Serum- and Glucocorticoid-inducible Kinase-1 Regulation of Human Cystic Fibrosis transmembrane Conductance Regulator

摘要

Serum- and glucocorticoid-inducible kinase-1 (SGK1) has been shown by heterologous expression in Xenopus oocytes to regulate several transport proteins including epithelial sodium channels (ENaC), sodium-hydrogen exchanger isoform 3 (NHE3), and the human cystic fibrosis trans-membrane conductance regulator (CFTR). This serine/threonine kinase shares a high degree of similarity in its catalytic region with the kinases protein kinase A (PKA), protein kinase C (PKC), protein kinase B (PKB)/Akt, phosphatidylinositol-dependent kinase-1 (PDK1), and p70 S6 kinase. Because SGK increases the plasma membrane expression of other ion channels, we tested the hypothesis that SGK1 stimulates CFTR-mediated Cl currents by increasing the number of CFTR Cl channels in the plasma membrane. CFTR Cl currents were measured in Xenopus oocytes by the two-electrode voltage clamp technique, and CFTR in the plasma membrane was determined by laser scanning confocal microscopy. Wild-type SGK1 stimulated CFTR Cl currents by 42% and increased the amount of CFTR in the plasma membrane by 35%. A kinase-dead SGK mutant (K127N) had a dominant-negative effect on CFTR, reducing CFTR Cl currents by 38%. In addition, deletion of the C-terminal PDZ-interacting motif (SGK1-?SFL) increased CFTR Cl currents by 108%. Thus, SGK1-?SFL was 2.7-times as effective than wt-SGK1 in stimulating CFTR Cl currents. Neither wt-SGK nor the K127N mutant had any effect on Cl currents in oocytes when expressed alone in the absence of CFTR. We conclude that SGK1 stimulates CFTR Cl currents in Xenopus oocytes by increasing the number of channels in the plasma membrane. In addition, the effect of SGK may be mediated by protein-protein interactions involving the PDZ interacting motif. The finding that SGK1 regulates CFTR function in Xenopus oocytes may open up new approaches to discover therapies for cystic fibrosis if these results can be replicated in human airway epithelial cell lines or other relevant experimental models. This research was supported by grant RO1-DK45881 from NIDDK to BAS, a Cystic Fibrosis Foundation research development program grant to BAS, grant R01-DK68196 from NIDDK to RAF, NIEHS Center grant P30-ES03828, and by INBRE grant P20-RR016463 from NCRR. RT was supported by NSF Research Experience for Undergraduates grant NSF DBI-0453391.