|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 15, 10566-10570, April 9, 1999
From the Department of Biochemistry, Kyoritsu College of Pharmacy,
Shibakoen 1-5-30, Minato-ku, Tokyo, Japan 105-8512
Protein phosphorylation in a human glioblastoma
cell line, T98G, was examined after exposure to oxidative stress
in vitro. Hydrogen peroxide (1 mM) markedly
induced tyrosine phosphorylation of focal adhesion kinase (FAK) and
serine phosphorylation of Akt at 1 h after stimulation.
Concommitantly, the association of FAK with phosphatidylinositide
3'-OH-kinase (PI 3-kinase) was also observed by the hydrogen peroxide
stimulation. When T98G cells were incubated with wortmannin, a PI
3-kinase inhibitor, both PI 3-kinase activity and phosphorylation of
Akt were inhibited, whereas apoptosis by oxidative stress was
accelerated. Concomitant with apoptosis, elevated level of CPP32
protease activity (caspase-3) was observed, with decreases in Bcl-2
protein and increases in Bax protein. These results suggested that in
the signal transduction pathway from FAK to PI 3-kinase, Akt promotes
survival. Thus, it became apparent that FAK is the upstream signal
protein of the PI 3-kinase-Akt survival pathway in hydrogen
peroxide-induced apoptosis in T98G cells.
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 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.
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 [ 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.
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.
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).
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).
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).
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.
We are grateful to Masaya Ueno for technical
support and are grateful to Dr. Kazushige Yokoyama for the advice on
the antisense design. We also thank Dr. Howard Young, NCI-Frederick
Cancer Research and Development Center, for kind reviewing of this manuscript.
*
This study was supported in part by grants from the Human
Science Project of Japan and from the Science Reserch Promotion Fund,
Japan Private School Promotion Foundation.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.
The abbreviations used are:
ROS, reactive oxygen
species;
FAK, focal adhesion kinase;
PI 3-kinase, phosphatidylinositide
3'-OH kinase;
PtdIns, phosphatidylinositol;
Ab, antibody;
mAb, monoclonal antibody.
FAK Is the Upstream Signal Protein of the
Phosphatidylinositol 3-Kinase-Akt Survival Pathway in Hydrogen
Peroxide-induced Apoptosis of a Human Glioblastoma Cell Line*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (28K):
[in a new window]
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.
View larger version (22K):
[in a new window]
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.
View larger version (20K):
[in a new window]
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.
View larger version (26K):
[in a new window]
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.
View larger version (49K):
[in a new window]
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.
View larger version (21K):
[in a new window]
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.
View larger version (26K):
[in a new window]
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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biochemistry,
