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INTRODUCTION |
Phosphorylation and dephosphorylation on tyrosine residues play
critical roles in the signal transduction pathways that regulate cell
activation, proliferation, and differentiation. Reactive oxygen species
(ROS)1 have been reported to
induce increased tyrosine phosphorylation of several proteins, such as
p77btk, p72syk,
p56/59hck, and p56lck
(1-4).
Many growth factors and cytokines promote cell survival, including
insulin-like growth factor 1 (5) and platelet-derived growth factor
(6). Phosphatidylinositide 3'-OH-kinase (PI 3-kinase) has recently been
shown to be involved in cell survival. Growth factors activate PI
3-kinase, and p85 subunit of PI 3-kinase associates with specific
phosphotyrosine either on the cytoplasmic domain of growth factor
receptors or on receptor-associated adaptor proteins. One target of PI
3-kinase is the serine-threonine kinase Akt, also named PKB
(5). Akt
is a general mediator of growth factor-induced survival and has been
shown to suppress apoptotic death by a variety of stimuli (5).
Signaling via growth factor receptor activation leads to the sequential
activation of PI 3-kinase and Akt. Recently, Datta et al.
(7) reported that Akt phosphorylates BAD in vitro and
in vivo and blocks BAD-induced death of primary neurons. In eukaryotes, Bcl-2 family are central to the regulation of cell death.
Several members of the Bcl-2 family (Bcl-2, Bcl-XL, MCl-1, and A1)
promote survival, whereas other members (Bcl-Xs, BAD, Bax, Bak) promote
cell death (8-13). Bcl-2 family proteins homo- and heterodimerize, and
the balance between homo- and heterodimers appears to be critical to
the maintenance of cell survival and cell death. The mechanisms of
Bcl-2 family members function yet remains to be determined.
In a previous study (14), we reported that hydrogen peroxide markedly
induced rapid tyrosine phosphorylation of focal adhesion kinase (FAK)
followed by the decrease of phosphorylation concomitant with apoptosis.
Further, the inhibition of tyrosine phosphorylation of FAK by
herbimycin A, a tyrosine kinase inhibitor, accelerated apoptosis, and
antisense oligonucleotides of FAK decreased cell viability. From these
studies, we proposed an anti-apoptotic role of FAK in hydrogen
peroxide-induced apoptosis. Based on these findings, we hypothesized
FAK may be an upstream signal protein in the PI 3-kinase and Akt
pathway and promotes cell survival against stresses in some cell types.
Therefore, we examined the relationships between FAK, PI 3-kinase, Akt,
Bcl-2 family proteins, and CPP32 protease (caspase-3) using a human
glioblastoma cell line, T98G.
To investigate the role of PI 3-kinase and Akt in hydrogen
peroxide-induced apoptosis, T98G cells were treated with hydrogen peroxide (1 mM) which caused tyrosine phosphorylation of
FAK and serine phosphorylation of Akt. We also found that the
association of FAK with PI 3-kinase was stimulated by hydrogen
peroxide. Interestingly, the PI 3-kinase inhibitor wortmannin
accelerated apoptosis and inhibited serine phosphorylation of Akt.
Decreases in Bcl-2 protein and increases in Bax protein and CPP32
protease activity were observed concomitantly with apoptosis. These
data suggested that the signal transduction from FAK to PI 3-kinase and
Akt exerts an anti-apoptotic effect on apoptosis induced by oxidative
stress and FAK locates in the upstream signal of the PI 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis of T98G cells.
