From the Department of Molecular and Cell Biology and
The Cancer Research Laboratory, University of California,
Berkeley, California 94720-3200, ¶ CompuCyte Corporation,
Cambridge, Massachusetts 02139, and the
Department of
Nutritional Science, University of California,
Berkeley, California 94720
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ABSTRACT |
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The serum- and glucocorticoid-inducible kinase
(sgk) is a novel serine/threonine protein kinase that is
transcriptionally regulated in rat mammary tumor cells by serum under
proliferative conditions or by glucocorticoids that induce a
G1 cell cycle arrest. Our results establish that the
subcellular distribution of Sgk is under stringent cell cycle and
hormonal control. Sgk is localized to the perinuclear or cytoplasmic
compartment as a 50-kDa hypophosphorylated protein in cells arrested in
G1 by treatment with the synthetic glucocorticoid
dexamethasone. In serum-stimulated cells, Sgk was transiently
hyperphosphorylated and resided in the nucleus. Laser scanning
cytometry, which monitors Sgk localization and DNA content in
individual mammary tumor cells of an asynchronously growing population,
revealed that Sgk actively shuttles between the nucleus (in S and
G2/M) and the cytoplasm (in G1) in
synchrony with the cell cycle. In cells synchronously released from the
G1/S boundary, Sgk localized to the nucleus during
progression through S phase. The forced retention of exogenous Sgk in
either the cytoplasmic compartment, using a wild type sgk
gene, or the nucleus, using a nuclear localization signal-containing
sgk gene (NLS-Sgk), suppressed the growth and DNA synthesis
of serum-stimulated cells. Thus, our study implicates the
nuclear-cytoplasmic shuttling of sgk as a requirement for cell cycle
progression and represents a novel convergence point of
anti-proliferative and proliferative signaling in mammary tumor cells.
A dynamic balance of steroid hormones, protein growth factors, and
other environmental cues coordinately regulates an intricate network of
intracellular processes that stringently control mammalian cell
proliferation (1-5). A large body of literature has characterized the
individual cellular events activated by either steroid hormones (6-10)
or by protein hormones and growth factors (3, 11-15), which trigger
the two principal signal transduction pathways that eukaryotic cells
employ to respond to external stimuli. To understand the functional
connections between the transcriptional events regulated by nuclear
steroid receptors (7, 8, 10, 16, 17) and the cascades of
phosphorylation-dephosphorylation reactions mediated by the
cell-surface growth factor receptors (3, 11, 13, 15, 18), a crucial
issue was to define the key steps at which these signal transduction
pathways converge. There are a variety of potential mechanisms of
cross-talk between growth factor and steroid-responsive pathways (5,
19). These regulatory steps include steroid-mediated changes in the
expression of growth factors, their cognate receptors, and components
of phosphorylation and dephosphorylation cascades (20-29). In other
cellular contexts, the phosphorylation of steroid receptors can alter
their function and target gene specificity (30, 31). The regulation of
cell signaling events in the nucleus for the coordinate control of target genes allows cells to respond to external stimuli in a physiologically appropriate manner. The nuclear import of protein kinases provides one mechanism for modulating cellular signal transduction pathways that functionally complement or potentially couple multiple growth factor pathways to steroid receptor signaling. Several classes of protein kinases, such as protein kinase A, certain
isoforms of protein kinase C, mitogen-activated protein kinase, Jun
N-terminal kinase, and p90rsk, can translocate to the
nucleus and phosphorylate a select group of transcription factors, such
as c-Jun and c-Fos components of the AP-1 complex, whose expression
and/or activity is targeted by steroid receptor signaling (14, 32-36).
Several of the protein kinases implicated in the nuclear cross-talk
between steroid receptor signaling and growth factor-induced pathways
are involved in the regulation of proliferative or anti-proliferative
responses. For example, in T cells, glucocorticoids down-regulate the
phosphorylation of the basal and activated forms of the
p70S6K/p85S6K kinase (37), a protein kinase
whose activity and phosphorylation is regulated during cell cycle
progression (38). However, relatively little is known about the
functional coordination between the expression and localization of
protein kinases involved in cell signaling and the hormonal control of
cell proliferation.
Glucocorticoids have been shown to inhibit the growth and regulate cell
cycle progression of many different cell types (5, 9), with their most
characterized effects shown in mammary tumor cells (39-41), hepatoma
cells (42, 43), osteosarcoma cells (44), and lymphoma cells (45). To
investigate directly mechanistic relationships between nuclear growth
factor and steroid receptor signaling events associated with the
control of cellular proliferation, we have been characterizing the
Con8.hd6 rat mammary epithelial tumor cell line (39, 40), which is
derived from the hormone-responsive
7,12-dimethylbenz(a)anthracene-induced 13762NF rat mammary
adenocarcinoma (46). We have documented that glucocorticoid hormones
strongly suppress Con8.hd6 cell growth by inducing a specific
G1 block in cell cycle progression (41), inhibit the
in vivo growth of Con8-cell derived tumors (47), induce the
formation of tight junctions (48, 49), and inhibit the production of an
autocrine acting transforming growth factor- The sgk gene encodes a 50-kDa protein with 431 amino acids
that shows strong homology (45-54% amino acid identity) to the catalytic domains of other serine/threonine protein kinases, such as
Akt (54%), the rat p70S6K/p85S6K kinases
(50%), rat protein kinase C- Cell Culture Conditions and Radiolabeling--
Con8.dh6 mammary
epithelial tumor cells were routinely cultured on standard tissue
culture plates in DMEM/F-12 (BioWhittaker) supplemented with 10% calf
serum (BioWhittaker) and penicillin/streptomycin (BioWhittaker) and
maintained at 37 °C and 5% CO2 as described (39, 40).
The cells were incubated with serum-free medium prior to their
treatment with the indicated concentrations of serum and/or 1 µM dexamethasone for the indicated times. In the appropriate experiments, 5-bromo-2'-deoxyuridine (BrdUrd) (Sigma) was
added to a final concentration 100 µM for 30 min. For
cell synchronization, hydroxyurea was added to a final concentration of
1 mM to 30% confluent cells for 24 h, after which
95% of the cell population was found to be in G1 phase of
the cell cycle, as determined by flow cytometry. For radiolabeling with
[32P]orthophosphate (10 mCi/ml H2O, NEN Life
Science Products), Con8.hd6 cells were grown to 30% confluency before
they were serum-deprived for 48 h. The cells were then incubated
in phosphate-free DMEM for 1 h, followed by incubation in
phosphate-free DMEM supplemented with the indicated combinations of
serum-free conditions, 10% dialyzed calf serum, and/or 1 µM dexamethasone for 4 h. Cells were pulse-labeled
for 1 h with 0.5 mCi of carrier-free
[32P]orthophosphate in 4 ml of incubation medium. Cells
were harvested, and Sgk was immunoprecipitated using the affinity
purified polyclonal anti-Sgk antibodies and protein A beads (Pharmacia
Biotech, Sweden). Final beads were resolved by SDS-PAGE, followed by autoradiography.
Generation of Polyclonal Anti-Sgk Antibodies and Affinity
Purification--
To express Sgk in bacteria, the pET HAX-Sgk
expression vector was generated for
isopropyl-1-thio- Western Blot Analysis--
Soluble whole cell extracts (20-50
µg of protein) were electrophoretically resolved by SDS-PAGE, and the
proteins were transferred to Nitran Plus membranes (Schleicher & Schuell). The membrane was probed with a 1:5000 dilution of polyclonal
anti-Sgk antibody in 50 mM Tris, pH 8.0, 150 mM
NaCl, 0,05% Tween 20 with 1% nonfat dry milk. The secondary antibody
was a goat anti-rabbit IgG horseradish peroxidase-conjugated antibody
(Bio-Rad) and was used at a dilution of 1:1,000. The Western blot was
developed by using the Renaissance developing kit (NEN Life Science
Products) and exposed to x-ray film.
