From the Dana-Farber Cancer Institute and the Department of
Pathology, Harvard Medical School, Boston, Massachusetts 02115
Growth factor-dependent survival of a
variety of mammalian cells is dependent on the activation of
phosphatidylinositol (PI) 3-kinase and its downstream effector, the
protein kinase Akt. Glycogen synthase kinase-3 (GSK-3) has been
previously identified as a physiological target of Akt, which is
inhibited by phosphorylation, so we have investigated the role of GSK-3
in cell survival. Overexpression of catalytically active GSK-3 induced
apoptosis of both Rat-1 and PC12 cells, whereas dominant-negative GSK-3
prevented apoptosis following inhibition of PI 3-kinase. GSK-3 thus
plays a critical role in regulation of apoptosis and represents a key
downstream target of the PI 3-kinase/Akt survival signaling
pathway.
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INTRODUCTION |
Although many types of mammalian cells are dependent upon growth
factors for survival (1), the intracellular signaling pathways that
control cell survival by preventing apoptosis have only begun to be
elucidated. A role for PI1
3-kinase in the regulation of cell survival was first indicated by
experiments showing that PI 3-kinase was required to prevent apoptosis
of PC12 rat pheochromocytoma cells maintained in nerve growth factor
(NGF) (2). These findings have been extended by observations
demonstrating that PI 3-kinase is required for survival of several
other growth factor-dependent cell types, including
fibroblasts, epithelial cells, hematopoietic cells, and primary neurons
(3-12). In addition, the protein kinase Akt has been identified as a
key effector of PI 3-kinase in signaling cell survival (5, 13, 14).
The principal characterized physiological substrate of Akt is glycogen
synthase kinase-3 (GSK-3) (15), which was initially identified as an
enzyme that regulates glycogen synthesis in response to insulin (16).
GSK-3 is a ubiquitously expressed protein-serine/threonine kinase whose
activity is inhibited by Akt phosphorylation in response to growth
factor stimulation. In addition to glycogen synthase, GSK-3
phosphorylates a broad range of substrates, including several transcription factors and translation initiation factor eIF2B (16).
GSK-3 has also been implicated in the regulation of cell fate in
Dictyostelium (17) and is a component of the Wnt signaling pathway required for Drosophila and Xenopus
development (18-21). These studies suggest that GSK-3 is involved in
multiple cellular processes, including metabolism, proliferation, and
differentiation. Here we show that GSK-3 is also involved in the
regulation of apoptosis, identifying it as a critical downstream
element of the PI 3-kinase/Akt cell survival pathway.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
PC12 cells were grown in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and
5% horse serum. Rat-1 cells were grown in DMEM supplemented with 10%
calf serum.
Assay of GSK-3--
PC12 cells were plated in 100-mm culture
dishes (3 × 105 cells/plate) in DMEM containing 10%
fetal bovine serum and 5% horse serum. On the next day the medium was
changed to DMEM containing 0.5% horse serum. One day later, cells were
stimulated by addition of 100 ng/ml of NGF (Life Technologies, Inc.)
for 5, 15, or 30 min with or without preincubation with 50 µM LY294002 (Biomol) or 100 nM wortmannin
(Sigma). Cells were washed with phosphate-buffered saline and lysed in
extraction buffer (100 mM Tris-HCl, pH 7.4, 100 mM KCl, 2 mM EDTA, 0.1% Triton X-100, 1 mM benzamidine, 0.1 mM
Na3VO4, 1 mg/ml glycogen, 10 µg/ml pepstatin,
10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 nM okadaic
acid) for 20 min at 4 °C with constant rotation. The cell extracts
were passed through a 27-gauge needle and then centrifuged at 14,000 rpm in an Eppendorf microcentrifuge for 30 min at 4 °C. Protein
concentrations were determined, and 10 µg of protein were diluted to
150 µl by addition of immunprecipitation buffer (50 mM
sodium glycerophosphate, pH 7.3, 1 mM EGTA, 1 mM benzamidine, 1 mM dithiothreitol, 0.1 mM Na3VO4, 1 µg/ml pepstatin, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 100 nM okadaic
acid) and incubated with 0.375 µg of GSK-3
antibody (Transduction
Laboratories) for 2 h at 4 °C with rotation. 5 µg of rabbit
anti-mouse IgG (Upstate Biotechnology Inc.) was added for 30 min.
Protein A-Sepharose (60 µl of a 30% suspension) was then added, and
the incubation was continued for 1 h at 4 °C with rotation.
Immune complexes were recovered by centrifugation at 4 °C and washed
once with extraction buffer and twice with immunoprecipitation buffer.
Kinase activity of the immunoprecipitated GSK-3 was assayed in a total volume of 20 µl containing 250 mM sodium
glycerophosphate, pH 7.4, 1 M NaCl, 100 mM
MgCl2, 5 mM EGTA, 5 mM benzamidine,
5 mM dithiothreitol, 0.5 mM
Na3VO4, 100 nM okadaic acid, 20 µM phosphoglycogen synthase peptide-2 (Upstate
Biotechnology), and 50 µM [
-32P]ATP (1 µCi). After 10 min of incubation at 30 °C, reaction mixtures were
centrifuged for 1 min, and 20 µl of the supernatant was spotted onto
Whatman P81 phosphocellulose paper. Filters were washed in four changes
of 175 mM phosphoric acid for a total of 20 min, rinsed in
acetone, dried, and counted in a liquid scintillation counter.
Transient Transfection--
3 × 105 cells were
plated on poly-L-lysine treated coverslips in 35-mm plates
24 h before transfection. Transient transfections were performed
with 1 µg of expression vectors using the LipofectAMINE Reagent (Life
Technologies, Inc.) according to the manufacturer's instructions.
Wild-type rat GSK-3 (pGSK3
) and catalytically inactive GSK-3
(pR85GSK3
) (21) are transcribed from the EF1
promoter (22);
wild-type p53 (pWTp53) and dominant-negative p53 (p143p53) are
transcribed from the cytomegalovirus promoter (23); Bcl-2 (pSG5-bcl-2)
and Bcl-xL (pSG5-Bcl-xL) are transcribed from
the early SV40 promoter (provided by C. Nalin, Sandoz Pharmaceuticals); dominant-negative PI 3-kinase (p
p85) (24) is transcribed from the
SR
promoter (provided by C. Rudd, Dana-Farber Cancer Institute). Cells were cotransfected with 1 µg of a green fluorescent protein (GFP) expression construct (pEGFP-C1) (CLONTECH).
After 2 days cells were fixed and nuclei were stained with the DNA dye
bisbenzimide (Hoechst 33258). Transfected cells were identified by GFP
fluorescence and scored for apoptosis by nuclear morphology.
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RESULTS AND DISCUSSION |
Inhibition of GSK-3 Activity Is Mediated by the PI 3-Kinase/Akt
Signaling Pathway during Cell Survival--
Previous studies have
demonstrated that GSK-3 is inhibited as a result of growth factor
stimulation and activation of the PI 3-kinase/Akt signaling pathway in
several cell types (15, 25-27). We therefore sought to determine
whether stimulation of cells with growth factors known to signal cell
survival by suppression of apoptosis similarly resulted in inhibition
of GSK-3 activity. Treatment of PC12 cells with NGF, which signals cell
survival via the PI 3-kinase pathway (2), resulted in a 30-40%
inhibition of GSK-3 activity (Fig. 1),
which is similar to that reported in other cell systems (15, 25-27).
This inhibition of GSK-3 was prevented by preincubation with the PI
3-kinase inhibitors LY294002 (28) and wortmannin (29, 30) (Fig. 1),
indicating that NGF inhibition of GSK-3 was mediated by the PI 3-kinase
pathway.

