©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Interferon-inducible Protein Kinase PKR Modulates the Transcriptional Activation of Immunoglobulin Gene (*)

(Received for publication, June 13, 1995)

Antonis E. Koromilas (1)(§) Claude Cantin (2) Andrew W. B. Craig (3) Rosemary Jagus (4) John Hiscott (5) Nahum Sonenberg (3)

From the  (1)Departments of Oncology and Medicine, McGill University, Montreal, Quebec H3T 1E2, Canada, the (2)Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada, the (3)Department of Biochemistry and McGill Cancer Centre, McGill University, Montreal, Quebec H3G 1Y6, Canada, the (4)Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202, and the (5)Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3G 1Y6, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 IkappaBalpha upon double-stranded RNA treatment resulting in activation of NF-kappaB 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 kappa-chain transcriptional activation induced by lipopolysaccharide or IFN-. However, expression of the dominant negative PKR mutant inhibited kappa gene transcription independently of NF-kappaB activation. Phosphorylation of eIF-2alpha was not increased by lipopolysaccharide or IFN-, suggesting that PKR mediates kappa 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-kappaB-dependent and NF-kappaB-independent.


INTRODUCTION

IFNs (^1)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 alpha 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-2alpha(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 (PKRDelta6) (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.^2 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 IkappaBalpha leading to activation of NF-kappaB(28) . Furthermore, cells depleted of PKR activity were unresponsive to activation of NF-kappaB by dsRNA(29) . Other mechanisms which are NF-kappaB 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 PKRDelta6(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 kappa gene. Transcription of the kappa 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 kappa gene is mediated by PKR. Expression of the dominant negative PKRDelta6 resulted in inhibition of kappa-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.


EXPERIMENTAL PROCEDURES

Cell Culture

70Z/3 cells (ATCC TIB 158) were grown at 37 °C in complete RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (heat-inactivated), 2 mML-glutamine, 50 µM beta-mercaptoethanol, penicillin (100 units/ml), streptomycin (100 units/ml), and humidified with 5%/95% CO(2)/air gas mixture. HeLa S3 cells (ATCC CCL 2.2) were grown in complete Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 2 mML-glutamine, penicillin (100 units/ml), streptomycin (100 units/ml), and humidified with 5%/95% CO(2)/air gas mixture.

Transfection and Selection of Stable Transfectants

Plasmids containing wt PKR, and PKRDelta6 cDNAs under the control of human cytomegalovirus promoter in the pcDNAI/neo vector (16) were used for expression in 70Z/3 cells. Plasmid DNA (10 µg) was linearized with KpnI and electroporated into 1 times 10^7 cells at 300 V-960 µF (Bio-Rad) as described previously(33) . After electroporation, 70Z/3 cells were cultured in nonselective medium and grown for 24 h to allow for expression of the transfected genes. Cells were recultured in 24-well plates at a concentration of 1 times 10^5/ml (1 ml/well) in medium containing G418 (Life Technologies, Inc.) at a final concentration 400 µg/ml. Medium was replenished every 3 days. Cells (polyclonal populations) were expanded and characterized 15 days postselection. Independent clones were selected by a limiting dilution method as described previously(34) .

