From the Lady Davis Institute for Medical Research,
Sir Mortimer B. Davis-Jewish General Hospital, Montréal,
Québec H3T 1E2, Canada and the § Departments of
Oncology, Medicine, Microbiology and Immunology, and Cell Biology and
Anatomy, McGill University, Montréal, Québec H3A
2B4, Canada
Received for publication, September 6, 2000, and in revised form, January 19, 2001
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ABSTRACT |
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The double-stranded RNA (dsRNA)-activated protein
kinase PKR (protein kinase
dsRNA-dependent) plays an important role in the regulation of protein synthesis by phosphorylating the In eukaryotes, RNA translation plays an important role in cell
proliferation induced by various extracellular stimuli (reviewed in
Ref. 1). Among many regulatory proteins involved in this process, the
interferon (IFN)1-inducible
double-stranded RNA (dsRNA)-activated protein kinase PKR
(protein kinase
dsRNA-dependent) is an important regulator of
translation initiation through its capacity to phosphorylate the
Several reports have assigned to PKR a tumor suppressor function
in vitro (2). Specifically, expression of wild type (WT) human PKR in yeast (5) or in mouse cells (6) results in cell growth
inhibition and in some cases in the induction of cell death by
apoptosis (7). On the other hand, expression of PKR mutants in NIH3T3
cells that are catalytically inactive or dsRNA binding-defective causes
malignant transformation and induction of tumorigenesis after injection
of the transformed cells in nude mice (6, 8-10). Contrary to these
in vitro functions, deletion of the pkr gene by
homologous recombination is not tumorigenic (11, 12). In addition, PKR
knockout (PKR Work in many laboratories using in vitro mutagenesis of
human and mouse PKR has led to an extensive characterization of the structure-function relationship of the molecule (2). Briefly, in both
species the amino-terminal half of PKR contains two RNA-binding motifs
(dsRBMs) (2), which are conserved among most of the RNA-binding
proteins (18). On the other hand, the carboxyl-terminal half of PKR is
divided into 11 subdomains, which are required for catalytic activity
(2) and are conserved among many serine/threonine protein kinases (19).
At the genomic level, the human PKR gene contains 17 exons that vary in size from 18 nucleotides in exon 1 to 840 nucleotides in exon 17 (20), whereas the mouse gene contains 16 exons
varying from 35 nucleotides in exon 8 to 750 nucleotides in exon 16 (21).
Despite the tumor suppressor function in vitro, naturally
occurring mutants of PKR in tumor cells have not as yet been identified with the exemption of a mutant form of mouse PKR in a pro-B leukemia cell line (22). Here, we report the cloning of a point mutant of human
PKR (Y176H) from Jurkat leukemia cells encoding for a protein that
retains the RNA-binding and catalytic properties of wild type PKR
in vitro. We also describe the cloning and characterization of an alternatively spliced form of PKR (PKR RNA Isolation and RT-PCRs--
The primers used in RT-PCRs
are summarized in Table I. For sequencing
of the protein-coding sequence of PKR, 1 µg of RNA isolated by the
guanidium thiocyanate method (23) was reverse transcribed using the
P303 primer. The single-stranded PKR cDNA was then amplified by PCR
using the P504/P304, P501/P301, P502/P302, and P503/P303 sets of
primers with denaturing at 94 °C for 1 min, annealing at 60 °C
for 1 min, and extension at 72 °C for 1 min for a total of 30 cycles. The PCR products, which spanned the entire protein-coding
sequence of PKR, were subcloned into pCRTMII (Invitrogen) and sequenced
with T7 DNA Polymerase (U.S. Biochemical Corp.) or subjected to direct
sequencing using the Deaza T7SequencingTM Kit
according to the supplier's instructions (Amersham Pharmacia Biotech).
The human WT PKR cDNA was subcloned into
HindIII/BamHI sites of either pcDNA3.0-neo
(Invitrogen) or pFLAG-CMV-2 vector (Eastman Kodak Co.). For transient
PKR
For PKR Cell Culture--
HeLa S3 cells (ATCC CCL-2.2) and Jurkat cells
(ATCC TIB-152) were maintained in Dulbecco's modified Eagle's medium
(Life Technologies, Inc.) and RPMI 1640 (Life Technologies),
respectively, with 10% heat-inactivated fetal bovine serum (Life
Technologies), 2 mM L-glutamine (Life
Technologies), and 100 units/ml penicillin/streptomycin (Life
Technologies). Immortalized PKR Protein Expression with the Vaccinia/T7 Virus System--
One
day before transfection, 0.8 × 106 HeLa S3 or
PKR Protein Extraction and PKR Autophosphorylation--
Cells were
washed twice with ice-cold 1× phosphate buffer saline, and proteins
were extracted with a lysis buffer containing 10 mM
Tris-HCl, pH 7.5, 50 mM KCl, 2 mM
MgCl2, 1% Triton X-100, 1 mM dithiothreitol,
0.2 mM phenylmethylsulfonyl fluoride, 3 µg/ml aprotinin,
1 µg/ml leupeptin, and 1 µg/ml pepstatin. After incubation on ice
for 20 min, the cell lysate was centrifuged at 10,000 × g for 10 min. The cytoplasmic supernatant (S10
fraction) was transferred to a fresh tube, the protein concentration
was measured by Bradford assay (Bio-Rad), and stored at
For PKR autophosphorylation in vitro, 50-500 µg of
protein extracts were subjected to immunoprecipitation with mouse
monoclonal anti-human PKR antibodies (clone F9 or E8; Ref. 26) and
anti-mouse IgG-agarose beads. PKR immunoprecipitates were equilibrated
in 1 × PKR kinase buffer consisting of 10 mM
Tris-HCl, pH 7.7, 50 mM KCl, 2 mM
MgCl2, 1 mM dithiothreitol, 0.2 mM
phenylmethylsulfonyl fluoride, 3 µg/ml aprotinin, 1 µg/ml
leupeptin, and 1 µg/ml pepstatin. PKR autophosphorylation was
performed in the presence of 0.1 µg/ml activator reovirus dsRNA and 1 µCi of [ Generation of an Anti-phosphoserine 51-specific eIF-2 Western Blotting--
Protein extracts or PKR immunoprecipitates
were subjected to SDS-PAGE and proteins were transferred onto nylon
ImmobilonTM P membrane (Millipore Corp.). Immunoblots were
performed with mouse monoclonal anti-human PKR, anti-FLAG (Kodak;
catalog no. IB13025), anti-human eIF-2 Yeast Strains and Growth Analysis--
The yeast strains used in
this study are summarized in Table II.
