Differential Regulation of Na-K-Cl Cotransport

描述(申请人提供)：NKCC1(Na-K-2Cl共转运体)是一种电解质转运体，定位于肺衬里上皮细胞的基侧膜，对最佳的粘液纤毛清除至关重要，因为它为分泌提供氯。NKCC1基因突变导致跨上皮液异常分泌。NKCC1的调控是复杂的，涉及非哺乳动物细胞、cAMP依赖的PKA和蛋白磷酸酶PP1。PI发现PKC-？是人类呼吸道NKCC1功能所必需的，最近发现靶向PKC-？TO NKCC1涉及一种新的多蛋白复合体，它包括NKCC1、PKC-β、肌动蛋白和NHERF。人们对这种多蛋白复合体知之甚少。结合研究揭示了PKC-？对肌动蛋白和功能的研究表明了PP2A的作用和肌动蛋白聚合状态在NKCC1功能中的作用。这笔赠款的假设是PKC-？通过肌动蛋白细胞骨架参与调节NKCC1的功能。这将在以下特定目的中进行检验：1)检验肌动蛋白细胞骨架对NKCC1功能是必需的假说。肌动蛋白的组织、动态和功能将使用海洋毒素来操纵。将确定对基础和激动剂刺激的NKCC1功能和转换的功能影响，以及PKC-？活性。以及PP1和PP2A。PKC-？而PP2A将使用反义方法下调。2)检验PKC-？通过多蛋白复合体与NKCC1相互作用。将使用下拉、免疫共沉淀和结合分析来研究蛋白质对的相互作用。在竞争实验中，将使用标记的融合蛋白和突变的蛋白来确定特定的相互作用部位，抑制肽将被输送到细胞中进行功能研究。我们将首次确定PP2A是否是多蛋白复合体中的合作伙伴。3)检验PKC-？通过磷酸化事件调节NKCC1的功能。我们将确定PKC-？直接磷酸化或通过NKCC1相关蛋白间接磷酸化NKCC1(S)。PKC-？并将使用从NKCC1的细胞质结构域预测的标记融合蛋白来研究PP2A的去磷酸化。这些结果将为调控NKCC1功能的蛋白质和受NKCC1调控的蛋白质提供新的信息。长期的结果是，在分子水平上操纵蛋白质-蛋白质相互作用，促进NKCC1的功能，并可能导致NKCC1在病理生理状态下的功能改善。

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.