Kyoritsu College of Pharmacy, Shibakoen, Minato-ku, Tokyo, 105, Japan. Tel./Fax: 81-3-5400-2697; E-mail: kasahara-td{at}kyoritsu-ph.ac.jp.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  R. E. Schweppe, A. A. Kerege, J. D. French, V. Sharma, R. L. Grzywa, and B. R. Haugen Inhibition of Src with AZD0530 Reveals the Src-Focal Adhesion Kinase Complex as a Novel Therapeutic Target in Papillary and Anaplastic Thyroid Cancer J. Clin. Endocrinol. Metab., June 1, 2009; 94(6): 2199 - 2203. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. P. Del Re, S. Miyamoto, and J. H. Brown Focal Adhesion Kinase as a RhoA-activable Signaling Scaffold Mediating Akt Activation and Cardiomyocyte Protection J. Biol. Chem., December 19, 2008; 283(51): 35622 - 35629. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Ren, H. Jin, C. Bian, H. He, X. Liu, S. Zhang, Y. Wang, and R.-g. Shao MR-1 Modulates Proliferation and Migration of Human Hepatoma HepG2 Cells through Myosin Light Chains-2 (MLC2)/Focal Adhesion Kinase (FAK)/Akt Signaling Pathway J. Biol. Chem., December 19, 2008; 283(51): 35598 - 35605. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. A. Beierle, N. A. Massoll, J. Hartwich, E. V. Kurenova, V. M. Golubovskaya, W. G. Cance, P. McGrady, and W. B. London Focal Adhesion Kinase Expression in Human Neuroblastoma: Immunohistochemical and Real-time PCR Analyses Clin. Cancer Res., June 1, 2008; 14(11): 3299 - 3305. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Niikura, A. Dixit, R. Scott, G. Perkins, and K. Kitagawa BUB1 mediation of caspase-independent mitotic death determines cell fate J. Cell Biol., July 10, 2007; 178(2): 283 - 296. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Thamilselvan, D. H. Craig, and M. D. Basson FAK association with multiple signal proteins mediates pressure-induced colon cancer cell adhesion via a Src-dependent PI3K/Akt pathway FASEB J, June 1, 2007; 21(8): 1730 - 1741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Lunn, R. Jacamo, and E. Rozengurt Preferential Phosphorylation of Focal Adhesion Kinase Tyrosine 861 Is Critical for Mediating an Anti-apoptotic Response to Hyperosmotic Stress J. Biol. Chem., April 6, 2007; 282(14): 10370 - 10379. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Huang, M. Khoe, M. Befekadu, S. Chung, Y. Takata, D. Ilic, and M. Bryer-Ash Focal adhesion kinase mediates cell survival via NF-{kappa}B and ERK signaling pathways Am J Physiol Cell Physiol, April 1, 2007; 292(4): C1339 - C1352. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Murakami, E. Aizu-Yokota, Y. Sonoda, S. Ohta, and T. Kasahara Suppression of Endoplasmic Reticulum Stress-induced Caspase Activation and Cell Death by the Overexpression of Bcl-xL or Bcl-2 J. Biochem., March 1, 2007; 141(3): 401 - 410. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Yamagata, S. L. Rook, Y. Sassa, R. C. Ma, P. Geraldes, L. Goddard, A. Clermont, B. Gao, H. Salti, R. Gundel, et al. Bactericidal/permeability-increasing protein's signaling pathways and its retinal trophic and anti-angiogenic effects FASEB J, October 1, 2006; 20(12): 2058 - 2067. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. van Nimwegen, M. Huigsloot, A. Camier, I. B. Tijdens, and B. van de Water Focal Adhesion Kinase and Protein Kinase B Cooperate to Suppress Doxorubicin-Induced Apoptosis of Breast Tumor Cells Mol. Pharmacol., October 1, 2006; 70(4): 1330 - 1339. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Alfano, I. Iaccarino, and M. P. Stoppelli Urokinase Signaling through Its Receptor Protects against Anoikis by Increasing BCL-xL Expression Levels J. Biol. Chem., June 30, 2006; 281(26): 17758 - 17767. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Lu, L. Wang, C. Stehlik, D. Medan, C. Huang, S. Hu, F. Chen, X. Shi, and Y. Rojanasakul Phosphatidylinositol 3-Kinase/Akt Positively Regulates Fas (CD95)-Mediated Apoptosis in Epidermal Cl41 Cells. J. Immunol., June 1, 2006; 176(11): 6785 - 6793. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Ramos, M. Sirisawad, R. Miller, and L. Naumovski Motexafin gadolinium modulates levels of phosphorylated Akt and synergizes with inhibitors of Akt phosphorylation Mol. Cancer Ther., May 1, 2006; 5(5): 1176 - 1182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. Zhang, K. M. Keledjian, J. N. Rao, T. Zou, L. Liu, B. S. Marasa, S. R. Wang, L. Ru, E. D. Strauch, and J.-Y. Wang Induced focal adhesion kinase expression suppresses apoptosis by activating NF-{kappa}B signaling in intestinal epithelial cells Am J Physiol Cell Physiol, May 1, 2006; 290(5): C1310 - C1320. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Piantadosi and H. B. Suliman Mitochondrial Transcription Factor A Induction by Redox Activation of Nuclear Respiratory Factor 1 J. Biol. Chem., January 6, 2006; 281(1): 324 - 333. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-W. Liu, M. E. Taube, K.-K. Jung, Z. Dong, Y. J. Lee, S. Roshy, B. F. Sloane, R. Fridman, and H.-R. C. Kim Tissue Inhibitor of Metalloproteinase-1 Protects Human Breast Epithelial Cells from Extrinsic Cell Death: A Potential Oncogenic Activity of Tissue Inhibitor of Metalloproteinase-1 Cancer Res., February 1, 2005; 65(3): 898 - 906. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Seo, Y. Ahn, S.-R. Lee, C. Y. Yeo, and K. C. Hur The Major Target of the Endogenously Generated Reactive Oxygen Species in Response to Insulin Stimulation Is Phosphatase and Tensin Homolog and Not Phosphoinositide-3 Kinase (PI-3 Kinase) in the PI-3 Kinase/Akt Pathway Mol. Biol. Cell, January 1, 2005; 16(1): 348 - 357. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Dahmani, A. Tesniere, D. Rouelle, J.-M. Desmonts, and J. Mantz Thiopental and isoflurane attenuate the decrease in hippocampal phosphorylated Focal Adhesion Kinase (pp125FAK) content induced by oxygen-glucose deprivation Br. J. Anaesth., August 1, 2004; 93(2): 270 - 274. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. D. Nair, T. Yuen, C. W. Olanow, and S. C. Sealfon Early Single Cell Bifurcation of Pro- and Antiapoptotic States during Oxidative Stress J. Biol. Chem., June 25, 2004; 279(26): 27494 - 27501. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-Y. Sun, N. Hail Jr, and R. Lotan Apoptosis as a Novel Target for Cancer Chemoprevention J Natl Cancer Inst, May 5, 2004; 96(9): 662 - 672. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Zhougang and R. G. Schnellmann H2O2-induced transactivation of EGF receptor requires Src and mediates ERK1/2, but not Akt, activation in renal cells Am J Physiol Renal Physiol, May 1, 2004; 286(5): F858 - F865. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-W. Liu, M. M. Bernardo, R. Fridman, and H.-R. C. Kim Tissue Inhibitor of Metalloproteinase-1 Protects Human Breast Epithelial Cells Against Intrinsic Apoptotic Cell Death via the Focal Adhesion Kinase/Phosphatidylinositol 3-Kinase and MAPK Signaling Pathway J. Biol. Chem., October 10, 2003; 278(41): 40364 - 40372. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. M. Golubovskaya, S. Gross, A. S. Kaur, R. I. Wilson, L.-H. Xu, X. H. Yang, and W. G. Cance Simultaneous Inhibition of Focal Adhesion Kinase and Src Enhances Detachment and Apoptosis in Colon Cancer Cell Lines Mol. Cancer Res., August 1, 2003; 1(10): 755 - 764. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-C. Hung, T. Ichimura, J. L. Stevens, and J. V. Bonventre Protection of Renal Epithelial Cells against Oxidative Injury by Endoplasmic Reticulum Stress Preconditioning Is Mediated by ERK1/2 Activation J. Biol. Chem., August 1, 2003; 278(31): 29317 - 29326. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Funakoshi-Tago, Y. Sonoda, S. Tanaka, K. Hashimoto, K. Tago, S.-i. Tominaga, and T. Kasahara Tumor Necrosis Factor-induced Nuclear Factor {kappa}B Activation Is Impaired in Focal Adhesion Kinase-deficient Fibroblasts J. Biol. Chem., August 1, 2003; 278(31): 29359 - 29365. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Shibukawa, M. Takahashi, I. Laffont, K. Honke, and N. Taniguchi Down-regulation of Hydrogen Peroxide-induced PKCdelta Activation in N-Acetylglucosaminyltransferase III-transfected HeLaS3 Cells J. Biol. Chem., January 24, 2003; 278(5): 3197 - 3203. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Golubovskaya, L. Beviglia, L.-H. Xu, H. S. Earp III, R. Craven, and W. Cance Dual Inhibition of Focal Adhesion Kinase and Epidermal Growth Factor Receptor Pathways Cooperatively Induces Death Receptor-mediated Apoptosis in Human Breast Cancer Cells J. Biol. Chem., October 4, 2002; 277(41): 38978 - 38987. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Feng, L. Li, P. Y. Ng, and A. G. Porter Neuronal Differentiation and Protection from Nitric Oxide-Induced Apoptosis Require c-Jun-Dependent Expression of NCAM140 Mol. Cell. Biol., August 1, 2002; 22(15): 5357 - 5366. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Williamson, T. Scales, B. R. Clark, G. Gibb, C. H. Reynolds, S. Kellie, I. N. Bird, I. M. Varndell, P. W. Sheppard, I. Everall, et al. Rapid Tyrosine Phosphorylation of Neuronal Proteins Including Tau and Focal Adhesion Kinase in Response to Amyloid-beta Peptide Exposure: Involvement of Src Family Protein Kinases J. Neurosci., January 1, 2002; 22(1): 10 - 20. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Chakravarti, J. S. Loeffler, and N. J. Dyson Insulin-like Growth Factor Receptor I Mediates Resistance to Anti-Epidermal Growth Factor Receptor Therapy in Primary Human Glioblastoma Cells through Continued Activation of Phosphoinositide 3-Kinase Signaling Cancer Res., January 1, 2002; 62(1): 200 - 207. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Su, M. Overholtzer, D. Besser, and A. J. Levine WISP-1 attenuates p53-mediated apoptosis in response to DNA damage through activation of the Akt kinase Genes & Dev., January 1, 2002; 16(1): 46 - 57. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Goldshmit, S. Erlich, and R. Pinkas-Kramarski Neuregulin Rescues PC12-ErbB4 Cells from Cell Death Induced by H2O2. REGULATION OF REACTIVE OXYGEN SPECIES LEVELS BY PHOSPHATIDYLINOSITOL 3-KINASE J. Biol. Chem., November 30, 2001; 276(49): 46379 - 46385. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. N. Hanna, L. G. Berthiaume, Y. Kikuchi, D. Begg, S. Bourgoin, and D. N. Brindley Tumor Necrosis Factor-alpha Induces Stress Fiber Formation through Ceramide Production: Role of Sphingosine Kinase Mol. Biol. Cell, November 1, 2001; 12(11): 3618 - 3630. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Huang, J. Li, M. Ding, S. S. Leonard, L. Wang, V. Castranova, V. Vallyathan, and X. Shi UV Induces Phosphorylation of Protein Kinase B (Akt) at Ser-473 and Thr-308 in Mouse Epidermal Cl 41 Cells through Hydrogen Peroxide J. Biol. Chem., October 19, 2001; 276(43): 40234 - 40240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-A. Bae, J.-E. Pie, and B. J. Song Acetaminophen Induces Apoptosis of C6 Glioma Cells by Activating the c-Jun NH2-Terminal Protein Kinase-Related Cell Death Pathway Mol. Pharmacol., October 1, 2001; 60(4): 847 - 856. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-h. Tang and N. F. Shay Zinc Has an Insulin-Like Effect on Glucose Transport Mediated by Phosphoinositol-3-Kinase and Akt in 3T3-L1 Fibroblasts and Adipocytes J. Nutr., May 1, 2001; 131(5): 1414 - 1420. [Abstract] [Full Text]
|
|
|
|
 |
|
 F. H. Pham, P. H. Sugden, and A. Clerk Regulation of Protein Kinase B and 4E-BP1 by Oxidative Stress in Cardiac Myocytes Circ. Res., June 23, 2000; 86(12): 1252 - 1258. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Qin, E. R. Stadtman, and P. B. Chock Regulation of oxidative stress-induced calcium release by phosphatidylinositol 3-kinase and Bruton's tyrosine kinase in B cells PNAS, June 6, 2000; (2000) 130198197. [Abstract] [Full Text]
|
|
|
|
|
|
Y. Sonoda, Y. Matsumoto, M. Funakoshi, D. Yamamoto, S. K. Hanks, and T. Kasahara Anti-apoptotic Role of Focal Adhesion Kinase (FAK). INDUCTION OF INHIBITOR-OF-APOPTOSIS PROTEINS AND APOPTOSIS SUPPRESSION BY THE OVEREXPRESSION OF FAK IN A HUMAN LEUKEMIC CELL LINE, HL-60 J. Biol. Chem., May 19, 2000; 275(21): 16309 - 16315. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wang, K. D. McCullough, T. F. Franke, and N. J. Holbrook Epidermal Growth Factor Receptor-dependent Akt Activation by Oxidative Stress Enhances Cell Survival J. Biol. Chem., May 5, 2000; 275(19): 14624 - 14631. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Gilmore, A. D. Metcalfe, L. H. Romer, and C. H. Streuli Integrin-mediated Survival Signals Regulate the Apoptotic Function of Bax through Its Conformation and Subcellular Localization J. Cell Biol., April 17, 2000; 149(2): 431 - 446. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. V. Chuenkova and M. A. Pereira A Trypanosomal Protein Synergizes with the Cytokines Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor to Prevent Apoptosis of Neuronal Cells Mol. Biol. Cell, April 1, 2000; 11(4): 1487 - 1498. [Abstract] [Full Text]
