CFTR REGULATION BY TARGETED KINASE AND PHOSPHATASE

靶向激酶和磷酸酶对 CFTR 的调节

• 批准号: 6499598
• 负责人: Neil A Bradbury
• 金额: $ 12.41万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6499598
• 关键词: biological signal transduction; cellular polarity; chloride channels; clathrin; cyclic AMP; cystic fibrosis; cytoskeletal proteins; enzyme activity; enzyme mechanism; immunocytochemistry; isozymes; phosphoprotein phosphatase; phosphorylation; protein kinase; protein localization; protein structure function; second messengers

Polarized epithelial cells segregate the function and activities of their apical and basolateral membranes. This necessitates the translation of hormonal and neurotransmitter signals at the basolateral membrane into signals which can be utilized at the apical membrane. Transduction of the cyclic AMP second message relies on multifunctional serine-threonine protein kinases and phosphoprotein phosphatases. These proteins regulate events such cellular events as ion channel activation and enzyme activity by altering the phosphorylation status of these enzymes and ion channels. Recently it has been proposed that targeting and sequestration of kinases and phosphatases at sites close to specific substrates influences the specificity of these seemingly multifunctional enzymes. Recently, it has been recognized that CFTR chloride ion channels bind EBP50 through PDZ domain interactions. In other systems, it is known that EBP50 also binds ezrin, an AKAP initially identified in gastric parietal cells. We have isolated clathrin coated vesicles as a membrane fraction enriched in CFTR. These vesicles also contain EBP50, ezrin and type II PKA, leading us to speculate that such a multimeric protein complex may be involved in targeting PKA to CFTR. Although ezrin has been identified as an AKAP in polarized cells, it is by no means the only AKAP known. Thus, we have identified other AKAPs present in clathrin coated vesicles using an RII overlay assay. Recent studies have also indicated that certain AKAPs may be multifunctional proteins, serving as scaffolds to target both kinases and phosphatase activities. Therefore we propose that targeting of kinase and phosphatase activities to CFTR allows for the selective and specific regulation of CFTR via phosphorylation and dephosphorylation. Thus the overall aim of the proposal is to identify the biochemical and molecular basis for the anchoring of kinase and phosphatase isoforms at the apical membrane of polarized epithelial cells necessary for the regulation of CFTR. Aims 1 and 2 are directed at elucidating the scaffold molecules that target kinase activity to CFTR. In aim 1 we test the hypothesis that ezrin serves this function by establishing the association of ezrin, RII, EBP50 and CFTR by performing co-immunoprecipitation and immunocytochemical co-localization. Aim 2 is directed at evaluating the role of other AKAPs in targeting kinase the role of other AKAPs in targeting kinase activity to CFTR. In aim 1 we test the hypothesis that ezrin serves this function by establishing the association of ezrin, RII, EBP50 and CFTR by performing co-immunoprecipitation and immunocytochemical co-localization. Aim 2 is directed at evaluation the role of other AKAPs in targeting kinase activity to CFTR. RII overlay and cAMP-affinity chromatography will be performed to delineate AKAPs present in clathrin coted vesicles. Aim 3 is directed at elucidating the scaffold proteins that target phosphatase activity to CFTR. Immunogold electron microscopy and immunoblot on subcellular fractionations (including isolation of clathrin coated vesicles) will be performed using antibodies against the B56alpha subunit isoform which is expressed in lung and pancreas. These investigations will provide important insights into the general mechanisms by which epithelial cells segregate their second messenger-dependent signaling pathways, and specifically into the mechanisms by which CFTR is regulated.

极化的上皮细胞分离其顶膜和基底侧膜的功能和活动。这就需要将基底膜上的激素和神经递质信号转化为可在根尖膜上利用的信号。环状AMP第二信息的转导依赖于多功能丝氨酸-苏氨酸蛋白激酶和磷酸蛋白磷酸酶。这些蛋白质通过改变这些酶和离子通道的磷酸化状态来调节细胞事件，如离子通道激活和酶活性。最近，有人提出，在靠近特定底物的位置靶向和隔离激酶和磷酸酶会影响这些看似多功能的酶的特异性。最近，人们认识到CFTR氯离子通道通过PDZ结构域相互作用与EBP50结合。在其他系统中，已知EBP50还与Ezrin结合，Ezrin是最初在胃壁细胞中发现的一种AKAP。我们已经分离出被包裹在囊泡上的囊泡，作为富含CFTR的膜部分。这些囊泡还含有EBP50、Ezrin和II型PKA，我们推测这种多聚体蛋白复合体可能参与了靶向PKA到CFTR的过程。虽然Ezrin已被鉴定为极化细胞中的AKAP，但它绝不是唯一已知的AKAP。因此，我们已经使用RII重叠分析确定了存在于笼状蛋白包被的囊泡中的其他AKAP。最近的研究也表明，某些AKAP可能是多功能蛋白，作为靶向激酶和磷酸酶活性的支架。因此，我们认为，通过将激酶和磷酸酶活性靶向于cftr，可以通过磷酸化和去磷酸化来选择性和特异性地调节cftr。因此，该提案的总体目标是确定在极化上皮细胞的顶膜上锚定激酶和磷酸酶异构体的生化和分子基础，这是调节CFTR所必需的。目标1和目标2旨在阐明靶向cftr的激酶活性的支架分子。在目标1中，我们通过执行免疫共沉淀和免疫细胞化学共定位来建立Ezrin、RII、EBP50和CFTR之间的关联，从而检验Ezrin发挥这一功能的假设。目的2旨在评估其他AKAP在靶向蛋白激酶中的作用，以及其他AKAP在靶向CFTRK活性中的作用。在目标1中，我们通过执行免疫共沉淀和免疫细胞化学共定位来建立Ezrin、RII、EBP50和CFTR之间的关联，从而检验Ezrin发挥这一功能的假设。目的2旨在评估其他AKAP在靶向CFTRK活性方面的作用。RII覆盖和cAMP亲和层析将被用来描述存在于笼罩囊泡中的AKAP。目的3旨在阐明针对CFTR的磷酸酶活性的支架蛋白。将使用在肺和胰腺中表达的B56alpha亚基亚型的抗体，对亚细胞进行免疫金电子显微镜和免疫印迹分析(包括分离包被笼罩的小泡)。这些研究将为上皮细胞分离其第二信使依赖的信号通路的一般机制提供重要的见解，特别是对CFTR的调控机制。