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(Received for publication, June 13, 1995) From the
PKR is an interferon (IFN)-induced serine/threonine protein
kinase that regulates protein synthesis through phosphorylation of
eukaryotic translation initiation factor-2 (eIF-2). In addition to its
demonstrated role in translational control, recent findings suggest
that PKR plays an important role in regulation of gene transcription,
as PKR phosphorylates I
IFNs ( One of the best characterized
IFN-stimulated proteins is the double-stranded RNA-dependent protein
kinase, PKR (also known as dsRNA-PK, dsI, and DAI)(3) . PKR is
a 68-kDa polypeptide in humans and 65-kDa in mice. There is also a
yeast homologue, termed GCN2, that is involved in regulation of amino
acid biosynthesis under starvation conditions(4) . PKR is a
serine/threonine-specific protein kinase (3) that displays two
distinct kinase activities (i) activation by autophosphorylation upon
treatment with dsRNA and (ii) phosphorylation of the Cloning of
the human and mouse PKR cDNAs (7, 8, 9, 10) enabled a detailed
analysis of the structure-function relationship of the proteins (8, 9, 10, 11, 12, 13) .
The dsRNA binding domain has been localized to the N-terminal half of
the kinase(9, 11, 12, 13) . The
C-terminal half of the molecule contains all 11 conserved domains that
are present in protein kinases (14) . A single amino acid
substitution in the invariant lysine 296 in catalytic domain II of
human PKR (this invariant lysine is directly involved in ATP binding
and the phosphotransfer reaction) (14) causes the inactivation
of the human PKR, but the protein retains the ability to bind
dsRNA(11) . Studies on the role of PKR in regulation of cell
growth suggest that it may function as a tumor suppressor. Expression
of wt PKR in yeast inhibits cell growth, which correlates with
increased phosphorylation of eIF-2 The mechanism(s) of growth
suppression by wt PKR remains to be established. In addition to its
role in translational control, several reports have suggested a role
for PKR in regulation of gene
transcription(21, 22, 23, 24) . For
example, the PKR inhibitor 2-aminopurine inhibits gene transcription
that is induced by virus infection or dsRNA
treatment(25, 26, 27) . Moreover, PKR
activation by dsRNA results in phosphorylation of I To investigate the role of
PKR in signaling to the transcriptional machinery, we expressed wt
human PKR, or the dominant negative catalytically inactive mutant
PKR
The surface staining of induced 70Z/3 cells expressing sIgM was
performed with fluorescein isothiocyanate (FITC)-conjugated rabbit
anti-mouse
For transcriptional assay, nuclei (
For in vivo phosphorylation
of eIF-2
Figure 1:
A,
expression of human wt PKR and PKR
Figure 2:
Surface IgM expression is decreased in
cells expressing PKR
To examine whether the
decrease in sIgM expression was due to inhibition of
Figure 3:
LPS- or IFN-
Figure 4:
PKR
Effects on stability of mRNA
were tested by the following experiment. Following actinomycin D
treatment, total RNA from control or PKR
Figure 5:
Phosphorylation of eIF-2
Figure 6:
NF-
Genes
encoding The interaction of mitogens and cytokines with their
receptors triggers signaling cascades through the activation of kinases
which result in the phosphorylation and activation of numerous
proteins. The LPS-induced protein phosphorylation is mediated by
mitogen-activated protein kinases(56, 57) , protein
kinase C(58) , protein kinase A(58) , and tyrosine
phosphorylation(59, 60) . Interaction of IFN- PKR has also been implicated in several other
signaling pathways. For example (i) activation of PKR is required for
gene transcription induced by dsRNA(28, 29) ; (ii)
stimulation of cell growth by interleukin-3 results in a decrease of
PKR activity and eIF-2 Like other eukaryotic genes,
the A second enhancer element, which
lies 8.5 kilobases downstream of the
Volume 270,
Number 43,
Issue of October 27, 1995 pp. 25426-25434
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Gene (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B
upon double-stranded RNA treatment
resulting in activation of NF-B DNA binding in vitro (Kumar, A., Haque, J., Lacoste, J., Hiscott, J., and Williams, B.
R. G.(1994) Proc. Natl. Acad. Sci. U. S. A. 91,
6288-6292). To further investigate the role of PKR in
transcriptional signaling, we expressed the wild type human PKR and a
catalytically inactive dominant negative PKR mutant in the murine pre-B
lymphoma 70Z/3 cells. Here, we report that expression of wild type PKR
had no effect on -chain transcriptional activation induced by
lipopolysaccharide or IFN-. However, expression of the dominant
negative PKR mutant inhibited
gene transcription independently of
NF-B activation. Phosphorylation of eIF-2 was not increased
by lipopolysaccharide or IFN-, suggesting that PKR mediates
gene transcriptional activation without affecting protein synthesis.
Our findings further support a transcriptional role for PKR and
demonstrate that there are at least two distinct PKR-mediated signal
transduction pathways to the transcriptional machinery depending on
cell type and stimuli, NF-B-dependent and NF-B-independent.
)induce a large number of genes whose products
either singly or coordinately mediate antiviral, growth-inhibitory, or
immunoregulatory activities(1, 2) . IFN-mediated gene
induction is accomplished by a cascade of events in which many positive
and negative regulatory factors are involved. IFN-inducible proteins
initiate a cascade of activation of a second set of genes, whose
expression requires continued protein
synthesis(1, 2) . subunit of
the eukaryotic translation factor eIF-2(5) , a modification
that causes inhibition of protein synthesis (6) .(15) . Expression of
catalytically inactive mutants of human PKR in NIH 3T3 cells results in
malignant transformation(16, 17) . The mutants studied
consisted of either a deletion of 6 amino acids
(Leu-Phe-Ile-Gln-Met-Glu; amino acids 361-366) in subdomain V
(PKR
6) (16) or substitution of the invariant lysine 296 to
arginine (PKR K296R)(11, 17) . These findings suggest
that wt PKR is a tumor suppressor gene product whose activity can be
inhibited by the presence of catalytically inactive PKR mutants. In
this regard, a form of murine lymphoblastic leukemia is associated with
an in-frame deletion in the PKR gene, which results in expression of an
inactive protein.
