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*

Meredith L. L. Leong, Anita C. Maiyar, Brian KimDagger, Bridget A. O'Keeffe, and Gary L. Firestone§

From the Department of Molecular and Cell Biology and The Cancer Research Laboratory, The University of California at Berkeley, Berkeley, California 94720-3200

Received for publication, November 15, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1-3). 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 (4-7). 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 (4, 8-10). 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),1 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 (5, 8, 11-14). Known substrates and other downstream targets of these stress-activated protein kinases include transcription factors and other protein kinases (5, 8, 12). 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 (15, 16). 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 (16). All of these protein kinases, including Sgk, are directly phosphorylated and activated by phosphoinositide-dependent kinase 1 (PDK1) (17-23), which acts in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner (20). 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 (24, 25). 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 (26). Sgk has also been implicated in stress signaling in other cell systems through its induction by osmotic changes (27, 28), cytokines (29, 30), TGF-beta (31-33), cortical brain injury (34), changes in cell volume (35, 36), chronic viral hepatitis (33), aldosterone (37, 38), DNA-damaging agents (28), and hypotonic conditions (39). Sgk is also transcriptionally induced by growth pathway signaling by serum (16), insulin and insulin-like growth factor-1 (17, 40), follicle-stimulating hormone (41), cAMP (42), and activators of extracellular signal-regulated kinase (Erk) signaling pathways, fibroblast growth factor, platelet-derived growth factor, and TPA (12-O-tetradecanoylphorbol-13-acetate) (43).

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 (17, 20, 40). 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 (17, 40). 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 (44) and known to respond to stress signals (8). Activated Sgk can phosphorylate GSK-3 (40), b-Raf (45), and the forkhead transcription factor family member, FKHRL1 (46), also known as FOXO3a (47). FKHRL1 has a pro-apoptotic function by stimulating expression of FasL (48), Bim (49), cell cycle inhibitor p27/KIP1 (50, 51), and DNA damage response gene GADD45 (52). 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 (46, 49). 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 (53). 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 (54, 55).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Cell Culture and Materials-- 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.

Stress and Inhibitor Treatments-- 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 mM MgCl2, 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.

Generation of Stable Transfectants under Lac Repressor Control-- 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 into XhoI/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 (56). 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 mM IPTG 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.

Western Blot Analysis-- To visualize Sgk protein levels, membranes were probed with a 1:2500 dilution of affinity-purified anti-Sgk antibody as described previously (26, 57). 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.

Indirect Immunofluorescence-- 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 (26). 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.

Luciferase Reporter Plasmid Activity Assay-- 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 µM D-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.

Immunoprecipitation and in Vitro Kinase Assay of Sgk-- 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 (17). 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.

DNA Fragmentation Assay for Apoptotic Cells-- 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 (17). Transiently transfected NMuMg cells and IPTG-inducible Con8 cells were stressed to induce cell death as described under "Experimental Procedures" and assessed by propidium iodide staining for DNA fragmentation as described previously (58). At least 10,000 nuclei were analyzed by flow cytometry with the excitation set at 488 nm and emission at 610 nm. Data are shown as percentage of cells with subdiploid DNA and are mean ± S.D. of three experiments. Analysis was performed with the Multicycle computer program provided by Phoenix Flow Systems in the Cancer Research Laboratory Microchemical Facility of the University of California, Berkeley.

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ABSTRACT
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Induction and Localization of Sgk After Exposure to Environmental Stress Cues or Glucocorticoids-- We have previously shown that hyperosmotic stress strongly stimulates expression of the serum- and glucocorticoid-inducible protein kinase, Sgk, in non-tumorigenic murine mammary (NMuMg) cells through a p38/MAPK-activated pathway (26), which suggests that Sgk production may be induced by other types of environmental stress cues. To test this possibility, NMuMg cells were exposed to 40 J/m2 ultraviolet irradiation, treated with 0.5 mM hydrogen peroxide to generate reactive oxygen species (oxidative stress), heat shocked at 42 °C temperature for 0.5 h, or incubated with 300 mM sorbitol to induce hyperosmotic stress. One set of cells was treated with 1 µM dexamethasone, a synthetic glucocorticoid, which rapidly stimulates Sgk expression in other cell systems and is considered representative of a physiological stress hormone. In the absence of stress, NMuMg cells display very low levels of detectable Sgk. Western blot analysis of total cell lysates from cells exposed to each stress for various amounts of time revealed that all four environmental stress conditions induced Sgk protein levels. As a control, tubulin protein levels remained unchanged throughout the time course (Fig. 1). The kinetics of Sgk induction differed with each of the stress treatments. UV irradiation, oxidative stress, and heat shock each rapidly and transiently stimulated Sgk production with peak expression observed at 30 min for heat shock, 2 h for UV radiation, and 1 h for oxidative stress (Fig. 1). Consistent with our previous results (26), 4 h after exposure to hyperosmotic shock (300 mM sorbitol) there was no induction of Sgk, but at 8 h post-treatment Sgk protein levels were significantly increased and remained high through at least 48 h (data not shown). Dexamethasone rapidly (within 1 h) stimulated Sgk protein that remained at a high level over an extended time frame (Fig. 1). Similar to what we previously showed with rat mammary tumor cells (16, 57), in NMuMg mouse mammary epithelial cells high levels of Sgk were maintained after dexamethasone treatment for at least 72 h (data not shown). Thus, distinct types of environmental stress as well as glucocorticoids, a physiological stress hormone, stimulate Sgk protein suggesting that this protein kinase may play a key role in the cellular stress response.


