From the Department of Biological Sciences, Columbia University, New York, New York 10027
Received for publication, December 7, 2000, and in revised form, January 22, 2001
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ABSTRACT |
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The serine/threonine kinase PAK4 was identified
first as an effector molecule for the Rho GTPase Cdc42. PAK4 differs
from other members of the PAK family both in sequence and function. Previously we have shown that an important function of this kinase is
to mediate the induction of filopodia in response to activated Cdc42.
Studies with a constitutively active PAK4 mutant have shown that it
also has a role in promoting anchorage-independent growth, an important
hallmark of oncogenic transformation. Here we show that another
function of PAK4 is to protect cells against apoptotic cell death.
Expression of wild-type or constitutively active PAK4 delays the onset
of apoptosis in response to tumor necrosis factor Normal development requires a carefully controlled balance between
cell survival and cell death. Throughout development excess cells are
eliminated by the process of programmed cell death, or apoptosis. A
number of different stimuli can trigger apoptosis in cells in culture
including Fas ligand, UV irradiation, serum deprivation, and cytokines
such as tumor necrosis factor Caspase-8 can also activate a signaling pathway that leads to
cytochrome c release from the mitochondria. Release of
cytochrome c leads to activation of caspase-9 followed by
cleavage and activation of caspase-3, leading to apoptosis (2, 3). The
release of cytochrome c from the mitochondria is governed
largely by Bid and members of the Bcl-2 family. Members of the Bcl-2
family can have both proapoptotic and antiapoptotic activities. For
example Bcl-2, Bcl-xL, Mcl-1, A1, and Bag-1 promote
survival, whereas Bcl-xS, Bad, Bax, and Bak promote
apoptosis (4). The interactions between the different BCL-2 family
members play an important role in determining cell fate. For example
Bcl-2 and Bcl-xL, which are located at the mitochondrial
outer membrane, promote cell survival by inhibiting cytochrome
c release. In contrast Bad can interact with Bcl-2 and
Bcl-xL and prevent their inhibitory activities. Thus
binding of Bad to Bcl-2 and Bcl-xL stimulates the release of cytochrome c from the mitochondria. One way that the
activities of the Bcl-2 family members are regulated is by
phosphorylation. When Bad is phosphorylated on serines 136 and 112 it
can no longer interact with Bcl-2 or Bcl-xL, cytochrome
c release is inhibited, and apoptosis is prevented (5). A
number of survival signals including growth factors can lead to
phosphorylation of Bad at both serines 136 and 112. Some of these
signals are mediated by the survival factor Akt, which phosphorylates
Bad on serine 136 (6). In addition to Akt other protein kinases can
phosphorylate Bad. For example constitutively active PAK1, a member of
the PAK family of serine/threonine kinases, was shown recently to
stimulate Bad phosphorylation at both serines 136 and 112 (7, 8).
The PAK family members were identified originally as
molecular targets for the Rho GTPases Rac and Cdc42 (Refs. 9-14; for review see Refs. 15-17). At present four major members of the PAK family have been identified in mammalian cells, which seem to fall into
two categories based on their structures. The first category includes
the closely related human PAK1, human PAK2, and mouse PAK3 and the
corresponding rat homologues One important function of the PAK proteins is the regulation of
cytoskeletal architecture (24-27). PAK1 has been reported to induce
filopodia and membrane ruffles by a mechanism that is independent of
its ability to bind the Rho GTPases and partially independent of its
kinase activity (28, 29). Others have found that PAK4 was identified originally on the basis of its role as a
cytoskeletal regulatory protein. It is the first member of the PAK
family that was shown to be a link between Cdc42 and filopodia formation (14). More recently we have found that PAK4 also has a role
in regulating cell growth. In fact a constitutively active mutant of
PAK4 can induce anchorage-independent growth, one of the hallmarks of
oncogenic transformation.3
Here we have investigated another role for PAK4: the induction of cell survival pathways in response to apoptotic stimuli. We have
found that cells overexpressing either wild-type or constitutively active PAK4 have a survival advantage during apoptosis. PAK4 protects cells from apoptosis not only in response to serum withdrawal but also
in response to TNF Plasmids--
To construct pLPC-HA-PAK4wt, HA-PAK4wt (14) was
removed form pBluescript KS II(+) as a
HindIII-StuI fragment and inserted into the
HindIII-EcoRI (blunted) site of the pLPC vector.
