Enhanced Antiviral and Antiproliferative Properties of a STAT1 Mutant Unable to Interact with the Protein Kinase PKR*

Andrew Hoi-Tao WongDagger §, Joan E. Durbin, Suiyang LiDagger ||, Thomas E. Dever**, Thomas DeckerDagger Dagger , and Antonis E. KoromilasDagger §§

From the Dagger  Terry Fox Molecular Oncology Group, Lady Davis Institute, Jewish General Hospital, Montreal H3T 1E2, Canada, the  Children's Hospital Research Foundation and Department of Pediatrics, Ohio State University, Columbus, Ohio 43205, the ** Laboratory of Eukaryotic Gene Expression, NICHD, National Institutes of Health, Bethesda, Maryland 20892, and the Dagger Dagger  Vienna Biocenter, Institute for Microbiology and Genetics, Vienna 1030, Austria

Received for publication, December 13, 2000, and in revised form, January 24, 2001




    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously reported a physical association between STAT1 and the protein kinase double-stranded RNA-activated protein kinase (PKR). PKR inhibited STAT1 function in a manner independent of PKR kinase activity. In this report, we have further characterized the properties of both molecules by mapping the sites of their interaction. A STAT1 mutant unable to interact with PKR displays enhanced interferon gamma  (IFN-gamma )-induced transactivation capacity compared with STAT1. This effect appears to be mediated by the higher capacity of STAT1 mutant to heterodimerize with STAT3. Furthermore, expression of STAT1 mutant in STAT1-/- cells enhances both the antiviral and antiproliferative effects of IFNs as opposed to STAT1. We also provide evidence that STAT1 functions as an inhibitor of PKR in vitro and in vivo. That is, phosphorylation of eIF-2alpha is enhanced in STAT1-/- than STAT1+/+ cells in vivo, and this correlates with higher activation capacity of PKR in STAT1-/- cells. Genetic experiments in yeast demonstrate the inhibition of PKR activation and eIF-2alpha phosphorylation by STAT1 but not by STAT1 mutant. These data substantiate our previous findings on the inhibitory effects of PKR on STAT1 and implicate STAT1 in translational control through the modulation of PKR activation and eIF-2alpha phosphorylation.




    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cytokines and growth factors exert a diverse range of biological activities, from host defense, growth regulation, to immunomodulation. Upon ligand binding to cell-surface receptors, JAK1 kinases are activated and proceed to phosphorylate the receptor on tyrosine residues, which then function as docking sites for cytoplasmic transcription factors of the STAT family (1, 2). STATs are subsequently activated by tyrosine phosphorylation, dimerize by phosphotyrosyl·SH2 interactions, and translocate to the nucleus to induce transcription of cytokine-responsive genes (3). A single tyrosine phosphorylation site in the carboxyl-terminal activation domain is absolutely essential for STAT dimerization and DNA binding (3), whereas phosphorylation of a serine residue in this region is important for transactivational activity (4).

One of the major STATs intimately involved in both the innate and acquired immune responses is STAT1. Upon virus infection or exposure to interferons (IFNs), STAT1 is found in protein complexes that bind specific DNA sequences upstream to genes responsible for host resistance. For instance, IFN-alpha /beta induces formation of the heterodimeric ISGF3, whereas IFN-gamma induces binding of homodimeric STAT1 (2, 3). Moreover, dsRNA, an intermediate produced during virus replication, can also activate STAT1 DNA binding (5, 6). The non-redundant role of STAT1 in the antiviral response is further appreciated by findings that stat1 null mice (STAT1-/-) are highly susceptible to microbial infection (7, 8). IFN signaling leads to the expression of a number of genes, one of which encodes for the dsRNA-dependent protein kinase, PKR (9, 10). PKR is a serine/threonine protein kinase that displays two distinct activities: (i) autophosphorylation upon dsRNA binding (9, 10) and (ii) phosphorylation of the eukaryotic translation initiation factor eIF-2alpha (9, 10), a modification resulting in inhibition of protein synthesis (11). Several studies with cultured cells provide evidence for antiviral (12, 13), antiproliferative, and tumor suppressor functions of PKR (9, 10). However, pkr null (PKR-/-) mice exhibit a modest susceptibility to viral infection (14-17) and show no signs of tumor formation (14, 17), suggesting that the lack of PKR can be compensated by other PKR-like molecules (9, 10). This hypothesis is supported by the recent identification of the PKR-related genes, PERK/PEK (18) and the mouse homolog of the yeast eIF-2alpha kinase, GCN2 (19).

We previously described an association between PKR and STAT1 (6). This interaction takes place in unstimulated cells and diminishes upon treatment with IFNs or dsRNA. Increased levels of PKR·STAT1 complex have a negative effect on STAT1 DNA-binding and transactivation capacities. In this report we have mapped the interaction sites between the two proteins and have identified a novel function of STAT1. Specifically, we demonstrate that STAT1 functions as an inhibitor of PKR activation and eIF-2alpha phosphorylation in vitro and in vivo. A mutant of STAT1, which was unable to interact with PKR, could not inhibit PKR function in yeast and was better able to mediate the transcriptional, antiviral, and antiproliferative responses of IFNs compared with STAT1. Taken together, these findings not only support our previous observations, but they also provide strong evidence for tight regulation of PKR and STAT1 functions by virtue of their interaction. In addition, our data suggest that STAT1 has a dual role in regulation of gene expression by functioning as a transcriptional factor and possibly as translational regulator through PKR activation and eIF-2alpha phosphorylation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Transfections-- HeLa S3, U3A, STAT1+/+, STAT1-/-, PKR+/+, and PKR-/- cells were maintained in Dulbecco's modification of Eagle's medium supplemented with 10% calf serum, 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin (Life Technologies). For IFN treatment, cells were incubated with 1000 IU/ml of recombinant murine IFN-alpha /beta (Lee Biomolecules) or 100 IU/ml of IFN-gamma (PharMingen). Double-stranded RNA transfections were performed as previously described (6). Transient transfections were performed with LipofectAMINE Plus reagent (Life Technologies). T7 vaccinia virus transient transfections were carried out using the recombinant vaccinia virus, vTF7-3, encoding the bacteriophage T7 RNA polymerase (20). In vivo [35S]methionine experiments were performed as previously described (21). pBABE, pBABE-HA-STAT1alpha WT, or TM STAT1-/- cells were generated as previously described (22). Cell cycle analyses and CPE assays were performed as described in previous studies (Refs. 23 and 24, respectively).