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Fig. 1.   Time course of induction of Sgk protein by extracellular stresses and dexamethasone. NMuMg mouse mammary epithelial cells were treated with different stress conditions and harvested at indicated time points. Expression of Sgk protein and tubulin protein was evaluated by Western blot analysis using affinity-purified polyclonal anti-Sgk and monoclonal anti-tubulin antibodies.

The subcellular localization of Sgk is regulated in a stimulus-dependent manner in mammary epithelial cells and in ovarian cells, with a nuclear form of Sgk predominantly observed in proliferating cells after serum treatment and in proliferating follicle cells, whereas a cytoplasmic form is detected in glucocorticoid or hyperosmotic stressed cells and non-growing granulosa cells (26, 57, 59). Indirect immunofluorescence using anti-Sgk polyclonal primary antibodies was utilized to determine the compartmentalization of endogenous Sgk protein in NMuMg cells treated with each environmental stress for a duration that allows maximal protein expression. The cells were exposed to hyperosmotic shock for 8 h, to heat shock for 0.5 h, to UV irradiation for 2 h, to oxidative stress for 1 h, and treated with dexamethasone for 24 h. In all sets of cell cultures, the unstressed cells displayed low background levels of Sgk protein (Fig. 2, left panels). In heat-shocked, UV-irradiated, or H2O2-treated conditions, Sgk protein was heterogeneously expressed throughout the cells (Fig. 2, right panels). Consistent with our previous studies (26, 57), in sorbitol- or dexamethasone-treated cells, Sgk was detected predominantly in the cytoplasm (Fig. 2, right panels).


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Fig. 2.   Subcellular localization of stress- and steroid-induced Sgk. NMuMg cells were grown to 70% confluency on 2-well LabTek slides. Cells were stressed for optimal length of time as determined by Sgk induction (for sorbitol, 24 h; for 42 °C, 0.5 h; for UV, 2 h; for H2O2, 1 h; and for dex, 24 h). Subcellular localization of Sgk either in the absence (-Stimulus) or presence (+Stimulus) of each stress or hormonal condition was examined by indirect immunofluorescence microscopy using anti-Sgk polyclonal antibodies. The secondary antibodies used were fluorescein isothiocyanate-conjugated goat anti-rabbit antibodies.

Sgk Protein Is Induced by Extracellular Stresses via p38/MAPK Pathway-- One of the major cell signaling cascades that can be activated in response to diverse extracellular stresses is the p38/MAPK pathway (8, 12). For example, we previously documented that in NMuMg cells the hyperosmotic stress stimulation of Sgk gene expression requires a functioning p38/MAPK pathway (26). One test of the role of p38/MAPK in stress-induced signaling in a cellular context is the use of pharmacological inhibitors of p38/MAPK enzymatic activity such as SB202190 (60). NMuMg cells were treated with 10 µM SB202190 for 30 min prior to exposure to each of the environmental or hormonal stress cues, and total cell extracts harvested at the duration of maximal Sgk protein induction for each condition. Western blot analysis revealed that the stress induction of Sgk protein by hyperosmotic stress, heat shock, UV radiation, and H2O2 treatment is nearly ablated in the presence of the SB202190 p38/MAPK inhibitor (Fig. 3). These results indicate that the induction of Sgk protein after exposure to four distinct stress cues is a p38/MAPK-dependent response. In contrast, an inhibition of p38/MAPK function had no effect on the dexamethasone stimulation of Sgk production (Fig. 3, lower left panel). The level of Sgk protein was compared in all conditions to tubulin production, which did not change. Also, treatment with SB202190 had no effect on the near background level of Sgk observed in the absence of environmental or hormonal stress (data not shown).


