Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase.

The double-stranded RNA-dependent protein kinase (dsRNA-PK) is thought to be a key mediator of the antiviral and antiproliferative effects of interferons (IFNs). Studies examining the physiological function of the kinase suggest that it participates in cell growth and differentiation by regulating protein synthesis. Autophosphorylation and consequent activation of dsRNA-PK in vitro and in vivo result in phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2) and inhibition of protein synthesis. Expression of a functionally defective mutant of human dsRNA-PK in NIH 3T3 cells resulted in malignant transformation, suggesting that dsRNA-PK may function as a suppressor of cell proliferation and tumorigenesis.

Double-stranded-RNA-activated protein kinase PKR enhances transcriptional activation by tumor suppressor p53.

The tumor suppressor p53 plays a key role in inducing G1 arrest and apoptosis following DNA damage. The double-stranded-RNA-activated protein PKR is a serine/threonine interferon (IFN)-inducible kinase which plays an important role in regulation of gene expression at both transcriptional and translational levels. Since a cross talk between IFN-inducible proteins and p53 had already been established, we investigated whether and how p53 function was modulated by PKR. We analyzed p53 function in several cell lines derived from PKR+/+ and PKR-/- mouse embryonic fibroblasts (MEFs) after transfection with the temperature-sensitive (ts) mutant of mouse p53 [p53(Val135)]. Here we report that transactivation of transcription by p53 and G0/G1 arrest were impaired in PKR-/- cells upon conditions that ts p53 acquired a wild-type conformation. Phosphorylation of mouse p53 on Ser18 was defective in PKR-/- cells, consistent with an impaired transcriptional induction of the p53-inducible genes encoding p21(WAF/Cip1) and Mdm2. In addition, Ser18 phosphorylation and transcriptional activation by mouse p53 were diminished in PKR-/- cells after DNA damage induced by the anticancer drug adriamycin or gamma radiation but not by UV radiation. Furthermore, the specific phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 inhibited the induction of phosphorylation of Ser18 of p53 by adriamycin to a higher degree in PKR+/+ cells than in PKR-/- cells. These novel findings suggest that PKR enhances p53 transcriptional function and implicate PKR in cell signaling elicited by a specific type of DNA damage that leads to p53 phosphorylation, possibly through a PI-3 kinase pathway.

Control of interferon signaling in human papillomavirus infection.

Human papillomaviruses (HPV) infect mucosal and cutaneous epithelium resulting in several types of pathologies, most notably, cervical cancer. Persistent infection with sexually transmitted oncogenic HPV types represents the major risk factor for the development of cervical cancer. The development of HPV-associated cervical cancer has been closely linked to the expression of the viral oncogenes E6 and E7 in the tumor cells. The major viral oncoproteins, E6 and E7, target the cellular tumor suppressor gene products p53 and Rb, respectively. As detailed within, these interactions result in the stimulation of proliferation and the inhibition of apoptosis, thus representing major oncogenic insults to the infected cell. In addition to mediating transformation, the E6 and E7 genes also play significant roles in altering the immune response against infected cells by suppressing interferon (IFN) expression and signaling. At the clinical level, IFNs have been used in the treatment of HPV-associated cervical intraepithelial neoplasia (CIN) or cervical cancers with mixed results. The success of the treatment is largely dependent on the subtype of HPV and the immune response of the patients. Despite this inefficiency, the increasing knowledge about the regulation of IFN signaling pathways at molecular level may hold a promise for the use of new therapeutic strategies against HPV infection. Studies on the regulation of the function of IFN-inducible gene products by the E6 and E7 may lead to the development of new therapeutic approaches based on strategies that modify the function of the HPV oncoproteins and restore IFN-signaling pathways through endogenous control mechanisms.

Identification of the interferon-inducible double-stranded RNA-dependent protein kinase as a regulator of cellular response to bulky adducts.

