Enhancement of the interferon-induced double-stranded RNA-dependent protein kinase activity by Sindbis virus infection and heat-shock stress

In extracts of FL cells that were infected with Sindbis virus or treated with heat-shock stress, dsRNA-dependent phosphorylation of 77K protein was markedly increased. The 77K phosphoprotein was indistinguishable from the autophosphorylated and activated form of interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) by two-dimensional gel electrophoresis, and was immunologically related to P68 (Galabru, J. and Hovanessian, A., J. Biol. Chem. 262, 15538 (1987], the HeLa cell counterpart of PK-I. Immunoblotting experiments using monoclonal antibody against PK-I revealed that control cell extracts contained a substantial amount of PK-I protein, although they showed no measurable PK-I activity even when dsRNA was added. The amount of PK-I protein did not increase during a transient dsRNA-dependent enhancement of PK-I activity caused by Sindbis virus infection and heat-shock stress. This implies that the conversion of PK-I protein from a dsRNA-unresponsive form to a responsive form may be important in the regulation of PK-I activity. A similar mode of PK-I regulatory mechanism was operative in the early stages of IFN treatment, although after a prolonged treatment a net synthesis of the PK-I protein did take place.     

RAX, a cellular activator for double-stranded RNA-dependent protein kinase during stress signaling.

The double-stranded (ds) RNA-dependent protein kinase (PKR) regulates protein synthesis by phosphorylating the alpha subunit of eukaryotic initiation factor-2. PKR is activated by viral induced dsRNA and thought to be involved in the host antiviral defense mechanism. PKR is also activated by various nonviral stresses such as growth factor deprivation, although the mechanism is unknown. By screening a mouse cDNA expression library, we have identified an ubiquitously expressed PKR-associated protein, RAX. RAX has a high sequence homology to human PACT, which activates PKR in the absence of dsRNA. Although RAX also can directly activate PKR in vitro, overexpression of RAX does not induce PKR activation or inhibit growth of interleukin-3 (IL-3)-dependent cells in the presence of IL-3. However, IL-3 deprivation as well as diverse cell stress treatments including arsenite, thapsigargin, and H2O2, which are known to inhibit protein synthesis, induce the rapid phosphorylation of RAX followed by RAX-PKR association and activation of PKR. Therefore, cellular RAX may be a stress-activated, physiologic activator of PKR that couples transmembrane stress signals and protein synthesis.     

Inhibitor of interferon-induced double-stranded RNA-dependent protein kinase and its relevance to alteration of cellular protein kinase activity level in response to external stimuli.

In FL cells, interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) was found to be present in a form complexed with a potent inhibitor of its dsRNA-dependent activation. The inhibitor was readily dissociated from PK-I by DEAE-cellulose chromatography to yield a dsRNA-responsive PK-I. The inhibitor was also dissociated easily from PK-I by gel filtration through Sephacryl S-200. The apparent molecular mass of the inhibitor as estimated by gel filtration was more than 160 kilodaltons. Activity of the inhibitor was decreased on IFN treatment for 8.5 hr or on Sindbis virus infection with concomitant increase in the amount of dsRNA-activatable form of PK-I. This result implies that the inhibitor may be one of the regulatory factors of cellular PK-I activity. Longer IFN treatment (24 hr) led to recovery of the inhibitor activity, but it was overridden by an extensive net synthesis of the PK-I protein.     

Tobacco mosaic virus infection stimulates the phosphorylation of a plant protein associated with double-stranded RNA-dependent protein kinase activity

The influence of tobacco mosaic virus (TMV) infection on nucleotide binding and phosphorylation of an Mr 68,000 host-encoded protein (p68) was examined. The phosphorylation of p68 in homogenates from TMV-infected tissues was 4-fold greater than in homogenates from mock inoculated tissues. Phosphorylation of p68 in extracts from mock inoculated tissues was enhanced by the addition of double-stranded (ds) RNA. Nucleotide photoaffinity labeling experiments indicate that p68 contains an ATP binding site with characteristics consistent with protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase from interferon-treated human cells immunoprecipitated p68 from extracts of TMV-infected tissue, and p68-containing immunocomplexes catalyzed the phosphorylation of endogenous p68. These data suggest that p68 may be an autophosphorylating, dsRNA-dependent protein kinase involved in viral pathogenesis. Based upon analogous functions demonstrated for dsRNA-dependent protein kinases in mammalian systems, p68 may have a role in the regulation of protein synthesis and viral replication in infected cells.     

