SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr346/356/366 phosphorylation

Studies of HeLa cells and serum- and glucocorticoid-regulated kinase 1 (SGK1) knockout mice identified threonine residues in the n-myc downstream-regulated gene 1 protein (NDRG1-Thr^346/356/366) that are phosphorylated by SGK1 but not by related kinases (Murray et al., Biochem J 385:1–12, 2005). We have, therefore, monitored the phosphorylation of NDRG1-Thr^346/356/366 in order to explore the changes in SGK1 activity associated with the induction and regulation of the glucocorticoid-dependent Na⁺ conductance (G _Na) in human airway epithelial cells. Transient expression of active (SGK1-S422D) and inactive (SGK1-K127A) SGK1 mutants confirmed that activating SGK1 stimulates NDRG1-Thr^346/356/366 phosphorylation. Although G _Na is negligible in hormone-deprived cells, these cells displayed basal SGK1 activity that was sensitive to LY294002, an inhibitor of 3-phosphatidylinositol phosphate kinase (PI3K). Dexamethasone (0.2 μM) acutely activated SGK1 and the peak of this response (2–3 h) coincided with the induction of G _Na, and both responses were PI3K-dependent. While these data suggest that SGK1 might mediate the rise in G _Na, transient expression of the inactive SGK1-K127A mutant did not affect the hormonal induction of G _Na but did suppress the activation of SGK1. Dexamethasone-treated cells grown on permeable supports formed confluent epithelial sheets that generated short circuit current due to electrogenic Na⁺ absorption. Forskolin and insulin both stimulated this current and the response to insulin, but not forskolin, was LY294002-sensitive and associated with the activation of SGK1. While these data suggest that SGK1 is involved in the control of G _Na, its role may be minor, which could explain why sgk1 knockout has different effects upon different tissues.