Phosphatase Treatment of Immunoprecipitated Sgk--
Cells
ectopically expressing His-tagged Sgk were harvested in HEMGN buffer
containing 10 mM imidazole and the lysate incubated with
Ni2+-nitrilotriacetic acid-agarose. The manufacturer's
recommendations were followed to affinity purify His-tagged Sgk. The
beads were equilibrated in 40 mM PIPES, pH 6.0, 1 mM dithiothreitol, 20 µg/ml aprotinin, 20 µM leupeptin and incubated in the presence and absence of
100 µg/ml potato acid phosphatase (Sigma) for 30 min at 30 °C.
Final beads were washed once and resolved by SDS-PAGE, followed by
Western blotting with polyclonal anti-Sgk antibodies.
Indirect Immunofluorescence Microscopy for Sgk Localization and
Incorporation of Bromodeoxyuridine--
Con8.dh6 cells were cultured
on 8-well Lab-Tek Permanox slides (Nalgene Nunc International,
Naperville, IL) or on sterile cover slides and grown to 30% confluency
before the indicated combinations of serum and/or dexamethasone were
added for the indicated time frame. Cell confluency prior to fixation
did not exceed 60%. For indirect immunofluorescence microscopy, cells were washed with PBS, fixed for 15 min in 3.7% formaldehyde, 0.1% glutaraldehyde, rinsed with PBS, and permeabilized with 50% methanol, 50% acetone for 1 min. Following a rinse in PBS, the cells were preabsorbed for 5 min in PBS containing 4% normal goat serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Cells were incubated with a 1:300 dilution of affinity purified rabbit polyclonal anti-Sgk antibody for 1-2 h at 25 °C. After 5 washes with PBS, the
cells were treated for 5 min with PBS containing 4% normal goat serum.
The cells were incubated with a 1:300 dilution of anti-rabbit
fluorescein isothiocyanate-conjugated secondary antibody (Cappel
Research Products, Durham, NC) in PBS and then incubated for 30 min at
25 °C. Cells were washed 5 times with PBS and mounted with 50%
glycerol, 50 mM Tris, pH 8.0, containing 4 mg/ml
n-propyl gallate, and examined on a Nikon Optiphot
fluorescence microscope. Images were captured using Adobe Photoshop
3.0.5 (Adobe Systems, Inc., Mountain View, CA) and a Sony DKC-5000
digital camera. Nonspecific fluorescence was determined by incubation
with the secondary antibody alone and shown to be negligible. Due to
the use of an affinity purified antibody, preimmune serum showed a
relatively higher background and was not used as a negative control.
Double labeling for Sgk localization and the incorporation of
5-bromo-2'-deoxyuridine (BrdUrd) utilized the above procedures with the
following modifications. During the final hour of incubation in medium
containing the indicated combinations of serum and/or dexamethasone,
the cells were incubated in medium containing a final concentration of
100 µM BrdUrd (Sigma) at 37 °C for 30 min. Following
fixation, the cells were rinsed with PBS, and the DNA was denatured by
incubation in 0.12 N HCl at 37 °C for 1 h. After neutralization in two changes of 0.1 M borate buffer over
10 min, cells were washed 3 times for 30 min and then incubated in PBS containing 4% normal goat serum (Jackson ImmunoResearch). In addition to the Sgk antibody incubation detailed above, the cells were also
incubated with a 1:80 dilution of mouse monoclonal anti-BrdUrd antibody
(Dako Corp., Carpinteria, CA) in PBS for 60 min at 25 °C. After the
washes, secondary antibodies were added as described above with the
addition of a 1:300 dilution of anti-mouse rhodamine-conjugated secondary antibody (Jackson ImmunoResearch) in PBS and incubated at
25 °C for 30 min.
Flow Cytometry--
Con8.hd6 cells were cultured in 6-cm plates,
not exceeding 35% confluency. The cells were washed twice with PBS and
suspended in 500 µl of propidium iodide solution (75 µM
propidium iodide, 0.1% sodium citrate, 0.1% Triton X-100). Finally,
cells were filtered through a polyacrylamide mesh and maintained on ice
until use. Nuclear-emitted fluorescence was measured with a Coulter
Elite instrument with laser output adjusted to deliver 15 milliwatts at
488 nm. Cell nuclei (104) were analyzed from each sample at
a rate of 300-500 nuclei/s. The percentages of cells within the
G1, S, and G2/M phases of the cell
cycle were determined by analysis with the Multicycle computer program
provided by Phoenix Flow Systems in the Cancer Research Laboratory
Microchemical Facility of the University of California, Berkeley. The
data were collected and analyzed with a Becton Dickinson FACScan and
Lysis II software (Becton Dickinon).
Laser Scanning Cytometry--
The cells were analyzed on a laser
scanning cytometer (CompuCyte Corp., Cambridge MA) using argon ion
laser excitation at 488 nm. Fluorescence was collected by
photomultiplier tubes with green (530 DF 30) and red (600 DF 60) band
pass filters, digitized to 0.25 µm by 0.5-µm pixel resolution, and
temporarily stored as computer memory images. When complete memory
images were obtained, they were segmented into nuclei using image
processing techniques, and 20 features were calculated for each cell,
including the total DNA content, and amount of green fluorescence, and
stored in a list mode computer data file. Green fluorescence was
calculated twice, once for the region containing the cell nucleus and
second for a torus extending out from the nucleus to sample the
cytoplasm of the cell. By plotting these features in two parameter dot
plots, it was possible to define regions around clusters of cells with nuclear fluorescence, and regions around cells with cytoplasmic fluorescence, and color gate cells from these regions onto
one-parameter DNA histograms. Cells from the specific regions were also
relocated and visualized by epifluorescence illumination. The scanning
is done in an automated fashion, with 5,000-10,000 cells analyzed per sample.
Generation of Wild Type and Nuclear Localization
Signal-containing sgk Expression Vectors--
The pcDNA3 mammalian
expression vector (Invitrogen, Carlsbad, CA) with the full-length sgk
cDNA was utilized for the ectopic expression of Sgk in cell
culture. Standard PCR techniques were used starting with the
full-length wild type rat sgk cDNA (22). The final
expression vector contains an Asp-718/XbaI insert which consists of the full cDNA of sgk fused in frame to a
poly-8-His tag at the C terminus. To generate this chimeric construct
with the full-length sgk gene in frame with the poly-His tag
and with compatible restriction sites for the cloning sites of the
vector, the two PCR primers used were
5'-AGGTACCGCCACCATGACCGTCAAAACCGAGGCTG-3', which contains an Asp-718
site and the first codons of the sgk sequence, and
5'-TTCTAGATCAATGATGATGATGATGATGATGATGGAGGAAGGAGTCCATAGGAGGG-3', which contains the XbaI site, the Stop-codon, eight
His-residues, and the final amino acids of the Sgk C terminus.
The pcDNA3 NLS-sgk-His expression vector was generated by fusing
the nuclear localization signal (NLS) of the SV40 large T antigen in
frame to the C terminus of the full-length sgk cDNA, followed in frame by eight His residues. The PCR primers used were 5'-AGGTACCGCCACCATGACCGTCAAAACCGAGGCTG-3', which contains an
Asp-718 site and the first codons of the sgk sequence and
5'-TTCTAGATCAATGATGATGATGATGATGATGATGGAACTTTACCTTCCTCTTCTTCTTTGGGAGGAAGGAGTCCATAGG-3'. This second primer contains an XbaI site, a stop
codon, eight His-residues, the nuclear localization signal (NLS), which
is specified as the amino acid sequence FKVKRKKKP, and the last amino acids of the Sgk C terminus. The PCR-generated insert in both vectors
was fully sequenced to confirm the fidelity of the PCR reactions and ligations.