Immunoprecipitation and Immunoblotting

Cells (1 times 10^7) were washed three times with cold phosphate-buffered saline (PBS, 140 mM NaCl, 15 mM KH(2)PO4 (pH 7.2), and 2.7 mM KCl) and incubated on ice with an equal volume of 2 times lysis RIPA (100 mM TrisbulletCl (pH 7.5), 300 mM NaCl, 2% Nonident P-40, 1% sodium deoxycholate, and 0.2% SDS) supplemented with 2 mM dithiothreitol (DTT), 0.4 mM phenylmethylsulfonyl fluoride (PMSF), and 4 µg/ml aprotinin. The lysate was centrifuged at 10,000 times g for 10 min, and the supernatant was incubated with 2.5 µl of anti-PKR polyclonal antibody for 2 h at 4 °C. Then, 50 µl of 50% suspension of protein A-Sepharose 4L (Pharmacia Biotech Inc.) in 1 times RIPA were added, and incubation was continued for additional 4 h at 4 °C under rotation. The Sepharose beads were washed with 1 times RIPA plus 1 M NaCl twice and 1 times RIPA twice. Immunoprecipitates were subjected to electrophoresis on SDS-8% polyacrylamide gel. The separated proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell) in 25 mM TrisbulletCl (pH 7.5), 190 mM glycine, and 20% (v/v) methanol for 2 h at 1 Å. The filter was first incubated with 5% (w/v) non-fat dried skimmed milk powder in PBS for 1 h at room temperature and then with 25% fetal bovine serum and 0.5% (v/v) Triton X-100 in PBS containing a mouse monoclonal antibody to human PKR (13B8-F9). The blot was incubated with peroxidase-conjugated rabbit antibody to mouse immunoglobulin G, and proteins were visualized using the enhanced chemiluminescence system (Amersham Corp.) according to the manufacturer's specifications.

PKR Dephosphorylation Assay

For PKR dephosphorylation, 2 times 10^7 cells were washed with ice-cold PBS twice and lysed with an equal volume of 2 times lysis buffer (20 mM TrisbulletCl (pH 7.5), 100 mM KCl, 4 mM MgCl(2), 2% Triton X-100, 2 mM DTT, 0.4 mM PMSF, and 4 µg/ml aprotinin). The lysate was centrifuged at 10,000 times g for 10 min, and the supernatant was incubated with 2.5 µl of anti-PKR polyclonal antibody for 2 h at 4 °C. Then, 50 µl of 50% suspension of protein A-Sepharose 4L (Pharmacia) in 1 times lysis buffer were added, and incubation was continued for an additional 4 h at 4 °C under rotation. The Sepharose beads were washed with high salt buffer (20 mM TrisbulletCl (pH 7.5), 400 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM DTT, 0.2 mM PMSF, 2 µg/ml aprotinin, and 20% glycerol) twice and low salt buffer (20 mM TrisbulletCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 2 µg/ml aprotinin, and 20% glycerol) twice. Immunoprecipitates were subjected to dephosphorylation by adding 3 µl of 10 times phosphatase buffer (0.5 M TrisbulletCl (pH 9.0), 10 mM MgCl(2), 1 mM ZnCl(2), and 10 mM spermidine) and 3 units of calf intestine phosphatase (Promega) in 30 µl total volume and incubating at 37 °C for 2 h. Then, immunoprecipitates were washed with 1 times RIPA plus 1 M NaCl twice, 1 times RIPA twice, and subjected to immunoblotting analysis as described above.

Cell Induction and Immunofluorescent Analysis

Cells were incubated at concentration 5 times 10^5 cells/ml with appropriate concentrations of inducing agents: 10 µg/ml of Salmonella typhosa LPS (Sigma) or 100 U/ml murine recombinant IFN- (Cedarlane, Canada). For prolonged inductions, cells were diluted daily to 5 times 10^5 cells/ml with fresh medium supplemented with the inducing agent.

The surface staining of induced 70Z/3 cells expressing sIgM was performed with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse kappa antibodies (BioCan) as described elsewhere (35) . Immediately after staining, cells were analyzed on a cell sorter (FACStar, Becton Dickinson, Mountain View, CA) as previously described (35) .