Plasmid Construction-- GST-PKRK296R, GST-PKR 1-262 (PKR N), and GST-PKR K296R 263-551 (PKR C) were generated as previously described (25). GST-PKRLS4K296R was generated by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene) with the following primer pairs (Sheldon Biotechnology): 5'-d[CCAGAAGGTGAAGGTGGAGCATTGAAGGAAGCAAAAAATGCCGC]-3' and 5'-d[GCGGCATTTTTTGCTTCCTTCAATGCTCCACCTTCACCTTCTGG]-3'. GST-PKRLS9K296R was generated by subcloning an EcoNI and AflII fragment of PKRLS9. Truncated GST-PKR proteins were generated with the following primers: pGEX forward primer (Amersham Pharmacia Biotech); 5'-PKR 263, 5'-d[GGGGGATCCTAAAACCTCTTGTCCACAGTATAC]-3'; 5'-PKR 325, 5'-d[GGGGGGATCCGGCTGTTGGGATGGATTTGATTAT]-3'; 5'-PKR 367, 5'-d[GGGGGGATCCTTCTGTGATAAAGGGACCTTGGAA]-3'; 3'-PKR 262, 5'-d[GGGGGATCCTAAAACCTCTTGTCCACAGTATAC]-3'; 3'-PKR 324, 5'-d[CCCGGGCTAATTGTAGTGAACAATATTTACATGAT]-3'; 3'-PKR 366, 5'-d[CCCGGGCTATTCCATTTGGATGAAAAGGCACT]-3'; 3'-PKR 415, 5'-d[CCCGGGCTACTTAAGATCTCTATGAATTAATTTTTT]-3'; and pGEX reverse primer (Amersham Pharmacia Biotech). PCR fragments were cut with BamHI and/or SmaI and ligated to pGEX-2T. Truncated STAT1 proteins were generated by PCR from the template pGEX-5X-3-HA-STAT1 (26) using the following primers: 5'-HA-STAT1, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTATGTCTCAGTGGTACGAACTTCAG]-3'; 5'-HA-STAT1 304, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTCGCACCTTCAGTCTTTTCCAG]-3'; 5'-HA-STAT1 343, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTGTGAAGTTGAGACTGTTGGTGAAA]-3'; 5'-HA-STAT1 365, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTGATAAAGATGTGAATGAGAGAAATAC]-3'; 5'-HA-STAT1 380, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTTTCAACATTTTGGGCACGCACAC]-3'; 5'-HA-STAT1 414, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTAATGCTGGCACCAGAACGAATG]-3'; 5'-HA-STAT1 519, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTCTGAACATGTTGGGAGAGAAGC]-3'; 5'-HA-STAT1 611, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTGCCATCACATTCACATGGGTG]-3'; 3'-STAT1 303, 5'-d[GGGGCGGCCGCCTAGTCCCATAACACTTGTTTGTTTTT]-3'; 3'-STAT1 342, 5'-d[GGGGCGGCCGCCTAAGTGAACTGGACCCCTGTCTTC]-3'; 3'-STAT1 348, 5'-d[GGGGCGGCCGCCTACAACAGTCTCAACTTCACAGTGAA]-3'; 3'-STAT1 357, 5'-d[GGGGCGGCCGCCTAATTATAATTCAGCTCTTGCAATTTCA]-3'; 3'-STAT1 364, 5'-d[GGGGCGGCCGCCTAAAATAAGACTTTGACTTTCAAATTATAAT]-3'; 3'-STAT1 379, 5'-d[GGGGCGGCCGCCTACTTCCTAAATCCTTTTACTGTATTTC]- 3'; 3'-STAT1 413, 5'-d[GGGGCGGCCGCCTATTTCTGTTCTTTCAATTGCAGGTG]-3'; 3'-STAT1 518, 5'-d[GGGGCGGCCGCCTACTGGTCCACATTGAGACCTCT]-3'; 3'-STAT1 610, 5'-d[GGGGCGGCCGCCTACCCTTCCCGGGAGCTCTCA]-3'; and the pGEX reverse primer. PCR products were BamHI-NotI-digested, subcloned into the mammalian expression vector, pcDNA3.1/zeo, and confirmed by sequencing. Site-directed mutagenesis was carried out with the following primer pairs: R346A, 5'-d[CAGTTCACTGTGAAGTTGGCACTGTTGGTGAAATTGCAAG]-3' and 5'-[CTTGCAATTTCACCAACAGTGCCAACTTCACAGTGAACTG]-3'; R346A/L347D, 5'-[CAGTTCACTGTGAAGTTGGCAGACTTGGTGAAATTGCAAGAGCTG]-3' and 5'-[CAGCTCTTGCAATTTCACCAAGTCTGCCAACTTCACAGTGAACTG]-3'; and R346A/L347D/L348D, 5'-[GTTCACTGTGAAGTTGGCAGACGACGTGAAATTGCAAGAGCTG]-3' and 5'-[CAGCTCTTGCAATTTCACGTCGTCTGCCAACTTCACAGTGAAC]-3'. PCR products were ligated to pcDNA3.1/zeo-HA-STAT1 by EcoNI restriction digest. pBABE-HA- STAT1alpha WT and TM were generated by ligation into BamHI restriction sites.

Cell Extract Preparation, Immunoprecipitation, and Immunoblot Analysis-- Cell extract preparation, immunoprecipitation, and immunoblotting were performed as previously described (6). The following antibodies were used: STAT1alpha (Santa Cruz Biotechnology); HA (12CA5, Roche Molecular Biochemicals); STAT2 (Upstate Biotechnology Inc.); STAT3 (Santa Cruz); PKR; GST (Amersham Pharmacia Biotech); Myc (9E10, Roche Molecular Biochemicals); eIF-2alpha ; phosphoserine 51 of eIF-2alpha (Research Genetics Inc.); FLAG (M2, Kodak); HA horseradish peroxidase antibody (3F10, Roche Molecular Biochemicals); phosphotyrosine (4G10/PY20, UBI and Transduction Laboratories); and phosphoserine 727 of STAT1alpha (27). Proteins were visualized by ECL (Amersham Pharmacia Biotech).