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Fig. 3.   Induction of Sgk by stress stimuli occurs via p38/MAPK pathway. NMuMg cells were exposed to the indicated stimuli for optimal times of Sgk induction (for sorbitol, 24 h; for 42 °C, 0.5 h; for UV, 2 h; for H2O2, 1 h; and for dex, 24 h) in the presence or absence of p38/MAPK inhibitor SB202190. Western blot analysis was performed on total cell extracts with affinity purified anti-Sgk and anti-tubulin antibodies to monitor Sgk and tubulin protein expression.

Extracellular Stress Cues Induce a Hyperphosphorylated Sgk Protein, but Not of the Sgk Homolog Akt-- The most homologous protein kinase to Sgk is Akt, which is a constitutively expressed kinase with a well characterized role in cell survival pathways (61-63). Based on studies identifying peptide substrates and protein targets, Sgk and Akt have been shown to have similar, but distinct substrate specificity (17, 40). For example, both protein kinases inactivate the forkhead transcription factor FKHRL1 by phosphorylation at a different but overlapping set of phosphorylation sites (46, 49). Both kinases also phosphorylate GSK-3 and B-raf (40, 45). Sgk and Akt are both enzymatically activated by phosphorylation in their respective activation loops (Thr-256 for Sgk and Thr-308 for Akt) by PDK1, which is directly downstream of PI 3-kinase (17, 20, 40). In addition, both Sgk and Akt are phosphorylated by a PDK-like kinase in a PI 3-kinase-dependent manner at a carboxyl-terminal site (Ser-422 for Sgk and Ser-473 for Akt) (17, 20, 40). To determine whether the environmental and hormone stress stimuli induce the hyperphosphorylated states of Sgk and Akt, NMuMg cells were exposed to individual stresses or dexamethasone in the presence or absence of LY294002, a PI 3-kinase inhibitor (64). Sgk protein production was analyzed in Western blots of cell extracts harvested at times that correspond to maximal Sgk induction.

The hyperphosphorylated, active Sgk can be visualized by Western blot analysis as a slower-migrating protein band, and a faster-migrating species represents the hypophosphorylated, inactive protein (16, 17, 57). As shown in Fig. 4, all four environmental stress stimuli and dexamethasone induce a hyperphosphorylated slower migrating form of Sgk, indicating that each condition produces an enzymatically active Sgk (17, 57). Moreover, in each case, treatment with LY294002 collapsed the hyperphosphorylated Sgk protein bands into a faster-migrating species (Fig. 4). This result implicates the PI 3-kinase pathway as the upstream regulator of stress-induced Sgk enzymatic activity in these mammary epithelial cells. In the absence of stress or dexamethasone treatment, negligible amounts of Sgk protein were produced (Fig. 4), and cells treated only with the inhibitor did not express detectable levels of Sgk (data not shown).


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Fig. 4.   Cellular stresses induce hyperphosphorylated Sgk through the PI 3-kinase pathway. NMuMg cells were stressed for optimal times of Sgk induction (for sorbitol, 24 h; for 42 °C, 0.5 h; for UV, 2 h; for H2O2, 1 h; and for dex, 24 h) in the presence or absence of PI 3-kinase inhibitor LY294002. Western blot analysis was performed on total cell extracts with affinity purified anti-Sgk and anti-tubulin antibodies to assess Sgk and tubulin protein expression.

To determine whether the stress conditions shown to induce hyperphosphorylated Sgk can similarly activate Akt, Western blots of isolated NMuMg cell extracts were probed with primary antibodies to total Akt, or with antibodies specific for either the phosphorylated threonine 308 or phosphorylated serine 473 forms of Akt. Under conditions in which hyperphosphorylated Sgk is induced (Fig. 5, bottom panel), Akt protein was not phosphorylated (Fig. 5, upper two panels), and the total Akt level remained unchanged (Fig. 5, third panel). As a positive control for stress-induced Akt phosphorylation, HEK293T cells were treated with 5 mM H2O2 for 5 min and Western blots probed with antibodies specific for either total or phosphorylated Akt (65). As also shown in Fig. 5 (left lanes), oxidative stress in HEK293T cells rapidly stimulated the production of phosphorylated Akt without altering total Akt protein levels. Interestingly, under these conditions, oxidative stress had no effect on Sgk protein levels in HEK293T cells. Thus, although Akt and Sgk share upstream activators and certain substrates, the use of these related protein kinases in the stress response is likely to be regulated in a cell type- and tissue-specific manner.