The human PKR gene maps to chromosome
region 2p21-22(18, 19, 20) , and
abnormalities involving this region are observed among patients with
acute myelogenous leukemia (20) , raising the possibility of a
role for PKR in leukemogenesis.B leading
to activation of NF-B(28) . Furthermore, cells depleted of
PKR activity were unresponsive to activation of NF-B by
dsRNA(29) . Other mechanisms which are NF-B independent
cannot be excluded, however(27) .6(16) , in 70Z/3 cells. 70Z/3 is a mouse pre-B lymphoma
cell line which has been used successfully as a model system to study
transcriptional regulation of the immunoglobulin gene.
Transcription of the gene, which is thought to be the
rate-limiting event for differentiation of pre-B to B
cells(30) , is induced by a variety of mitogens and
lymphokines(31, 32) , leading to the expression of
surface immunoglobulin M (sIgM). Here, we demonstrate that
transcriptional activation of gene is mediated by PKR. Expression
of the dominant negative PKR6 resulted in inhibition of
-chain transcription induced by either LPS or IFN-. In
addition to cell growth regulation by
PKR(15, 16, 17) , these findings also provide
evidence for a role of PKR in lymphoid cell differentiation.
Cell Culture
70Z/3 cells (ATCC TIB 158) were
grown at 37 °C in complete RPMI 1640 medium (Life Technologies,
Inc.) supplemented with 10% fetal bovine serum (heat-inactivated), 2
mML-glutamine, 50 µM 
-mercaptoethanol, penicillin (100 units/ml), streptomycin
(100 units/ml), and humidified with 5%/95% CO/air gas
mixture. HeLa S3 cells (ATCC CCL 2.2) were grown in complete
Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 10% fetal bovine serum, 2 mML-glutamine, penicillin (100 units/ml), streptomycin (100
units/ml), and humidified with 5%/95% CO/air gas mixture.Transfection and Selection of Stable
Transfectants
Plasmids containing wt PKR, and PKR6 cDNAs
under the control of human cytomegalovirus promoter in the pcDNAI/neo
vector (16) were used for expression in 70Z/3 cells. Plasmid
DNA (10 µg) was linearized with KpnI and electroporated
into 1 
10
cells at 300 V-960 µF (Bio-Rad) as
described previously(33) . After electroporation, 70Z/3 cells
were cultured in nonselective medium and grown for 24 h to allow for
expression of the transfected genes. Cells were recultured in 24-well
plates at a concentration of 1 10
/ml (1 ml/well) in
medium containing G418 (Life Technologies, Inc.) at a final
concentration 400 µg/ml. Medium was replenished every 3 days. Cells
(polyclonal populations) were expanded and characterized 15 days
postselection. Independent clones were selected by a limiting dilution
method as described previously(34) .Immunoprecipitation and Immunoblotting
Cells (1
10) were washed three times with cold
phosphate-buffered saline (PBS, 140 mM NaCl, 15 mM KHPO4 (pH 7.2), and 2.7 mM KCl) and incubated
on ice with an equal volume of 2 lysis RIPA (100 mM Tris
Cl (pH 7.5), 300 mM NaCl, 2% Nonident P-40, 1%
sodium deoxycholate, and 0.2% SDS) supplemented with 2 mM dithiothreitol (DTT), 0.4 mM phenylmethylsulfonyl
fluoride (PMSF), and 4 µg/ml aprotinin. The lysate was centrifuged
at 10,000 g for 10 min, and the supernatant was
incubated with 2.5 µl of anti-PKR polyclonal antibody for 2 h at 4
°C. Then, 50 µl of 50% suspension of protein A-Sepharose 4L
(Pharmacia Biotech Inc.) in 1 RIPA were added, and incubation
was continued for additional 4 h at 4 °C under rotation. The
Sepharose beads were washed with 1 RIPA plus 1 M NaCl
twice and 1 RIPA twice. Immunoprecipitates were subjected to
electrophoresis on SDS-8% polyacrylamide gel. The separated proteins
were transferred to a nitrocellulose membrane (Schleicher &
Schuell) in 25 mM TrisCl (pH 7.5), 190 mM glycine, and 20% (v/v) methanol for 2 h at 1 Å. The filter
was first incubated with 5% (w/v) non-fat dried skimmed milk powder in
PBS for 1 h at room temperature and then with 25% fetal bovine serum
and 0.5% (v/v) Triton X-100 in PBS containing a mouse monoclonal
antibody to human PKR (13B8-F9). The blot was incubated with