PKR, PKR Cloning of a Point Mutant and an Alternatively Spliced Form of PKR
from the Human Leukemia Jurkat T Cells--
We have reported a
diminished PKR activation in various human leukemia cell lines
including Jurkat T lymphocytes (26). We speculated that PKR
inactivation in these cells might be caused by mutations in the
PKR gene. To test this possibility, we amplified and
sequenced the PKR cDNA from Jurkat cells and normal PBMCs after
RT-PCR as described under "Materials and Methods." Direct sequencing of the PCR products verified the presence of a T to C point
mutation in Jurkat PKR cDNA at nucleotide 526 downstream from the
initiator ATG (Fig. 1A), which
results in a single substitution of tyrosine 176 to histidine. When the
PCR products were subcloned into pCRII vector and sequenced (see
"Materials and Methods"), nine out of 10 clones contained the T to
C mutation. One clone, however, harbored a 77-bp deletion, which
corresponds to the entire exon 7 of the human PKR gene (Fig.
1B). Deletion of exon 7 leads to the conjunction of exons 6 and 8 with a frameshift that introduces a stop codon (TGA) within exon
8 and produces an RNA encoding for a 174 amino acid protein. This
truncated protein contains the two dsRBMs of PKR and herein is named
PKR
We confirmed the expression of the PKR Biochemical Characterization of PKR PKR
Similar observations were made when the dominant negative function of
PKR
The dominant negative function of PKR Functional Characterization of the Dominant Negative Function of
PKR
The expression of PKR, FLAG-PKR
Next, we examined the dominant negative effect of PKR Activation of Reporter Gene Expression by PKR Tissue Distribution of PKR In this paper, we have characterized the function of an
alternatively spliced form of PKR produced by a deletion of exon 7 (PKR Analysis of the biochemical characteristics of PKR We have seen that self-association of PKR-subunit of
eukaryotic initiation factor 2. Through this activity, PKR is thought
to mediate the antiviral and antiproliferative actions of interferon.
Here, we show that the human T cell leukemia Jurkat cells express an
alternatively spliced form of PKR with a deletion of exon 7 (PKR
E7),
resulting in a truncated protein that retains the two dsRNA-binding
motifs. PKR
E7 exhibits a dominant negative function by inhibiting
both PKR autophosphorylation and eukaryotic initiation factor 2
-subunit phosphorylation in vitro and in vivo. Reverse transcriptase-polymerase chain reaction assays
showed that PKR
E7 is expressed in a broad range of human tissues at variable levels. Interestingly, expression of PKR
E7 is higher in
Jurkat cells than in normal peripheral blood mononuclear cells, raising
the possibility of a role in cell proliferation and/or transformation.
Thus, expression of alternatively spliced forms of PKR may represent a
novel mechanism of PKR autoregulation with important implications in
the control of cell proliferation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunit of eukaryotic initiation factor 2 (eIF-2
) (reviewed in
Ref. 2). Binding of PKR to dsRNA results in its activation by
autophosphorylation and subsequently in the phosphorylation of eIF-2
(2). Phosphorylation of eIF-2
by PKR on serine 51 leads to an
increased affinity of the initiation factor for eukaryotic translation
initiation factor 2B, also known as guanine exchange factor, and thus
increases the proportion of the latter that is trapped as an inactive
complex with eIF-2 and GDP (reviewed in Ref. 3). The reduction in free
eukaryotic translation initiation factor 2B results in a fall of the
overall rate of guanine nucleotide exchange on the remaining
unphosphorylated eIF-2, eventually leading to an inhibition of
translation initiation (3). In addition to translational control, PKR
has been implicated in signaling pathways leading to transcriptional
activation by dsRNA, virus infection, various cytokines, or genotoxic
stress (reviewed in Ref. 4).
/
) mice are not susceptible to
virus infection (11, 12) with the exemption of encephalomyelocarditis
virus after priming with IFN-
(12) or vesicular stomatitis virus
after intranasal infection (13, 14). Therefore, it has been
suggested that the lack of PKR may be compensated by the expression of
other PKR-like molecules whose function is possibly blocked by the
expression of the PKR mutants in vitro (2, 11). This is
supported by the cloning and characterization of PKR-related genes,
such as PKR-like endoplasmic reticulum kinase/pancreatic
eIF-2
kinase, which functions as an eIF-2
kinase (15), and the
mouse homologue of the yeast eIF-2
kinase GCN2 (16). Thus, PKR may
be the prototype of a family of kinases with overlapping biochemical
and biological functions (17).
E7) from Jurkat cells. PKR
E7 is a splicing product of exon 7 of the human PKR
gene, which contains the two dsRBMs and exhibits a dominant negative function in vitro an in vivo. PKR
E7 is
differentially expressed in various types of normal human tissue, and
we provide evidence that its expression may play a role in induction of
cell proliferation as a result of PKR inactivation.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
E7 expression, the 900-bp NcoI/AccI
fragment of the WT PKR in pcDNA3.0-neo or pFLAG-CMV-2 vector was
replaced with the corresponding fragment from pCRTMII vector carrying
the
E7 deletion to produce PKR
E7 or FLAG-PKR
E7 under the
control of the human cytomegalovirus promoter. For expression in yeast,
the HindIII/BamHI fragments of PKR
E7 and
FLAG-PKR
E7 were subcloned into the corresponding sites of pYES2
vector (Invitrogen).
PCR primers
E7 RNA expression, 1 µg of RNA was subjected to RT using
the P305 primer and PCR amplification using the P505/P305 set of
primers. For expression of PKR
E7 in human tissues, the human
multiple tissue cDNA (K1420-1) and the human immune system panel (K1426-1) from CLONTECH were used. The
normalized cDNAs were amplified by PCR-denaturation at 94 °C for
1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min for a total of 30 cycles using the P505/P305 set of primers. The
PCRs were subjected to agarose or polyacrylamide gel electrophoresis
and visualized by ethidium bromide or silver staining (24), respectively.
/
fibroblasts (11, 12) were grown in Dulbecco's modified Eagle's medium
supplemented with 10% heat-inactivated calf serum, 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin.
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy
human adults using the Ficoll-Paque® method according to the
supplier's instructions (Amersham Pharmacia Biotech). Newly isolated
lymphocytes were stimulated with 10 units/ml phytohemagglutinin (Life
Technologies) and maintained in RPMI 1640 (Life Technologies) medium
supplemented with 10% fetal bovine serum (Life Technologies), 2 mM L-glutamine (Life Technologies), and 100 units/ml penicillin-streptomycin (Life Technologies) for 3 days.
/
cells were seeded in 6-cm plates. One
h before transfection, the cells were infected with recombinant
vaccinia virus containing the bacterial T7 RNA polymerase gene (25).
Transfection was performed using the ratio of 1 µg DNA to 2.5 µg of
LipofectAMINE (Life Technologies) per plate, and cells were incubated
in serum-free medium at 37 °C for 5 h. Subsequently,
complete medium was added, and cells were grown for an additional
16 h before analysis.
85 °C.
