(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 IB
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.
IFNs ()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) .
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 subunit of
the eukaryotic translation factor eIF-2(5) , a modification
that causes inhibition of protein synthesis (6) .
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(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.
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 IB
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) .
To investigate the role of
PKR in signaling to the transcriptional machinery, we expressed wt
human PKR, or the dominant negative catalytically inactive mutant
PKR6(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 PKR
6 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.
The surface staining of induced 70Z/3 cells expressing sIgM was
performed with fluorescein isothiocyanate (FITC)-conjugated rabbit
anti-mouse 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) .
For transcriptional assay, nuclei (1
10
nuclei/reaction) were resuspended in 100 µl of 0.3 M ammonium sulfate, 100 mM Tris
Cl (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) .
For in vivo phosphorylation
of eIF-2 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 Na
MoO
and lysed in 10 mM Tris
Cl (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 Na
MoO
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) .
Figure 1:
A,
expression of human wt PKR and PKR6 proteins in 70Z/3 cells. wt
PKR and PKR
6 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, PKR
6-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).
Figure 2:
Surface IgM expression is decreased in
cells expressing PKR6. 70Z/3 cells expressing the neomycin
resistance gene (A and B), wt PKR (C and D), or PKR
6 (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.
To examine whether the
decrease in sIgM expression was due to inhibition of - or
µ-chain immunoglobulin expression, the level of
- and
µ-chain mRNAs in a control clone (CON-8) and several
PKR
6-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 PKR
6-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
PKR
6-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).
Figure 3:
LPS- or IFN--induced
-chain mRNA
expression is inhibited by PKR
6. 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).
Figure 4:
PKR6 inhibits LPS or IFN-
induction of
-chain expression at the transcriptional level. A and B, run-on assay. PKR
6-1 (A) and
PKR
6-20 (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
PKR
6 (PKR
6-1; 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).
Effects on stability of mRNA
were tested by the following experiment. Following actinomycin D
treatment, total RNA from control or PKR6 cells was isolated, and
the levels of
-chain and µ-chain mRNA were compared by
Northern blotting. Although
-chain mRNA expression was decreased
in PKR
6 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 PKR
6
cells after actinomycin D treatment. This is consistent with previous
studies showing that
-chain mRNA is very stable (49) and
indicates that PKR
6 does not affect
-chain mRNA stability.
Figure 5:
Phosphorylation of eIF-2 in 70Z/3
cells expressing PKR
6. 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
PKR
6 (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).
Figure 6:
NF-B DNA binding activity is not
affected by expression of PKR
6. 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
PKR
6 clones (PKR
6-1 and PKR
6-20) 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 PKR
6-1 clone; lane 5, LPS-induced
PKR
6-20 clone. B, control (CON-8), wt PKR
(polyclonal populations) and PKR
6 (clone PKR
6-1)-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).
Genes
encoding 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 PKR
6 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 PKR
6 clone (PKR
6-1) 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 PKR
6 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 PKR
6-expressing cells
is apparently not mediated through NF-
B.
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-
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 PKR
6 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.
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 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) .
Like other eukaryotic genes,
the 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 I
B
phosphorylation and degradation(71, 72) .
Interestingly, I
B 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.
A second enhancer element, which
lies 8.5 kilobases downstream of the 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.