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Fig. 1.
Inhibition of GSK-3 is mediated by PI
3-kinase in response to NGF. PC12 cells were incubated for 24 h in DMEM containing 0.5% horse serum and stimulated with NGF (100 ng/ml) for 5-30 min. Where indicated, cells were preincubated with the
PI 3-kinase inhibitors 50 µM LY294002 (LY) or
100 nM wortmannin (WT). Following NGF treatment,
cells were harvested and assayed for GSK-3 activity in
immunoprecipitation kinase assays as described under "Experimental
Procedures." Data are presented as percentages of untreated control
cultures. Standard deviations are indicated.
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Induction of Apoptosis by Overexpression of GSK-3--
Because
GSK-3 was inhibited by PI 3-kinase signaling in NGF-stimulated PC12
cells, we investigated whether overexpression of active GSK-3 induced
apoptosis. Both PC12 cells and Rat-1 fibroblasts, which are also
dependent upon PI 3-kinase signaling for survival (3), were used as
recipients in transient transfection assays. Cells were
transfected with a construct expressing GFP and cotransfected with
plasmids expressing either wild-type GSK-3
or catalytically inactive
R85 GSK-3
cDNAs (21) from the EF1
promoter (22). Transfected
cells were identified by fluorescence microscopy to detect GFP
expression, and apoptotic cells were scored by nuclear morphology after
staining with Hoechst dye.
Fluorescence micrographs of a representative experiment are shown in
Fig. 2, and data from several experiments
with both PC12 and Rat-1 cells are presented in Fig.
3. Approximately 20% of either PC12 and
Rat-1 cells were apoptotic in control cultures transfected either with
the pEF1
vector alone or with catalytically inactive R85 GSK-3. In
contrast, 60-70% of both cell types underwent apoptosis following
transfection with wild-type GSK-3, similar to the percentage of cells
that underwent apoptosis following transfection with wild-type p53.
Thus, overexpression of active GSK-3 was sufficient to induce
apoptosis.

