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Expression of the Serum- and Glucocorticoid-inducible Protein Kinase, Sgk, Is a Cell Survival Response to Multiple Types of Environmental Stress Stimuli in Mammary Epithelial Cells

2003; Elsevier BV; Volume: 278; Issue: 8 Linguagem: Inglês

10.1074/jbc.m211649200

ISSN

1083-351X

Autores

Meredith L. Leong, Anita C. Maiyar, Brian Kim, Bridget O’Keeffe, Gary L. Firestone,

Tópico(s)

Cytokine Signaling Pathways and Interactions

Resumo

The effects of multiple stress stimuli on the cellular utilization of the serum- and glucocorticoid-inducible protein kinase (Sgk) were examined in NMuMg mammary epithelial cells exposed to hyperosmotic stress induced by the organic osmolyte sorbitol, heat shock, ultraviolet irradiation, oxidative stress induced by hydrogen peroxide, or to dexamethasone, a synthetic glucocorticoid that represents a general class of physiological stress hormones. Each of the stress stimuli induced Sgk protein expression with differences in the kinetics and duration of induction and in subcellular localization. The environmental stresses, but not dexamethasone, stimulated Sgk expression through a p38/MAPK-dependent pathway. In each case, a hyperphosphorylated active Sgk protein was produced under conditions in which Akt, the close homolog of Sgk, remained in its non-phosphorylated state. Ectopic expression of wild type Sgk or of the T256D/S422D mutant Sgk that mimics phosphorylation conferred protection against stress-induced cell death in NMuMg cells. In contrast, expression of the T256A/S422A Sgk phosphorylation site mutant has no effect on cell survival. Sgk is known to phosphorylate and negatively regulate pro-apoptotic forkhead transcription factor FKHRL1. The environmental stress stimuli that induce Sgk, but not dexamethasone, strongly inhibited the nuclear transcriptional activity and increased the cytoplasmic retention of FKHRL1. Also, the conditional IPTG inducible expression of wild type Sgk, but not of the kinase dead T256A mutant Sgk, protected Con8 mammary epithelial tumor cells from serum starvation-induced apoptosis. Taken together, our study establishes that induction of enzymatically active Sgk functions as a key cell survival component in response to different environmental stress stimuli. The effects of multiple stress stimuli on the cellular utilization of the serum- and glucocorticoid-inducible protein kinase (Sgk) were examined in NMuMg mammary epithelial cells exposed to hyperosmotic stress induced by the organic osmolyte sorbitol, heat shock, ultraviolet irradiation, oxidative stress induced by hydrogen peroxide, or to dexamethasone, a synthetic glucocorticoid that represents a general class of physiological stress hormones. Each of the stress stimuli induced Sgk protein expression with differences in the kinetics and duration of induction and in subcellular localization. The environmental stresses, but not dexamethasone, stimulated Sgk expression through a p38/MAPK-dependent pathway. In each case, a hyperphosphorylated active Sgk protein was produced under conditions in which Akt, the close homolog of Sgk, remained in its non-phosphorylated state. Ectopic expression of wild type Sgk or of the T256D/S422D mutant Sgk that mimics phosphorylation conferred protection against stress-induced cell death in NMuMg cells. In contrast, expression of the T256A/S422A Sgk phosphorylation site mutant has no effect on cell survival. Sgk is known to phosphorylate and negatively regulate pro-apoptotic forkhead transcription factor FKHRL1. The environmental stress stimuli that induce Sgk, but not dexamethasone, strongly inhibited the nuclear transcriptional activity and increased the cytoplasmic retention of FKHRL1. Also, the conditional IPTG inducible expression of wild type Sgk, but not of the kinase dead T256A mutant Sgk, protected Con8 mammary epithelial tumor cells from serum starvation-induced apoptosis. Taken together, our study establishes that induction of enzymatically active Sgk functions as a key cell survival component in response to different environmental stress stimuli. A diverse set of environmental stress and hormonal signals inundate mammalian cells and can potentially lead to mutations and other cellular changes that ultimately influence the transformed state of cells (1Cadet J. Berger M. Douki T. Morin B. Raoul S. Ravanat J.L. Spinelli S. Biol. Chem. 1997; 378: 1275-1286Google Scholar, 2Burg M.B. Kwon E.D. Kultz D. Annu Rev Physiol. 