DNA Binding and Transactivation Assays-- Electrophoretic mobility shift analysis was performed using the dsDNA c-Fos c-sis-inducible element (SIE, 5'-GATCGTGCATTTCCCGTAAATCTTGTCTACAATTC-3') according to protocols previously described (6, 28). The Dual Luciferase system (Promega) was used to assess the transactivation potential of STAT1. Briefly, STAT1-/- cells or cells expressing STAT1 WT or TM were transfected with Renilla luciferase (pRL-TK) and pGL-2XIFP53 GAS luciferase. Twenty-four hours after transfection, cells were replated and treated with IFN-gamma for 18 h before harvesting. The results presented represent quadruplicate experiments where GAS luciferase activity was normalized to Renilla luciferase activity.

Isoelectric Focusing and PKR in Vitro Kinase Assays-- Isoelectric focusing and immunoblot analysis of yeast eIF-2alpha were performed as previously described (29). PKR in vitro kinase assays were carried as previously described (6).

GST Pull-down Assays-- Protein production and extraction were performed according to previously described protocols (25, 30). Normalized GST fusion proteins were co-incubated with HeLa whole cell lysates or [35S]methionine in vitro translated proteins, washed, subjected to SDS-PAGE, and visualized by fluorography (25).

Yeast Plasmids, Transformations, Growth Protocols, and Protein Extractions-- Wild-type and mutants of HA-STAT1 1-413 were subcloned by restriction digest of BamHI-NotI sites into a modified form of the yeast expression vector, pEMBL/yex4 (29), containing a NotI site in the multiple cloning site. Transformation of yeast strains H2544 and J110 and growth analyses were performed as previously described (31).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The catalytic domain of PKR specifically associates with the DNA-binding domain of STAT1. To map the PKR·STAT1 interaction we performed a series of binding assays using full-length GST-PKRK296R mutant (GST-PKR WT could not be overexpressed in bacteria (32)) or truncations of PKR bearing either the dsRNA-binding (GST-PKR N, amino acids 1-262) or catalytic (GST-PKR C, amino acids 263-551) domain (Fig. 1A). HeLa S3 extracts expressing human HA-STAT1alpha were incubated with GST-PKR fusion proteins, subjected to SDS-PAGE, and immunoblotted with an anti-HA antibody. As shown in Fig. 1B (top panel), both full-length GST-PKR (lane 5) and the carboxyl terminus of PKR (lane 4) interacted with STAT1 whereas binding to the amino terminus was not detectable (lane 3). Furthermore, this interaction is specific for STAT1, because we could not detect binding of GST-PKR proteins with other STATs (Fig. 1B).



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Fig. 1.   The kinase domain of PKR specifically interacts with STAT1 in vitro. A, GST alone, GST-PKR N, GST-PKR C, and GST-PKR FL were subjected to SDS-PAGE and visualized by Coomassie Blue staining. B, GST-PKR fusion proteins were co-incubated with protein extracts of HeLa S3 cells transfected with HA-STAT1alpha (top), STAT2 (second panel), STAT3 (third panel), or myc-STAT5B (bottom panel). Bound proteins were revealed by immunoblotting with HA (top panel), STAT2 (second panel), STAT3 (third panel), or Myc (bottom panel) antibodies. C, GST alone, GST-PKRK296R, GST-PKRK296RLS4, and GST-PKRK296RLS9 were co-incubated with protein extracts from HeLa S3 cells transfected with HA-STAT1alpha . After GST-pull down, proteins were detected by immunoblotting with HA (top panel) whereas the GST fusion proteins were visualized by Coomassie Blue staining (bottom panel).

We previously reported that binding of PKR to STAT1 is independent of RNA but requires an intact RNA-binding domain, because the dsRNA-binding-defective mutant PKRLS4 (Arg58-Ser59-Lys60 to Gly58-Ala59-Leu60) (33) does not interact with mouse STAT1 in NIH3T3 cell extracts stably expressing this PKR mutant (6). Based on this, we proposed that the integrity of the dsRNA-binding domain of PKR plays a role in PKR·STAT1 interaction in vivo (6). The observation, however, that the carboxyl terminus of PKR is required for binding to STAT1 in vitro (Fig. 1B) prompted us to examine the interaction of STAT1 with GST-PKR bearing either the LS4 or the LS9 mutation (Ala66-Ala68 to Gly66-Pro68), which also abolishes RNA binding (33) (Fig. 1C). The RNA-binding-defective mutants were expressed and purified in the GST-PKRK296R background, because expression of LS4 and LS9 in the GST-PKR WT background could not be achieved (data not shown). HeLa S3 extracts containing HA-STAT1alpha were incubated with GST-PKRK296R, GST-PKRK296RLS4, or GST-PKRK296RLS9, and GST-PKR bound proteins were subjected to immunoblotting with anti-HA antibody. All GST-PKR mutants interacted with STAT1 equally well (Fig. 1C, top panel), suggesting that mutations within the dsRNA-binding domain do not interfere with PKR binding to STAT1 in vitro.

To map the region on STAT1 that facilitates its interaction with PKR, we used truncated STAT1 proteins corresponding to its amino-terminal, DNA-binding, linker, SH2, or transactivation domains (Fig. 2A). The pull-down assays were performed with GST-PKR C, because this protein binds to STAT1 (Fig. 1A) and is more stable than GST-PKRK296R (data not shown). We observed that GST-PKR C specifically interacted with the DNA-binding domain of STAT1 (Fig. 2B, lane 14). Upon truncation of the STAT1 DNA-binding domain from the carboxyl-terminal end, a critical junction is reached when binding to PKR is retained (middle panel, HA-STAT1 1-364, lane 13) and when binding is abolished (HA-STAT1 1-342, lane 12). Further truncation of STAT1 from the amino terminus also presented a similar junction between amino acids 343-365 (Fig. 2C, lower panel, compare lanes 25 and 26). Moreover, truncation of the carboxyl terminus of the DNA-binding domain of STAT1 at positions 348 and 357 still retained binding to GST-PKR (Fig. 2D, lanes 10 and 11), suggesting that the region of interaction lies between amino acids 343 and 348 (beta -sheet 3) on the DNA-binding domain of STAT1 (33, 34).



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Fig. 2.   Mapping of amino acids on STAT1 required for PKR-binding. A, STAT1 was truncated into five portions represented by the various shades of the molecule depicted. B, GST alone or GST-PKR C was co-incubated with [35S]methionine-labeled, in vitro translated STAT1 proteins and subjected to SDS-PAGE. C, GST alone or GST-PKR C was incubated with in vitro translated STAT1 proteins truncated from either the amino or carboxyl terminus and subjected to SDS-PAGE. D, similar pull-down assays were performed using GST or GST-PKR C with in vitro translated STAT1 proteins further truncated from the carboxyl terminus. E, GST alone or GST-PKR C was co-precipitated with in vitro translated STAT1 1-413 wild-type (WT) or triple-mutant (TM). F, U3A cells were transiently transfected with HA-STAT1alpha WT or TM in the presence or absence of PKRK296R. Extracts were immunoprecipitated with anti-PKR antibodies and immunoblotted with HA (top panel) and PKR (bottom panel) antibodies.

