The interferon-inducible, double-stranded (ds)
RNA-dependent serine/threonine protein kinase (PKR) plays a
role in viral pathogenesis, cell growth, and differentiation and is
implicated as a tumor suppressor gene. Expression of a
trans-dominant negative, catalytically inactive mutant PKR
protected NIH3T3 cells from apoptosis in response to either
treatment with tumor necrosis factor
(TNF
), serum deprivation.
In cells expressing mutant PKR, TNF
, but not dsRNA induced
transcription from a nuclear factor
B-dependent
promoter, demonstrating specificity for dsRNA in signaling
through the PKR pathway. Serum or platelet-derived growth factor
addition to serum-deprived mutant PKR-expressing cells induced
transcription of the early response genes c-fos and
c-jun, indicating that the immediate early response
signaling was intact. Overexpression of wild-type PKR in a transient
DNA transfection system was sufficient to induce apoptosis.
TNF
-induced apoptosis correlated with increased phosphorylation of
the
subunit of eukaryotic translation initiation factor 2 (eIF-2
), the primary physiological substrate of the PKR.
Furthermore, forced expression of a nonphosphorylatable S51A mutant
eIF-2
partially protected cells from TNF
-induced apoptosis, and
expression of a S51D mutant eIF-2
, a mutant that mimics
phosphorylated eIF-2
, was sufficient to induce apoptosis. Taken
together, these studies identify a novel requirement for PKR in
stress-induced apoptosis that is mediated through eIF-2
phosphorylation.
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INTRODUCTION |
The interferon-inducible, double-stranded
(ds)1
RNA-dependent serine/threonine protein kinase (PKR) is a
ubiquitously expressed protein in mammalian cells that was first
identified as a mediator of the antiproliferative and antiviral actions
of interferon (1, 2). A variety of dsRNA molecules generated during
viral infection, specific single-stranded RNAs, and various agents,
such as heparin, activate PKR from its latent form. Upon activation,
PKR displays two known activities: autophosphorylation and
phosphorylation of its physiological substrate, the
subunit of the
heterotrimeric eukaryotic translational initiation factor 2 (eIF-2
).
eIF-2
phosphorylation impairs its ability to recycle and thus
inhibits protein synthesis at the level of initiation (3, 4). As a
consequence, activated PKR inhibits viral replication (5, 6) and cell
growth (7, 8) and may promote cell differentiation (9, 10). In
addition, expression of catalytically inactive mutants of PKR
transforms NIH3T3 cells to induce tumors in nude mice. This observation
is ascribed to a trans-dominant inhibitory effect of the
mutant enzyme on the endogenous wild-type PKR and implicates PKR as a
tumor suppressor gene (11-14). PKR is also implicated in the
transcriptional regulation of dsRNA-activated genes, such as interferon
, by activation of nuclear transcription factor
B (NF
B) by
phosphorylating its inhibitor, I
B, in response to dsRNA
(15-17).
Polypeptide chain synthesis is initiated when the ternary complex of
heterotrimeric eIF-2, GTP, and initiator met-tRNA bind the 40S
ribosomal subunit to generate a 43S preinitiation complex (for reviews,
see Refs. 3 and 4). Subsequently, mRNA binds and the 60S ribosomal
subunit joins to form the 80S initiation complex with the concomitant
hydrolysis of GTP to GDP. For eIF-2 to promote another round of
initiation, GDP bound to eIF-2 must be exchanged for GTP, a reaction
catalyzed by the guanine nucleotide exchange factor (eIF-2B).
Phosphorylation of the
subunit of eIF-2 (eIF-2
) stabilizes the
eIF-2·GDP·eIF-2B complex. Because eIF-2B is obligatory for the
exchange of GTP for bound GDP and because eIF-2B exists in cells in
relatively low molar quantities with respect to eIF-2, the exchange
process can be inhibited when only a fraction (i.e.
10-20%) of eIF-2
is phosphorylated (18). Thus, phosphorylation of
eIF-2
is part of a key regulatory process that is proposed to result
in the quantitative sequestering of eIF-2B that can virtually shut down
protein synthesis initiation.
TNF
is an inflammatory cytokine that, upon binding to its receptors
TNFR1 and TNFR2, initiates signaling pathways that play important roles
in cell activation, differentiation, and apoptosis, a physiologically
controlled process important in development and pathogenesis of a
variety of human diseases (for review, see Ref. 19). Upon binding of
TNF
, TNFR1 becomes trimerized and recruits the adapter protein, TNF
receptor-associated death domain, which subsequently recruits
Fas-associating protein with death domain to activate the interleukin-1
converting enzyme family of cysteine proteases that induce an apoptotic
signal. TNF
provides an antiviral function for the host by targeting
virus-infected cells for apoptotic cell death (20-22). In
addition, upon trimerization of TNFR1, TNFR-associated factor is
recruited to lead to activation of NF
B through phosphorylation of
its inhibitor, I
B. Activation of NF
B induces the expression of
survival genes. Because PKR is implicated in dsRNA-activation of NF
B
through phosphorylation of its inhibitor, I
B (16, 17), and because
PKR induces apoptosis in the context of a vaccinia virus infection (6),
we studied the ability of PKR to induce programmed cell death in
response to TNF
by analyzing fibroblasts expressing a
trans-dominant negative K296P mutant PKR to inhibit the PKR
signaling pathway(s). Our results indicate that PKR can induce
apoptosis through phosphorylation of eIF-2
.
