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INTRODUCTION |
Cells respond to stress stimulants by activating two opposing
processes; cell growth and survival pathways are activated for protection and repair, whereas programmed cell death pathways are
activated to eliminate damaged cells. The decision between cell
survival and cell death depends on the balance of constitutive and
extracellular signal-induced pro- and anti-apoptotic factors. Disturbances in the balance of pro- and anti-apoptotic factors can lead
to either increased cell death or increased cell survival and are
involved the development of diseases such as AIDS and cancer.
Programmed cell death can occur by multiple pathways with the apoptotic
and the necrotic pathways at the extremes (1). Some cells are competent
to follow one cell death pathway but lack required components for
another or contain survival factors that block a particular pathway. In
addition, different cell death pathways can co-exist within the same
cell and be activated by different stimulants. The regulation of cell
survival and cell death involves many signaling pathways, and the
decision between cell survival and cell death requires cross-talk
between these pathways and checkpoints where pro- and anti-apoptotic
signals converge (2).
p21-activated protein kinases
(PAK)1 are a growing family
of serine/threonine protein kinases, which are activated by binding of
monomeric (p21) G-proteins such as Cdc42 and Rac and subsequent autophosphorylation at a threonine residue in the activation loop (3-8). PAKs have been implicated in a wide range of biological functions such as cell morphology and motility, stress response, programmed cell death, and malignant transformation. At least three
isoforms of PAK exist in mammals.
-PAK (PAK1), a 68-kDa protein, is
present in brain, muscle, and spleen (9, 10).
-PAK (PAK3), a 65-kDa
protein, is also present in brain but in different areas than
-PAK
(11, 12).
-PAK (PAK2, PAK I, PAK65), a 58- to 62-kDa protein, is
present ubiquitously in all tissues and cell types (10, 13-15).
PAKs have been linked to mitogen-activated protein kinase and
stress-activated protein kinase pathways. PAKs can stimulate JNK and
p38 activity in some cell types but the degree of stimulation is modest
and may reflect indirect effects (4, 16-18).
-PAK phosphorylates
and positively regulates c-Raf in the ERK pathway (19). (The reference
states that PAK-3 (
-PAK) phosphorylates c-Raf, but it is actually
PAK-2 (
-PAK) (20). This error in nomenclature is due to temporary
mistakes in the GenBankTM annotations for PAK.)
Phosphorylation of c-Raf by
-PAK is required for activation of the
ERK pathway in response to growth factors or by oncogenic Ras (19).
MEK-1, another protein kinase in the ERK pathway, has been shown to be
phosphorylated by
-PAK (21). In addition to a role in mitogenic
stimulation PAKs are also involved in cell transformation.
Catalytically inactive mutants of PAK inhibit Ras transformation of rat
Schwann cells and cooperative transformation of Rat-1 cells by Ras,
Rac, and Rho (22-24). Hyperactive forms of
-PAK and
-PAK have
been detected in highly proliferative breast cancer cell lines (25).
Hyperactivity of PAK is required to convey the highly proliferative
phenotype. Recently,
-PAK has been shown to mediate signals from Ras
through phosphoinositide 3-kinase and Akt to sustain cell
transformation (26). Activated Akt stimulates
-PAK activity through
a p21 G-protein-independent mechanism. One of the downstream targets of
Akt is the pro-apoptotic Bcl-2 family protein Bad. Bad dimerizes with
anti-apoptotic Bcl-2 or Bcl-XL and inhibits their ability
to block the release of cytochrome c from mitochondria (27).
Phosphorylation of Bad at Ser-112 and Ser-136 results in dissociation
from Bcl-2 or Bcl-XL and association with 14-3-3.
-PAK
has been shown to phosphorylate Bad in vitro and in
vivo at Ser-112 and Ser-136, and phosphorylation at these residues correlates with stimulation of cell survival (28). The authors
also report that
-PAK phosphorylates Bad in vitro, but
phosphorylation and inhibition of Bad in vivo has not yet been demonstrated for
-PAK.
The mammalian PAK isoforms have a high degree of sequence homology,
especially within their catalytic domain (3-8). The regulatory domains
contain regions of high sequence homology but also nonconserved regions
that may reflect important differences in functions. A unique
characteristic of the ubiquitous
-PAK is the existence of a cleavage
site for caspase 3 or a caspase 3-like protease within the regulatory
domain (29, 30). Cleavage by caspase 3 removes most of the regulatory
domain and results in the irreversibly activated
-PAKp34 fragment.
Caspase cleavage of
-PAK is correlated with Fas- and
ceramide-induced cell death of Jurkat cells, TNF-
-induced cell death
of MCF-7 cells, heat shock-induced cell death of BALB3T3 and Hep 3B
cells, and UVC light-induced cell death of A431 cells (29, 31, 32).
Ectopic expression of
-PAKp34 induces morphological changes, which
are typical for the initial stages of apoptosis, and is sufficient to
trigger apoptosis in CHO, HeLa, and Jurkat cells (33, 34). Therefore,
caspase-activated
-PAK appears to have a pro-apoptotic role in
programmed cell death. Here, we show that the ubiquitous
-PAK can
also act as a survival factor and protect cells from programmed cell
death. Ectopic expression of constitutively active full-length
-PAK
stimulates survival of BALB3T3 fibroblasts in response to stress
stimulants such as tumor necrosis factor
(TNF-
), growth factor
withdrawal, and UVC light. Cell survival is stimulated, because
constitutively active
-PAK protects BALB3T3 cells from cell death.
Phosphorylation and inhibition of the pro-apoptotic Bcl-2 family
protein Bad is one mechanism by which
-PAK mediates stimulation of
cell survival. Other potential survival mechanisms mediated by
-PAK
include regulation of ERK, JNK, and p38 activation.
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EXPERIMENTAL PROCEDURES |
Materials--
The inducible retroviral expression system
pRevTet-On was obtained from CLONTECH. The
constitutive retroviral expression vector pBMNZ was a gift from Dr.
Gary Nolan at Stanford University. Cloned Pfu DNA polymerase
was purchased from Stratagene. An ABI PRISM BigDye Terminator Cycle
Sequencing Ready Reaction Kit was obtained from PE Applied Biosystems.
Restriction enzymes, T4 DNA ligase, phospho-p44/42 MAPK
(Thr-202/Tyr-204) and phospho-p38 MAPK (Thr-180/Tyr-182) antibodies,
and the PhosphoPlus Bad (Ser-112/Ser-136) antibody kit were
purchased from New England BioLabs. The anti-
-PAK antibodies
PAK
(N-19) and
PAK (V-19), the anti-
-PAK antibody
PAK (N-20), and
the phospho-specific anti-p-JNK antibody were from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated secondary antibodies, ECL reagents, and protein G-Sepharose were from Amersham Pharmacia Biotech. Alexa Fluor 568-conjugated secondary antibody was from Molecular Probes. Bromodeoxyuridine (BrdUrd), monoclonal
anti-bromodeoxyuridine antibody, ATP, GTP
S, and histone 4 were
purchased from Roche Molecular Biochemicals. Dulbecco's modified
Eagle's medium, fetal bovine serum (FBS), LipofectAMINE 2000, and
customized primers were obtained from Life Technologies. Tumor necrosis
factor (TNF)-
and caspase inhibitor ZVAD-FMK were from Calbiochem.