-32P]ATP (ICN). After incubation at 30 °C
for 20 min, the reactions were subjected to SDS-PAGE, and radioactive
bands were visualized by autoradiography. Alternatively, the in
vitro kinase assay of PKR was performed with S10
protein extracts in 1× PKR kinase buffer in the conditions described
above. The autophosphorylated PKR was then immunoprecipitated with
mouse monoclonal anti-human PKR antibodies, subjected to SDS-PAGE, and
visualized by autoradiography.
Antibody--
Rabbit antiserum was produced against a chemically
synthesized phosphopeptide ILLSELpSRRRIRS (where pS represents
phosphoserine) that contains serine 51 of human eIF-2
. The
antibody was purified from rabbit serum by sequence-specific
chromatography and was negatively preadsorbed using a nonphosphopeptide
corresponding to the site of phosphorylation to remove antibody that is
reactive with nonphosphorylated eIF-2
protein. The final product was
generated by affinity chromatography using an eIF-2
-derived peptide
phosphorylated at serine 51.
antibodies, rabbit polyclonal
anti-phosphoserine 51 eIF-2
antibodies (homemade or from
BIOSOURCE, catalog no. 44-728), and rabbit
antiserum to TrpE-yeast eIF-2
fusion protein (CM-217) at a
concentration of 1 µg/ml using the standard protocol (27). After
incubation with horseradish peroxidase-conjugated anti-mouse or
anti-rabbit IgG antibodies (1:1000 dilution; Amersham Pharmacia
Biotech), proteins were visualized with the enhanced chemiluminescence
(ECL) detection system according to the manufacturer's instructions
(Amersham Pharmacia Biotech). Quantification of the bands in the linear
range of exposure was performed by densitometry using the NIH Image
1.54 software.
E7, and PKRLS9
E7 in pYES2 vector were transformed into
these strains and selected in SD-Trp medium as described (28).
Single colonies were picked up and grew in SD-Trp liquid medium at
30 °C to A600 = 1.5, and the liquid
cultures were streaked on SGLU and SGAL minimal plates containing 10%
glucose and 20% galactose, respectively, and incubated at 30 °C for
up to 72 h. The yeast growth was measured by diluting the liquid
cultures to A600 = 0.01 in synthetic medium
containing 10% galactose, 2% raffinose, and the required amino acid
supplement (SGAL). The cultures were incubated at 30 °C for
various time periods, and a 0.5-ml liquid culture from each point was
used to measure A600.
Yeast strains
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
E7 (Fig. 2).
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Fig. 1.
Identification of a point mutant and an
alternatively spliced form of PKR in Jurkat cells. A,
direct sequencing of the PCR products of the PKR gene from
normal PBMCs and Jurkat cells. The single point mutation on the
nucleotide sequence, which results in the change of amino acid tyrosine
176 to histidine, is indicated with an asterisk.
B, an alternatively spliced form of PKR revealed by DNA
sequencing. Shown is a comparison of DNA sequence between full-length
Jurkat PKR and the alternatively spliced form PKR E7. The deletion of
77 bp (exon 7) in PKR
E7 cDNA is indicated.
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Fig. 2.
Schematic illustration of the alternatively
spliced form PKR E7. A, the
wild type human PKR cDNA contains sequences from 17 exons, where
the translation initiation codon ATG and the translation termination
codon TAG are located in exon 3 and exon 17, respectively. The Y176H
mutation in exon 7 of Jurkat gene is indicated. When exon 7 is deleted
by splicing, the reading frame is shifted thereafter and terminates by
a newly introduced stop codon TGA. The resulting truncated protein,
PKR
E7, contains the two dsRNA-binding motifs (dsRBM I and II).
B, the nucleotide and amino acid sequence translated in the
vicinity of the alternative splicing site.
E7 RNA in Jurkat cells by an
RT-PCR assay (see "Materials and Methods"). The PKR
E7 PCR
product contains a 77-bp deletion and therefore migrates faster than
the WT PKR PCR product on agarose gel electrophoresis (Fig. 3A, compare lanes
3 and 4). The ratio of the band intensities of
the two PCR products is proportional to the amount of each PKR
transcript within the cells. To examine whether PKR
E7 expression is
unique for Jurkat cells, we performed an RT-PCR assay with RNA from
normal PBMCs. We found that PBMCs contain very low levels of PKR
E7
RNA (<1% of full-length PKR RNA; see also Fig. 8), whereas PKR
E7
RNA levels in Jurkat cells is about ~10% of WT PKR transcript (Fig.
3A). We also verified the PKR
E7 protein expression by
immunoblot analysis (Fig. 3B). To facilitate the detection
of PKR
E7, Jurkat cells were treated with IFN-
/
to induce PKR
RNA expression. The protein extracts before (lane
2) and after IFN treatment (lane 3)
were subjected to immunoprecipitation and immunoblotting with antibodies specific to the N terminus domain of PKR to detect both
full-length PKR (top panel) and PKR
E7
(bottom panel). These experiments showed that
PKR
E7 protein is expressed in Jurkat cells at low levels
(lane 2), and its expression is induced after IFN
treatment (Fig. 3B, lane 3).
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Fig. 3.
Detection of PKR E7
RNA and protein expression in Jurkat cells. A, RNA
isolated from normal PBMCs (lane 5) and Jurkat
(lane 6) was subjected to RT-PCR. PCR products
were fractionated in 2% agarose gel and stained with ethidium bromide.
A DNA molecular weight (M.W.) marker was loaded to indicate
the size of the PCR products (lane 1). As a
negative control, a PCR containing no DNA template was used
(lane 2). As positive controls, PCR products from
wild type PKR (lane 3) and PKR
E7
(lane 4) cDNAs were 162 and 85 bp,
respectively. B, 200 µg of Jurkat S10 protein
extracts before (lane 2) or after
IFN-
2b treatment (1000 IU/ml; 18 h)
(lane 3) were immunoprecipitated with a rabbit
polyclonal anti-human PKR antibody specific for the N-terminal domain
of PKR. Immunoprecipitates were then subjected to Western blotting with
the mouse monoclonal anti-human PKR (F9) antibody. The expression
levels of full-length PKR (top panel) and
PKR
E7 (bottom panel) are shown. Note that PKR
levels were visualized by ECL after film exposure for 10 s,
whereas PKR
E7 levels were visualized after 1 min. As a positive
control (Ctl) to PKR
E7, 10 µg of S10
protein extracts from HeLa cells transfected with PKR
E7 were run in
lane 1. Detection of endogenous HeLa PKR
(top panel, lane 1) was
possible after long film exposure (data not shown).