The pLPC vector is a retroviral expression vector with a puromycin resistance marker (a gift from R. Prywes). pCAN-Myc-PAK4(S445N,S474E) is cloned into the EcoRI site of the pCAN-Myc2 vector as
described.3 This constitutively active PAK4 mutant
contains a serine to glutamate mutation at amino acid 474, the putative
autophosphorylation site, and a serine to asparagine mutation at amino
acid 445. Both mutations were generated using site-directed mutagenesis
(Stratagene QuickChange kit). The mutation at amino acid 474 is thought
to mimic a phosphorylated amino acid, and the mutation at amino acid
445 is thought to stabilize the catalytic loop as
described.3 The Bad-glutathione S-transferase
(GST) fusion mammalian expression plasmid (pEBG-Bad) was obtained from
New England Biolabs.
Reagents and Antibodies--
Cycloheximide (CHX), propidium
iodide, and Hoechst 33258 reagent (bis-benzimide) were from Sigma.
Human recombinant TNF Cell Culture and Transfection--
NIH3T3 stably transfected
with either pLPC, PAK4, or PAK4(S445N,S474E) as described3
were grown at 37 °C in 5% CO2 and cultured in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
containing 10% bovine calf serum in the presence of 2 µg/ml
puromycin. HeLa cells were grown in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum in the presence of 1 µg/ml puromycin for stably transfected PAK4 and pLPC clones. All
media were supplemented with 50 units/ml penicillin, 50 µg/ml
streptomycin, and 4 mM glutamine. Transient transfection
assays were carried out in HeLa cells using the calcium phosphate
precipitation method. HeLa stable cell lines were generated by
retroviral infection. Briefly, Survival and Apoptosis Assays--
To estimate apoptosis and
survival of stably transfected cell lines, equal numbers of cells were
seeded in growth medium in 3.5-, 6-, or 10-cm plates. Two days later,
cells were stimulated as follows: for UV irradiation cells were washed
twice in phosphate-buffered saline (PBS). After removal of the PBS,
cells were exposed to 50 J/m2 UV-light in a UV cross-linker
(Fisher) followed by addition of fresh medium. For TNF
After stimulation cells were collected at the indicated time points
(attached and floating dead cells unless otherwise indicated) and fixed
for flow cytometry analysis or used to prepare total cell extracts. To
determine the percentage of cell death by flow cytometry analysis cells
with DNA content lower than the G1 peak (sub-G1) were considered to be apoptotic. Caspase-3-like
activity was examined by Western blot analysis of the caspase-3
substrate PARP in equal amounts of cell lysate. Detection of the
Mr 85,000 proteolytic product of PARP was
used as an indication of caspase activity.
To examine nuclear condensation, cells stained with Hoechst 33258 were
analyzed by fluorescence microscopy. Cells that displayed condensed
chromatin and blebbed nuclei were considered apoptotic. To determine
the amount of cell death in each clone apoptotic cells were counted in
the same number of viewing fields.
To determine survival rates cells were seeded in 6-well plates and
treated as described above. At the indicated time point the medium was
aspirated, and floating cells were removed by washing twice with PBS.
Attached cells were collected and counted. The survival rate is
expressed in percentage of surviving cells in treated cells compared
with the untreated control.
Flow Cytometry--
After stimulation cells were detached from
the plates and combined with the floating cells present in the growth
medium, collected by low speed centrifugation, washed in PBS, and fixed
in ice-cold methanol for 30 min. After washing in PBS, DNA was stained
with propidium iodide (50 µg/ml) in the presence of 50 µg/ml RNase A for 30 min at room temperature.
The DNA content was analyzed using a FACSCalibur flow cytometer (Becton Dickinson).