Selection of peptide inhibitors for double-stranded RNA-dependent protein kinase PKR

Protein kinase inhibitors have been developed and applied as antitumor drugs. The majority of these inhibitors are derived from ATP analogs with limited specificity towards the kinase target. Here we present our proof-of-principle study on peptide inhibitors for kinases. Two peptides were selected by phage display against double-stranded RNA-dependent protein kinase (PKR). In vitro assay revealed that these peptides exhibit an inhibitory effect on PKR-catalyzed phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). The peptides also interrupt PKR activity in cells infected by viruses, as PKR activation is one of the hallmarks of host response to viral infection. Kinetic study revealed that one of the peptides, named P1, is a competitive inhibitor for PKR, while the other, named P2, exhibits a more complicated pattern of inhibition on PKR activity. Fragment-based docking of the PKR-peptide complex suggests that P1 occupies the substrate pocket of PKR and thus inhibits the binding between PKR and eIF2α, whereas P2 sits near the substrate pocket. The computational model of PKR-peptide complex agrees with their kinetic behavior. We surmise that peptide inhibitors for kinases have higher specificity than ATP analogs, and that they provide promising leads for the optimization of kinase inhibitors.     

Growth-related expression of a double-stranded RNA-dependent protein kinase in 3T3 cells

Cultured mouse 3T3-F442A and 3T3-C2 fibroblasts exhibit a transient double-stranded RNA (dsRNA)-dependent phosphorylation of a 67,000-dalton protein (67K) without prior treatment with interferon (IFN). This phosphoprotein is similar but not identical to the dsRNA-dependent eukaryotic initiation factor-2 (eIF-2) alpha protein kinase (dsI), which regulates protein synthesis in rabbit reticulocytes. We have studied the relationship between cell growth and phosphorylation of the 67K protein (designated 3T3-dsRNA-dependent eIF-2 alpha kinase). A low level of dsRNA-dependent phosphorylation of 3T3-dsI was detectable in extracts prepared from cells not treated with IFN and grown at a low cell density. The phosphorylation of dsI and the phosphorylation of a 38K protein identified as the alpha-subunit (38K) of 3T3-eIF-2 (eIF-2 alpha) occurred concomitantly; the levels of these phosphorylations confluent and thereafter decreased markedly. Treatment of cells with IFN at all stages of growth resulted in an increase in phosphorylation of dsI. 3T3-F442A and 3T3-C2 fibroblasts were found to produce and secrete IFN at levels sufficient to induce an elevated dsI activity.     

Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase.

Double-stranded RNA-dependent protein kinase (PKR) has been implicated in interferon (IFN) induction, antiviral response and tumor suppression. We have generated mice devoid of functional PKR (Pkr%). Although the mice are physically normal and the induction of type I IFN genes by poly(I).poly(C) (pIC) and virus is unimpaired, the antiviral response induced by IFN-gamma and pIC was diminished. However, in embryo fibroblasts from Pkr knockout mice, the induction of type I IFN as well as the activation of NF-kappa B by pIC, were strongly impaired but restored by priming with IFN. Thus, PKR is not directly essential for responses to pIC, and a pIC-responsive system independent of PKR is induced by IFN. No evidence of the tumor suppressor activity of PKR was demonstrated.     

PACT, a stress-modulated cellular activator of interferon-induced double-stranded RNA-activated protein kinase, PKR

The interferon (IFN)-induced, double-stranded (ds)RNA-activated serine-threonine protein kinase, PKR, is a key mediator of the antiviral activities of IFNs. In addition, PKR activity is also involved in regulation of cell proliferation, apoptosis, and signal transduction. In virally infected cells, dsRNA has been shown to bind and activate PKR kinase function. Implication of PKR activity in normal cellular processes has invoked activators other than dsRNA because RNAs with perfectly duplexed regions of sufficient length that are able to activate PKR are absent in cellular RNAs. We have recently reported cloning of PACT, a novel protein activator of PKR. PACT heterodimerizes with PKR and activates it by direct protein-protein interaction. Overexpression of PACT in mammalian cells leads to phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha), the cellular substrate for PKR, and leads to inhibition of protein synthesis. Here, we present evidence that endogenous PACT acts as a protein activator of PKR in response to diverse stress signals such as serum starvation, and peroxide or arsenite treatment. Following exposure of cells to these stress agents, PACT is phosphorylated and associates with PKR with increased affinity. PACT-mediated activation of PKR leads to enhanced eIF2alpha phosphorylation followed by apoptosis. Based on the results presented here, we propose that PACT is a novel stress-modulated physiological activator of PKR.     