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EXPERIMENTAL PROCEDURES |
Cells and Materials--
T98G cells were suspended in RPMI 1640 medium containing 5% fetal bovine serum (Nippon Bio-Supply Center,
Tokyo, Japan). For oxidative stress experiments, growing cells were
subcultured at a density of 2 × 105/ml cell in medium
containing 1% fetal bovine serum. Monoclonal anti-phosphotyrosine
antibody (mAb: 4G10) and rabbit anti-PI 3-kinase (p85) Ab were
purchased from Upstate Biotechnology Inc. (NY), anti-FAK mAb from
Transduction Laboratories (KY), goat anti-Bcl-2 Ab, rabbit anti-Bax Ab,
and goat anti-CPP32 Ab from Santa Cruz Biotechnology, rabbit anti-Akt
and phospho-Akt Abs from New England Biolabs. Inc. (MA), and the
horseradish peroxidase-conjugated secondary Ab from DAKO (Denmark).
OPTI-MEM, Lipofectin reagent, and prestained molecular marker were
obtained from Life Technologies, Inc. (MD). Enhanced chemiluminescence
reagents were obtained from Amersham Pharmacia Biotech (Tokyo, Japan).
Substrates for protease activity, YVAD-MCA and DEVD-MCA, were obtained
from Peptide Institute, Inc. (Osaka, Japan).
PI 3-Kinase Activity in Immunoprecipitates--
T98G cells
(5 × 106 cells) were incubated with hydrogen peroxide
at various times, washed once with ice-cold phosphate-buffered saline,
and lysed with lysis buffer as described previously (15). Insoluble
material was removed by centrifugation at 4 °C for 20 min at
10,000 × g. The supernatants were incubated with
anti-FAK mAb at 4 °C overnight. Immunocomplex was precipitated with
protein G-Sepharose (Amersham Pharmacia Biotech) and washed as
described previously (15). The immunoprecipitates were incubated with phosphatidylinositol (PtdIns) and 1 µCi [
-32P]ATP in
the reaction buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 µM ATP, 200 µg/ml
phosphatidylserine) at 25 °C for 10 min and terminated by addition
of 100 µl of 1 M HCl. Phospholipids were then extracted
with 200 µl of CHCl3/MeOH (1:1). The organic layer was
spotted onto a silica gel 60 plate (Merck, Darmstadt, Germany) pretreated with 1% potassium oxalate. Thin-layer chromatography plates
were developed with
CHCl3/MeOH/acetone/CH3COOH/H2O
(7:5:2:2:2), dried, visualized, and analyzed by Fuji image analyzer
(Tokyo, Japan).
Electrophoresis and Immunoblotting--
For the preparation of
cell lysate, 1 × 106 packed cells were lysed with
lysis buffer as described previously (16). After centrifugation,
Laemmli sample buffer was added to the cell lysate. Samples were boiled
for 5 min, and equal amounts of protein were separated by
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes. The membranes were blocked in 3% bovine
serum albumin in phosphate-buffered saline for 1 h and then
incubated with primary Ab for 1 h at room temperature. After incubation with secondary Ab coupled to horseradish peroxidase, detection was made using the enhanced chemiluminescence system (Amersham Pharmacia Biotech). Molecular sizes were determined by the
relative mobilities of prestained molecular weight markers. Densitometric analysis was performed on a Macintosh computer using the
public domain NIH Image program.
Analysis of DNA Fragmentation--
DNA fragmentation study was
performed as described elsewhere (14). In brief, cells were gently
lysed for 30 min at 4 °C in a buffer containing 5 mM
Tris buffer (pH 7.4), 20 mM EDTA, and 0.5% Triton X-100.
After centrifugation at 15,000 rpm for 15 min, supernatants containing
soluble fragmented DNA were collected and treated with RNase (20 µg/ml), followed by proteinase K (20 µg/ml). DNA fragments were
precipitated in ethanol. Each sample was then electrophoresed on a 2%
agarose gel and visualized by staining with 0.1% ethidium bromide.
Quantitation of Apoptosis--
Cell viability was determined by
Trypan blue dye exclusion, and the existence of apoptotic cells was
confirmed by the appearance of sub-G0/G1 peak
fractions in cell cycle analysis. For the cell cycle analysis,
ethanol-fixed cells were stained with propidium iodide (50 µg/ml) in
the presence of RNase A (100 µg/ml, Wako Pure Chemical, Osaka, Japan)
and then analyzed by fluorescence-activated cell sorter Calibur using a
CELLQuest program (Becton Dickinson, CA).