The CellTiter 96 AQueous One Solution cell proliferation assay was
purchased from Promega. M-PER Mammalian Protein Extraction Reagent and
Gelcode Blue staining reagent were from Pierce. Butylated hydroxyanisol (BHA), propidium iodine, and myelin basic protein were from Sigma Chemical Co. [
-32P]ATP was purchased from PerkinElmer
Life Sciences.
Subcloning and Site-directed Mutagenesis--
cDNAs encoding
the protein coding region of wild-type
-PAK and the kinase-deficient
mutant
-PAK-K278R were subcloned into pKoz/EGFP to form a fusion
with enhanced green fluorescent protein (EGFP). Site-directed
mutagenesis according to the megaprimer PCR method (35, 36) was
performed with Pfu DNA polymerase to obtain the
constitutively active mutant
-PAK-T402E, where Thr-402 was replaced
with glutamic acid to mimic a phosphate group incorporated by
autophosphorylation. The resulting mutant was subcloned into pKoz/EGFP
and sequenced to confirm the T402E mutation and to ensure the absence
of accidental mutations due to misincorporation during PCR. EGFP fusion
constructs for wild-type
-PAK, kinase-deficient
-PAK-K278R, and
constitutively active
-PAK-T402E were subcloned into the inducible
retroviral expression vector pRevTRE. Mouse Bad was subcloned by PCR
using the pEBG-mBad plasmid from New England BioLabs as a template into
the constitutive retroviral expression vector pBMNZ.
Cell Culture, Transfection, and Retroviral Transduction--
The
ecotropic 293T-cell-derived packaging cell line Phoenix Eco (37), the
normal mouse fibroblast cell line BALB3T3, and the K-Ras sarcoma
virus-transformed K-BALB cell line (38, 39) were obtained from American
Tissue Culture Collection (ATCC). Phoenix Eco, BALB3T3, and K-BALB
cells were maintained in Dulbecco's modified Eagle's medium
containing 10% heat-inactivated FBS, 2 mM glutamine, and
100 units/ml penicillin/100 µg/ml streptomycin at 37 °C in a
humidified atmosphere of 5% CO2. For long term storage, cells were frozen overnight at
80 °C and stored in liquid
nitrogen. Ecotropic retroviruses for the transduction of murine cells
were obtained by transient transfection of the retroviral vectors into the packaging cell line Phoenix Eco. Phoenix Eco cells were grown to
~50% confluency in 100-mm culture dishes and then transfected with 4 µg of plasmid DNA using LipofectAMINE 2000. The culture medium was
replaced at 24 h after transfection, and retrovirus-containing medium was collected at 48 h after transfection and filtered
through a 0.45-µm filter. For long term storage the
retrovirus-containing medium was frozen in liquid nitrogen and stored
at
80 °C. BALB3T3 fibroblasts were grown to ~10-20% confluency
in 100-mm culture dishes and then transduced by addition of 0.5-2 ml
of retrovirus-containing medium from packaging cells in the presence of
4 µg/ml Polybrene. The culture medium was replaced at 24 h after
transfection and culture medium containing 750 µg/ml Geneticin or 300 µg/ml hygromycin B was added at 48 h to select stable cell
populations for pRevTet-On or pRevTRE, respectively.
Cell Viability, Proliferation, and Cell Death Assays--
Cells
were treated with TNF-
by changing to growth medium containing 1 or
10 ng/ml TNF-
. Growth factor withdrawal was achieved by changing to
growth medium without fetal bovine serum or with 0.1% fetal bovine
serum. For UVC light treatment, growth medium was removed, cells were
exposed to 50 J/m2 in an UV-cross-linker at 254 nm, and
then new growth medium was added. Cell viability in response to stress
stimulants was determined with the CellTiter 96 AQueous One Solution
cell proliferation assay. Quadruplicate cell samples were grown in
96-well plates, and cell viability was measured at 24 h after
treatment by addition of the MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2M) tetrazolium compound. Over a 2 h incubation at 37 °C, MTS was converted to the colored formazan, which was detected at 490 nm with a
plate reader. Background absorbance was corrected by subtraction of
blanks with an equal volume of growth medium. Background caused by
cellular debris was corrected by subtraction of sample absorbance at
the reference wavelength of 630 nm. Cell proliferation was measured as
the percentage of cells in DNA synthesis by BrdUrd incorporation. Cells
treated with stress stimulants or untreated control cells were
incubated for 24 h. BrdUrd at 10 µM was then added
for the final 30 min. Cells were detached and collected by
centrifugation. Collected cells were fixed with 70% ethanol, stained
with a monoclonal anti-BrdUrd antibody and an Alexa-568-conjugated secondary antibody according to the manufacturer's instructions, and
analyzed by flow cytometry on a Becton-Dickinson FACScan. Cell death
was measured by the uptake of propidium iodine, which indicates loss of
membrane integrity. Stress stimulant-treated cells or control cells
were detached and collected by centrifugation. Collected cells were
stained with 0.2 µg/ml propidium iodine and analyzed by flow
cytometry on a Becton-Dickinson FACScan.
Immunoprecipitation and Western Blot--
Cells were lysed in
M-PER Mammalian Protein Extraction Reagent containing 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin, and 200 µM sodium vanadate. Protein concentrations were
determined by a Bradford protein assay using bovine
-globulin as a
standard protein. Recombinant EGFP-tagged
-PAK was
immunoprecipitated with the polyclonal anti-EGFP antibody. Endogenous
-PAK and
-PAK were immunoprecipitated with the polyclonal
agarose-conjugated anti-
-PAK (N19) and anti-
-PAK (N20)
antibodies, respectively. For immunoprecipitations 0.4 µg of antibody
was used per 100 µg of cell lysate. Western blots were performed with
cell lysates (30 µg) or immunoprecipitates (from 50 to 100 µg of
cell lysate) by SDS-polyacrylamide gel electrophoresis, semidry
transfers onto polyvinylidene membranes, and detection by ECL using
horseradish peroxidase-conjugated secondary antibodies. Recombinant
EGFP-tagged
-PAK was detected with the monoclonal anti-EGFP
antibody. Endogenous
-PAK and
-PAK were detected with the
polyclonal anti-
-PAK (V19) and anti-
-PAK (N20) antibodies,
respectively. Phosphorylated forms of Bad, ERK, JNK, and p38 were
detected with phospho-specific antibodies. Antibodies that detect both
unphosphorylated and phosphorylated forms were used to determine the
protein levels of Bad, ERK, JNK, and p38.