Ectopic Expression of Sgk--
Sgk was expressed in Con8.dh6
mammary tumor cells that had been transiently transfected with
pcDNA3-sgk-His using LipofectAMINE (Life Technologies, Inc.) for
24 h. The procedure was performed exactly as recommended by the
manufacturer. The CMV-driven mammalian expression plasmid
constitutively expresses Sgk protein which is tagged on the C terminus
with an 8-mer His epitope. The coding sequence is flanked by Asp-718
and XbaI sites for insertion into the multiple cloning sites
of pcDNA3 (Invitrogen, Carlsbad, CA).
Cell Foci Growth Assay of Transiently Transfected
Cells--
Cells were transfected exactly as described above with
pcDNA3-sgk-His, pcDNA3 NLS-sgk-His, or the pcDNA3 vector
control. Twenty-four hours after transfection, the cell number of the
two cell populations was normalized, and cells were seeded at 1:1,000
and 1:5,000 dilutions. A small aliquot of cells was removed for
analysis by Western blotting to confirm the presence of overexpressed
Sgk in the pcDNA3-sgk-His- or the pcDNA3
NLS-sgk-His-transfected cells. Twenty-four to forty-eight hours
post-transfection, the cells were incubated in medium containing 750 µg/ml neomycin (Sigma). The medium was changed every 48 h. One
week later, the neomycin-resistant colonies were stained with 10%
formalin, 0.5% crystal violet in PBS. The plates were washed in
deionized water and air-dried, and the colonies on each plate were counted.
Differential Regulation of Sgk Subcellular Distribution by
Treatment with Glucocorticoids and Serum Stimulation--
Treatment of
rat Con8.hd6 mammary tumor cells with glucocorticoids or serum rapidly
and strongly stimulates the transcription of the sgk gene by
directly affecting sgk promoter activity (22, 51). Serum
stimulation induces the mammary tumor cells to progress through the
cell cycle, whereas treatment with glucocorticoids results in a
stringent growth arrest. Thus, distinct signal transduction pathways
with opposite proliferative responses appear to converge on
sgk gene expression, suggesting that this protein kinase may potentially play a role in both proliferative and anti-proliferative signaling in mammary tumor cells. Conceivably, the cellular utilization of Sgk protein, such as its localization, may be altered in a manner
that is crucial for the distinct proliferative conditions induced by
glucocorticoids or serum. To examine the cellular distribution of Sgk
under serum-stimulated proliferative and glucocorticoid growth-arrested
conditions, mammary tumor cells were preincubated for 48 h in
serum-deprived conditions and then treated with combinations of 10%
serum and/or 1 µM dexamethasone, a synthetic
glucocorticoid. Sgk localization was monitored by indirect
immunofluorescence microscopy using an affinity purified anti-Sgk
polyclonal antibody (Fig. 1). Remarkably,
treatment with either serum or dexamethasone resulted in Sgk being
localized into distinct subcellular compartments. After a 3-h
stimulation of confluent cells with serum, Sgk started to accumulate in
the nucleus of a large fraction of cells (Fig. 1, top right
panel). A significant proportion of serum-stimulated cells also
displayed a homogeneous staining pattern throughout the cells; the
proportion of homogeneously stained cells increased after 18 h of
serum treatment (data not shown). In contrast to the staining pattern
observed in cells stimulated only with serum, treatment of cells with
dexamethasone in the presence (Fig. 1, lower right panel) or
absence of serum (Fig. 1, lower left panel) for 3 h
caused Sgk to display a mostly perinuclear or cytoplasmic fluorescent
pattern, as observed by a brightly staining ring around the nuclear
membrane. Similar to cells treated only with glucocorticoids, mammary
tumor cells treated with dexamethasone and serum harbor Sgk exclusively
in a periplasmic/cytoplasmic location (Fig. 1, lower right
panel). Cells maintained in a serum-deprived state have a low
level of diffuse staining of Sgk (Fig. 1, top left panel)
that is only slightly more intense than that observed when fixed cells
are incubated only with secondary antibodies in the absence of primary
anti-Sgk antibody (data not shown). The faint homogeneous staining
observed in serum-deprived conditions may be due to a low level of
expressed Sgk since significant overexposure of Western blots revealed
a small amount of the 50-kDa Sgk protein.
The differential effects of serum and dexamethasone on Sgk localization
was quantitated by visually assessing the subcellular distribution of
Sgk in at least 500 cells per condition. In order to visually quantify
individual cells, the mammary tumor cells were cultured at very low
confluency (10-20%) in the indicated combinations of serum and
dexamethasone. This experiment employed 18- and 24-h treatments,
compared with the shorter time course shown in Fig. 1, because of the
lower starting confluency of the cells, which increases the time
required to observe nuclear-associated Sgk. Sgk subcellular
localization was categorized as having either nuclear,
perinuclear/cytoplasmic, or homogeneous staining. As shown in Fig.
2, the proportion of cells in each
category was expressed as the percentage of Sgk immunostaining cells.
After a prolonged serum deprivation in the absence of steroid, only 12% of cells displayed specific nuclear staining, whereas a smaller percentage of cells was categorized with cytoplasmically localized Sgk.
In the absence of serum and dexamethasone, only a small percentage of
mammary tumor cells stain positive for Sgk, and therefore 83% of the
cells is not accounted for in the quantitation (Fig. 2, left set
of bar graphs). An 18-h serum stimulation caused Sgk to
specifically accumulate in the nuclei of 40% of the cells. In
approximately 50% of the cells, Sgk was localized equally between the
nucleus and the cytoplasm of a given cell and therefore was classified
as "homogeneous" staining. Thus, in approximately 85% of
serum-stimulated cells, Sgk displayed a significant nuclear distribution, whereas less than 15% of the cells harbored Sgk in the
cytoplasm alone.
Treatment of serum-deprived cells with a combination of serum and
dexamethasone for 18 h resulted in a heterogeneous staining pattern in which 25% of cells harbored Sgk in the nucleus, 15% were
found to have Sgk in their nuclei as well as in the cytoplasm (homogeneous staining), and 65% of cells displayed exclusively perinuclear-cytoplasmic staining of Sgk (Fig. 2, middle set of bar graphs). A 48-h treatment with both serum and dexamethasone resulted in virtually all cells producing Sgk as a
perinuclear-cytoplasmic staining protein that coincided with the loss
of the hyperphosphorylated form of Sgk. The exclusive localization of
Sgk in the perinuclear-cytoplasmic compartment of cells treated for
48 h with both serum and dexamethasone was approximately equal to
that observed in cells treated only with dexamethasone for 18 h in
the absence of serum (Fig. 2, far right set of bar
graphs). The opposing effects of serum and glucocorticoid on Sgk
localization were completely reversible. For example, incubation of
serum-treated cells with dexamethasone caused Sgk to redistribute from
a predominantly nuclear localization to a perinuclear-cytoplasmic location, whereas additional treatment of these cells with a potent glucocorticoid antagonist, RU486, in the presence of serum resulted in
the movement of Sgk back to the nuclear compartment (data not shown).
Sgk Is Phosphorylated after Serum Stimulation but Not after
Glucocorticoid Treatment--
Because sgk is differentially localized
in serum-stimulated cells compared with glucocorticoid-treated cells,
the potential modification of Sgk was examined in mammary tumor cells
stimulated with high (10%) or low (0.5%) serum in the presence or
absence of 1 µM dexamethasone. Cells were initially
serum-deprived for 48 h prior to treatment with serum and/or
dexamethasone for an 8-h time course. Western blots of
SDS-PAGE-fractionated total cell lysates were probed for Sgk with the
affinity purified polyclonal anti-Sgk antibody. Treatment with
dexamethasone in low serum rapidly stimulated the expression of a
50-kDa Sgk species that was continuously present in cells during the
8-h time course (Fig. 3, lower left of panel
A) and was maintained at high levels even after 48 h steroid
treatment (data not shown). In contrast, stimulation of serum-deprived
cells with 10% serum is accompanied by the rapid and transient
induction of differentially migrating forms of Sgk, including the
50-kDa species as well as larger forms (up to 52 kDa) of Sgk (Fig. 3,
top right of A). The peak expression of the larger Sgk
species was observed after 2 h in serum, and by 5 h of serum
treatment, the 50-kDa Sgk protein was the predominant species. This
same pattern of multiple Sgk species, although at a lower level of
expression, was observed when serum-starved cells were stimulated with
transforming growth factor-
To test whether the higher molecular weight forms of Sgk observed in
serum-stimulated cells represent hyperphosphorylated forms of the
protein, 32P-labeled Sgk was immunoprecipitated from
extracts of cells that had been treated at very low confluency for
5 h with combinations of serum and/or dexamethasone and
radiolabeled with [32P]orthophosphate during the last
hour of treatment. Under these conditions, the highest molecular weight
form of Sgk is visible on the immunoblots. Electrophoretic
fractionation of the immunoprecipitated 32P-labeled Sgk
revealed that the higher molecular weight forms of Sgk produced in
serum-treated cells are phosphorylated, whereas the 50-kDa sgk species
produced in dexamethasone-treated cells did not incorporate
[32P]phosphate and is therefore hypophosphorylated (Fig.