peroxidase-conjugated rabbit antibody to mouse immunoglobulin G, and
proteins were visualized using the enhanced chemiluminescence system
(Amersham Corp.) according to the manufacturer's specifications.PKR Dephosphorylation Assay
For PKR
dephosphorylation, 2 10 cells were washed with
ice-cold PBS twice and lysed with an equal volume of 2 lysis
buffer (20 mM TrisCl (pH 7.5), 100 mM KCl, 4
mM MgCl, 2% Triton X-100, 2 mM DTT, 0.4
mM PMSF, and 4 µg/ml aprotinin). The lysate was
centrifuged at 10,000 g for 10 min, and the
supernatant was incubated with 2.5 µl of anti-PKR polyclonal
antibody for 2 h at 4 °C. Then, 50 µl of 50% suspension of
protein A-Sepharose 4L (Pharmacia) in 1 lysis buffer were
added, and incubation was continued for an additional 4 h at 4 °C
under rotation. The Sepharose beads were washed with high salt buffer
(20 mM TrisCl (pH 7.5), 400 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM DTT, 0.2 mM PMSF, 2
µg/ml aprotinin, and 20% glycerol) twice and low salt buffer (20
mM TrisCl (pH 7.5), 100 mM KCl, 0.1 mM
EDTA, 1 mM DTT, 0.2 mM PMSF, 2 µg/ml aprotinin,
and 20% glycerol) twice. Immunoprecipitates were subjected to
dephosphorylation by adding 3 µl of 10 phosphatase buffer
(0.5 M TrisCl (pH 9.0), 10 mM MgCl,
1 mM ZnCl, and 10 mM spermidine) and 3
units of calf intestine phosphatase (Promega) in 30 µl total volume
and incubating at 37 °C for 2 h. Then, immunoprecipitates were
washed with 1 RIPA plus 1 M NaCl twice, 1 RIPA
twice, and subjected to immunoblotting analysis as described above.Cell Induction and Immunofluorescent
Analysis
Cells were incubated at concentration 5
10 cells/ml with appropriate concentrations of inducing
agents: 10 µg/ml of Salmonella typhosa LPS (Sigma) or 100
U/ml murine recombinant IFN- (Cedarlane, Canada). For prolonged
inductions, cells were diluted daily to 5
10 cells/ml with fresh medium supplemented with the inducing agent. antibodies (BioCan) as described elsewhere (35) . Immediately after staining, cells were analyzed on a
cell sorter (FACStar, Becton Dickinson, Mountain View, CA) as
previously described (35) .RNA Extraction and Northern Blotting
Total RNA was
isolated by the guanidinium thiocyanate method(36) . RNA (10
µg) was denatured with glyoxal and dimethyl sulfoxide and subjected
to electrophoresis on a 1% agarose gel in 10 mM sodium
phosphate buffer (pH 7.0). For RNA stability experiments, total RNA was
isolated from cells treated with actinomycin D (10 µg/ml) for 30,
60, and 120 min. RNA was transferred onto a nylon membrane (BioTrans,
ICN). Hybridization was performed at 65 °C for 16 h with
[-P]dATP-labeled random-primed cDNA probes
(5
10
cpm/ml)(37) , consisting of either
the 750-base pair SmaI-PstI fragment of the
µ-chain cDNA together with a 3.0-kilobase HindIII fragment
of the -chain cDNA (38) or the entire coding sequence of
mouse -actin. After hybridization, the filter was washed with 0.1
SSC (150 mM NaCl and 15 mM sodium citrate (pH
7.0)) plus 1% SDS for 1 h at 45 °C. The filter was dried and
exposed to an x-ray film for 10 h.Nuclear Run-on Analysis
A modification of the
Larner et al.(39) method was used. Cells were washed
in ice-cold PBS twice and resuspended in ice-cold buffer consisting of
0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 15
mM Hepes (pH 7.5), 2 mM EDTA, 0.5 mM EGTA,
0.15 mM spermine, 0.5 mM spermidine, 14 mM
-mercaptoethanol, and 0.1% Nonidet P-40 at 3-5
10 cells/ml. After swelling for 5 min, the nuclei were
pelleted by centrifugation at 2,000 g for 5 min,
resuspended at 10 nuclei/ml in buffer containing 20 mM TrisCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA,
50% glycerol, 0.85 mM DTT, 0.125 mM PMSF, and 100
units/ml RNase inhibitor (Promega). Nuclei were stored at -85
°C.1
10 nuclei/reaction) were resuspended in 100 µl of 0.3 M ammonium sulfate, 100 mM TrisCl (pH 7.9), 4
mM MgCl, 4 mM MnCl, 40 mM NaCl, 0.4 mM EDTA, 0.125 mM PMSF, 1.2 mM DTT, 1 mM UTP, 1 mM ATP, 1 mM CTP,
0.2-0.5 µCi of [-P]GTP (3,000
Ci/mmol; 1 Ci = 37 GBq), 10 mM creatine phosphate, and
30% glycerol. Nuclei were incubated for 30 min at 26-28 °C.