ICE (-like) and CPP32 (-like) Protease Activity--
After
washing with phosphate-buffered saline, cell lysate was prepared as
described by Nicholson et al. (17). Cell lysate (50 µg of
protein) was incubated at 37 °C with 50 mM DEVD-MCA as a
substrate for apopain/CPP32, for 30 min, or YVAD-MCA as a substrate for
ICE, for 60 min. The amounts of released 7-amino-4-methylcoumarin were
measured with fluorescence spectrofluorometer (Hitachi F-4000, Tokyo,
Japan), with excitation at 380 nm and emission at 460 nm.
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RESULTS |
Tyrosine Phosphorylation of FAK and Serine Phosphorylation of Akt
and the Association with PI 3-Kinase in Hydrogen Peroxide-treated T98G
Cells--
We have previously described that hydrogen peroxide induced
significant tyrosine phosphorylation of p125FAK in a human glioblastoma cell line, T98G (14). To investigate the signal transduction from FAK,
we examined whether FAK associates with PI 3-kinase, which has been
reported to associate with FAK by stimulation of platelet-derived
growth factor in NIH3T3 mouse fibroblast (18). T98G cells were treated
with or without 1 mM hydrogen peroxide for various times.
Lysates were prepared from these cells and immunoprecipitated by
anti-FAK mAb, followed by the detection of its associated PI 3-kinase
activities, as well as the p85 subunit of PI 3-kinase, and the FAK
tyrosine phosphorylation. In response to hydrogen peroxide stimulation,
a significant increase of PI 3-kinase activity (i.e.
increases of 3-fold at 1 h and 10-fold at 2-4 h) was found in the
anti-FAK immunoprecipitates (Fig.
1A). The product was confirmed
as PtdIns 3-phosphate (PtdIns 3-P) by a comparison with the product of
kinase assay using the immunoprecipitates with anti-PI 3-kinase Ab. The
formation of PtdIns 3-P was completely inhibited (> 95% inhibition)
by the 0.5 µM wortmannin (data not shown). The PI
3-kinase activity gradually increased and reached maximal at 2 h.
Western blotting of anti-FAK immunoprecipitates with anti-p85 Ab
paralleled with this observation (Fig. 1B). Simultaneously, tyrosine phosphorylation of the anti-FAK immunoprecipitates were determined by using anti-phosphotyrosine mAb, indicating that FAK
phosphorylation increased significantly at 1-2 h and maintained constant till 4 h (Fig. 1C). Thus, the tyrosine
phosphorylation of FAK preceded the PI 3-kinase association with FAK in
response to hydrogen peroxide stimulation. Of note is that blotting
with anti-FAK mAb revealed the same amounts of FAK precipitated from all five samples (Fig. 1D). These results suggested that PI
3-kinase associates clearly with tyrosine-phosphorylated FAK. It was
recently reported that insulin-like growth factor 1 promotes cell
survival by activating PI 3-kinase and its down-stream target, the
serine-threonine kinase Akt (5). We therefore examined the effect of
hydrogen peroxide on serine phosphorylation of Akt using
anti-phospho-Akt Ab. The cell lysates were subjected to immunoblotting
with anti-phospho-Akt or anti-Akt Abs. When lysates of hydrogen
peroxide-treated cells were electrophoresed and immunoblotted with
anti-Akt Ab, the corresponding band of Akt was consistently detected up
to 4 h after stimulation with hydrogen peroxide (Fig.
2). Immunoblotting using anti-phospho-Akt Ab, which recognizes phosphorylated Ser-473 of Akt, revealed
phosphorylation of Akt at 1 h after stimulation with hydrogen
peroxide, which increased markedly till 4 h. Pretreatment with
wortmannin (0.5 µM), a specific PI 3-kinase inhibitor for
1 h, completely inhibited Akt phosphorylation. These findings
suggested that FAK-associated PI 3-kinase activity is prerequisite for
Akt activation.