Fluorescence Microscopy--
Cells were seeded in 6-well plates
containing 22-mm glass coverslips and treated with TNF Western Blots--
Cell extracts were obtained in M2 lysis
buffer (20 mM Tris, pH 7.6, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, 20 mM Protein Kinase Assays--
Cell lysates and
immunoprecipitations for kinase assays were performed as described in
Ref. 14 using histone H4 as a substrate. For the detection of
phosphorylated Bad, cells were lysed in M2 buffer (34), and equal
amounts of proteins were used for immunoprecipitation. Kinase assays
were then performed essentially as described in Ref. 35 with slight
modifications: immunopurified proteins were washed four times in lysis
buffer, once in ice-cold water, and once with Akt kinase buffer (20 mM Hepes, pH 7.4, 10 mM MgCl2,10 mM MnCl2; Ref. 35). Kinase reactions were
performed in kinase buffer with 1 mM dithiothreitol and 5 µM ATP for 20 min at 30 °C in the presence of 10 µCi
of [ Expression of PAK4 Results in a Delay in Caspase Activation and
Apoptosis in TNF Delay in Apoptosis in UV-treated HeLa Cells--
To see whether
the PAK4 protective effect could be extended to other stimuli cell
survival and apoptosis were analyzed after exposure of cells to UV
irradiation. UV irradiation induced PARP cleavage in control cells by
6 h after exposure to UV, and near complete cleavage of PARP was
observed by 18 h. In contrast in the PAK4-expressing cells there
was no PARP cleavage after 6 h, and only partial cleavage was
detectable after 18 h (see Fig. 3A). Thus similar to the
TNF Delay in Apoptosis in Serum-deprived NIH3T3 Cells--
Another
trigger of apoptosis in many cells is serum withdrawal. Serum
withdrawal induces apoptosis most likely by preventing Akt activation
and thereby preventing Bad phosphorylation (37). To see whether PAK4
protects cells from serum withdrawal-induced apoptosis NIH3T3 cells
expressing either empty vector, wild-type PAK4, or a hyperactive PAK4
mutant, PAK4(S445N,S474E)3 were grown in either complete
medium or medium containing low serum. DNA content was examined after
24 h. Significantly fewer cells expressing PAK4 wild type
displayed a sub-G1 DNA content compared with the control
cells, and even fewer of the PAK4(S445N,S474E)-expressing cells had a
sub-G1 DNA content (Fig.
4).
TNF PAK4 Phosphorylates Bad--
To analyze Bad phosphorylation by
PAK4, additional cells were transfected with either empty vector or
activated PAK4(S445N,S474E). In vitro kinase assays then
were carried out in which recombinant Bad was used as a substrate for
immunopurified PAK4. The results revealed that PAK4 phosphorylates Bad
specifically on serine 112 (see Fig. 6).
Phosphorylation of serine 136 that we observed in PAK4 cell lines
therefore is more likely to be an indirect effect rather than direct
phosphorylation by PAK4.
The PAK4 serine/threonine kinase was identified originally as a
molecular target of the Rho GTPase Cdc42, and one function of PAK4 is
to mediate the induction of filopodia in response to Cdc42 (14). Here
we have shown that in HeLa and NIH3T3 cells, another function of PAK4
is to protect cells against apoptosis. We have found that
overexpression of PAK4 protects cells against apoptosis induced by
three different stimuli: serum withdrawal, TNF Although all members of the PAK family are thought to be involved in
the apoptotic response, the different family members have quite
different roles. During apoptosis Because PAK4-overexpressing cell lines were resistant to apoptotic
stimuli we have examined a number of different signaling pathways that
have important roles in cell growth and apoptosis including the c-Jun
NH2-terminal kinase, p38, extracellular signal-regulated kinase, and NF- Caspase activation triggered by either TNF The finding that PAK4 can cause a protective effect in cells is
especially interesting because PAK4 is a target for Cdc42. Cdc42 and
other members of the Rho GTPase family have important roles in the
regulation of cell growth, proliferation, and oncogenic transformation
(45-49). Recently we have found that constitutively active
PAK4(S445N,S474E) can induce anchorage-independent growth3
an important hallmark of oncogenic transformation similar to activated
mutants of Cdc42 (50-52). Furthermore, a dominant negative PAK4 mutant
can inhibit transformation by oncogenic Dbl, an exchange factor for
Cdc423 These results suggest that similar to Cdc42, PAK4
also has an important role in oncogenic transformation. The mechanisms
by which PAK4 can regulate oncogenic transformation are not entirely clear. Although changes in cytoskeletal architecture and cell adhesion
are likely to be involved,3 another important aspect of
transformation is the inhibition of apoptosis. It will be interesting
to determine whether the antiapoptotic function of PAK4 plays a
direct role in the oncogenic process.