RNA Extraction and Northern Blotting

Total RNA was isolated by the guanidinium thiocyanate method(36) . RNA (10 µg) was denatured with glyoxal and dimethyl sulfoxide and subjected to electrophoresis on a 1% agarose gel in 10 mM sodium phosphate buffer (pH 7.0). For RNA stability experiments, total RNA was isolated from cells treated with actinomycin D (10 µg/ml) for 30, 60, and 120 min. RNA was transferred onto a nylon membrane (BioTrans, ICN). Hybridization was performed at 65 °C for 16 h with [alpha-P]dATP-labeled random-primed cDNA probes (5 times 10^6 cpm/ml)(37) , consisting of either the 750-base pair SmaI-PstI fragment of the µ-chain cDNA together with a 3.0-kilobase HindIII fragment of the kappa-chain cDNA (38) or the entire coding sequence of mouse beta-actin. After hybridization, the filter was washed with 0.1 times SSC (150 mM NaCl and 15 mM sodium citrate (pH 7.0)) plus 1% SDS for 1 h at 45 °C. The filter was dried and exposed to an x-ray film for 10 h.

Nuclear Run-on Analysis

A modification of the Larner et al.(39) method was used. Cells were washed in ice-cold PBS twice and resuspended in ice-cold buffer consisting of 0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 15 mM Hepes (pH 7.5), 2 mM EDTA, 0.5 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 14 mM beta-mercaptoethanol, and 0.1% Nonidet P-40 at 3-5 times 10^7 cells/ml. After swelling for 5 min, the nuclei were pelleted by centrifugation at 2,000 times g for 5 min, resuspended at 10^6 nuclei/ml in buffer containing 20 mM TrisbulletCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA, 50% glycerol, 0.85 mM DTT, 0.125 mM PMSF, and 100 units/ml RNase inhibitor (Promega). Nuclei were stored at -85 °C.

For transcriptional assay, nuclei (1 times 10^7 nuclei/reaction) were resuspended in 100 µl of 0.3 M ammonium sulfate, 100 mM TrisbulletCl (pH 7.9), 4 mM MgCl(2), 4 mM MnCl(2), 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 [alpha-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 kappa-chain cDNAs, glyceraldehyde-3-phosphate dehydrogenase cDNA, and KS Bluescript vector DNA was immobilized on a nylon membrane (BioTrans, ICN) and hybridized with [alpha-P]GTP-labeled RNA (5 times 10^6 cpm/ml) for 48 h at 65 °C as described elsewhere(39) .

PKR Autophosphorylation and eIF-2alpha Phosphorylation Analysis

For in vitro autophosphorylation of PKR, 10 µg of extracts from untreated HeLa S3 cells or HeLa S3 cells treated with human IFN-beta for 18 h (1000 IU/ml; Lee Biomolecules) were suspended in kinase reaction buffer (10 mM TrisbulletCl, pH 7.7, 50 mM KCl, 2 mM MgCl(2), 5 mM beta-mercaptoethanol, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 0.1 mM PMSF) and 10 µCi of [-P]ATP. Reovirus dsRNA was added to the final concentration of 0.1 µg/ml. After incubation at 30 °C for 30 min, the reaction was diluted 5-fold with RIPA and split equally into two fractions. In one of the fractions, 2.5 µl of anti-PKR monoclonal antibody (13B8-F9) were added, and immunoprecipitation of the autophosphorylated PKR was performed as described above. In the other fraction, 5 µl of sheep anti-eIF-2alpha polyclonal antibody were added, and eIF-2alpha immunoprecipitation was performed as for PKR using protein G-Sepharose (Pharmacia) as a carrier. PKR immunoprecipitates were subjected on SDS-8% polyacrylamide gels, whereas eIF-2alpha immunoprecipitates were on SDS-10% polyacrylamide gels.

For in vivo phosphorylation of eIF-2alpha 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 beta-glycerophosphate, and 20 mM Na(2)MoO(4) and lysed in 10 mM TrisbulletCl (pH 7.5), 50 mM KCl, 2 mM MgCl(2), 1% Triton X-100, 1 mM DTT, 0.2 mM PMSF, and 2 µg/ml aprotinin. The lysate was centrifuged at 10,000 times 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-2alpha 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 beta-glycerophosphate, and 20 mM Na(2)MoO(4) 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 times 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-2alpha and subjected to immunoblotting using a monoclonal antibody to eIF-2alpha as described previously(41) .