1997; 59: 437-455Google Scholar, 3Gabai V.L. Sherman M.Y. J Appl Physiol. 2002; 92: 1743-1748Google Scholar). The ability of a cell to sense and appropriately respond to adverse conditions is determined by an integrated network of intracellular signaling pathways that trigger proliferative, adaptive, and survival responses or mediate events leading to cell death (4Martindale J.L. Holbrook N.J. J. Cell. Physiol. 2002; 192: 1-15Google Scholar, 5De Zutter G.S. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6168-6173Google Scholar, 6Cross T.G. Scheel-Toellner D. Henriquez N.V. Deacon E. Salmon M. Lord J.M. Exp. Cell Res. 2000; 256: 34-41Google Scholar, 7Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Google Scholar). Environmental stresses as divergent as osmotic shock, ionizing radiation, and nutrient deprivation activate intracellular protein kinase cascades, which are generally conserved between metazoans and mammals, culminating in the induced or repressed transcription of specific sets of target genes (4Martindale J.L. Holbrook N.J. J. Cell. Physiol. 2002; 192: 1-15Google Scholar, 8Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Google Scholar, 9Tyrrell R.M. Exs. 1996; 77: 255-271Google Scholar, 10Tyrrell R.M. Photochem. Photobiol. 1996; 63: 380-383Google Scholar). For example, it is well established that members of the stress activated protein kinase family, c-Jun N-terminal kinase and p38/mitogen-activated protein kinase (MAPK), 1The abbreviations used are: MAPK, mitogen-activated protein kinase; Sgk, serum and glucocorticoid inducible protein kinase; dex, dexamethasone; PI 3-kinase, phosphatidylinositol 3-kinase; FKHRL1, forkhead transcription factor; FHRE, forkhead responsive element; IPTG, isopropyl-1-thio-β-d-galactopyranoside; PDK1, 3-phosphoinositide-dependent kinase 1; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HEK, human embryonic kidney are enzymatically activated by a wide range of environmental and cytotoxic stresses, as well as ischemic injury, which in many cell systems leads to apoptosis (5De Zutter G.S. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6168-6173Google Scholar, 8Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Google Scholar, 11Irving E.A. Bamford M. J Cereb. Blood Flow Metab. 2002; 22: 631-647Google Scholar, 12Obata T. Brown G.E. Yaffe M.B. Crit. Care Med. 2000; 28: N67-N77Google Scholar, 13Tibbles L.A. Woodgett J.R. Cell Mol. Life Sci. 1999; 55: 1230-1254Google Scholar, 14Karin M. Ann. N. Y. Acad. Sci. 1998; 851: 139-146Google Scholar). Known substrates and other downstream targets of these stress-activated protein kinases include transcription factors and other protein kinases (5De Zutter G.S. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6168-6173Google Scholar, 8Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Google Scholar, 12Obata T. Brown G.E. Yaffe M.B. Crit. Care Med. 2000; 28: N67-N77Google Scholar). Thus, the appropriate utilization of protein kinases is critical for the cell to respond to individual extracellular stress stimuli. However, relatively little is known about intracellular protein kinases whose expression is regulated in a stimulus-dependent manner to help trigger and mediate the selectivity of the stress response to environmental cues. We have reported the original isolation of the serum and glucocorticoid inducible protein kinase gene, Sgk, that is under acute transcriptional control by both serum and glucocorticoids (15Webster M.K. Goya L. Firestone G.L. J. Biol. Chem. 1993; 268: 11482-11485Google Scholar,16Webster M.K. Goya L. Ge Y. Maiyar A.C. Firestone G.L. Mol. Cell. Biol. 1993; 13: 2031-2040Google Scholar). Sgk is a serine/threonine protein