3B). This result demonstrates that Sgk was produced in
dexamethasone-treated cells under conditions in which it did not
incorporate radiolabeled [32P]phosphate. Consistent with
the kinetic experiments, serum stimulation results in the expression of
three forms of Sgk that migrate as a triplet of proteins, and likely
represent distinct phosphorylated forms of Sgk. To establish further
that the differentially migrating forms of Sgk produced in
serum-treated cells varied by their degree of phosphorylation and did
not represent distinct isoforms, poly-His-tagged Sgk was transiently
overexpressed in the mammary tumor cells, affinity purified by binding
to a nickel matrix, and incubated in the presence or absence of potato
acid phosphatase. Western blot analysis of Sgk showed that potato acid
phosphatase digestion caused the multiple forms of Sgk to collapse into
a single protein species with a molecular mass of approximately 50 kDa
(Fig. 3B, blot). This apparent loss of Sgk immunoreactive
protein following phosphatase treatment is likely due to a change in
antigenicity resulting from its complete dephosphorylation in
vitro. Furthermore, we have recently observed that incubation with
either wortmannin or LY294002 inhibited the production of the slower
migrating forms of Sgk, which suggests that the regulation of Sgk
phosphorylation is a downstream target of the cytoplasmic
phosphatidylinositol 3-kinase
pathway.2 Taken together, our
results show that the distinct electrophoretic migration of Sgk
produced in serum-treated mammary tumor cells resulted from
differential phosphorylation. Moreover, the formation of the
hyperphosphorylated forms of Sgk correlated with the localization of
sgk into the nucleus under serum-stimulated conditions, whereas a
hypophosphorylated form of sgk was detected under conditions in which
sgk was localized to the cytoplasm in glucocorticoid treated cells.
Nuclear Localization of Sgk in S Phase Mammary Tumor
Cells--
The differential subcellular distribution and
phosphorylation of Sgk in serum-stimulated versus
glucocorticoid growth-arrested mammary tumor cells suggested that the
regulation of Sgk localization may be closely linked to the steroid and
growth factor control of the cell cycle. As an initial test whether the
nuclear localization of Sgk during serum stimulation coincides with
entry into S phase, cells were labeled with the thymidine nucleotide
analog BrdUrd, and cells were stained for both Sgk and for the
incorporation of BrdUrd. Double staining of individual cells was
achieved by using fluorescein-labeled secondary antibodies that
recognize the anti-Sgk primary antibodies and rhodamine-conjugated
secondary antibodies that selectively bind to the anti-BrdUrd primary
antibodies. As shown in Fig. 4
(left column of panels), essentially all of the 18-h
serum-stimulated mammary tumor cells that are actively synthesizing DNA
harbor Sgk in the nucleus as shown by the striking co-staining of Sgk
and BrdUrd. A fraction of the cells did not incorporate BrdUrd but
still localized Sgk to the nucleus and, as shown in a later section,
represent cells in G2/M. In contrast, cells
that were growth-suppressed after treatment with both serum and
dexamethasone for 24 h failed to incorporate BrdUrd and
selectively expressed Sgk as a perinuclear-cytoplasmic protein (Fig. 4,
right column of panels).
The subcellular distribution of Sgk was quantitated in the S phase
cells within a large population of mammary tumor cells. The mammary
tumor cells were initially serum-deprived for 48 h. BrdUrd-positive cells were then scored after treatment with only serum
for 18 h, with serum and dexamethasone for 18 h, and with both extracellular signals for 48 h. At least 500 cells per
condition were examined, and the relative numbers of BrdUrd-positive
cells were found to match closely the relative number of cells with an
S phase DNA content (48) and the level of [3H]thymidine
incorporation (41). After an 18-h serum stimulation, 410 cells stained
positive for BrdUrd incorporation. Of those cells, 50% exhibited
nuclear staining of Sgk, 38% of cells were homogeneously stained, and
12% displayed periplasmic or cytoplasmic staining (Fig.
5, left bar graphs). The
homogeneously stained cells expressed Sgk to approximately the same
extent in the nucleus and in the cytoplasm. Combining the nuclear and
homogeneous categories revealed that 88% of the BrdUrd-positive cells
harbor Sgk in the nucleus. Treatment with dexamethasone and serum for
18 h reduced by approximately one-half the number of
BrdUrd-positive cells, which reflects the partially growth-arrested
state. In this smaller number of S phase cells, the same relative
proportion of nuclear-localized Sgk was maintained as that observed
with the serum-stimulated cells. Ninety percent of the S phase cells
expressed Sgk in the nucleus with 40% of the BrdUrd-positive cells
displaying exclusively nuclear Sgk and 50% of cells showing
homogeneous staining of Sgk (Fig. 5, middle bar graphs).
Under G1 cell cycle-arrested conditions in which cells are
treated with serum and dexamethasone for 48 h, only 3% of the
cells detectably incorporated BrdUrd, and the Sgk staining in these few
cells was equally divided between a nuclear and a homogeneous pattern
(Fig. 5, right bar graphs). Taken together, our results
indicate that Sgk localization may be regulated in a cell
cycle-dependent manner because this protein kinase is
distributed to the nucleus in cells that are actively synthesizing DNA
and into a perinuclear/cytoplasmic compartment in G1
growth-arrested cells.
Determination of Sgk Subcellular Localization and Cell Cycle Phase
in an Asynchronous Cell Population by Laser Scanning
Cytometry--
Conceivably, the nuclear localization of Sgk observed
in serum-stimulated cells and its perinuclear-cytoplasmic localization in glucocorticoid-treated cells arrested in G1 results from
the cell cycle phase-regulated compartmentalization of this protein kinase. To test directly this possibility, laser scanning cytometry was
utilized to simultaneously determine the subcellular distribution of
Sgk and the DNA content of a large number of individual cells in an
asynchronously growing population. Serum-stimulated cells were fixed
and then simultaneously stained for Sgk and for DNA content by
propidium iodide. Five thousand to ten thousand mammary tumor cells
were individually examined for Sgk localization in the nuclear (green
profile) or perinuclear (red profile) compartments and for the
corresponding DNA content (black profile) by laser scanning cytometry.
As shown in Fig. 6, the overall
distribution of cellular DNA content (shown in black) is of
an asynchronous growing population of cells. The nuclear-associated Sgk
(green) strongly correlated with cells containing a DNA content
indicative of S and G2/M phase cells.
Interestingly, Sgk was primarily localized to the nucleus in S phase
cells, whereas cells in G2/M displayed a more
diffuse nuclear staining pattern. The nuclear localization of Sgk in
this population of mitotic cells explains the observation that Sgk can
be nuclear-associated in a subset of cells that do not incorporate
BrdUrd (see Figs. 4 and 5). As also shown in Fig. 6, in contrast, the
cells with a G1 DNA content in the growing population of
serum-treated cells expressed Sgk in the cytoplasmic compartment (red)
suggesting that the phase of the cell cycle regulates Sgk localization.
