Dominant Negative Function by an Alternatively Spliced Form of the Interferon-inducible Protein Kinase PKR*

Suiyang LiDagger and Antonis E. KoromilasDagger §

From the Dagger  Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montréal, Québec H3T 1E2, Canada and the § Departments of Oncology, Medicine, Microbiology and Immunology, and Cell Biology and Anatomy, McGill University, Montréal, Québec H3A 2B4, Canada

Received for publication, September 6, 2000, and in revised form, January 19, 2001




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The double-stranded RNA (dsRNA)-activated protein kinase PKR (protein kinase dsRNA-dependent) plays an important role in the regulation of protein synthesis by phosphorylating the alpha -subunit of eukaryotic initiation factor 2. Through this activity, PKR is thought to mediate the antiviral and antiproliferative actions of interferon. Here, we show that the human T cell leukemia Jurkat cells express an alternatively spliced form of PKR with a deletion of exon 7 (PKRDelta E7), resulting in a truncated protein that retains the two dsRNA-binding motifs. PKRDelta E7 exhibits a dominant negative function by inhibiting both PKR autophosphorylation and eukaryotic initiation factor 2 alpha -subunit phosphorylation in vitro and in vivo. Reverse transcriptase-polymerase chain reaction assays showed that PKRDelta E7 is expressed in a broad range of human tissues at variable levels. Interestingly, expression of PKRDelta E7 is higher in Jurkat cells than in normal peripheral blood mononuclear cells, raising the possibility of a role in cell proliferation and/or transformation. Thus, expression of alternatively spliced forms of PKR may represent a novel mechanism of PKR autoregulation with important implications in the control of cell proliferation.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In eukaryotes, RNA translation plays an important role in cell proliferation induced by various extracellular stimuli (reviewed in Ref. 1). Among many regulatory proteins involved in this process, the interferon (IFN)1-inducible double-stranded RNA (dsRNA)-activated protein kinase PKR (protein kinase dsRNA-dependent) is an important regulator of translation initiation through its capacity to phosphorylate the alpha -subunit of eukaryotic initiation factor 2 (eIF-2alpha ) (reviewed in Ref. 2). Binding of PKR to dsRNA results in its activation by autophosphorylation and subsequently in the phosphorylation of eIF-2alpha (2). Phosphorylation of eIF-2alpha by PKR on serine 51 leads to an increased affinity of the initiation factor for eukaryotic translation initiation factor 2B, also known as guanine exchange factor, and thus increases the proportion of the latter that is trapped as an inactive complex with eIF-2 and GDP (reviewed in Ref. 3). The reduction in free eukaryotic translation initiation factor 2B results in a fall of the overall rate of guanine nucleotide exchange on the remaining unphosphorylated eIF-2, eventually leading to an inhibition of translation initiation (3). In addition to translational control, PKR has been implicated in signaling pathways leading to transcriptional activation by dsRNA, virus infection, various cytokines, or genotoxic stress (reviewed in Ref. 4).

Several reports have assigned to PKR a tumor suppressor function in vitro (2). Specifically, expression of wild type (WT) human PKR in yeast (5) or in mouse cells (6) results in cell growth inhibition and in some cases in the induction of cell death by apoptosis (7). On the other hand, expression of PKR mutants in NIH3T3 cells that are catalytically inactive or dsRNA binding-defective causes malignant transformation and induction of tumorigenesis after injection of the transformed cells in nude mice (6, 8-10). Contrary to these in vitro functions, deletion of the pkr gene by homologous recombination is not tumorigenic (11, 12). In addition, PKR knockout (PKR-/-) mice are not susceptible to virus infection (11, 12) with the exemption of encephalomyelocarditis virus after priming with IFN-gamma (12) or vesicular stomatitis virus after intranasal infection (13, 14). Therefore, it has been suggested that the lack of PKR may be compensated by the expression of other PKR-like molecules whose function is possibly blocked by the expression of the PKR mutants in vitro (2, 11). This is supported by the cloning and characterization of PKR-related genes, such as PKR-like endoplasmic reticulum kinase/pancreatic eIF-2alpha kinase, which functions as an eIF-2alpha kinase (15), and the mouse homologue of the yeast eIF-2alpha kinase GCN2 (16). Thus, PKR may be the prototype of a family of kinases with overlapping biochemical and biological functions (17).

Work in many laboratories using in vitro mutagenesis of human and mouse PKR has led to an extensive characterization of the structure-function relationship of the molecule (2). Briefly, in both species the amino-terminal half of PKR contains two RNA-binding motifs (dsRBMs) (2), which are conserved among most of the RNA-binding proteins (18). On the other hand, the carboxyl-terminal half of PKR is divided into 11 subdomains, which are required for catalytic activity (2) and are conserved among many serine/threonine protein kinases (19). At the genomic level, the human PKR gene contains 17 exons that vary in size from 18 nucleotides in exon 1 to 840 nucleotides in exon 17 (20), whereas the mouse gene contains 16 exons varying from 35 nucleotides in exon 8 to 750 nucleotides in exon 16 (21).

Despite the tumor suppressor function in vitro, naturally occurring mutants of PKR in tumor cells have not as yet been identified with the exemption of a mutant form of mouse PKR in a pro-B leukemia cell line (22). Here, we report the cloning of a point mutant of human PKR (Y176H) from Jurkat leukemia cells encoding for a protein that retains the RNA-binding and catalytic properties of wild type PKR in vitro. We also describe the cloning and characterization of an alternatively spliced form of PKR (PKRDelta E7) from Jurkat cells. PKRDelta E7 is a splicing product of exon 7 of the human PKR gene, which contains the two dsRBMs and exhibits a dominant negative function in vitro an in vivo. PKRDelta E7 is differentially expressed in various types of normal human tissue, and we provide evidence that its expression may play a role in induction of cell proliferation as a result of PKR inactivation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RNA Isolation and RT-PCRs-- The primers used in RT-PCRs are summarized in Table I. For sequencing of the protein-coding sequence of PKR, 1 µg of RNA isolated by the guanidium thiocyanate method (23) was reverse transcribed using the P303 primer. The single-stranded PKR cDNA was then amplified by PCR using the P504/P304, P501/P301, P502/P302, and P503/P303 sets of primers with denaturing at 94 °C for 1 min, annealing at 60 °C for 1 min, and extension at 72 °C for 1 min for a total of 30 cycles. The PCR products, which spanned the entire protein-coding sequence of PKR, were subcloned into pCRTMII (Invitrogen) and sequenced with T7 DNA Polymerase (U.S. Biochemical Corp.) or subjected to direct sequencing using the Deaza T7SequencingTM Kit according to the supplier's instructions (Amersham Pharmacia Biotech). The human WT PKR cDNA was subcloned into HindIII/BamHI sites of either pcDNA3.0-neo (Invitrogen) or pFLAG-CMV-2 vector (Eastman Kodak Co.). For transient PKRDelta E7 expression, the 900-bp NcoI/AccI fragment of the WT PKR in pcDNA3.0-neo or pFLAG-CMV-2 vector was replaced with the corresponding fragment from pCRTMII vector carrying the Delta E7 deletion to produce PKRDelta E7 or FLAG-PKRDelta E7 under the control of the human cytomegalovirus promoter. For expression in yeast, the HindIII/BamHI fragments of PKRDelta E7 and FLAG-PKRDelta E7 were subcloned into the corresponding sites of pYES2 vector (Invitrogen).