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MATERIALS AND METHODS |
Cell Culture--
NIH3T3 cell lines that were transfected and
selected for expression of the catalytically inactive K296P mutant PKR
were previously described (23). Cells were co-transfected with the
pEDmtxrVA
empty vector and
pSV2Neo, selected for G418 resistance, pooled, and used as
a control. NIH3T3 cells transfected and selected for wild-type and S51A
mutant eIF-2
expression were previously described (24). Cells were
cultured at 37 °C with 10% CO2 in Dulbecco's modified
essential medium containing 10% fetal bovine serum (FBS) and
penicillin/streptomycin in 100-mm tissue culture dishes (for cell cycle
and DNA fragmentation analysis) or on coverslips (for fluorescence
microscopy). Cells were incubated in the presence or absence of TNF
or in the presence of 10% FBS or 0.1% FBS for times indicated in the
figure legends. Where indicated, cells were treated with interferon
(1000 units/ml; Life Technologies, Inc.) for 20 h and then treated
with poly(I)·poly(C) (100 µg/ml; Pharmacia Biotech Inc.) for 6 h prior to analysis by phase contrast microscopy.
DNA Fragmentation Analysis--
Cells were collected and
harvested in 0.5 ml of lysis buffer (10 mM Tris-Cl, pH 7.4, 1 mM EDTA, 400 mM NaCl, 1% SDS). Lysates were
incubated at 50 °C for 2 h with 0.2 mg/ml proteinase K. After extraction with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and reextraction with chloroform:isoamyl alcohol, the DNA was
precipitated by addition of 1 volume of isopropanol at
70 °C
overnight. Precipitated DNA was collected by centrifugation at
14,000 × g for 30 min, washed with ice-cold 70%
ethanol, resuspended in Tris-EDTA buffer (pH 8.0), and treated with
RNase (DNase free, 20 µg/ml; Boehringer Mannheim). DNA samples were
fractionated by electrophoresis on a 1.6% agarose gel and visualized
by ethidium bromide staining. Band intensities of DNA fragments (size
ranging between 200 and 600 base pairs) were quantified using the
National Institutes of Health Image 1.55b program.
Transient DNA Transfection and Analysis--
NIH3T3 cell lines
were co-transfected with the NF
B-chloramphenicol acetyltransferase
(CAT) expression plasmid (4x
B-CAT; provided by Dr. Gary Nabel,
Howard Hughes Medical Institute, Ann Arbor, MI; 25) and a Rous sarcoma
virus promoter-driven luciferase construct (to measure transfection
efficiency) by the DEAE-dextran method (26). At 48 h
posttransfection, cells were treated either with poly(I)·poly(C) (100 µg/ml; Pharmacia) or TNF
(10 ng/ml; Life Technologies, Inc.) for
20 h at 37 °C. Cell extracts were harvested by freeze-thawing,
and CAT and luciferase activities were measured essentially as
described in the Promega Protocol and Applications Guide (Madison,
WI).
COS-1 cells were transfected with expression plasmids encoding either
wild-type PKR, K296P mutant PKR, the dsRNA binding domain of PKR (amino
acid residues 1-243), wild-type eIF-2
, S51A eIF-2
, or S51D
eIF-2
contained in the pMT2VA
expression
vector (27, 28). The Bcl-2 expression plasmid was kindly provided by
Dr. S. J. Korsmeyer (Howard Huges Medical Institute, St. Louis,
MO). Transfections were performed in the presence of an equal amount of
DNA encoding the jellyfish green fluorescence protein (GFP) (expression
vector psynGFPS65T (29)) to identify transfected cells. At 24 h
posttransfection, cells were fixed and nuclei were stained with Hoechst
33258 (Molecular Probes, Eugene, OR). Stained cells were washed in cold
phosphate-buffered saline, mounted on slides, and visualized using an
Olympus BX60 fluorescence microscope.