The reaction was stopped by adding 100 µg of calf liver tRNA (RNase
free, Sigma) and 50 units of DNase I (RNase free, Life Technologies,
Inc.). Nuclear RNA was extracted and freed of unincorporated
triphosphates by trichloroacetic acid precipitation(40) . DNA
(3 µg) of µ- and
-chain cDNAs, glyceraldehyde-3-phosphate
dehydrogenase cDNA, and KS Bluescript vector DNA was immobilized on a
nylon membrane (BioTrans, ICN) and hybridized with
[-P]GTP-labeled RNA (5
10 cpm/ml) for 48 h at 65 °C as described
elsewhere(39) .PKR Autophosphorylation and eIF-2
For in vitro autophosphorylation of PKR, 10
µg of extracts from untreated HeLa S3 cells or HeLa S3 cells
treated with human IFN- Phosphorylation
Analysis for 18 h (1000 IU/ml; Lee Biomolecules)
were suspended in kinase reaction buffer (10 mM TrisCl,
pH 7.7, 50 mM KCl, 2 mM MgCl, 5 mM -mercaptoethanol, 2 µg/ml aprotinin, 1 µg/ml
leupeptin, 1 µg/ml pepstatin, and 0.1 mM PMSF) and 10
µCi of [-
P]ATP. Reovirus dsRNA was
added to the final concentration of 0.1 µg/ml. After incubation at
30 °C for 30 min, the reaction was diluted 5-fold with RIPA and
split equally into two fractions. In one of the fractions, 2.5 µl
of anti-PKR monoclonal antibody (13B8-F9) were added, and
immunoprecipitation of the autophosphorylated PKR was performed as
described above. In the other fraction, 5 µl of sheep
anti-eIF-2
polyclonal antibody were added, and eIF-2
immunoprecipitation was performed as for PKR using protein G-Sepharose
(Pharmacia) as a carrier. PKR immunoprecipitates were subjected on
SDS-8% polyacrylamide gels, whereas eIF-2 immunoprecipitates were
on SDS-10% polyacrylamide gels. two different assays were used. (i) 70Z/3 cells were
serum starved in Dulbecco's modified Eagle's medium lacking
phosphate (Life Technologies, Inc.) for 3 h followed by
[P]orthophosphate (200 µCi/ml; DuPont)
labeling in the same medium for 3 h. Then, LPS (10 µg/ml) or
IFN-
(100 IU/ml) was added, and cells were labeled for an
additional 3 h. Cells were washed in ice-cold PBS supplemented with 100
mM NaF, 20 mM
-glycerophosphate, and 20
mM NaMoO
and lysed in 10 mM TrisCl (pH 7.5), 50 mM KCl, 2 mM
MgCl, 1% Triton X-100, 1 mM DTT, 0.2 mM PMSF, and 2 µg/ml aprotinin. The lysate was centrifuged at
10,000 g for 10 min, and equal counts of P-labeled proteins (10% trichloroacetic acid precipitates)
from the supernatants were incubated with 5 µl of sheep
anti-eIF-2
polyclonal antibody for 2 h at 4 °C. Then, 50
µl of 50% suspension of protein G-Sepharose were added, and
incubation was continued for overnight at 4 °C under rotation.
Immunoprecipitates were washed five times with ice-cold RIPA (plus
protease inhibitors) plus 1 M NaCl buffer followed by five
washings with ice-cold RIPA (plus protease inhibitors) and subjected to
SDS-10% polyacrylamide gel electrophoresis. (ii) Exponentially grown
70Z/3 cells were induced by LPS (10 µg/ml) or IFN- (100 IU/ml)
for 24 h. Cells at similar densities were washed in ice-cold PBS
supplemented with 100 mM NaF, 20 mM
-glycerophosphate, and 20 mM NaMoO and lysed in 20 mM Hepes (pH 7.2), 2 mM EDTA,
100 mM KCl, 0.5% elugent, 0.05% SDS, 10% glycerol, 20
µg/ml chymostatin, 50 nM microcystin, and 1 mM DTT. The lysate was centrifuged at 10,000 g for
10 min and clarified with BPA-1000 (Toso-Haas, Philadelphia). Protein
extracts (50 µg) were analyzed by isoelectric focusing on vertical
slab gel electrophoresis to separate the phosphopshorylated and
nonphosphorylated forms of eIF-2 and subjected to immunoblotting
using a monoclonal antibody to eIF-2 as described
previously(41) .Electrophoretic Mobility Shift Assays
Nuclear
protein extracts were prepared as described elsewhere(42) .
Five µg of protein extracts were tested for NF-B activity by
binding to 80 pg of a P-5`-end-labeled dsDNA
oligonucleotide (1
10 cpm/ng;
5`-GATCCAAGGGGACTTTCCATGGATCCAAGGGGACTTTCCATG-3`; Life Technologies,
Inc.; the underlined sequences correspond to the NF-B-binding
sites) as described previously(43) . Antibody mobility
supershift assays were performed by incubating 10 µg of protein
extracts together with 200 pg of the P-5`-end-labeled
dsDNA HIV-
B oligonucleotide (44) (5 10 cpm/ng; 5`-AGCTGGGACTTTCCGCTA-3`; the underlined sequence
corresponds to the NF-B-binding site) and 1 µl of the stock of
affinity-purified rabbit polyclonal antibodies against
rel(45) , p65 (45) , or p50 (46) proteins. For
cold competition an 125-fold excess of unlabeled dsDNA oligonucleotides
was added. The specificity of the supershifted bands was tested by
antibody binding competition with 1 µg of the epitope peptide (45, 46) used for antisera preparation.
Expression of Wild Type PKR and Catalytically Inactive
PKR
70Z/3 cells were transfected with wt
human PKR or PKR6 in 70Z/3 Cells6 (originally termed p686) (16) cDNA
and selected in G418. Polyclonal populations of G418-resistant cells
were expanded and characterized for protein expression by
immunoblotting using a monoclonal antibody (13B8-F9) specific for the
human PKR. (
)As expected, the anti-human PKR antibody failed
to detect the endogenous mouse PKR in control 70Z/3 cells transfected
with the neomycin-resistant gene only (Fig. 1, A and B, lane 2). Two bands were detected in wt
PKR-transfected cells (1, A and B, lane 3)
which correspond to the phosphorylated (upper band) and
nonphosphorylated (lower band) forms of the
kinase(5, 47) . Phosphatase treatment of wt PKR
yielded the slower migrating nonphosphorylated form of the molecule (Fig. 1B, compare lanes 3 and 4). In
contrast, expression of the mutant PKR6 yielded one polypeptide
species which is the nonphosphorylated form (Fig. 1A, lane 4). This is consistent with the dominant negative
character of PKR6(16) , as PKR6 is neither
autophosphorylated nor is it phosphorylated by endogenous mouse PKR.