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Fig. 5.   Akt expressed in NMuMg cells is not phosphorylated in response to stress stimuli. NMuMg mouse mammary epithelial cells were treated with different stress conditions and harvested at optimal times of Sgk induction (for sorbitol, 24 h; for heat shock, 0.5 h; for UV, 2 h; for H2O2, 1 h; and for dex, 24 h). As a positive control for stress induced Akt phosphorylation, HEK293T cells were treated with 5 mM hydrogen peroxide for 5 min. Upper panels show Western blot analyses were performed with anti-phospho-Thr-308-Akt, anti-phospho-Ser-473-Akt, and anti-Akt antibodies to evaluate levels of phosphorylated Akt and total Akt expression. The bottom panel depicts a parallel Western blot was probed with anti-Sgk antibodies to show corresponding Sgk protein induction by stress in both NMuMg and HEK293T cells.

Catalytically Active Sgk Protects Mammary Epithelial Cells from Stress-induced Death-- To determine the role of enzymatically active Sgk in controlling the decision between cell survival and apoptosis in response to cellular stress, wild type or phosphorylation site mutants of Sgk were expressed in NMuMg cells, and the cells were assayed for cell death after exposure to each of the environmental or hormone stress cues. NMuMg cells were transfected with a vector control or with HA epitope-tagged expression constructs encoding wild type Sgk (Wt Sgk), catalytically inactive Sgk with phosphorylation sites mutated to alanine (T256A/S422A Sgk), or a mutant Sgk that mimics a constitutively phosphorylated state (T256D/S422D Sgk) (17, 40). After exposure to each of the stress stimuli under conditions that induce programmed cell death, the number of cells undergoing apoptosis was assayed by staining with propidium iodide and measuring hypodiploid nuclei by flow cytometry (58, 66, 67). After exposure to hyperosmotic stress (sorbitol treatment), heat shock, UV irradiation, or oxidative stress (H2O2 treatment), populations of cells transfected either with the wild type or with T256D/S422D Sgk displayed a decreased level of apoptosis compared with those transfected with a vector control (Fig. 6, solid and hatched bars). In comparison, the overexpression of the catalytically inactive Sgk (T256A/S422A Sgk) had either no effect on the level of apoptosis, as in the cases of sorbitol treatment and heat shock, or slightly increased the number of apoptotic cells, as in the cases of H2O2 and UV treatments, compared with cells transfected with an empty vector (Fig. 6, open bars). Each set of cells produced approximately equal levels of exogenous Sgk protein as determined by Western blots (Fig. 6, bottom panel). As expected, the addition of dexamethasone did not induce cell death, and there was no effect of the ectopically expressed Sgk constructs in glucocorticoid-treated cells. These data suggest that the expression of active Sgk, either wild type or the constitutively phosphorylated form, can protect cells from the apoptotic response to heat, UV, hyperosmotic or oxidative stresses.


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Fig. 6.   Sgk protects from stress-induced cell death. NMuMg cells were transiently transfected with an empty vector or an expression vector encoding wild type Sgk (Wt Sgk), kinase dead Sgk (T256A/S422A), or constitutively phosphorylated Sgk (T256D/S422D). Twenty-four hours after transfection, cells were stressed to induce apoptosis, and cell survival was quantified as change in the hypodiploid DNA content between cells transfected with empty vector or with wild type or mutant Sgk constructs. Data are the means and variances for three independent experiments conducted in triplicate. The lower panel depicts Western blot analysis of the transfected proteins after sorbitol treatment using an anti-HA monoclonal antibody and tubulin expression was used as a control. Protein expression with sorbitol treatment is representative of the ectopic Sgk protein expression seen with other stress treatments.

Environmental Stress Cues Decrease Forkhead Transcriptional Activity and Alter Its Subcellular Localization-- Consistent with a cell survival role, catalytically active Sgk has recently been shown to phosphorylate and inactivate the FKHRL1 forkhead transcription factor (46). FKHRL1 is a member of the winged helix FOXO subfamily of transcription factors that binds to a forkhead-responsive element (FHRE) in the promoters of FKHRL1-inducible pro-apoptotic genes, such as Bim (48) and Fas ligand (49, 68, 69). Because environmental stress cues stimulate expression of enzymatically active Sgk that provides protection against stress induced apoptosis (Fig. 6), and since FKHRL1 is a known substrate of Sgk (46), we therefore tested whether the various stress stimuli inhibit FKHRL1 transcriptional activity. NMuMg cells were co-transfected with a luciferase reporter plasmid driven by an FHRE with either an FKHRL1 expression vector or an empty vector, then exposed to the same stress conditions that induce Sgk protein. Cells co-transfected with the empty expression vector and the FHRE-luciferase reporter plasmid displayed a low level of background reporter activity under all tested conditions (Fig. 7, hatched bars). Co-expression of wild type FKHRL1 with the FHRE-luciferase reporter plasmid greatly stimulated luciferase reporter activity either in the absence of any environmental stress or in dexamethasone-treated cells (Fig. 7, Untreated or Dex). Exposure to hyperosmotic stress, heat shock, UV irradiation, or oxidative stress strongly inhibited FKHRL1-induced reporter activity compared with untreated cells (Fig. 7, filled bars). The level of exogenous FKHRL1 protein expressed remained constant in all the conditions as determined by anti-HA immunoblotting, and the levels of Sgk protein were induced in response to the stress treatments (Fig. 7, lower panel). Similar data were observed with the co-expression of constructs encoding FKHR, a FKHRL1 gene family member, with the FHRE-luciferase reporter plasmid (data not shown).