Cell Cycle Analysis--
Cells were incubated for the indicated
times in the presence or absence of TNF
or in the presence of 10%
FBS or 0.1% FBS. Cells were then washed with cold phosphate-buffered
saline containing 0.1% bovine serum albumin and fixed in 70% ethanol
in phosphate-buffered saline at 4 °C overnight. Cells were
rehydrated in phosphate-buffered saline, incubated with RNase (20 µg/ml) for 30 min at 37 °C, and stained with propidium iodide (50 µg/ml) for 30 min. DNA content was analyzed using a FACStar Flow
Cytometer (Immunocytometry System, Becton Dickinson, Mountain View,
CA). Cells were analyzed for scatter-gated fluorescence to prevent
scoring of dead cells and debris. Green fluorescence was measured using
a 530/30-nm bandpass filter, and red fluorescence was measured using a
625/35-nm bandpass filter.
eIF-2
Phosphorylation--
eIF-2
phosphorylation was
measured by [32P]phosphate incorporation as described
(23) or by Western blot analysis. Steady state levels of eIF-2
and
phosphorylated eIF-2
were measured in cell extracts by
SDS-polyacrylamide gel electrophoresis under reducing conditions and
electroblotting to nitrocellulose. Filters were sequentially incubated
and developed with rabbit anti-phosphopeptide antibody specific to
phosphorylated eIF-2
(kindly provided by Gary Krause, Wayne State
University) and then with anti-eIF-2
monoclonal antibody (provided
by Dr. Henshaw). After incubation with antibody, the filters were
washed and developed using the ECL kit (Amersham Corp.).
RNA Analysis--
Total cellular RNA was isolated using TRIAzol
(Life Technologies, Inc.). Total RNA samples (10 µg) were analyzed by
RNA Northern blot hybridization following electrophoresis on
formaldehyde-formamide 1% agarose gels (30, 31). RNA was transferred
to nitrocellulose membranes and baked at 80 °C under vacuum. The
blots were prehybridized, hybridized to either 32P-labeled
c-fos or 32P-labeled c-jun cDNA
(provided by Dr. Tom Kerpolla, Howard Hughes Medical Institute, Ann
Arbor, MI) probes prepared by [
-32P]dCTP labeling
using the Rediprime DNA labeling system (Amersham Corp.) and washed two
times with 0.1× SSC (1× = 150 mM sodium chloride, 15 mM sodium citrate) for 15 min. As a control for loading equal amounts of RNA, the blots were treated with 0.1% sodium dodecyl
sulfate at 95 °C and rehybridized to a 32P-labeled
-actin probe. Band intensities were quantified using the National
Institutes of Health Image 1.55b program.
Total RNA (150 ng) was reverse transcribed using oligo(dT) primer and
superscript II reverse transcriptase (Life Technologies, Inc.) in a
20-µl reaction volume essentially as described by the supplier. PCR
was performed using 2 µl from each cDNA reaction with primer sets
for detecting Fas mRNA (5
-CATCTCCGAGAGTTTAAAGCTGAGG and
5
-GTTTCCTGCAGTTTGTATTGCTGGTTCC) or glyceraldehyde-3-phosphate dehydrogenase mRNA (5
-CCATGGAGAAGGCTGGGG and
5
-CAAAGTTGTCATGGATGACC). Control experiments demonstrated that the PCR
was performed in the linear range to detect Fas and
glyceraldehyde-3-phosphate dehydrogenase mRNAs.
 |
RESULTS |
A Functional PKR Pathway Is Required for TNF
- and Serum
Deprivation-induced Apoptosis--
The requirement for a functional
PKR pathway in apoptosis was studied in NIH3T3 cells stably transfected
with empty vector alone or in NIH3T3 cells expressing mutant K296P PKR.
The expression of K296P mutant PKR in these cells was previously
characterized (23). TNF
- and serum deprivation-induced apoptosis
were studied by TNF
treatment or growth in 0.1% FBS. Both of these
conditions induced characteristic ladders of DNA fragments in NIH3T3
cells stably transfected with empty vector alone that were not observed in a cells that overexpress mutant PKR (Fig.
1A). Similar results were
obtained with three additional independently isolated clones expressing
K296P PKR (data not shown), implying a requirement for a functional PKR
pathway in both of these apoptotic responses. The resistance to TNF
-
or serum deprivation-induced apoptosis in mutant PKR-expressing cells
was further confirmed by quantitating the percentage of apoptotic cells
by flow cytometry. NIH3T3 cells transfected with vector alone contained
a range of 2-4 N DNA representing cells in the
G0/G1, S, and G2/M phases of the
cell cycle. Following TNF
treatment or serum deprivation, the number
of cells in the G0/G1 phase of cell cycle
decreased in parallel to the appearance of a peak of cells containing
hypodiploid DNA (Fig. 1B). In contrast, cells that express
mutant PKR showed a reduced number of cells containing hypodiploid DNA
following TNF
(6%) or serum deprivation (8%). These results
support the involvement of PKR in TNF
- and serum deprivation-induced
apoptosis.

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Fig. 1.
