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，其遗传基础是囊性纤维化跨膜调节剂（CFTR）的突变，CFTR是一种顶端分泌Cl通道。CF患者气道功能障碍的纠正主要集中在激活CFTR以外的根尖Cl通道和改变CFTR的基因来纠正异常的Cl分泌，从而抵消过多的粘液积聚。CFTR主要由CAMP调控，然而，PI发现PKC-epsilon的组成活性对于CAMP依赖性的CFTR调控是必需的。因此，CFTR功能的调控比仅仅camp依赖性的CFTR磷酸化更为复杂。新的试点研究表明PKC-epsilon和CFTR之间的关联可能代表直接或间接的结合。除CFTR外，其他气管上皮蛋白与PKC-epsilon相关，包括支架蛋白，其可能形成多蛋白复合物，将PKC-epsilon系在其靶点的近端。本课题假设PKC-epsilon通过磷酸化调控CFTR功能。具体目的如下：1)确定PKC- epsilon的活性是否调控其与CFTR的相互作用。活性可以使用PKC抑制剂来控制，或者使用PKC-epsilon的反义寡核苷酸来降低质量。酶活性将与PKC-epsilon与CFTR的共纯化和野生型CFTR的磷酸化有关。将确定转运称职和不称职突变体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。2)确定PKC-epsilon的活性是否受多蛋白复合物的调控。PKC-epsilon与重组蛋白或内源性蛋白（CFTR， RACK, actin）的结合将通过直接结合或覆盖试验进行测量，并以K-m进行定量。将确定活化和/或失活酶的结合以及PKC同型的特异性。活性可以通过省略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控制。PKC抑制剂导致CFTR功能的快速丧失是否与丝氨酸/苏氨酸蛋白磷酸酶活性相关，以及其与PKC-epsilon寻找伴侣的关系将被确定。3)确定PKC- epsilon与靶蛋白/结合蛋白相互作用的位点。PKC-epsilon和/或结合的特定区域的序列基序将使用肽来预测和测试，以抑制结合和camp依赖性的CFTR激活。

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.