Protein Kinase Assays--
Autophosphorylation and activity of
immunoprecipitated
-PAK (from 50 to 100 µg of cell lysate) toward
histone 4 (1 µg) was determined in 50 mM Tris-HCl (pH
7.4), 10 mM MgCl2, 2 mM
dithiothreitol, and 200 µM [
-32P]ATP
(1000-2000 cpm/pmol) for 30 min at 30 °C in absence and presence of
Cdc42(GTP
S) (1 µg). Cdc42 was expressed in Escherichia coli, purified, and preloaded with GTP
S as described previously (13). 32P incorporation into
-PAK or histone 4 was
analyzed by electrophoresis on 15% SDS-polyacrylamide gels followed by
autoradiography and quantified by scintillation counting of excised
protein bands. In-gel assays were performed with 0.2 mg/ml myelin basic
protein co-polymerized in the separating gel of 11% SDS-polyacrylamide gels (40). Cell lysates were prepared as described above. Cell lysates
(30 µg) or immunoprecipitates (from 50 to 100 µg of cell lysate)
were separated by electrophoresis in the substrate-containing gels.
After electrophoresis, gels were washed twice for 1 h in 50 mM Tris-HCl pH (8.0), 20% 2-propanol, once for 1 h in
50 mM Tris-HCl pH (8.0), 2 mM 2-mercaptoethanol
(buffer A), and denatured twice for 1 h in buffer A containing 6 M guanidine-HCl at room temperature. Renaturation was
performed with five changes of buffer A containing 0.04% Tween 40 for
16-24 h at 4 °C. Phosphorylation was carried out in 20 mM Tris-HCl (pH 7.4), 10 mM MgCl2,
0.4 mM EGTA, 30 mM 2-mercaptoethanol, and 50 µM [
-32P]ATP (500 cpm/pmol) for 1 h
at room temperature. Excess of [
-32P]ATP was removed
by washing in 5% trichloroacetic acid, 1% sodium pyrophosphate. Gels
are stained with Gelcode Blue, dried, and subjected to autoradiography.
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RESULTS |
Recombinant Expression of Wild-type and Mutant
-PAK--
Wild-type
-PAK, constitutively active
-PAK-T402E,
and kinase-deficient
-PAK-K278R were subcloned with an N-terminal
EGFP tag into the retroviral expression vector pRev-TRE. Expression of
EGFP-tagged
-PAK is controlled by the Tet-response element (TRE)
upstream of a minimal CMV promoter. A reversed tetracycline-controlled transactivator is expressed from a second retroviral vector pRevTet-On. After addition of tetracycline or doxycycline to the culture medium, the reversed tetracycline-controlled transactivator binds to the TRE
and induces expression of recombinant
-PAK. Recombinant retroviruses were obtained with the 293T cell-derived ecotropic packaging cell line
Phoenix Eco. The use of the retroviral system has the advantage that
constructs are efficiently transduced into populations of cells rather
than a few selected clones, which may show unspecific effects due to
disruption or activation of genes by the integration into the host
genome. BALB3T3 mouse fibroblasts were transduced with pRevTet-On and
selected with Geneticin to obtain a stable BALB3T3-On cell population,
which expressed the reversed tetracycline-controlled transactivator.
BALB3T3-On cells where then transduced with pRev-TRE containing
EGFP-tagged wild-type
-PAK, constitutively active
-PAK-T402E,
kinase-deficient
-PAK-K278R, or the EGFP-tag alone. Stable cell
populations for the expression of EGFP-
-PAK, EGFP-
-PAK-T402E, EGFP-
-PAK-K278R, or EGFP alone were obtained by selection with hygromycin B.
To determine the optimal concentration and incubation time for the
induction of expression, EGFP-
-PAK-T402E cells were treated with
increasing amounts of doxycycline. Expression was monitored by
fluorescence microscopy and by Western blot with an anti-EGFP antibody
(Fig. 1). Without addition of
doxycycline, a basal level of EGFP-
-PAK-T402E was observed, which
might be due to translation from transcripts initiated by the 5'-viral
long terminal repeat. The expression was stimulated with increasing
amounts of doxycycline and reached saturation at ~2 µg/ml.
Doxycycline-stimulated expression increased with time and reached a
steady-state level at ~48 h (data not shown). Similar results were
obtained for EGFP-
-PAK, EGFP-
-PAK-K278R, and EGFP alone.
Therefore, expression was stimulated with 2 µg/ml doxycycline for
48 h in all subsequent experiments. Because basal levels were
present without addition of doxycycline, BALB3T3-On cells, which
contain only the pRevTet-On vector, were used as controls rather than
uninduced cells.

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Fig. 1.
Induced expression of recombinant
EGFP-tagged -PAK. Cells transduced with
EGFP- -PAK-T402E were grown for 48 h in growth medium containing
the indicated amounts of doxycycline (Dox). A,
expression of EGFP- -PAK-T402E was analyzed by Western blot with a
monoclonal anti-EGFP antibody. Results shown are representative of
three independent experiments. B, fluorescence microscopy
image showing expression of EGFP- -PAK-T402E at 200× magnification
was taken from a representative experiment.
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Protein kinase activity of recombinant wild-type and mutant EGFP-tagged
-PAK was determined by immunoprecipitation and immunocomplex protein
kinase assays (Fig. 2). BALB3T3 cells
expressing EGFP-
-PAK, EGFP-
-PAK-T402E, EGFP-
-PAK-K278R, or
EGFP alone, which were dividing at approximately the same time
intervals of 22 h, were grown to ~70% confluency. Cells were
lysed, and EGFP-
-PAK, EGFP-
-PAK-T402E, EGFP-
-PAK-K278R, or
EGFP alone were immunoprecipitated with a polyclonal anti-EGFP antibody
using equal amounts of cell lysate protein. Equal amounts of cell
lysate protein and equal aliquots of immunoprecipitate for each
construct were analyzed by Western blot with a monoclonal anti-EGFP
antibody (Fig. 2A). The Western blot showed a specific
protein band of 85 kDa for EGFP-
-PAK, EGFP-
-PAK-T402E, and
EGFP-
-PAK-K278R and of 27 kDa for EGFP in the cell lysates and the
immunoprecipitates. Approximately equal amounts of EGFP fusion protein
was detected with the different constructs. This suggests that
EGFP-
-PAK, EGFP-
-PAK-T402E, and EGFP-
-PAK-K278R are expressed
at very similar levels in BALB3T3 fibroblasts. Equal aliquots of
immunoprecipitate from cells expressing EGFP-
-PAK,
EGFP-
-PAK-T402E, and EGFP-
-PAK-K278R were assayed for
autophosphorylation and activity toward histone 4 (Fig. 2, B
and C). Samples without immunoprecipitate were used as
controls and subtracted from the activity values. Activation of
-PAK
involves autophosphorylation at Thr-402 in the activation loop (30,
41). Once
-PAK is activated, autophosphorylation also occurs at
several serine residues in the regulatory domain, and exogenous
substrates such as histone 4 and myelin basic protein can be
phosphorylated. No autophosphorylation and low levels of histone
4 phosphorylation were observed with the kinase-deficient
EGFP-
-PAK-K278R in the absence and presence of Cdc42(GTP
S). The
low level of histone 4 phosphorylation with the EGFP-
-PAK-K278R
immunoprecipitate could be due to co-precipitating protein kinases,
including endogenous
-PAK. EGFP-
-PAK showed a low basal level of
autophosphorylation and no significant increase in phosphorylation of
histone 4 as compared with kinase-deficient EGFP-
-PAK-K278R.