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Fig. 7.   Cellular stresses decrease FKHRL1-dependent transcription. NMuMg cells were transiently transfected with the FHRE-luciferase construct and an empty vector or a vector encoding wild type FKHRL1. Twenty-four hours after the transfections, cells were exposed to the indicated stressors, and at the time of optimal Sgk induction (for sorbitol, 24 h; for heat shock, 0.5 h; for UV, 2 h; for H2O2, 1 h; and for dex, 24 h), luciferase reporter activity was assayed. Data are the means and variances for three independent experiments conducted in triplicate. Lower panels depict Western blot analysis of the transfected FKHRL1 proteins and the induced Sgk proteins, using an anti-HA monoclonal antibody and anti-Sgk polyclonal antibody, respectively.

Previous studies have established that unphosphorylated FKHRL1 is imported into the nucleus, whereas, phosphorylated FKHRL1 is sequestered by 14-3-3 proteins in the cytoplasm and are less likely to undergo apoptosis (70, 71). To determine if there is a change in the localization of FKHRL1 in response to individual stress treatments, indirect immunofluorescence with anti-HA antibodies was used to visualize the subcellular location of ectopically expressed FKHRL1 protein. In the absence of stress, HA-FKHRL1 protein is localized either predominantly in the cytoplasm (48% of tested cells) or is found to be heterogeneous throughout the cell (52% of tested cells) (Fig. 8, Untreated). In cells exposed to hyperosmotic stress, heat shock, UV irradiation, or oxidative stress, a significantly greater percentage of cells displayed predominantly a cytoplasmic form of HA-FKHRL1 (60-65% of cells tested), with fewer cells displaying FKHRL1 equally distributed between the cytoplasm and the nucleus (35-40% of tested cells) (Fig. 8). The immunofluorescence data showed similar results with each stress condition (Fig. 8, upper panels). In dexamethasone-treated cells, the distribution of FKHRL1 remained relatively unchanged compared with untreated cells (Fig. 8, Dex). Taken together, these data show that under conditions in which environmental stress stimuli induce production of active Sgk, the FKHRL1 forkhead transcription factor is inactive and predominantly cytoplasmic.


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Fig. 8.   Stress stimuli alter the subcellular localization of FKHRL1. NMuMg cells were grown to 50% confluency on 2 well LabTek slides and were transiently transfected with a FKHRL1 expression vector. Twenty-four hours after the transfections, cells were exposed to the indicated stressors, and subcellular localization of FKHRL1 was examined by indirect immunofluorescence microscopy using anti-HA monoclonal antibodies (upper panels). The secondary antibodies used were Texas red-conjugated goat anti-mouse antibodies. In the lower panel, the number of cells displaying cytoplasmic (filled bars) or homogenous staining (hatched bars) for FKHRL1 were quantified as the percentage of total immunostaining cells. Data represent the means and variances for three independent experiments.

