©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Platelet-derived Growth Factor Signal Transduction through the Interferon-inducible Kinase PKR
IMMEDIATE EARLY GENE INDUCTION (*)

(Received for publication, August 8, 1994; and in revised form, November 16, 1994)

Laura J. Mundschau (§) Douglas V. Faller (¶)

From the Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The interferon-inducible, double-stranded RNA (dsRNA)-dependent eukaryotic initiation factor-2alpha 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.


INTRODUCTION

Treatment of quiescent Balb/c/3T3 fibroblasts with peptide platelet-derived growth factor (PDGF)(^1)-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.

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)bulletpoly(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-2alpha 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-2alpha (Petryshyn et al., 1983). This phosphorylation of eIF-2alpha 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).

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(0) and early G(1) (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).

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)bulletpoly(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.


MATERIALS AND METHODS

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^6-2`-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)bulletpoly(C) was obtained in soluble form and as a conjugate to agarose beads (poly(I)bulletpoly(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 beta-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(2), 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)bulletpoly(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)bulletpoly(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-beta), 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).


RESULTS

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.


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 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.



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-beta) 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)bulletpoly(C) for 15 min at 37 °C. Proteins that could bind dsRNA were partially affinity-purified on poly(I)bulletpoly(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.



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), (^2)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.

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)bulletpoly(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.

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)bulletpoly(C), induces c-fos, c-myc, and JE, it remained to be seen if poly(I)bulletpoly(C) could induce egr-1. Total cellular RNA from BALB cells treated with 50 µg/ml poly(I)bulletpoly(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).


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)bulletpoly(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)bulletpoly(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.

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-beta (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.




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 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.

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 [-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)bulletpoly(C) for 15 or 30 min at 37 °C. The cells were then rinsed and lysed, and affinity precipitation with poly(I)bulletpoly(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)bulletpoly(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.


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, [-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)bulletpoly(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)bulletpoly(C)-agarose. Poly(I)bulletpoly(C)-bound, P-labeled proteins were analyzed by SDS-PAGE. An autoradiogram is shown here.



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)bulletpoly(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)bulletpoly(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.


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-beta, 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)bulletpoly(C) as indicated, followed by precipitation on poly(I)bulletpoly(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) (^3)(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).^3 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.


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 P-labeled probes specific for c-fos or JE. An autoradiogram is shown.




DISCUSSION

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)bulletpoly(C), other polyanions have also been shown to have the capacity to induce PKR to autophosphorylate in vitro.

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-kappaB 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).

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-2alpha, 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.(^4)(^5)(^6)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).

In conclusion, we have presented direct evidence that activation of the interferon-induced, dsRNA-activated eIF-2alpha 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-2alpha, is unlikely to be involved since serine to alanine mutations of the residues normally phosphorylated on eIF-2alpha 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Cardiovascular Biology Training Grant from the National Institutes of Health.

Supported by research grants from the National Institutes of Health and the Council for Tobacco Research. To whom correspondence should be addressed: Boston University School of Medicine, Cancer Research Center, 80 E. Concord St., E-124, Boston, MA 02118. Tel.: 617-638-4173; Fax: 617-638-4176.

(^1)
The abbreviations used are: PDGF, platelet-derived growth factor; IE, immediate early; dsRNA, double-stranded RNA; 2-AP, 2-aminopurine; eIF-2alpha, eukaryotic initiation factor-2alpha; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis.

(^2)
M. Zubiaur and D. V. Faller, manuscript in preparation.

(^3)
L. J. Mundschau, unpublished data.

(^4)
A. Shatkin, A., personal communication.

(^5)
R. Kaufman, personal communication.

(^6)
L. J. Mundschau and D. V. Faller, unpublished results.


ACKNOWLEDGEMENTS

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.


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