Addition of Cdc42(GTP
S) enhanced autophosphorylation and increased
phosphorylation of histone 4 by 2- to 3-fold. High levels of
autophosphorylation and a 5-fold increase in phosphorylation of histone
4 were observed with EGFP-
-PAK-T402E. Addition of Cdc42(GTP
S)
enhanced autophosphorylation but only slightly increased histone 4 phosphorylation. Therefore, EGFP-
-PAK-T402E was constitutively
active and expression of EGFP-
-PAK-T402E was used to mimic
endogenously activated
-PAK.

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Fig. 2.
Autophosphorylation and activity of
recombinant EGFP-tagged -PAK.
EGFP- -PAK (PAK), EGFP- -PAK-T402E (T402E),
EGFP- -PAK-K278R (K278R), and EGFP alone were
immunoprecipitated from BALB3T3 cells with a polyclonal anti-EGFP
antibody. A, immunoprecipitates and cell lysates were
analyzed by Western blot with a monoclonal anti-EGFP antibody.
Positions of molecular mass standard proteins in kilodaltons are
indicated at the left; positions of EGFP-tagged -PAK and
EGFP are indicated at the right. B and
C, autophosphorylation and activity toward histone 4 (H4) of immunoprecipitates were determined in the absence
and presence of Cdc42(GTP S) and analyzed by electrophoresis on a
15% SDS-polyacrylamide gel followed by autoradiography.
Autophosphorylation is shown as the autoradiograph; phosphorylation of
histone 4 was quantified by scintillation counting of excised histone 4 bands. Results shown are representative of three independent
experiments.
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Effects of Activated
-PAK on Cell Survival of BALB3T3
Fibroblasts--
BALB3T3 cells, which express EGFP-
-PAK or
EGFP-
-PAK-T402E, were analyzed for effects on cell survival in
response to TNF-
, growth factor withdrawal, and UVC light. Parental
BALB3T3-On cells and transformed K-BALB cells, which express a
constitutively active K-Ras oncogene, were used as controls. Cell
viability was measured after 24 h with a colorimetric
proliferation/viability assay. Cell viability levels of untreated
K-BALB cells, BALB3T3-On cells, and cells that express EGFP-
-PAK or
EGFP-
-PAK-T402E were very similar to each other and used as
reference values at 100% (Fig. 3A). In addition, cell
viability was also analyzed by phase contrast microscopy (Fig.
3B). BALB3T3-On cells were highly sensitive to TNF-
,
growth factor withdrawal, and UVC light. Cell viability decreased
significantly to 30-40% of that of untreated control cells. Similar
results were obtained with BALB3T3 cells, indicating that transduction
with the pRevTet-On vector does not effect cell viability (data not
shown). There were no significant differences in cell viability between
BALB3T3-On cells and cells expressing EGFP-
-PAK, but in response to
1 ng/ml TNF-
, complete growth factor withdrawal (0% FBS), and UVC
light, cells expressing EGFP-
-PAK showed a trend of slightly higher
cell survival than BALB3T3-On cells. In contrast, cells expressing
constitutively active EGFP-
-PAK-T402E showed increased cell survival
in response to TNF-
, growth factor withdrawal, and UVC light. Cell
viability decreased only to 60-85% of that of untreated control
cells. Similar results were obtained with transformed K-BALB cells,
which express constitutively active K-Ras. In response to TNF-
,
growth factor withdrawal and UVC light cell viability decreased only to
65-90% of that of untreated control cells.

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Fig. 3.
Active EGFP-tagged
-PAK stimulates cell survival.
v-K-Ras-transformed BALB3T3 cells (K-BALB), BALB3T3 cells
transduced with the reverse tetracycline-controlled transactivator
alone (BALB3T3-On), and BALB3T3-On cells transduced
with EGFP- -PAK (PAK) or EGFP- -PAK-T402E
(T402E) were grown for 48 h in the presence of 2 µg/ml doxycycline. Then, cells were treated with TNF- at 1 or 10 ng/ml, growth factor withdrawal at 0.1 or 0% FBS, or UVC light of 254 nm at 50 J/m2 and incubated for 24 h. Cell survival
was analyzed by a colorimetric proliferation/viability assay and by
phase contrast microscopy. A, viability levels of cells in
growth medium without treatment were used as reference values at 100%.
Cell viability of treated cells was calculated as percentage of these
reference values. Results are shown as the mean ± S.D.
(n = 4) of a representative of three independent
experiments. B, phase contrast images at 100× magnification
were taken from a representative experiment.
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Increased cell viability in response to stress stimulants could be the
result of stimulation of proliferation and/or protection from cell
death. Proliferation and cell death was measured at 24 h after
treatment with 1 ng/ml TNF-
or complete growth factor withdrawal to
0% fetal bovine serum. Untreated cells were used as controls.
Proliferation was measured as numbers of cells in DNA synthesis by
BrdUrd incorporation, whereas cell death was measured by the uptake of
propidium iodine, which enters cells that have lost membrane integrity
(Fig. 4). The total numbers of K-BALB
cells, BALB3T3-On cells, and cells that express EGFP-
-PAK or
EGFP-
-PAK-T402E were very similar to each other and used as reference values at 100%. Without any treatment, parental BALB3T3-On cells and cells expressing EGFP-
-PAK or EGFP-
-PAK-T402E showed similar proliferation with 46-53% of BrdUrd-incorporating cells (Fig.
4A). K-BALB cells, which express an activated K-Ras
oncogene, showed the fastest proliferation with 68% of cells
incorporating BrdUrd. Treatment with TNF-
did not affect
proliferation, whereas growth factor withdrawal resulted in a
significant decrease of BrdUrd-incorporating cells, which is probably
the result of arrested cell growth due to the lack of growth factor
stimulation. For BALB3T3-On cells and cells expressing EGFP-
-PAK,
growth factor withdrawal decreased the number of BrdUrd-incorporating
cells to 21% and 26%, respectively. For K-BALB cells and cells
expressing EGFP-
-PAK-T402E, growth factor withdrawal decreased the
number of BrdUrd-incorporating cells only to ~40%. Without
treatment, the rates of cell death for K-BALB cells, BALB3T3-On cells,
and cells expressing EGFP-
-PAK or EGFP-
-PAK-T402E were similar at 7-12% (Fig. 4B). Treatment with TNF-
or growth factor
withdrawal increased cell death of parental BALB3T3-On cells to 43% or
49%, respectively. Cells expressing EGFP-
-PAK were partially
protected; cell death was at 23% with TNF-
and at 38% with growth
factor withdrawal. Cells expressing EGFP-
-PAK-T402E were highly
protected; cell death was at 16% with TNF-
and at 23% with growth
factor withdrawal. The transformed K-BALB cells, which express an
activated K-Ras oncogene, also showed protection against cell death.
But in comparison with cells expressing EGFP-
-PAK or
EGFP-
-PAK-T402E protection of K-BALB cells was more efficient in
response to growth factor withdrawal than after treatment with TNF-
.
