PROTEIN PHOSPHORYLATION AND AIRWAY CHLORIDE SECRETION

本提案的目的是确定cAMP的作用- C1跨气管分泌依赖性蛋白磷酸化 上皮细胞 使用培养的气管上皮细胞，蛋白质将被 用32 PO 4孵育一小时磷酸化。 细胞溶质和 膜蛋白将被分离。 变化的时间过程 在个体蛋白质的磷酸化反应中 增加细胞内cAMP，并进行比较 与Cl分泌变化的时间进程有关。 催化 cAMP依赖性蛋白激酶的亚基AT 32 P和cAMP将 被引入顶端膜囊泡。 变化 磷酸化将与Cl流入的变化相关 用C1敏感的荧光探针SPQ测量。 心尖 将使用膜，因为它们相对富含 膜蛋白 为了同样的目的，我们将努力重复 这些关于细胞骨架成分的囊泡的研究 被移除了 正常培养细胞的磷酸化变化 并将囊泡与CF细胞的囊泡进行比较。 水平 cAMP依赖性蛋白激酶及其对cAMP的敏感性将 在正常和CF细胞中进行比较。 内源性底物将 在细胞提取物中测量。 RI和RII的各种形式 cAMP依赖性蛋白激酶的调节亚基将被 用cAMP的光亲和衍生物共价标记，和 在二维凝胶上分离后测定其水平。 所有 实验最初将在狗或牛细胞上进行。 一旦 建立了技术，非CF和CF人类细胞将被 比较了 因为CF可能涉及有缺陷的交互 在钙依赖性和cAMP依赖性系统之间， 依赖cAMP的蛋白激酶和磷酸化 将在钙调蛋白依赖性蛋白激酶上进行， 蛋白激酶C 我们希望能得到信息， C1的分泌受到调节。 膜蛋白改变其 磷酸化的速度与C1分泌的变化一样快， 顶膜C1通道的候选者。 差异 CF中的激酶水平或磷酸化模式将提供 关于这种疾病的基本代谢缺陷的信息。

The purpose of this proposal is to determine the role of cAMP- dependent protein phosphorylation in C1 secretion across tracheal epithelia. Using cultured tracheal epithelial cells, proteins will be phosphorylated by an hour's incubation with 32PO4. Cytosolic and membrane proteins will be separated. The time course of changes in phosphorylation of individual proteins in response to agents which increase intracellular cAMP will be followed, and compared to the time course of change in Cl secretion. The catalytic subunit of cAMP-dependent protein kinase, AT32P and cAMP will be introduced into apical membrane vesicles. Changes in phosphorylation will be correlated with changes in Cl influx measured with the C1-sensitive fluorescent probe, SPQ. Apical membranes will be used because they are relatively enriched in membrane proteins. To this same end, we will try and repeat these studies on vesicles from which cytoskeletal elements have been removed. Phosphorylation changes in normal cultured cells and vesicles will be compared with those of CF cells. Levels of cAMP-dependent protein kinase and its sensitivity to cAMP will be compared in normal and CF cells. Endogenous substrates will be measured in cell extracts. The various form of the RI and RII regulatory subunits of cAMP-dependent protein kinase will be covalently labelled with a photo-affinity derivative of cAMP, and their levels determined following separation on two-D gels. All experiments will be performed initially on dog or cow cells. Once the techniques are established, non-CF and CF human cells will be compared. Because, CF may involve a defective interaction between Ca- and cAMP-dependent systems, similar experiments to those on cAMP-dependent protein kinase and phosphorylation will be performed on the calmodulin-dependent protein kinase and protein kinase C. We hope to obtain information on how normal C1 secretion is regulated. Membrane proteins which change their phosphorylation as rapidly as the change in C1 secretion are candidates for the apical membrane C1 channel. Differences in kinase levels or phosphorylation patterns in CF would provide information as to the basic metabolic defect in this disease.