E7--
Characterization of
Y176H mutation showed that PKRY176H retains both the dsRNA binding and
catalytic activities of WT PKR in vitro (data not shown). As
a result of it, we concentrated our efforts on characterizing the
function of PKR
E7. The N terminus domain of PKR is involved in
dsRNA-binding (29). PKR
E7 is similar to an artificially made N
terminus-truncated form of PKR, known as p20, which contains the two
contiguous dsRBMs. p20 can bind to dsRNA (30-33) and heterodimerize
with WT PKR in yeast two-hybrid assays (32). Based on this, we wished
to examine the ability of PKR
E7 to self-associate and associate with
WT PKR in the presence and absence of dsRNA. To do so, we constructed a
fusion protein of PKR
E7 bearing the FLAG epitope in the N terminus
end. When FLAG-PKR
E7 and PKR
E7 were transiently co-expressed into
HeLa cells, an equal amount of the two proteins was
co-imunoprecipitated with anti-FLAG antibodies (Fig.
4A, lane
8), confirming their ability to self-associate. In similar
assays, an equal amount of endogenous WT PKR (Fig. 4B,
top panel, lane 1) and
PKR
E7 (bottom panel, lane
1) was found to associate FLAG-PKR
E7 (middle
panel, lane 1). However, treatment
with micrococcal nuclease (MN) diminished the association of
FLAG-PKR
E7 with WT PKR (top panel,
lane 2) without affecting its association with
PKR
E7 (bottom panel, lane 2). These data suggested that self-association of PKR
E7
may take place in the absence of dsRNA, whereas its association with
full-length PKR is dsRNA-dependent. To further investigate
this possibility, we used a FLAG-PKR
E7 construct bearing the LS9
mutation (substitutions of alanine 66 and alanine 68 to glycine 66 and
proline 68), which completely abolishes dsRNA binding (34).
Co-expression of FLAG-PKRLS9
E7 and PKR
E7 in HeLa cells and
immunoprecipitation with anti-FLAG antibody revealed the lack of
association of FLAG-PKR
E7 with either PKR
E7 (lanes
3 and 4, bottom row) or the
endogenous PKR (lanes 3 and 4,
top row). Since the LS9 mutation may affect the conformation of the dsRNA-binding domain of PKR (34), these data
suggested that the integrity of dsRNA-binding domain is essential for
PKR
E7 self-association and association with full-length PKR. In
these experiments, we noticed that a higher amount of FLAG-PKR
E7 was
immunoprecipitated with anti-FLAG antibodies after MN treatment. One
plausible explanation is that binding of RNA to FLAG-PKR
E7 impedes
the accessibility of the antibody to FLAG epitope, and this inhibition
may be alleviated by MN treatment.
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Fig. 4.
Self-association and association of
PKR E7 with wild type PKR. A,
PKR
E7 and FLAG-PKR
E7 were expressed in HeLa cells with the
vaccinia virus/T7 system. 100 µg of S10 protein extracts
were immunoprecipitated (IP) with mouse monoclonal anti-FLAG
antibody (lanes 5-8). Proteins were separated in
SDS-14% PAGE and subjected to Western blotting (WB) using
mouse monoclonal anti-human PKR (F9) antibody. In parallel, 20 µg of
S10 protein extracts from each transfection was used to
show the PKR
E7 expression levels (whole cell extracts
(WCE), lanes 1-4). B,
PKR
E7 and FLAG-PKR
E7 or PKR
E7 and FLAG-PKRLS9
E7 were
co-expressed in HeLa cells as above. Protein extracts were left
untreated (lanes 1 and 3) or treated
(lanes 2 and 4) with MN and
immunoprecipitated with mouse monoclonal anti-FLAG antibody. Proteins
were separated in SDS-14% PAGE and subjected to Western blotting using
mouse monoclonal anti-PKR (F9) antibody (top and
bottom panels) followed by immunoblotting with
mouse monoclonal anti-FLAG antibody (middle
panel).
E7 Exhibits a Dominant Negative Function--
The ability of
PKR
E7 to associate with WT PKR prompted us to examine for a possible
dominant negative function in PKR activation. To this end, first we
assessed the ability of FLAG-PKR
E7 to inhibit the
autophosphorylation of endogenous PKR when transiently expressed in
HeLa cells using the vaccinia/T7 virus system (25). Because the
vaccinia/T7 virus system is a two-step procedure utilizing transfection
with LipofectAMINE and infection with recombinant virus (see
"Materials and Methods"), we measured PKR activation in cells
treated with LipofectAMINE and vector DNA (Fig
5A, mock, lane 1), LipofectAMINE plus vector DNA plus virus
(mock, lane 2), or LipofectAMINE plus
FLAG-PKR
E7 cDNA plus virus (lane 3). The
activation of endogenous PKR was measured first by autophosphorylation in the protein extracts in vitro followed by
immunoprecipitation with an anti-human PKR antibody (Fig.
5A). We observed that PKR autophosphorylation, after
normalization to protein levels, was more highly induced by the virus
(Fig. 5A, compare lane 2 with lane 1) presumably by the production of activator
dsRNA during infection. On the other hand, FLAG-PKR
E7 expression
resulted in the inhibition of PKR autophosphorylation (Fig.
5A, lane 3) compared with
mock-transfected cells in the presence (lane 2) or absence (lane 1) of vaccinia/T7 virus. The
levels of endogenous PKR or FLAG-PKR
E7 were detected by immunoblot
analysis with the anti-human PKR (middle panel)
or anti-FLAG antibody (bottom panel), respectively. These data argued for a dominant negative effect of
PKR
E7 on PKR activation in vitro.
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Fig. 5.
Dominant negative function of
PKR E7 in vitro and in
vivo. A, HeLa cells were transfected with
pFLAG-CMV-2 vector and LipofectAMINE in the absence (lane
1) or presence (lane 2) of recombinant
vaccinia/T7 virus (mock transfections). FLAG-PKR
E7 cDNA was
overexpressed using LipofectAMINE and vaccinia/T7 virus
(lane 3). 100 µg of S10 protein
extracts was subjected to in vitro PKR kinase assay in the
presence of reovirus activator dsRNA (0.1 µg/ml) and
[
-32P]ATP (1 µCi). PKR was then immunoprecipitated
with a rabbit polyclonal anti-PKR antibody, and 32P-labeled
proteins were separated in SDS-10% PAGE and visualized by
autoradiography (top panel). In parallel, 20 µg
of S10 protein extracts used in the in vitro
kinase assay were separated in SDS-10% PAGE (middle
panel) or SDS-14% PAGE (bottom panel)
and subjected to Western blotting with mouse monoclonal anti-PKR (F9)
antibody or mouse anti-FLAG antibody, respectively. B and
C, PKR
/
fibroblasts were
transfected with vector DNA (lane 1), WT human
PKR cDNA (lane 2), PKR
E7 cDNA
(lane 3), or WT human PKR and PKR
E7 cDNAs
(lane 4) using the vaccinia/T7 virus system. 100 µg of S10 protein extracts was immunoprecipitated with a
rabbit polyclonal anti-human PKR antibody and subjected to in
vitro PKR kinase assay as described above. Proteins were separated
in SDS-10% PAGE, and the PKR autophosphorylation was visualized by
autoradiography (top panel). In parallel, 50 µg
of S10 protein extracts from transfected cells was used to
show the expression levels of PKR (middle panel)
or PKR
E7 (lower panel) by Western blotting
using mouse monoclonal anti-PKR (F9) antibody. C, HeLa cells
were transfected with WT eIF-2
cDNA (lane
2), the serine 51 to alanine mutant of eIF-2
cDNA
(lane 3), or wild type eIF-2
cDNA together
with PKR
E7 cDNA (lane 4) using the
vaccinia/T7 virus system. 20 µg of S10 protein extracts
was subjected to Western blotting first with a homemade rabbit
polyclonal anti-phosphoserine 51 eIF-2
specific antibody
(top panel). An equal amount of the same extracts
was used for immunoblotting with a mouse monoclonal anti-eIF-2
antibody (middle panel) or mouse monoclonal
anti-PKR (F9) antibody (bottom panel).