Cell death of K-BALB cells was at 26% with TNF-
and at 16% with
growth factor withdrawal. Therefore, stimulation of cell survival by
expression of EGFP-
-PAK-T402E, and to a lower degree by
EGFP-
-PAK, is a result of protection from cell death rather than by
stimulation of the proliferation rate. However, cells expressing
EGFP-
-PAK-T402E were able to grow under growth factor-deprived
conditions to a similar degree as K-BALB cells, which express an
activated K-Ras oncogene.

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Fig. 4.
Stimulation of cell survival by active
EGFP-tagged -PAK is caused by inhibition of
cell death. Cells were grown as described in Fig. 3 and then
treated with TNF- at 1 ng/ml, by growth factor withdrawal at 0%
FBS, or left untreated for 24 h. A, proliferation was
analyzed as percentage of cells in DNA synthesis by incorporation of
BrdUrd. Total cell numbers were used as reference values at 100%.
Results are shown as the mean ± S.D. (n = 4) of a
representative of three independent experiments. B, cell
death was analyzed as percentage of cells that lost membrane integrity
by uptake of propidium iodine. Total cell numbers were used as
reference values at 100%. Results are shown as the mean ± S.D.
(n = 4) of a representative of four independent
experiments.
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Effects of Caspases and Reactive Oxygen Species on Cell Survival of
BALB3T3 Fibroblasts--
The role of caspases and reactive oxygen
species (ROS) in cell death of BALB3T3 cells in response to 1 ng/ml
TNF-
, growth factor withdrawal to 0% fetal bovine serum, and UVC
light at 50 J/m2 were determined with the general caspase
inhibitor ZVAD-FMK and the ROS scavenger butylated hydroxyanisol (BHA).
Parental BALB3T3-On cells and cells expressing EGFP-
-PAK-T402E were
preincubated for 1 h in ZVAD-FMK or BHA and then treated with
TNF-
, growth factor withdrawal, or UVC light. After 24 h cells
were assayed for cell viability with a colorimetric
proliferation/viability assay. Cell viability levels of control cells
in growth medium alone were very similar to each other and used as
reference values at 100%. The solvent Me2SO, BHA,
and ZVAD-FMK had no significant effects on cell viability (Fig.
5A). Treatment with TNF-
,
growth factor withdrawal, or UVC light resulted in a decrease of cell viability to ~50% for parental BALB3T3-On cells. In contrast, cells
expressing EGFP-
-PAK-T402E maintained cell viability at similar
levels as untreated control. BHA and ZVAD-FMK had no effect on cell
survival in response to growth factor withdrawal and UVC light.
However, BHA stimulated cell survival of parental BALB3T3-On cells by
~20% in response to TNF-
. For cells expressing
EGFP-
-PAK-T402E, cell viability was already above 90% in response
to TNF-
and was not stimulated further by addition of BHA. ZVAD-FMK
even sensitized cell death in response to TNF-
and further reduced
cell survival of BALB3T3-On cells to 15-25% and completely blocked
stimulation of cell survival by expression of EGFP-
-PAK-T402E. In a
control experiment ZVAD-FMK inhibited cell death of PC12 cells in
response to growth factor withdrawal (data not shown).

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Fig. 5.
Effects of caspases and reactive oxygen
species (ROS) on cell survival. BALB3T3 cells transduced with the
reverse tetracycline-controlled transactivator alone
(BALB3T3-On) and BALB3T3-On cells transduced with
EGFP- -PAK-T402E (T402E) were grown for 48 h in the
presence of 2 µg/ml doxycycline. Then, cells were pretreated for
1 h with the ROS scavenger butylated hydroxyanisol
(BHA), the caspase inhibitor ZVAD-FMK or with a dimethyl
sulfoxide solvent control (DMSO), as indicated. Cells were
treated with stress stimulants and incubated for 24 h in the
presence of the indicated inhibitor or solvent control. Cell survival
was analyzed by a colorimetric proliferation/viability assay, and
viability levels of cells in growth medium without treatment were used
as reference values at 100%. Cell viability of treated cells was
calculated as a percentage of these reference values. Results are shown
as the mean ± S.D. (n = 4) of a representative of
three independent experiments. A, effects of BHA and
ZVAD-FMK on cell survival were determined in response to TNF- (1 ng/ml), growth factor withdrawal (0% FBS), and UVC light (50 J/m2). B, effects of increasing concentrations
of BHA and ZVAD-FMK on cell survival of BALB3T3-On cells in response to
TNF- (1 ng/ml) were analyzed. C, effects of co-treatment
of BHA and ZVAD-FMK on cell survival in response to TNF- was
examined.
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To determine if the stimulation of cell survival by BHA and the
sensitization of cell death by ZVAD-FMK are significant, we performed
dose-response experiments with BALB3T3-On cells (Fig. 5B).
The solvent Me2SO and up to 100 µM BHA or
ZVAD-FMK had no significant effects on cell viability in the absence of
TNF-
. The stimulation of cell survival by BHA was
concentration-dependent in response to TNF-
. Decreasing
concentrations of BHA from 100, to 10, and to 1 µM
resulted in decreasing numbers of viable BALB3T3-On cells at 81, 69, and 62%, respectively. Also the sensitizing effect of ZVAD-FMK on
TNF-
-induced cell death was concentration-dependent. Decreasing concentrations of ZVAD-FMK from 100, to 10, and to 1 µM resulted in increasing numbers of viable BALB3T3-On
cells at 17, 23 and 38%, respectively. In addition, we determined if co-treatment with BHA reduces the sensitizing effect of ZVAD-FMK on
TNF-
-induced cell death (Fig. 5C). The solvent
Me2SO, 100 µM BHA, 10 µM
ZVAD-FMK, and 100 µM BHA with 10 µM
ZVAD-FMK had no significant effects on cell viability in the absence of
TNF-
. In response to TNF-
, treatment with 10 µM
ZVAD-FMK and 100 µM BHA partially increased cell
viability of BALB3T3-On cells to 49% as compared with 33% with 10 µM ZVAD-FMK alone and completely restored the stimulation
of cell survival by EGFP-
-PAK-T402E. The protection by BHA indicates
that TNF-
-induced cell death requires the generation of ROS, whereas
the lack of protection by BHA indicates that ROS are not required for
cell death induced by growth factor withdrawal and UVC light. A caspase
or a caspase-like protease appears to protect BALB3T3 fibroblasts from
TNF-
-induced cell death by counteracting the ROS-mediated
stimulation of cell death.
Cell death in BALB3T3 cells does not follow a classical apoptotic
pathway. A study on cell death by growth factor withdrawal reported
that BALB3T3 cells exhibit some features of apoptosis such as cell
rounding and nuclear chromatin condensation but not internucleosomal
DNA fragmentation (42). We also did not observe internucleosomal DNA
fragmentation in BALB3T3 cells treated with TNF-
, growth factor
withdrawal, UVC light or cisplatin, whereas controls of human mesangial
cells treated with cisplatin showed internucleosomal DNA fragmentation
(data not shown). In addition, we did not observe stimulation of
caspase 3 activity in BALB3T3 cells in response to stress stimulants
(data not shown).
