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
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ABSTRACT |
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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 (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- 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.
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 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.
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.
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).
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).
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).
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.
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.
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).
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.
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,
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.
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).
INTRODUCTION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
(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).
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DISCUSSION
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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.
RESULTS
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DISCUSSION
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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
[ -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.
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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.
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DISCUSSION
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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- pathway (86) and that the Sgk protein induced by glucocorticoids, sorbitol, or UV irradiation interacts with importin-
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.
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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.
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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.
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
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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--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.
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