D, HeLa cells were transfected with vector DNA and
LipofectAMINE in the absence (lane 1) or presence
(lane 2) of recombinant vaccinia/T7 virus (mock
transfections) or with PKR
E7 cDNA, LipofectAMINE, and
vaccinia/T7 virus (lane 3). Protein extracts
(S10, 50 µg) were subjected to SDS-10% PAGE and
immunoblot analysis with a phosphospecific anti-eIF-2
serine 51 antibody (BIOSOURCE) (top
panel). An equal amount of protein was used for Western
blotting with a mouse monoclonal anti-eIF-2
antibody
(middle panel) or the mouse monoclonal anti-human
PKR (F9) antibody (bottom panel).
A-D, the intensity of the bands was quantified with the NIH
Image 1.54 software, and the ratios of autophosphorylated PKR to PKR
protein levels (A and B) or eIF-2
serine 51 phosphorylation to the amount of eIF-2
protein (C and
D) are indicated. vv/T7, vaccinia/T7 virus
system.
E7 was tested in fibroblasts derived from a mouse with targeted
disruption of the catalytic domain of PKR
(C-PKR
/
cells; Ref. 11) (Fig.
5B). Transient expression of PKR
E7 with human WT PKR into
C-PKR
/
cells resulted in inhibition of PKR
autophosphorylation (top panel, lane
4) compared with expression of WT PKR alone (top
panel, lane 2). Immunoblot analysis
with an anti-human PKR antibody verified the expression of WT PKR
(middle panel) and PKR
E7 (bottom
panel). The partial down-regulation of PKR
E7
(bottom panel, lane 4)
could possibly be explained by differences in transfection efficiency between the various samples.
E7 in PKR autophosphorylation
prompted us to examine the inhibition of eIF-2
phosphorylation. To
this end, we performed transient transfections of PKR
E7 cDNA together with either WT eIF-2
or a phosphorylation-defective mutant
of eIF-2
(serine 51 to alanine; Ref. 35) cDNA (Fig. 5C). Phosphorylation of eIF-2
in vivo was then
detected by immunoblot analysis using a homemade rabbit polyclonal
antibody specific to phosphoserine 51 of eIF-2
(see "Materials and
Methods"). Transient expression of WT eIF-2
resulted in induction
of phosphorylation on serine 51 caused by the activation of endogenous
HeLa PKR (36) (top panel, lane
2). Co-expression of WT eIF-2
with PKR
E7
(lanes 4) resulted in inhibition of eIF-2
serine 51 phosphorylation (top panel, compare
lane 4 with lane 2),
whereas the expression levels of eIF-2
remained stable
(middle panel, lanes 2 and
6). Note the low (undetectable) levels of endogenous
eIF-2
phosphorylation with this antibody (lane
1) and the lack of its cross-reactivity with the serine 51 to alanine mutant of eIF-2
(lane 3). The
phosphorylation levels of endogenous HeLa eIF-2
were detected,
however, when a commercially available phosphoserine 51-specific
antibody was used (Fig. 5D). We measured eIF-2
phosphorylation in cells treated with LipofectAMINE plus vector DNA
alone (mock, lane 1), LipofectAMINE plus virus
plus vector DNA (mock, lane 2), or LipofectAMINE
plus vaccinia virus plus PKR
E7 cDNA (lane
3). Infection with vaccinia virus (lane
2) induced the phosphorylation of eIF-2
(compare lanes 1 and 2), which was diminished
by PKR
E7 expression (lane 3) through the
inhibition of endogenous PKR. Taken together, the above data
demonstrate the dominant negative function of PKR
E7 in both PKR
activation and eIF-2
phosphorylation.
E7 in Yeast--
It has been shown that high level of PKR
expression in Saccharomyces cerevisae is toxic
due to inhibition of general translation (5). However, at a lower level
of expression, PKR can substitute the function of GCN2, the only
eIF-2
kinase known to exist in yeast (37), by phosphorylating
eIF-2
on serine 51 and stimulating GCN4 translation, a transcription
factor involved in amino acid biosynthesis (38). To verify the dominant
negative function of PKR
E7 in vivo, we used a yeast
strain that lacks endogenous GCN2 (J110) (39) and two strains that
contain one (H2544) and two (H2543) alleles of WT human PKR,
respectively, under the control of the galactose-inducible promoter
(40). Induction of PKR expression in H2544 strain partially inhibits
growth, whereas PKR induction in H2543 completely abolishes growth.
Strains J110, H2544, and H2543 were transformed with vector alone,
FLAG-PKR
E7, or FLAG-PKR
E7LS9. As positive control, we used the
PKR inhibitor vaccinia virus K3L (40). The transformants were streaked
onto minimal medium plates containing either glucose or
galactose as a carbon source, and the effect of each of these proteins
on PKR-mediated growth inhibition was monitored. All transformants of
the isogenic J110 strain grew well in either glucose or galactose,
indicating that expression of these exogenous proteins did not perturb
normal yeast growth characteristics (Fig.
6A). In agreement with
previous studies (40), H2544 transformants containing vector DNA
without an insert demonstrated a slow growth
phenotype after PKR induction (Fig. 6A, bottom
plate). However, expression of K3L reversed this growth-inhibitory phenotype (bottom plate).
Likewise, expression of FLAG-PKR
E7 also rescued yeast growth
consistent with previous findings that the N terminus domain of PKR
from amino acid 1 to 262 rescues yeast growth inhibition by WT PKR (5).
In contrast to this, FLAG-PKRLS9
E7 was unable to counteract the
growth-inhibitory effects of PKR (bottom plate).
Growth curves showed that the ability of FLAG-PKR
E7 transformants to
rescue growth was equally potent to K3L (Fig. 6B,
middle and bottom graphs).
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Fig. 6.