                              
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Table I
PCR primers

For PKRDelta E7 RNA expression, 1 µg of RNA was subjected to RT using the P305 primer and PCR amplification using the P505/P305 set of primers. For expression of PKRDelta E7 in human tissues, the human multiple tissue cDNA (K1420-1) and the human immune system panel (K1426-1) from CLONTECH were used. The normalized cDNAs were amplified by PCR-denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min for a total of 30 cycles using the P505/P305 set of primers. The PCRs were subjected to agarose or polyacrylamide gel electrophoresis and visualized by ethidium bromide or silver staining (24), respectively.

Cell Culture-- HeLa S3 cells (ATCC CCL-2.2) and Jurkat cells (ATCC TIB-152) were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and RPMI 1640 (Life Technologies), respectively, with 10% heat-inactivated fetal bovine serum (Life Technologies), 2 mM L-glutamine (Life Technologies), and 100 units/ml penicillin/streptomycin (Life Technologies). Immortalized PKR-/- fibroblasts (11, 12) were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated calf serum, 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy human adults using the Ficoll-Paque® method according to the supplier's instructions (Amersham Pharmacia Biotech). Newly isolated lymphocytes were stimulated with 10 units/ml phytohemagglutinin (Life Technologies) and maintained in RPMI 1640 (Life Technologies) medium supplemented with 10% fetal bovine serum (Life Technologies), 2 mM L-glutamine (Life Technologies), and 100 units/ml penicillin-streptomycin (Life Technologies) for 3 days.

Protein Expression with the Vaccinia/T7 Virus System-- One day before transfection, 0.8 × 106 HeLa S3 or PKR-/- cells were seeded in 6-cm plates. One h before transfection, the cells were infected with recombinant vaccinia virus containing the bacterial T7 RNA polymerase gene (25). Transfection was performed using the ratio of 1 µg DNA to 2.5 µg of LipofectAMINE (Life Technologies) per plate, and cells were incubated in serum-free medium at 37 °C for 5 h. Subsequently, complete medium was added, and cells were grown for an additional 16 h before analysis.

Protein Extraction and PKR Autophosphorylation-- Cells were washed twice with ice-cold 1× phosphate buffer saline, and proteins were extracted with a lysis buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 2 mM MgCl2, 1% Triton X-100, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 3 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin. After incubation on ice for 20 min, the cell lysate was centrifuged at 10,000 × g for 10 min. The cytoplasmic supernatant (S10 fraction) was transferred to a fresh tube, the protein concentration was measured by Bradford assay (Bio-Rad), and stored at -85 °C.

For PKR autophosphorylation in vitro, 50-500 µg of protein extracts were subjected to immunoprecipitation with mouse monoclonal anti-human PKR antibodies (clone F9 or E8; Ref. 26) and anti-mouse IgG-agarose beads. PKR immunoprecipitates were equilibrated in 1 × PKR kinase buffer consisting of 10 mM Tris-HCl, pH 7.7, 50 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 3 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin. PKR autophosphorylation was performed in the presence of 0.1 µg/ml activator reovirus dsRNA and 1 µCi of [gamma -32P]ATP (ICN). After incubation at 30 °C for 20 min, the reactions were subjected to SDS-PAGE, and radioactive bands were visualized by autoradiography. Alternatively, the in vitro kinase assay of PKR was performed with S10 protein extracts in 1× PKR kinase buffer in the conditions described above. The autophosphorylated PKR was then immunoprecipitated with mouse monoclonal anti-human PKR antibodies, subjected to SDS-PAGE, and visualized by autoradiography.

Generation of an Anti-phosphoserine 51-specific eIF-2alpha Antibody-- Rabbit antiserum was produced against a chemically synthesized phosphopeptide ILLSELpSRRRIRS (where pS represents phosphoserine) that contains serine 51 of human eIF-2alpha . The antibody was purified from rabbit serum by sequence-specific chromatography and was negatively preadsorbed using a nonphosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with nonphosphorylated eIF-2alpha protein. The final product was generated by affinity chromatography using an eIF-2alpha -derived peptide phosphorylated at serine 51.

Western Blotting-- Protein extracts or PKR immunoprecipitates were subjected to SDS-PAGE and proteins were transferred onto nylon ImmobilonTM P membrane (Millipore Corp.). Immunoblots were performed with mouse monoclonal anti-human PKR, anti-FLAG (Kodak; catalog no. IB13025), anti-human eIF-2alpha antibodies, rabbit polyclonal anti-phosphoserine 51 eIF-2alpha antibodies (homemade or from BIOSOURCE, catalog no. 44-728), and rabbit antiserum to TrpE-yeast eIF-2alpha fusion protein (CM-217) at a concentration of 1 µg/ml using the standard protocol (27). After incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (1:1000 dilution; Amersham Pharmacia Biotech), proteins were visualized with the enhanced chemiluminescence (ECL) detection system according to the manufacturer's instructions (Amersham Pharmacia Biotech). Quantification of the bands in the linear range of exposure was performed by densitometry using the NIH Image 1.54 software.

Yeast Strains and Growth Analysis-- The yeast strains used in this study are summarized in Table II. PKR, PKRDelta E7, and PKRLS9Delta E7 in pYES2 vector were transformed into these strains and selected in SD-Trp medium as described (28). Single colonies were picked up and grew in SD-Trp liquid medium at 30 °C to A600 = 1.5, and the liquid cultures were streaked on SGLU and SGAL minimal plates containing 10% glucose and 20% galactose, respectively, and incubated at 30 °C for up to 72 h. The yeast growth was measured by diluting the liquid cultures to A600 = 0.01 in synthetic medium containing 10% galactose, 2% raffinose, and the required amino acid supplement (SGAL). The cultures were incubated at 30 °C for various time periods, and a 0.5-ml liquid culture from each point was used to measure A600.