The double-stranded RNA-dependent protein kinase PKR plays a central role in IFN-mediated antiviral response. The ability of PKR mutants to transform rodent fibroblasts led to the hypothesis that PKR acts as a tumor suppressor. Recent studies have identified an expanding network of PKR signaling partners, including signal transducers and activators of transcription 1 (STAT1), p53, and IkappaB-kinase. Here we demonstrate that PKR is involved in the cellular response to genotoxic stress. PKR-deficient mouse-embryonic fibroblasts (PKR-/-) are hypersensitive to bulky adduct DNA damage caused by cisplatin, melphalan, and UV radiation but not to other DNA-damaging agents such as Adriamycin. PKR-deficient cells are highly susceptible to cisplatin-induced apoptosis. They demonstrate retarded cisplatin adduct removal kinetics. Most strikingly, PKR localizes to the nucleus rapidly upon cisplatin treatment. Restoration of PKR in PKR-/- cells results in resistance to cisplatin and enhanced cell capacity to remove cisplatin DNA adducts. We conclude that PKR has a function in the regulation of cellular response to bulky adduct-inducing agents, possibly by modulating DNA repair mechanisms.

Activation of the I kappa B alpha kinase (IKK) complex by double-stranded RNA-binding defective and catalytic inactive mutants of the interferon-inducible protein kinase PKR.

The interferon (IFN)-inducible double stranded (ds) RNA-activated protein kinase PKR plays an important role in protein synthesis by modulating the phosphorylation of the alpha-subunit of eukaryotic initiation fact 2 (eIF-2 alpha). In addition to translational control, PKR has been implicated in several signaling pathways leading to gene transcription. For example, PKR induces I kappa B alpha kinase (IKK) activity and I kappa B alpha phosphorylation leading to the induction of NF-kappa B-mediated gene transcription. Recent findings suggested that NF-kappa B activation by PKR does not require the catalytic activity of the kinase. Here, we provide novel evidence that induction of IKK and NF-kappa B activities proceeds independently of the dsRNA-binding properties of PKR and also verify the kinase-free role of PKR in this process. We also show that the effects of PKR mutants on IKK and NF-kappa B activation are independent of cell transformation but are dependent on the amount of the mutant PKR proteins expressed in cells. These data strongly support an indirect role of PKR in I kappa B alpha phosphorylation by modulating IKK activity through pathways that do not utilize the enzymatic and dsRNA-binding properties of PKR.

IRF-1 induced cell growth inhibition and interferon induction requires the activity of the protein kinase PKR.

Expression of the tumor suppressor IRF-1 results in the inhibition of cell growth and transcriptional activation of the IFN-beta gene. IFN production is not responsible for the IRF-1 mediated cell growth inhibition. It is shown here that activation of the IRF-1 causes induction of PKR expression. PKR is a serine/threonine kinase with tumor suppressor activity. IRF-1 mediated cell growth inhibition and IFN induction correlates with PKR expression. A catalytically inactive dominant negative PKR mutant abolishes both activities of IRF-1. These data demonstrate that the tumor suppressor activity of IRF-1 is mediated, at least in part, by PKR.

Disruption of I kappa B alpha regulation by antisense RNA expression leads to malignant transformation.

NF-kappa B transcription factors regulate the expression of a variety of genes involved in immune regulation and cell growth. In most cell types NF-kappa B proteins are localized in an inactive form in the cytoplasm coupled to the inhibitory I kappa B proteins. Viruses, cytokines, lipopolysaccharides and other stimulating agents promote the dissociation of the cytosolic NF-kappa B/I kappa B complexes, via phosphorylation and degradation of I kappa B, resulting in the translocation of DNA binding, NF-kappa B complexes to the nucleus. To further understand the association of I kappa B with cell growth regulation, the effect of ectopic expression of sense and antisense I kappa B genes was examined in NIH3T3 cells. Overexpression of I kappa B alpha antisense RNA but not I kappa B gamma antisense RNA decreased the steady state levels of I kappa B alpha protein, altered NF-kappa B DNA binding and gene activity and, most importantly, induced malignant transformation as measured by saturation density, growth in soft agar and tumorigenicity in nude mice. In contrast, overexpression of I kappa B alpha resulted in decreased saturation density, a flattened cellular morphology and decreased NF-kappa B dependent reporter gene activity. These results indicate that overexpression of an I kappa B alpha antisense RNA may disrupt the NF-kappa B/I kappa B autoregulatory loop, leading to cellular transformation. Our results raise the interesting possibility that I kappa B alpha represents a potential tumor suppressor activity.