Functional characterization of
PKR E7 in yeast. A, the yeast
strains J110, H2543, and H2544 were transformed with pYES2 vector,
pEMBL/yex4 vector containing K3L, and pYES2 vector containing
FLAG-PKR
E7 or FLAG-PKR LS9
E7. The transformants were streaked on
SD agar plates containing either glucose or galactose and maintained at
30 °C for 72 h. B, the transformants were incubated
in SD liquid medium containing 10% galactose and 2% raffinose at
30 °C. The growth rate was quantified by measuring the optical
density (A600) values at different time
points. C, expression levels of PKR, FLAG-PKR
E7, and
FLAG-PKRLS9
E7 in yeast. The transformants of strains J110
(lanes 1 and 2), H2544
(lanes 3 and 4), and H2543
(lanes 5 and 6) were incubated in SD
liquid medium containing 10% galactose and 2% raffinose. 20 µg of
whole protein extracts was used for Western blotting with a rabbit
polyclonal anti-human PKR antibody (top panel) or
a mouse monoclonal anti-human FLAG antibody (bottom
panel). The band above PKR that is also present in J110
cells, which lack PKR (lanes 1 and 2),
is nonspecific (NS). D, protein extracts (20 µg) from the J80 (WT eIF-2
) and J82 (serine 51 to alanine eIF-2
mutant) transformants expressing WT PKR (lanes 3,
6, 9, and 12), FLAG-PKR
E7
(lanes 2, 3, 5, and
6), or FLAG-PKRLS9
E7 (lanes 8,
9, 11, and 12) were subjected to
Western blotting analysis using a mouse monoclonal anti-FLAG antibody.
E, protein extracts from H2544 and H2543 cell (20 µg)
transformed with vector DNA only (lanes 1 and
5), vaccinia virus K3L DNA (lanes 2 and 6), PKR
E7 cDNA (lanes 3 and
7), or PKRLS9
E7 DNA (lanes 4 and
8) were subjected to immunoblot analysis using a rabbit
polyclonal anti-human PKR antibody (top panel),
the homemade phosphoserine 51 eIF-2
-specific antibody
(middle panel), or mouse monoclonal
anti-eIF-2
-specific antibody (bottom panel).
The intensity of the bands was quantified with the NIH Image 1.54 software, and the ratio of eIF-2
serine 51 phosphorylation to the
amount of eIF-2
protein is indicated.
E7, and FLAG-PKRLS9
E7 in yeast was
then examined by Western blotting (Fig. 6C). PKR expression was detected in H2544 (top panel,
lanes 3 and 4) and H2543
(top panels, lanes 5 and
6) but not in the control J110 strain (lanes 1 and 2). However, expression of WT PKR in H2544
and H2543 strains was more highly induced in the presence of
FLAG-PKR
E7 than FLAG-PKRLS9
E7 (top panel,
compare lane 3 with lane 4 and lane 5 with lane 6). This higher PKR induction could be translational in nature and caused
by the dominant negative function of FLAG-PKR
E7 but not FLAG-PKRLS9
E7. The migration of PKR in polyacrylamide gels as a
doublet could have been a result of partial degradation or could represent the phosphorylated (upper band) and
nonphosphorylated forms of PKR (lower band) as
previously reported for human PKR expressed in mouse cells (41). On the
other hand, both FLAG-PKR
E7 and FLAG-PKRLS9
E7 were equally
expressed in strain J110 (bottom panel,
lanes 1 and 2). However, expression of
FLAG-PKRLS9
E7 in strains H2544 and H2543 was very low under
growth conditions in which PKR expression was induced
(bottom panel, lanes 4 and
6). Since FLAG-PKRLS9
E7 does not exhibit a dominant
negative function, we speculated that its low expression was due to
translation inhibition by PKR. To further investigate this possibility,
WT human PKR and FLAG-PKR
E7 or FLAG-PKRLS9
E7 were co-expressed
into yeast strains J80 and J82, which lack GCN2 but contain wild type
eIF-2
and the serine 51 to alanine mutant eIF-2
, respectively
(38). As shown in Fig. 6D, FLAG-PKR
E7 was equally
expressed in both strains in the absence (lanes 2 and 5) or presence of WT PKR (lanes 3 and 6). On the other hand, expression of FLAG-PKRLS9
E7
was significantly reduced in both strains when WT PKR was induced (lanes 9 and 12). These data indicated
that the inhibition of FLAG-PKRLS9
E7 expression by WT PKR may not be
translational in nature, since PKR-mediated inhibition of protein
synthesis cannot take place in the eIF-2
mutant-containing strain
(38). The mechanism of down-regulation of FLAG-PKRLS9
E7 by PKR is
not presently known.
E7 on
PKR-mediated eIF-2
phosphorylation in H2544 and H2543 strains (Fig.
6E). To do so, H2544 and H2543 strains were transformed with
vector DNA alone (lanes 1 and 5), the
vaccinia virus inhibitor K3L (lanes 2 and
6), PKR
E7 (lanes 3 and
7), or PKRLS9
E7 (lanes 4 and
8) followed by the induction of WT PKR in the presence of galactose. WT PKR expression was detected by immunoblot analysis using
an anti-human PKR specific antibody (top panel).
Phosphorylation of eIF-2
was detected by immunoblotting using the
homemade phosphospecific antibody (middle panel)
and normalized to eIF-2
protein levels using a rabbit polyclonal
antibody to yeast eIF-2
(bottom panel). These
experiments proved that expression of K3L or PKR
E7 inhibit the
eIF-2
phosphorylation in both yeast strains (middle
panel, compare lane 1 with
lane 2 or 3 and compare
lane 5 with lane 6 or
7). On the other hand, PKRLS9
E7 expression did not
affect eIF-2
phosphorylation by PKR (compare lane
1 with lane 4, and compare
lane 5 with lane 8). Due to
the dominant negative functions of K3L and PKR
E7, expression of WT
PKR was more highly induced in the presence of these inhibitors
compared with PKRLS9
E7 (top panel, compare
lane 4 with lane 2 or
3, and compare lane 8 with lane 6 or 7). These data clearly
demonstrate the dominant negative function of PKR
E7 in PKR
activation and eIF-2
phosphorylation in yeast.
E7--
The
dominant negative function of PKR
E7 was further verified in reporter
assays in HeLa cells or in mouse fibroblasts derived from two different
PKR knockout (PKR
/
) mice (11, 12) (Fig.
7). The first
PKR
/
mouse was generated by the disruption
of the N terminus domain of the kinase (deletion of exons 2 and 3;
N-PKR
/
; Ref. 12), whereas the second was
generated by the disruption of the catalytic domain of the molecule
(deletion of exon 12; C-PKR
/
; Ref. 11).
