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 muM) 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.
Inglis
S K
SK
Lung Membrane Transport Group, Division of Maternal and Child Health Sciences, College of Medicine, Dentistry and Nursing, University of Dundee, Dundee DD1 9SY, Scotland.
Gallacher
M
M
Brown
S G
SG
McTavish
N
N
Getty
J
J
Husband
E M
EM
Murray
J T
JT
Wilson
S M
SM
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000166:cytology; D012964:Sodium / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D000099184:Serum-Glucocorticoid Regulated Kinases; D000099259:N-myc Downstream-Regulated Gene 1 Protein
D018797:Cell Cycle Proteins; D005576:Colforsin; D017362:Cyclic AMP Response Element-Binding Protein; D003907:Dexamethasone; D017874:Immediate-Early Proteins; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D012964:Sodium; D013912:Threonine; D000099184:Serum-Glucocorticoid Regulated Kinases; D000099259:N-myc Downstream-Regulated Gene 1 Protein
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