The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase

The protein kinase from human cells dependent on double-stranded (ds) RNA is a 68-kDa protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by low concentrations of dsRNA, the p68 kinase becomes phosphorylated and thereby catalyzes the phosphorylation of the protein-synthesis initiation factor, eIF2. Here, we have purified the p68 kinase to homogeneity using a specific monoclonal antibody to investigate its capacity to bind dsRNA, poly(I).poly(C). Our study suggest that p68 kinase has high- and low-affinity binding sites: the high-affinity binding site is responsible for the activation and the low-affinity binding site for the inhibition of kinase activity. This is in accord with the fact that autophosphorylation of p68 kinase occurs at low concentrations of dsRNA whereas high concentrations of dsRNA inhibit its autophosphorylation. We have also investigated the binding of adenoviral VAI RNA to the purified p68 kinase and have found that the affinity of this binding is lower than that of poly(I).poly(C). We show that VAI RNA can activate or inhibit autophosphorylation of p68 kinase in a dose-dependent manner, i.e. activation at less than or equal to 1 microgram/ml or inhibition at greater than 1 microgram/ml of VAI RNA. In spite of its lower affinity of binding, VAI RNA cannot be displaced by poly(I).poly(C) or reovirus dsRNA. These data confirm our previous results to illustrate that VAI RNA can bind p68 kinase and cause its inactivation irreversably.     

Inhibition of glioma growth by tumor-specific activation of double-stranded RNA-dependent protein kinase PKR

Activated double-stranded RNA (dsRNA-dependent protein kinase PKR is a potent growth inhibitory protein that is primarily activated in virally infected cells, inducing cell death. Here we investigate whether selective activation of PKR can be used to kill cancer cells that express mutated genes containing deletions or chromosomal translocations. We show that antisense (AS) RNA complementary to fragments flanking the deletion or translocation can produce a dsRNA molecule of sufficient length to activate PKR and induce cell death following hybridization with mutated but not wild-type mRNA. Using the U87MG Delta EGFR cell line, which expresses a truncated form of epidermal growth factor receptor (EGFR), Delta(2-7) EGFR, we found that expression of a 39-nucleotide (nt) AS RNA complementary to the unique exon 1 to 8 junction caused selective death of cells harboring the truncated EGFR both in vitro and in vivo but did not affect cells expressing wild-type EGFR. A lentiviral vector expressing the 39-nt AS sequence strongly inhibited glioblastoma growth in mouse brain when injected after tumor cell implantation. This PKR-mediated killing strategy may be useful in treating many cancers that express a unique RNA species.     

DRBP76, a double-stranded RNA-binding nuclear protein, is phosphorylated by the interferon-induced protein kinase, PKR.

The interferon-induced double-stranded RNA-activated protein kinase PKR is the prototype of a class of double-stranded (dsRNA)-binding proteins (DRBPs) which share a dsRNA-binding motif conserved from Drosophila to humans. Here we report the purification of DRBP76, a new human member of this class of proteins. Sequence from the amino terminus of DRBP76 matched that of the M phase-specific protein, MPP4. DRBP76 was also cloned by the yeast two-hybrid screening of a cDNA library using a mutant PKR as bait. Analysis of the cDNA sequence revealed that it is the full-length version of MPP4, has a bipartite nuclear localization signal, two motifs that can mediate interactions with both dsRNA and PKR, five epitopes for potential M phase-specific phosphorylation, two potential sites for phosphorylation by cyclin-dependent kinases, a RG2 motif present in many RNA-binding proteins and predicts a protein of 76 kDa. DsRNA and PKR interactions of DRBP76 were confirmed by analysis of in vitro translated and purified native proteins. Cellular expression of an epitope-tagged DRBP76 demonstrated its nuclear localization, and its co-immunoprecipitation with PKR demonstrated that the two proteins interact in vivo. Finally, purified DRBP76 was shown to be a substrate of PKR in vitro, indicating that this protein's cellular activities may be regulated by PKR-mediated phosphorylation.     

Mechanism of interferon action. The interferon-induced phosphoprotein P1 possesses a double-stranded RNA-dependent ATP-binding site

Protein P1, the interferon-induced protein phosphorylated in the presence of dsRNA in human amnion U-cells, was covalently labeled with [alpha-32P]ATP following ultraviolet irradiation. The photoaffinity labeling of protein P1 was dependent upon double-stranded RNA. Antibody prepared against phosphorylated protein P1 immunoprecipitated the double-stranded RNA-dependent photoaffinity-labeled product. The extent of photoaffinity labeling was significantly decreased by the addition of unlabeled ATP, GTP, or AMP; adenosine had little effect on the photoaffinity labeling of protein P1. These results suggest that protein P1 possesses a site capable of binding an adenine nucleotide in a double-stranded RNA-dependent manner.     