Cells were co-transfected with the
-galactosidase reporter gene and
K3L or PKR
E7 cDNA in the absence or presence of WT human PKR
cDNA. Expression of K3L or PKR
E7 alone induced
-galactosidase
activity in HeLa cells (Fig. 7A), most likely due to the
relief of translational inhibition caused by the activation of the
endogenous PKR during transfection (36). As expected, expression of WT
PKR in HeLa cells resulted in the inhibition of
-galactosidase
activity compared with control, which was relieved by the co-expression
of either K3L or PKR
E7. Note that
-galactosidase activity was
more highly inhibited when HeLa cells were transfected with a higher
amount of WT PKR cDNA (data not shown). On the other hand, in
N-PKR
/
(Fig. 7B) and
C-PKR
/
cells (Fig. 7C) K3L but
not PKR
E7 expression resulted in an induction of
-galactosidase
activity. This effect of K3L may indicate the presence of other
eIF-2
kinase(s) that can be activated during transfection. Whether
this is PKR-like endoplasmic reticulum kinase (15), GCN2 (16), or
another PKR-like kinase is not presently known. Expression of WT human
PKR in both knockout cells led to the inhibition of
-galactosidase
activity (Fig. 7, B and C), which was relieved by
the co-expression of either K3L or PKR
E7. Taken together, these data
demonstrate the dominant negative function of PKR
E7 in PKR-mediated
inhibition of reporter gene expression. Consistent with our data, Tian
and Mathews (42) have recently reported that induction of reporter gene
expression by p20 in transient transfection assays in human 293 cells.
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Fig. 7.
Induction of gene expression by
PKR E7. HeLa cells (A) and
immortalized fibroblasts derived from mice with a targeted deletion of
exons 2 and 3 (N-PKR
/
cells; B)
or exon 12 of PKR (C-PKR
/
cells;
C) were transfected with the
-galactosidase gene (0.2 µg) in the presence of the inhibitor K3L DNA (0.5 µg), PKR
E7
cDNA (0.5 µg), WT human PKR cDNA (0.5 µg), K3L DNA and WT human
PKR cDNA (0.5 µg each) or PKR
E7 and WT human PKR cDNAs
(0.5 µg each) using the vaccinia/T7 virus system. The -fold induction
of
-galactosidase activity for each transfection is shown. The
values represent the average of four independent experiments performed
in triplicates.
E7 RNA--
To investigate the
physiological relevance of PKR
E7 expression, we examined the
expression levels of PKR
E7 relative to WT PKR RNA in various types
of normal human tissue by a RT-PCR assay. As shown in Fig.
8, PKR
E7 RNA was expressed in a broad
range of human tissues but at variable levels. In most tissues,
expression was below 5% of WT PKR RNA, whereas expression in heart,
placenta, liver, and skeletal muscle was as high as 5.3, 5.7, 5.3, and
8.4%, respectively. Expression of PKR
E7 RNA in spleen was
undetectable.
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Fig. 8.
Tissue distribution of
PKR E7 RNA. Human multiple normal tissue
cDNA (Stratagene) was used for the detection of PKR
E7 RNA
expression by PCR amplification followed by 10% polyacrylamide gel
electrophoresis and silver staining (upper
panel). The proportion of PKR
E7 RNA compared with
full-length PKR RNA in each tissue was quantified using the NIH Image
1.54 software (lower panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
E7). Although alternative splicing has been previously described for the 5'-untranslated region of PKR mRNA (43), to our knowledge this is the first study to describe the expression of an alternatively spliced product of human PKR with a dominant negative function. PKR
E7 is composed of the two copies of the dsRBMs of PKR, a sequence motif found in many dsRNA-binding proteins (18).
E7 has shown that
it binds to dsRNA and is capable of both self-associating and
associating with full-length WT PKR. Both properties of PKR
E7 appear
to require the presence of dsRNA, since treatment with MN
abolishes its association with WT PKR but partially diminishes its
self-association (Fig. 4). These findings are in accord with previous
observations by Wu and Kaufman (44) that dimerization of intact PKR
with p20 requires the dsRNA binding activity. Consistent with this,
recent studies by Tian and Mathews have shown that the efficacy or rate
of p20/PKR dimerization through a protein/protein interaction is
considerably less than that of their dsRNA-mediated dimerization (42).
Our data show that self-association of PKR
E7 can also take place
independently of dsRNA, and this is in line with previous data showing
that dimerization of p20 is independent of RNA (45). In accord with
this, the intrinsic ability of RNA-free preparations of p20 to dimerize
in the absence of dsRNA has been recently reported (42). Therefore,
PKR
E7 dimerization in vivo may take place in the absence
of dsRNA, whereas the presence of dsRNA may induce conformational
changes that facilitate heterodimerization and/or heterodimer
stabilization between PKR
E7 and WT PKR (42, 45).
E7 is stronger than its
association with full-length PKR, in agreement with a previous study
showing that homodimerization of p20 in yeast two-hybrid assays is
better than its heterodimerization with the full-length PKR (32). The
requirement of dsRNA for PKR
E7 binding to PKR might give the
specificity for PKR
E7 to selectively associate with PKR that is
bound to dsRNA supporting the notion that dsRNA binding is required for
the dominant negative activity of the N terminus truncated form of PKR
(44). In regard to this, the dominant negative function of PKR
E7
could be mediated through its interaction with the WT PKR, resulting in
the formation of inactive heterodimers (Fig.
9). This may be limited to those latent PKR molecules that are accessible to dsRNA, and this could relieve possible localized inhibitory effects of PKR on protein synthesis. Alternatively, expression of PKR
E7 may lead to the sequestration of
cellular activator dsRNA, resulting in the inhibition of WT PKR
activation (Fig. 9). Consistent with this notion, Tian and Mathews have
recently shown that the dominant negative function of p20 correlates
with the ability of the two dsRBMs to bind to dsRNA but not with their
ability to dimerize, supporting the view that dsRNA sequestration may
underlie the dominant negative effect (42).
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Fig. 9.
Possible biochemical and biological functions
of PKR E7 in vivo. In
inactive form, the N terminus dsRNA-binding domain (DRBD) of
PKR folds over the C terminus kinase domain (KD) keeping it
in a "closed" conformation (61). Binding of dsRNA induces PKR
homodimerization and exposes the kinase domain, resulting in activation
by autophosphorylation (61). Activated PKR induces the phosphorylation
of eIF-2
on serine 51, leading to the inhibition of translation
initiation, cell growth, and/or virus replication (step
1). PKR
E7 may exert the dominant negative function by
binding to and blocking PKR homodimerization (step
2) and/or by sequestering dsRNA from binding to and
activating PKR (step 3). The low levels of
PKR
E7 (10% compared with full-length PKR) are compatible with the
notion for a local dominant negative function in PKR activation and
eIF-2
phosphorylation. Such local effects have been thought to be
maintained by compartmentalization or anchoring of translational
factors to cytoskeletal framework and be critical for the translation
of specific mRNAs encoding for proteins that play an important role
in cell growth, transformation, or differentiation (1). Also,
PKR
E7 may exhibit functions that are independent of PKR as an
RNA-binding protein (step 4; see
"Discussion").
PKR localizes in the cytoplasm, strongly in the nucleolus, and
diffusely throughout the nucleoplasm (46-48). Studies by Tian and
Mathews (42) recently showed that the dsRBMs are required for the
localization of PKR, and this activity correlates with dsRNA binding.