The double-stranded RNA activated protein kinase PKR physically associates with the tumor suppressor p53 protein and phosphorylates human p53 on serine 392 in vitro.

The tumor suppressor p53 is a multifunctional protein that plays a critical role in modulating cellular responses upon DNA damage or other stresses. These functions of p53 are regulated both by protein-protein interactions and phosphorylation. The double-stranded RNA activated protein kinase PKR is a serine/threonine kinase that modulates protein synthesis through the phosphorylation of translation initiation factor eIF-2alpha. PKR is an interferon (IFN)-inducible protein that is thought to mediate the anti-viral and anti-proliferative effects of IFN via its capacity to inhibit protein synthesis. Here we report that PKR physically associates with p53. The interaction of PKR with p53 is enhanced by IFNs and upon conditions that p53 acquires a wild type conformation. PKR/p53 complex formation in vitro requires the N-terminal regulatory domain of PKR and the last 30 amino acids of the C-terminus of human p53. In addition, p53 may function as a substrate of PKR since phosphorylation of human p53 on serine392 is induced by activated PKR in vitro. These novel findings raise the possibility of a functional interaction between PKR and p53 in vivo, which may account, at least in part, for the ability of each protein to regulate gene expression at both the transcriptional and the translational levels.

The human papilloma virus (HPV)-18 E6 oncoprotein physically associates with Tyk2 and impairs Jak-STAT activation by interferon-alpha.

We have examined the effects of human papilloma virus (HPV) E6 proteins on interferon (IFN) signaling. Here we show that expression of the 'malignant' HPV-18 E6 in human HT1080 cells results in inhibition of Jak-STAT activation in response to IFN-alpha but not IFN-gamma. This inhibitory effect is not shared by the 'benign' HPV-11 E6. The DNA-binding and transactivation capacities of the transcription factor ISGF3 are diminished in cells expressing HPV-18 E6 after IFN-alpha treatment as a result of decreased tyrosine phosphorylation of Tyk2, STAT2 and STAT1. However, HPV-18 E6 does not affect the induction of tyrosine phosphorylation and DNA-binding of STAT1 by IFN-gamma. In addition, HPV E6 proteins physically interact with Tyk2. This interaction takes place preferably with HPV-18 E6 and to a lesser extent with HPV-11 E6. The E6/Tyk2 interaction requires the JH6-JH7 domains of Tyk2, which are important for Tyk2 binding to the cytoplasmic portion of IFN-alpha receptor 1 (IFNAR1). These findings demonstrate an inhibitory role of HPV-18 E6 in the IFN-alpha-induced Jak-STAT pathway, which may be explained, at least in part, by the ability of E6 to interact with and impair Tyk2 activation.

Phosphorylation of the translation initiation factor eIF2α at serine 51 determines the cell fate decisions of Akt in response to oxidative stress.