Overexpression of trans-dominant
negative mutant K296P PKR protects cells from TNF - and serum
deprivation-induced apoptosis. A, DNA fragmentation analysis
of NIH3T3 cells stably transfected with empty vector alone and the
vector encoding K296P mutant PKR (clone KP). DNA was isolated from
cells treated with TNF (left panel) or from cells growing
in 10 or 0.1% FBS (right panel), fractionated by
electrophoresis in a 1.6% agarose gel, and visualized by ethidium
bromide staining. B, flow cytometric analysis of NIH3T3 cells stably transfected with empty vector and KP cells expressing the
mutant K296P PKR. For TNF experiments (left panel),
subconfluent cells were treated with TNF (10 ng/ml for 20 h) or
left alone for indicated time and harvested for flow cytometric
analysis as described under "Materials and Methods." For serum
deprivation experiments (right panel), confluent cells were
propagated in the presence of 10 or 0.1% serum and harvested for flow
cytometric analysis as described under "Materials and
Methods."
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Recently, dsRNA (poly(I)·poly(C)) was shown to induce apoptosis
in a PKR-dependent manner using mouse embryo fibroblasts
from PKR wild-type and PKR knockout mice (32). Therefore, we studied the requirement for PKR in apoptosis mediated by interferon and poly(I)·poly(C) treatment in NIH3T3 cells. NIH3T3 cells stably transfected with empty vector or NIH3T3 cells transfected with K296P
mutant PKR were treated with interferon, poly(I)·poly(C), or
poly(I)·poly(C) in combination with interferon. Under these experimental conditions, interferon or poly(I)·poly(C) alone did not
induce significant morphological changes in either cell line (Fig.
2). However, poly(I)·poly(C) in
combination with interferon induced significant cell death in NIH3T3
cells stably transfected with empty vector, whereas NIH3T3 cells that
were overexpressing mutant PKR were protected under identical
conditions. It is most likely that poly(I)·poly(C) alone did not
affect NIH3T3 cells due to their low levels of PKR. These results are
in agreement with previous observations (32, 33) and support the idea
that dsRNA is toxic to NIH3T3 cells that have a functional PKR
pathway.

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Fig. 2.
Overexpression of K296P mutant PKR protects
cells from death induced by poly(I)·poly(C) and interferon.
NIH3T3 cells stably transfected with empty vector or stably transfected
with K296P mutant PKR were treated with interferon or with
poly(I)·poly(C) or poly(I)·poly(C) in combination with interferon
as described under "Materials and Methods." Following treatment,
micrographs were taken at × 200 magnification.
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Signaling by dsRNA and not by TNF
or Platelet-derived Growth
Factor (PDGF) Is Selectively Abrogated in Cells Expressing Mutant
PKR--
Whether PKR function was abrogated in the cells expressing
the trans-dominant negative mutant PKR was tested by
analyzing the dsRNA-dependent activation of NF
B because
previous studies support the idea that PKR is required in this
signaling (16, 34). Control and mutant PKR-expressing NIH3T3 cells were
transiently transfected with an NF
B-specific CAT reporter plasmid
(25). In control cells, CAT activity was induced 5-fold in an
NF
B-dependent manner in response to poly(I)·poly(C).
Mutation of the NF
B binding sites within the vector prevented the
induction (Fig. 3). Cells expressing the
trans-dominant negative mutant PKR did not respond to
poly(I)·poly(C), and in addition, the basal transcription of the
NF
B-dependent promoter in these cells was reduced
compared with control NIH3T3 cells (Fig. 3). In this experiment, the
transfection efficiency was monitored by co-transfection with a
luciferase expression vector. The amount of luciferase activity varied
less than 10% between different plates of transfected cells. The
defective dsRNA response was specific, because both control and the
trans-dominant negative mutant PKR-expressing cells
responded to TNF
as indicated by induction of
NF
B-dependent CAT activity by 29- and 27-fold, respectively (Fig. 3). The greater response of these cells to TNF
compared with poly(I)·poly(C) may reflect the inefficient delivery of
poly(I)·poly(C) to the cells or differences in the general
responsiveness of these two different signal transduction pathways.
These results are consistent with a specific abrogation of PKR
signaling by expression of mutant K296P PKR.

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Fig. 3.
Overexpression of mutant PKR blocks the
double-stranded RNA-dependent signal transduction
pathway. NIH3T3 cells stably transfected with empty vector or KP
cells expressing trans-dominant negative mutant PKR were
transiently transfected with a 4x B-CAT vector or a mutant B-CAT
vector in the presence of an RSV-luciferase construct. At 48 h
posttransfection, cells were treated either with poly(I)·poly(C) (100 µg/ml) or TNF (10 ng/ml) for 20 h at 37 °C. Cell extracts
were harvested by freeze-thawing, and CAT activity was measured
essentially as described under "Materials and Methods."
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Results of recent studies suggest that PKR-mediated Fas mRNA
induction may be one mechanism for PKR-mediated cell death in response
to a variety of stresses (32, 35). We therefore measured Fas mRNA
levels in response to TNF
, poly(I)·poly(C), and interferon with poly(I)·poly(C) using reverse transcription-PCR with Fas mRNA-specific primers. After treatment with any of these agents, an
increase in Fas mRNA level either in NIH3T3 cells transfected with
vector or cells transfected with mutant PKR was not detected (data not
shown). The results demonstrated that apoptosis in response to either
TNF
or dsRNA with interferon is not secondary to an induction of Fas
mRNA.