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Fig. 1.
Induction of FAK-PI 3-kinase association by
hydrogen peroxide in T98G cells. T98G cells were treated with
hydrogen peroxide (1 mM) for various times as indicated.
Lysates (200 µg of protein) were prepared and immunoprecipitated by
anti-FAK mAb, and the associated PI 3-kinase was assayed by thin-layer
chromatography (A) as described under "Experimental
Procedures." The anti-FAK immunoprecipitates were also analyzed by
Western blotting with anti-PI 3-kinase (p85) Ab (B),
anti-phosphotyrosine mAb (C), or anti-FAK mAb
(D). Results were representative of two independent
experiments.
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Fig. 2.
Time course of Akt phosphorylation. T98G
cells were treated with hydrogen peroxide (1 mM) with or
without wortmannin (Wo) (0.5 µM) for various
periods. Lysates were prepared and analyzed by Western blotting using
anti-phospho-Akt Ab or anti-Akt Ab.
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Wortmannin Accelerates Hydrogen Peroxide-induced Apoptosis in T98G
Cells--
We demonstrated previously that 1 mM hydrogen
peroxide treatment for 15 h induced apoptosis in T98G cells (14).
When T98G cells were treated with 1 mM hydrogen peroxide
for various periods, less than 12% of the cells died within 4 h
by the estimation with trypan blue dye exclusion (Fig.
3). To investigate the role of PI
3-kinase in apoptosis, we examined the effects of wortmannin. When T98G
cells were pretreated with 0.5 µM wortmannin for 1 h, followed by treatment with 1 mM hydrogen peroxide, more
than 34% of the cells died within 4 h. Simultaneously, we
estimated the DNA content of the cells using flow cytometry after
staining with PI. DNA histogram of the PI-stained cells in Fig.
4 indicated that 10% of hydrogen
peroxide-treated cells (4 h) had hypodiploid DNA (Fig. 4A),
indicative of apoptosis, whereas untreated control cells contained less
than 4% in this area. In the presence of wortmannin, hydrogen peroxide
treatment for 4 h induced 40% of the cells with a hypodiploid DNA
pattern, indicating that wortmannin enhanced hydrogen peroxide-induced
apoptosis significantly (Fig. 4B). Wortmannin alone did not
induce apoptosis during these incubation periods (data not shown). In
addition, when DNA fragmentation was analyzed, marked DNA ladder was
observed after pretreatment with wortmannin for 1 h followed by
hydrogen peroxide for 4 h (Fig.
5B), suggesting that these
cells were apoptotic. No significant DNA fragmentation was induced by
treatment with wortmannin alone (data not shown).

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Fig. 3.
Time course of the viability of T98G cells
after treatment with hydrogen peroxide. Cells were treated with 1 mM hydrogen peroxide, hydrogen peroxide and wortmannin
(Wo) (0.5 µM), or wortmannin alone (0.5 µM). Results are shown as mean ± S.D. from three
independent experiments. Cont, control.
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Fig. 4.
Flow cytometric analysis of T98G cells.
PI-stained DNA histograms of drug-treated cells (black)
compared with that of untreated cells (white) are shown, and
Apo indicates the cells with hypodiploid DNA. A,
cells were treated with hydrogen peroxide (1 mM) for 4 h. B, cells were treated with hydrogen peroxide (1 mM) and wortmannin (0.5 µM) for 4 h.
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Fig. 5.
DNA fragmentation assay. A,
cells were treated with hydrogen peroxide (1 mM) for
indicated periods. DNA was extracted and analyzed as described under
"Experimental Procedures." The molecular size markers are indicated
on the right lane (M). B, wortmannin
accelerated apoptosis induced by hydrogen peroxide. Cells were treated
with wortmannin for 1 h before hydrogen peroxide exposure and then
treated with hydrogen peroxide for indicated periods.
