Regulation of CFTR by Protein Kinase C

蛋白激酶 C 对 CFTR 的调节

• 批准号: 6537977
• 负责人: CAROLE M LIEDTKE
• 金额: $ 26.78万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-10 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537977
• 关键词: antisense nucleic acid; chloride channels; cystic fibrosis; enzyme activity; enzyme inhibitors; isozymes; protein binding; protein isoforms; protein kinase C; protein protein interaction; respiratory epithelium; tissue /cell culture

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.

囊性纤维化是一种电解质转运异常疾病，其遗传基础是囊性纤维化跨膜调节因子（CFTR）突变，即顶端分泌型Cl通道。CF中气道功能障碍的纠正集中在激活除CFTR之外的顶端Cl通道的治疗方法和CFTR的遗传改变，以纠正异常Cl分泌，从而抵消多余的粘液积聚。CFTR主要由cAMP调节，然而，PI发现PKC-β的组成性活性对于CFTR的cAMP依赖性调节是必需的。因此，CFTR功能的调节比CFTR的cAMP依赖性磷酸化更复杂。新的初步研究表明，PKC-β和CFTR之间的关联可能代表直接或间接的结合。除了CFTR，其他气管上皮蛋白与PKC-β相关，包括支架蛋白，其可能形成多蛋白复合物，将PKC-β束缚在其靶标附近。这项拨款提案的假设是PKC-β通过磷酸化调节CFTR功能。这将在以下具体目标中详细研究：1）确定PKC-β的活性是否调节其与CFTR的相互作用。将使用PKC抑制剂或也为了降低质量，使用PKC-β的反义寡核苷酸来操纵活性。酶活性将与PKC-β与CFTR的共纯化和野生型CFTR的磷酸化相关。将确定有运输能力和无运输能力的突变CFTR（分别为G551 D、deltaF 508）是否改变CFTR与PKC-β的相互作用及其CFTR的磷酸化。2)确定PKC-β的活性是否受多蛋白复合物的调节。将通过直接结合或覆盖测定测量PKC-β与重组或内源性蛋白（CFTR、RACK、肌动蛋白）的结合，并定量为结合的Km。将测定活化和/或失活酶的结合以及对PKC同种型的特异性。通过省略PKC激活剂或添加PKC抑制剂以及通过使用反义方法下调结合蛋白来操纵活性。将确定PKC抑制剂导致的CFTR功能快速丧失是否与丝氨酸/苏氨酸蛋白磷酸酶的活性相关及其与PKC-β发现配偶体的关联。3)确定PKC-β与靶蛋白/结合蛋白之间的相互作用位点。将使用抑制CFTR的结合和cAMP依赖性活化的肽来预测和测试PKC-β和/或结合的特定结构域中的序列基序。