Mutations of STAT1 That Affect Binding to PKR-- To identify amino acids in STAT1 that form contacts with PKR, we next constructed mutations within amino acids 343-348 of HA-STAT1 1-413 that abolished binding to PKR. Alanine-scan mutagenesis of each of the six amino acids did not yield a point mutant of HA-STAT1 1-413 that disrupted interaction with PKR (data not shown). However, a three-amino acid substitution (TM; Arg346-Leu347-Leu348 to Ala346-Asp347-Asp348) within this region disrupted the ability of STAT1 to interact with GST-PKR C (Fig. 2E, lane 6). Interestingly, HA-STAT1 1-413 TM possessed faster mobility on SDS-PAGE gels compared with WT most likely through changes in the overall charge of the molecule.

To further characterize the interaction of full-length HA-STAT1alpha TM with PKR, we utilized the human fibrosarcoma, U3A cell line, which lacks endogenous STAT1 (35). HA-STAT1alpha WT or TM were co-transfected with PKRK296R into U3A cells, after which, the protein extracts were immunoprecipitated against PKR and immunoblotted with HA antibodies. As seen in Fig. 2F, STAT1alpha WT associated with both transfected and endogenous PKR (upper panel, lanes 2 and 5). Conversely, STAT1 TM binding with endogenous PKR was completely abolished (lane 3) and displayed very marginal binding to transfected PKR, which was detectable only after long exposures (lane 6). In contrast to HA-STAT1 1-413 TM, full-length STAT1alpha TM did not display any difference in its migration pattern compared with STAT1alpha WT. These in vitro findings were also verified in vivo by the yeast two-hybrid assay (data not shown). Taken together, it appears that the DNA-binding domain of STAT1 interacts with PKR in vitro and in vivo.

DNA Binding and Transcriptional Properties of STAT1 TM-- To test the ability of STAT1 TM to respond to IFN-gamma treatment, we performed transient transfection assays in STAT1-/- cells using STAT1 WT or STAT1 TM and a luciferase reporter construct driven by two copies of the GAS element from the IFP53 gene (28). As shown in Fig. 3A, we observed that luciferase expression in cells transfected with STAT1 WT was induced by IFN-gamma treatment. However, in cells transfected with STAT1 TM, we observed, after normalization to Renilla luciferase, a much higher basal luciferase activity (~5-fold) that could be slightly induced by IFN-gamma stimulation.



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Fig. 3.   STAT1 TM displays elevated transcriptional properties. A, STAT1-/- cells were transiently transfected with pRL-TK, pGL-2XIFP53-GAS, and control vector, HA-STAT1alpha WT, or HA-STAT1alpha TM. Transfected cells were left untreated (white bars) or stimulated with IFN-gamma for 18 h (black bars) and harvested. B, STAT1-/- cells stably transduced with control vector, HA-STAT1alpha WT, or HA-STAT1alpha TM were transfected with pRL-TK and pGL-2×IFP53-GAS. Transfected cells were left untreated (white bars) or stimulated with IFN-gamma for 18 h (black bars) and harvested. Luciferase activity was measured and normalized to Renilla luciferase activity. The data represent the average of quadruplicate experiments. The relative protein levels of STAT1 WT and STAT1 TM were compared by immunoblotting with antibodies against HA. C, control, WT, and TM cells were treated with IFN-gamma for 30 min or left untreated. Protein extracts were incubated with 32P-radiolabeled c-sis-inducible element from the c-Fos promoter and subjected to DNA binding assays. Competition reactions were performed using a 200-fold excess of unlabeled oligonucleotide. Supershift experiments were performed using non-reactive mouse IgG1, STAT1alpha , or STAT3 antibodies. D, control, STAT1 WT, and STAT1 TM cells were left untreated, or stimulated with IFN-alpha /beta or IFN-gamma for 30 min. STAT1 WT and TM protein levels were normalized with the appropriately stimulated or unstimulated control cell extracts (STAT1-/-) and subsequently immunoprecipitated with STAT1alpha antibody and immunoblotted against phosphotyrosine (top panel), STAT1alpha phosphoserine 727 (middle panel), and HA (bottom panel) antibodies. E, normalized protein extracts were immunoprecipitated with antibodies against STAT1 and immunoblotted with STAT3 (top panel) or HA (bottom panel) antibodies. F, the above extracts were immunoprecipitated with antibodies against STAT3 and immunoblotted against phosphotyrosine (top panel) or STAT3 (bottom panel).

To better characterize STAT1 TM, we infected STAT1-/- fibroblasts with retroviruses harboring the puromycin-resistant gene and HA-STAT1alpha WT or HA-STAT1alpha TM. As a control, retroviruses containing only the puromycin-resistant gene were used. After puromycin selection, polyclonal populations of STAT1 WT-expressing cells showed ~5-fold greater expression over STAT1 TM pools (Fig. 3B, compare lanes 2 and 3). Transactivation assays using the 2XIFP53-GAS luciferase reporter correlated with our findings in transient transfection experiments that STAT1 TM confers higher basal reporter activity, which can be induced by IFN-gamma treatment (Fig. 3B). We next tested whether STAT1 TM could bind DNA after IFN treatment. Although we could not detect ISGF3 formation in STAT1 TM cells in response to IFN-alpha /beta (data not shown), IFN-gamma stimulation resulted in the formation of DNA-binding complexes consisting of either STAT1·STAT3 heterodimers or STAT3 homodimers, but not that of STAT1 homodimers (Fig. 3C, compare lanes 7-16). This finding is consistent with previous reports that STAT3 is activated following IFN treatment (3). Moreover, the intensity of the STAT3 homodimer appears to be higher compared with control or STAT1 WT cells (compare lanes 2, 4, and 6).