The native PKR is nonphosphorylated (Fig. 1, A and B, lane 1). It is noteworthy that wt PKR can be
overexpressed in several transformed cell lines, ()including
70Z/3 cells, without apparent inhibition of cell growth, in contrast to
NIH 3T3 cells(16) . This may be explained by modulation of PKR
activity by a specific inhibitor(s) as reported for
v-ras-transformed cells(48) .
6 proteins in 70Z/3 cells. wt
PKR and PKR6 proteins were immunoprecipitated with a polyclonal
antibody to human PKR protein and electrophoresed on an SDS-8%
polyacrylamide gel. Cell extraction and immunoblot analysis using a
mouse monoclonal antibody to human PKR (13B8-F9) were performed as
described under ``Experimental Procedures.'' Lane 1,
native PKR; lane 2, control cells (expressing neomycin
resistance gene only); lane 3, human wt PKR-expressing cells; lane 4, PKR6-expressing cells. B, human wt PKR
is expressed in phosphorylated and nonphosphorylated forms (A and B, lane 3); treatment with calf intestine
phosphatase (CIP) results in the nonphosphorylated form of PKR (lane 4). Native PKR is in nonphosphorylated form (A and B, lane 1).
Inhibition of sIgM Expression in 70Z/3 Cells Expressing
PKR
The sIgM expression in 70Z/3 cells expressing
wt PKR or PKR6 Mutant6 was examined by cell surface staining with FITC
anti-mouse antibody followed by cell analysis on a cell
sorter(35) . Upon LPS or IFN- treatment, sIgM expression
was diminished in 70Z/3 cells expressing PKR
6 (2-3-fold; Fig. 2, E and F), but not in cells expressing
the neomycin-resistant gene only (herein referred as control 70Z/3
cells; Fig. 2, A and B). Expression of sIgM in
response to LPS or IFN- was not affected in 70Z/3 cells
overexpressing wt PKR relative to control 70Z/3 cells (Fig. 2, C and D). This is presumably because the endogenous
mouse PKR elicits the maximal IgM expression upon LPS or IFN-
treatment.
6. 70Z/3 cells expressing the neomycin
resistance gene (A and B), wt PKR (C and D), or PKR6 (E and F) were incubated
either with medium alone (1), 10 µg/ml LPS or 100 IU/ml
IFN- for 23 h (2) and 35 h (3). Cells were
stained with fluorescein isothiocyanate (FITC)-conjugated rabbit
anti-mouse
antibody, and sIgM levels were determined by flow
cytometry analysis on a cell sorter after propidium iodide
staining.
PKR
To understand the molecular basis of the inhibition
of sIgM expression by the dominant-negative PKR6 Inhibits Immunoglobulin -Chain RNA
Expression6, we isolated
single clones from the polyclonal population of control 70Z/3 cells
(herein referred as control clones) and 70Z/3 cells expressing
PKR6 (herein referred as PKR6 clones) by the limiting
dilution method(34) . Several clones were examined for sIgM
expression upon LPS or IFN- treatment. All 12 of the control
clones showed the same pattern of high levels of sIgM expression upon
LPS or IFN-
treatment (data not shown). However, from the
PKR
6 clones tested, 12 out of 16 showed a significant decrease
(between 40 and 70% compared to control cells) in sIgM expression,
whereas the rest of the PKR6 clones (4 out of 16) showed a smaller
but measurable effect (20-40% decrease in sIgM expression) upon
LPS or IFN- treatment (data not shown).
- or
µ-chain immunoglobulin expression, the level of - and
µ-chain mRNAs in a control clone (CON-8) and several
PKR6-expressing clones was examined by Northern analysis using
mouse - and µ-chain immunoglobulin (38) and
-actin cDNA probes. Expression of -chain was observed neither
in resting control clone nor in resting PKR6-expressing clones (Fig. 3, A, lanes 1, 4, and 7, and B, lanes 1 and 4), but was
induced upon treatment with LPS (Fig. 3, A, lanes
2, 5, and 8, and B, lanes 2 and 5) or IFN- (Fig. 3, A, lanes
3, 6, and 9, and B, lanes 3 and 6) for 24 h. However, expression of the
-chain
immunoglobulin relative to the µ-chain was significantly lower in
PKR6-expressing clones than in the control 70Z/3 clone treated for
24 h with either LPS (40-60% decrease; Fig. 3, A, lanes 2, 5, and 8, and B, lanes
2 and 5) or IFN- (60-80% decrease; Fig. 3, A, lanes 3, 6, and 9, and B, lanes 3 and 6).
Similarly, expression of
-chain relative to -actin was
decreased by 25-60% for the different clones after LPS treatment
and 30-65% after IFN- treatment (Fig. 3, A and B).
-induced
-chain mRNA
expression is inhibited by PKR6. Northern analysis. Expression of
-chain, µ-chain, and/or -actin mRNAs was determined
before (A, lanes 1, 4, and 7; B, lanes 1 and 4) and after treatment with
LPS (10 µg/ml; A, lanes 2, 5, and 8; B, lanes 2 and 5) or IFN-
(100 IU/ml; A, lanes 3, 6, and 9; B, lanes 3 and 6) for 24 h. RNA extraction
and Northern analysis were performed as described under
``Experimental Procedures.'' Quantitation of labeled bands
was performed by scanning autoradiograms in the linear range of
exposure with a Bio-Image system
(Millipore).