The same authors demonstrated that p20 exhibits a localization
indistinguishable from that of WT PKR, suggesting a similar function
for PKRE7 (42). Interestingly, nuclear localization of PKR was
recently shown to be rapidly induced upon treatment with DNA-damaging
agents (49), but it is not known whether this process requires dsRNA
binding. The two dsRBMs of PKR were reported to be required for its
association with ribosomes, and targeting to ribosomes may bring PKR
closer to the translation machinery, thus facilitating phosphorylation
of eIF-2
(50). In the same study, a PKR mutant with deletion of the
entire kinase catalytic domain from amino acid residue 271 to 551 was
found to associate strongly with ribosomes (50). Based on this finding,
it is reasonable to speculate that PKR
E7 competes with WT PKR for
binding to ribosomes, thus preventing the accessibility of PKR to
eIF-2
. However, expression of PKR
E7 compared with PKR is less
than 10% in most tissues, suggesting that the competition between the
two molecules for binding to ribosomes could be local. In fact, several
observations have supported the theory of localized activation of PKR
in regulation of translation of specific genes (2).
Expression of PKRE7 RNA was detected in a broad range of human
tissues at variable levels (Fig. 8). Heart, brain, placenta, liver, and
skeletal muscle were among the top five tissues that exhibited
expression over 5% compared with WT PKR RNA, with the highest levels
of PKR
E7 RNA expression in the skeletal muscle (8.4%). These five
tissues are all energy-demanding, and this could possibly indicate a
role of PKR in energy/intermediate metabolism pathways. The other
tissues contained less than 5%, whereas expression of PKR
E7 RNA in
spleen was undetectable. Interestingly, expression of PKR
E7 was
higher in Jurkat cells than in normal PBMCs (Fig. 3). Whether or not
this difference is a cause or an effect of the transformed phenotype of
Jurkat cells is an issue that requires further investigation.
We have shown that PKRE7 exhibits a dominant negative function in
PKR activation in mouse, human, and yeast cells, which results in
growth inhibition through the induction of eIF-2
phosphorylation. Whether the dominant negative function of PKR
E7 plays a role in
pathways other than inhibition of eIF-2
phosphorylation is currently
under investigation. For example, we have shown that PKR phosphorylates
human p53 on serine 392 in vitro, which may account for some
of the translational properties of p53 (51). Therefore, PKR
E7 may
also exhibit a dominant negative function in p53 phosphorylation
through its capacity to physically associate with PKR and p53 (data not
shown). Also, PKR
E7 contains the two dsRBMs, which have been found
in many dsRNA-binding proteins (18). These proteins bind dsRNA in a
largely sequence-independent fashion and are involved in a myriad of
biological processes such as RNA editing (52), RNA trafficking (53),
RNA processing (54), transcriptional regulation (55), and the
interferon antiviral response (56). Also, the dsRBMs of PKR and other
dsRBM-containing proteins have been shown to possess dsRNA annealing
activity and may play a role as chaperones by facilitating the folding
of cellular RNAs (42, 57). Therefore, the possibility that PKR
E7
exhibits functions that are independent of PKR cannot be excluded (Fig. 9).
Given the potential importance of the tight regulation of PKR activity
in growth control, it is not surprising that several cellular
inhibitors of PKR have been identified and characterized. For example,
the human immunodeficiency virus-1 TAR RNA-binding protein (TRBP) is a
dsRNA-binding protein that inhibits PKR (58) by binding to dsRNA and
forming heterodimers with endogenous PKR (32). Interestingly, TRBP
overexpression can transform mouse NIH3T3 cells in culture through the
inactivation of endogenous PKR (59). However, unlike TRBP, PKRE7
does not exhibit a dominant negative effect on mouse PKR (data not
shown), providing evidence for differences in the specificity between
the two dsRNA-binding inhibitors of PKR.
The question arises as to what is the physiological significance of
inactivation of PKR by PKRE7 and how the inhibitory function of
PKR
E7 differs from that of other dsRNA-binding PKR inhibitors (e.g. TRBP). One possibility is that association of each of
these dsRNA-binding proteins with PKR requires a specific RNA
structure, resulting in a local and specific inhibition of PKR
activation and eIF-2
phosphorylation. Another possibility is that
each of the heterodimers between PKR and dsRNA-binding proteins plays a
role in RNA-mediated biological processes other than translation (i.e. RNA trafficking, editing, splicing, or transport). In
this regard, activation of PKR has been implicated in the splicing of
human TNF-
mRNA (60).
In conclusion, the data presented here demonstrate the expression and
the dominant negative function of a dsRNA-binding alternatively spliced
product of human PKR. Further understanding of the basis of regulation
and function of alternatively spliced PKR products may yield important
insights into biological function of the kinase in regard to RNA
metabolism, transcription, translation, and regulation of signaling
pathways that affect cell proliferation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank T. Dever for the yeast strains J110,
H2544, H2543, J80, and J82, the pEMBLyes4/K3L DNA, and the rabbit
polyclonal anti-yeast eIF-2 antibody (CM-217); A. Darveu and G. N. Barber for mouse monoclonal anti-human PKR antibodies (clones E8 and F9); M. Mathews for PKRLS9 cDNA; N. Sonenberg for the wild
type and serine 51 to alanine mutant of eIF-2
cDNAs; and M. Clemens for mouse anti-eIF-2
monoclonal antibody.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant and a postdoctoral fellowship from the Cancer Research Society Inc. (to A. E. K. and S. L., respectively).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.
¶ Member of the Terry Fox Group of Molecular Oncology and a recipient of a Canadian Institutes of Health Research Scientist Award. To whom correspondence should be addressed: Lady Davis Institute for Medical Research, Rm. 508, Jewish General Hospital, 3755 Côte-Ste-Catherine St., Montréal, Québec, Canada H3T 1E2. Tel.: 514-340-8260 (ext. 3697); Fax: 514-340-7576; E-mail: akoromil@ldi.jgh.mcgill.ca.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M008140200
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ABBREVIATIONS |
---|
The abbreviations used are:
IFN, interferon;
dsRNA, double-stranded RNA;
GCN2 and -4, general control
nonderepressible-2 and -4, respectively;
eIF-2, eukaryotic
translation initiation factor 2
-subunit;
PKR
E7, alternatively
spliced product of human PKR with deletion of exon 7;
dsRBM, dsRNA-binding motif;
p20, a 20-kDa N terminus-truncated form of human
PKR containing the two contiguous dsRBMs;
PBMC, peripheral blood
monocyte;
RT, reverse transcription;
PCR, polymerase chain reaction;
WT, wild type;
bp, base pair;
PAGE, polyacrylamide gel electrophoresis;
MN, micrococcal nuclease;
TRBP, TAR RNA-binding protein.
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REFERENCES |
---|
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