Previous studies suggested that induction of the early response genes
requires a functional PKR pathway (15, 36). For example, 2-aminopurine,
an adenosine analog that is known to inhibit PKR, was shown to inhibit
serum induction of early response genes (15). To elucidate whether the
early response gene transcriptional induction is mediated through PKR,
we studied induction of c-fos and c-jun in
response to serum or PDGF addition to growth-arrested, serum-depleted
control NIH3T3 cells or NIH3T3 cells expressing K296P mutant PKR.
Analysis of total RNA by Northern blot hybridization demonstrated that
c-fos and c-jun mRNAs were induced in
response to the addition of either serum or PDGF to serum-starved
control NIH3T3 cells (Fig. 4, compare
lanes 1, 3, and 5), as well as in serum-starved
NIH3T3 cells expressing K296P mutant PKR (Fig. 4, lanes 7, 9, and 11). Importantly, addition of 2-aminopurine
inhibited c-fos and c-jun induction by serum and
PDGF in both control (Fig. 4, lanes 2, 4, and 6)
and mutant PKR-expressing cells (Fig. 4, lanes 8, 10, and
12). These results indicate that a functional PKR pathway is
not required for the immediate early response and that 2-aminopurine
inhibition of the immediate early response is likely a consequence of
inhibiting a PKR-independent pathway. The observation that PKR is not
required for the immediate early response is in contrast to the
findings of Mundschau and Faller (36), who used antisense
oligonucleotides to suppress PKR levels in BALB/c 3T3 cells to show a
defect in the serum/PDGF-mediated transcriptional induction of
c-fos and c-myc. Further studies are required to
determine whether the observed differences in the apparent requirement
for PKR in the immediate early response are due to the different cell
culture systems or due to the different methods utilized to inhibit the
PKR pathway. Our results, showing that c-fos and
c-jun transcriptional induction were not inhibited by mutant
PKR expression, demonstrate that apoptotic response and not the
serum growth response requires a functional PKR pathway. This also
indicates that protection from apoptosis in these mutant PKR-expressing
cells is specific and not due to general dysfunction of signal
transduction pathway(s).

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Fig. 4.
Induction of early response genes is not
inhibited by mutant K296P PKR. NIH3T3 cells stably transfected
with empty vector or cells transfected with K296P mutant PKR were grown
to confluency, and the medium was changed to 0.5% serum. After 48 h, cells were fed with medium containing 10% serum or 0.5% serum in
the absence or presence of PDGF for 1 h. For 2-aminopurine experiments, 10 mM 2-aminopurine was added to the medium at
the same time. Total cellular RNA was isolated and analyzed by Northern blot hybridization using 32P-labeled c-fos or
c-jun probes. As a control for loading equal amounts of RNA,
the blots were treated with 0.1% SDS at 95 °C and rehybridized to a
32P-labeled -actin probe. The fold induction in response
to serum or PDGF was measured by densitometry analysis. In NIH3T3 cells transfected with empty vector alone, c-fos and
c-jun mRNAs were induced 10- and 4-fold in response to
serum and 12- and 6-fold in response to PDGF, respectively. In NIH3T3
cells expressing mutant PKR, c-fos and c-jun
mRNAs were induced 6- and 8-fold in response to serum and 8- and
10-fold in response to PDGF, respectively.
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PKR-dependent Phosphorylation of eIF-2
Is Necessary
and Sufficient to Mediate Apoptosis--
To elucidate whether
TNF
-induced apoptosis is mediated through phosphorylation of
eIF-2
by PKR, in vivo analysis of eIF-2
phosphorylation was performed in NIH3T3 cells that overexpress wild-type eIF-2
or S51A mutant eIF-2
, the site of PKR
phosphorylation (27). The expression of wild-type and S51A mutant
eIF-2
in these NIH3T3 cells was previously characterized (24).
Immunoprecipitation of eIF-2
from
[32P]phosphate-labeled NIH3T3 cells that overexpress
wild-type eIF-2
detected [32P]phosphate incorporation
into eIF-2
(Fig. 5A, lane
2). Treatment with TNF
increased the
[32P]phosphate incorporation into wild-type eIF-2
by
2.5 fold (Fig. 5A, lane 3). In contrast, cells that express
the S51A mutant eIF-2
did not exhibit increased
[32P]phosphate incorporation in response to TNF
(Fig.
5A, lanes 4 and 5). Cells treated with TNF
showed increased background labeling, as is evident by the use of an
irrelevant monoclonal antibody (Fig. 5A, lanes 6 and
7). The increase in eIF-2
phosphorylation upon TNF
treatment was also demonstrated by Western blot analysis using
antibodies against phosphorylated and nonphosphorylated eIF-2
P (Fig.