The regulation of apoptosis also has important implications in normal
development, where extra cells are being discarded continuously. This
process is especially pronounced during neuronal development. It is
interesting that the closest known homologue to PAK4 is a
Drosophila protein called Mushroom Body Tiny (MBT; Ref. 53). Drosophila lacking the mbt gene show a reduced
number of Kenyon cells in the mushroom body, a structure in
Drosophila brain. These studies suggest that MBT has a role
in promoting either proliferation or survival of cells in the mushroom
body. Although PAK4 is ubiquitously expressed, it will be interesting
to see whether PAK4 and MBT share a common role in regulating cell
survival either in neuronal or nonneuronal cells.
Finally, the extracellular stimuli that regulate the activity of
endogenous PAK4 are not yet known. Although PAK4 is a target for Cdc42,
we do not rule out the possibility that it can also be regulated by
Cdc42-independent stimuli. Other members of the PAK family can be
regulated in fact by different types of stimuli and can be activated by
Rho GTPase-dependent and -independent mechanisms (15, 16).
In future work it will be important to determine what types of stimuli
can activate kinase activity in endogenous PAK4, and specifically
whether it is activated by stimuli that have a protective effect
against apoptosis.
stimulation, UV
irradiation, and serum starvation. Consistent with an antiapoptotic
function, expression of PAK4 leads to an increase in phosphorylation of
the proapoptotic protein Bad and an inhibition of caspase activation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
(TNF
).1 Exposure to
apoptosis-inducing agents triggers a series of events, often involving
the caspase cysteine proteases. For example, Fas ligand induces the
activation and cleavage of initiating caspases such as caspase-8, which
in turn leads to activation of downstream caspases including caspases-3
and -9. Caspase-3 as well as other effector caspases in turn cleave a
number of different target proteins that play important roles in
mediating the apoptotic response (1, 2).
PAK,
PAK, and
PAK, respectively
(9, 11-13). Each of these proteins contains an amino-terminal
regulatory domain and a carboxyl-terminal kinase domain. Within the
regulatory domain is a GTPase binding domain (GBD) that mediates
binding to Cdc42 and Rac. Also within the regulatory domain are two to
five proline-rich regions that bind to SH3 domain-containing proteins
such as the adaptor protein Nck (18, 19) and the exchange factors of
the PIX/COOL families (20, 21). Carboxyl-terminal to the kinase domain
is a motif that can interact with yeast G protein
subunits,
suggesting that the PAKs might be regulated by heterotrimeric G
proteins in mammalian cells (22, 23). The most recently identified member of the PAK family is PAK4, which falls into a second category of
PAKs (14). Like the other PAKs, PAK4 contains an amino-terminal GBD and
a carboxyl-terminal kinase domain. Unlike other PAKs, however, PAK4
does not have SH3-domain recognition sites or a G protein
binding domain, and it does not bind to PIX or Nck ( Ref.
14).2 Furthermore the GBD and
kinase domains of PAK4 have only ~50% identity with those of PAKs 1, 2, and 3, and the regulatory domain of PAK4 outside of the GBD is
completely different from the other PAKs.
PAK and PAK2 do not
induce filopodia or lamellipodia but instead have a role in the
dissolution of stress fibers, down-regulation of focal adhesions, and
cell retraction (30). More recently the PAKs have been shown also to
have important roles in regulating the apoptotic response, although the
different PAKs have different functions. For example
PAK and PAK2
get cleaved during apoptosis most likely by caspase-3. This leads to
their activation, and the activated kinases in turn are thought to
contribute to morphological and membrane changes that occur during
apoptosis (31-33). In contrast PAK1, which is not cleaved by caspases,
was reported to protect cells from apoptosis induced by serum
withdrawal in fibroblasts and interleukin-3 withdrawal in lymphoid
cells (7, 8). The survival signal induced by PAK1 is apparently due to
phosphorylation of Bad on both serines 112 and 136 (7, 8).
treatment or UV irradiation. Expression of PAK4
inhibits activation of caspase-3-like enzymes and specifically promotes
the phosphorylation of Bad on serine 112.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
was from R&D Systems. AlexaTM488
conjugated phalloidin (A-12379) was from Molecular Probes, Inc. Histone
H4 was from Roche Molecular Biochemicals. Bacterially expressed,
purified recombinant GST fusion-human Bad was kindly provided by T. Franke. Mouse monoclonal anti-HA antibody (HA.11, clone 16B12) was from
Covance, and mouse monoclonal anti-c-Myc antibody (sc-40, clone 9E10)
was from Santa Cruz Biotechnology. Mouse monoclonal anti-human PARP
(clone 4C10-5) was from PharMingen. Rabbit polyclonal antibodies to
phosphorylated Bad were from New England Biolabs. Mouse monoclonal
anti-GST antibody (clone GST-2) and secondary antibodies conjugated to
horseradish peroxidase were from Sigma.