                              
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Table II
Yeast strains



    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of a Point Mutant and an Alternatively Spliced Form of PKR from the Human Leukemia Jurkat T Cells-- We have reported a diminished PKR activation in various human leukemia cell lines including Jurkat T lymphocytes (26). We speculated that PKR inactivation in these cells might be caused by mutations in the PKR gene. To test this possibility, we amplified and sequenced the PKR cDNA from Jurkat cells and normal PBMCs after RT-PCR as described under "Materials and Methods." Direct sequencing of the PCR products verified the presence of a T to C point mutation in Jurkat PKR cDNA at nucleotide 526 downstream from the initiator ATG (Fig. 1A), which results in a single substitution of tyrosine 176 to histidine. When the PCR products were subcloned into pCRII vector and sequenced (see "Materials and Methods"), nine out of 10 clones contained the T to C mutation. One clone, however, harbored a 77-bp deletion, which corresponds to the entire exon 7 of the human PKR gene (Fig. 1B). Deletion of exon 7 leads to the conjunction of exons 6 and 8 with a frameshift that introduces a stop codon (TGA) within exon 8 and produces an RNA encoding for a 174 amino acid protein. This truncated protein contains the two dsRBMs of PKR and herein is named PKRDelta E7 (Fig. 2).



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Fig. 1.   Identification of a point mutant and an alternatively spliced form of PKR in Jurkat cells. A, direct sequencing of the PCR products of the PKR gene from normal PBMCs and Jurkat cells. The single point mutation on the nucleotide sequence, which results in the change of amino acid tyrosine 176 to histidine, is indicated with an asterisk. B, an alternatively spliced form of PKR revealed by DNA sequencing. Shown is a comparison of DNA sequence between full-length Jurkat PKR and the alternatively spliced form PKRDelta E7. The deletion of 77 bp (exon 7) in PKRDelta E7 cDNA is indicated.



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Fig. 2.   Schematic illustration of the alternatively spliced form PKRDelta E7. A, the wild type human PKR cDNA contains sequences from 17 exons, where the translation initiation codon ATG and the translation termination codon TAG are located in exon 3 and exon 17, respectively. The Y176H mutation in exon 7 of Jurkat gene is indicated. When exon 7 is deleted by splicing, the reading frame is shifted thereafter and terminates by a newly introduced stop codon TGA. The resulting truncated protein, PKRDelta E7, contains the two dsRNA-binding motifs (dsRBM I and II). B, the nucleotide and amino acid sequence translated in the vicinity of the alternative splicing site.

We confirmed the expression of the PKRDelta E7 RNA in Jurkat cells by an RT-PCR assay (see "Materials and Methods"). The PKRDelta E7 PCR product contains a 77-bp deletion and therefore migrates faster than the WT PKR PCR product on agarose gel electrophoresis (Fig. 3A, compare lanes 3 and 4). The ratio of the band intensities of the two PCR products is proportional to the amount of each PKR transcript within the cells. To examine whether PKRDelta E7 expression is unique for Jurkat cells, we performed an RT-PCR assay with RNA from normal PBMCs. We found that PBMCs contain very low levels of PKRDelta E7 RNA (<1% of full-length PKR RNA; see also Fig. 8), whereas PKRDelta E7 RNA levels in Jurkat cells is about ~10% of WT PKR transcript (Fig. 3A). We also verified the PKRDelta E7 protein expression by immunoblot analysis (Fig. 3B). To facilitate the detection of PKRDelta E7, Jurkat cells were treated with IFN-alpha /beta to induce PKR RNA expression. The protein extracts before (lane 2) and after IFN treatment (lane 3) were subjected to immunoprecipitation and immunoblotting with antibodies specific to the N terminus domain of PKR to detect both full-length PKR (top panel) and PKRDelta E7 (bottom panel). These experiments showed that PKRDelta E7 protein is expressed in Jurkat cells at low levels (lane 2), and its expression is induced after IFN treatment (Fig. 3B, lane 3).



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Fig. 3.   Detection of PKRDelta E7 RNA and protein expression in Jurkat cells. A, RNA isolated from normal PBMCs (lane 5) and Jurkat (lane 6) was subjected to RT-PCR. PCR products were fractionated in 2% agarose gel and stained with ethidium bromide. A DNA molecular weight (M.W.) marker was loaded to indicate the size of the PCR products (lane 1). As a negative control, a PCR containing no DNA template was used (lane 2). As positive controls, PCR products from wild type PKR (lane 3) and PKRDelta E7 (lane 4) cDNAs were 162 and 85 bp, respectively. B, 200 µg of Jurkat S10 protein extracts before (lane 2) or after IFN-alpha 2b treatment (1000 IU/ml; 18 h) (lane 3) were immunoprecipitated with a rabbit polyclonal anti-human PKR antibody specific for the N-terminal domain of PKR. Immunoprecipitates were then subjected to Western blotting with the mouse monoclonal anti-human PKR (F9) antibody. The expression levels of full-length PKR (top panel) and PKRDelta E7 (bottom panel) are shown. Note that PKR levels were visualized by ECL after film exposure for 10 s, whereas PKRDelta E7 levels were visualized after 1 min. As a positive control (Ctl) to PKRDelta E7, 10 µg of S10 protein extracts from HeLa cells transfected with PKRDelta E7 were run in lane 1. Detection of endogenous HeLa PKR (top panel, lane 1) was possible after long film exposure (data not shown).

Biochemical Characterization of PKRDelta E7-- Characterization of Y176H mutation showed that PKRY176H retains both the dsRNA binding and catalytic activities of WT PKR in vitro (data not shown). As a result of it, we concentrated our efforts on characterizing the function of PKRDelta E7. The N terminus domain of PKR is involved in dsRNA-binding (29). PKRDelta E7 is similar to an artificially made N terminus-truncated form of PKR, known as p20, which contains the two contiguous dsRBMs. p20 can bind to dsRNA (30-33) and heterodimerize with WT PKR in yeast two-hybrid assays (32). Based on this, we wished to examine the ability of PKRDelta E7 to self-associate and associate with WT PKR in the presence and absence of dsRNA. To do so, we constructed a fusion protein of PKRDelta E7 bearing the FLAG epitope in the N terminus end. When FLAG-PKRDelta E7 and PKRDelta E7 were transiently co-expressed into HeLa cells, an equal amount of the two proteins was co-imunoprecipitated with anti-FLAG antibodies (Fig. 4A, lane 8), confirming their ability to self-associate. In similar assays, an equal amount of endogenous WT PKR (Fig. 4B, top panel, lane 1) and PKRDelta E7 (bottom panel, lane 1) was found to associate FLAG-PKRDelta E7 (middle panel, lane 1). However, treatment with micrococcal nuclease (MN) diminished the association of FLAG-PKRDelta E7 with WT PKR (top panel, lane 2) without affecting its association with PKRDelta E7 (bottom panel, lane 2). These data suggested that self-association of PKRDelta E7 may take place in the absence of dsRNA, whereas its association with full-length PKR is dsRNA-dependent. To further investigate this possibility, we used a FLAG-PKRDelta E7 construct bearing the LS9 mutation (substitutions of alanine 66 and alanine 68 to glycine 66 and proline 68), which completely abolishes dsRNA binding (34). Co-expression of FLAG-PKRLS9Delta E7 and PKRDelta E7 in HeLa cells and immunoprecipitation with anti-FLAG antibody revealed the lack of association of FLAG-PKRDelta E7 with either PKRDelta E7 (lanes 3 and 4, bottom row) or the endogenous PKR (lanes 3 and 4, top row). Since the LS9 mutation may affect the conformation of the dsRNA-binding domain of PKR (34), these data suggested that the integrity of dsRNA-binding domain is essential for PKRDelta E7 self-association and association with full-length PKR. In these experiments, we noticed that a higher amount of FLAG-PKRDelta E7 was immunoprecipitated with anti-FLAG antibodies after MN treatment. One plausible explanation is that binding of RNA to FLAG-PKRDelta E7 impedes the accessibility of the antibody to FLAG epitope, and this inhibition may be alleviated by MN treatment.