kinase that is ∼45–55% homologous to Akt/PKB, cAMP-dependent protein kinase, p70S6 kinase, and protein kinase C (PKC) isoforms in their respective catalytic domains (16Webster M.K. Goya L. Ge Y. Maiyar A.C. Firestone G.L. Mol. Cell. Biol. 1993; 13: 2031-2040Google Scholar). All of these protein kinases, including Sgk, are directly phosphorylated and activated by phosphoinositide-dependent kinase 1 (PDK1) (17Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Google Scholar, 18Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Curr. Biol. 1999; 9: 186-197Google Scholar, 19Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Google Scholar, 20Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Google Scholar, 21Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Google Scholar, 22Cheng X. Ma Y. Moore M. Hemmings B.A. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9849-9854Google Scholar, 23Storz P. Toker A. Front. Biosci. 2002; 7: d886-d902Google Scholar), which acts in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner (20Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Google Scholar). The expression, enzymatic activity, and subcellular localization of Sgk is regulated in a stimulus-dependent manner in a variety of cell types and experimental conditions that have implicated Sgk as a key component of the cellular stress response. Glucocorticoids, a class of physiological stress hormones, stimulate Sgk promoter activity through a glucocorticoid response element and Sgk is a transcriptional target of the p53 tumor suppressor gene, a known target of genotoxic stress (24Maiyar A.C. Huang A.J. Phu P.T. Cha H.H. Firestone G.L. J. Biol. Chem. 1996; 271: 12414-12422Google Scholar,25Maiyar A.C. Phu P.T. Huang A.J. Firestone G.L. Mol. Endocrinol. 1997; 11: 312-329Google Scholar). We recently uncovered a hyperosmotic stress regulated element in the Sgk promoter that is activated as a downstream target of the MKK3/MKK6-p38/MAPK stress-signaling cascade (26Bell L.M. Leong M.L. Kim B. Wang E. Park J. Hemmings B.A. Firestone G.L. J. Biol. Chem. 2000; 275: 25262-25272Google Scholar). Sgk has also been implicated in stress signaling in other cell systems through its induction by osmotic changes (27Waldegger S. Barth P. Raber G. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4440-4445Google Scholar, 28Waldegger S. Gabrysch S. Barth P. Fillon S. Lang F. Cell Physiol. Biochem. 2000; 10: 203-208Google Scholar), cytokines (29Cowling R.T. Birnboim H.C. J. Leukoc. Biol. 2000; 67: 240-248Google Scholar, 30Cooper M.S. Bujalska I. Rabbitt E. Walker E.A. Bland R. Sheppard M.C. Hewison M. Stewart P.M. J. Bone Miner. Res. 2001; 16: 1037-1044Google Scholar), TGF-β (31Waldegger S. Klingel K. Barth P. Sauter M. Rfer M.L. Kandolf R. Lang F. 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Pflugers Arch. 2002; 443: 617-624Google Scholar), chronic viral hepatitis (33Fillon S. Warntges S. Matskevitch J. Moschen I. Setiawan I. Gamper N. Feng Y.X. Stegen C. Friedrich B. Waldegger S. Broer S. Wagner C.A. Huber S.M. Klingel K. Vereninov A. Lang F. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 130: 367-376Google Scholar), aldosterone (37Naray-Fejes-Toth A. Canessa C. Cleaveland E.S. Aldrich G. Fejes-Toth G. J. Biol. Chem. 1999; 274: 16973-16978Google Scholar, 38Chen S.Y. Bhargava A. Mastroberardino L. Meijer O.C. Wang J. Buse P. Firestone G.L. Verrey F. Pearce D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2514-2519Google Scholar), DNA-damaging agents (28Waldegger S. Gabrysch S. Barth P. Fillon S. Lang F. Cell Physiol. Biochem. 2000; 10: 203-208Google Scholar), and hypotonic conditions (39Rozansky D.J. Wang J. Doan N. Purdy T. Faulk T. Bhargava A. Dawson K. Pearce D. Am. J. Physiol. Renal Physiol. 2002; 283: F105-F113Google Scholar). Sgk is also transcriptionally induced by growth pathway signaling by serum (16Webster M.K. Goya L. Ge Y. Maiyar A.C. Firestone G.L. Mol. Cell. Biol. 1993; 13: 2031-2040Google Scholar), insulin and insulin-like growth factor-1 (17Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Google Scholar, 40Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Google Scholar), follicle-stimulating hormone (41Alliston T.N. Maiyar A.C. Buse P. Firestone G.L. Richards J.S. Mol. Endocrinol. 