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：
8136971
负责人：
NURIA M. PASTOR-SOLER
金额：
$ 29.7万
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/8136971
关键词：
5&apos -AMP-activated protein kinase 6-carboxyfluorescein AMP-activated protein kinase kinase 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 Functional disorder Generations Genitourinary system Health 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 reproductive sensor sperm cell vacuolar H+-ATPase water channel

5&apos -AMP-activated protein kinase 6-carboxyfluorescein AMP-activated protein kinase kinase 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 Functional disorder Generations Genitourinary system Health 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 reproductive sensor sperm cell vacuolar H+-ATPase water channel

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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（由申请人提供）：肾脏中的V-ATPase功能对于排泄由代谢产生的非易失性酸的排泄很重要。肾脏插入细胞（IC）中V-ATPase活性缺陷的个体可能患有严重的慢性代谢酸中毒，骨，肾脏和其他器官中潜在的严重后遗症。 V-ATPase活性对于上皮质子分泌附子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，理解V-ATPase调节可能会提供涉及泌尿生殖道质子分泌生理学和病理生理学的见解，并为严重的代谢性酸中毒和男性不育症提供更好的治疗。 V-ATPase活性可能是通过多种调节机制精心策划的。然而，仅少数研究已经解决了哺乳动物质子分泌上皮细胞中的直接V-ATPase磷酸化。我们和其他人表明，在透明细胞中，V -ATPase转移到根尖膜上对碱性腔pH的反应，腔内HCO3浓度增加，可溶性腺苷酸环化酶（SAC）和蛋白激酶A（PKA）活性。我们的初步研究表明，在IC中，顶端V-ATPase也需要PKA，并且SAC调节在分离的灌注外髓外收集管（OMCD）中调节V-ATPase介导的细胞内pH（PHI）恢复。此外，代谢传感器AMP激活的蛋白激酶（AMPK）的激活可防止透明细胞和ICS中的V-ATPase易位，并且PKA和AMPK磷酸化V-ATPase A和C2B亚基在体外和VIVO中均可阻止V-ATPase 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-ATPase活性取决于PKA和AMPK对其亚基的直接磷酸化，并且V-ATPase活性可以与通过PKA和通过AMPK代谢的细胞外酸基酶状态的感觉耦合。该提议的目的是：1）确定PKA在IC中调节V-ATPase亚细胞定位和活性的机制； 2）确定IC中AMPK调节V-ATPase亚细胞定位和活性的机制。最初，我们将集中精力绘制V1部门的PKA和AMPK候选磷酸化位点。为了确定相关的磷酸化位点，我们将使用位置定向的诱变，然后使用体外和体内磷酸化测定法以及质谱法。此外，我们将检查相关的PKA和AMPK磷酸化位点在A亚基在V-ATPase亚基亚基定位和使用ICS在细胞培养中的活性的作用。我们将确定PKA或AMPK激活对OMCD中V-ATPase依赖性PHI变化的影响。最后，我们将研究AMPK激活对V-ATPase A亚基的PKA介导的体外和体内PKA介导的磷酸化的影响。该应用程序的成功完成将有助于治疗肾脏和泌尿生殖道中酸化疾病的新策略。公共卫生相关性：液泡ATPase（V-ATPase）是一种重要的蛋白质复合物，在肾脏中起作用，可帮助人体代谢产生的排泄酸。肾脏插入细胞中V-ATPase活性有缺陷的患者可能患有严重的慢性代谢性酸中毒，骨，肾脏和其他器官中潜在的后遗症潜在的后遗症。我们假设V-ATPase函数取决于与酸/碱和代谢状态的感测的酶对其亚基的直接磷酸化。我们认为，成功完成本应用程序中详细介绍的特定目标将有助于治疗肾脏和泌尿生殖道中酸化疾病的新策略。