Effects of Activated
-PAK on the Pro-apoptotic Bcl-2 Family
Protein Bad--
The role of the pro-apoptotic Bcl-2 family protein
Bad as a target of
-PAK in the protection of BALB3T3 fibroblasts
from cell death was examined. Phosphorylation of Bad at Ser-112 and Ser-136 inhibits its pro-apoptotic activity by preventing
heterodimerization with Bcl-2 and Bcl-XL. Both
-PAK and
-PAK have been shown to phosphorylate Bad at Ser-112 and Ser-136
in vitro (28). The phosphorylation of endogenous Bad at
Ser-112 and Ser-136 in response to TNF-
and growth factor withdrawal
was analyzed by Western blot with phospho-specific antibodies. In
parental BALB3T3-On cells, little or no stimulation of Ser-112
phosphorylation was observed in response to TNF-
and growth factor
withdrawal (Fig. 6A). However,
expression of EGFP-
-PAK-T402E increased phosphorylation of Bad at
Ser-112. Phosphorylation of endogenous Bad at Ser-136 could not be
detected in BALB3T3-On cells or cells expressing EGFP-
-PAK-T402E,
probably because the anti-phospho-Ser-136 antibody is less sensitive
than the anti-phospho-Ser-112 antibody. Western blots, with an
antibody that detects unphosphorylated and phosphorylated Bad equally,
showed that the protein levels of Bad did not change significantly.

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Fig. 6.
Effects of activated
-PAK on the pro-apoptotic activity of Bad. The
phosphorylation and pro-apoptotic activity of the Bcl-2 family protein
Bad was examined in BALB3T3-On cells and cells expressing
constitutively active EGFP-tagged -PAK-T402E. A,
phosphorylation of endogenous Bad at Ser-112 was monitored in
BALB3T3-On cells and cells expressing EGFP-tagged -PAK-T402E in
response to TNF- (1 ng/ml) or growth factor withdrawal (0% FBS).
Lysates from untreated cells and cells treated for the indicated time
points were analyzed by Western blot with a phospho-specific antibody
for Ser-112 of Bad. Total levels of Bad protein were determined with an
anti-Bad antibody. The positions of phospho-Bad (Ser-112) or total Bad
are indicated at the right. Results shown are representative
of two independent experiments. B, analysis of cell death
induced by ectopic expression of Bad. BALB3T3-On cells and cells
expressing EGFP- -PAK-T402E (T402E) were transduced with
pBMNZ-mBad and incubated for the indicated time points. Cell death was
analyzed by loss of membrane integrity and uptake of propidium iodine
and measured as percentage of total cells. Results are shown as the
mean ± S.D. (n = 4) of a representative of three
independent experiments. C, analysis of Bad phosphorylation
in BALB3T3-On cells and cells expressing EGFP- -PAK-T402E (T402E)
transduced with recombinant Bad and incubated for 72 h. The
positions of phospho-Bad (Ser-112), phospho-Bad (Ser-136), and total
Bad are indicated at the right. Results shown are
representative of two independent experiments.
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To determine if phosphorylation of Bad by constitutively active
EGFP-
-PAK-T402E protects BALB3T3 fibroblasts from cell death, we
generated the constitutive retroviral expression construct pBMNZ-mBad.
Ecotropic retroviruses for pBMNZ-mBad or the pBMNZ vector alone were
transduced into BALB3T3-On cells and cells expressing EGFP-
-PAK-T402E. Cell death was analyzed at 24, 48, and 72 h after transduction by uptake of propidium iodine into cells that lost
membrane integrity (Fig. 6B). BALB3T3-On cells and cells expressing EGFP-
-PAK-T402E transduced with the pBMNZ vector alone showed basal levels of cell death. BALB3T3-On cells transduced with Bad
started to show increased levels of cell death at 48 h and reached
~35% of cell death at 72 h. Cells expressing EGFP-
-PAK-T402E transduced with Bad showed no significantly increased levels of cell
death. Phosphorylation of recombinant Bad at Ser-112 and Ser-136 was
greatly stimulated by expression of EGFP-
-PAK-T402E as compared with
parental BALB3T3-On cells (Fig. 6C). Under the same
conditions, phosphorylation of endogenous Bad in cells transduced with
pBMNZ vector alone was not detectable (data not shown). Therefore, phosphorylation of Bad appears to be one of the mechanisms by which
constitutively activated
-PAK-T402E protects cells from cell death.
Effects of Activated
-PAK on Activation of ERK, JNK, and
p38--
The stimulation of ERK, JNK, and p38 in BALB3T3-On cells and
cells expressing EGFP-
-PAK-T402E was measured in Western blots using
phospho-specific antibodies, which specifically detect the active forms
of these protein kinases (Fig. 7). In
BALB3T3-On cells, TNF-
activated ERK, JNK, and p38 within 15 min,
and activation was sustained up to 24 h. Expression of
EGFP-
-PAK-T402E changed the activation profile of ERK, JNK, and p38.
In cells expressing EGFP-
-PAK-T402E, ERK activation was more
increased between 15 and 30 min as compared with parental BALB3T3-On
cells, but ERK activation was significantly decreased at later time
points. EGFP-
-PAK-T402E expression increased the basal activity of
JNK-p46 and further stimulated the JNK-p46 activation between 15 and 30 min as compared with parental BALB3T3-On cells, but activation of
JNK-p46 was significantly decreased at later time points. Activation of
JNK-p54 was greatly reduced by EGFP-
-PAK-T402E throughout all time
points. EGFP-
-PAK-T402E stimulated p38 activation at 15 min as
compared with parental BALB3T3-On cells, but p38 activation was greatly decreased at later time points. Western blots with antibodies that
detect unphosphorylated and phosphorylated forms of ERK, JNK, and p38
equally showed that the protein levels did not change (data not shown).
Growth factor withdrawal did not activate JNK and p38 as compared with
basal levels and only resulted in slightly increased stimulation of ERK
(data not shown).

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Fig. 7.
Effects of activated
-PAK on stimulation of ERK, JNK, and p38. The
stimulation of ERK, JNK, and p38 pathways in response to TNF- (1 ng/ml) was examined in BALB3T3-On cells and cells expressing
EGFP-tagged -PAK-T402E. Lysates from untreated cells and cells
treated for the indicated time points were analyzed by Western blot
using phospho-specific antibodies for ERK, JNK, or p38, which
specifically detect the active forms of these protein kinases. The
positions of phospho-ERK p44 and p42, phospho-JNK p54 and p46, and
phospho-p38 are indicated at the right. Results shown are
representative of three independent experiments.
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Activation of Endogenous
-PAK in Response to TNF-
and Growth
Factor Withdrawal--
To analyze if endogenous
-PAK is activated
in response to stress stimulants, we performed in-gel assays with
myelin basic protein as substrate (Fig.