Electrophoretic Mobility Shift Assays

Nuclear protein extracts were prepared as described elsewhere(42) . Five µg of protein extracts were tested for NF-kappaB activity by binding to 80 pg of a P-5`-end-labeled dsDNA oligonucleotide (1 times 10^6 cpm/ng; 5`-GATCCAAGGGGACTTTCCATGGATCCAAGGGGACTTTCCATG-3`; Life Technologies, Inc.; the underlined sequences correspond to the NF-kappaB-binding sites) as described previously(43) . Antibody mobility supershift assays were performed by incubating 10 µg of protein extracts together with 200 pg of the P-5`-end-labeled dsDNA HIV-kappaB oligonucleotide (44) (5 times 10^6 cpm/ng; 5`-AGCTGGGACTTTCCGCTA-3`; the underlined sequence corresponds to the NF-kappaB-binding site) and 1 µl of the stock of affinity-purified rabbit polyclonal antibodies against rel(45) , p65 (45) , or p50 (46) proteins. For cold competition an 125-fold excess of unlabeled dsDNA oligonucleotides was added. The specificity of the supershifted bands was tested by antibody binding competition with 1 µg of the epitope peptide (45, 46) used for antisera preparation.


RESULTS

Expression of Wild Type PKR and Catalytically Inactive PKRDelta6 in 70Z/3 Cells

70Z/3 cells were transfected with wt human PKR or PKRDelta6 (originally termed p68Delta6) (16) cDNA and selected in G418. Polyclonal populations of G418-resistant cells were expanded and characterized for protein expression by immunoblotting using a monoclonal antibody (13B8-F9) specific for the human PKR. (^3)As expected, the anti-human PKR antibody failed to detect the endogenous mouse PKR in control 70Z/3 cells transfected with the neomycin-resistant gene only (Fig. 1, A and B, lane 2). Two bands were detected in wt PKR-transfected cells (1, A and B, lane 3) which correspond to the phosphorylated (upper band) and nonphosphorylated (lower band) forms of the kinase(5, 47) . Phosphatase treatment of wt PKR yielded the slower migrating nonphosphorylated form of the molecule (Fig. 1B, compare lanes 3 and 4). In contrast, expression of the mutant PKRDelta6 yielded one polypeptide species which is the nonphosphorylated form (Fig. 1A, lane 4). This is consistent with the dominant negative character of PKRDelta6(16) , as PKRDelta6 is neither autophosphorylated nor is it phosphorylated by endogenous mouse PKR. The native PKR is nonphosphorylated (Fig. 1, A and B, lane 1). It is noteworthy that wt PKR can be overexpressed in several transformed cell lines, (^4)including 70Z/3 cells, without apparent inhibition of cell growth, in contrast to NIH 3T3 cells(16) . This may be explained by modulation of PKR activity by a specific inhibitor(s) as reported for v-ras-transformed cells(48) .


Figure 1: A, expression of human wt PKR and PKRDelta6 proteins in 70Z/3 cells. wt PKR and PKRDelta6 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, PKRDelta6-expressing cells. B, human wt PKR is expressed in phosphorylated and nonphosphorylated forms (A and B, lane 3); treatment with calf intestine phosphatase (CIP) results in the nonphosphorylated form of PKR (lane 4). Native PKR is in nonphosphorylated form (A and B, lane 1).



Inhibition of sIgM Expression in 70Z/3 Cells Expressing PKRDelta6 Mutant

The sIgM expression in 70Z/3 cells expressing wt PKR or PKRDelta6 was examined by cell surface staining with FITC anti-mouse kappa antibody followed by cell analysis on a cell sorter(35) . Upon LPS or IFN- treatment, sIgM expression was diminished in 70Z/3 cells expressing PKRDelta6 (2-3-fold; Fig. 2, E and F), but not in cells expressing the neomycin-resistant gene only (herein referred as control 70Z/3 cells; Fig. 2, A and B). Expression of sIgM in response to LPS or IFN- was not affected in 70Z/3 cells overexpressing wt PKR relative to control 70Z/3 cells (Fig. 2, C and D). This is presumably because the endogenous mouse PKR elicits the maximal IgM expression upon LPS or IFN- treatment.