1997; 11: 1934-1949Google Scholar), cAMP (42Perrotti N. He R.A. Phillips S.A. Haft C.R. Taylor S.I. J. Biol. Chem. 2001; 276: 9406-9412Google Scholar), and activators of extracellular signal-regulated kinase (Erk) signaling pathways, fibroblast growth factor, platelet-derived growth factor, and TPA (12-O-tetradecanoylphorbol-13-acetate) (43Mizuno H. Nishida E. Genes Cells. 2001; 6: 261-268Google Scholar). The regulation of Sgk signaling is also consistent with a role for this protein kinase in cell survival pathways. Sgk, along with Akt, is a downstream target of the PI 3-kinase pathway that is known to activate cell survival pathways in response to growth factor stimulation as well as stress stimuli (17Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Google Scholar, 20Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Google Scholar, 40Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Google Scholar). In the case of Sgk, the enzymatic activity, phosphorylation, and subcellular localization of the protein kinase is controlled in a PI 3-kinase-dependent manner (17Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Google Scholar,40Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Google Scholar). Sgk has been shown to be further phosphorylated by big mitogen-activated kinase-1, BMK1 (also known as Erk5), a member of the MAPK family that is required for growth factor cell proliferation (44Hayashi M. Tapping R.I. Chao T.H. Lo J.F. King C.C. Yang Y. Lee J.D. J. Biol. Chem. 2001; 276: 8631-8634Google Scholar) and known to respond to stress signals (8Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Google Scholar). Activated Sgk can phosphorylate GSK-3 (40Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Google Scholar), b-Raf (45Zhang B.H. Tang E.D. Zhu T. Greenberg M.E. Vojtek A.B. Guan K.L. J. Biol. Chem. 2001; 276: 31620-31626Google Scholar), and the forkhead transcription factor family member, FKHRL1 (46Brunet A. Park J. Tran H. Hu L.S. Hemmings B.A. Greenberg M.E. Mol. Cell. Biol. 2001; 21: 952-965Google Scholar), also known as FOXO3a (47Kaestner K.H. Knochel W. Martinez D.E. Genes Dev. 2000; 14: 142-146Google Scholar). FKHRL1 has a pro-apoptotic function by stimulating expression of FasL (48Dijkers P.F. Medema R.H. Lammers J.W. Koenderman L. Coffer P.J. Curr. Biol. 2000; 10: 1201-1204Google Scholar), Bim (49Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Google Scholar), cell cycle inhibitor p27/KIP1 (50Medema R.H. Kops G.J. Bos J.L. Burgering B.M. Nature. 2000; 404: 782-787Google Scholar, 51Dijkers P.F. Medema R.H. Pals C. Banerji L. Thomas N.S. Lam E.W. Burgering B.M. Raaijmakers J.A. Lammers J.W. Koenderman L. Coffer P.J. Mol. Cell. Biol. 2000; 20: 9138-9148Google Scholar), and DNA damage response gene GADD45 (52Tran H. Brunet A. Grenier J.M. Datta S.R. Fornace Jr., A.J. DiStefano P.S. Chiang L.W. Greenberg M.E. Science. 2002; 296: 530-534Google Scholar). Notably, FKHRL1 transcriptional activity is inhibited after phosphorylation by either Sgk or Akt, suggesting that both Sgk and Akt may be involved in promoting cell survival (46Brunet A. Park J. Tran H. Hu L.S. Hemmings B.A. Greenberg M.E. Mol. Cell. Biol. 2001; 21: 952-965Google Scholar, 49Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Google Scholar). In MCF-7 breast cancer cells, the addition of glucocorticoids, which stimulates Sgk expression, or the overexpression of wild type Sgk protein were shown to protect these cells from growth factor starvation-induced apoptosis (53Mikosz C.A. Brickley D.R. Sharkey M.S. Moran T.W. Conzen S.D. J. Biol. Chem. 2001; 276: 16649-16654Google Scholar). The newly discovered Sgk family member, cytokine-independent survival kinase (CISK), can phosphorylate and negatively regulate pro-apoptotic BAD to protect against IL-3 withdrawal-induced death (54Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Google Scholar, 55Liu D. Yang X. Songyang