8). First,
-PAK bands in the in-gel assays were identified by immunoprecipitations and by Western blots
with specific anti-
-PAK antibodies. BALB3T3-On cell lysates were
immunoprecipitated with specific anti-
-PAK and anti-
-PAK antibodies. Immunoprecipitates and BALB3T3-On cell lysate were analyzed
by in-gel assay with myelin basic protein (Fig. 8A). Protein
kinase bands at 62 and 58 kDa were detected with the anti-
-PAK immunoprecipitate, whereas the anti-
-PAK immunoprecipitate did not
result in detectable protein kinase bands. Rat brain and BALB3T3-On cell lysates were analyzed by Western blots with specific anti-
-PAK and anti-
-PAK antibodies (Fig. 8B). Protein bands at 62 and 58 kDa were detected in rat brain and BALB3T3-On cells with the
specific anti-
-PAK antibody, whereas the anti-
-PAK antibody
detected a protein band at 68 kDa. The results show that the protein
kinase bands at 62 and 58 kDa both represent
-PAK. Because the
predicted molecular mass of rat and rabbit
-PAK is ~58 kDa,
both activity bands may represent full-length mouse
-PAK, which
differs in post-translational modifications.
-PAK protein is present
at low levels in BALB3T3 fibroblasts, but active
-PAK was not
detected.

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Fig. 8.
Activation of endogenous
-PAK in response to TNF-
and growth factor withdrawal. In-gel assays with myelin
basic protein as substrate were performed as described under
"Experimental Procedures" to monitor activation of endogenous
-PAK. PAK bands were identified by immunoprecipitation with specific
anti- -PAK or anti- -PAK antibody followed by in-gel assay, and by
Western blots using specific anti- -PAK or anti- -PAK antibody.
A, in-gel assays of -PAK and -PAK, which were
immunoprecipitated from BALB3T3-On cell lysates with specific
anti- -PAK or anti- -PAK antibodies. BALB3T3-On cell lysate was
used as a control. Positions of -PAK protein kinase bands are shown
at the right. B, Western blot analysis of cell
lysates from rat brain and BALB3T3-On cells with specific anti- -PAK
or anti- -PAK antibody. Positions of -PAK and -PAK are shown at
the right. C, in-gel assays to analyze activation
of endogenous -PAK in BALB3T3-On cells and cells expressing
EGFP-tagged -PAK-T402E, which were treated with TNF- (1 ng/ml) or
growth factor withdrawal (0% FBS). Cell lysates of untreated cells and
cells treated for the indicated time points were analyzed. Positions of
EGFP-tagged -PAK, -PAK, and -PAKp34 are shown at the
right. Results shown are representative of three independent
experiments.
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In-gel assays were carried out to examine activation of the 62- and
58-kDa
-PAK bands in response to TNF-
and growth factor withdrawal (Fig. 8C). The 62-kDa
-PAK band did not change
significantly, but the 58-kDa
-PAK band showed a biphasic
activation. In response to TNF-
, the early activation phase of the
58-kDa
-PAK band was between 30 min and 1 h and the second
activation phase was between 3 and 12 h. In response to growth
factor withdrawal, the early activation phase was between 15 and 30 min
and the second activation phase was between 12 and 24 h. In cells
expressing EGFP-
-PAK-T402E, a faint protein kinase band was visible
at ~87 kDa, which probably represents EGFP-
-PAK-T402E. It appears
that the EGFP tag reduces the ability of EGFP-
-PAK-T402E to refold in the in-gel assay. However, protein levels of endogenous
-PAK and
recombinant EGFP-
-PAK-T402E were similar (data not shown) and
immunoprecipitated EGFP-
-PAK-T402E showed a high level of constitutive activity (Fig. 2). Expression of EGFP-
-PAK-T402E stimulated the activation of the 58-kDa band of endogenous
-PAK in
response to TNF-
or growth factor withdrawal, especially in the late
activation phase (3-24 h). Therefore, activated EGFP-
-PAK-T402E appears to, directly or indirectly, stimulate the activation of the
endogenous full-length
-PAK. An activity at 34 kDa appeared at
6 h and increased at 12 and 24 h after treatment with TNF-
or growth factor withdrawal in both parental BALB3T3-On cells and cells
expressing EGFP-
-PAK-T402E. Western blots with a C-terminal PAK
antibody identified this band as caspase-cleaved
-PAKp34 (data not
shown). Therefore, stress stimulants induce activation of full-length
-PAK (58 kDa) and at later time points activation of
-PAKp34 by
caspase cleavage. Activation of
-PAK was not detected in BALB3T3-On
cells or cells expressing EGFP-
-PAK-T402E in response to TNF-
and
growth factor withdrawal.
 |
DISCUSSION |
The coordination and balance between cell growth, cell
survival, and cell death requires a complex signaling network,
including multiple checkpoints to determine cell fate. Stress
stimulants activate pathways for cell survival as well as pathways for
programmed cell death, which start a racetrack for life or death (2). The final decision to live or to die depends on the integration of all
activated cell survival and programmed cell death pathways. These
signaling pathways include key-point regulators that act as molecular
switches at checkpoints between cell survival and cell death. The
ubiquitous p21-activated protein kinase
-PAK appears to be such a
key-point regulator.
-PAK has been shown to be reversibly activated
as a full-length enzyme in response to ionizing radiation in 3T3-L1
fibroblasts and U937 leukemia cells, and by UV light, DNA-damaging
drugs, and hyperosmolarity in 3T3-L1 fibroblasts (43, 44). Irreversible
activation of
-PAK by caspase 3 or a caspase 3-like protease has
been shown in response to Fas and ceramide in Jurkat cells, TNF-
in
MCF-7 cells, heat shock in BALB3T3 and Hep 3B cells, and UVC light in A431 cells (29, 31, 32). Proteolytic cleavage correlates with cell
death and ectopic expression of the active
-PAKp34 fragment is
sufficient to trigger apoptosis in CHO, HeLa, and Jurkat cells (33,
34). Here, we report that activated full-length
-PAK acts as a cell
survival factor and protects cells from stress-induced cell death in
the contact-inhibited mouse fibroblast cell line BALB3T3.
BALB3T3 fibroblasts are highly sensitive to TNF-
and growth factor
withdrawal. In these cells TNF-
induces cell death without the need
to inhibit protein synthesis. This indicates that BALB3T3 fibroblasts
lack the ability to induce expression of survival factors in response
to TNF-
. As little as 1 ng/ml TNF-
was sufficient to reduce cell
viability of BALB3T3-On cells to ~50% within 24 h. Growth
factor withdrawal by incubation in FBS-deprived medium also results in
reduction of cell viability to ~50% within 24 h. Therefore,
BALB3T3 fibroblasts do not only require serum factors for proliferation
but also for cell survival. Expression of constitutively active
EGFP-
-PAK-T402E results in a dramatic stimulation of cell survival
in response to TNF-
, growth factor withdrawal, and UVC light whereas
expression of wild-type EGFP-
-PAK has a much lower effect.
Stimulation of cell survival by expression of EGFP-
-PAK-T402E, and
to a lower degree by wild-type EGFP-
-PAK, is mainly due to protection from cell death rather than by stimulation of proliferation. However, suppression of cell death results in increased net cell growth.