Catalytically Active Sgk Protects Cells from Growth Factor Starvation-induced Apoptosis in Con8 Mammary Tumor Cells-- The potential cell survival role for catalytically active Sgk was also examined in Con8 mammary epithelial tumor cells. The extracellular stress stimuli applied to NMuMg cells, a nontumorigenic mammary cell line, have no apparent effects on Sgk induction or function in the Con8 cells (data not shown). However, this tumor cell line is susceptible to stress by growth factor starvation, which are conditions in which virtually no Sgk is produced. The addition of serum to the Con8 cells rapidly induces an active Sgk protein (16, 17, 57). To determine if catalytically active Sgk protects Con8 cells from growth factor starvation-induced apoptosis, stable cell lines expressing either wild type or kinase dead Sgk (T256A Sgk) on an IPTG-inducible promoter were constructed. To accomplish this, stable cell lines expressing the lac repressor protein were obtained by transfecting the lac repressor vector containing the hygromycin resistance gene and selecting for their resistance to the cytotoxic effects of hygromycin. Cell lines were then selected for their high expression of the lac repressor protein and subsequently, transfected with the lac operator vector containing the neomycin-resistant gene and either the wild type or mutant T256A Sgk. Two representative subclones from either the wild type Sgk- or the T256A mutant Sgk-expressing cells were screened for the expression of the conditionally expressed Sgk proteins in the presence of 0.5 mM IPTG for 24 h. Western blot analysis using an anti-Sgk primary antibody revealed that in the absence of IPTG and serum for 72 h Sgk proteins were undetectable in both cell lines (Fig. 9, upper panel, -IPTG). IPTG strongly induced the exogenous wild type and T256A Sgk proteins in the absence of serum (Fig. 9, upper panel, +IPTG). Under these conditions, the lac repressor protein levels remained constant in the absence or presence of IPTG in both stable transfected cell lines. Also, the addition of IPTG to parental Con8 cells had no effect on the Sgk levels and no lac repressor immunoreactive protein was observed (data not shown).


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Fig. 9.   IPTG-induced expression of wild type and kinase dead Sgk in Con8 cells. In the top panel, Con8 cell lines stably transfected with the lac repressor and the IPTG inducible Sgk sequences, as described in the text, were serum-starved in the presence or absence of IPTG (0.5 mM) for 120 h. Western blot analysis was performed with affinity-purified polyclonal anti-Sgk antibodies to verify that IPTG induces production of the wild type and kinase dead Sgk proteins. Expression of lac repressor (LacR) protein was assessed in a parallel blot using anti-LacR antibodies. The bottom panel shows the catalytic activity of exogenous wild type and kinase dead T256A Sgk proteins immunoprecipitated from IPTG-treated and untreated cells were tested in vitro with Sgktide as a peptide substrate in the presence of [gamma -32P]ATP. The level of radiolabeled Sgktide was quantified as described under "Experimental Procedures." The Sgk kinase activity is the average of two independent experiments.

The catalytic activity of the IPTG-inducible wild type and T256A Sgk proteins was examined by an in vitro kinase assay of immunoprecipitated Sgk using the Sgktide peptide as the substrate (17, 26). As shown in Fig. 9 (bottom panel), the IPTG induced wild type Sgk kinase is catalytically active, and no active Sgk is detected in the absence of IPTG. In contrast, the ectopically expressed T256A Sgk was catalytically inactive (Fig. 9, bottom panel), under conditions where each exogenous Sgk protein was induced at similar levels (Fig. 9, top panel).

To determine the effects of the catalytically active versus inactive forms of Sgk on the survival of transfected Con8 cells from growth factor starvation induced apoptosis, cells were serum-starved for 120 h in the presence or absence of IPTG. Adherent and non-adherent cells were harvested and assayed by flow cytometry for apoptotic nuclei by measuring subdiploid DNA content. As shown in Fig. 10 (upper panel, Wt Sgk), IPTG-induced ectopic expression of wild type Sgk greatly decreased the number of apoptotic nuclei as compared with cells not treated with IPTG. In contrast, the induction of the T256A catalytically inactive form of Sgk had no significant effect on the amount of hypodiploid nuclei detected after 120 h of serum starvation (Fig. 10, upper panel, T256A Sgk). The data from multiple experiments are quantified in the lower panel of Fig. 10. These results demonstrate that the presence of a catalytically active Sgk protein can protect mammary tumor cells from cell death induced by serum starvation and further support a role for Sgk in cell survival.


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Fig. 10.   Conditional induction of wild type Sgk protects Con8 cells from growth factor starvation-induced cell death. Con8 cell lines that ectopically expressed IPTG-inducible forms of wild type Sgk or kinase dead Sgk were serum starved in the presence or absence of IPTG (0.5 mM) for 120 h. Cell death was measured by flow cytometry to determine the presence of hypodiploid DNA content. The upper panel shows data from one representative flow cytometry experiment of cell lines expressing either wild type Sgk or kinase dead Sgk in the presence and absence of IPTG (0.5 mM). The lower panel represents the compilation of data from three experiments. These data represent the difference in hypodiploid nuclei with the addition of IPTG after 120 h of serum starvation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mammalian cells survive exposure to a wide range of adverse environmental stimuli by the activation of specific combinations of intracellular signal transduction pathways. One key stress response is the initiation of intracellular protein kinase cascades that lead to the control of gene expression (6-8, 12, 13). Notably, the activation of p38/MAPK-mediated phosphorylation events, which ultimately target other cell signaling components and transcription factors, has been implicated in transducing a variety of stress stimuli (5, 8, 12). In the present study, we document that the p38/MAPK-dependent stimulation of Sgk expression is a cellular response shared by several distinct types of environmental stress signals and that Sgk signaling plays a key role in the ability of mammary epithelial cells to survive these adverse conditions. As summarized in Fig. 11, UV irradiation, heat shock, oxidative stress, and hyperosmotic stress each induce Sgk expression through a p38/MAPK-dependent pathway. We previously demonstrated that hyperosmotic stress activation of p38/MAPK requires the MKK3/MKK6 upstream kinases and that the Sgk promoter contains a hyperosmotic stress regulated element in its promoter that binds to the Sp1 transcription factor (26). In addition, pharmacological evidence suggests that the sorbitol induction of Sgk transcripts in human HepG2 hepatoma cells is a p38/MAPK-dependent response (28), which is likely a transcriptional response. We hypothesize that the other tested environmental stresses stimulate Sgk expression through pathways that ultimately target the Sgk promoter. Glucocorticoids, which are considered a physiological stress hormone, induced Sgk expression independent of p38/MAPK, and stimulated Sgk promoter activity through a glucocorticoid response element (25).