NX cells were transfected with pLPC
empty vector, or pLPC-HA-PAK4, by the calcium phosphate precipitation
method. Supernatants containing the released viruses were collected
from cells 2 days after transfection and filtered through 0.45-µm
filters. The virus was used then to infect HeLa cells. Cells were
selected with puromycin (1 µg/ml), and colonies were picked ~2
weeks after selection. Expression of PAK4 was determined by Western
blot and immunofluorescence microscopy using a monoclonal antibody
against the HA tag.
-CHX treatment
cells were washed once with fresh medium that was replaced by medium
containing TNF
and CHX either alone or together at a concentration
of 10 ng/ml and 10 µg/ml, respectively. For serum deprivation
experiments cells were washed once with medium without serum, followed
by addition of fresh medium containing 0.1, 0.5, or 10% serum for
24 h.
-CHX as
described. At the indicated time point cells were fixed by adding 1 volume of 4% paraformaldehyde (in PBS) to the medium and stored for
16 h at 4 °C. Cells were washed in PBS and permeabilized with
0.1% Triton X-100 in PBS for 20 min at room temperature. Permeabilized
cells were treated then with 10 µg/ml Hoechst 33258 in PBS (to stain
the DNA) and AlexaTM488-phalloidin (to visualize F-actin
structures) for 45 min at room temperature. Nuclear and cellular
morphology was then determined using a Carl Zeiss, Inc. fluorescence
microscope with appropriate filters. Images of representative fields
were taken with a digital imaging device.
-glicerophosphate, 1 mM
Na3VO4, 1 µg/ml leupeptin) (34) and equal
amounts of proteins were separated by SDS-polyacrylamide gel
electrophoresis (PAGE), transferred to polyvinylidene difluoride
membranes (Immobilon P, Millipore Corp.), and incubated with antibodies
as described in Ref. 14.
-32P]ATP (3000 Ci/mmol).
[
-32P]ATP was omitted when phosphorylation was
examined by Western blotting. Reactions were stopped by the addition of
SDS-loading buffer, and denatured proteins were separated by SDS-PAGE.
Gels were dried and exposed to x-ray films for the detection of
radioactive-labeled proteins or processed for Western blotting.
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ABSTRACT
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DISCUSSION
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/Cycloheximide-treated Cells--
To examine the
function of PAK4 in cell survival pathways NIH3T3 cells and HeLa cells
were generated in which either wild-type PAK4, constitutively active
PAK4(S445N,S474E),3 or empty vector were stably expressed.
Even wild-type PAK4 had kinase activity in these cells without prior
stimulation by extracellular factors. The expression and activity of
PAK4 in the stable cell lines are illustrated in Fig.
1. To examine cell survival in the stable
cell lines HeLa cells were treated with TNF
and CHX to induce
apoptosis. (CHX was used to block the NF-
B-mediated survival pathway
induced by TNF
; Ref. 36.) As shown in Fig.
2A, in control cells, TNF
and CHX induced caspase activation within 2 h after treatment, as
demonstrated by the partial cleavage of the caspase-3 substrate PARP.
By 4 h PARP was cleaved almost completely. In contrast in cells
expressing wild-type PAK4, PARP remained completely uncleaved 2 h
after treatment and was cleaved only partly at 4 h, indicating
that induction of caspase-3-like activity was delayed in the
PAK4-overexpressing cells. At 2 h a significant proportion of the
control cells displayed condensed nuclei indicating that they were
undergoing apoptosis, as determined by Hoechst staining (see Fig.
2B). At this time point approximately five times more control cells had condensed nuclei as compared with the PAK4-expressing cells. At later time points many of the control cells had detached from
the plate, and viability was assessed by survival assays in which the
number of living cells that remained attached to the surface of the
dish was determined after TNF
/CHX treatment. The survival assays
revealed that ~5-fold more PAK4-expressing cells from two independent
cell lines were surviving 6 h after TNF
and CHX treatment,
compared with the control cells (Fig. 2C). Another assay for
apoptosis is DNA fragmentation, which can be assessed by flow cytometry
analysis of propidium iodide-stained cells. A significant proportion of
control cells displayed a sub-G1 DNA content after TNF
and CHX treatment, indicative of DNA fragmentation and apoptosis,
whereas only a small percentage of PAK4-expressing cells displayed a
sub-G1 DNA content (Fig. 2D).
