Differential Regulation of Na-K-Cl Cotransport

描述（由申请人提供）：NKCC1 （Na-K-2Cl共转运）是一种定位于肺上皮细胞基底外膜的电解质转运蛋白，它为分泌提供Cl，对优化纤毛黏液清除至关重要。基因改变的NKCC1导致上皮液分泌异常。NKCC1的调控是复杂的，涉及非哺乳动物细胞、camp依赖性PKA和蛋白磷酸酶PP1。PI发现PKC-？是人类气道NKCC1功能所必需的，最近发现靶向PKC-？NKCC1与一种新的多蛋白复合物有关，该复合物包括NKCC1、PKC-？， actin和NHERF。人们对这种多蛋白复合物知之甚少。结合研究揭示PKC-？对肌动蛋白和功能的研究表明PP2A和肌动蛋白聚合状态在NKCC1功能中的作用。这项拨款的假设是PKC-？通过参与肌动蛋白细胞骨架调节NKCC1功能。这将在以下具体目的中进行测试：1)验证肌动蛋白细胞骨架是NKCC1功能所必需的假设。肌动蛋白的组织，动态和功能将被操纵使用海洋毒素。对基础和激动剂刺激的NKCC1功能和转换的功能影响将被确定，PKC-？PP1和PP2A。PKC-?的质量和活性而PP2A将通过反义方式下调。2)验证PKC-？通过多蛋白复合物与NKCC1相互作用。蛋白对的关联将通过拉下、共免疫沉淀和结合试验进行研究。相互作用的特定位点将在竞争实验中使用标记融合蛋白和突变蛋白来确定，抑制肽将被递送到细胞中进行功能研究。我们将首次确定PP2A是否是多蛋白复合物中的伙伴。3)验证PKC-？通过磷酸化事件调节NKCC1的功能。我们会决定PKC-？直接磷酸化或通过NKCC1相关蛋白间接磷酸化NKCC1。PKC-?磷酸化位点将使用从NKCC1细胞质结构域预测的标记融合蛋白来研究PP2A对PP2A去磷酸化的影响。该结果将为研究NKCC1功能调控蛋白和受NKCC1调控的蛋白提供新的信息。长期结果是在分子水平上操纵蛋白质-蛋白质相互作用，促进NKCC1功能，并可能导致病理生理状态下NKCC1功能的改善。

DESCRIPTION (provided by applicant): NKCC1 (Na-K-2Cl co-transport) is an electrolyte transporter localized to the basolateral membrane of lining epithelial cells of the lung and is crucial for optimal mucociliary clearance because it supplies Cl for secretion. Genetically altered NKCC1 leads to abnormal transepithelial fluid secretion. Regulation of NKCC1 is complex, involving in non-mammalian cells, cAMP-dependent PKA and protein phosphatase PP1. The PI discovered that PKC-? is necessary for human airway NKCC1 function and recently found that targeting of PKC-? to NKCC1 involves a novel multiprotein complex that includes NKCC1, PKC-?, actin and NHERF. Very little is known about this multiprotein complex. Binding studies reveal direct association of PKC-? to actin and functional studies indicate a role for PP2A and the state of actin polymerization in NKCC1 function. The hypothesis of this grant is that PKC-? modulates NKCC1 function through the involvement of the actin cytoskeleton. This will be tested in the following specific aims: 1) to test the hypothesis that actin cytoskeleton is necessary for NKCC1 function. Actin organization, dynamics, and function will be manipulated using marine toxins. The functional effects on basal and agonist-stimulated NKCC1 function and turnover will be determined as will the activity of PKC-? and PP1 and PP2A. Mass and activity of PKC-? and PP2A will be downregulated using an antisense approach. 2) To test the hypothesis that PKC-? interacts with NKCC1 through a multiprotein complex. Association of protein pairs will be studied using pulldown, co-immunoprecipitation and binding assays. Specific sites of interaction will be determined using tagged fusion proteins and mutated proteins in competition experiments, Inhibitory peptides will be delivered into cells for functional studies. We will determine, for the first time, whether PP2A is a partner in the multiprotein complex. 3) To test the hypothesis that PKC-? modulates NKCC1 function through a phosphorylation event. We will determine whether PKC-? directly phosphorylates NKCC1 or indirectly through NKCC1-associated protein(s). Sites of phosphorylation by PKC-? and of dephosphorylation by PP2A will be investigated using tagged fusion proteins predicted from the cytoplasmic domains of NKCC1. The results will provide new information on proteins that regulate NKCC1 function and proteins regulated by NKCC1. The long-term outcome is manipulation, at the molecular level, of protein-protein interactions that facilitate NKCC1 function and could lead to improved NKCC1 function in pathophysiological states.