PKR
The effect of PKR6 Mediates Inhibition of -Chain Expression
at Transcriptional Level6 on -chain
expression could be explained by either a transcriptional or a
post-transcriptional mechanism(s). To distinguish between these
possibilities, a nuclear run-on analysis of - and µ-chain
transcription was performed with two of the PKR6-expressing clones
(PKR6-1 and PKR6-20). Run-on experiments with PKR6-1
clone (Fig. 4A) showed that inhibition of -chain
transcription relative to µ was 60% (compare lanes 2 and 5) and 65% (compare lanes 3 and 5) upon LPS
and IFN- treatment, respectively (the experiment was repeated
twice and the results varied by no more than 10%). Inhibition of
-chain transcription relative to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was 60% (lanes 2 and 5) and 70% (lanes 3 and 6) upon LPS and
IFN- treatment, respectively. In the case of PKR
6-20, 55 and
60% inhibition of transcription of -chain relative to the
µ-chain was seen upon LPS and IFN- treatment, respectively (Fig. 4B, compare lane 2 to 5 and lane 3 to 6; the experiment was repeated three times,
and the results varied by less than 10%). These findings indicate that
PKR
6 expression leads to inhibition of -chain expression at
the level of transcription initiation.
6 inhibits LPS or IFN-
induction of
-chain expression at the transcriptional level. A and B, run-on assay. PKR6-1 (A) and
PKR6-20 (B) and control (CON-8) clones were treated with
LPS (10 µg/ml; lanes 2 and 5) or IFN- (100
IU/ml; lanes 3 and 6) for 24 h. Preparation of nuclei
and run-on assays were carried out as described under
``Experimental Procedures.'' Quantitation of radioactive band
intensities was performed as described in Fig. 3. C mRNA stability assay. Expression of
- and µ-chain mRNAs
was determined in control (CON-8; lanes 1-8) or
PKR6 (PKR6-1; lanes 9-16)-expressing cells
treated with LPS (10 µg/ml; lanes 1-4 and 9-12) or IFN- (100 IU/ml; lanes 5-8 and 13-16) for 24 h. actinomycin (10 µg/ml) was
added to the cultures and cells were harvested after 0 (lanes
1, 5, 9, and 13), 30 (lanes 2, 6, 10, and 14), 60 (lanes 3, 7, 11, and 15), or 120 min (lanes
4, 8, 12, and 16). RNA extraction and
Northern analysis were performed as described under ``Experimental
Procedures.'' Quantitation of labeled bands was performed by
scanning autoradiograms in the linear range of exposure with a enhanced
laser densitometer Ultroscan XL (LKB).
6 cells was isolated, and
the levels of -chain and µ-chain mRNA were compared by
Northern blotting. Although -chain mRNA expression was decreased
in PKR6 cells upon LPS treatment (Fig. 4C, compare lanes 1 and 9) or IFN- treatment (compare lanes 5 and 13), the ratio of
-chain to
µ-chain mRNA did not change either in control cells or in PKR6
cells after actinomycin D treatment. This is consistent with previous
studies showing that -chain mRNA is very stable (49) and
indicates that PKR6 does not affect -chain mRNA stability.Phosphorylation of eIF-2
The regulation of
Is Not Required for the
Transcriptional Activation of Gene-chain transcription by PKR is the second example of regulation of
transcription by an eIF-2 kinase. The yeast eIF-2 kinase,
GCN2, regulates expression of the transcriptional activator GCN4 at the
translational level upon amino acid starvation conditions(4) .
It is possible that PKR mediates -chain transcription indirectly
by regulating the protein synthesis of a transcriptional factor(s)
through eIF-2 phosphorylation as does GCN2. To examine this
possibility, we measured the extent of eIF-2 phosphorylation in vivo upon LPS or IFN- treatment. We used two different
assays for eIF-2
phosphorylation: (i) immunoprecipitation of
eIF-2 from [P]orthophosphate-labeled cells (Fig. 5B) and (ii) isoelectric focusing followed by
eIF-2
immunoblotting (Fig. 5C). In the first assay
we used a sheep anti-eIF-2 polyclonal antibody, whose suitability
was tested first (Fig. 5A). HeLa S3 cell extracts were
incubated with reovirus dsRNA and [P-
]ATP
followed by immunoprecipitation with either anti-PKR antibody (Fig. 5A, lanes 1-4) or anti-eIF-2
antibody (Fig. 5A, lanes 5-8). Induction
of PKR autophosphorylation by dsRNA before (lane 2) or after
treatment with IFN- (lane 4) resulted in increased
phosphorylation (lanes 6 and 8, respectively) of a
protein immunoprecipitated by anti-eIF-2 antibody, whose molecular
size corresponds to eIF-2 (38 kDa). Based on these results,
we used the anti-eIF-2
polyclonal antibody to immunoprecipitate
the in vivoP-labeled eIF-2
. Phosphorylation
of eIF-2 did not significantly differ between control (Fig. 5B, lanes 1-3) and PKR6 cells (lanes 4-6), which were stimulated either with LPS (lanes 2 and 5) or IFN- (lanes 3 and 6). This experiment was performed three times with no
significant variations in eIF-2
phosphorylation. Similarly, no
significant differences in the levels of eIF-2 phosphorylation
were observed when the isoelectric focusing and eIF-2
immunoblotting assay was used (Fig. 5C). These
experiments suggest that eIF-2 is not a substrate for PKR
activated by LPS or IFN- in 70Z/3 cells.
in 70Z/3
cells expressing PKR6. A, phosphorylation of eIF-2
by PKR in vitro. Cell extracts (10 µg) from HeLa S3 cells
before (lanes 1, 2, 5, and 6) or
after IFN- treatment for 18 h (1000 IU/ml; lanes 3, 4, 7, and 8) were incubated in absence (lanes 1, 3, 5, and 7) or presence
of reovirus dsRNA (0.1 µg/ml; lanes 2, 4, 6, and 8) and [P-
]ATP as described
under ``Experimental Procedures.'' After incubation, samples
were subjected to immunoprecipitation either with a monoclonal antibody
to human PKR (13B8-F9; lanes 1-4) or with a sheep
anti-eIF-2
polyclonal antibody (lanes 5 and 6)
followed by SDS-polyacrylamide gel electrophoresis analysis. B and C, phosphorylation of eIF-2 in vivo.
