kidney, and other organs. V-ATPase activity is also critical to the function of epithelial proton-secreting epididymal clear cells, which contribute to the acidic environment where spermatozoa mature. Therefore, understanding V-ATPase regulation may provide insights involving the physiology and pathophysiology of proton secretion in the urogenital tract and lead to better treatments for severe metabolic acidosis and male infertility. V-ATPase activity is likely orchestrated by a number of regulatory mechanisms; direct V-ATPase phosphorylation in mammalian proton-secreting epithelial cells, however, has been addressed by only a few studies. We and others have shown that in clear cells, V-ATPase translocation to the apical membrane responds to alkaline luminal pH, increased luminal HCO3 - concentration, soluble adenylyl cyclase (sAC), and protein kinase A (PKA) activity. Our preliminary studies demonstrate that PKA is also required for apical V-ATPase in ICs and that sAC regulates V-ATPase-mediated intracellular pH (pHi) recovery in isolated perfused outer medullary collecting ducts (OMCDs). In addition, activation of the metabolic sensor AMP-activated protein kinase (AMPK) prevents V-ATPase translocation in clear cells and ICs, and that both PKA and AMPK phosphorylate V-ATPase A and C2b subunits in vitro and in vivo. Based on these findings, we hypothesize that subcellular localization and V-ATPase activity in ICs depend on the direct phosphorylation of its subunits by PKA and AMPK, and that V-ATPase activity may be coupled to the sensing of extracellular acid-base status via PKA and metabolic status via AMPK. The Aims of this proposal are to: 1) determine the mechanisms involved in the modulation of V-ATPase subcellular localization and activity by PKA in ICs; and 2) determine the mechanisms involved in the modulation of V-ATPase subcellular localization and activity by AMPK in ICs. Initially, we will focus our efforts toward mapping PKA and AMPK candidate phosphorylation sites in the V1 sector A subunit. To identify relevant phosphorylation sites, we will use site-directed mutagenesis followed by in vitro and in vivo phosphorylation assays, as well as mass spectrometry. Additionally, we will examine the roles of relevant PKA and AMPK phosphorylation sites in the A subunit on V-ATPase subcellular localization and activity using ICs in cell culture. We will determine the effects of PKA or AMPK activation on V-ATPase-dependent pHi changes in OMCDs. Finally, we will examine the effects of AMPK activation on PKA-mediated phosphorylation of the V-ATPase A subunit in vitro and in vivo. The successful completion of the Aims of this application will contribute to new strategies to treat disorders of acidification in the kidney and in the urogenital tract. PUBLIC HEALTH RELEVANCE: The vacuolar ATPase (V-ATPase) is an important protein complex that functions in the kidney to help excrete acid generated by the body's metabolism. Patients with defective V-ATPase activity in kidney intercalated cells may suffer from severe chronic metabolic acidosis with potential serious sequelae in bone, kidney, and other organs. We hypothesize that V-ATPase function depends on direct phosphorylation of its subunits by enzymes that are coupled to the sensing of acid/base and metabolic status. The successful completion of the Specific Aims detailed in this application will, we believe, contribute to new strategies to treat disorders of acidification in the kidney and in the urogenital tract.

描述（申请人提供）：肾脏中的V-ATP酶功能对于代谢产生的非挥发性酸的排泄很重要。肾闰细胞 (IC) 中 V-ATP 酶活性缺陷的个体可能患有严重的慢性代谢性酸中毒，并可能在骨、肾和其他器官中产生严重的后遗症。 V-ATP酶活性对于分泌质子的上皮附睾透明细胞的功能也至关重要，这有助于精子成熟的酸性环境。因此，了解 V-ATP 酶调节可能提供涉及泌尿生殖道质子分泌的生理学和病理生理学的见解，并为严重代谢性酸中毒和男性不育症提供更好的治疗。 V-ATP酶活性可能是由多种调节机制协调的；然而，只有少数研究涉及哺乳动物质子分泌上皮细胞中 V-ATP 酶的直接磷酸化。我们和其他人已经证明，在透明细胞中，V-ATP酶易位到顶膜会对碱性管腔 pH 值、增加的管腔 HCO3 浓度、可溶性腺苷酸环化酶 (sAC) 和蛋白激酶 A (PKA) 活性做出反应。我们的初步研究表明，IC 中的顶端 V-ATP 酶也需要 PKA，并且 sAC 调节分离的灌注外髓集合管 (OMCD) 中 V-ATP 酶介导的细胞内 pH (pHi) 恢复。此外，代谢传感器 AMP 激活蛋白激酶 (AMPK) 的激活可防止透明细胞和 IC 中的 V-ATP 酶易位，并且 PKA 和 AMPK 均可在体外和体内磷酸化 V-ATP 酶 A 和 C2b 亚基。基于这些发现，我们假设 IC 中的亚细胞定位和 V-ATP 酶活性取决于 PKA 和 AMPK 对其亚基的直接磷酸化，并且 V-ATP 酶活性可能与通过 PKA 感知细胞外酸碱状态相关联。和通过 AMPK 的代谢状态。该提案的目的是： 1) 确定 IC 中 PKA 调节 V-ATP 酶亚细胞定位和活性所涉及的机制； 2) 确定 IC 中 AMPK 调节 V-ATP 酶亚细胞定位和活性的机制。最初，我们将集中精力绘制 V1 区 A 亚基中的 PKA 和 AMPK 候选磷酸化位点。为了识别相关的磷酸化位点，我们将使用定点诱变，然后进行体外和体内磷酸化测定以及质谱分析。此外，我们将在细胞培养中使用 IC 检查 A 亚基中相关 PKA 和 AMPK 磷酸化位点对 V-ATP 酶亚细胞定位和活性的作用。我们将确定 PKA 或 AMPK 激活对 OMCD 中 V-ATP 酶依赖性 pHi 变化的影响。最后，我们将在体外和体内检查 AMPK 激活对 PKA 介导的 V-ATPase A 亚基磷酸化的影响。本申请目标的成功完成将有助于制定治疗肾脏和泌尿生殖道酸化疾病的新策略。公共健康相关性：液泡 ATP 酶（V-ATP 酶）是一种重要的蛋白质复合物，在肾脏中发挥作用，帮助排出身体代谢产生的酸。肾闰细胞中 V-ATP 酶活性缺陷的患者可能患有严重的慢性代谢性酸中毒，并可能在骨、肾和其他器官中产生严重的后遗症。我们假设 V-ATP 酶功能依赖于与酸/碱和代谢状态传感相结合的酶对其亚基的直接磷酸化。我们相信，成功完成本申请中详述的具体目标将有助于制定治疗肾脏和泌尿生殖道酸化疾病的新策略。