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Fig. 1.
Expression and activity of PAK4 and
PAK4(S445N,S474E) in HeLa and NIH3T3 stable cell lines. Whole-cell
lysates were prepared from HeLa stable cell lines containing either
empty vector (pLPC) or HA-PAK4 wt (left panels)
or NIH (right panels) stable cell lines containing empty
vector (pLPC), Myc-PAK4wt, or Myc-PAK4(S445N,S474E). Equal
amounts of lysates were analyzed for the expression of PAK4 proteins by
Western blotting (WB) using anti-HA antibodies (left
panels) or anti-Myc antibodies (right panels). To
measure kinase activity, immunocomplex kinase assays were carried out
using histone H4 (HH4) as a substrate. Phosphorylated
histone H4 was detected by autoradiography after SDS-PAGE.
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Fig. 2.
Stable cell lines expressing PAK4 are
resistant to TNF -induced apoptosis.
A, delay in caspase activation in PAK4 cells treated with
TNF
: HeLa pLPC (control) and PAK4 clones were treated with TNF
(10 ng/ml) and CHX (10 µg/ml) alone or together as indicated or left
untreated (
). Cells were harvested at the indicated number of hours.
Whole-cell lysates were prepared and analyzed for PARP cleavage by
immunoblotting with anti-PARP antibodies (top panel).
Caspase activation is indicated by the appearance of the
Mr 85,000 PARP cleavage product. The same
lysates were probed with anti-HA antibodies (bottom panel)
to visualize HA-PAK4 expression levels. B, nuclear
condensation is reduced in TNF
-treated PAK4 clones. Nuclear
condensation after 2 h of TNF
and CHX treatment was detected by
fluorescence microscopy analysis of pLPC and PAK4 cells stained with
Hoechst 33258. AlexaTM488-phalloidin was used to stain
F-actin structures to help visualize cell shape. Fields with
representative numbers of apoptotic cells are shown. C, PAK4
cells show higher survival after TNF
treatment. HeLa pLPC cells and
two independent PAK4 cell lines (PAK4 clones 8 and 9) were either
treated with TNF
and CHX for 6 h or left untreated. Floating
dead cells were washed away, and the cells that remained attached to
the plates were trypsinized and counted. The graph shows (in
percentage) the relative numbers of cells in the treated
versus untreated samples. D, the
sub-G1 population is reduced in TNF
-treated PAK4 cells.
HeLa pLPC (light bars) and PAK4 (dark bars) cells
were treated with TNF
and CHX as described above, cells were
collected at the indicated time points, fixed, and stained with
propidium iodide, and DNA content was analyzed by flow cytometry. The
percentage of cells displaying DNA content lower than the
G1 peak (sub-G1%) is shown in the
graph.
-treated cells, caspase activation seemed to be delayed in the
PAK4-expressing cells. Also similar to the TNF
-treated cells, the
percentage of cells that displayed a sub-G1 DNA content was
significantly lower for the PAK4-expressing cells compared with control
cells (Fig. 3B).
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Fig. 3.
Stable cell lines expressing PAK4 are
resistant to apoptosis induced by UV irradiation.
A, delay in caspase activation in UV-treated PAK4 cells.
HeLa pLPC and PAK4 clones were exposed (+) to 50 J/m2 UV
radiation (UV) or left untreated ( ) and harvested at the
indicated time points. Whole-cell lysates were prepared and analyzed
for PARP cleavage (top panel). PAK4 expression levels were
assessed by Western blot analysis using anti-HA antibody (bottom
panel) as described in Fig. 2A. B, the
sub-G1 population is reduced in UV-treated PAK4 cells. HeLa
pLPC and PAK4 clones were UV-treated as described above and collected
at the indicated time points. DNA content was analyzed by flow
cytometry as described in Fig. 2D. The percentage of cells
displaying DNA content lower than the G1 peak
(sub-G1%) is shown in the graph.
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Fig. 4.
NIH3T3 cell lines expressing PAK4 are
resistant to apoptosis induced by serum deprivation. The
sub-G1 population is reduced in serum-deprived PAK4 cells.