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Fig. 11.   Model for the stress and hormone stimuli control of Sgk expression, activity, and localization. The expression of Sgk gene products is stimulated by multiple extracellular stresses via the p38/MAPK cascade, whereas, the dexamethasone-induced expression of Sgk proceeds independent of p38/MAPK signaling. We propose that all of these stimuli induce Sgk promoter activity; a functional glucocorticoid response element and a hyperosmotic stress-regulated element were previously identified in the Sgk promoter. The stress-induced Sgk, but not the dexamethasone-induced Sgk, decreases FKHRL1 forkhead transcriptional activity and increases its cytoplasmic localization. We hypothesize that the stress-induced Sgk phosphorylates FKHRL1 to inactivate its transcriptional activity and contribute to the cell survival signaling to protect cells from stress-induced death.

One notable difference in the regulated expression of Sgk is that the kinetics, duration, and peak times of induction differ between the various stimuli. For example, the response to oxidative stress, heat shock and UV irradiation was rapid and transient, whereas, in response to exposure to sorbitol causing hyperosmotic stress there was a sustained accumulation of Sgk gene products after a several hour time lag. Dexamethasone treatment caused a rapid and sustained accumulation of Sgk protein (16, 25). The sustained induction of Sgk by sorbitol and glucocorticoids can be explained in part by the experimental design that required that the stress reagents remained in the cell culture media during the entire time course. In contrast, the UV and heat treatments were pulses, in which the cells were exposed to the stimuli for a short amount of time and then allowed to recover in normal growing conditions. Hydrogen peroxide is converted to water and oxygen in cells, also resulting in a transient exposure (72). Fundamental differences in the signaling pathways and downstream targets activated by each stress condition, such as changes in transcription factor expression and activation, likely also play a role in the transient versus sustained nature of the Sgk induction profile. We are currently investigating the underlying mechanism that drives these kinetic differences in Sgk expression.

As also summarized in Fig. 11, each of the tested environmental stresses and glucocorticoids induce Sgk that is phosphorylated in a PI 3-kinase dependent manner, which was previously shown to be the enzymatically active form of this protein kinase (17, 40). Our functional studies demonstrated that ectopic expression of either the hyperphosphorylated wild type Sgk or the T256D/S422D Sgk, in which both of the PI 3-kinase dependent phosphorylation sites were changed to aspartic acids to mimic the phosphorylation charge, provided protection against the apoptotic effects of oxidative stress, hyperosmotic stress, heat shock or UV irradiation. In contrast, mutation of the PI 3-kinase-dependent phosphorylation sites (T256A/S422A) eliminated the cell survival signaling by Sgk. Furthermore, in Con8 mammary tumor cells, the conditional expression of active wild type Sgk protected cells from growth factor starvation induced cell death, whereas the conditional expression of a kinase dead form of Sgk was incapable of conferring protection against the growth factor deprivation stress. Taken together, these data strongly suggest that the cell survival response to the environmental stress cues involves the induction of an enzymatically active Sgk.

Interestingly, Akt, a protein kinase highly homologous to Sgk, remained constitutively expressed but not phosphorylated after exposure to the same stress stimuli that stimulate production of hyperphosphorylated Sgk, highlighting the importance of the Sgk cell survival pathway in the NMuMg mammary epithelial cells. Akt, which is also phosphorylated by the PI 3-kinase dependent pathway, has been shown to be activated by a number of extracellular stresses in other cell systems (65, 73, 74). The effect of particular stresses on Akt activity can vary significantly in a cell type specific manner (73, 75-80). For example, after UV irradiation, Akt is activated in human epithelial cells (81) (77) and in JB6 mouse epidermal cell line Cl41 (74) but remains inactive in HEK293T, Swiss 3T3, (65), or NIH 3T3 cells (73).