5B). Previous detailed characterization of the anti-eIF-2
antibody demonstrated that it specifically reacts with phosphorylated eIF-2
and not with nonphosphorylated eIF-2
(37). Whereas TNF
treatment increased the relative amount of phosphorylated eIF-2
by
approximately 2-fold in cells expressing wild-type eIF-2
, there was
no increase in cells expressing the S51A mutant eIF-2
. These results
support the idea that TNF
induces eIF-2
phosphorylation.

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Fig. 5.
Expression of S51A mutant eIF-2 prevents
TNF -induced eIF-2 phosphorylation. NIH3T3 cells that
overexpress wild-type eIF-2 or S51A mutant eIF-2 were treated
with TNF (10 ng/ml for 20 h) and labeled with
[32P]orthophosphoric acid for 4 h as described
previously (23). Cell extracts were prepared and immunoprecipitated
with anti-eIF-2 monoclonal antibody and analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography
(A). In parallel, cells were harvested and analyzed by
SDS-polyacrylamide gel electrophoresis and by Western blot analysis
using antibody that reacts with total eIF-2 or antibody that reacts
with eIF-2 -P (B). The fold induction of eIF-2
phosphorylation in response to TNF was measured by densitometry analysis and is presented as a relative ratio of phosphorylated eIF-2 to nonphosphorylated eIF-2 (*).
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The requirement for eIF-2
phosphorylation for TNF
-induced
apoptosis was analyzed by TNF
treatment of NIH3T3 cells expressing either wild-type or S51A mutant eIF-2
. Flow cytometry analysis of
DNA content (Fig. 6A), as well
as the DNA fragmentation analysis (Fig. 6B), demonstrated
that expression of the phosphorylation-resistant S51A mutant eIF-2
partially protected cells from apoptosis upon TNF
treatment. We
expect that the incomplete protection may be due to the presence of
endogenous wild-type eIF-2
, which may become phosphorylated and act
in a dominant manner to inhibit protein synthesis.

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Fig. 6.
Expression of S51A mutant eIF-2 protects
cells from TNF -induced apoptosis. Control NIH3T3 cells and
NIH3T3 cells that express either wild-type or S51A mutant eIF-2 were
treated with TNF (10 ng/ml for 20 h) and analyzed by flow
cytometry as described under "Materials and Methods"
(A). In parallel, DNA was prepared and analyzed by
electrophoresis on an agarose gel as described under "Materials and
Methods" (B). Quantitation by densitometry demonstrated a
3-fold increase in the level of fragmented DNA in NIH3T3 cells stably
transfected with vector alone compared with cells transfected with S51A
mutant eIF-2 (lane 3 versus lane 7 and lane 4 versus lane 8).
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To test the hypothesis that activation of PKR is sufficient to induce
apoptosis, a wild-type PKR expression vector was introduced into cells
by transient DNA transfection. In these studies, COS-1 monkey cells
were co-transfected with an expression vector encoding the GFP (26) to
identify the transfected cells. Co-transfection with wild-type PKR
reduced expression of GFP, as indicated by the reduced fluorescence
emitted by the co-transfected cells compared with control vector
co-transfected cells (Fig. 7A,
pETF.VA
versus PKRwt). This is consistent with
an inhibition of protein synthesis mediated by PKR overexpression.
Transfection of the wild-type PKR expression vector, but not that of
expression vector alone, induced apoptosis in the majority of cells
that received GFP as detected by nuclear fragmentation (Fig.
7A). To test whether phosphorylation of eIF-2
itself is
sufficient to induce apoptosis, COS-1 monkey kidney cells were
co-transfected with an expression vector encoding GFP and S51D mutant
eIF-2
, a mutant that mimics phosphorylated eIF-2
(27), into cells
by transient DNA transfection. Transfection of S51D mutant eIF-2
reproducibly induced apoptosis in cells that received GFP compared with
cells transfected with the vector alone (Fig. 7A;
pETF.VA
versus eIF2
S51D) or cells
transfected with an expression vector encoding wild-type eIF-2
(data
not shown). In addition, flow cytometric analysis of S51D mutant
eIF-2
transfected cells demonstrated a higher percentage of
hypodiploid cells (55%) compared with the cells transfected with the
vector control (33%) (data not shown). Apoptosis induced by
overexpression of wild-type PKR or S51D mutant eIF-2
was also
confirmed by DNA fragmentation analysis (Fig. 7B).
Expression of either wild-type PKR or S51D mutant eIF-2
increased
the amount of degraded DNA by 3-fold compared with expression of K296P
mutant PKR, S51A mutant eIF-2
, the PKR dsRNA binding domain (amino
acid residues 1-243), or Bcl-2. Under the transfection conditions,
approximately 30% of the cells were transfected, and this may account
for the small increase in the amount of degraded DNA. These results
indicate that overexpression of either wild-type PKR or S51D mutant
eIF-2
is sufficient to induce apoptosis.