Consistent with our previous flow cytometry results (41, 48),
dexamethasone growth-arrested cells accumulated with a predominantly
G1 DNA content and expressed Sgk as a
perinuclear-cytoplasmic protein (data not shown). Based on these
results, we propose that Sgk localization is regulated by the phase of
the cell cycle as well as by the cell cycle effects of growth factor
and glucocorticoid treatment. Sgk is observed to be
perinuclear-cytoplasmic during progression through the G1 phase, whereas during the onset of S phase and progression into G2/M of the cell cycle, Sgk is mostly
nuclear.
Sgk Localizes to the Nucleus during Progression through S
Phase--
To verify that the nuclear localization of Sgk can be
regulated by progression through S phase of the cell cycle, Sgk
distribution was examined in serum-treated mammary tumor cells after
release from a G1/S block in cell cycle progression. The
cells were first blocked at the G1/S boundary by a 24-h
incubation with hydroxyurea and then allowed to progress through S
phase by replacing the hydroxyurea-containing medium with fresh
serum-containing medium without hydroxyurea. Flow cytometry of
propidium iodide-stained nuclei showed that the 24-h incubation in
hydroxyurea resulted in 64% of cells displaying a G1 DNA
content, and 35% of cells had an S phase DNA content with minimal cell
death (Fig. 7, 0 h release).
Replacing the hydroxyurea-containing media with fresh serum-containing
media without hydroxyurea allowed the cell population to synchronously
progress through the cell cycle. Four hours after release from
hydroxyurea treatment, approximately 90% of the cell population was in
S phase, and by 8 h release a large fraction of the cell
population had progressed into G2/M phase. At
each time of release, cells were co-stained by immunofluorescence for Sgk localization and for the incorporation of BrdUrd. As shown in Fig.
7, at the G1/S boundary (0 h release time) Sgk displayed a
diffuse cytoplasmic staining pattern with negligible incorporation of
BrdUrd. After 4 h of serum stimulation and subsequent release from
hydroxyurea treatment, Sgk was localized predominantly to the nuclear
compartment under conditions in which virtually all of the cells had
entered S phase and incorporated BrdUrd. Eight hours after release,
significantly fewer cells incorporated BrdUrd and the sgk-staining
pattern was still mostly nuclear with a more diffuse staining pattern.
In a control set of plates, in which the cells were also exposed to
fresh serum-containing medium, but with continued incubation with
hydroxyurea, the cells failed to enter S phase and Sgk remained as a
weakly staining, cytoplasmic protein (data not shown). Thus,
progression through S phase, rather than the growth factor signaling
stimulated by exposure to fresh serum, caused Sgk to become localized
to the nucleus which likely accounts for the observed serum regulation
of Sgk distribution and formation of hyperphosphorylated forms of
Sgk.
The Anti-proliferative Effects of Sgk Ectopically Expressed in the
Nuclear or Cytoplasmic Compartments--
To determine if the targeted
Sgk localization affects the proliferative state, mammary tumor cells
were transfected with either a wild type sgk cDNA
expression vector or with an sgk expression vector in which
the SV40 large T antigen nuclear localization signal is fused to the C
terminus of Sgk (forming NLS-Sgk). The presence of the nuclear
translocation signal should force Sgk to be nuclear-associated in
transfected cells. Each form of the sgk gene was His-tagged
at the C terminus and driven by the CMV promoter. Western blots
confirmed that the exogenously expressed Sgk proteins were full-length
and modified similarly to the endogenous protein, including the
presence of the more slowly migrating phosphorylated forms of Sgk after
serum treatment (data not shown). The subcellular distribution of Sgk
and BrdUrd incorporation were monitored by co-immunofluorescence in a
serum-treated population of transiently transfected cells. Since
commercially available anti-His antibodies failed to detect the
His-tagged Sgk, immunocytochemistry was performed using a 1:800
dilution of the affinity purified polyclonal antibodies to Sgk which is
a concentration below the threshold level required to detect
endogenously expressed Sgk but is sufficient to detect the high levels
of overexpressed Sgk protein. As shown in the upper panels
of Fig. 8, exogenously expressed wild
type Sgk (WT-sgk) was exclusively localized to the
cytoplasmic compartment and did not harbor Sgk in the nucleus. However,
the NLS-Sgk was localized primarily to the nuclear compartment with
virtually no staining observed in the cytoplasm. Approximately 5% of
the subconfluent monolayer of cells produced the ectopically expressed
forms of Sgk (Fig. 8, upper panels). For either the WT-Sgk
or NLS-Sgk constructs, the subset of cells that ectopically express Sgk
generally failed to incorporate BrdUrd (Fig. 8, upper versus
lower panels). Conversely, the cells that stain positive for
BrdUrd do not produce either the WT-Sgk or the NLS-Sgk. A small subset
of the NLS-Sgk-expressing cells were shown to incorporate a low level
of BrdUrd; however, in general, the production of nuclear-associated
Sgk did not drive the overexpressing cells into S phase. Based on the
laser scanning cytometric analysis of endogenous Sgk, in which this
kinase is nuclear-associated in either the S or
G2 phases of the cell cycle, it is possible
that the cells that ectopically express NLS-Sgk may reside in
G2 phase and therefore do not incorporate
BrdUrd. These results show that cells ectopically expressing either a cytoplasmic-residing Sgk or a nuclear-residing Sgk are not observed to
enter S phase and that the nuclear association of Sgk is not sufficient
to induce proliferation of the mammary tumor cells.
A colony formation assay was employed to determine if ectopic
expression of the WT-Sgk or NLS-Sgk can suppress the growth of a
population of transfected mammary tumor cells. In this assay, cells
were co-transfected with either WT-Sgk or NLS-Sgk expression vectors or
with an empty vector (vector control) along with the neomycin
resistance gene, and identical numbers of transfected cells were plated
in selective medium. After a week, the cell cultures were stained to
visualize the surviving cell colonies (Fig.
9). The combined results from five
independent experiments were quantitated. Ectopic expression of either
the WT-Sgk or NLS-Sgk reduced the formation of cell foci by
approximately 60% compared with cells that received only the empty
vector (Fig. 9). The final recovered cell colonies do not express
exogenous Sgk, and consistent with the growth-suppressing effects of
this kinase, we have not been able to recover cells that stably
overexpress Sgk in these mammary tumor cells (data not shown). Our
results from both the BrdUrd labeling of individual cells and from the
cell foci population assay show that Sgk can function in an
anti-proliferative pathway when ectopically expressed and retained in
either the cytoplasmic or nuclear compartments. Since Sgk is observed
in the nucleus in S and G2/M phase cells and in
the cytoplasmic compartment in G1 phase cells, our results
further imply that the nuclear-cytoplasmic shuttling of Sgk may be
required for the cell to progress through the cell cycle.
The growth of normal and transformed cells is controlled by an
intricate network of intracellular processes that converge at key steps
and target genes within each pathway, thereby allowing cells to
appropriately and coordinately respond to specific sets of
extracellular signals (1-5). The types of cellular processes under
control of proliferative signals, as well as the activities of the
regulated cellular components, define the functional connections between pathways that either inhibit or stimulate cell proliferation. We have previously documented that treatment of mammary tumor cells
with either serum or glucocorticoids induce sgk
transcription (22, 23), although each extracellular signal has opposite effects on cell proliferation. Serum stimulates cell cycle progression (22, 23), whereas glucocorticoids cause a G1 cell cycle
arrest (41, 48). The stimulation of sgk gene expression by
each extracellular signal implicates this protein kinase as a
functioning component of either the serum-stimulated proliferative
pathway or of the anti-proliferative response to glucocorticoids.
Consistent with this notion, our results have now established that
nuclear-cytoplasmic distribution of sgk is differentially regulated
depending on the proliferative state of the cells in that sgk is
localized to the cytoplasmic compartment in mammary tumor cells
growth-arrested by glucocorticoids and is predominantly localized in
the nucleus after serum stimulation.