Phosphorylation of the α subunit of the translation initiation factor eIF2 at serine 51 (eIF2αP) is a master regulator of cell adaptation to various forms of stress with implications in antitumor treatments with chemotherapeutic drugs. Herein, we demonstrate that genetic loss of the eIF2α kinases PERK and GCN2 or impaired eIF2αP by genetic means renders immortalized mouse fibroblasts as well as human tumor cells increasingly susceptible to death by oxidative stress. We also show that eIF2αP facilitates Akt activation in cells subjected to oxidative insults. However, whereas Akt activation has a pro-survival role in eIF2αP-proficient cells, the lesser amount of activated Akt in eIF2αP-deficient cells promotes death. At the molecular level, we demonstrate that eIF2αP acts through an ATF4-independent mechanism to control Akt activity via the regulation of mTORC1. Specifically, eIF2αP downregulates mTORC1 activity, which in turn relieves the feedback inhibition of PI3K resulting in the upregulation of the mTORC2-Akt arm. Inhibition of mTORC1 by rapamycin restores Akt activity in eIF2αP-deficient cells but renders them highly susceptible to Akt-mediated death by oxidative stress. Our data demonstrate that eIF2αP acts as a molecular switch that dictates either cell survival or death by activated Akt in response to oxidative stress. Hence, we propose that inactivation of eIF2αP may be a suitable approach to unleash the killing power of Akt in tumor cells treated with pro-oxidant drugs.

Induction of PG G/H synthase-2 in bovine myometrial cells by interferon-tau requires the activation of the p38 MAPK pathway.

PGs are regulators of a plethora of uterine functions during reproductive processes, including uterine contractility. In bovine uterus, the rate-limiting step in PG synthesis is catalyzed by the PG endoperoxide G/H synthase (PGHS) enzymes. It has previously been established that PGHS-2 isoform expression is affected by the ruminant-specific interferon (IFN)-tau in bovine endometrial cells. Here, we show that PGHS-2 mRNA and protein levels are induced by IFN-tau in primary cell cultures from bovine myometrium. Treatment with recombinant bovine IFN-tau induces the activation of the JAK-STAT and p38 MAPK pathways in bovine myometrial cells. Inhibition of the p38 pathway by the specific inhibitor SB203580 strongly decreases PGHS-2 mRNA and protein expression without affecting the phosphorylation and DNA-binding of transcription factors STAT-1 and STAT-2. The p38 pathway regulates PGHS-2 expression at the posttranscriptional level, because the presence of SB203580 results in the destabilization of IFN-tau-induced PGHS-2 mRNA. Taken together, these data demonstrate the ability of IFN-tau to induce the activation of the JAK-STAT pathway in a manner similar to other types of IFN (i.e. alpha, beta, and gamma) and to regulate PGHS-2 mRNA stability through the activation of the p38 pathway. These findings provide new insights into the physiological function of IFN-tau, in regard to regulation of specific genes associated with myometrial contractility.

PKR protection against intranasal vesicular stomatitis virus infection is mouse strain dependent.

The interferon-induced antiviral state is mediated by interferon-stimulated genes that are upregulated in concert after stimulation by type I interferons. Because so many viruses encode strategies to inactivate the interferon-inducible double-stranded RNA (dsRNA)-dependent protein kinase PKR, this protein is likely to be a major player in antiviral defense. Here we demonstrate the increased susceptibility of PKR-/- animals to vesicular stomatitis virus (VSV) by the intranasal route, but also demonstrate that the protective effects of PKR are mouse strain dependent. We have found the difference between wild-type-BALB/c and 129SvEv animals to be on the order of 5 logs, with high levels of virus present in the lungs of BALB/c but not 129SvEv animals. To evaluate the sensitivity of PKR-/- mice to VSV clearly, the PKR mutation was bred onto the resistant 129SvEv background. The increased sensitivity of PKR-/- mice, compared to PKR+/+ strain-matched controls, is on the order of 10-fold as measured by median lethal dose (LD50). PKR-/- 129 mice support VSV replication in the lung unlike controls. While this result clearly demonstrates an important role for PKR in protection against VSV infection of the lung, it also underlines the importance of other host factors in containing a viral infection.

Doxorubicin bypasses the cytoprotective effects of eIF2α phosphorylation and promotes PKR-mediated cell death.