Figure 2: Surface IgM expression is decreased in cells expressing PKRDelta6. 70Z/3 cells expressing the neomycin resistance gene (A and B), wt PKR (C and D), or PKRDelta6 (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 kappa antibody, and sIgM levels were determined by flow cytometry analysis on a cell sorter after propidium iodide staining.



PKRDelta6 Inhibits Immunoglobulin kappa-Chain RNA Expression

To understand the molecular basis of the inhibition of sIgM expression by the dominant-negative PKRDelta6, we isolated single clones from the polyclonal population of control 70Z/3 cells (herein referred as control clones) and 70Z/3 cells expressing PKRDelta6 (herein referred as PKRDelta6 clones) by the limiting dilution method(34) . Several clones were examined for sIgM expression upon LPS or IFN- treatment. All 12 of the control clones showed the same pattern of high levels of sIgM expression upon LPS or IFN- treatment (data not shown). However, from the PKRDelta6 clones tested, 12 out of 16 showed a significant decrease (between 40 and 70% compared to control cells) in sIgM expression, whereas the rest of the PKRDelta6 clones (4 out of 16) showed a smaller but measurable effect (20-40% decrease in sIgM expression) upon LPS or IFN- treatment (data not shown).

To examine whether the decrease in sIgM expression was due to inhibition of kappa- or µ-chain immunoglobulin expression, the level of kappa- and µ-chain mRNAs in a control clone (CON-8) and several PKRDelta6-expressing clones was examined by Northern analysis using mouse kappa- and µ-chain immunoglobulin (38) and beta-actin cDNA probes. Expression of kappa-chain was observed neither in resting control clone nor in resting PKRDelta6-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 kappa-chain immunoglobulin relative to the µ-chain was significantly lower in PKRDelta6-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 kappa-chain relative to beta-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 kappa-chain mRNA expression is inhibited by PKRDelta6. Northern analysis. Expression of kappa-chain, µ-chain, and/or beta-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).



PKRDelta6 Mediates Inhibition of kappa-Chain Expression at Transcriptional Level

The effect of PKRDelta6 on kappa-chain expression could be explained by either a transcriptional or a post-transcriptional mechanism(s). To distinguish between these possibilities, a nuclear run-on analysis of kappa- and µ-chain transcription was performed with two of the PKRDelta6-expressing clones (PKRDelta6-1 and PKRDelta6-20). Run-on experiments with PKRDelta6-1 clone (Fig. 4A) showed that inhibition of kappa-chain transcription relative to µ was 60% (compare lanes 2 and 5) and 65% (compare lanes 3 and 5) upon LPS and IFN- treatment, respectively (the experiment was repeated twice and the results varied by no more than 10%). Inhibition of kappa-chain transcription relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was 60% (lanes 2 and 5) and 70% (lanes 3 and 6) upon LPS and IFN- treatment, respectively. In the case of PKRDelta6-20, 55 and 60% inhibition of transcription of kappa-chain relative to the µ-chain was seen upon LPS and IFN- treatment, respectively (Fig. 4B, compare lane 2 to 5 and lane 3 to 6; the experiment was repeated three times, and the results varied by less than 10%). These findings indicate that PKRDelta6 expression leads to inhibition of kappa-chain expression at the level of transcription initiation.