The ability of STAT1 to be phosphorylated upon IFN stimulation was also examined (Fig. 3D). To compare STAT1 phosphorylation per equal amounts of STAT1 protein, we used a 5-fold higher amount of STAT1 TM extracts versus STAT1 WT before and after IFN stimulation. These reactions were also normalized to total protein concentration by the addition of treated or untreated STAT1-/- control extracts. Although STAT1 WT was tyrosine-phosphorylated following IFN-alpha /beta or IFN-gamma treatment (top panel, lanes 5 and 6), we failed to detect STAT1 TM tyrosine phosphorylation (lanes 8 and 9). In contrast, phosphorylation of serine 727 did not significantly differ between STAT1 WT and STAT1 TM after IFN treatment (middle panel, lanes 5-6 and 8-9). Reprobing of the membrane with antibodies to HA revealed the hypo- and hyperphosphorylated forms of STAT1 usually observed after IFN treatment (lower panel). Because STAT1 is also known to form heterodimers with STAT3 following IFN stimulation (3), we tested whether STAT1 TM could associate with STAT3. A much higher amount of STAT3 co-precipitated with STAT1 TM before and after IFN treatment (Fig. 3E, top panel, lanes 7-9), although STAT1 protein levels were approximately equal (bottom panel). We also analyzed expression and activation of STAT3 in the same protein extracts used for the STAT1/STAT3 co-immunoprecipitation. STAT3 phosphorylation was slightly elevated (~50%) in cells expressing STAT1 TM before or after treatment with either IFN-alpha /beta or IFN-gamma (Fig. 3F, lanes 7-9). This increase in STAT3 activity may account for increased STAT3 DNA binding in STAT1 TM cells.

STAT1 TM Enhances the Antiviral and Antiproliferative Effects of IFNs-- The biological effects of STAT1 TM activation were examined by cell cycle analysis after treatment with IFN-alpha /beta or IFN-gamma (Fig. 4A). A greater proportion of STAT1 TM-expressing cells (IFN-alpha /beta , 6-8%; IFN-gamma , 10-11%) were arrested in G0/G1 phase after treatment with either type I or type II IFNs (right panel) compared with control (left panel) or STAT1 WT-expressing (middle panel) cells. In addition, the ability of STAT1 TM cells to resist virus infection was also investigated. Control, STAT1 WT, and STAT1 TM cells were primed with IFNs and subsequently infected with serially diluted VSV. The amount of virus needed to induce CPE was qualitatively measured. As shown in Fig. 4B (upper panel), STAT1 TM cells that were treated with IFN-gamma were ~50-fold more resistant to VSV infection compared with STAT1 WT cells, and ~104-fold more resistant versus control STAT1-/- cells. In contrast, IFN-alpha /beta -treated STAT1 TM cells were 10-fold more susceptible to VSV CPE compared with control, STAT1 WT, and STAT1+/+ cells. Interestingly, even untreated STAT1 TM cells provided a greater degree of protection compared with STAT1 WT and STAT1+/+ cells. This enhanced ability of STAT1 TM cells to resist virus infection was also observed after encephalomyelocarditis virus infection (data not shown). Western blotting against STAT1alpha revealed that STAT1 TM is expressed at much lower levels than STAT1 WT and endogenous STAT1 from STAT1+/+ cells (bottom panel). Taken together, these data suggest that STAT1 TM enhances the antiproliferative and antiviral effects of IFNs on a per molecule basis.



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Fig. 4.   STAT1 TM has enhanced antiproliferative and antiviral properties. A, polyclonal STAT1-/- cell lines transduced with control vector, HA-STAT1alpha WT, or HA-STAT1alpha TM were synchronized by serum starvation for 48 h and stimulated with IFN-alpha /beta or IFN-gamma for 12 and 24 h. Cells were fixed, stained with propidium iodide, and subjected to cell cycle analysis. The data were processed using the WINMDI v2.8 application. The relative cell-cycle distributions presented in the table represent two averaged experiments. B, cells were pretreated with IFN-alpha /beta or IFN-gamma for 18 h before being infected with serially diluted VSV for 24 h, after which viable cells were stained with crystal violet (top panel). Whole-cell extracts from the cell lines were immunoblotted with antibodies against STAT1alpha and immunoblotted with an antibody against the HA epitope (bottom panel).

STAT1 Functions as a PKR Inhibitor in Vitro-- To gain better insight into the PKR·STAT1 interaction, we next mapped the STAT1-binding site on PKR (Fig. 5A). In agreement with earlier results, the full-length kinase domain (lane 4), but not the dsRNA-binding domain (lane 3), bound PKR. Truncation of the kinase domain from either the amino or carboxyl terminus (Fig. 5A) defined a region critical for STAT1 binding: amino acids 367-415 (lanes 5-9). Interestingly, this corresponds to the same region in the large lobe of the PKR kinase domain, to which the vaccinia virus K3L protein binds to block PKR activation (36, 37). In view of this fact, we tested whether the two proteins could compete for binding to PKR. Bacterially expressed FLAG-K3L was co-incubated with GST-PKR C and extracts from HeLa cells expressing increasing amounts of HA-STAT1alpha . Immunoblotting was performed to detect either binding of HA-STAT1alpha or FLAG-K3L to GST-PKR C. As seen in Fig. 5B, STAT1 displaced K3L from PKR in a dose-dependent manner (bottom panel, lanes 10-13), indicating that K3L and STAT1 bind to the same region on PKR.



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Fig. 5.   STAT1 inhibits PKR activity in vitro. A, GST-PKR truncated proteins were generated according to the schematic shown. GST-PKR proteins were co-incubated with extracts expressing HA-STAT1alpha and binding to PKR was determined by immunoblotting with STAT1alpha antibodies (top panel) and reprobed with GST antibody (bottom panel) to determine the levels of fusion protein. B, GST alone or GST-PKR C was incubated with a constant amount of recombinant FLAG-K3L and cell extracts expressing increasing amounts of HA-STAT1alpha (mock, 0.1 µg, 0.5 µg, and 5 µg of DNA). SDS-PAGE was performed followed by immunoblot analysis with HA (top panel) or FLAG (bottom panel) antibodies. C, PKR was immunoprecipitated from 50 µg of HeLa S3 protein extracts, and PKR was activated in vitro in the presence of activator reovirus dsRNA, [gamma -32P]ATP, and 500 ng of GST protein or increasing amounts of purified STAT1alpha protein (5, 50, and 500 ng of STAT1alpha ; lanes 4, 5, and 6, respectively). The reactions were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membranes, exposed (top panel), and immunoblotted with anti- STAT1alpha (middle panel) or anti-PKR (bottom panel) antibodies.