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Fig. 4.   Self-association and association of PKRDelta E7 with wild type PKR. A, PKRDelta E7 and FLAG-PKRDelta E7 were expressed in HeLa cells with the vaccinia virus/T7 system. 100 µg of S10 protein extracts were immunoprecipitated (IP) with mouse monoclonal anti-FLAG antibody (lanes 5-8). Proteins were separated in SDS-14% PAGE and subjected to Western blotting (WB) using mouse monoclonal anti-human PKR (F9) antibody. In parallel, 20 µg of S10 protein extracts from each transfection was used to show the PKRDelta E7 expression levels (whole cell extracts (WCE), lanes 1-4). B, PKRDelta E7 and FLAG-PKRDelta E7 or PKRDelta E7 and FLAG-PKRLS9Delta E7 were co-expressed in HeLa cells as above. Protein extracts were left untreated (lanes 1 and 3) or treated (lanes 2 and 4) with MN and immunoprecipitated with mouse monoclonal anti-FLAG antibody. Proteins were separated in SDS-14% PAGE and subjected to Western blotting using mouse monoclonal anti-PKR (F9) antibody (top and bottom panels) followed by immunoblotting with mouse monoclonal anti-FLAG antibody (middle panel).

PKRDelta E7 Exhibits a Dominant Negative Function-- The ability of PKRDelta E7 to associate with WT PKR prompted us to examine for a possible dominant negative function in PKR activation. To this end, first we assessed the ability of FLAG-PKRDelta E7 to inhibit the autophosphorylation of endogenous PKR when transiently expressed in HeLa cells using the vaccinia/T7 virus system (25). Because the vaccinia/T7 virus system is a two-step procedure utilizing transfection with LipofectAMINE and infection with recombinant virus (see "Materials and Methods"), we measured PKR activation in cells treated with LipofectAMINE and vector DNA (Fig 5A, mock, lane 1), LipofectAMINE plus vector DNA plus virus (mock, lane 2), or LipofectAMINE plus FLAG-PKRDelta E7 cDNA plus virus (lane 3). The activation of endogenous PKR was measured first by autophosphorylation in the protein extracts in vitro followed by immunoprecipitation with an anti-human PKR antibody (Fig. 5A). We observed that PKR autophosphorylation, after normalization to protein levels, was more highly induced by the virus (Fig. 5A, compare lane 2 with lane 1) presumably by the production of activator dsRNA during infection. On the other hand, FLAG-PKRDelta E7 expression resulted in the inhibition of PKR autophosphorylation (Fig. 5A, lane 3) compared with mock-transfected cells in the presence (lane 2) or absence (lane 1) of vaccinia/T7 virus. The levels of endogenous PKR or FLAG-PKRDelta E7 were detected by immunoblot analysis with the anti-human PKR (middle panel) or anti-FLAG antibody (bottom panel), respectively. These data argued for a dominant negative effect of PKRDelta E7 on PKR activation in vitro.



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Fig. 5.   Dominant negative function of PKRDelta E7 in vitro and in vivo. A, HeLa cells were transfected with pFLAG-CMV-2 vector and LipofectAMINE in the absence (lane 1) or presence (lane 2) of recombinant vaccinia/T7 virus (mock transfections). FLAG-PKRDelta E7 cDNA was overexpressed using LipofectAMINE and vaccinia/T7 virus (lane 3). 100 µg of S10 protein extracts was subjected to in vitro PKR kinase assay in the presence of reovirus activator dsRNA (0.1 µg/ml) and [gamma -32P]ATP (1 µCi). PKR was then immunoprecipitated with a rabbit polyclonal anti-PKR antibody, and 32P-labeled proteins were separated in SDS-10% PAGE and visualized by autoradiography (top panel). In parallel, 20 µg of S10 protein extracts used in the in vitro kinase assay were separated in SDS-10% PAGE (middle panel) or SDS-14% PAGE (bottom panel) and subjected to Western blotting with mouse monoclonal anti-PKR (F9) antibody or mouse anti-FLAG antibody, respectively. B and C, PKR-/- fibroblasts were transfected with vector DNA (lane 1), WT human PKR cDNA (lane 2), PKRDelta E7 cDNA (lane 3), or WT human PKR and PKRDelta E7 cDNAs (lane 4) using the vaccinia/T7 virus system. 100 µg of S10 protein extracts was immunoprecipitated with a rabbit polyclonal anti-human PKR antibody and subjected to in vitro PKR kinase assay as described above. Proteins were separated in SDS-10% PAGE, and the PKR autophosphorylation was visualized by autoradiography (top panel). In parallel, 50 µg of S10 protein extracts from transfected cells was used to show the expression levels of PKR (middle panel) or PKRDelta E7 (lower panel) by Western blotting using mouse monoclonal anti-PKR (F9) antibody. C, HeLa cells were transfected with WT eIF-2alpha cDNA (lane 2), the serine 51 to alanine mutant of eIF-2alpha cDNA (lane 3), or wild type eIF-2alpha cDNA together with PKRDelta E7 cDNA (lane 4) using the vaccinia/T7 virus system. 20 µg of S10 protein extracts was subjected to Western blotting first with a homemade rabbit polyclonal anti-phosphoserine 51 eIF-2alpha specific antibody (top panel). An equal amount of the same extracts was used for immunoblotting with a mouse monoclonal anti-eIF-2alpha antibody (middle panel) or mouse monoclonal anti-PKR (F9) antibody (bottom panel). D, HeLa cells were transfected with vector DNA and LipofectAMINE in the absence (lane 1) or presence (lane 2) of recombinant vaccinia/T7 virus (mock transfections) or with PKRDelta E7 cDNA, LipofectAMINE, and vaccinia/T7 virus (lane 3). Protein extracts (S10, 50 µg) were subjected to SDS-10% PAGE and immunoblot analysis with a phosphospecific anti-eIF-2alpha serine 51 antibody (BIOSOURCE) (top panel). An equal amount of protein was used for Western blotting with a mouse monoclonal anti-eIF-2alpha antibody (middle panel) or the mouse monoclonal anti-human PKR (F9) antibody (bottom panel). A-D, the intensity of the bands was quantified with the NIH Image 1.54 software, and the ratios of autophosphorylated PKR to PKR protein levels (A and B) or eIF-2alpha serine 51 phosphorylation to the amount of eIF-2alpha protein (C and D) are indicated. vv/T7, vaccinia/T7 virus system.

Similar observations were made when the dominant negative function of PKRDelta E7 was tested in fibroblasts derived from a mouse with targeted disruption of the catalytic domain of PKR (C-PKR-/- cells; Ref. 11) (Fig. 5B). Transient expression of PKRDelta E7 with human WT PKR into C-PKR-/- cells resulted in inhibition of PKR autophosphorylation (top panel, lane 4) compared with expression of WT PKR alone (top panel, lane 2). Immunoblot analysis with an anti-human PKR antibody verified the expression of WT PKR (middle panel) and PKRDelta E7 (bottom panel). The partial down-regulation of PKRDelta E7 (bottom panel, lane 4) could possibly be explained by differences in transfection efficiency between the various samples.

The dominant negative function of PKRDelta E7 in PKR autophosphorylation prompted us to examine the inhibition of eIF-2alpha phosphorylation. To this end, we performed transient transfections of PKRDelta E7 cDNA together with either WT eIF-2alpha or a phosphorylation-defective mutant of eIF-2alpha (serine 51 to alanine; Ref. 35) cDNA (Fig. 5C). Phosphorylation of eIF-2alpha in vivo was then detected by immunoblot analysis using a homemade rabbit polyclonal antibody specific to phosphoserine 51 of eIF-2alpha (see "Materials and Methods"). Transient expression of WT eIF-2alpha resulted in induction of phosphorylation on serine 51 caused by the activation of endogenous HeLa PKR (36) (top panel, lane 2). Co-expression of WT eIF-2alpha with PKRDelta E7 (lanes 4) resulted in inhibition of eIF-2alpha serine 51 phosphorylation (top panel, compare lane 4 with lane 2), whereas the expression levels of eIF-2alpha remained stable (middle panel, lanes 2 and 6). Note the low (undetectable) levels of endogenous eIF-2alpha phosphorylation with this antibody (lane 1) and the lack of its cross-reactivity with the serine 51 to alanine mutant of eIF-2alpha (lane 3). The phosphorylation levels of endogenous HeLa eIF-2alpha were detected, however, when a commercially available phosphoserine 51-specific antibody was used (Fig. 5D). We measured eIF-2alpha phosphorylation in cells treated with LipofectAMINE plus vector DNA alone (mock, lane 1), LipofectAMINE plus virus plus vector DNA (mock, lane 2), or LipofectAMINE plus vaccinia virus plus PKRDelta E7 cDNA (lane 3). Infection with vaccinia virus (lane 2) induced the phosphorylation of eIF-2alpha (compare lanes 1 and 2), which was diminished by PKRDelta E7 expression (lane 3) through the inhibition of endogenous PKR. Taken together, the above data demonstrate the dominant negative function of PKRDelta E7 in both PKR activation and eIF-2alpha phosphorylation.

Functional Characterization of the Dominant Negative Function of PKRDelta E7 in Yeast-- It has been shown that high level of PKR expression in Saccharomyces cerevisae is toxic due to inhibition of general translation (5). However, at a lower level of expression, PKR can substitute the function of GCN2, the only eIF-2alpha kinase known to exist in yeast (37), by phosphorylating eIF-2alpha on serine 51 and stimulating GCN4 translation, a transcription factor involved in amino acid biosynthesis (38). To verify the dominant negative function of PKRDelta E7 in vivo, we used a yeast strain that lacks endogenous GCN2 (J110) (39) and two strains that contain one (H2544) and two (H2543) alleles of WT human PKR, respectively, under the control of the galactose-inducible promoter (40). Induction of PKR expression in H2544 strain partially inhibits growth, whereas PKR induction in H2543 completely abolishes growth. Strains J110, H2544, and H2543 were transformed with vector alone, FLAG-PKRDelta E7, or FLAG-PKRDelta E7LS9. As positive control, we used the PKR inhibitor vaccinia virus K3L (40). The transformants were streaked onto minimal medium plates containing either glucose or galactose as a carbon source, and the effect of each of these proteins on PKR-mediated growth inhibition was monitored. All transformants of the isogenic J110 strain grew well in either glucose or galactose, indicating that expression of these exogenous proteins did not perturb normal yeast growth characteristics (Fig. 6A). In agreement with previous studies (40), H2544 transformants containing vector DNA without an insert demonstrated a slow growth phenotype after PKR induction (Fig. 6A, bottom plate). However, expression of K3L reversed this growth-inhibitory phenotype (bottom plate). Likewise, expression of FLAG-PKRDelta E7 also rescued yeast growth consistent with previous findings that the N terminus domain of PKR from amino acid 1 to 262 rescues yeast growth inhibition by WT PKR (5). In contrast to this, FLAG-PKRLS9Delta E7 was unable to counteract the growth-inhibitory effects of PKR (bottom plate). Growth curves showed that the ability of FLAG-PKRDelta E7 transformants to rescue growth was equally potent to K3L (Fig. 6B, middle and bottom graphs).



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Fig. 6.   Functional characterization of PKRDelta E7 in yeast. A, the yeast strains J110, H2543, and H2544 were transformed with pYES2 vector, pEMBL/yex4 vector containing K3L, and pYES2 vector containing FLAG-PKRDelta E7 or FLAG-PKR LS9Delta E7. The transformants were streaked on SD agar plates containing either glucose or galactose and maintained at 30 °C for 72 h. B, the transformants were incubated in SD liquid medium containing 10% galactose and 2% raffinose at 30 °C. The growth rate was quantified by measuring the optical density (A600) values at different time points. C, expression levels of PKR, FLAG-PKRDelta E7, and FLAG-PKRLS9Delta E7 in yeast. The transformants of strains J110 (lanes 1 and 2), H2544 (lanes 3 and 4), and H2543 (lanes 5 and 6) were incubated in SD liquid medium containing 10% galactose and 2% raffinose. 20 µg of whole protein extracts was used for Western blotting with a rabbit polyclonal anti-human PKR antibody (top panel) or a mouse monoclonal anti-human FLAG antibody (bottom panel). The band above PKR that is also present in J110 cells, which lack PKR (lanes 1 and 2), is nonspecific (NS). D, protein extracts (20 µg) from the J80 (WT eIF-2alpha ) and J82 (serine 51 to alanine eIF-2alpha mutant) transformants expressing WT PKR (lanes 3, 6, 9, and 12), FLAG-PKRDelta E7 (lanes 2, 3, 5, and 6), or FLAG-PKRLS9Delta E7 (lanes 8, 9, 11, and 12) were subjected to Western blotting analysis using a mouse monoclonal anti-FLAG antibody. E, protein extracts from H2544 and H2543 cell (20 µg) transformed with vector DNA only (lanes 1 and 5), vaccinia virus K3L DNA (lanes 2 and 6), PKRDelta E7 cDNA (lanes 3 and 7), or PKRLS9Delta E7 DNA (lanes 4 and 8) were subjected to immunoblot analysis using a rabbit polyclonal anti-human PKR antibody (top panel), the homemade phosphoserine 51 eIF-2alpha -specific antibody (middle panel), or mouse monoclonal anti-eIF-2alpha -specific antibody (bottom panel). The intensity of the bands was quantified with the NIH Image 1.54 software, and the ratio of eIF-2alpha serine 51 phosphorylation to the amount of eIF-2alpha protein is indicated.

The expression of PKR, FLAG-PKRDelta E7, and FLAG-PKRLS9Delta E7 in yeast was then examined by Western blotting (Fig. 6C). PKR expression was detected in H2544 (top panel, lanes 3 and 4) and H2543 (top panels, lanes 5 and 6) but not in the control J110 strain (lanes 1 and 2). However, expression of WT PKR in H2544 and H2543 strains was more highly induced in the presence of FLAG-PKRDelta E7 than FLAG-PKRLS9Delta E7 (top panel, compare lane 3 with lane 4 and lane 5 with lane 6). This higher PKR induction could be translational in nature and caused by the dominant negative function of FLAG-PKRDelta E7 but not FLAG-PKRLS9Delta E7. The migration of PKR in polyacrylamide gels as a doublet could have been a result of partial degradation or could represent the phosphorylated (upper band) and nonphosphorylated forms of PKR (lower band) as previously reported for human PKR expressed in mouse cells (41). On the other hand, both FLAG-PKRDelta E7 and FLAG-PKRLS9Delta E7 were equally expressed in strain J110 (bottom panel, lanes 1 and 2). However, expression of FLAG-PKRLS9Delta E7 in strains H2544 and H2543 was very low under growth conditions in which PKR expression was induced (bottom panel, lanes 4 and 6). Since FLAG-PKRLS9Delta E7 does not exhibit a dominant negative function, we speculated that its low expression was due to translation inhibition by PKR. To further investigate this possibility, WT human PKR and FLAG-PKRDelta E7 or FLAG-PKRLS9Delta E7 were co-expressed into yeast strains J80 and J82, which lack GCN2 but contain wild type eIF-2alpha and the serine 51 to alanine mutant eIF-2alpha , respectively (38). As shown in Fig. 6D, FLAG-PKRDelta E7 was equally expressed in both strains in the absence (lanes 2 and 5) or presence of WT PKR (lanes 3 and 6). On the other hand, expression of FLAG-PKRLS9Delta E7 was significantly reduced in both strains when WT PKR was induced (lanes 9 and 12). These data indicated that the inhibition of FLAG-PKRLS9Delta E7 expression by WT PKR may not be translational in nature, since PKR-mediated inhibition of protein synthesis cannot take place in the eIF-2alpha mutant-containing strain (38). The mechanism of down-regulation of FLAG-PKRLS9Delta E7 by PKR is not presently known.

Next, we examined the dominant negative effect of PKRDelta E7 on PKR-mediated eIF-2alpha phosphorylation in H2544 and H2543 strains (Fig. 6E). To do so, H2544 and H2543 strains were transformed with vector DNA alone (lanes 1 and 5), the vaccinia virus inhibitor K3L (lanes 2 and 6), PKRDelta E7 (lanes 3 and 7), or PKRLS9Delta E7 (lanes 4 and 8) followed by the induction of WT PKR in the presence of galactose. WT PKR expression was detected by immunoblot analysis using an anti-human PKR specific antibody (top panel). Phosphorylation of eIF-2alpha was detected by immunoblotting using the homemade phosphospecific antibody (middle panel) and normalized to eIF-2alpha protein levels using a rabbit polyclonal antibody to yeast eIF-2alpha (bottom panel). These experiments proved that expression of K3L or PKRDelta E7 inhibit the eIF-2alpha phosphorylation in both yeast strains (middle panel, compare lane 1 with lane 2 or 3 and compare lane 5 with lane 6 or 7). On the other hand, PKRLS9Delta E7 expression did not affect eIF-2alpha phosphorylation by PKR (compare lane 1 with lane 4, and compare lane 5 with lane 8). Due to the dominant negative functions of K3L and PKRDelta E7, expression of WT PKR was more highly induced in the presence of these inhibitors compared with PKRLS9Delta E7 (top panel, compare lane 4 with lane 2 or 3, and compare lane 8 with lane 6 or 7). These data clearly demonstrate the dominant negative function of PKRDelta E7 in PKR activation and eIF-2alpha phosphorylation in yeast.

Activation of Reporter Gene Expression by PKRDelta E7-- The dominant negative function of PKRDelta E7 was further verified in reporter assays in HeLa cells or in mouse fibroblasts derived from two different PKR knockout (PKR-/-) mice (11, 12) (Fig. 7). The first PKR-/- mouse was generated by the disruption of the N terminus domain of the kinase (deletion of exons 2 and 3; N-PKR-/-; Ref. 12), whereas the second was generated by the disruption of the catalytic domain of the molecule (deletion of exon 12; C-PKR-/-; Ref. 11). Cells were co-transfected with the beta -galactosidase reporter gene and K3L or PKRDelta E7 cDNA in the absence or presence of WT human PKR cDNA. Expression of K3L or PKRDelta E7 alone induced beta -galactosidase activity in HeLa cells (Fig. 7A), most likely due to the relief of translational inhibition caused by the activation of the endogenous PKR during transfection (36). As expected, expression of WT PKR in HeLa cells resulted in the inhibition of beta -galactosidase activity compared with control, which was relieved by the co-expression of either K3L or PKRDelta E7. Note that beta -galactosidase activity was more highly inhibited when HeLa cells were transfected with a higher amount of WT PKR cDNA (data not shown). On the other hand, in N-PKR-/- (Fig. 7B) and C-PKR-/- cells (Fig. 7C) K3L but not PKRDelta E7 expression resulted in an induction of beta -galactosidase activity. This effect of K3L may indicate the presence of other eIF-2alpha kinase(s) that can be activated during transfection. Whether this is PKR-like endoplasmic reticulum kinase (15), GCN2 (16), or another PKR-like kinase is not presently known. Expression of WT human PKR in both knockout cells led to the inhibition of beta -galactosidase activity (Fig. 7, B and C), which was relieved by the co-expression of either K3L or PKRDelta E7. Taken together, these data demonstrate the dominant negative function of PKRDelta E7 in PKR-mediated inhibition of reporter gene expression. Consistent with our data, Tian and Mathews (42) have recently reported that induction of reporter gene expression by p20 in transient transfection assays in human 293 cells.



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Fig. 7.   Induction of gene expression by PKRDelta E7. HeLa cells (A) and immortalized fibroblasts derived from mice with a targeted deletion of exons 2 and 3 (N-PKR-/- cells; B) or exon 12 of PKR (C-PKR-/- cells; C) were transfected with the beta -galactosidase gene (0.2 µg) in the presence of the inhibitor K3L DNA (0.5 µg), PKRDelta E7 cDNA (0.5 µg), WT human PKR cDNA (0.5 µg), K3L DNA and WT human PKR cDNA (0.5 µg each) or PKRDelta E7 and WT human PKR cDNAs (0.5 µg each) using the vaccinia/T7 virus system. The -fold induction of beta -galactosidase activity for each transfection is shown. The values represent the average of four independent experiments performed in triplicates.

Tissue Distribution of PKRDelta E7 RNA-- To investigate the physiological relevance of PKRDelta E7 expression, we examined the expression levels of PKRDelta E7 relative to WT PKR RNA in various types of normal human tissue by a RT-PCR assay. As shown in Fig. 8, PKRDelta E7 RNA was expressed in a broad range of human tissues but at variable levels. In most tissues, expression was below 5% of WT PKR RNA, whereas expression in heart, placenta, liver, and skeletal muscle was as high as 5.3, 5.7, 5.3, and 8.4%, respectively. Expression of PKRDelta E7 RNA in spleen was undetectable.



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Fig. 8.   Tissue distribution of PKRDelta E7 RNA. Human multiple normal tissue cDNA (Stratagene) was used for the detection of PKRDelta E7 RNA expression by PCR amplification followed by 10% polyacrylamide gel electrophoresis and silver staining (upper panel). The proportion of PKRDelta E7 RNA compared with full-length PKR RNA in each tissue was quantified using the NIH Image 1.54 software (lower panel).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this paper, we have characterized the function of an alternatively spliced form of PKR produced by a deletion of exon 7 (PKRDelta E7). Although alternative splicing has been previously described for the 5'-untranslated region of PKR mRNA (43), to our knowledge this is the first study to describe the expression of an alternatively spliced product of human PKR with a dominant negative function. PKRDelta E7 is composed of the two copies of the dsRBMs of PKR, a sequence motif found in many dsRNA-binding proteins (18).

Analysis of the biochemical characteristics of PKRDelta E7 has shown that it binds to dsRNA and is capable of both self-associating and associating with full-length WT PKR. Both properties of PKRDelta E7 appear to require the presence of dsRNA, since treatment with MN abolishes its association with WT PKR but partially diminishes its self-association (Fig. 4). These findings are in accord with previous observations by Wu and Kaufman (44) that dimerization of intact PKR with p20 requires the dsRNA binding activity. Consistent with this, recent studies by Tian and Mathews have shown that the efficacy or rate of p20/PKR dimerization through a protein/protein interaction is considerably less than that of their dsRNA-mediated dimerization (42). Our data show that self-association of PKRDelta E7 can also take place independently of dsRNA, and this is in line with previous data showing that dimerization of p20 is independent of RNA (45). In accord with this, the intrinsic ability of RNA-free preparations of p20 to dimerize in the absence of dsRNA has been recently reported (42). Therefore, PKRDelta E7 dimerization in vivo may take place in the absence of dsRNA, whereas the presence of dsRNA may induce conformational changes that facilitate heterodimerization and/or heterodimer stabilization between PKRDelta E7 and WT PKR (42, 45).

We have seen that self-association of PKRDelta E7 is stronger than its association with full-length PKR, in agreement with a previous study showing that homodimerization of p20 in yeast two-hybrid assays is better than its heterodimerization with the full-length PKR (32). The requirement of dsRNA for PKRDelta E7 binding to PKR might give the specificity for PKRDelta E7 to selectively associate with PKR that is bound to dsRNA supporting the notion that dsRNA binding is required for the dominant negative activity of the N terminus truncated form of PKR (44). In regard to this, the dominant negative function of PKRDelta E7 could be mediated through its interaction with the WT PKR, resulting in the formation of inactive heterodimers (Fig. 9). This may be limited to those latent PKR molecules that are accessible to dsRNA, and this could relieve possible localized inhibitory effects of PKR on protein synthesis. Alternatively, expression of PKRDelta E7 may lead to the sequestration of cellular activator dsRNA, resulting in the inhibition of WT PKR activation (Fig. 9). Consistent with this notion, Tian and Mathews have recently shown that the dominant negative function of p20 correlates with the ability of the two dsRBMs to bind to dsRNA but not with their ability to dimerize, supporting the view that dsRNA sequestration may underlie the dominant negative effect (42).



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Fig. 9.   Possible biochemical and biological functions of PKRDelta E7 in vivo. In inactive form, the N terminus dsRNA-binding domain (DRBD) of PKR folds over the C terminus kinase domain (KD) keeping it in a "closed" conformation (61). Binding of dsRNA induces PKR homodimerization and exposes the kinase domain, resulting in activation by autophosphorylation (61). Activated PKR induces the phosphorylation of eIF-2alpha on serine 51, leading to the inhibition of translation initiation, cell growth, and/or virus replication (step 1). PKRDelta E7 may exert the dominant negative function by binding to and blocking PKR homodimerization (step 2) and/or by sequestering dsRNA from binding to and activating PKR (step 3). The low levels of PKRDelta E7 (10% compared with full-length PKR) are compatible with the notion for a local dominant negative function in PKR activation and eIF-2alpha phosphorylation. Such local effects have been thought to be maintained by compartmentalization or anchoring of translational factors to cytoskeletal framework and be critical for the translation of specific mRNAs encoding for proteins that play an important role in cell growth, transformation, or differentiation (1). Also, PKRDelta E7 may exhibit functions that are independent of PKR as an RNA-binding protein (step 4; see "Discussion").

PKR localizes in the cytoplasm, strongly in the nucleolus, and diffusely throughout the nucleoplasm (46-48). Studies by Tian and Mathews (42) recently showed that the dsRBMs are required for the localization of PKR, and this activity correlates with dsRNA binding. The same authors demonstrated that p20 exhibits a localization indistinguishable from that of WT PKR, suggesting a similar function for PKRDelta E7 (42). Interestingly, nuclear localization of PKR was recently shown to be rapidly induced upon treatment with DNA-damaging agents (49), but it is not known whether this process requires dsRNA binding. The two dsRBMs of PKR were reported to be required for its association with ribosomes, and targeting to ribosomes may bring PKR closer to the translation machinery, thus facilitating phosphorylation of eIF-2alpha (50). In the same study, a PKR mutant with deletion of the entire kinase catalytic domain from amino acid residue 271 to 551 was found to associate strongly with ribosomes (50). Based on this finding, it is reasonable to speculate that PKRDelta E7 competes with WT PKR for binding to ribosomes, thus preventing the accessibility of PKR to eIF-2alpha . However, expression of PKRDelta E7 compared with PKR is less than 10% in most tissues, suggesting that the competition between the two molecules for binding to ribosomes could be local. In fact, several observations have supported the theory of localized activation of PKR in regulation of translation of specific genes (2).

Expression of PKRDelta E7 RNA was detected in a broad range of human tissues at variable levels (Fig. 8). Heart, brain, placenta, liver, and skeletal muscle were among the top five tissues that exhibited expression over 5% compared with WT PKR RNA, with the highest levels of PKRDelta E7 RNA expression in the skeletal muscle (8.4%). These five tissues are all energy-demanding, and this could possibly indicate a role of PKR in energy/intermediate metabolism pathways. The other tissues contained less than 5%, whereas expression of PKRDelta E7 RNA in spleen was undetectable. Interestingly, expression of PKRDelta E7 was higher in Jurkat cells than in normal PBMCs (Fig. 3). Whether or not this difference is a cause or an effect of the transformed phenotype of Jurkat cells is an issue that requires further investigation.

We have shown that PKRDelta E7 exhibits a dominant negative function in PKR activation in mouse, human, and yeast cells, which results in growth inhibition through the induction of eIF-2alpha phosphorylation. Whether the dominant negative function of PKRDelta E7 plays a role in pathways other than inhibition of eIF-2alpha phosphorylation is currently under investigation. For example, we have shown that PKR phosphorylates human p53 on serine 392 in vitro, which may account for some of the translational properties of p53 (51). Therefore, PKRDelta E7 may also exhibit a dominant negative function in p53 phosphorylation through its capacity to physically associate with PKR and p53 (data not shown). Also, PKRDelta E7 contains the two dsRBMs, which have been found in many dsRNA-binding proteins (18). These proteins bind dsRNA in a largely sequence-independent fashion and are involved in a myriad of biological processes such as RNA editing (52), RNA trafficking (53), RNA processing (54), transcriptional regulation (55), and the interferon antiviral response (56). Also, the dsRBMs of PKR and other dsRBM-containing proteins have been shown to possess dsRNA annealing activity and may play a role as chaperones by facilitating the folding of cellular RNAs (42, 57). Therefore, the possibility that PKRDelta E7 exhibits functions that are independent of PKR cannot be excluded (Fig. 9).

Given the potential importance of the tight regulation of PKR activity in growth control, it is not surprising that several cellular inhibitors of PKR have been identified and characterized. For example, the human immunodeficiency virus-1 TAR RNA-binding protein (TRBP) is a dsRNA-binding protein that inhibits PKR (58) by binding to dsRNA and forming heterodimers with endogenous PKR (32). Interestingly, TRBP overexpression can transform mouse NIH3T3 cells in culture through the inactivation of endogenous PKR (59). However, unlike TRBP, PKRDelta E7 does not exhibit a dominant negative effect on mouse PKR (data not shown), providing evidence for differences in the specificity between the two dsRNA-binding inhibitors of PKR.

The question arises as to what is the physiological significance of inactivation of PKR by PKRDelta E7 and how the inhibitory function of PKRDelta E7 differs from that of other dsRNA-binding PKR inhibitors (e.g. TRBP). One possibility is that association of each of these dsRNA-binding proteins with PKR requires a specific RNA structure, resulting in a local and specific inhibition of PKR activation and eIF-2alpha phosphorylation. Another possibility is that each of the heterodimers between PKR and dsRNA-binding proteins plays a role in RNA-mediated biological processes other than translation (i.e. RNA trafficking, editing, splicing, or transport). In this regard, activation of PKR has been implicated in the splicing of human TNF-alpha mRNA (60).

In conclusion, the data presented here demonstrate the expression and the dominant negative function of a dsRNA-binding alternatively spliced product of human PKR. Further understanding of the basis of regulation and function of alternatively spliced PKR products may yield important insights into biological function of the kinase in regard to RNA metabolism, transcription, translation, and regulation of signaling pathways that affect cell proliferation.


    ACKNOWLEDGEMENTS

We thank T. Dever for the yeast strains J110, H2544, H2543, J80, and J82, the pEMBLyes4/K3L DNA, and the rabbit polyclonal anti-yeast eIF-2alpha antibody (CM-217); A. Darveu and G. N. Barber for mouse monoclonal anti-human PKR antibodies (clones E8 and F9); M. Mathews for PKRLS9 cDNA; N. Sonenberg for the wild type and serine 51 to alanine mutant of eIF-2alpha cDNAs; and M. Clemens for mouse anti-eIF-2alpha monoclonal antibody.


    FOOTNOTES

* This work was supported by a grant and a postdoctoral fellowship from the Cancer Research Society Inc. (to A. E. K. and S. L., respectively).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Member of the Terry Fox Group of Molecular Oncology and a recipient of a Canadian Institutes of Health Research Scientist Award. To whom correspondence should be addressed: Lady Davis Institute for Medical Research, Rm. 508, Jewish General Hospital, 3755 Côte-Ste-Catherine St., Montréal, Québec, Canada H3T 1E2. Tel.: 514-340-8260 (ext. 3697); Fax: 514-340-7576; E-mail: akoromil@ldi.jgh.mcgill.ca.

Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M008140200


    ABBREVIATIONS

The abbreviations used are: IFN, interferon; dsRNA, double-stranded RNA; GCN2 and -4, general control nonderepressible-2 and -4, respectively; eIF-2alpha , eukaryotic translation initiation factor 2 alpha -subunit; PKRDelta E7, alternatively spliced product of human PKR with deletion of exon 7; dsRBM, dsRNA-binding motif; p20, a 20-kDa N terminus-truncated form of human PKR containing the two contiguous dsRBMs; PBMC, peripheral blood monocyte; RT, reverse transcription; PCR, polymerase chain reaction; WT, wild type; bp, base pair; PAGE, polyacrylamide gel electrophoresis; MN, micrococcal nuclease; TRBP, TAR RNA-binding protein.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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