In an asynchronous growing population of mammary tumor cells, Sgk
distributed to either the nucleus or the cytoplasmic compartment in
synchrony with the phase of the cell cycle. Laser scanning cytometry,
which simultaneously monitors DNA content and Sgk localization in
individual cells, revealed that Sgk resides in the cytoplasm during the
G1 phase of the cell cycle and then is compartmentalized to
the nucleus in the S and G2/M phases. This
result indicates that the cytoplasmic localization of Sgk in
glucocorticoid-treated cells may be due to the specific stage of the
cell cycle in which these cells are arrested, rather than by the direct
actions of this steroid on sgk localization. The precise location of
Sgk within the cytoplasm has not been determined, although based on the
subcellular distribution of homologous kinases (33, 34, 53-56), Sgk
may either be anchored to cytoplasmic membranes or free in the
cytoplasm. Immunofluorescence co-staining for Sgk localization and
incorporation of BrdUrd in serum-stimulated cells, or in cells
synchronously released from a G1/S block, revealed that
virtually all of the cells in S phase express sgk as a
nuclear-associated protein. We propose that during cell cycle
progression, Sgk shuttles between the nuclear and cytoplasmic
compartments at the beginning of G1 and at the
G1/S boundary. It is tempting to consider that the
shuttling between the nucleus and cytoplasm is required for sgk
function in cellular proliferative pathways, whereas its regulated retention in the cytoplasm in growth-arrested cells, for example after
the G1 cell cycle arrest mediated by glucocorticoids,
allows sgk to function within an anti-proliferative pathway or be
sequestered away from a crucial proliferative cascade. Thus, the
regulation of Sgk compartmentalization represents a previously
uncharacterized convergence point for proliferative or
anti-proliferative signaling cascades.
A key prediction for the requirement of nuclear-cytoplasmic shuttling
of Sgk in proliferating cells is that the forced retention of this
kinase in either the nucleus or the cytoplasm should suppress cell
growth. Ectopic expression of either WT-Sgk, the wild type kinase that
stably resides in the cytoplasmic compartment, or NLS-Sgk, which stably
resides in the nucleus as a result of its nuclear localization signal,
suppressed mammary tumor cell growth in a cell foci population assay.
Also, immunofluorescence analysis of individual cells producing either
WT-Sgk or NLS-Sgk revealed that the cells do not enter S phase. These
results showed that the forced retention of Sgk in either the nuclear
or cytoplasmic compartments is sufficient to suppress cell growth and
that the shuttling of this kinase between these two cell compartments
is likely to be important for the mammary tumor cells to proliferate. Conceivably, the regulated translocation of Sgk between the nuclear and
cytoplasmic compartments controls the availability of stimulus-specific or cell cycle-regulated signaling components that may modulate the
activity of Sgk and potential access to specific substrates. For
example, nuclear Sgk could interact with and potentially phosphorylate specific sets of transcription factors that regulate the expression of
genes that control proliferative functions, or phosphorylate essential
cell cycle factors required during S and G2.
The cytoplasmic compartmentalization of Sgk during G1 may
exclude Sgk from having access to specific nuclear components during
this phase of the cell cycle or may recruit this kinase to a new set of
targets in the cytoplasm that help the cells progress through
G1. One explanation for the growth suppression induced
after ectopic expression of Sgk into either the nucleus or the
cytoplasm is that high levels of exogenous Sgk may competitively
sequester key components into an inactive complex, which are needed for
cell cycle progression, or prevent the active forms from localizing to
their site of function. In other cell systems, the ectopic expression
of certain Ser/Thr protein kinases has been shown to attenuate cellular
proliferation and/or decrease the tumor formation (57-60) but does not
induce a stringent growth arrest.
Our studies show for the first time that the post-translational
nuclear-cytoplasmic shuttling of a transcriptionally regulated protein
kinase can be regulated at specific stages of the cell cycle. There are
only a few precedents for the nuclear localization of Ser/Thr protein
kinases that are activated as part of growth factor receptor signaling.
Protein kinase C, mitogen-activated protein kinase, p90rsk,
and Akt/PKB have been shown to translocate into the nucleus in order to
phosphorylate and activate a number of transcription factors (34, 55,
61). The p70S6K/p85S6K protein kinases, which
are 50% identical to Sgk in their catalytic domains, have been shown
to be phosphorylated and localized in a cell
cycle-dependent manner (38, 53). In S phase in their respective systems, Sgk and p70S6K/p85S6K are
predominantly nuclear with a speckled staining pattern indicative of
co-localization with heterochromatin (38), whereas both kinases are
enriched in the cytoplasm in the G1 phase. Microinjection of rat embryo fibroblasts with antibodies that inhibit
p70S6K/p85S6K activity abolished the
serum-induced entry into S phase (53), whereas we have shown that
ectopic expression of Sgk inhibited cell growth. Similar to Sgk, the
phosphorylation of p70S6K/p85S6K has been shown
to be reduced after glucocorticoid treatment of T cells (37). The C
terminus of Sgk contains a Ser/Thr-Pro recognition motif for cell
cycle-regulated kinases, and the same motif with similar spacing
between polar amino acid residues is the site of cell
cycle-dependent phosphorylation in the homologous
p70S6K/p85S6K kinase. Thus, Sgk and
p70S6K/p85S6K may be associated with
functionally analogous phosphorylation-dephosphorylation pathways that
direct progression through the cell cycle.
In serum-stimulated cells, the formation of a hyperphosphorylated form
of sgk generally correlated with its localization to the nuclear
compartment, whereas at steady state in glucocorticoid-treated cells,
sgk was hypophosphorylated and distributed to the
cytoplasmic compartment. Phosphorylation or dephosphorylation has been
shown to affect directly or to correlate with the nuclear import or export of a wide variety of proteins, including several protein kinases
and cell cycle-regulated factors (32, 56, 62-66). Thus, one potential
function for the phosphorylation of Sgk in serum-stimulated cells may
be to facilitate or alter its nuclear transport at particular phases of
the cell cycle or in a stimulus-dependent manner. However, the phosphorylation of sgk per se does not appear to
regulate directly its subcellular distribution because the exogenously introduced wild type sgk, which remains cytoplasmic, and the
NLS-sgk, which is retained in the nucleus, are both
hyperphosphorylated (data not shown). Also, cellular stress, such as
osmotic shock, induces a hyperphosphorylated form of sgk that resides
in the cytoplasm.3 In this
regard, it is likely that the phosphorylation of sgk occurs in the
cytoplasm because our recent preliminary studies have shown that the
phosphorylation of Sgk is regulated by the phosphatidylinositol
3-kinase pathway,2 and based on the homology of Sgk to Akt
(22, 23), it is likely to be a direct target of the
3-phosphoinositide-dependent protein kinase family of cytoplasmic kinases.