The PKR pseudosubstrate, K3L, has been shown to bind PKR and block access of eIF-2alpha to the catalytic pocket of PKR (29, 37). To address the importance of STAT1 binding to PKR, we investigated the ability of STAT1 to act as a cellular inhibitor of PKR. A series of in vitro assays were performed to assess the effect of STAT1 on PKR activation. Human PKR was activated by reovirus dsRNA in vitro from HeLa S3 cells in the presence of increasing amounts of recombinant full-length STAT1 (Fig. 5C, middle panel). We observed that PKR autophosphorylation diminished in a dose-dependent manner by the addition of STAT1 (top panel), although PKR protein levels were equal (bottom panel).

STAT1 Inhibits the Antiproliferative Properties of PKR in Yeast-- To date, the best approach to test for the translational function of PKR is in Saccharomyces cerevisiae. It has been shown that high levels of PKR expression in yeast are toxic due to inhibition of general translation (38). However, at lower levels of expression PKR can substitute the function of GCN2 (39), the only eIF-2alpha kinase known to exist in S. cerevisiae (40), by phosphorylating eIF-2alpha on serine 51 to inhibit protein synthesis. Through this approach, a number of PKR inhibitors have been identified and characterized (31).

Given that PKR expression in yeast results in inhibition of cell growth (38, 39), we wanted to analyze whether STAT1 could block this effect when co-expressed with PKR. Yeast strain H2544, which lacks the yeast eIF-2alpha kinase GCN2, contains a stable integration of human PKR WT cDNA downstream to a galactose-inducible promoter, whereas the isogenic strain J110 is identical except that the PKR cDNA was not inserted. Previous studies have shown that induction of PKR expression in H2544 results in inhibition of cell growth through phosphorylation of eIF-2alpha (39). Conversely, co-expression of a PKR inhibitor, such as K3L, leads to rescue of PKR-mediated growth inhibition (29). To test the inhibitory activity of STAT1 on PKR, strains J110 and H2544 were transformed with vector alone, K3L, HA-STAT1 1-413 WT, or HA-STAT1 1-413 TM. Transformants were streaked onto minimal media 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, upper plate, and Fig. 6B, upper graph). H2544 transformants containing only empty plasmid DNA demonstrated a slow-growth phenotype after PKR induction (Fig. 6A, lower plate, labeled C). However, expression of K3L reversed this growth inhibitory phenotype (lower plate, labeled K3L). Likewise, expression of STAT1 1-413 WT also rescued yeast growth (lower plate, labeled WT), although in contrast, the interaction mutant of STAT1 was unable to counteract the growth inhibitory effects of PKR (lower plate, labeled TM). Growth curves to assess the degree of rescue showed that the ability of HA-STAT1 1-413 WT transformants to rescue growth was half as potent relative to K3L (Fig. 6B, lower graph). Because it was not possible to quantify the relative levels of K3L and STAT1 1-413 WT expression, the degree of rescue may be dependent on their different levels of expression. Efforts to rescue growth by full-length HA-STAT1alpha were unsuccessful, because we could not detect HA-STAT1alpha expression in yeast (data not shown). However, the truncated HA-STAT1 1-413 proteins were readily detectable in yeast protein extracts (Fig. 6C) as was the expression of human PKR in H2544 transformants (Fig. 6D, lanes 5-8). In correlation with the growth curves, the extent of eIF-2alpha phosphorylation, as assessed by isoelectric focusing experiments (29), was diminished in H2544 transformants expressing either K3L or HA-STAT1 1-413 WT (Fig. 6E, lanes 3 and 4). The induction of PKR levels in Fig. 6D, lane 6, was probably translational in nature as a result of inhibition of eIF-2alpha phosphorylation and up-regulation of PKR protein synthesis by K3L (Fig. 6E, lane 3). This PKR up-regulation was not evident in lane 7 most likely due to the weaker inhibitory effect of STAT1 1-413 WT on eIF-2alpha phosphorylation compared with K3L (Fig. 6E, compare lanes 3 and 4).



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Fig. 6.   STAT1 inhibits PKR activity in yeast. A, strains J110 (bottom half of both plates) and H2544 (top half of both plates) were transformed with control vector (labeled C), K3L (labeled K3L), HA-STAT1 1-413 WT (labeled WT), or HA-STAT1 1-413 TM (labeled TM). Transformants were streaked on control 10% glucose (upper plate) or 10% galactose agar plates and monitored for slow growth phenotype. B, transformants were grown in galactose medium, and relative growth rates were monitored at the indicated times by trypan blue cell counting. The upper graph represents the growth curves of control J110 transformants whereas the bottom graph shows the growth curves of H2544 transformants. C, J110 and H2544 were grown in galactose medium to induce HA-STAT1 1-413 WT and TM expression. 2 mg of protein extracts were immunoprecipitated with HA antibodies and immunoblotted with HA antibodies. D, J110 and H2544 were grown in galactose medium to induce PKR expression. Total protein extracts were subjected to immunoblot analysis with rabbit antisera to human PKR. The upper band, which is present in J110 extracts lacking PKR, is nonspecific (NS). E, extracts from J110 control and H2544 strains transformed with vector alone, K3L, HA-STAT1 1-413 WT, or HA-STAT1 1-413 TM were subjected to isoelectric focusing after induction with galactose and probed with antibodies against yeast eIF-2alpha .

Increased PKR Activation and eIF-2alpha Phosphorylation in STAT1-/- Cells-- Next we investigated whether the loss of STAT1 would augment PKR activity. To do so, protein extracts from untreated or IFN-treated STAT1+/+ and STAT1-/- cells were used to assess PKR activity in vitro. We noticed that the basal activity of PKR was ~5-fold higher in STAT1-/- cells compared with STAT1+/+ cells (Fig. 7A, upper panel, compare lanes 1 and 3), although PKR protein levels were equivalent, as assessed by in vivo [35S]methionine labeling (lower panel, compare lanes 1 and 2). The increase in PKR activity after IFN treatment in STAT1+/+ cells (top panel) reflects increased PKR expression, whereas no such increase in PKR activity/protein was observed in STAT1-/- cells. This is consistent with the notion that transcriptional up-regulation of PKR after IFN treatment is dependent on the JAK/STAT pathway (upper panel, compare lanes 2 and 4). To further substantiate our findings that PKR activity is elevated in STAT1-/- cells, we compared the levels of eIF-2alpha phosphorylation in STAT1+/+ and STAT1-/- cells in vivo. The effect of STAT1 on eIF-2alpha phosphorylation in STAT1+/+ and STAT1-/- cells was examined by treatment with dsRNA and immunoblotting with a phosphospecific antibody to phosphoserine 51 of eIF-2alpha (Fig. 7B, top panel). These experiments showed that a higher amount of eIF-2alpha was phosphorylated in STAT1-/- cells compared with STAT1+/+ cells (top panel, compare lanes 1 and 3) and that this phosphorylation was more highly induced after dsRNA treatment (compare lanes 2 and 4). Taken together, the inhibition of PKR activity by STAT1 in mammalian and yeast cells supports our findings that STAT1 can inhibit PKR activity in vitro and in vivo.