NIH pLPC (light bars), PAK4wt (dark bars), or
PAK4(S445N,S474E) (gray bars) clones was cultured for
24 h in medium containing 10, 0.5, or 0.1% serum. Cells were
collected, and DNA content was analyzed by flow cytometry as described
in Fig. 2D. The percentage of cells displaying DNA content
lower than the G1 peak (sub-G1%) is
shown in the graph.
-induced Dephosphorylation of Bad Is Delayed in Cell Lines
Expressing PAK4--
Activated PAK1 was shown recently to induce a
protective effect against serum deprivation and cytokine withdrawal by
phosphorylating the apoptotic regulatory protein Bad on serines 112 and
136. We found that the basal level of Bad phosphorylation on both
serines 112 and 136 was higher in PAK4-expressing HeLa cells compared with control cells when the cells were grown in low serum (see Fig.
5, left panels). When cells
were grown in the presence of 10% serum this difference was less
noticeable because in this case the basal levels of Bad phosphorylation
were quite high even in the control cells. However, even in the
presence of serum a significant difference between the two HeLa clones
was detected when the cells were treated with TNF
and CHX. When
control cells were treated with TNF
and CHX Bad phosphorylation on
both serines 112 and 136 decreased over time (Fig. 5, right
panels), suggesting that as cells undergo apoptosis Bad
phosphorylation decreases, cytochrome c is released, and
caspase-3 is activated. In cells expressing PAK4, however, there was a
significant delay in the decrease in Bad dephosphorylation on both
sites, suggesting that one way that PAK4 can inhibit apoptosis is by
causing an increase in Bad phosphorylation.
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Fig. 5.
TNF induced
activation of Bad is delayed in cell lines expressing PAK4.
Left panels, levels of phospho-Bad in low serum: HeLa pLPC
and PAK4 clones, transfected with GST-Bad expression vector
(pEBG-Bad), were cultured in 0.5% serum for
24 h. Equal amounts of cell extracts were analyzed by Western
blotting with antibodies directed against Bad that is phosphorylated on
Ser-112 or Ser-136 either together (B) or separately
(C and D, respectively). GST-Bad expression
levels were detected with anti-GST antibodies (A).
Right panels, TNF
-induced dephosphorylation of Bad is
delayed in PAK4 cells. HeLa pLPC and PAK4 clones were transfected with
a control plasmid (
) or GST-Bad expression vector (+). After 36 h, cells were treated with TNF
and CHX
(TNF-CHX) for the indicated number of hours or
left untreated (
). Equal amounts of protein cell extracts were
analyzed for Bad phosphorylation by Western blotting as described
above.
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Fig. 6.
PAK4 phosphorylates Bad on serine 112. HeLa cells were transfected either with empty vector or
Myc-PAK4(S445N,S474E), as indicated, and equal amounts of cell extracts
were used for in vitro kinase assays (KA):
immunoprecipitates (IP) were used in kinase reactions with
recombinant GST-Bad as a substrate. After SDS-PAGE, phosphorylated Bad
was detected by autoradiography
(32P-GST-Bad)
or by Western blotting (WB) using phospho-specific
antibodies (anti-p-Bad) recognizing
residue Ser-112 (p-S112) or Ser-136
(p-S136) separately or combined
(S112+S136). Equal amounts of
substrate (GST-Bad) were used in each
reaction, as detected with anti-GST antibodies. PAK4(S445N, S474E)
levels were detected in whole-cell lysates (WCL) by
Western blotting with anti-c-Myc antibodies.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/CHX treatment, and UV
irradiation. PAK4 causes a delay in caspase activation, a corresponding
delay in apoptosis, and an increased overall cell survival rate in
response to these stimuli. The antiapoptotic function of PAK4 seems to
be correlated with its kinase activity because a constitutively active
PAK4 mutant has a stronger protective effect than wild-type PAK4 (see
Fig. 4), although even wild-type PAK4 has kinase activity and protects cells from apoptosis.
PAK and PAK2 are activated by
caspase cleavage and are thought to be involved in promoting the
apoptotic response because activated forms induce morphological changes
that are typical of the onset of apoptosis (31-33, 38). In contrast
PAK1 is not cleaved by caspases, and expression of activated PAK1
protects cells from apoptosis triggered by serum and interleukin-3
withdrawal (7, 8). We have found that PAK4 cleavage is not induced upon
apoptotic stimulation and that it is not cleaved by caspase-3 in
vitro (data not shown). Likewise, we have found that both
wild-type and activated PAK4 protects cells from apoptosis. Not only
does PAK4 protect cells from serum withdrawal, but it also protects
cells from TNF
- or UV irradiation-induced apoptosis.