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Fig. 7.
Overexpression of wild-type PKR or S51D
mutant eIF-2 induces apoptosis. COS-1 monkey kidney cells were
co-transfected with expression vectors encoding the indicated products
with an expression vector encoding the jellyfish GFP to identify
transfected cells. After 48 h, nuclei were stained with Hoechst
33258 and were analyzed for nuclear fragmentation by immunofluorescence analysis (A). In addition, DNA from transfected cells was
prepared and analyzed by electrophoresis on an agarose gel as described under "Material and Methods" (B).
|
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DISCUSSION |
PKR was first identified as a mediator of the antiproliferative
and antiviral action of interferon (1, 38). The antiproliferative action of PKR was previously demonstrated through expression of wild-type and catalytically inactive mutant PKR. Overexpression of
wild-type PKR inhibited protein synthesis and cell growth (7, 8),
whereas overexpression of a catalytically inactive mutant PKR
transformed NIH3T3 cells (11-13). The mechanism for mutant PKR-induced
transformation is likely either through formation of inactive
heterodimers with endogenous PKR or through competition for endogenous
PKR activators. Both of these mechanisms invoke disruption of the
proper regulation of endogenous PKR activity. Using this
trans-dominant negative property of the catalytically inactive PKR, we have shown that a functional PKR pathway is required to induce apoptosis in response to serum deprivation or TNF
treatment. Our results confirm and extend recent observations that
implicate PKR as a general transducer of the apoptotic response (6, 32, 33, 35). Although it was appreciated for many years that dsRNA in the
presence of interferon is toxic to cells, it was only recently suggested that PKR mediates the apoptotic response to dsRNA upon vaccinia virus (6, 35, 39) and influenza virus (40) infection. Recent
observations also support the idea that TNF
-induced apoptosis requires a functional PKR pathway, whereas either expression of antisense PKR mRNA or deletion of the PKR gene reduced the
apoptotic response to TNF
(32, 33). We have demonstrated that
apoptotic responses to TNF
, serum deprivation, and interferon
treatment with poly(I)·poly(C) require a functional PKR pathway by
expression of a trans-dominant negative mutant of PKR. In
addition, forced expression of wild-type PKR was sufficient to induce
apoptosis; this is also consistent with previous observations (33). PKR activation was also previously demonstrated to occur in response to
stress conditions, such as activation of the heat shock response, presence of unfolded protein in the endoplasmic reticulum, growth factor depletion, and increases in cytosolic calcium (23, 41-43). All
of these stress responses are frequently accompanied by apoptosis. These results support the idea that PKR is a general transducer of
apoptosis in response to a variety of different stimuli. It seems
probable that the tumor suppressor activity previously attributed to
PKR (11, 12) is mediated through its proapoptotic function.
Two fundamental questions remain regarding the mechanism of PKR-induced
apoptosis: 1) what mechanism(s) activates PKR in the absence of a viral
infection? and 2) what are the immediate substrates downstream of PKR
that lead to apoptosis? Although it is most likely that PKR activation
upon viral infection occurs through expression of dsRNA from the viral
genome, it is less obvious what activates PKR in the absence of a viral
infection, and numerous possibilities exist. Ligand binding-induced
trimerization of the TNF
receptor may elicit activation of an
upstream protein kinase, such as the TNF
receptor-interacting
protein RIP (44), that phosphorylates and activates PKR. Alternatively,
activation of an interleukin-1 converting enzyme-like protease in
response to TNF
or other stress inducers may cleave the dsRNA
binding domain from PKR to generate a kinase domain fragment that is
known to act as a constitutively active kinase (28). It is also
possible that induced synthesis of a cellular dsRNA molecule or a
change in secondary structure of a preexisting RNA molecule may elicit PKR activation. Previous studies demonstrated that PKR activation in
response to increased cytosolic calcium required the dsRNA binding
activity of PKR (23). The requirement for the dsRNA binding activity of
PKR suggests that increased cytosolic calcium activates PKR through an
RNA binding-dependent mechanism. Alternatively, the
activation of PKR may be modulated by cellular inhibitors (45-47) that
are regulated by external stimuli. For example, it was recently
demonstrated that the ubiquitously expressed PKR inhibitor identified
as p58 is inhibited by hsp40 (48), a molecular chaperone that interacts
with the translational machinery and may play a role in nascent chain
polypeptide folding (49).