Control (CON-8) 70Z/3 cells (B, lanes 1-3; C, lanes 3-5) and 70Z/3 cells expressing
PKR6 (B, lanes 4-6; C, lanes
6-8) were analyzed for eIF-2 phosphorylation either
after P-labeling in vivo and immunoprecipitation (B) or after isoelectric focusing and immunoblotting (C) as described under ``Experimental Procedures.''
Lanes marked NC or PC in C represent either
purified nonphosphorylated eIF-2
(Negative Control) only or purified eIF-2 phosphorylated in
vitro by the heme regulated eIF-2 kinase (Positive Control) to indicate the position of phosphorylated and
nonphosphorylated forms of eIF-2. Quantitation of labeled bands
was performed by scanning autoradiograms in the linear range of
exposure with a enhanced laser densitometer Ultroscan XL
(LKB).
Induction of NF-
The induction of B Activity Is Not Inhibited by
PKR6-chain transcription in 70Z/3
cells upon LPS treatment requires the activation of NF-B, which
binds to the -chain enhancer motif, GGGACTTTCC(50) . It
has been recently shown that the transcription inhibitor IB can be
phosphorylated by PKR in vitro resulting in induction of
NF-B DNA binding(28) . Based on this finding, we examined
the possibility that PKR6 inhibits phosphorylation of IB by
PKR resulting in inhibition of NF-B activation and consequently in
a decrease of -chain transcription. To this end, we tested
NF-B activity in nuclear extracts of 70Z/3 cells induced with LPS
only since IFN- induces
-chain transcription in the absence
of NF-B activation(51, 52) . NF-B binding to
a DNA fragment containing two repeats of NF-B consensus sequence
-GGGACTTTCC- was analyzed by the gel retardation assay(43) . No
detectable NF-B activity was observed in resting 70Z/3 control
cells, as no protein-DNA complexes were formed (Fig. 6A, lane 1), but DNA binding was evident
after LPS treatment (Fig. 6A, lane 2). The two
inducible bands most likely correspond to the binding of one (lower
band) or two NF-B complexes (upper band) to one or
two B sites, respectively, in the DNA probe (see also below).
Formation of these complexes was drastically reduced by competition
with an unlabeled oligonucleotide containing the two NF-B-binding
sites (Fig. 6A, lane 3). Significantly,
indistinguishable NF-BDNA complexes were observed in control
and two independent PKR6-expressing clones (PKR6-1 and
PKR6-20) upon LPS treatment (Fig. 6A,
compare lane 2 to lanes 4 and 5).
B DNA binding activity is not
affected by expression of PKR6. A, an equal number of
cells (1 10) was treated with 10 µg/ml LPS for
24 h. Nuclear extracts (5 µg) from a control clone (CON-8) and two
PKR6 clones (PKR6-1 and PKR6-20) were used for NF-B
DNA binding assays with a dsDNA oligonucleotide containing two B
sites. Lane 1, uninduced control clone (CON-8); lane
2, LPS-induced control clone; lane 3, cold competition
with 125-fold excess of unlabeled oligonucleotide; lane 4,
LPS-induced PKR6-1 clone; lane 5, LPS-induced
PKR6-20 clone. B, control (CON-8), wt PKR
(polyclonal populations) and PKR6 (clone PKR6-1)-expressing
cells were treated with LPS for 6 h. Nuclear extracts (10 µg) were
tested for NF-B DNA binding by gel supershift assays using
specific antisera and the HIV-B site. Lanes 1-4, 11-14, and 21-24, no antiserum was added; lanes 5, 15, and 25, antiserum to p65 was
added; lanes 6, 16, and 26, excess of
epitope peptide to p65 added to show specificity of the supershifts
seen in lanes 5, 15, and 25; lanes
7, 17, and 27, antiserum to rel was added; lanes 8, 18, and 28, epitope peptide and
antiserum to rel were incubated together; lanes 9, 19, and 29, incubation with antiserum to p50; lanes 10, 20, and 30, incubation of p50
antiserum together with epitope peptide to p50. For cold competition, a
125-fold excess of unlabeled HIV-B dsDNA oligonucleotide was added (lanes 3, 13, and 23).
B-binding proteins form a family of related genes that
include NFKB1 (p50/p105), NFKB2 (p52/p100),
v-rel, c-rel, relA (p65), relA
(p65), and relB (for review, see (53) ). Recent
findings suggest that treatment of pre-B cells with LPS changes the
subunit composition of B-binding complexes from p50-p65 to
p50-rel(54, 55) . Based on this observation we wished
to investigate whether PKR6 expression had an effect on NF-B
subunit composition upon LPS induction. To examine which of the two
B-binding complexes, p50-p65 or p50-rel, were involved in the
binding to B site, we performed gel supershift assays by
incubating nuclear extracts from a control clone (CON-8), wt PKR cells
(polyclonal populations), and a PKR6 clone (PKR6-1) together
with antibodies against p65, rel, or p50 protein. As shown in Fig. 6B, no differences in NF-B subunit
composition between control, wt PKR, or PKR6 cells were observed.