B pathways (Refs. 39 and 40; data not shown). However, none of these pathways were significantly elevated or inhibited in the PAK4-overexpressing cells. In contrast phosphorylation of the proapoptotic protein Bad was elevated in the cell lines expressing PAK4. Phosphorylation of Bad on two critical sites, serines
112 and 136, inhibits its activity and therefore protects cells against
apoptosis (5). A number of different stimuli including growth factors
lead to phosphorylation of Bad via a pathway that requires the
serine/threonine kinase Akt, which phosphorylates Bad on serine 136 (5). Recently PAK1 was shown to be activated by Akt and to
phosphorylate Bad on both serines 112 and 136 (7, 8). In contrast to
PAK1 we have found that PAK4 phosphorylates Bad specifically on serine
112 in in vitro kinase assays. In the stable PAK4-expressing
cell lines, however, Bad phosphorylation on both serines 112 and 136 are increased. The reason for the increase in phosphorylation of serine
136 in the PAK4-expressing cell lines is not clear but is more likely
to be an indirect effect rather than direct phosphorylation by PAK4.
One possibility is that Akt may be responsible for Bad phosphorylation
on this residue. In fact we have found that in contrast to
PAK1-expressing cells (7, 8) cells expressing PAK4 show an increase in
Akt phosphorylation (data not shown), suggesting a possible increase in
Akt activity. Thus although expression of PAK4 leads to Bad
phosphorylation on both serines 112 and 136, the two sites are most
likely phosphorylated by different mechanisms. Although serine 112 can
be phosphorylated directly by PAK4, serine 136 is most likely
phosphorylated by an indirect mechanism.
or UV irradiation is
prevented or delayed in PAK4 cell lines, as detected by PARP cleavage.
This is the first demonstration that caspase activity can be regulated
by expression of a PAK protein. Because PAK4 phosphorylates Bad the
most likely explanation for this is that PAK4 prevents the
release of cytochrome c from the mitochondria by inhibiting
Bad activity and thereby inhibits the activation of caspase-3, an
effector caspase in this pathway. Another possibility, however, is that
initiator caspases are affected. Death ligands such as TNF
activate
initiator caspases such as caspase-8, which in turn can trigger
apoptosis by either mitochondria-dependent or -independent
mechanisms (41). Therefore protection from TNF
-induced cell death by
PAK4 could also potentially occur at the level of initiator caspases
such as caspase-8 by a mechanism that does not strictly require Bad
phosphorylation. Although the mechanism by which TNF
induces
apoptosis is relatively well understood (41), the molecular events that
mediate UV irradiation-induced apoptosis are quite complex. UV
irradiation can stimulate apoptosis by triggering the activation of
death receptors such as TNF receptor and thereby activate a pathway
similar to the one described above for TNF
(42). However, UV can
also induce apoptosis by other mechanisms such as by DNA
damage-dependent pathways (43, 44). Thus although we have
found that PAK4 inhibits caspase cleavage induced by UV irradiation,
the exact role for PAK4 in UV-induced apoptosis remains to be fully clarified.
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ACKNOWLEDGEMENTS |
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We thank T. Franke for GST-Bad protein and D. Sreedharan and J. Kirkland for technical assistance.
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FOOTNOTES |
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* This work was supported by Grant R01 CA76342 and an American Scientist Development Grant Award from the American Heart Association (to A. M.).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.
To whom correspondence should be addressed: Dept. of Biological
Sciences, Columbia University, Sherman Fairchild Center, Rm. 813, 1212 Amsterdam Ave., New York, New York 10027. Tel.: 212-854-5632; Fax:
212-854-7655; E-mail: agm24@columbia.edu.
Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M011046200
2 C. Dan and A. Minden, unpublished results.
3 Qu, J., Cammarano, M. S., Shi, Q., Ha, K., de Lanerolle, P., and Minden, A., (2001) Mol. Cell. Biol. in press.
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ABBREVIATIONS |
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The abbreviations used are:
TNF, tumor
necrosis factor
;
GST, glutathione S-transferase;
CHX, cycloheximide;
HA, hemagglutinin;
PAGE, polyacrylamide gel
electrophoresis;
GBD, GTPase binding domain;
PBS, phosphate-buffered
saline;
PARP, Poly(ADP-ribose) polymerase.
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