Figure 4: PKRDelta6 inhibits LPS or IFN- induction of kappa-chain expression at the transcriptional level. A and B, run-on assay. PKRDelta6-1 (A) and PKRDelta6-20 (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 kappa- and µ-chain mRNAs was determined in control (CON-8; lanes 1-8) or PKRDelta6 (PKRDelta6-1; 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 PKRDelta6 cells was isolated, and the levels of kappa-chain and µ-chain mRNA were compared by Northern blotting. Although kappa-chain mRNA expression was decreased in PKRDelta6 cells upon LPS treatment (Fig. 4C, compare lanes 1 and 9) or IFN- treatment (compare lanes 5 and 13), the ratio of kappa-chain to µ-chain mRNA did not change either in control cells or in PKRDelta6 cells after actinomycin D treatment. This is consistent with previous studies showing that kappa-chain mRNA is very stable (49) and indicates that PKRDelta6 does not affect kappa-chain mRNA stability.

Phosphorylation of eIF-2alpha Is Not Required for the Transcriptional Activation of kappa Gene

The regulation of kappa-chain transcription by PKR is the second example of regulation of transcription by an eIF-2alpha kinase. The yeast eIF-2alpha kinase, GCN2, regulates expression of the transcriptional activator GCN4 at the translational level upon amino acid starvation conditions(4) . It is possible that PKR mediates kappa-chain transcription indirectly by regulating the protein synthesis of a transcriptional factor(s) through eIF-2alpha phosphorylation as does GCN2. To examine this possibility, we measured the extent of eIF-2alpha phosphorylation in vivo upon LPS or IFN- treatment. We used two different assays for eIF-2alpha phosphorylation: (i) immunoprecipitation of eIF-2alpha from [P]orthophosphate-labeled cells (Fig. 5B) and (ii) isoelectric focusing followed by eIF-2alpha immunoblotting (Fig. 5C). In the first assay we used a sheep anti-eIF-2alpha polyclonal antibody, whose suitability was tested first (Fig. 5A). HeLa S3 cell extracts were incubated with reovirus dsRNA and [P-]ATP followed by immunoprecipitation with either anti-PKR antibody (Fig. 5A, lanes 1-4) or anti-eIF-2alpha antibody (Fig. 5A, lanes 5-8). Induction of PKR autophosphorylation by dsRNA before (lane 2) or after treatment with IFN-beta (lane 4) resulted in increased phosphorylation (lanes 6 and 8, respectively) of a protein immunoprecipitated by anti-eIF-2alpha antibody, whose molecular size corresponds to eIF-2alpha (38 kDa). Based on these results, we used the anti-eIF-2alpha polyclonal antibody to immunoprecipitate the in vivoP-labeled eIF-2alpha. Phosphorylation of eIF-2alpha did not significantly differ between control (Fig. 5B, lanes 1-3) and PKRDelta6 cells (lanes 4-6), which were stimulated either with LPS (lanes 2 and 5) or IFN- (lanes 3 and 6). This experiment was performed three times with no significant variations in eIF-2alpha phosphorylation. Similarly, no significant differences in the levels of eIF-2alpha phosphorylation were observed when the isoelectric focusing and eIF-2alpha immunoblotting assay was used (Fig. 5C). These experiments suggest that eIF-2alpha is not a substrate for PKR activated by LPS or IFN- in 70Z/3 cells.


Figure 5: Phosphorylation of eIF-2alpha in 70Z/3 cells expressing PKRDelta6. A, phosphorylation of eIF-2alpha by PKR in vitro. Cell extracts (10 µg) from HeLa S3 cells before (lanes 1, 2, 5, and 6) or after IFN-beta 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-2alpha polyclonal antibody (lanes 5 and 6) followed by SDS-polyacrylamide gel electrophoresis analysis. B and C, phosphorylation of eIF-2alpha in vivo. Control (CON-8) 70Z/3 cells (B, lanes 1-3; C, lanes 3-5) and 70Z/3 cells expressing PKRDelta6 (B, lanes 4-6; C, lanes 6-8) were analyzed for eIF-2alpha 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-2alpha (Negative Control) only or purified eIF-2alpha phosphorylated in vitro by the heme regulated eIF-2alpha kinase (Positive Control) to indicate the position of phosphorylated and nonphosphorylated forms of eIF-2alpha. Quantitation of labeled bands was performed by scanning autoradiograms in the linear range of exposure with a enhanced laser densitometer Ultroscan XL (LKB).