The eukaryotic cell responds to various forms of environmental stress by adjusting the rates of mRNA translation thus facilitating adaptation to the assaulting stress. One of the major pathways that control protein synthesis involves the phosphorylation of the α-subunit of eukaryotic initiation factor eIF2 at serine 51. Different forms of DNA damage were shown to induce eIF2α phosphorylation by using PERK, GCN2 or PKR. However, the specificity of the eIF2α kinases and the biological role of eIF2α phosphorylation pathway in the DNA damage response (DDR) induced by chemotherapeutics are not known. Herein, we show that PKR is the eIF2α kinase that responds to DDR induced by doxorubicin. We show that activation of PKR integrates two signaling pathways with opposing biological outcomes. More specifically, induction of eIF2α phosphorylation has a cytoprotective role, whereas activation of c-jun N-terminal kinase (JNK) by PKR promotes cell death in response to doxorubicin. We further show that the proapoptotic effects of JNK activation prevail over the cytoprotection mediated by eIF2α phosphorylation. These findings reveal that PKR can be an important inducer of cell death in response to chemotherapies through its ability to act independently of eIF2α phosphorylation.

Dominant negative function by an alternatively spliced form of the interferon-inducible protein kinase PKR.

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 (PKRDeltaE7), resulting in a truncated protein that retains the two dsRNA-binding motifs. PKRDeltaE7 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 PKRDeltaE7 is expressed in a broad range of human tissues at variable levels. Interestingly, expression of PKRDeltaE7 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.

mRNAs containing extensive secondary structure in their 5' non-coding region translate efficiently in cells overexpressing initiation factor eIF-4E.

Cellular eukaryotic mRNAs (except organellar) contain at the 5' terminus the structure m7(5')Gppp(5')N (where N is any nucleotide), termed cap. Cap recognition by eukaryotic initiation factor eIF-4F plays an important role in regulating the overall rate of translation. eIF-4F is believed to mediate the melting of mRNA 5' end secondary structure and facilitate 43S ribosome binding to capped mRNAs. eIF-4E, the cap-binding subunit of eIF-4F, plays an important role in cell growth; its overexpression results in malignant transformation of rodent cells, and its phosphorylation is implicated in signal transduction pathways of mitogens and growth factors. The molecular mechanism by which eIF-4E transforms cells is not known. Here, we report that overexpression of eIF-4E facilitates the translation of mRNAs containing excessive secondary structure in their 5' non-coding region. This effect may represent one mechanism by which eIF-4E regulates cell growth and transforms cells in culture.

A diminished activation capacity of the interferon-inducible protein kinase PKR in human T lymphocytes.

The double-stranded (ds) RNA activated protein kinase PKR is an interferon (IFN)-inducible serine/threonine protein that regulates protein synthesis through the phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2alpha). PKR activation in cells is induced by virus infection or treatment with dsRNA and is modulated by a number of viral and cellular factors. To better understand the mechanisms of PKR action we have analyzed and compared the mode of PKR activation in a number of cell lines of different histological origin. Here we show that PKR activation and phosphorylation of eIF-2alpha are both diminished in various virus-transformed and nontransformed human T cells. Priming of T cells with IFN does not restore PKR activation. In vitro kinase assays show that the diminished PKR activation in T cells correlates with the presence of a 60-kDa (p60) phosphoprotein coimmunoprecipitated with PKR. P60 is absent from PKR immunoprecipitates from non T cells. Incubation of active PKR with T cell extracts results in inhibition of PKR autophosphorylation, which is proportional to the amount of phosphorylated p60 in the kinase reactions. Treatment of T cells with proteasome inhibitors or incubation of PKR immunoprecipitates with phosphatase inhibitors does not restore PKR activation. However, phosphorylation of p60 is enhanced upon treatment with the phosphatase inhibitor microcystin. These data show that the impaired activation capacity of PKR in human T cells is exerted at the post-translational levels in a manner that is independent of cell transformation or virus infection.

The interferon-inducible protein kinase PKR modulates the transcriptional activation of immunoglobulin kappa gene.