The two best characterized substrates that mediate the downstream
effects of PKR are eIF-2
and I
B. Because TNF
-mediated activation of NF
B elicits transcriptional induction of antiapoptotic genes (50-52) and because PKR-deleted mouse embryo fibroblasts exhibit
normal TNF
activation of NF
B but are defective in their TNF
apoptotic response (17, 32), a direct role for activation of NF
B
would seem unlikely. In these latter studies, the
PKR-dependent apoptotic response to TNF
could be
attributed to Fas mRNA induction, possibly through the
transcription factor interferon regulatory factor 1. In contrast, the
TNF
apoptotic response that we have studied in NIH3T3 cells did not
induce Fas mRNA. However, it has been shown that PKR expression is
induced by TNF
(33), and if TNF
is activated, this may increase
the level of phosphorylated eIF-2
. We have provided four
observations that support the idea that eIF-2
is the downstream
target for PKR-induced apoptosis. First, TNF
increased the
phosphorylation status of eIF-2
in NIH3T3 cells. Second, expression
of a trans-dominant negative mutant PKR prevented the
TNF
-mediated increase in eIF-2
phosphorylation, as well as the
apoptotic response. Third, cells that express a nonphosphorylatable
S51A mutant of eIF-2
were partially protected from TNF
-induced
apoptosis. It is possible that only partial protection was observed
because these cells also express endogenous wild-type eIF-2
that
could be phosphorylated and inhibit protein synthesis in a dominant
manner. Alternatively, other mechanisms may also be involved in
addition to phosphorylation of eIF-2
. Fourth, expression of a S51D
mutant eIF-2
, which mimics a phosphorylated serine, was sufficient
to induce apoptosis. These observations support a role for eIF-2
phosphorylation in the proapoptotic response and suggest a role for
translation initiation in apoptosis. A requirement for inhibition of
new protein synthesis is frequently observed for TNF
-induced
apoptosis (53), and inhibition of protein synthesis itself is
sufficient to induce apoptosis in some systems (54, 55). Upon TNF
treatment, it is possible that PKR-mediated phosphorylation of eIF-2
and subsequent translational inhibition counteracts the protective
effect of NF
B-dependent induced transcription of genes
encoding proteins that prevent apoptosis.
Previous studies also suggest that eIF-2
is the downstream target
for PKR-mediated apoptosis. PKR mediates phosphorylation of eIF-2
in
response to calcium depletion from the endoplasmic reticulum mediated
by ionophore (23, 41), a treatment used to induce apoptosis.
Furthermore, removal of the growth factor interleukin 3 from an
interleukin 3-dependent cell line induces the
autophosphorylation of PKR, which in turn phosphorylates eIF-2
with
subsequent inhibition of protein synthesis and cell death (43).
Finally, overexpression of a nonphosphorylatable S51A mutant eIF-2
was reported to transform NIH3T3 cells (56), suggesting that wild-type
eIF-2
may act as a tumor suppressor, possibly through its
phosphorylation and subsequent inhibition of translation initiation and
induction of apoptosis. Although phosphorylation of eIF-2
is
generally thought to be a general inhibitor of global protein
synthesis, it is possible that translation of mRNAs encoding proapoptotic functions is selectively enhanced, whereas translation of
mRNAs encoding antiapoptotic functions is suppressed when levels of
eIF-2 become limited, i.e. when eIF-2
becomes
phosphorylated. The best characterized paradigm for specific mRNA
translational control mediated by eIF-2
phosphorylation is the
translation of the mRNA encoding the transcriptional activator
GCN4, which is selectively translated in response to amino acid
deprivation through activation of the eIF-2
kinase GCN2 in
Saccharomyces cerevisaie (57).
Numerous viruses have evolved gene products that inhibit apoptosis
(58). Among these antideath mechanisms are the modulation of Bcl-2
(59), inactivation of the tumor suppressor p53 (60), and inhibition of
the interleukin-1 converting enzyme-like proteases (61). PKR was
originally identified as an antiviral response mechanism of the host.
The protective response is mediated by inhibition of protein synthesis
to prohibit viral replication. However, the study presented here
indicates that activation of PKR in the context of a viral infection
may provide additional antiviral activity through induction of
apoptosis and subsequent autodigestion to prevent viral spread. Viruses
have evolved numerous mechanisms to circumvent PKR activation (62),
suggesting that PKR is an additional target for which viruses have
evolved antiapoptotic responses. The best characterized viral inhibitor
of PKR is adenovirus VAI RNA, which specifically binds the dsRNA
binding site on PKR (63). Many viruses, such as reovirus (64, 65),
rotavirus (66), and vaccinia virus (6, 39), have evolved specific dsRNA-binding proteins that can sequester dsRNA and prevent PKR activation. Vaccinia virus actually produces two gene products, E3L and K3L, that block PKR activity through different mechanisms (67,
68), and vaccinia virus deletion mutants lacking E3L activate
apoptosis (6). In contrast, influenza virus activates the cellular
inhibitor of PKR, p58 (13). Interestingly, the herpes simplex virus
gene product
34.5 inhibits virus-induced apoptosis by restoring
cellular protein synthesis, possibly through dephosphorylation of
eIF-2
(69, 70). This implies that viral PKR antagonists will prove
to be general inhibitors of apoptosis. It is possible that pathogenesis
for those viruses that do not have effective means of preventing PKR
activation is attributed to apoptotic cell death.