Induction of NF-kappaB Activity Is Not Inhibited by PKRDelta6

The induction of kappa-chain transcription in 70Z/3 cells upon LPS treatment requires the activation of NF-kappaB, which binds to the kappa-chain enhancer motif, GGGACTTTCC(50) . It has been recently shown that the transcription inhibitor IkappaB can be phosphorylated by PKR in vitro resulting in induction of NF-kappaB DNA binding(28) . Based on this finding, we examined the possibility that PKRDelta6 inhibits phosphorylation of IkappaB by PKR resulting in inhibition of NF-kappaB activation and consequently in a decrease of kappa-chain transcription. To this end, we tested NF-kappaB activity in nuclear extracts of 70Z/3 cells induced with LPS only since IFN- induces kappa-chain transcription in the absence of NF-kappaB activation(51, 52) . NF-kappaB binding to a DNA fragment containing two repeats of NF-kappaB consensus sequence -GGGACTTTCC- was analyzed by the gel retardation assay(43) . No detectable NF-kappaB activity was observed in resting 70Z/3 control cells, as no protein-DNA complexes were formed (Fig. 6A, lane 1), but DNA binding was evident after LPS treatment (Fig. 6A, lane 2). The two inducible bands most likely correspond to the binding of one (lower band) or two NF-kappaB complexes (upper band) to one or two kappaB sites, respectively, in the DNA probe (see also below). Formation of these complexes was drastically reduced by competition with an unlabeled oligonucleotide containing the two NF-kappaB-binding sites (Fig. 6A, lane 3). Significantly, indistinguishable NF-kappaBbulletDNA complexes were observed in control and two independent PKRDelta6-expressing clones (PKRDelta6-1 and PKRDelta6-20) upon LPS treatment (Fig. 6A, compare lane 2 to lanes 4 and 5).


Figure 6: NF-kappaB DNA binding activity is not affected by expression of PKRDelta6. A, an equal number of cells (1 times 10^7) was treated with 10 µg/ml LPS for 24 h. Nuclear extracts (5 µg) from a control clone (CON-8) and two PKRDelta6 clones (PKRDelta6-1 and PKRDelta6-20) were used for NF-kappaB DNA binding assays with a dsDNA oligonucleotide containing two kappaB 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 PKRDelta6-1 clone; lane 5, LPS-induced PKRDelta6-20 clone. B, control (CON-8), wt PKR (polyclonal populations) and PKRDelta6 (clone PKRDelta6-1)-expressing cells were treated with LPS for 6 h. Nuclear extracts (10 µg) were tested for NF-kappaB DNA binding by gel supershift assays using specific antisera and the HIV-kappaB 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-kappaB dsDNA oligonucleotide was added (lanes 3, 13, and 23).



Genes encoding kappaB-binding proteins form a family of related genes that include NFKB1 (p50/p105), NFKB2 (p52/p100), v-rel, c-rel, relA (p65), relADelta (p65Delta), and relB (for review, see (53) ). Recent findings suggest that treatment of pre-B cells with LPS changes the subunit composition of kappaB-binding complexes from p50-p65 to p50-rel(54, 55) . Based on this observation we wished to investigate whether PKRDelta6 expression had an effect on NF-kappaB subunit composition upon LPS induction. To examine which of the two kappaB-binding complexes, p50-p65 or p50-rel, were involved in the binding to kappaB site, we performed gel supershift assays by incubating nuclear extracts from a control clone (CON-8), wt PKR cells (polyclonal populations), and a PKRDelta6 clone (PKRDelta6-1) together with antibodies against p65, rel, or p50 protein. As shown in Fig. 6B, no differences in NF-kappaB subunit composition between control, wt PKR, or PKRDelta6 cells were observed. The NF-kappaB 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 kappa-chain transcription in PKRDelta6-expressing cells is apparently not mediated through NF-kappaB.