PKR is an interferon (IFN)-induced serine/threonine protein kinase that regulates protein synthesis through phosphorylation of eukaryotic translation initiation factor-2 (eIF-2). In addition to its demonstrated role in translational control, recent findings suggest that PKR plays an important role in regulation of gene transcription, as PKR phosphorylates I kappa B alpha upon double-stranded RNA treatment resulting in activation of NF-kappa B DNA binding in vitro (Kumar, A., Haque, J., Lacoste, J., Hiscott, J., and Williams, B.R.G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6288-6292). To further investigate the role of PKR in transcriptional signaling, we expressed the wild type human PKR and a catalytically inactive dominant negative PKR mutant in the murine pre-B lymphoma 70Z/3 cells. Here, we report that expression of wild type PKR had no effect on kappa-chain transcriptional activation induced by lipopolysaccharide or IFN-gamma. However, expression of the dominant negative PKR mutant inhibited kappa gene transcription independently of NF-kappa B activation. Phosphorylation of eIF-2 alpha was not increased by lipopolysaccharide or IFN-gamma, suggesting that PKR mediates kappa gene transcriptional activation without affecting protein synthesis. Our findings further support a transcriptional role for PKR and demonstrate that there are at least two distinct PKR-mediated signal transduction pathways to the transcriptional machinery depending on cell type and stimuli, NF-kappa B-dependent and NF-kappa B-independent.

Abrogation of translation initiation factor eIF-2 phosphorylation causes malignant transformation of NIH 3T3 cells.

The interferon induced double-stranded RNA-activated kinase, PKR, has been suggested to act as a tumor suppressor since expression of a dominant negative mutant of PKR causes malignant transformation. However, the mechanism of transformation has not been elucidated. PKR phosphorylates translation initiation factor eIF-2 alpha on Ser51, resulting in inhibition of protein synthesis and cell growth arrest. Consequently, it is possible that cell transformation by dominant negative PKR mutants is caused by inhibition of eIF-2 alpha phosphorylation. Here, we demonstrate that in NIH 3T3 cells transformed by the dominant negative PKR mutant (PKR delta 6), eIF-2 alpha phosphorylation is dramatically reduced. Furthermore, expression of a mutant form of eIF-2 alpha, which cannot be phosphorylated on Ser51 also caused malignant transformation of NIH 3T3 cells. These results are consistent with a critical role of phosphorylation of eIF-2 alpha in control of cell proliferation, and indicate that dominant negative PKR mutants transform cells by inhibition of eIF-2 alpha phosphorylation.

Genomic organization of mouse J kappa recombination signal binding protein (RBP-J kappa) gene.

We have isolated a cDNA clone (RBP-2) for the protein (RBP-J kappa) which binds to immunoglobulin recombination signals with 23-base pair spacers (Matsunami, N., Hamaguchi, Y., Yamamoto, Y., Kuze, K., Kangawa, K., Matsuo, H., Kawaichi, M., and Honjo, T. (1989) Nature 342, 934-937). During further screening of a cDNA library from the same mouse pre-B cell line (38B9), we have isolated a second cDNA clone (RBP-2N) which differs from RBP-2 in its 5' sequence. RNase protection assays indicated that the RBP-2N type mRNA was produced in 10-20 times the quantity as RBP-2 mRNA. To elucidate the relationship between these two mRNAs, we analyzed the genomic organization of the RBP-J kappa gene. Southern hybridization of mouse genomic DNA detected at least 7 EcoRI fragments hybridizing to an RBP-2 cDNA probe, suggesting a complex structure for the RBP-J kappa gene. Cloning of each EcoRI fragment revealed one functional RBP-J kappa gene and three related genes. The functional gene was composed of 11 exons and spanned at least 50 kilobase pairs. The sequence of exon 1 and its 5'-flanking region contained a GC-rich promoter-like region but no apparent TATA box. The initiation site of transcription was heterogeneous, and the two types of mRNA are produced from the same exon by transcription initiation at different sites and by different usage of splice signals. Two of the three related genes were processed pseudogenes with scattered stop codons. The other was also a processed gene with a sequence exactly the same as that of RBP-2, except that this gene lacked the sequence corresponding to the first exon of the functional gene.