RNase L and double-stranded RNA-dependent protein kinase exert complementary roles in islet cell defense during coxsackievirus infection

Coxsackievirus (CV) is an important human pathogen that has been linked to the development of autoimmunity. An intact pancreatic beta cell IFN response is critical for islet cell survival and protection from type 1 diabetes following CV infection. In this study, we show that IFNs trigger an antiviral state in beta cells by inducing the expression of proteins involved in intracellular antiviral defense. Specifically, we demonstrate that 2',5'-oligoadenylate synthetases (2-5AS), RNase L, and dsRNA-dependent protein kinase (PKR) are expressed by pancreatic islet cells and that IFNs (IFN-alpha and IFN-gamma) increase the expression of 2-5AS and PKR, but not RNase L. Moreover, our in vitro studies uncovered that these pathways play important roles in providing unique and complementary antiviral activities that critically regulate the outcome of CV infection. The 2-5AS/RNase L pathway was critical for IFN-alpha-mediated islet cell resistance from CV serotype B4 (CVB4) infection and replication, whereas an intact PKR pathway was required for efficient IFN-gamma-mediated repression of CVB4 infection and replication. Finally, we show that the 2-5AS/RNase L and the PKR pathways play important roles for host survival during a challenge with CVB4. In conclusion, this study has dissected the pathways used by distinct antiviral signals and linked their expression to defense against CVB4.     

Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the α-subunit of eukaryotic initiation factor 2 (eIF2α), inhibiting the function of the eIF2 complex and continued initiation of translation. When bound to an activating RNA and ATP, PKR undergoes autophosphorylation reactions at multiple serine and threonine residues. This autophosphorylation reaction stimulates the eIF2α kinase activity of PKR. The binding of certain viral RNAs inhibits the activation of PKR. Wild-type PKR is obtained as a highly phosphorylated protein when overexpressed in Escherichia coli. We report here that treatment of the isolated phosphoprotein with the catalytic subunit of protein phosphatase 1 dephosphorylates the enzyme. The in vitro autophosphorylation and eIF2α kinase activities of the dephosphorylated enzyme are stimulated by addition of RNA. Thus, inactivation by phosphatase treatment of autophosphorylated PKR obtained from overexpression in bacteria generates PKR in a form suitable for in vitro analysis of the RNA-induced activation mechanism. Furthermore, we used gel mobility shift assays, methidiumpropyl-EDTA·Fe footprinting and affinity chromatography to demonstrate differences in the RNA-binding properties of phospho- and dephosphoPKR. We found that dephosphorylation of PKR increases binding affinity of the enzyme for both kinase activating and inhibiting RNAs. These results are consistent with an activation mechanism that includes release of the activating RNA upon autophosphorylation of PKR prior to phosphorylation of eIF2α.     

Cooperative roles of fish protein kinase containing Z-DNA binding domains and double-stranded RNA-dependent protein kinase in interferon-mediated antiviral response

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) inhibits protein synthesis by phosphorylating eukaryotic translation initiation factor 2α (eIF2α). In fish species, in addition to PKR, there exists a PKR-like protein kinase containing Z-DNA binding domains (PKZ). However, the antiviral role of fish PKZ and the functional relationship between fish PKZ and PKR remain unknown. Here we confirmed the coexpression of fish PKZ and PKR proteins in Carassius auratus blastula embryonic (CAB) cells and identified them as two typical interferon (IFN)-inducible eIF2α kinases, both of which displayed an ability to inhibit virus replication. Strikingly, fish IFN or all kinds of IFN stimuli activated PKZ and PKR to phosphorylated eIF2α. Overexpression of both fish kinases together conferred much more significant inhibition of virus replication than overexpression of either protein, whereas morpholino knockdown of both made fish cells more vulnerable to virus infection than knockdown of either. The antiviral ability of fish PKZ was weaker than fish PKR, which correlated with its lower ability to phosphorylate eIF2α than PKR. Moreover, the independent association of fish PKZ or PKR reveals that each of them formed homodimers and that fish PKZ phosphorylated eIF2α independently on fish PKR and vice versa. These results suggest that fish PKZ and PKR play a nonredundant but cooperative role in IFN antiviral response.