CFTR REGULATION BY TARGETED KINASE AND PHOSPHATASE

极化上皮细胞分离其顶膜和基底膜的功能和活动。这就需要将基底外侧膜上的激素和神经递质信号转化为可以在顶膜上利用的信号。环AMP第二信使的转导依赖于多功能丝氨酸-苏氨酸蛋白激酶和磷蛋白磷酸酶。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诸如离子通道激活和酶活性的细胞事件。最近有人提出，在接近特定底物的位点上靶向和螯合激酶和磷酸酶会影响这些看似多功能的酶的特异性。 内切酶最近，已经认识到CFTR氯离子通道通过PDZ结构域相互作用结合EBP 50。在其他系统中，已知EBP 50也结合ezrin，一种最初在胃壁细胞中鉴定的AKAP。我们已经分离出网格蛋白包被的囊泡作为富含CFTR的膜部分。这些囊泡还含有EBP 50，埃兹蛋白和II型PKA，使我们推测，这种多聚体蛋白复合物可能参与靶向PKA CFTR。虽然ezrin已被确定为极化细胞中的AKAP，但它绝不是已知的唯一AKAP。因此，我们已经使用RII重叠测定法鉴定了网格蛋白包被的囊泡中存在的其他AKAP。最近的研究还表明，某些AKAP可能是多功能蛋白质，作为支架靶向激酶和磷酸酶活性。因此，我们提出，针对CFTR的激酶和磷酸酶活性允许通过磷酸化和去磷酸化选择性和特异性调节CFTR。因此，该提案的总体目标是确定激酶和磷酸酶亚型锚定在极化上皮细胞的顶端膜上的生化和分子基础，这是CFTR调节所必需的。目的1和2是针对阐明靶向CFTR的激酶活性的支架分子。在目的1中，我们通过进行免疫共沉淀和免疫细胞化学共定位来建立ezrin、RII、EBP 50和CFTR的关联，从而测试ezrin发挥这种功能的假设。目的2旨在评价其他AKAP在靶向激酶中的作用，其他AKAP在靶向CFTR的激酶活性中的作用。在目的1中，我们通过进行免疫共沉淀和免疫细胞化学共定位来建立ezrin、RII、EBP 50和CFTR的关联，从而测试ezrin发挥这种功能的假设。目的2旨在评价其他AKAP在将激酶活性靶向CFTR中的作用。将进行RII覆盖和cAMP亲和色谱法，以描述网格蛋白包被囊泡中存在的AKAP。目的3旨在阐明靶向CFTR磷酸酶活性的支架蛋白。将使用抗肺和胰腺中表达的B56 α亚基同种型的抗体对亚细胞组分（包括网格蛋白包被囊泡的分离）进行免疫金电子显微镜检查和免疫印迹。这些调查将提供重要的见解上皮细胞隔离其第二信使依赖性信号通路的一般机制，特别是CFTR的调节机制。

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.