Cell death in BALB3T3 cells does not follow a classical apoptotic
pathway (42). We did not observe internucleosomal DNA fragmentation in
BALB3T3 cells treated with TNF-
, growth factor withdrawal, UVC
light, or cisplatin (data not shown). The general caspase inhibitor
ZVAD-FMK did not protect BALB3T3-On cells and cells expressing
EGFP-
-PAK-T402E from cell death by TNF-
, growth factor
withdrawal, and UVC light. In addition, we did not observe stimulation
of caspase 3 activity in BALB3T3 cells in response to stress stimulants
(data not shown). However, cell death is executed by intracellular
signaling pathways that involve the Bcl-2 family protein Bad and
therefore is programmed and not accidental. In response to TNF-
, the
caspase inhibitor ZVAD-FMK sensitizes cell death in response to TNF-
and inhibits protection from cell death by EGFP-
-PAK-T402E. Such a
sensitization of TNF-
-induced cell death by caspase inhibitors also
occurs in mouse L929 fibrosarcoma cells (1, 45). The mitochondrial ROS
scavenger BHA protects BALB3T3-On cells from TNF-
-induced cell
death, abrogates the sensitizing effect of ZVAD-FMK partially for
BALB3T3-On cells, and completely abrogates the effect for cells
expressing EGFP-
-PAK-T402E. BHA also protects L929 cells from
TNF-
-induced cell death by abrogating the sensitizing effect of
caspase inhibitors (1, 45). It is suggested that the TNF-
-induced
cell death pathway in L929 and BALB3T3 cells involves the formation of
mitochondrial ROS and that a putative caspase acts as a negative
regulator in this cell death pathway possibly by removing damaged
mitochondria. BHA has no effect on cell death induced by growth factor
withdrawal and UVC light, indicating that mitochondrial ROS formation
is not required in these death pathways. Our results show that
expression of EGFP-
-PAK-T402E protects BALB3T3 fibroblasts from cell
death induced by several signaling pathways. Therefore, constitutively active
-PAK appears to act at a central position where several death
pathways converge.
Mitochondria play a central role in many cell death signaling pathways.
The interactions of pro- and anti-apoptotic Bcl-2 family proteins at
the outer mitochondrial membrane appear to control the release of
cytochrome c and other pro-apoptotic factors such as
apoptosis-inducing factor (2, 46). Phosphorylation of the pro-apoptotic
Bcl-2 family protein Bad inhibits cell death by abrogating dimerization
with anti-apoptotic Bcl-2 and Bcl-XL. Both
-PAK and
-PAK have been shown to phosphorylate Bad at the critical Ser-112
and Ser-136 residues in vitro (28). However, we did not
detect
-PAK activation in BALB3T3-On cells or cells expressing
EGFP-
-PAK-T402E in response to TNF-
or growth factor withdrawal.
Therefore, phosphorylation of Bad in BALB3T3 fibroblasts appears to be
due to
-PAK and not
-PAK. We have found that expression of
EGFP-
-PAK-T402E increases phosphorylation of endogenous Bad in
response to TNF-
and growth factor withdrawal. Ectopic expression of
Bad by retroviral transduction induces cell death in BALB3T3-On cells
whereas cells expressing constitutively active EGFP-
-PAK-T402E are
greatly protected. Protection from cell death by recombinant Bad
correlates with increased phosphorylation of Bad at Ser-112 and
Ser-136. Therefore, phosphorylation of Bad appears to be at least one
of the mechanisms by which activated full-length
-PAK protects
BALB3T3 fibroblasts from cell death. Phosphorylation of Bad by
-PAK
appears to involve a signaling pathway from Ras via phosphoinositide
3-kinase and Akt to PAK. Interestingly, we did not detect activated Akt
with a phospho-specific antibody in BALB3T3 cells (data not shown). The
lack of Akt activation might explain why BALB3T3 cells are highly
sensitive to TNF-
and growth factor withdrawal and why expression of
constitutively active
-PAK-T402E so effectively protects BALB3T3
fibroblasts from cell death by these stimuli.
Another mechanism by which
-PAK may affect cell survival is by
acting on ERK, JNK, and p38 signaling pathways. ERK, JNK, and p38
pathways are involved in the response to growth factors, cytokines, and
stress stimulants.
-PAK phosphorylates and positively regulates
c-Raf, the MEKK in the ERK pathway (19). Expression of constitutively
active EGFP-
-PAK-T402E does not affect basal activity of ERK. In
response to TNF-
, EGFP-
-PAK-T402E stimulates ERK activation
within the first 30 min but reduces ERK activity after 1 h.
Transient overexpression of PAK in some cell types has been shown to
activate JNK and p38 (4, 16-18). Stable expression of
EGFP-
-PAK-T402E stimulates basal activity of JNK-p46 but not JNK-p54. In response to TNF-
, EGFP-
-PAK-T402E expression also stimulates JNK-p46 activation within the first 30 min but reduces JNK-p46 activity after 1 h. Although stable expression of
EGFP-
-PAK-T402E does not affect basal p38 activity, it stimulates
p38 activation within the first 15 min of treatment with TNF-
and
greatly reduces p38 activity after 30 min. ERK, JNK, and p38 do not
appear to be involved in pro- or anti-apoptotic signaling in response
to growth factor withdrawal. Stimulation of early and/or reduction of
late activation of ERK, JNK-p46, and p38 by expression of
EGFP-
-PAK-T402E might be crucial for stimulation of cell survival in
response to TNF-
but not growth factor withdrawal.
Treatment with TNF-
and growth factor withdrawal induces rapid
activation of endogenous full-length
-PAK and at later time points
results in proteolytic activation of
-PAK by caspases or
caspase-like proteases. The early activation of full-length
-PAK
coincides with the phosphorylation of Bad and the early activation of
ERK, JNK, and p38. Caspase-activated
-PAKp34 appears at 6-24 h
after treatment and correlates with cell death in BALB3T3-On cells.
Because stimulation of caspase 3 activity could not be detected in
BALB3T3 fibroblasts in response to stress stimulants, the proteolytic
activation of
-PAK appears to occur by another caspase or
caspase-like protease. In MCF-7 breast cancer cells, proteolytic
activation of
-PAK occurs independent of caspase 3. MCF-7 cells are
deficient in caspase 3 because of a deletion in the caspase 3 gene
(47). However, treatment with TNF-
results in proteolytic activation
of
-PAK in MCF-7 cells (29). Ectopic expression of
-PAKp34 has
been shown to cause cell death in CHO, HeLa, and Jurkat cells (33, 34).
Cells expressing EGFP-
-PAK-T402E exhibit similar levels of
-PAKp34 as parental BALB3T3-On cells but show greatly reduced cell
death. Therefore, expression of EGFP-
-PAK-T402E appears to abrogate
the action of
-PAKp34 rather than preventing proteolytic activation
of
-PAKp34.
Our results show that, in addition to the documented pro-apoptotic
function after caspase cleavage,
-PAK has an anti-apoptotic function
if activated as a full-length enzyme. This is the first report that
demonstrates such an anti-apoptotic activity for the ubiquitous
-PAK. Activated full-length
-PAK protects fibroblasts from cell
death induced by different pathways and, therefore, appears to play a
central role in the decision between cell survival and cell death. One
mechanism for protection from cell death by activated full-length
-PAK is through phosphorylation of the pro-apoptotic Bcl-2 family
protein Bad, and other survival mechanisms might include regulation of
the stress-induced activation of ERK, JNK, and p38 pathways.