The precise mechanism by which sgk is transported between the nucleus
and the cytoplasm in a stimulus- or cell cycle-regulated manner is not
known, although the cellular control of this shuttling process is
likely to be functionally linked to the growth factor, steroid, and
cellular stress signal transduction pathways that target sgk. Many
proteins that are imported into the nucleus have an identifiable NLS
defined by a dense cluster of basic amino acids (62, 67, 68). Sgk does
not contain a complete NLS, but there is a short stretch of basic
residues between amino acid residues 29 and 32 (KQRR) that has some
NLS-like qualities, and linkage of a canonical NLS at the C terminus of
Sgk (forming NLS-Sgk) was necessary to force this protein to reside
exclusively in the nucleus in serum-treated cells. Similar to Sgk,
several of the Ser/Thr protein kinases that translocate into the
nucleus, such as ERK, p90RSK, and protein kinase A, also do
not contain a known NLS and can reside in the nucleus in a
stimulus-regulated manner (32-34, 38, 56). The nuclear-cytoplasmic
shuttling of this class of protein kinases is likely driven by their
association with or tethering to other proteins that are directly
recognized by the nuclear import/export machinery. Specific anchor
proteins have been shown to localize their tethered partners, which
include certain kinases (69, 70), to specific subcellular compartments
or serve to target enzymes to specific substrates (62, 71). Recently, a
novel anchoring protein, Jip-1, has been identified that retains Jun
N-terminal kinase in the cytoplasm and thereby prevents access of Jun
N-terminal kinase to its target transcription factors ATF2 and c-Jun in
the nucleus (72). Sgk localization could potentially be regulated by a
multivalent adaptor protein, such as Ste5 (73) and AKAP79 (74), which
serves as a platform for the simultaneous association and ordered
activation of kinases, phosphatases, and other components of a specific
signaling unit, to coordinate the compartment-specific response to a
biological stimulus. Some of these adaptor proteins harbor SH3 domains
and bind to a PXXP motif in their target proteins. Sgk
contains a proline-rich region (30% proline in a 33-amino acid
stretch) in its N-terminal regulatory domain that includes three such
PXXP motifs which could be involved in protein-protein
interactions with SH3-containing proteins and thereby potentially
regulate its nuclear-cytoplasmic shuttling.
The results from this study, and our previous reports (22, 23, 51, 52,
75), have shown that the transcriptional and post-translational control
of Sgk expression and localization represent unique points of
cross-talk that couple glucocorticoid receptor and growth factor
receptor signaling pathways. This multilevel regulation permits several
layers of control that can simultaneously or individually converge on
the same cellular component in response to appropriate extracellular
signals. Although glucocorticoids or serum can each induce
transcription of the sgk gene, the Sgk protein is
selectively localized to the cytoplasmic compartment in
glucocorticoid-treated cells that undergo a G1 cell cycle
arrest and shuttles between the nucleus and the cytoplasmic compartment in proliferating cells. Thus, Sgk could maintain the cells in a
growth-suppressed state while localized to the cytoplasm and facilitate
proliferative processes as a result of its nuclear-cytoplasmic shuttling during progression through the cell cycle. We would predict
that in various tissues that express high levels of sgk and that
undergo hormone-regulated changes in cell proliferation, such as the
mammary gland and the ovary (22, 23, 75), sgk would be exclusively
localized to the cytoplasm in growth-arrested cells. We are currently
attempting to uncover the regulatory molecules acting on Sgk, the
mechanism of nuclear-cytoplasm transport, and the compartment-specific
regulators and substrates of Sgk that allow this protein kinase to be
involved in both proliferative and anti-proliferative signal
transduction pathways in normal and transformed cells.
INTRODUCTION
Top
Abstract
Introduction
References
(21) which acts through
epidermal growth factor receptor-mediated phosphorylation cascades
(50). Subtractive cloning of glucocorticoid-responsive genes from a
Con8 mammary tumor cell cDNA library was used to identify genes
involved in the glucocorticoid-mediated growth suppression response. We
isolated a novel serum and
glucocorticoid-inducible serine/threonine protein
kinase gene,
sgk,1 which is
transcriptionally regulated by glucocorticoids and serum (22, 23). The
sgk promoter contains a functional glucocorticoid response
element that accounts for its glucocorticoid inducibility (51) and is a
transcriptional target of the p53 tumor suppressor protein (52). The
existence of this novel transcriptionally regulated protein kinase
suggests a new pathway of cross-talk by which glucocorticoid
receptor-mediated and phosphorylation-dependent cell signaling
can be coordinated and therefore implicates Sgk as a potential target
in the control of cell proliferation.
(48%), and the mouse protein kinase A
(45%) (22). Several members of these protein kinase gene families that
are homologous to sgk are involved in cell signaling events
that are associated with the control of cell growth and differentiation
(3, 5, 15, 34, 53). The phosphorylation, activity, and in some cases
the cellular location of these protein kinases can be regulated in
response to specific extracellular stimuli. Thus, even though
sgk gene expression is under an acute transcriptional
control by several distinct pathways, the cellular utilization of Sgk
protein may also be highly regulated under conditions in which the
mammary tumor cells are either growth-arrested by glucocorticoids or
actively proliferating in the presence of serum. In this study, we
demonstrate that the nuclear versus cytoplasmic localization
of Sgk is under stringent hormonal and cell cycle control, and we also
provide evidence that the nuclear-cytoplasmic shuttling of Sgk may be
required for the ability of the mammary tumor cells to be actively
progressing through the cell cycle.
EXPERIMENTAL PROCEDURES
-D-galactopyranoside-induced Sgk
expression in HMS 174. For this purpose, an NdeI fragment containing the full-length and sequenced sgk open reading
frame was inserted into pET-HAX vector. The vector harbors the sequence that encodes a hemagglutinin (HA) tag. The sgk cDNA
beginning with the initial Met was inserted into the NdeI
site, and its N terminus was thus fused in frame to the HA tag.
Milligram quantities of HA-tagged Sgk were expressed in HMS174 bacteria
that had been transformed with the pET-HA-Sgk expression construct.
After induction with
isopropyl-1-thio-
-D-galactopyranoside, cells were
harvested and lysed in HEMGN (25 mM Hepes, 100 mM KCl, 12.5 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0.1% Nonidet P-40, pH 7.9)
homogenization buffer; the lysate was size-fractionated by SDS-PAGE,
and the major Sgk band at 50 kDa was excised. The SDS-PAGE gel slice
was then quick-frozen and homogenized. The gel particles were mixed with Freund's adjuvant, filtered, and inoculated into female New Zealand White rabbits. Serum was extracted from the immunized rabbits
at standard intervals to monitor antibody titer (Babco, Richmond, CA).
The final titer of the unfractionated serum was high enough to detect
sgk at a 1:10,000 dilution on a Western blot of whole cell lysate from
glucocorticoid-treated Con.hd6 cells not overexpressing Sgk. For the
purification of anti-Sgk antibodies by affinity column chromatography,
bacterially expressed His-tagged Sgk was coupled to a nickel column
(Ni2+-nitrilotriacetic acid-agarose) (Qiagen, Chatsworth,
CA). The anti-Sgk serum was repeatedly applied to the column, and the
specifically bound anti-Sgk antibodies were eluted with 50 mM glycine, 0.15 M NaCl, pH 2.3 into 1 M Tris, pH 9.0, for neutralization and dialyzed against
HEMGN. The purified antibodies were tested by Western blot analysis and
shown to recognize multiple forms of sgk that migrated between 50 and
52 kDa.
RESULTS
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Fig. 1.
Effects of serum and dexamethasone on the
subcellular localization of Sgk. Con8.hd6 mammary tumor cells were
treated with the indicated combinations of 10% serum and/or 1 µM dexamethasone (Dex) for 3 h. The
subcellular distribution of Sgk was examined by indirect
immunofluorescence microscopy using affinity purified rabbit polyclonal
antibodies to Sgk as the primary antibodies. The secondary antibodies
were fluorescein isothiocyanate-conjugated goat anti-rabbit.
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Fig. 2.
Quantitation of hormone-regulated
relocalization of Sgk. Con8.hd6 mammary tumor cells were treated
with combinations of 10% serum and/or 1 µM dexamethasone
(Dex) for the indicated periods. The subcellular
distribution of Sgk was examined by indirect immunofluorescence
microscopy using affinity purified rabbit polyclonal antibodies to Sgk
as described in the text. The number of individual cells with nuclear,
cytoplasmic/perinuclear, or homogeneous staining of Sgk was quantitated
and expressed as the percentage of total immunostaining cells. The
error bars indicate the standard deviations.
instead of serum (data not shown),
indicating that the serum response is mediated by growth factor
signaling pathways. By 8 h in 10% serum, the overall intensity of
the Sgk signal was diminished, indicating that Sgk is either
synthesized at a lower rate or degraded more rapidly. Simultaneous
treatment with both serum and dexamethasone resulted in an additive
effect of both extracellular signals in that multiple forms of Sgk
protein are rapidly and transiently induced with only the 50-kDa Sgk
protein remaining as a stable species at the later time points (Fig. 3,
bottom right of A). Consistent with the transcriptional
effects of serum and glucocorticoids (22), virtually no Sgk was
detected in cells maintained in low serum without dexamethasone (Fig.
