SGK1 activity in NaSuperscript+/Superscript ab

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

Publish Date: 2008/09/12

Volume: 457, Issue: 6, Pages: 1287-1301

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Abstract

Studies of HeLa cells and serum and glucocorticoidregulated kinase 1 SGK1 knockout mice identified threonine residues in the nmyc downstreamregulated gene 1 protein NDRG1Thr346/356/366 that are phosphorylated by SGK1 but not by related kinases Murray et al Biochem J 3851–12 2005 We have therefore monitored the phosphorylation of NDRG1Thr346/356/366 in order to explore the changes in SGK1 activity associated with the induction and regulation of the glucocorticoiddependent Na+ conductance G Na in human airway epithelial cells Transient expression of active SGK1S422D and inactive SGK1K127A SGK1 mutants confirmed that activating SGK1 stimulates NDRG1Thr346/356/366 phosphorylation Although G Na is negligible in hormonedeprived cells these cells displayed basal SGK1 activity that was sensitive to LY294002 an inhibitor of 3phosphatidylinositol phosphate kinase PI3K Dexamethasone 02 μM acutely activated SGK1 and the peak of this response 2–3 h coincided with the induction of G Na and both responses were PI3Kdependent While these data suggest that SGK1 might mediate the rise in G Na transient expression of the inactive SGK1K127A mutant did not affect the hormonal induction of G Na but did suppress the activation of SGK1 Dexamethasonetreated 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 LY294002sensitive 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 tissuesMany responses to glucocorticoid and mineralocorticoid hormones involve the activation of serum and glucocorticoidregulated kinase 1 SGK1 and genetic deletion of the sgk1 gene prevents the aldosteronemediated increase in renal Na+ retention that normally occurs when dietary Na+ is reduced 53 However in the distal colon sgk1 knockout also leads to a paradoxical stimulation of Na+ absorption and does not prevent the increased absorption of fecal Na+ seen in Na+restricted animals 39 It is also clear that sgk1 deletion does not cause overt lung symptoms despite the fact that the integrated functioning of the respiratory tract is dependent upon glucocorticoidinduced absorption of Na+ from the liquid film that covers the lung/airway epithelia 3 9 30 and so although SGK1 does seem to be involved in the control of epithelial Na+ absorption see eg 5 6 17 26 44 the way in which this kinase contributes to this regulated ion transport process is not fully understood 12 19 40 49 51SGK1 belongs to the AGC subfamily of protein kinases that also includes protein kinase B PKB ribosomal S6 kinases RSKs S6Ks and the mitogen/stressactivated kinases However SGK1 most closely resembles PKB and these enzymes can phosphorylate the same proteins especially if their activities are assayed in vitro or using cellular over expression systems 25 34 However it is also clear that SGK1 and PKB selectively phosphorylate certain proteins in vivo 31 and the product of nmyc downstream gene 1 NDRG1 has recently been identified as a physiological substrate for SGK1 but not PKB RSK1 or S6K1 31 32 This was initially surprising since the NDRG1 residues that are phosphorylated by SGK1 Thr346/356/366 form part of canonical AGC substrate motifs within a Cterminal nonapeptide ArgSerArgSerHisThrSerGluGly that is repeated three times and this location predicts phosphorylation by other AGC kinases However studies of synthetic peptides identified residues within the nonapeptide that prevented phosphorylation by PKB and RSK1 32 and NDRG1Thr346/356/366 phosphorylation in HeLa cells and mouse tissues is strictly dependent upon the expression of SGK1 31 Although SGK1 also phosphorylates other residues within this protein Thr228 and Ser330 antibodies that recognize the phosphorylation of these residues crossreact with the equivalent residues in the closely related NDRG2 protein Thr330 Ser332 In contrast the SGK1 consensus sequences that include Thr346/356/366 are found within a repeated nonapeptide that is not found in NDRG2 NDRG3 or NDRG4 Antibodies against this nonapeptide thus show almost no crossreactivity with other phosphoproteins 31Since the phosphorylation of NDRG1Thr346/356/366 can be assayed using wellcharacterized antibodies 31 these data effectively establish a new way of monitoring cellular SGK1 activity which could simplify physiological studies of this enzyme 31 In the present study we therefore use this new approach to monitor the changes in SGK1 activity associated with the induction and hormonal regulation of the glucocorticoiddependent Na+ conductance G Na in a human airway epithelial cell line 11 13H441 cells were routinely cultured in RPMI 1640 medium supplemented with fetal bovine serum FBS 85 newborn calf serum NCS 85 glutamine 2 mM transferrin 5 μg mL−1 selenium 5 ng mL−1 and an antibiotic/antimycotic mixture Sigma Chemical Poole Dorset England For experiments