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Fig. 2.
Effects of wild-type and catalytically
inactive GSK-3 in Rat-1 cells. Cells were transfected with the
indicated expression vectors. Transfected cells were identified by
fluorescence microscopy (panels A and C) and
scored for apoptosis by nuclear morphology (panels B and
D). Nonapoptotic nuclei are indicated with straight
arrows, and apoptotic nuclei are indicated with zigzag
arrows.
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Fig. 3.
Induction of apoptosis by overexpression of
GSK-3. Cells were transfected with the indicated expression
vectors. Data are averaged from three experiments with PC12 cells and
seven experiments with Rat-1 cells. Typically, 100-200 cells
transfected with each vector were counted per experiment. The
error bars are standard deviations.
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We next examined whether apoptosis induced by GSK-3 could be blocked by
known inhibitors of apoptotic cell death (Fig.
4). Cotransfection with a
dominant-negative mutant of p53 (V143A) (23) protected cells from
apoptosis induced by GSK-3, consistent with the involvement of p53 in
apoptosis induced by inhibition of PI 3-kinase in both PC12 and Rat-1
cells (31). Induction of apoptosis by GSK-3 was also blocked by a
peptide inhibitor of CPP32-like caspases, which are effectors of cell
death activated by diverse apoptotic stimuli (32), including PI
3-kinase inhibition (4, 31). Finally, apoptosis induced by GSK-3 was
inhibited by cotransfection with plasmids expressing either Bcl-2 or
Bcl-xL, which protect cells from apoptosis induced by a
variety of agents, including inhibitors of PI 3-kinase (4). Apoptosis
induced by GSK-3 thus required caspase activity and was modulated by
p53 and Bcl-2 family members, consistent with a role for GSK-3 as a
downstream element in the PI 3-kinase signaling pathway.

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Fig. 4.
Effects of known inhibitors of cell death on
apoptosis induced by GSK-3. Cells were transfected with the
indicated expression vectors. The peptide CPP32 inhibitor (DEVD-CHO,
100 µM) (35) was added immediately after the
transfection. Data are averaged from three experiments with both PC12
and Rat-1 cells, presented as in Fig. 3.
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Dominant-negative GSK-3 Inhibits Apoptosis Resulting from
Inhibition of PI 3-Kinase--
We next sought to determine whether
GSK-3 activity was required for apoptosis induced by inhibition of PI
3-kinase. PC12 and Rat-1 cells were transfected with the catalytically
inactive GSK-3 R85 mutant, which acts as a dominant-negative inhibitor
of GSK-3 activity (21), and then treated with the specific PI 3-kinase inhibitor LY294002 (Fig. 5). Treatment
with LY294002 induced apoptosis of approximately 60% of control cells
that had been transfected with the pEF1
vector alone. Transfection
with the R85 GSK-3 mutant significantly inhibited apoptosis induced by
LY294002 to levels of 30-40%, approaching the background level of
20% apoptosis observed in control cells that were not treated with the
PI 3-kinase inhibitor. The activity of the R85 GSK-3 mutant in
inhibiting apoptosis in these experiments was similar to that of
dominant-negative p53.

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Fig. 5.
Effect of dominant-negative GSK-3 on
apoptosis resulting from inhibition of PI 3-kinase. Cells were
transfected with the indicated expression vectors. After 48 h
cells were treated with LY294002 (50 µM) for 24 h
and scored for apoptosis 3 days after transfection. Data are averaged
from five experiments with PC12 cells and four experiments with Rat-1
cells.
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Similar results were obtained when cells were transfected with a
plasmid expressing dominant-negative PI 3-kinase (24) rather than being
treated with LY294002 (Fig. 6).
Expression of dominant-negative PI 3-kinase induced apoptosis in
approximately 60% of transfected cells, and this was significantly
inhibited by cotransfection with dominant-negative R85 GSK-3. Thus,
GSK-3 activity was necessary for apoptosis resulting from PI 3-kinase
inhibition.

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Fig. 6.
Effect of dominant-negative GSK-3 on
apoptosis induced by dominant-negative PI 3-kinase. Cells were
transfected with the indicated expression vectors. Data are averaged
from three experiments with both PC12 and Rat-1 cells.
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Taken together, these results indicate that GSK-3 is a key target of PI
3-kinase signaling leading to prevention of apoptosis. Overexpression of active GSK-3 is sufficient to induce apoptosis, whereas expression of dominant-negative GSK-3 effectively protects cells from apoptosis resulting from inhibition of PI 3-kinase. Although
other targets of the Akt protein kinase, including the Bcl-2 family
member Bad (33, 34), may also be important in the regulation of cell
survival, these findings implicate GSK-3 as a central element in the PI
3-kinase/Akt survival pathway, with phosphorylation of one or more
targets of GSK-3 presumably serving to activate apoptotic cell death.
Identification of the GSK-3 targets that regulate apoptosis thus poses
a critical next step to understanding the signaling pathways by which
growth factors control cell survival.
We are grateful to I. Dominguez for GSK-3
plasmids, to C. E. Rudd for the dominant-negative PI 3-kinase
plasmid, and to P. Erhardt for kind help and advice.