3, top left of A).
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Fig. 3.
Effects of glucocorticoids and serum on
protein expression and phosphorylation of Sgk. A,
Con8.hd6 mammary tumor cells were first incubated for 48 h in low
(0.5%) serum and then treated with the indicated combinations of 0.5%
serum, 10% serum, and/or 1 µM dexamethasone
(Dex). At the indicated times, cells were harvested, and
Western blots of electrophoretically fractionated total cell extracts
were probed for the expression of Sgk. B, left
gel, serum-deprived Con8.hd6 cells plated at low confluency were
treated with the indicated combinations of 10% serum and/or 1 µM dexamethasone for 5 h. During the last hour of
treatment, cells were radiolabeled with
[32P]orthophosphate, and cell extracts were
immunoprecipitated with affinity purified polyclonal antibodies to Sgk.
The immunoprecipitated material was electrophoretically fractionated
and 32P-Sgk protein visualized by autoradiography. B,
right gel, cells were transiently transfected with an expression
vector encoding His-tagged Sgk. After 24 h culture in 10% serum,
His-Sgk was affinity purified on nickel beads, treated with or without
potato acid phosphatase (PAP), and the electrophoretic
migration of the phosphatase-digested and undigested proteins were
determined by Western blots.
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Fig. 4.
Co-staining of serum- or
glucocorticoid-treated cells for Sgk localization and incorporation of
BrdUrd. Serum-deprived Con8.hd6 mammary tumor cells were treated
with 10% serum in the absence or presence of 1 µM
dexamethasone (Dex) for 24 h. The subcellular
distribution of Sgk was examined by indirect immunofluorescence
microscopy using affinity purified rabbit polyclonal antibodies to Sgk
as described in the text (middle panels). The incorporation
of BrdUrd (BrdU) was examined in the same cells by indirect
immunofluorescence microscopy using anti-BrdUrd primary antibodies and
rhodamine-conjugated secondary antibodies (lower panels). A
phase image of the cells is shown in the top panels.
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Fig. 5.
Quantitation of Sgk localization in cells
that incorporate BrdUrd. Con8.hd6 mammary tumor cells were treated
with combinations of 10% serum and/or 1 µM dexamethasone
(Dex) for the indicated times. Cells were co-stained for the
incorporation of BrdUrd and for Sgk by indirect immunofluorescence
microscopy as described in Fig. 4. The total number of
BrdUrd-incorporating cells was determined and shown in the black
bar graphs. The subcellular distribution of Sgk was quantitated
only in individual BrdUrd-positive cells, and the bar graphs
display the number of cells with nuclear, cytoplasmic/perinuclear, or
homogeneous staining of Sgk under each of the culture conditions.
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Fig. 6.
Laser scanning flow cytometric analysis of
Sgk localization in an asynchronously growing population of mammary
tumor cells. A growing population of serum-treated cells was fixed
and stained for Sgk localization using affinity purified rabbit
polyclonal primary antibodies to Sgk. During the incubation with the
fluorescein isothiocyanate-conjugated secondary antibodies, the fixed
cells were also treated with propidium iodide to stain the nuclear DNA.
Individual cells within the cell population (10,000 cells per sample)
were simultaneously examined for Sgk localization and DNA content using
a laser scanning cytometer with an argon ion laser excitation at 488 nm. On an individual cell basis, the localization of Sgk was determined
and compared with the corresponding cellular DNA content. The
quantitation of cellular DNA content within the cell population is
shown in the black profile. The number of cells with cytoplasmically
localized Sgk is shown in red, and the nuclear localized Sgk
is shown in green. The lower panel represents a
magnified portion of the upper panel.
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Fig. 7.
Characterization of Sgk localization in cells
synchronously progressing through the cell cycle. Serum-deprived
Con8.hd6 mammary tumor cells were blocked at the G1/S
border by 24 h treatment with hydroxyurea. To induce synchronous
progression through the cell cycle, hydroxyurea was removed by
replacing the cell culture medium with fresh medium containing 10%
serum. At the indicated times after hydroxyurea removal, the cell cycle
phase distribution of the cells was determined by staining with
propidium iodide, and the nuclei were analyzed for DNA content by flow
cytometry with a Coulter Elite Laser. A total of 10,000 nuclei were
analyzed from each sample. The percentages of cells within the
G1, S, and G2/M phases of the cell
cycle were determined as described in the text (panels in
left column). The subcellular distribution of Sgk was
examined by indirect immunofluorescence microscopy using affinity
purified rabbit polyclonal antibodies to Sgk as described in the text
(panels in center column). Co-staining for the
incorporation of BrdUrd was accomplished in the same cells by indirect
immunofluorescence microscopy using anti-BrdUrd primary antibodies and
rhodamine-conjugated secondary antibodies (panels in
right column).
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Fig. 8.
Ectopic expression and growth states of wild
type Sgk and Sgk constructed with a nuclear localization signal.
Serum-treated Con8.hd6 mammary tumor cells were transiently transfected
with CMV-driven expression vectors containing either the wild type
sgk gene (WT-sgk) or an sgk gene
containing the SV40 large T antigen nuclear localization signal in its
C terminus (NLS-sgk). Twenty-four hours after transfection,
the cells were co-stained for Sgk localization and for the
incorporation of BrdUrd (BrdU) by indirect
immunofluorescence microscopy as described in the text.
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Fig. 9.
Cell colony formation assay in mammary tumor
cells ectopically expressing wild type or nuclear localized forms of
Sgk. Con8.hd6 mammary tumor cells were transfected with CMV-driven
expression vectors containing either the wild type sgk gene
(WT-sgk), an sgk gene containing the SV40 large T
antigen nuclear localization signal in its C terminus
(NLS-Sgk), or with pcDNA3 vector (V) control
DNA. Each expression also encodes the neomycin resistance gene driven
by a constitutive promoter. Twenty hours after transfection,
105 cells were re-plated on 100-mm tissue culture dishes
and were cultured for 1 week in medium containing 750 µg/ml G418.
Cells were then washed and stained with formalin/crystal violet to
visualize the cell colonies. The number of foci observed from five
independent experiments after transfection of each expression vector or
vector control were counted.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We express our appreciation to Lisa M. Bell and Anita C. Maiyar for their critical evaluation of this manuscript and helpful experimental suggestions. We also thank Hei-Sook Sul and Jeremy Thorner for their helpful critique of P. B.'s Ph.D. thesis, portions of which are included in this manuscript. We thank Wei-Ming Kao, Huy Nguyen, and Minnie Wu for their technical assistance. We are also grateful to Jerry Kapler for excellent photography, Isabel Reichert for competent assistant with the photographic imaging, and to Anna Fung and Tran Van for their help in the preparation of this manuscript.
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FOOTNOTES |
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* This work was supported by Public Health Service Grant CA-71514 (to G. L. F.) from the National Cancer Institute.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.
§ Submitted portions of this work to fulfill the requirements for a doctorate of philosophy at the University of California, Berkeley.
** To whom correspondence and reprint requests should be addressed: Dept. of Molecular and Cell Biology, 591 LSA, University of California, Berkeley, CA 94720-3200. Tel.: 510-642-8319; Fax: 510-643-6791; E-mail: glfire{at}uclink4.berkeley.edu.
2 P. Buse and G. L. Firestone, unpublished results.
3 L. M. Bell and G. L. Firestone, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: sgk, serum- and glucocorticoid-inducible protein kinase; DMEM, Dulbecco's modified Eagle's medium; BrdUrd, 5-bromo-2'-deoxyuridine; NLS, nuclear localization signal; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; CMV, cytomegalovirus; WT, wild type.
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REFERENCES |
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