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Hydrogen Peroxide in the Presence of Wortmannin Down-regulates the
Amount of Bcl-2 Protein and Up-regulates the Amount of Bax
Protein--
We next examined whether hydrogen peroxide-induced
apoptosis is modulated by Bcl-2 family proteins. Western blotting
indicated the amounts of Bcl-2 and Bax in hydrogen peroxide-treated
T98G cells were constant until 4 h after stimulation with hydrogen peroxide (Fig. 6). In the presence of
wortmannin, however, Bcl-2 gradually decreased to one-tenth at
4 h, whereas Bax showed a 15-fold increase at 4 h. Thus,
there appears to be an inverse correlation between expression of Bcl-2
and Bax in hydrogen peroxide- and wortmannin-induced apoptosis of T98G
cells. The above data confirmed that Bcl-2 is down-regulated, whereas
apoptosis-inducing Bax protein is up-regulated during apoptosis
in some cell types (19, 20).

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Fig. 6.
Expression of Bcl-2 family (Bcl-2 and Bax)
proteins in the hydrogen peroxide-treated cells with or without
wortmannin. Cells were treated with wortmannin for 1 h and
then followed by hydrogen peroxide treatment for indicated periods,
lysed, and analyzed by Western blotting using anti-Bcl-2 Ab or anti-Bax
Ab. Proteins (40 µg) were separated on 12% SDS-polyacrylamide gel
electrophoresis and analyzed as described under "Experimental
Procedures." Densitometric analysis revealed that the Bcl-2 band
reduced to one-fifth (3 h) and to one-tenth (4 h), whereas Bax
increased up to 15-fold at 4 h, compared with time 0.
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Hydrogen Peroxide in the Presence of Wortmannin Activates CPP32
Protease--
ICE family proteases have been reported to be activated
in apoptosis (21). To examine the possible involvement of ICE family proteases in hydrogen peroxide-induced apoptosis, we measured the
activity of ICE protease and CPP32 protease using peptide substrates in
cells treated with hydrogen peroxide, hydrogen peroxide and wortmannin,
or wortmannin alone. Although ICE protease activity was not elevated
(data not shown), CPP32 protease activity was significantly elevated in
T98G cells when treated with wortmannin followed by hydrogen peroxide
treatment for 4 h (Fig.
7A). CPP32 protease activity
was not elevated in T98G cells treated with either hydrogen peroxide or
wortmannin alone. Because it is known that CPP32 protease is
synthesized as a 32-kDa inactive precursor which is proteolytically
cleaved to produce a mature enzyme with 17- and 12-kDa subunits, we
examined the cleavage of the CPP32 protein in response to apoptosis. As
shown in Fig. 7B, a 32-kDa CPP32 protein band disappeared
upon the induction of apoptosis by the treatment with wortmannin and
hydrogen peroxide. These results indicated that wortmannin and hydrogen
peroxide treatment induced CPP32 cleavage to generate an active CPP32
fragment. Hydrogen peroxide alone had no significant effect on CPP32
protein levels (Fig. 7B).

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Fig. 7.
Activation of CPP32 protease by hydrogen
peroxide and wortmannin. A, increases in CPP32 protease
activity in cells treated with hydrogen peroxide and wortmannin.
Results were shown as means ± S.D. of values obtained from three
independent experiments. B, Western blot analysis of
pro-CPP32 protein. Cells were treated with hydrogen peroxide in the
presence or absence of wortmannin. Lysed proteins (40 µg) were
analyzed by Western blotting using anti-CPP32 Ab.
