SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr(346/356/366) phosphorylation

S. K. Inglis, M. Gallacher, S. G. Brown, N. McTavish, J. Getty, E. M. Husband, J. T. Murray, S. M. Wilson

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    Abstract

    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 mu M) 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.

    Original languageEnglish
    Pages (from-to)1287-1301
    Number of pages15
    JournalPflugers Archiv-European Journal of Physiology
    Volume457
    Issue number6
    DOIs
    Publication statusPublished - Apr 2009

    Keywords

    • Glucocorticoids
    • Epithelial Na+ channel
    • n-myc downstream-regulated gene 1
    • Cell signaling
    • CHANNEL ALPHA-SUBUNIT
    • REGULATED PROTEIN-KINASE
    • SODIUM-CHANNEL
    • A6 CELLS
    • FLUID TRANSPORT
    • LUNG-DISEASE
    • MICE LACKING
    • ENAC
    • SERUM
    • ACTIVATION

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