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.
| Shen, B Q; Yang, C M; Widdicombe, J H (1991) Rapid procedure for obtaining tracheal apical membranes. Am J Physiol 261:L102-5 |
| Verkman, A S; Takla, R; Sefton, B et al. (1989) Quantitative fluorescence measurement of chloride transport mechanisms in phospholipid vesicles. Biochemistry 28:4240-4 |
