Differential Regulation of Na-K-Cl Cotransport

描述(由申请人提供)：呼吸道上皮细胞NKCC1与分泌形式的基侧Na-K-2Cl共转运，通过提供分泌到气道腔的氯来共享特性。NKCC1还在维持纤毛周围纤毛周围的纤毛周围液体层方面发挥非分泌性作用，这是最佳粘液纤毛清除所必需的。纤毛周围液层受损被认为与肺部疾病的病理生理学有关，如哮喘、支气管炎和囊性纤维化。我们以前的研究证实，蛋白激酶C-增量(PKC4)及其与支架肌动蛋白的结合，在多种刺激(包括高渗应激、细胞内低氯和Gs偶联受体)激活气道上皮细胞NKCC1中起着关键作用。我们发现了一个由NKCC1的N端、蛋白磷酸酶2A(PP2A)、应激激活的丝氨酸苏氨酸激酶SPAK和PKC4组成的调控蛋白质组，但不了解该蛋白质组如何调控NKCC1的功能。我们还发现了NKCC1的C末端(CT)与铜转运蛋白Murr1(COMMD1)的相互作用，以及在Murr1下调后高渗胁迫下NKCC1的活性丧失。我们还没有确定Murr1是如何调控NKCC1的。我们研究的长期目标是了解蛋白质-蛋白质相互作用如何调节NKCC1。我们提出了以下具体目标：1)验证PKC4调节SPAK功能的假设。我们将使用沉默RNA将Spak的下调与NKCC1和PKC4的激活相关联。我们将研究PKC4与SPAK的结合，以及PKC4对SPAK的磷酸化和PP2A的去磷酸化。将确定蛋白质与SPAK相互作用的结合基序，并通过氧化足迹建立结合热力学。2)验证Murr1调控NKCC1表面表达的假说。我们将通过沉默RNA下调NKCC1的表面表达和功能，确定a)Murr1是否对NKCC1的表面表达和功能是必需的，b)NKCC1在基底侧膜的周转时间及其受肌动蛋白和Murr1的调节，以及c)Murr1和CT-NKCC1的结合基序，使用新的氧化足迹和质谱学方法，我们将从中开发抑制肽来改变末端反应，包括蛋白质相互作用、NKCC1活性和NKCC1表面表达。3)验证NKCC1磷酸化调节其活性的假说。我们将确定Spak和PP2A在NKCC1磷酸化中的作用。我们计划测试编码可能的磷酸化位点的多肽来抑制NKCC1的激活，并计划下调关键酶，并将质量和活性的丧失与对NKCC1功能的影响相关联。项目简介：这个项目是理解蛋白质相互作用在调节Na-K-2Cl活性中的重要性和突出作用的重要的第一步。从这些研究中获得的新信息将使我们在纠正NKCC1功能缺陷方面更进一步，并导致开发独特的治疗工具来促进NKCC1在疾病状态下的功能。更重要的是，这些研究将为预测和测试改变NKCC1功能的药物(包括药物和娱乐药物)的效果奠定基础。

DESCRIPTION (provided by applicant): Airway epithelial NKCC1 shares properties with secretory forms of basolateral Na-K-2Cl cotransport by supplying Cl for secretion into the airway lumen. NKCC1 also has a non-secretory role in the maintenance of a periciliary fluid layer surrounding cilia, which are necessary for optimal mucociliary clearance. Compromising the periciliary fluid layer is thought to contribute to the pathophysiology of lung disorders, such as asthma, bronchitis, and cystic fibrosis. Our previous studies established a key role for protein kinase C-delta (PKC4) and its binding to the scaffold actin, in the activation of airway epithelial NKCC1 by multiple stimuli, including hyperosmotic stress, low intracellular Cl, and Gs coupled receptors. We identified a regulatory proteome, consisting of the N terminus of NKCC1, protein phosphatase 2A (PP2A), stress activated serine-threonine kinase SPAK and PKC4, but do not understand how the proteome regulates NKCC1 function. We also discovered the interaction of the C terminus (CT) of NKCC1 with a copper transport protein Murr1 (COMMD1) and loss of NKCC1 activation by hyperosmotic stress after downregulation of Murr1. We have yet to ascertain how Murr1 regulates NKCC1. The long term goal of our research is to understand how protein-protein interactions regulate NKCC1. We propose the following specific aims: 1) To test the hypothesis that PKC4 regulates the function of SPAK. We will correlate downregulation of SPAK, using silencing RNA, with activation of NKCC1 and of PKC4. We will investigate binding of PKC4 to SPAK and phosphorylation of SPAK by PKC4 and dephosphorylation by PP2A. Binding motifs for proteins interacting with SPAK will be determined and the thermodynamics of binding established by oxidative footprinting. 2) To test the hypothesis that Murr1 regulates NKCC1 surface expression. We will determine a) whether Murr1 is necessary for surface expression and function of NKCC1 by downregulating Murr1 using silencing RNA, b) the turnover time of NKCC1 in the basolateral membrane and its regulation by actin and Murr1 and c) binding motifs for Murr1 and CT-NKCC1, using novel methods of oxidative footprinting and mass spectrometry, from which we will develop inhibitory peptides to alter end responses, including protein interaction, NKCC1 activity, and NKCC1 surface expression. 3) To test the hypothesis that phosphorylation of NKCC1 modulates its activity. We will determine a role for SPAK and PP2A in NKCC1 phosphorylation. We plan to test peptides encoding putative phosphorylation sites for inhibition of NKCC1 activation and plan to downregulate the critical enzyme and correlate loss of mass and activity with effects on NKCC1 function. Project Narrative: This project is an important first step in understanding the importance and prominent role of protein interactions in the regulation of Na-K-2Cl activity. New information gained from these studies will take us one step further toward correcting defective NKCC1 function and lead to the development of unique therapeutic tools to promote NKCC1 function in disease states. More importantly, the studies will lay the foundation for predicting and testing the effects of drugs, both pharmaceutical and recreational, that alter NKCC1 function.