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Fig. 7.   PKR activity and eIF-2alpha phosphorylation are elevated in STAT1-/- cells. A, STAT1+/+ (top panel) and STAT1-/- MEFs were treated with IFN-alpha /beta overnight or left untreated. Protein extracts were immunoprecipitated against mouse PKR, and in vitro kinase assays were performed in the presence of activator reovirus dsRNA and [gamma -32P]ATP. To monitor PKR protein levels, [35S]methionine in vivo labeling was performed (bottom panel) in STAT1+/+, STAT1-/-, PKR+/+, and PKR-/- MEFs. Mouse PKR was immunoprecipitated with a rabbit anti-mouse PKR polyclonal antibody, subjected to SDS-PAGE, and visualized by fluorography. B, STAT1+/+ or STAT1-/- cells were left untreated or transfected with dsRNA (i.e. poly(rI·rC), 100 µg/ml), and protein extracts were subjected to SDS-PAGE analysis. Immunoblotting was performed with antibodies against phosphoserine 51 of eIF-2alpha (top panel) or eIF-2alpha (bottom panel).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have mapped the sites of interaction between PKR and STAT1 in vitro using GST pull-down assays and also in vivo. We found that STAT1 interacts with PKR on amino acids 367-415, an area located within the large lobe of the kinase domain of PKR that is also bound by the vaccinia virus encoded PKR inhibitor, K3L (36, 37). Sequence comparison with other eIF-2alpha family members, GCN2, HRI, and the newly discovered PERK/PEK revealed that this part of the kinase domain is highly conserved and suggests that these members might be capable of interacting with STAT1. Our previous observations that the dsRNA-binding mutant PKRLS4 was unable to associate with STAT1 in vivo (6), at first, appear to be challenged by our in vitro studies herein, which show that the carboxyl terminus of PKR is required for binding to STAT1 and that GST-PKRK296RLS4 can interact with STAT1 (Fig. 1). One possible explanation for this difference may be inferred, based on structural data of the amino terminus of PKR (41) and also on previous mutational studies (33), that the LS4 and LS9 mutations could introduce conformational changes that would affect its interaction with STAT1 in vivo. Such conformational changes may not take place when GST-PKRK296RLS4 and GST-PKRK296RLS9 are purified from bacteria. Another conceivable explanation is that the association of PKR with STAT1 in vivo is facilitated by the presence of other protein(s) whose action is modulated by the amino terminus of PKR and may become limited when the interaction is tested in vitro.

In the case of STAT1, amino acids 343-348 provide a major site of interaction with PKR. This region of STAT1 corresponds to beta -sheet 3, a structure located on the back side of the DNA-binding domain of STAT1 (33, 34). Mutation of three amino acids within this region (STAT1 TM; Arg346-Leu347-Leu348 to Ala346-Asp347-Asp348) abolished binding to PKR in vitro and in vivo. Paradoxically, sequence alignment of this three-residue stretch of wild-type STAT1 with other STAT members showed almost complete conservation. Similarly, residues in beta -sheet 3 of the DNA-binding domain of STAT3 responsible for specific interaction with c-Jun are also nearly identical between STAT members; however, c-Jun specifically interacts with STAT3 (42). Thus, our observation that PKR specifically interacts with STAT1 can probably only be explained in the context of the presentation of beta -sheet 3 in STAT1 compared with other STAT molecules. In the end, structural analysis might be necessary to determine the exact points of contact between PKR and STAT1.

In light of our previous observations that the ability of PKR to interact with STAT1 can block STAT1 DNA binding (6), we examined whether release of STAT1 from this inhibitory mechanism might augment its transcriptional activity. In contrast to STAT1 WT, we were unable to observe tyrosine phosphorylation of STAT1 TM following IFN stimulation, and as a result, ISGF3 and STAT1 homodimer formation could not be detected in DNA binding experiments. We believe that the inability of STAT1 TM to be phosphorylated cannot be the result of a misfolded protein, because this mutant is phosphorylated on serine 727 after IFN treatment. Rather, it appears that some undefined negative regulatory mechanism is responsible for this phenomenon. The protein kinase that directly phosphorylates serine 727 of STAT1 in vivo is not as yet known (4). It is unlikely to be PKR because STAT1 is not phosphorylated by PKR in vitro (6). Interestingly, a recent report shows a defective serine 727 phosphorylation of STAT1 in PKR-/- fibroblasts after IFN stimulation providing evidence for an indirect role of the kinase in this process (43). Although unable to bind DNA as a homodimer, STAT1 TM was competent and more capable in forming DNA-binding complexes with STAT3 relative to STAT1 WT cells. Interestingly, tyrosine phosphorylation of STAT3 was elevated by 50% before and after IFN treatment in polyclonal cell populations expressing STAT1 TM. The mechanism behind this finding is not clear at this time, but it might be possible that recruitment of STAT3 to the JAK·receptor complex is more efficient in STAT1 TM cells compared with STAT1 WT cells. Nevertheless, the net effect of increased STAT1·STAT3 heterodimer and STAT3 homodimer formation probably contributes to up-regulation of STAT1·STAT3 DNA-binding and GAS-dependent transactivation. Because STAT3 homodimers appear to play a minimal role in the transactivation of IFN-dependent genes by IFN-gamma (44), it is likely that the transcriptional activity observed in our reporter assays is contributed by the STAT1·STAT3 heterodimer. This is the second instance where a transcriptional role of STAT1 has been shown to require serine 727 but not tyrosine 701 phosphorylation. An earlier study demonstrated that mutation of serine 727 to alanine, but not tyrosine 701 to phenylalanine, on STAT1 significantly ablated the TNF-alpha -dependent induction of caspase genes (45). It was speculated that STAT1 could potentially participate in signaling pathways independent of its tyrosine phosphorylation state (46); such a hypothesis is supported by our findings in here.

