Regulation of CFTR by Protein Kinase C

囊性纤维化是一种电解质转运异常的疾病，其遗传学基础是囊性纤维化跨膜调节因子(CFTR)突变，这是一种顶端分泌性氯通道。纠正慢性阻塞性肺疾病的气道功能障碍主要集中在通过治疗途径激活心尖氯通道，而不是通过改变cftr基因来纠正异常的氯离子分泌，从而抵消粘液的过度堆积。Cftr主要受cAMP调节，然而PI发现PKC-epsilon的结构性活性对于cAMP依赖的CFtr调节是必需的。因此，对cftr功能的调控比cAMP依赖的cAMP对cftr的磷酸化更为复杂。新的初步研究表明，PKC-epsilon和CFTR之间的关联可能代表直接或间接结合。除了CFTR，其他与PKC-epsilon相关的气管上皮蛋白，包括支架蛋白，可能形成一个多蛋白复合体，将PKC-epsilon近端捆绑在一起。这项资助计划的假设是，PKC-epsilon通过磷酸化来调节CFTR功能。我们将从以下几个方面对其进行详细的研究：1)确定PKC-epsilon的活性是否调节其与CFTR的相互作用。活性将使用PKC抑制剂来控制，或者也可以减少质量，使用PKC-epsilon的反义寡核苷酸来控制。酶活性与PKC-epsilon与cftr共纯化和野生型cftr的磷酸化有关。贩运有能力和不能突变的CFTR(分别为G551D和deltaF508)是否会改变CFTR与PKC-epsilon的相互作用及其对CFTR的磷酸化将被确定。2)确定PKC-epsilon的活性是否受多蛋白复合体的调节。PKC-epsilon与重组蛋白或内源性蛋白(CFTR、RACK、肌动蛋白)的结合将通过直接结合或重叠实验来测量，并以结合的K-m来定量。将确定激活和/或失活的酶的结合以及PKC同型的特异性。活性将通过省略PKC激活剂或添加PKC抑制剂以及使用反义方法下调结合蛋白来操纵。蛋白激酶C抑制剂快速丧失cftr功能是否与丝氨酸/苏氨酸蛋白磷酸酶(S)的活性有关及其与蛋白激酶C-epsilon寻找伴侣的关系将被确定。3)确定PKC-epsilon与靶/结合蛋白相互作用的位点(S)。将预测和测试PKC-epsilon和/或结合特定结构域中的序列基序，并使用抑制CFTR结合和cAMP依赖的激活的多肽进行测试。

Cystic fibrosis is a disease of electrolyte transport abnormalities, which has, as its genetic basis, a mutation of the cystic fibrosis transmembrane regulator (CFTR), an apical secretory Cl channel. Correction of airway dysfunction in CF has centered on therapeutic approaches to activate apical Cl channels other than CFTR and genetic alteration of CFTR to correct abnormal Cl secretion and thus offset excess mucus accumulation. CFTR is regulated primarily by CAMP, however, the PI discovered that constitutive activity of PKC-epsilon is necessary for CAMP-dependent regulation of CFTR. Thus, regulation of CFTR function is more complicated than just cAMP-dependent phosphorylation of CFTR. New pilot studies indicate an association between PKC-epsilon and CFTR that might represent direct or indirect binding. In addition to CFTR, other tracheal epithelial proteins associate with PKC-epsilon, including scaffold proteins which might form a multiprotein complex that tethers PKC-epsilon proximal to its target. The hypothesis of this grant proposal is that PKC-epsilon regulates CFTR function through phosphorylation. This will be studied in detail in the following specific aims: 1) To determine whether activity of PKC- epsilon regulates its interaction with CFTR. Activity will be manipulated using PKC inhibitors or, to also decrease mass, with antisense oligonucleotides to PKC-epsilon. Enzyme activity will be correlated with co-purification of PKC-epsilon with CFTR and phosphorylation of wild type CFTR. Whether trafficking competent and incompetent mutant CFTR (G551D, deltaF508, respectively) alter CFTR interaction with PKC-epsilon and its phosphorylation of CFTR will be determined. 2) To determine whether activity of PKC-epsilon is regulated by association with a multiprotein complex. Binding of PKC-epsilon with recombinant or endogenous proteins (CFTR, RACK, actin) will be measured by direct binding or overlay assay and quantitated as a K-m for binding. Binding of activated and/or inactive enzyme and specificity for PKC isotype will be determined. Activity will be manipulated by omitting PKC activators or adding PKC inhibitor and by downregulating binding protein using an antisense approach. Whether rapid loss of CFTR function by PKC inhibitor is correlated to activity of serine/threonine protein phosphatase(s) and its association with PKC-epsilon finding partners will be determined. 3) To identify site(s) of interaction between PKC- epsilon and target/binding protein. Sequence motifs in specific domains of PKC-epsilon and/or binding will be predicted and tested using peptides to inhibit binding and cAMP-dependent activation of CFTR.