|
|
|
|
|
|
G. L. Fox, I. Rebay, and R. O. Hynes Expression of DFak56, a Drosophila homolog of vertebrate focal adhesion kinase, supports a role in cell migration in vivo PNAS, December 21, 1999; 96(26): 14978 - 14983. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Li, R. Fridman, and H.-R. C. Kim Tissue Inhibitor of Metalloproteinase-1 Inhibits Apoptosis of Human Breast Epithelial Cells Cancer Res., December 1, 1999; 59(24): 6267 - 6275. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P.-C. Chan, J.-F. Lai, C.-H. Cheng, M.-J. Tang, C.-C. Chiu, and H.-C. Chen Suppression of Ultraviolet Irradiation-induced Apoptosis by Overexpression of Focal Adhesion Kinase in Madin-Darby Canine Kidney Cells J. Biol. Chem., September 17, 1999; 274(38): 26901 - 26906. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Tamura, J. Gu, E. H. J. Danen, T. Takino, S. Miyamoto, and K. M. Yamada PTEN Interactions with Focal Adhesion Kinase and Suppression of the Extracellular Matrix-dependent Phosphatidylinositol 3-Kinase/Akt Cell Survival Pathway J. Biol. Chem., July 16, 1999; 274(29): 20693 - 20703. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Nemoto and T. Finkel Redox Regulation of Forkhead Proteins Through a p66shc-Dependent Signaling Pathway Science, March 29, 2002; 295(5564): 2450 - 2452. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kim, Y. Jung, D. Kim, H. Koh, and J. Chung Extracellular Zinc Activates p70 S6 Kinase through the Phosphatidylinositol 3-Kinase Signaling Pathway J. Biol. Chem., August 18, 2000; 275(34): 25979 - 25984. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Ding, T. Takano, S. Gao, W. Han, C. Noda, S. Yanagi, and H. Yamamura Syk Is Required for the Activation of Akt Survival Pathway in B Cells Exposed to Oxidative Stress J. Biol. Chem., September 29, 2000; 275(40): 30873 - 30877. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Reddy, N. V. Prasadarao, C. A. Wass, and K. S. Kim Phosphatidylinositol 3-Kinase Activation and Interaction with Focal Adhesion Kinase in Escherichia coli K1 Invasion of Human Brain Microvascular Endothelial Cells J. Biol. Chem., November 17, 2000; 275(47): 36769 - 36774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. van de Water, F. Houtepen, M. Huigsloot, and I. B. Tijdens Suppression of Chemically Induced Apoptosis but Not Necrosis of Renal Proximal Tubular Epithelial (LLC-PK1) Cells by Focal Adhesion Kinase (FAK). ROLE OF FAK IN MAINTAINING FOCAL ADHESION ORGANIZATION AFTER ACUTE RENAL CELL INJURY J. Biol. Chem., September 21, 2001; 276(39): 36183 - 36193. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. H. Korn, E. F. M. Wouters, N. Vos, and Y. M. W. Janssen-Heininger Cytokine-induced Activation of Nuclear Factor-kappa B Is Inhibited by Hydrogen Peroxide through Oxidative Inactivation of Ikappa B Kinase J. Biol. Chem., September 14, 2001; 276(38): 35693 - 35700. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Poullet, A. Gautreau, G. Kadare, J.-A. Girault, D. Louvard, and M. Arpin Ezrin Interacts with Focal Adhesion Kinase and Induces Its Activation Independently of Cell-matrix Adhesion J. Biol. Chem., September 28, 2001; 276(40): 37686 - 37691. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Qin, E. R. Stadtman, and P. B. Chock Regulation of oxidative stress-induced calcium release by phosphatidylinositol 3-kinase and Bruton's tyrosine kinase in B cells PNAS, June 20, 2000; 97(13): 7118 - 7123. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |