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(Received for publication, August 8, 1994; and in revised form, November
16, 1994) From the
The interferon-inducible, double-stranded RNA (dsRNA)-dependent
eukaryotic initiation factor-2 Treatment of quiescent Balb/c/3T3 fibroblasts with peptide
platelet-derived growth factor (PDGF)( Since PDGF-mediated induction of IE genes requires no de novo protein synthesis, it presumably occurs by directed
modification or activation of pre-existing factors or ``second
messengers'' in the cell. A complex cascade of intracellular
events has been identified that takes place in response to exposure to
PDGF-BB. These include dimerization and autophosphorylation of the PDGF
receptor, association and/or tyrosine phosphorylation of several
proteins (including phospholipase C, phosphoinositol kinase, Raf-1, and
pp42), increased phosphoinositol and phosphatidylcholine turnover and
calcium mobilization, and activation of protein kinase C (Meisenhelder et al., 1989; Morrison et al., 1989; Williams, 1989;
Sultzman et al., 1991; Exton, 1990). Some causal and
sequential relationships of these PDGF-induced phenomena to each other
and to eventual DNA synthesis are beginning to be established. However,
the events linking early second messenger activation with subsequent IE
gene expression are unknown. While all of the known PDGF-activated
second messengers described above reach their peak level of activation
within 5-10 min of PDGF binding to its receptor, peak times of
expression for the IE genes range from 30 min to several hours after
PDGF stimulation (Muller et al., 1984; Sukhatme et
al., 1988; Rollins et al., 1988). We therefore sought to
identify second messengers that act later in the pathway. One
candidate for such a PDGF-activated second messenger was suggested by
several reports in which cells were treated with double-stranded RNA
(dsRNA) or with the guanine analog 2-aminopurine (2-AP). The addition
of dsRNA, in the form of poly(I) There are, however, several
reasons to suspect that PKR also plays a direct role in the regulation
of cell growth. As described above, activators and inhibitors of PKR
have profound effects on growth factor-inducible genes. In addition,
PKR protein levels are known to fluctuate during the cell cycle, being
highest in G Given this substantial indirect evidence
suggestive of PKR involvement in growth factor signal transduction, we
tested the proposition that PKR functions as a signal transducer in
PDGF-stimulated pathway(s) that induce transcription of the IE genes
c-fos, c-myc, and JE. Assuming a common
model for signal transduction pathways in which a pathway consists of a
sequential series of biochemical events, each triggered by the one
preceding it, we reasoned that the biochemical event of PKR activation
may be concluded to be a component of the PDGF signal transduction
pathway inducing c-fos, c-myc, and JE if 1)
specific inhibitors of PKR activation block c-fos,
c-myc, and JE gene induction; 2) specific
down-regulation of PKR protein levels by antisense inhibition of
translation results in a decrease or elimination of c-fos,
c-myc, and JE induction by PDGF; and 3) PDGF can be
shown to activate PKR in vivo. We demonstrate herein that
all of these requirements are met with respect to the participation of
the enzyme PKR in the PDGF signal transduction pathway resulting in the
induction of the IE genes c-fos, c-myc, and JE. In addition, these criteria were tested by analysis of
another PDGF-inducible IE gene, egr-1. egr-1 was
inducible by PDGF, but not by a direct activator of PKR
(poly(I)
Figure 1:
PDGF-mediated induction of some IE
genes is blocked by inhibitors of PKR activation. A, total
cellular RNA was extracted from confluent BALB monolayers after
incubation for 24 h in 0.5% serum-containing medium, followed by no
additions (first lane 1), 10 ng/ml PDGF-BB for 60 min (second lane), or 10 mM 2-AP for 2 h with 10 ng/ml
PDGF-BB added after the first 60 min of treatment (third lane ). Each lane contained 20 µg of total cellular RNA separated
on a 1% formaldehyde-agarose gel. The RNA was transferred to
nitrocellulose and simultaneously hybridized with
Figure 2:
IE gene induction and PKR activity in
v-ras-expressing and ras revertant cell lines. A, in vitro kinase assay of PKR in
v-ras-expressing and ras revertant cell lysates.
Cytoplasmic extracts were made from confluent, interferon-treated (18-h
pretreatment with 500 units/ml murine interferon-
Although utilizing v-ras as a PKR
inhibitor might be somewhat complicated by the fact that v-ras appears to affect several steps in PDGF signal transduction (Rake et al., 1991; Benjamin et al., 1987, 1988; Zullo and
Faller, 1989), ( A Krev cell line and KBALB cells
treated for 48 h with 2 mM dibutyryl-cAMP (KcAMP) were made
quiescent by incubation in 0.5% serum for 24 h and then stimulated with
10 ng/ml PDGF-BB for 60 min and harvested for RNA. As shown in parallel
with RNA from identically stimulated BALB and KBALB cells for
comparison, PDGF induction of the IE genes c-fos,
c-myc, and JE was found to be restored in the
revertant cells (Fig. 2B) (c-fos not shown).
Induction of egr-1 continued to be normal (data not shown).
When extracts of these revertant cells were used in the in vitro PKR kinase assay, they now autophosphorylated PKR to equal or
greater levels in response to exogenous poly(I) Having thus
demonstrated above or in previous reports that two inhibitors of PKR
activation block PDGF induction of the IE genes c-fos,
c-myc, and JE, but not egr-1, and that the
direct activator of PKR, poly(I)
Figure 3:
dsRNA does not induce egr-1
mRNA levels. Total cellular RNA was extracted from confluent BALB
monolayers after incubation for 24 h in 0.5% serum-containing medium,
followed by treatment for 60 min with no additions (lane 1);
200 nM 12-phorbol 13-myristate acetate, a strong inducer of egr-1 (lane 2); or 50 µg/ml poly(I)
Antisense
phosphorothioate oligonucleotides designed to be complementary to the
5`-end of the PKR message (see ``Materials and Methods'')
were synthesized and added to the medium of BALB cells to a
concentration of 5 µM. ``Missense''
oligonucleotides of identical base composition were added to some
control cultures. The depletion of PKR protein levels by antisense
inhibition of translation was monitored by Western blotting with
antisera specific for murine PKR. Incubation of confluent BALB cells
for 48 h in medium containing antisense oligonucleotide, but not in
medium containing missense or no added oligonucleotide, significantly
down-regulated PKR protein levels (Fig. 4). Such PKR-depleted
cells were treated with PDGF for 1 h and harvested for RNA, and the RNA
was analyzed by hybridization for IE gene induction. In experiments in
which down-regulation of PKR protein by antisense treatment was
verified, PDGF induction of c-myc and JE was found to
be inhibited, while egr-1 induction was unaffected, consistent
with the results obtained when other inhibitors of PKR (2-AP and
v-ras) were used (Fig. 5). The pattern of gene
induction by PDGF was not altered by pretreatment of the cells with
missense oligonucleotides (data not shown).
Figure 4:
Specific down-regulation of PKR protein
levels by antisense inhibition of translation. Confluent BALB cells
were left untreated (lanes 1, 3, and 5) or
were pretreated with 500 units/ml murine interferon-
Figure 5:
Antisense-mediated depletion of PKR
protein inhibits PDGF induction of IE genes myc and JE, but not egr-1. Total cellular RNA was extracted
from confluent BALB monolayers after incubation in the presence or
absence of an antisense oligonucleotide (5 µM)
complementary to the 5`-end of the PKR transcript for 48 h. Immediately
prior to RNA harvest, some cultures were treated with 10 ng/ml PDGF for
60 min. The RNA (20 µg/lane) was size-separated by electrophoresis
on a 0.9% formaldehyde-agarose gel, transferred to nitrocellulose, and
hybridized sequentially with
Instead, a protocol modified
from a method for monitoring receptor autophosphorylation in intact
cells was developed. In this strategy, the cell membrane was
sufficiently permeabilized with the detergent digitonin to allow
[
Figure 6:
PDGF-induced phosphorylation of PKR in
digitonin-permeabilized BALB cells. Confluent BALB cells in 35-mm wells
were made quiescent by 48 h of incubation in 0.5% bovine calf serum and
washed once with warm phosphate-buffered saline and once with warm
isotonic wash buffer. Washes were carefully drained, and the monolayers
were overlaid with isotonic wash buffer containing digitonin for
permeabilization of the cellular membrane,
[
Since time points beyond
30 min were not possible in the presence of digitonin, an indirect
assay that did not require permeabilization of cells was employed to
verify the time course of PKR phosphorylation in response to PDGF. If
intracellular PKR was phosphorylated to a significant extent in
response to PDGF, the amount of additional radioactive phosphate
incorporated in an in vitro stimulation assay (with
poly(I)
Figure 7:
In vitro PKR kinase assay of
cytoplasmic extracts from PDGF-stimulated cells. Cytoplasmic extracts
were made from BALB cells pretreated for 24 h with 1% bovine calf serum
+ 500 units/ml murine interferon-
Figure 8:
PMA
induction of JE and c-fos in the presence of PKR
inhibitors. Whole cell RNA was extracted from confluent KBALB cells
after incubation for 24 h in 0.5% serum-containing medium, followed by
treatment with one of the following: no additions (lane 1), 15
ng/ml PDGF-BB for 60 min (lane 2), 200 ng/ml PMA for 60 min (lane 3), 10 mM 2-AP for 90 min with 15 ng/ml PDGF-BB
added after the first 30 min of treatment (lane 4). or 10
mM 2-AP for 90 min with 200 ng/ml PMA added after the first 30
min of treatment (lane 5). RNA (20 µg/lane) was
size-fractionated on a 1% formaldehyde-agarose gel, transferred to
nitrocellulose membrane, and hybridized with
Previous reports utilizing activators and inhibitors of PKR
implicated this serine/threonine kinase in growth factor signal
transduction for the induction of some IE genes. This study confirms
those earlier results and further shows that they could not be
attributed to indiscriminate activation or inhibition of gene induction
pathways since at least one PDGF-inducible gene (egr-1) was
unaffected by these factors. Furthermore, specific depletion of PKR
protein by antisense oligonucleotide-mediated translational inhibition
blocked PDGF induction of the same genes, as did the chemical
inhibitors or protein inhibitors of PKR. Finally, PKR activation by
PDGF in intact cells, as measured by autophosphorylation of the enzyme,
was demonstrated to have a time course and magnitude similar to those
induced by dsRNA and was consistent with the time course of PDGF
induction of the relevant IE genes. How might a growth factor induce
activators of PKR? Activation of PKR by dsRNA is coincident with and
dependent upon autophosphorylation of the kinase on serine. Although
PKR is capable of autophosphorylation, a possible mechanism by which a
growth factor might activate PKR is by activating a kinase that has PKR
as its substrate. Indeed, our results show that a 2-AP- and
v-ras-inhibitable event appears to lie downstream of protein
kinase C activation, although PMA-activated protein kinase C does not
appear to directly phosphorylate PKR in the signal transduction pathway
examined in this study. Double-stranded RNA has been shown to be a
highly effective activator of PKR both in vitro (Kostura and
Matthews, 1989) and in vivo, as in cases of viral infection
(see Samuel(1991) for review) and others involving dsRNAs of cellular
origin (Li and Petryshyn, 1991; Judware and Petryshyn, 1991). PDGF
stimulation may generate a nonprotein molecule capable of activating
PKR. Such a nonprotein signaling factor need not be a dsRNA molecule.
In addition to viral dsRNA and poly(I) A third possible mechanism for
PKR activation by PDGF involves a pattern already demonstrated for
several gene induction pathways, which is the phosphorylation and
subsequent dissociation of a chaperone protein that retains the factor
in a particular cellular compartment as long as the chaperone is bound
to it, e.g. NF- A role for PKR in growth factor signal transduction may help to
explain the long-standing paradox with respect to PKR activation. Given
its ability to inhibit protein synthesis by phosphorylating the
rate-limiting translation initiation factor eIF-2 In conclusion, we have presented direct
evidence that activation of the interferon-induced, dsRNA-activated
eIF-2
Volume 270,
Number 7,
Issue of February 17, 1995 pp. 3100-3106
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
IMMEDIATE EARLY GENE INDUCTION (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
kinase PKR has primarily been
characterized as a component of the interferon-mediated cellular
antiviral response. Several lines of evidence now exist that suggest
that PKR plays a role in the regulation of growth in uninfected cells.
The most direct examples are the finding of an oncogenic variant of PKR
and the effects of activators and inhibitors of PKR phosphorylation on
the expression of platelet-derived growth factor (PDGF)-inducible
genes. Previous reports have shown that 1) dsRNA, a direct activator of
PKR, induces the genes c-myc, c-fos, and JE;
2) 2-aminopurine, a chemical inhibitor of PKR, blocks the induction of
these genes by serum; and 3) activated p21
induces a cellular inhibitor of PKR. We report here that
activation of PKR was correlated with the induction of the immediate
early genes c-fos, c-myc, and JE by PDGF in
the following situations: 1) PDGF induction of these genes, also
inducible by dsRNA, was blocked by two inhibitors of PKR activation:
2-aminopurine and v-ras; 2) PDGF induction of another
immediate early gene, egr-1, which could not be induced by
dsRNA, was not blocked by 2-aminopurine or v-ras; 3) agents
that reverse v-ras inhibition of PKR activation also reversed
the v-ras block of PDGF induction of c-myc,
c-fos, and JE; 4) down-regulation of PKR protein
levels by antisense inhibition of translation blocked the induction of
c-myc, c-fos, and JE by PDGF, but had no
effect on egr-1 induction; and finally, 5) PKR was
autophosphorylated in vivo in response to PDGF. These results
provide direct evidence that PKR activation functions as a second
messenger in a growth factor signal transduction pathway. Thus, PKR may
serve as a common mediator of growth-promoting and growth inhibitory
signals.
)-BB induces DNA
synthesis and mitosis in these cells within 24 h. An early step in this
growth process is stimulation of the expression of a set of genes known
as immediate early (IE) genes, so named because their induction by PDGF
requires no new protein synthesis. Whether the expression of any (or
all) of these genes is a prerequisite for PDGF-induced mitogenesis is
debated. The fact that many IE genes are proto-oncogenes, however,
strongly suggests that precise regulation of their expression is at
least important for cell growth regulation if not initiation. 
poly(C), to the medium of
quiescent fibroblasts was shown to induce transcription of the
PDGF-inducible IE genes c-myc, c-fos, and JE, with a somewhat faster time course than that seen with
PDGF (Zullo et al., 1985; Hall et al., 1989).
Conversely, treatment of fibroblasts with 2-AP prior to stimulation
with serum was shown to block induction of c-myc and c-fos (Zinn et al., 1988). The unifying aspect to these
observations is that the only known intracellular target of both dsRNA
and 2-AP is the dsRNA-dependent eIF-2 kinase PKR (Farrell et
al., 1977). PKR (formerly known as dsI, DAI, dsKinase, p68, and
p68
) is a highly conserved serine/threonine kinase,
constitutively present at low levels in the cytoplasm in a latent state
(Clemens et al., 1993; Dever et al., 1993; Petryshyn et al., 1983). First discovered as a component of the
interferon-inducible cellular antiviral defenses, levels of latent
enzyme are increased by interferons. In the presence of low
concentrations (<1 µM) of dsRNA, PKR
autophosphorylates, resulting in activation and the ability to
phosphorylate other substrates, most prominently eIF-2 (Petryshyn et al., 1983). This phosphorylation of eIF-2 is
responsible for the antiviral activity of PKR, inhibiting viral protein
production by preventing recycling of this limiting factor in protein
synthesis, thereby greatly reducing production of new virus particles
(O'Malley et al., 1986).
and early G (Petryshyn et
al., 1984). Oncogenic Ras proteins have been shown to induce an
inhibitor of PKR activation in BALB cells, suggesting that
p21 activation leads to down-regulation of PKR
activity (Mundschau and Faller, 1992). Furthermore, the expression of a
mutant and presumed dominant suppressor PKR protein containing a
deletion of 6 amino acids between catalytic domains V and VI of the
kinase results in malignant transformation in NIH3T3 cells (Koromilas et al., 1992).poly(C)), suggesting that egr-1 is induced by
PDGF via a PKR-independent, PDGF-stimulated pathway. As would be
predicted from the three propositions above, PDGF induction of egr-1 was found to be unaffected by inhibitors or
down-regulation of PKR. Finally, experiments measuring induction of the
PKR-dependent genes by direct activators of protein kinase C in the
presence and absence of 2-AP suggest that PKR activation lies
downstream of protein kinase C activation in the pathway for induction
of some IE genes.
Cell Culture
Clone A31 of Balb/c/3T3 cells and
KBALB cells, a nonproducing Kirsten sarcoma virus-transformed
BALB/c/3T3 line, were obtained from the American Tissue Culture
Collection (Rockville, MD). Morphological revertants of KBALB cells
were produced by pharmacologically elevating intracellular levels of
cAMP by treatment with 2 mMN
-2`-O-dibutyryl-cAMP (Sigma) for at
least 48 h or by stable transfection with the Krev-1 gene as
described previously (Quinones et al., 1991). All cell lines
were carried in Dulbecco's modified Eagle's medium
supplemented with 10% heat-inactivated donor bovine calf serum
(Hazelton Research Products, Inc., Lenexa, KS), 2 mML-glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin.Growth Factors and Reagents
PDGF-BB was obtained
from Amgen (Thousand Oaks, CA). 2-Aminopurine was obtained as a nitrate
salt 198.2 g/mol; Sigma) and was dissolved in 1.5 M Tris-HCl
(pH 8.8) and Dulbecco's modified Eagle's medium until a
neutral pH was obtained (as determined by the phenol red pH indicator
in Dulbecco's modified Eagle's medium) and the
2-aminopurine had dissolved. A 200 mM stock was made fresh for
each experiment. Poly(I)poly(C) was obtained in soluble form and
as a conjugate to agarose beads (poly(I)poly(C)-agarose, Type VI)
from Pharmacia Biotech Inc.RNA Blot Analysis of Total Cellular RNA
Total
cellular RNA was isolated by guanidine thiocyanate extraction
(Chomczynski and Sacchi, 1987), quantified, electrophoresed on
formaldehyde-agarose gels, and transferred to supported nitrocellulose.
Hybridizations and washes were carried out according to standard
protocols with 
P-labeled DNA probes made by the random
oligonucleotide primer method (Feinberg and Vogelstein, 1983). Murine
-actin, c-fos, JE, c-myc, and egr-1 and rat sodium potassium ATPase DNA probes have been
described previously (Mundschau and Faller, 1991).In Vitro PKR Phosphorylation Assays
Preparation of
cytoplasmic extracts and in vitro PKR phosphorylation assays
were carried out as described previously (Mundschau and Faller, 1992;
Katze et al., 1991).PKR Phosphorylation Assays in Digitonin-permeabilized
Cells
In a modification of the technique described by
Erusalimsky et al.(1988), cells were grown to confluence in
35- or 60-mm wells and then starved in 0.5% calf serum for 24 h or
allowed to become quiescent in 10% serum-containing medium. After
washing twice with phosphate-buffered saline and twice with isotonic
wash buffer (120 mM KCl, 30 mM NaCl, 2 mM MnCl
, 10 mM HEPES (pH 7.4)), the cells were
overlaid with a permeabilization buffer consisting of isotonic wash
buffer, 40 µM digitonin (Sigma), 1 µM ATP,
and 50 µCi/ml [
-P]ATP, with and without
20 ng/ml recombinant PDGF-BB or 10 µg/ml poly(I):poly(C), and
incubated at 37 °C for the periods of time indicated on the
figures. The radioactive overlay was discarded, and each well of cells
was gently washed once with 1 ml of isotonic wash buffer containing 5
mM EDTA. Finally, cells were scraped into 300 µl of EDTA
lysis buffer (20 mM HEPES (pH 7.4), 120 mM KCl, 5
mM EDTA, 1 mM dithiothreitol, 0.5% Nonidet P-40)
containing 2 µg/ml leupeptin and 50 µg/ml aprotinin (Sigma).
Precipitation of dsRNA-binding proteins on poly(I)poly(C)-agarose
was performed as described previously (Mundschau and Faller, 1992). The
rinsing step just prior to cell lysis with Nonidet P-40 was found to be
critical for achieving low background in this assay. Detergent lysis of
the cells without prior removal of most of the remaining
[-P]ATP stimulated PKR phosphorylation in
the absence of any exogenous activators. That this PDGF and
poly(I)poly(C)-independent phosphorylation was occurring after
lysis, and not before, was verified by control experiments in which
cells were overlaid with [-P]ATP in buffer
only, omitting digitonin.Down-regulation of PKR Protein by Antisense Inhibition of
Translation
Phosphorothioate DNA oligonucleotides with the
sequences 5`-TGG GGT ATC ACT GGC CAT-3` (antisense) and 5`-ACT CTA GTG
GCG GTG ACT-3` (missense control) were obtained from Marshall
University (Huntington, WV) or Midland Certified Reagent Co. (Midland,
TX). BALB cells were grown to confluence (with no treatment or with 18
h of pretreatment with 500 units/ml murine interferon-), and the
conditioned medium overlaying the cells was removed and mixed with
either antisense oligonucleotide or missense oligonucleotide to a final
concentration of 5 µM. The conditioned medium plus
oligonucleotide was then filter-sterilized and returned to the cells
for 48 h, at which time some cultures were stimulated with 10 ng/ml
PDGF-BB for an additional hour prior to harvesting of the cells for RNA
or for Western blot analysis of PKR protein levels. Western blot
analysis was performed as described previously (Faller et al.,
1994), except that the primary antibody was an anti-murine PKR rabbit
antiserum (Barber et al., 1993), generously provided by
Michael Katze and Glen Barber (University of Washington).
Inhibition of PDGF Induction of IE Genes c-fos, c-myc,
and JE, but Not egr-1, by 2-Aminopurine
If the genes
c-fos, c-myc, and JE are induced by PDGF via
a pathway dependent upon PKR activation, inhibitors of PKR activation
should block PDGF induction of these genes. Two methods of inhibiting
PKR activation while maintaining cell viability are known: treatment
with high doses of purine analogs, such as 2-aminopurine (Farrell et al., 1977), or expression of an oncogenic (activated) ras gene for at least 18 h (Mundschau and Faller, 1992). The
PKR inhibitor 2-AP has previously been shown to suppress serum
induction of the IE genes c-fos and c-myc in NIH3T3
cells (Zinn et al., 1988). Concordant with this result,
stimulation of quiescent BALB/c/3T3 murine fibroblasts (BALB cells)
with PDGF-BB instead of serum in the presence of 10 mM 2-AP
strongly inhibited induction of c-fos, c-myc, and JE (Fig. 1A and Table 1). However, PDGF
induction of another IE gene, egr-1, was unaffected by 2-AP in
BALB cells (Fig. 1A and Table 1). This verified
that 2-AP was not indiscriminately cytotoxic during the time course of
the experiment (90 min) and that it did not interfere with PDGF binding
to its receptor. In addition, the ability of PDGF to induce egr-1 in the presence of 2-AP suggested that at least two
PDGF-activated signal transduction pathways exist for IE gene induction
in BALB cells, distinguishable in their sensitivity to 2-AP and
possibly therefore in their dependence on PKR. The discovery of this
2-AP-insensitive signaling pathway would permit testing of predictions
made about the role of PKR in the 2-AP-sensitive pathway.
P-labeled probes specific for egr-1, actin, or JE. The actin probe serves as a control for equal loading of
RNA and even transfer. B, total cellular RNA was extracted
from BALB (first and second lanes) or KBALB (third and fourth lanes) confluent cell monolayers
after incubation for 24 h in 0.5% serum-containing medium, followed by
30 min of treatment with either no additions (first and third lanes) or 10 ng/ml PDGF-BB (second and forth lanes). The RNA was separated by electrophoresis and
transferred as described for A and then simultaneously
hybridized with P-labeled probes specific for c-fos or ATPase. The same filter was rehybridized for the egr-1
transcript. The ATPase transcript served as a control for equal loading
of RNA and even transfer. A and B are
autoradiograms.
Inhibition of PDGF Induction of IE Genes c-fos, c-myc,
and JE, but Not egr-1, by v-ras
The second method for inhibiting
PKR activation, expression of an oncogenic ras gene, has been
previously shown to act in BALB cells by inducing a cytoplasmic protein
that inhibits PKR autophosphorylation without down-regulating or
degrading the PKR protein itself (Mundschau and Faller, 1992). To test
the effect of this ras-induced inhibitor on IE gene induction,
we examined PDGF induction of IE genes in a BALB-derived cell line,
KBALB, chronically transformed by the activated ras gene
v-Ki-ras. The block to PKR autophosphorylation in KBALB cells
was verified by an in vitro kinase assay (Fig. 2A, lanes 1-4) discussed below.
RNA blot analysis of IE gene induction in KBALB cells in response to
PDGF is summarized in Table 1, and representative blots of egr-1 and c-fos (Fig. 1B) and
c-myc and JE (Fig. 2B) are shown. In
a pattern identical to the results observed in 2-AP-treated cells, PDGF
induction of the genes c-fos, c-myc, and JE was blocked or inhibited, while induction of egr-1
remained normal.
) BALB/c/3T3
cells (BALB), v-Ki-ras-transformed BALB/c/3T3 cells (KBALB),
and morphological revertants of KBALB cells induced either by
dibutyryl-cAMP (KcAMP) or by transfection with the Krev-1 gene
(Krev). After normalization for total protein concentration, the
lysates were incubated with 25 µM (10 µCi)
[-P]ATP in the presence (+) or
absence(-) of 20 ng/ml poly(I)poly(C) for 15 min at 37
°C. Proteins that could bind dsRNA were partially affinity-purified
on poly(I)poly(C)-agarose beads, separated by SDS-PAGE (10%
acrylamide), and exposed to film. PKR was readily identified in such an
assay as a 68-kDa band, which was phosphorylated in response to dsRNA. B, RNA blot of PDGF induction of IE genes in
v-ras-expressing (KBALB) and ras revertant (KcAMP and
Krev) cell lines. Total cellular RNA was extracted from BALB (lanes
1 and 2), KBALB (lanes 3 and 4), KcAMP (lanes 5 and 6), and Krev (lanes 7 and 8) confluent cell monolayers after incubation for 24 h in 0.5%
serum-containing medium, followed by 60 min of treatment with either no
additions (lanes 1, 3, 5, and 7) or
10 ng/ml PDGF-BB (lanes 2, 4, 6, and 8). The RNA (20 µg/lane) was separated by electrophoresis
and transferred to a nitrocellulose membrane, which was then
simultaneously hybridized with P-labeled probes specific
for c-myc, JE, or actin transcripts. A and B are
autoradiograms.
)it is also made particularly powerful by
the potential to create and study revertants of the ras-transformed phenotype (Kitayama et al., 1989;
Olinger et al., 1989; Carchman et al., 1974; Quinones et al., 1991). Two methods specific for reversion of ras-induced transformation, pharmacologic elevation of
intracellular cAMP levels and transfection with the Krev-1
gene, result in morphologically reverted cell lines derived from KBALB
cells (referred to as KcAMP and Krev cells, respectively) that have a
morphological phenotype intermediate to that of BALB and KBALB. Such
revertants have been shown previously to demonstrate partial
reconstitution of the PDGF signaling pathways (Quinones et
al., 1991). This has permitted some determination of which of the
phenomena associated with PDGF binding to its receptor revert as well
and may thus be causally related.poly(C) compared
with extracts from BALB (normal) cells (Fig. 2A),
consistent with PKR activity being causally related to induction of
c-fos, c-myc, and JE by PDGF.poly(C), induces c-fos,
c-myc, and JE, it remained to be seen if
poly(I)poly(C) could induce egr-1. Total cellular RNA
from BALB cells treated with 50 µg/ml poly(I)poly(C) was
therefore analyzed by RNA analysis with an egr-1 probe.
Consistent with egr-1 being induced by PDGF via a
PKR-independent pathway, dsRNA failed to induce egr-1 or
induced it very weakly with respect to induction by PDGF or PMA ( Fig. 3and Table 1).
poly(C) (lane 3). Each lane contained 20 µg of total cellular RNA
separated on a 1% formaldehyde-agarose gel. The RNA was transferred to
nitrocellulose and hybridized with P-labeled probes
specific for egr-1 or actin as a
control.
PKR Protein Depletion by Antisense-mediated Inhibition of
Translation
Although the reciprocal correlations between
apparent activation of PKR and PDGF induction of some IE genes, as
revealed by poly(I)poly(C) and two different PKR inhibitors,
presented a convincing circumstantial case for PKR involvement in PDGF
signal transduction, the strength of any such argument is dependent
entirely on the specificity of the inhibitors used. Activated c-ras or v-ras did not block all PDGF signal transduction
pathways since they did not interfere with induction of egr-1
by PDGF. Activated p21
, however, is not likely to be a
specific inhibitor of PKR as it has documented pleiotropic effects (as
discussed below) on PDGF signal transduction (Rake et al.,
1991; Quinones et al., 1991). Indirect evidence exists to
suggest that the specificity of 2-AP for inhibition of PKR activation,
however, is quite good (Giantini and Shatkin 1989; Kaufman and Murtha,
1987; Kalvakolanu et al., 1991; Mahadevan et al.,
1990; Zinn et al., 1988; Wathelet et al., 1989;
Tiwari et al., 1988). But, given that no work has been done to
rigorously assess the effect of 2-AP on the various serine/threonine
kinases, including the protein kinase C isoenzymes known to be involved
in PDGF signal transduction, it is not possible to conclude with
certainty that the 2-AP-inhibited event in the PDGF-activated signaling
cascade inducing IE gene expression is PKR activation. A more specific
inhibitor of PKR was therefore sought. When the murine PKR gene was
cloned and sequenced (Feng et al., 1992), it became possible
to attempt to directly deplete PKR protein levels by antisense
oligonucleotide inhibition of translation.
(lanes
2, 4, and 6) and then incubated in the presence
5 µM missense oligonucleotide (lanes 3 and 4) or 5 µM antisense oligonucleotide (lanes 5 and 6) complementary to the 5`-end of the PKR transcript
or left untreated (lanes 1 and 2) for 48 h.
Cytoplasmic extracts were then harvested, size-fractionated on a
5-15% gradient SDS-polyacrylamide gel, transferred to
nitrocellulose, probed for the PKR protein with a rabbit antiserum
specific for murine PKR, and developed with an alkaline
phosphatase-coupled second antibody. The PKR protein is indicated with
an arrowhead.
P-labeled probes specific for
c-myc (second exon), JE, actin, and egr-1
transcripts. Shown is an autoradiogram.
Activation of PKR in Response to PDGF
The PKR
kinase assay shown in Fig. 2A, and all assays in
previous reports demonstrating PKR autophosphorylation, have measured
the ability of PKR to phosphorylate itself in a cell lysate in response
to exogenously added dsRNA or other polyanions. If PKR is a
signal-transducing molecule for PDGF induction of IE genes, PDGF
treatment of cells might be expected to have a measurable effect on PKR
activation and therefore on its autophosphorylation state. Assaying for
activation of PKR in response to PDGF could not be performed in
vitro by the addition of PDGF to a cell lysate in place of dsRNA,
however, as many PDGF-induced second messenger events do not function
in such an in vitro system.-P]ATP to enter the cell while leaving
signal transduction pathways intact for at least a short period.
Confluent, serum-deprived BALB cells were loaded with
[-P]ATP by such permeabilization and
simultaneously stimulated with PDGF-BB or poly(I)poly(C) for 15
or 30 min at 37 °C. The cells were then rinsed and lysed, and
affinity precipitation with poly(I)poly(C)-agarose was carried
out. The 68-kDa kinase PKR was found to be phosphorylated in response
to PDGF within 15 min of PDGF addition and returned essentially to
base-line levels by 30 min (Fig. 6). Digitonin eventually caused
separation of the cell monolayers from the tissue culture plate
surface, such that it was not possible to assay time points beyond 30
min. The magnitude of the induction of PKR phosphorylation by PDGF was
comparable to the increases seen after stimulation with
poly(I)poly(C) (Fig. 6, lane 6). In repetitions
of this experiment, the time courses of PKR activation for these two
activators were similar. A 5-min time point was included in some
experiments (not shown here), and no detectable increase in PKR
autophosphorylation over background was observed at that time after
stimulation by PDGF or dsRNA. Therefore, PKR appears to become
activated, as determined by autophosphorylation, in response to
stimulation with PDGF-BB in intact cells.
-P]ATP, and one of the following: no
further additions (lane 2), 20 ng/ml PDGF-BB (lanes 3 and 4), or 10 µg/ml poly(I)poly(C) (lane
5). Also included was a control containing
[-P]ATP but no digitonin (no dig)
in order to monitor the degree of spontaneous PKR phosphorylation, if
any, occurring after cell lysis (lane 1). After incubation at
37 °C for the times indicated, the overlay was aspirated, the
monolayer was rinsed once with isotonic wash buffer containing 5 mM EDTA, and the cells were lysed in 0.5% Nonidet P-40. Particulate
matter was removed by microcentrifugation, and PKR was partially
affinity-purified from cytoplasmic extracts by precipitation on
poly(I)poly(C)-agarose. Poly(I)poly(C)-bound, P-labeled proteins were analyzed by SDS-PAGE. An
autoradiogram is shown here.
poly(C)) would therefore be predicted to be decreased. To
test this prediction, lysates from PDGF-stimulated cells were subjected
to a standard in vitro PKR kinase assay in the presence and
absence of exogenous dsRNA. In three independent experiments, reduction
in new dsRNA-inducible PKR phosphorylation was found in lysates from
cells treated with PDGF at 20 min, but not in lysates from cells
treated with PDGF at 40 min (Fig. 7). This finding suggested
pre-existing phosphorylation of PKR 20 min after PDGF exposure, which
was lost by 40 min, consistent with the result found in the digitonin
permeabilization assay. Levels of phosphorylation of a number of
proteins that were precipitated by the poly(I)poly(C)-agarose
from the 20-min lysate were reduced, a finding that may reflect a block
to new phosphorylation of cellular proteins in general due to
pre-existing phosphorylation induced by PDGF.
, followed by 15 ng/ml
PDGF-BB for zero min (first and second lanes), 20 min (third and fourth lanes 3), and 40 min (fifth and sixth lanes). An in vitro PKR kinase assay
was performed on each extract in the presence (+) and
absence(-) of exogenously added poly(I)poly(C) as
indicated, followed by precipitation on poly(I)poly(C)-agarose
and SDS-PAGE. Shown is an autoradiogram of the dried
gel.
A 2-Aminopurine- and v-ras-inhibited Event Downstream of
Protein Kinase C Activation
Having found direct evidence that
PKR functions as a signal transducer in a PDGF-stimulated pathway for
induction of some IE genes, we sought to locate the PKR activation
event in the PDGF signaling cascade with respect to known second
messengers for IE gene induction. Protein kinase C is a PDGF second
messenger for which both specific inhibitors and direct activators
exist, and these effectors can be used in intact cells. Phorbol
12-myristate 13-acetate (PMA) is a membrane-permeable, direct activator
of protein kinase C. All four IE genes examined in this investigation
were induced strongly by PMA. However, previous studies utilizing
protein kinase C inhibitors suggested that induction of egr-1,
c-fos, and c-myc by PDGF does not require protein
kinase C (Hall and Stiles, 1987; Wright et al., 1989) (
)(or alternatively utilizes isoforms not sensitive to the
inhibitors (Kikkawa et al., 1989)), whereas PDGF induction of
the JE gene appears to be completely dependent upon protein
kinase C activation (Hall and Stiles, 1987). PDGF induction
of the JE gene could thus be used to locate the relative
positions of PKR and protein kinase C in this PDGF signal transduction
pathway. Northern blot analysis was performed on total cellular RNA
from KBALB cells stimulated with PDGF or PMA in the presence or absence
of 2-AP. Analysis of both c-fos and JE gene
expression demonstrated that JE could not be induced by PDGF
or PMA in the presence of v-ras or v-ras and 2-AP (Fig. 8) or 2-AP alone (data not shown), thus localizing PKR
activation downstream of protein kinase C activation or indicating that
protein kinase C requires activated PKR to induce JE. In
contrast, induction of the c-fos gene differed for PDGF and
PMA. PDGF induction of c-fos was blocked by both v-ras and 2-AP as expected, while induction of c-fos by PMA was
unaffected by either inhibitor. This result verified that PDGF
induction of c-fos is not mediated through protein kinase C
and also served as a control to show that 2-AP does not inhibit protein
kinase C activation itself. However, since PDGF induction of all three
genes (c-fos, c-myc, and JE) appears to
require PKR activation even though their induction pathways seem to
diverge upstream of protein kinase C, protein kinase C would therefore
appear not to be the direct upstream activator of PKR in this PDGF IE
gene induction pathway.
P-labeled
probes specific for c-fos or JE. An autoradiogram is
shown.
poly(C), other polyanions
have also been shown to have the capacity to induce PKR to
autophosphorylate in vitro.
B and glucocorticoid receptors (Baeuerle
and Baltimore, 1988; Pratt et al., 1988; Denis et
al., 1988). Both PKR and its heme-regulated homolog PKH (Chen et al., 1991) have been shown to coprecipitate with another
protein species, an unidentified 90-kDa protein in the case of PKR
(Matts and Hurst, 1989; Rice et al., 1989). This 90-kDa
protein is not found to be associated with the autophosphorylated form
of PKR, and although there is no evidence that PKR translocates to the
nucleus upon dissociation of its chaperone, as is the usual case for
other proteins under this type of control, PKR has been localized to
the nucleus as well as the cytoplasm (Clemens et al., 1994). , PKR activation
would be expected to be growth inhibitory, at least in the short term.
However, attempts to inhibit PKR activity in normal cells by stable
expression of virally encoded or induced PKR inhibitors have repeatedly
failed due to lack of viability of the
transfectants.(
)(
)()Similarly,
prolonged exposure to the PKR inhibitor 2-AP arrests the growth of
murine fibroblasts. Only two PKR inhibitors have successfully been
expressed in cells over a long period: the endogenous cellular protein
induced by activated p21 (Mundschau and Faller, 1992,
1994) and the 58-kDa cellular inhibitor of PKR activated by influenza
virus (Lee et al., 1990). However, it is worthy of note that ras-transformed cells have long been known to be growth
factor-independent, and all cellular transfectants stably expressing
the 58-kDa cellular inhibitor of PKR were also found to be growth
factor-independent and transformed (Barber et al., 1994; Lee et al., 1990). serine/threonine kinase PKR is an essential component of the
PDGF signal transduction pathway for the induction of some IE genes.
The role of PKR in interferon-induced antiviral and antiproliferative
cellular responses is well established. Thus, PKR is a signaling
mediator common to both growth-promoting and growth inhibitory factors
and may provide a mechanism for cross-talk between these two pathways.
The mechanism of PDGF activation of PKR is unknown, but appears to lie
downstream of protein kinase C activation. Downstream targets of the
activated kinase are yet to be discovered. The only known PKR
substrate, eIF-2, is unlikely to be involved since serine to
alanine mutations of the residues normally phosphorylated on eIF-2
by active PKR have been shown to have no effect on normal cell growth
(Murtha-Riel et al., 1993). Finally, the requirement for
another kinase in the PDGF-activated cascade, one that is integral to
the pathway leading to the induction of certain genes by PDGF but not
others, may result in a better understanding of the ways in which
signaling pathways originating from a single, synchronized stimulus
diverge to produce a complex pattern of gene regulation.
), eukaryotic
initiation factor-2; PMA, phorbol 12-myristate 13-acetate; PAGE,
polyacrylamide gel electrophoresis.)))))
We thank Michael Katze and Glen Barber for generously
providing anti-PKR antibody, Michael Quinones for generating the Krev-1 reverted KBALB cells, Lora Wan Forman for expert
technical assistance, and Margaret Offermann for helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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