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DISCUSSION |
We have reached the following conclusions in this paper. 1)
Hydrogen peroxide stimulated the association of FAK with PI 3-kinase. 2) Wortmannin accelerated hydrogen peroxide-induced apoptosis in T98G
cells. 3) Hydrogen peroxide stimulated the phosphorylation of Akt. 4)
When apoptosis occurred, CPP32 protease was activated, concomitant with
the decrease of Bcl-2 protein and increase of Bax protein. 5)
Phosphorylation of Akt is inhibited by wortmannin. Recently, we
reported the anti-apoptotic role of FAK in hydrogen peroxide-induced
apoptosis (14). In this study, we demonstrated that tyrosine
phosphorylation of FAK, the association of FAK with PI 3-kinase, as
well as serine phosphorylation of Akt occur in T98G cells after
exposure to hydrogen peroxide. It should be mentioned that in the
presence of wortmannin, PI 3-kinase activity and serine phosphorylation
of Akt were inhibited with accelerating apoptosis. Putative
downstream effectors of PI 3-kinase are the ribosomal protein kinase
p70S6K, the Rho family Rac, and the
serine/threonine protein kinase Akt/PKB. Akt/PKB, which is a cellular
homolog of the retroviral oncogene v-akt, is also homologous
to the PKA and PKC families of protein kinases. Akt is involved in the
promotion of cell survival through inhibition of apoptosis, possibly
playing a role in PI 3-kinase-mediated neuronal cell survival (5). As
to a mode of survival signaling from Akt to BAD, Datta et
al. (7) proposed that in the case of insulin-like growth factor 1 stimulation, Akt is activated via PI 3-kinase and activated Akt
phosphorylated BAD, which dissociates from Bcl-XL or Bcl-2. Then,
phosphorylated BAD is sequestered in the cytosol bound to 14-3-3 (22).
As a result, Bcl-2 homodimer or Bcl-2-Bcl-XL heterodimer was formed, thereby leading to cell survival.
The above idea appears to be consistent with our observation on the
oxidative stress-induced apoptosis as shown in this study. In T98G
cells, after stimulation with hydrogen peroxide, FAK was tyrosine-phosphorylated followed by the association and activation of
PI 3-kinase. Activation of PI 3-kinase leads to the activation of Akt.
Although we could not determine whether the target of Akt in the
oxidative stress is BAD or not, Akt might regulate the balance of Bcl-2
family by phosphorylation of apoptosis-related proteins. In this study,
the presence of Bcl-2 led to survival, whereas the increase of Bax, a
BAD homolog, led to apoptosis. Although the mechanism of the function
of Bcl-2 and Bax in apoptosis remains to be determined, Aritomi
et al. (23) performed crystallographic studies indicating
that Bax possesses a greater potential for membrane insertion than
either Bcl-2 or Bcl-XL, and thus Bax is likely to form membrane pores.
They proposed that the roles of Bcl-XL and Bcl-2 are to inhibit pore
formation of Bax or other pore-forming proteins through
heterodimerization. Taken together, we speculated that in signal
transduction from FAK to PI 3-kinase, Akt plays a survival role by
regulating the balance of apoptosis-blocking protein (Bcl-2) and
apoptosis-inducing factor (Bax).
In this study, Akt was translocated to the plasma membrane after
stimulation with hydrogen peroxide, and this translocation was
sensitive to wortmannin (data not shown). We propose the following model for FAK, PI 3-kinase, Akt, in the apoptosis. After exposure to
hydrogen peroxide, FAK is activated by tyrosine phosphorylation, followed by PI 3-kinase activation and translocation of Akt to membrane. Akt is activated by serine phosphorylation and phosphorylates its target proteins in the cytosol, leading to the regulation of the
balance of Bcl-2 family. Akt phosphorylation almost disappeared by the
transfection of FAK antisense phosphorothioate oligonucleotides as
previously employed (14) (data not shown). Taken collectively, FAK is
an upstream signal protein of the PI 3-kinase-Akt survival pathway in
hydrogen peroxide-induced apoptosis. Further analysis of T98G
transfectants expressing active FAK or depletion of the FAK gene should
provide more information on the role of FAK in apoptosis.