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, James Murray, S.M. Wilson

Research output: Contribution to journalArticle

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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

Fingerprint

Phosphorylation
Epithelial Cells
Glucocorticoids
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Colforsin
serum-glucocorticoid regulated kinase
Phosphatidylinositol 3-Kinases
Dexamethasone
Phosphotransferases
Chemical activation
Phosphatidylinositol 3-Kinase
Insulin
Threonine
HeLa Cells
Knockout Mice
Short circuit currents

Cite this

Inglis, S.K. ; Gallacher, M. ; Brown, S.G. ; McTavish, N. ; Getty, J. ; Husband, E.M. ; Murray, James ; Wilson, S.M. / SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr(346/356/366) phosphorylation. In: PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY. 2009 ; Vol. 457, No. 6. pp. 1287-1301.
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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.",
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Inglis, SK, Gallacher, M, Brown, SG, McTavish, N, Getty, J, Husband, EM, Murray, J & Wilson, SM 2009, 'SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr(346/356/366) phosphorylation', PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY, vol. 457, no. 6, pp. 1287-1301. https://doi.org/10.1007/s00424-008-0587-1

SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr(346/356/366) phosphorylation. / Inglis, S.K.; Gallacher, M.; Brown, S.G.; McTavish, N.; Getty, J.; Husband, E.M.; Murray, James; Wilson, S.M.

In: PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY, Vol. 457, No. 6, 04.2009, p. 1287-1301.

Research output: Contribution to journalArticle

TY - JOUR

T1 - SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr(346/356/366) phosphorylation

AU - Inglis, S.K.

AU - Gallacher, M.

AU - Brown, S.G.

AU - McTavish, N.

AU - Getty, J.

AU - Husband, E.M.

AU - Murray, James

AU - Wilson, S.M.

PY - 2009/4

Y1 - 2009/4

N2 - 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.

AB - 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.

U2 - 10.1007/s00424-008-0587-1

DO - 10.1007/s00424-008-0587-1

M3 - Article

VL - 457

SP - 1287

EP - 1301

JO - Pflügers Archiv - European Journal of Physiology

JF - Pflügers Archiv - European Journal of Physiology

SN - 0031-6768

IS - 6

ER -