The antiproliferative and antiviral effects of IFNs were also investigated, because a number of cell cycle regulatory proteins are regulated at the transcriptional level through the JAK/STAT signaling pathway (44). IFN-dependent cell cycle arrest, as measured by FACS analysis, was significantly higher in cells expressing STAT1alpha TM versus control cells or cells expressing STAT1alpha WT. In addition, the ability of STAT1 TM cells to resist VSV infection was ~10-fold greater than control or STAT1 WT cells following IFN-gamma stimulation. These differences become more significant when the variation in the expression levels between STAT1 WT and TM is considered (i.e. 5-fold higher expression of WT than STAT1 TM; Fig. 3B, lanes 2 and 3; Fig. 4B, bottom panel). Interestingly, even untreated STAT1 TM cells provided greater protection relative to control and STAT1 WT cells, suggesting that the anti-viral responses available within STAT1 TM cells are already enhanced prior to IFN pretreatment.

Given the fact that STAT1 and K3L, a pseudosubstrate inhibitor of PKR, can compete for binding with PKR, we next analyzed whether STAT1 could inhibit PKR activation. We observed that recombinant STAT1 inhibited PKR activation in vitro in a dose-dependent manner. However, it is unlikely that STAT1 is a substrate or pseudosubstrate for PKR, because sequence alignment of STAT1 with K3L and eIF-2alpha did not reveal any significant similarities, findings that coincide with our earlier observations that PKR does not phosphorylate STAT1 in vitro or in vivo (6). Instead, the interaction of the two proteins might prevent opening of the cleft that separates the two lobes of PKR's kinase domain, thus inhibiting both PKR activation and eIF-2alpha phosphorylation.

The capacity of STAT1 to neutralize PKR activity is further appreciated from in vivo studies. PKR exhibits the same substrate specificity as the yeast GCN2 kinase that regulates protein synthesis by phosphorylating eIF-2alpha to inhibit cell growth and replacement of GCN2 with PKR leads to inhibition of translation and yeast growth (31). As such, this system has been consistently used as a means to probe for PKR activation, as well as for characterizing inhibitors of PKR, like K3L (29). The observation that HA-STAT1 1-413 WT, but not the interaction mutant, TM, was able to block PKR activity and subsequently relieve the growth inhibitory effects of PKR, supports our model whereby PKR activity is tightly regulated by its interaction with STAT1. These results from yeast were further substantiated in STAT1-/- cells where we observed augmentation of both PKR activity and in vivo eIF-2alpha phosphorylation in comparison to wild-type cells.

We rationalize that the interaction of PKR and STAT1 modulates cellular proliferation in normally growing cells and in cells challenged by viruses. In unstimulated cells, the balance of PKR·STAT1 heterodimer formation versus their free monomers may dictate cellular proliferative capacity. For example, perturbing this equilibrium by the expression of catalytic mutants of PKR would sequester a larger fraction of STAT1 (6) and could contribute to increased virus susceptibility and to the transformed phenotype of cells expressing such mutants of PKR (9, 10). Conversely, loss of PKR's repressive activity in PKR-/- mice would favor the antiviral and antiproliferative functions of STAT1. As such, PKR-/- mice are still capable of providing host resistance to many viruses (14, 17). It would thus appear that STAT1 provides a first line of defense against virus infection, whereas the role of PKR in viral resistance appears to be secondary. This is illustrated by the observation that STAT1-/- mice are exquisitely sensitive to a variety of pathogens (7, 8), whereas PKR-/- mice exhibit a modest sensitivity (14, 17). Increased eIF-2alpha phosphorylation in STAT1-/- cells could provide a compensatory mechanism for the loss of STAT1 and may play a role in differences in the susceptibility of STAT1-/- mice to virus infection (8, 24). Therefore, eIF-2alpha phosphorylation may play a role in the differential sensitivity of various viruses to interferon treatment, an effect that could be mediated through the interaction of STAT1 with PKR and other eIF-2alpha kinases, because they share high sequence homology in the STAT1 binding region. Generation of mice lacking multiple members of this family of kinases2 may be useful in determining such virus-specific responses.


    ACKNOWLEDGEMENTS

We thank T. Pannunzio and N. Tam for assistance in some of the experiments; C. Schindler for STAT1alpha and STAT2 cDNAs; M. Mathews for PKRLS4 and PKRLS9 cDNAs; A. Darveau, G. N. Barber, and J. C. Bell for PKR antibodies; M. Clemens for eIF-2alpha antibodies; C. Weissmann for PKR+/+ and PKR-/- cells; G. Stark for U3A cells; Y. Yoneda for the pGEX-5X-3-HA-STAT1alpha ; R. Schreiber for purified STAT1alpha protein; N. Sonenberg for pGEX2TK-FLAG-K3L; J. E. Darnell, Jr. for pRC/CMV STAT3; A. Veillette for pBABE/puro; N. Beauchemin for the psi 2 retroviral packaging cell line; and K. McDonald for assistance on flow cytometry. We thank J. J. M. Bergeron and members of our laboratory and of the Molecular Oncology Group for helpful discussions.


    FOOTNOTES

* This work was supported in part by research grants from the Canadian Institutes of Health Research and the Human Frontier Science Program (to A. E. K.).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.

§ Recipient of a National Cancer Institute of Canada, Terry Fox studentship. Present address: The Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, NY 10016.

|| Recipient of a post-doctoral award from the Cancer Research Society of Canada.

§§ A member of the Terry Fox Group in Molecular Oncology and recipient of a Canadian Institutes of Health Research Scientist Award. To whom correspondence should be addressed: Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote-Ste-Catherine Rd., Montreal, Quebec H3T 1E2, Canada. Tel.: 514-340-8260 (ext. 3697); Fax: 514-340-7576; E-mail: akoromil@ ldi.jgh.mcgill.ca.

Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M011240200

2 J. C. Bell and D. Ron, personal communication.


    ABBREVIATIONS

The abbreviations used are: JAK, Janus kinases; STAT, signal transducer and activator of transcription; SH2, Src homology 2; IFN, interferon; dsRNA, double-stranded RNA; PKR, interferon-induced dsRNA-activated protein kinase; HRI, heme-regulated inhibitor kinase; PERK, PKR-like endoplasmic reticulum kinase; PEK, pancreatic eIF-2alpha kinase; GCN2, general control non-derepressible-2; ISGF3, interferon-stimulated gene factor 3; HA, hemagglutinin; GST, glutathione S-transferase; GAS, gamma -interferon-activating sequence; VSV, vesicular stomatitis virus; CPE, cytopathic effect; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; TM, triple-mutant.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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