DISCUSSION

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 kappa gene transcription by PKRDelta6 suggests that PKR activation is necessary but not sufficient for the induction of kappa 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-2alpha 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-2alpha 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 kappa gene(50, 70) .

Like other eukaryotic genes, the kappa gene is regulated by the interaction of sequence-specific DNA-binding proteins with cis-acting DNA elements. NF-kappaB transcription factor binds to the kappaB site in the intron enhancer (J-C enhancer) of kappa gene(50) . Activation of NF-kappaB requires IkappaB phosphorylation and degradation(71, 72) . Interestingly, IkappaB can be phosphorylated by the two eIF-2alpha kinases, heme control repressor (73) and PKR (28) in vitro. In 70Z/3 cells, LPS but not IFN- induces NF-kappaB activity, which is necessary but not sufficient for kappa gene transcription(49, 63, 64, 74) . Our data show that PKR mediates kappa transcription independently of NF-kappaB. This is the second example of an NF-kappaB-independent pathway of kappa-gene transcription in 70Z/3 cells. Transforming growth factor-beta inhibits LPS-induced kappa gene transcription without affecting NF-kappaB activation(51) . Thus PKR activates at least two different pathways to the transcriptional machinery, NF-kappaB dependent for dsRNA and NF-kappaB independent for LPS or IFN-. However, it should be emphasized the difference in cell types used in these experiments. In this regard, IFN-beta expression by dsRNA in mouse F9 embryonal carcinoma cells does not require NF-kappaB activation(75) , indicating that dsRNA signaling clearly differs between cell lines.

A second enhancer element, which lies 8.5 kilobases downstream of the kappa gene, has been identified (kappa3` enhancer) and contains an IFN consensus sequence(76) . The kappa3` 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, (^5)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 kappa 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.


FOOTNOTES

*
This work was supported by grants from the National Cancer Institute of Canada (to A. E. K. and N. S.). 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.

§
Member of Terry Fox Group in Molecular Oncology and an awardee from the American Foundation for AIDS Research. To whom correspondence should be addressed: Depts. of Oncology and Medicine, McGill University, Lady Davis Institute-Jewish General Hospital, 3755 Cote-Ste-Catherine St., Montreal, H3T 1E2 Canada. Tel.: 514-340-8260 (ext. 4504); Fax: 514-340-7576; MDAK@MUSICA.MCGILL.CA.

(^1)
The abbreviations used are: IFN, interferon; PKR, double-stranded RNA-dependent protein kinase; eIF-2alpha, eukaryotic translation initiation factor-2alpha; LPS, lipopolysaccharide; sIg, surface immunoglobulin; kappa-chain, light immunoglobulin chain; µ-chain, heavy immunoglobulin chain; FITC, fluorescein isothiocyanate; NF-kappaB, transcription factor kappaB; IkappaB, protein inhibitor of NF-kappaB; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; RIPA, radioimmune precipitation buffer; wt, wild type; ds, double-stranded; PBS, phosphate-buffered saline.

(^2)
J. Bell, personal communication.

(^3)
G. N. Barber and M. G. Katze, personal communication.

(^4)
A. E. Koromilas and N. Sonenberg, unpublished data.

(^5)
M. L. Atchison, personal communication.


ACKNOWLEDGEMENTS

We thank Raymond Leung, Luc Chandonnet, and Anne Roulston for assistance in some of the experiments; Dr. R. Sekaly for providing the flow cytometry facility; Drs. G. Barber, M. Katze and A. Darveau for the anti-PKR monoclonal antibody (13B8-F9); Dr. M. Mathews for the anti-PKR polyclonal antibody; Dr. B. Safer for anti-eIF-2alpha polyclonal antibody; Dr. C. Paige for immunoglobulin kappa- and µ-chain cDNAs; Dr. Nancy Rice for anti-p65 and anti-c-rel polyclonal antibodies; and Drs. M. Szyf, C. Paige, H. Young, R. Sen, and J. Pelletier for comments on the manuscript.


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