Z. Curr. Biol. 2000; 10: 1233-1236Google Scholar). Emerging evidence indicates that the cellular utilization of Sgk is likely to be an important cell survival response to many types of adverse environmental conditions. In the present study, we directly compared the effects of multiple environmental stresses on the induction of Sgk protein expression in mammary epithelial cells and characterized the role of Sgk in cell survival signaling. We demonstrated that UV irradiation, heat shock, oxidative stress, and hyperosmotic stress induce active Sgk through a p38/MAPK-dependent pathway, although with varying kinetics of induction and subcellular localization, which results in the inactivation of the FKHRL1 forkhead transcription factor. Moreover, the ability of Sgk to mediate its cell survival function in response to these environmental stresses, or growth factor deprivation, depends upon its catalytic activity. Thus, Sgk has a key role in transducing intracellular signals in cell survival pathways in response to multiple types of adverse environmental stimuli. NMuMg nontumorigenic mouse mammary epithelial cells and human embryonic kidney (HEK) 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 10 μg/ml insulin, 50 units/ml penicillin, and 50 μg/ml streptomycin. Rat Con8 mammary epithelial tumor cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM-F12) containing 10% calf serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. All cells were propagated at 37 °C in humidified air containing 5% CO2. Cell culture reagents such as DMEM, DMEM-F12, calf serum, fetal bovine serum, calcium- and magnesium-free phosphate-buffered saline, and trypsin/EDTA were supplied by BioWhittaker, Inc. (Walkersville, MD). Insulin, d-sorbitol, hydrogen peroxide, and dexamethasone were purchased from Sigma. To induce hyperosmotic stress, NMuMg cells received equal volumes of DMEM as a vehicle control or 300 mm sorbitol in DMEM for the indicated time. To induce heat shock, cells were removed from the incubator and maintained at 42 °C for 0.5 h. Control cells were removed from incubator and kept at 37 °C for 0.5 h. For UV irradiation, cells had media removed, and UV-irradiated cells were treated in UV Stratalinker at 40 J/m2, while controls were kept out of the incubator for an equivalent amount of time. Both sets of cells had media replaced, and then were returned to the 37 °C incubator to resume culturing for the indicated amount of time. To expose cells to oxidative stress, cells were treated with 0.5 mm hydrogen peroxide in DMEM or DMEM alone as a vehicle control for the indicated amount of time. Dexamethasone-treated cells were exposed to 1 μm dexamethasone in ethanol, while vehicle control cells received an equal volume of ethanol. After treatments, all cells were transferred to the incubator and harvested after the indicated amount of time. As a positive control for phosphorylated Akt, HEK239T cells were treated with 5 mm hydrogen peroxide, transferred to the incubator for 5 min, and then harvested for subsequent analysis. For treatment with the PI 3-kinase inhibitor LY294002 (Calbiochem, La Jolla, CA), cells were pretreated with 50 μm LY294002 for 16 h. Half of the cell cultures was exposed to the above mentioned environmental stress treatments and the remaining half was left unstressed, while both sets of cells were cultured in the presence of LY294002. For treatment with the p38/MAPK inhibitor SB202190 (Calbiochem), cells were treated with 10 μm SB202190 for 0.5 h. One set of cells was exposed to the stress stimuli, whereas the other half was unstressed, while both remained in the presence of SB202190. Cells were harvested at the optimal time point based on protein induction (for sorbitol, 24 h; for heat shock, 0.5 h; for UV-irradiation, 2 h; for oxidative stress, 1 h; and for dexamethasone, 24 h). Cells were lysed in HEMGN lysis buffer (25 mm Hepes, 100 mm KCl, 12.5 mmMgCl2, 0.1 mm EDTA, 10% glycerol, 0.1% Nonidet P-40, pH 7.9), and whole cell extracts were normalized for protein levels using the Bradford assay (Bio-Rad, Hercules, CA). To induce