The NF-B binding complexes consisted of p65 (lanes 5, 15, and 25), rel (lanes 7, 17, and 27), and p50 (lanes 9, 19, and 29)
proteins. Similar results were obtained from three independent
experiments after different periods of LPS stimulation (data not
shown). These data are consistent with the existence of p50-p65 and
p50-rel heterodimers in 70Z/3 cells(54, 55) . Thus,
inhibition of -chain transcription in PKR6-expressing cells
is apparently not mediated through NF-B.
with its receptor elicits a cascade of tyrosine phosphorylation of
cytoplasmic and nuclear proteins resulting in transcriptional
activation of genes (61) . Recent findings suggest that serine
phosphorylation is also important in IFN-
signaling(62) .
Analysis of mutant variants of 70Z/3 cells has shown that LPS and
IFN-
share common signaling
pathways(63, 64, 65) . This is consistent
with our data which demonstrate that PKR is a mediator of LPS and
IFN-
signaling in 70Z/3 cells. However, the lack of complete
inhibition of
gene transcription by PKR6 suggests that PKR
activation is necessary but not sufficient for the induction of
gene transcription and indicates the existence of other pathways which
do not involve PKR. phosphorylation concomitant with a
stimulation of protein synthesis (66) ; (iii) induction of the
tumoricidal activity of macrophages by LPS requires PKR(67) ;
(iv) PKR mediates the induction of c-myc, c-fos, and
JE genes upon platelet-derived growth factor treatment(68) ;
and (v) induction of indoleamine 2,3-dioxygenase gene expression by
IFN- is mediated by PKR(69) . These findings together with
ours reveal a multifunctional and complex role for PKR in regulation of
gene expression at two different levels, translation and transcription.
It is not as yet clear how PKR activity is regulated by the different
stimuli. One possibility is that PKR activity is induced by cellular
dsRNA, whose nature and availability are dependent upon the cell type
and/or stimuli. Our data show that the PKR-mediated effect of LPS or
IFN-
is unlikely to proceed through eIF-2
phosphorylation,
suggesting that phosphorylation of other protein(s) is required for
this effect. This is consistent with earlier studies showing that new
protein synthesis is not required for transcriptional activation of
gene(50, 70) . gene is regulated by the interaction of sequence-specific
DNA-binding proteins with cis-acting DNA elements. NF-B
transcription factor binds to the B site in the intron enhancer
(J-C
enhancer) of
gene(50) . Activation of NF-B requires IB
phosphorylation and degradation(71, 72) .
Interestingly, IB can be phosphorylated by the two eIF-2
kinases, heme control repressor (73) and PKR (28) in vitro. In 70Z/3 cells, LPS but not IFN-
induces NF-
B activity, which is necessary but not sufficient for
gene
transcription(49, 63, 64, 74) . Our
data show that PKR mediates transcription independently of
NF-B. This is the second example of an NF-B-independent
pathway of -gene transcription in 70Z/3 cells. Transforming growth
factor- inhibits LPS-induced gene transcription without
affecting NF-B activation(51) . Thus PKR activates at
least two different pathways to the transcriptional machinery,
NF-B dependent for dsRNA and NF-B independent for LPS or
IFN-. However, it should be emphasized the difference in cell
types used in these experiments. In this regard, IFN-
expression
by dsRNA in mouse F9 embryonal carcinoma cells does not require
NF-B activation(75) , indicating that dsRNA signaling
clearly differs between cell lines. gene, has been identified
(3` enhancer) and contains an IFN consensus sequence(76) .
The 3` enhancer contains a binding site for B cell and
macrophage-specific factor PU.I(77) . PU.I recruits the binding
of a second B cell-restricted nuclear factor, NF-EM5. DNA binding by
NF-EM5 requires protein-protein interaction with PU.I and protein
phosphorylation of PU.I(77) . NF-EM5 is homologous to
interferon regulatory proteins, ()consistent with its
function in IFN- signaling. At the present time it is not known
what kinase(s) regulates PU.I phosphorylation in vivo, and PKR
is an intriguing possibility that remains to be examined. In
conclusion, our data demonstrate that PKR is a mediator of LPS and
IFN-
signaling to
gene transcription and substantiate the
transcriptional role of PKR in regulation of gene expression. Inasmuch
as PKR plays a role in many pathophysiological events such as virus
infections (78) including AIDS(79) , and possibly
cancer(16, 17) , the understanding of the mechanism of
action of PKR is important for devising strategies to combat these
diseases.
),
eukaryotic translation initiation factor-2; LPS,
lipopolysaccharide; sIg, surface immunoglobulin; -chain, light
immunoglobulin chain; µ-chain, heavy immunoglobulin chain; FITC,
fluorescein isothiocyanate; NF-B, transcription factor B;
IB, protein inhibitor of NF-B; DTT, dithiothreitol; PMSF,
phenylmethylsulfonyl fluoride; RIPA, radioimmune precipitation buffer;
wt, wild type; ds, double-stranded; PBS, phosphate-buffered saline.))))
We thank Raymond Leung, Luc Chandonnet, and Anne
Roulston for assistance in some of the experiments; Dr. R. Sekaly for
providing the flow cytometry facility; Drs. G. Barber, M. Katze and A.
Darveau for the anti-PKR monoclonal antibody (13B8-F9); Dr. M. Mathews
for the anti-PKR polyclonal antibody; Dr. B. Safer for anti-eIF-2
polyclonal antibody; Dr. C. Paige for immunoglobulin - and
µ-chain cDNAs; Dr. Nancy Rice for anti-p65 and anti-c-rel
polyclonal antibodies; and Drs. M. Szyf, C. Paige, H. Young, R. Sen,
and J. Pelletier for comments on the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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