cells removed from culture flasks using trypsin/ethylenediaminetetraacetic acid EDTA were plated onto glass cover slips 10 cm Petri dishes or Costar Snapwell Corning BV SchipolRijk The Netherlands culture membranes 37 After approximately 24 h incubation in standard medium the cells were washed and incubated in medium prepared using FBS 85 that had been dialyzed to remove hormones/growth factors Hormonedeprived cells were maintained in this medium for 24–36 h before being used in experiments while hormonestimulated cells were exposed to dexamethasone 02 μM as described in the textCells were transfected Lipofectamine Transfection Reagent Invitrogen UK with pGL3 plasmids Invitrogen 1–15 μg per well incorporating cDNA sequences encoding modified forms of SGK1 The construct used to enhance cellular SGK1 activity encoded a glutathione Stransferase GST fusion protein incorporating a truncated form of SGK1 lacking 60 N terminus amino acid residues that had been further modified by mutating Ser422 to Asp SGK1S422D The Nterminal truncation induces a 20fold to 250fold increase in protein expression by preventing protein degradation while the S422D mutation allows this truncated protein to be more readily activated by 3phosphoinositidedependent protein kinase 1 PDK1 25 26 Taken together these changes confer a constitutively active phenotype upon this mutant 25 We also explored the effects of expressing a construct encoding an analogous GST/truncated SGK1 protein in which Lys127 had been mutated to Ala SGK1K127A This disrupts the protein’s ATPbinding site creating a catalytically inactive form of the enzyme 25 Cells used for electrophysiological studies were cotransfected with a second plasmid pEGFP 01 μg encoding green fluorescent protein GFP which allowed successfully transfected cells to be identified by GFP fluorescence Membrane currents were recorded from these cells after approximately 24–26 h see below Nonspecific effects of the transfection procedure and/or expression of a heterologous protein were controlled for by recording currents from cells that had simply been transfected with the GFPexpressing plasmid The identity of all cDNA constructs was independently confirmed by sequencing at the DNA Analysis Facility Ninewells Hospital and Medical School University of DundeeProtein was extracted from cells on Petri dishes by scraping/sonication in a lysis buffer containing phosphatase and protease inhibitors 1 Triton 50 mM Tris–HCl pH 75 1 mM ethylene glycol tetraacetic acid 1 mM EDTA 1 mM Na orthovanadate 10 mM glycerol phosphate 50 mM NaF 5 mM Na pyrophosphate 270 mM sucrose 01 βmercaptoethanol 1 Roche Mini Protease Inhibitor tablet per 10 mL Extracted protein was then diluted 31 with 4x NuPage LDS sample buffer Invitrogen denatured at 70°C 10 min and aliquots of denatured protein normally 12 μg fractionated on sodium dodecyl sulfate polyacrylamide gels NuPage Novex BisTris Mini gels Invitrogen and blotted onto HybondP membranes Amersham Buckinghamshire UK Total NDRG1 abundance was then determined by Western analysis using an antibody 31 against the fulllength protein while expression of Thr346/356/366phosphorylated NDRG1 was quantified using a phosphospecific antibody against the SGK1phosphorylated form of the Cterminal nonapeptide 31 Preliminary experiments verified that these antibodies stained only a single substantive band in protein extracted from H441 cells The electrophoretic mobility of this protein indicated a molecular weight of 45 kDa which accords well with the previously reported value of 43–45 kDa 31 The phosphorylation of cyclic adenosine monophosphate cAMP response elementbinding protein CREB at Ser133 was similarly quantified using antibodies against total and Ser133phosphorylated CREB Upstate Dundee UK Results of such experiments were quantified by densitometry using the Syngene Genegenius Synoptics imagecapturing system GeneSnap imagecapturing program and GeneTools densitometry analysis programMembrane currents I m were recorded from single cells held under voltage clamp in the perforated patch recording configuration using bath and pipette solutions designed to maintain quasiphysiological ionic gradients 11 13 In each experiment the mean current evoked by driving the holding potential V Hold through a series of four ramps −113 to 87 mV in 2 s was recorded both under standard conditions and after Na+o had been lowered to 10 mM Nmethyldglucammonium substitution The current that persisted at low Na+o was then subtracted from the corresponding control current in order to isolate the Na+odependent component of I m I Na All reported potentials are corrected for the liquid junction potential between the pipette and bath solutions and membrane potentials V m were inferred from the values of V Hold at which I m was 0 Values of membrane conductance G Na were estimated by regression analysis ie ΔI/ΔV Hold These methods are described in detail elsewhere 11 13

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