apoptosis, a few alterations were made to the stress conditions. The sorbitol concentration was increased to 500 mm. The intensity of UV irradiation was increased to 100 J/m2. For oxidative stress, cells were exposed to 5 mm hydrogen peroxide. In these experiments, the final dexamethasone concentration was increased to 2 μm. All of these cells were harvested after a 24-h treatment period. For heat shock, cells were exposed to 42 °C for 2 h, while control cells remained at 37 °C for the same amount of time. These cells were then harvested 2 h following post-heat treatment. Growth factor starvation was achieved by incubating the cells for 120 h in serum-free DMEM-F12 containing penicillin/streptomycin. Serum-free media containing selective antibiotics with 0.5 mm IPTG or vehicle control was changed every day. The LacSwitch-inducible promoter system (Stratagene) was used for the inducible expression of wild type and the phosphorylation-deficient T256A Sgk. The full-length wild type Sgk and the T256A mutant Sgk were subcloned intoXhoI/NotI sites within the pOPI3 lac operator, mammalian expression vector using standard PCR techniques. The p3'SS lac repressor-expressing clones in Con8 cells were generated previously (56Woo P.L. Cercek A. Desprez P.Y. Firestone G.L. J. Biol. Chem. 2000; 275: 28649-28658Google Scholar). Clones that expressed high levels of lac repressor were subsequently transfected with 10 μg LipofectAMINE (Invitrogen) and 1 μg of either wild type (pOPI3-wt-Sgk) or phosphorylation-deficient mutant form of Sgk (pOPI3-T256A-Sgk), which contain the neomycin-resistant gene and according to the manufacturer's instructions. After selection with 300 μg/ml hygromycin B and 750 μg/ml neomycin analogue G418 (Invitrogen) for 2 weeks, 50 clones were selected, expanded, and tested for their ability to express either wild type Sgk or T256A Sgk in response to 0.5 mm IPTG (Sigma). To check the stable cell lines for expression, the Con8 clones were maintained serum-free for 72 h. After 48 h 0.5 mmIPTG or an equal volume of the vehicle control were added. Cells were harvested 24 h after IPTG addition, and the induction of Sgk proteins was analyzed using anti-Sgk Western blotting techniques. To visualize Sgk protein levels, membranes were probed with a 1:2500 dilution of affinity-purified anti-Sgk antibody as described previously (26Bell L.M. Leong M.L. Kim B. Wang E. Park J. Hemmings B.A. Firestone G.L. J. Biol. Chem. 2000; 275: 25262-25272Google Scholar, 57Buse P. Tran S.H. Luther E. Phu P.T. Aponte G.W. Firestone G.L. J. Biol. Chem. 1999; 274: 7253-7263Google Scholar). The anti-tubulin blots used 1:1,000 dilution of mouse monoclonal anti-tubulin antibody. The anti-HA blots used 1:1,000 dilution of mouse monoclonal antibody (Covance, Berkeley, CA). The anti-lac repressor blots used 1:10,000 dilution of mouse polyclonal antibody (Stratagene). The anti-Akt blots used 1:1,000 dilution of rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The anti-phospho-Thr-308 Akt rabbit polyclonal antibody and the anti-phospho-Ser-473 Akt mouse monoclonal antibody were both used at a dilution of 1:500 (Cell Signaling Technology, Beverly, MA). A goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody was used at a dilution of 1:10,000 (Bio-Rad) for Sgk and Akt Western blots. A goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody was used at a 1:10,000 dilution (Bio-Rad) for anti-HA blots. The Western blots were developed by using the Renaissance developing kit (PerkinElmer Life Sciences) and exposed to x-ray film. NMuMg cells were plated onto 8-well LabTek chamber slides (Nalgene, Rochester, NY) for indirect immunofluorescence of Sgk. The next day, the cells were treated with different stresses for the optimal amount of time to induce Sgk expression. For visualization of FKHRL1 by indirect immunofluorescence, NMuMg cells were plated in two-well LabTek chamber slides at 50% confluency. The next day the cells were transfected with 1 μg of HA-tagged forkhead cDNA (pCMV-HA-FKHRL1) (generously provided by Dr. Michael Greenberg's laboratory, Boston, MA) and 