The FKHRL1 forkhead transcription factor family member has been shown to be a substrate for Sgk and for Akt (46, 49). Based on their overlapping patterns of FKHRL1 phosphorylation and preferential phosphorylation of certain sites, Sgk and Akt have been postulated to have complementary rather than redundant roles in cell survival (46). Sgk selectively phosphorylates serine-315 within FKHRL1, while Akt prefers serine-253; threonine-32 is phosphorylated by both kinases (46). Phosphorylation of all three sites is required for growth factors to completely repress FKHRL1-induced transcription (46). Under each of the environmental stress conditions, but not after glucocorticoid treatment, ectopically expressed FKHRL1 was unable to activate a reporter plasmid driven by a forkhead-regulated element. In addition, exposure to stress increased the amount of exogenous FKHRL1 localized in the cytoplasm. FKHRL1 and other members of the forkhead transcription factor family induce the expression of several pro-apoptotic genes (48, 49). Therefore, we hypothesize that the cell survival function of stress-induced Sgk is that phosphorylation of FKHRL1 causes an increased cytoplasmic localization, which would sequester FKHRL1 away from its nuclear targets.

Treatment with dexamethasone caused only a slight decrease in FHRE reporter activity, suggesting that the dexamethasone induced Sgk likely targets protein substrates other than forkhead and has other cellular functions. The role of glucocorticoids in survival pathways is extremely cell type specific. Glucocorticoids have been shown to promote apoptosis in lymphocytes (82), but they are protective in human mammary epithelial cells (83) and rat hepatoma cells (84). Furthermore in MCF-7 human breast cancer cells, glucocorticoids protect against a growth factor starvation-induced death (53). However, the cellular role of glucocorticoid induced Sgk in NMuMg cells remains undefined. We are currently attempting to determine whether the glucocorticoid induced Sgk may afford protection against growth factor starvation conditions in NMuMg cells, perhaps by inducing a G1 cell cycle arrest similar to that observed with the Con8 mammary tumor cells (57, 85).

The induced Sgk is localized either primarily to the cytoplasmic compartment, as in the case of hyperosmotic stress or glucocorticoid treatment, or is detected throughout the cell, as with heat shock, oxidative stress or UV irradiation (Fig. 11). We have recently demonstrated that signal dependent shuttling of Sgk between the cytoplasmic and nuclear compartments is mediated by the importin-alpha pathway (86) and that the Sgk protein induced by glucocorticoids, sorbitol, or UV irradiation interacts with importin-alpha in vitro (data not shown). The retention of Sgk in the cytoplasm by glucocorticoids and osmotic shock may reflect the net export of Sgk from the nucleus induced by these stressors, whereas the transient stressors likely alter the equilibrium of shuttling between the cytoplasm and nucleus. The differences in the stress-induced subcellular localization of Sgk suggest that there are nuclear and cytoplasmic targets of Sgk in response to heat shock, UV irradiation, and oxidative stress that may be distinct from targets in dexamethasone- or sorbitol-treated cells.

The results presented in this study strongly support a cell survival role for Sgk in transducing a diverse set of cellular stress signals. The vast repertoire of extracellular signals that regulate Sgk transcription, activity and subcellular localization, including steroid hormones, growth factors, cytokines, and cellular stress stimuli, suggests wide ranging roles for Sgk and participation in a number of cell signaling cascades. The development of stably transfected mammary tumor cells that express either wild type or kinase dead forms of Sgk in an IPTG-inducible manner represents the first report of the conditional expression of Sgk. This unique cell system is being used to define Sgk specific signaling pathways and uncover key functional targets of Sgk.

    ACKNOWLEDGEMENTS

We thank Dr. Michael Greenberg for the FHRE reporter and FKHRL1 forkhead expression constructs. We also thank Terry Unterman for the FKHR forkhead expression constructs. We also greatly appreciate Hanh Garcia for critical evaluation of the manuscript and Cindy Huynh, Sophia Chung, and Jessie Young for technical assistance.

    FOOTNOTES

* This work was supported by a National Institutes of Health grant (to G. L. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported in part by a Haas Scholars Program undergraduate fellowship.

§ To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, 591 LSA, University of California at Berkeley, Berkeley, CA 94720-3200. Tel.: 510-642-8319; Fax: 510-643-6791; E-mail: glfire@uclink4.berkeley.edu.

Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M211649200

    ABBREVIATIONS

The 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-beta -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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