10 μg of LipofectAMINE (Invitrogen) according to the manufacturer's instructions. After 24 h, the cells were treated with different stressors to induce maximal Sgk expression, as described previously (26Bell L.M. Leong M.L. Kim B. Wang E. Park J. Hemmings B.A. Firestone G.L. J. Biol. Chem. 2000; 275: 25262-25272Google Scholar). Following washes, fixation, and permeabilization, affinity-purified anti-Sgk antibody diluted 1:150 in PBS or mouse monoclonal anti-HA antibody (Covance) diluted to 1:1,000 in PBS was added to samples and allowed to incubate for 1 h at room temperature. Goat anti-rabbit fluorescein isothiocyanate-conjugated or goat anti-mouse Texas red-conjugated secondary antibody was added at a dilution of 1:150 in PBS and allowed to incubate for 1 h at room temperature. The cells were then washed with PBS, and coverslips were mounted using Antifade (Molecular Probes, Inc., Eugene, OR) and then visualized on a Nikon Optiphot fluorescence microscope. Nonspecific fluorescence was determined by incubation with the secondary antibody alone and shown to be negligible. Expression plasmids encoding pCMV-HA-tagged FHKRL-1 and a luciferase reporter construct containing −743/−648 of the FasL promoter containing 3× forkhead responsive element (pGL3-FHRE-luciferase) were generously provided by Dr. Michael Greenberg's laboratory. NMuMg cells were plated in 35-mm plates and grown to 65–75% confluency. Cells were then transfected with 2 μg of FHRE-luciferase, 4 μg of pCMV-HA-FKHRL1, and 24 μg of LipofectAMINE (Invitrogen). Transfected cells were stressed to induce maximal Sgk protein expression, as described above, and harvested by washing twice in PBS and lysed in 100–200 μl of 1× reporter lysis buffer (Promega, Madison, WI). 10 μl of cell lysate was added to 12 × 75 mm cuvettes (Analytical Luminescence Laboratory, San Diego, CA) and subsequently loaded into a luminometer (Monolight 2010, Analytical Luminescence Laboratory). 100 μl of luciferase substrate buffer (20 mm Tricine, 1.07 mm(MgCO3)4Mg(OH)2·5H2O, 2.67 mm MgSO4, 0.1 mm EDTA, 33.3 mm dithiothreitol, 270 μm coenzyme A, 470 μmd-luciferin sodium salt, 530 μm ATP disodium salt, pH 7.8) was injected automatically into each sample, and luminescence was measured in relative light units. The luciferase specific activity was expressed as an average of relative light units produced per microgram of protein present in corresponding cell lysates as measured by the Bradford assay. These experiments were done in triplicate and repeated at least three times. Con8 clones were maintained serum-free for 72 h. After 48 h, 0.5 mm IPTG or an equal volume of the vehicle control were added. Cells were harvested 24 h after IPTG addition and placed on ice. Immunoprecipitations and kinase assays were performed as described previously (17Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Google Scholar). The amount of 32P-labeled Sgktide was quantitated by scintillation counting. A control set of immunoprecipitations employed nonimmune serum. The Sgk-specific transphosphorylation was determined by subtracting the filter-bound radioactivity observed with the nonimmune antibodies from that observed with the Sgk-specific antibodies. NMuMg mouse epithelial cells or Con8 IPTG-inducible clones were plated in 35-mm plates. The NMuMg cells were transfected with 10 μg of LipofectAMINE and 1 μg of either empty pCMV5 vector, pCMV5 wild type Sgk, double phosphorylation site Sgk mutant in which threonine 256 and serine 422 are substituted with alanine (pCMV5-HA-T256A/S422A Sgk), or mimicking constitutively phosphorylated Sgk with threonine 256 and alanine 422 substituted with aspartic acid (pCMV5-HA-T256D/S422D Sgk) according to the manufacturer's instructions. The construction of these mammalian expression plasmids encoding wild type or mutant Sgk in pCMV5 vector containing an N-terminal hemagglutinin (HA) tag have been described previously (17Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Google Scholar). Transiently transfected NMuMg cells and IPTG-inducibl

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