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PubMed1980

Purification and properties of the double-stranded RNA-activated eukaryotic initiation factor 3 kinase from rabbit reticulocytes.

The double-stranded RNA (dsRNA)-activated protein kinase (DAI) that phosphorylates the alpha subunit of the eukaryotic initiation factor eIF-2 and inhibits chain initiation has been isolated from rabbit reticulocyte lysates. The nonactivated enzyme or the enzyme partially activated by incubation with low levels of dsRNA (pro-DAI) could be purified only to a slight extent. However, the enzyme that was fully activated by incubation with both dsRNA and ATP was purified to near homogeneity. Active DAI is a phosphoprotein with an apparent subunit mass of 68,000 daltons. It can phosphorylate histone as well as the alpha subunit of eIF-2. Our results suggest that, after interaction with dsRNA, the enzyme phosphorylates itself and is thereby activated to phosphorylate alpha eIF-2 and histone.

Animals↗

PubMed1986

Expression of a cDNA derived from the yeast killer preprotoxin gene: implications for processing and immunity.

The type I killer strains of Saccharomyces cerevisiae secrete a dimeric 19-kDa protein that kills sensitive cells by disrupting cytoplasmic membrane function. This toxin is encoded by the double-stranded RNA plasmid M1-dsRNA, which also determines specific immunity to toxin. A preprotoxin, the 35-kDA in vitro translation product of denatured M1-dsRNA, is presumed to be the primary in vivo gene product. To facilitate studies on preprotoxin structure and maturation, we have inserted a partial cDNA copy of M1-dsRNA into the yeast vector p1A1, bringing it under control of the phosphate-repressible PHO5 promoter. This in-frame gene fusion encodes all of the preprotoxin sequence except for its N-terminal secretion leader, which is replaced by the leader sequence of PHO5. Transformation of sensitive yeast strains lacking M1-dsRNA with such fusion plasmids converts them to phosphate-repressible, immune killers, demonstrating that both toxin and immunity determinants are contained within the preprotoxin molecule. L-1-Tosylamido-2-phenylethyl chloromethyl ketone retards glycosylation of preprotoxin to toxin, facilitating size comparisons and indicating that processing of the normal precursor involves three glycosylation events but does not involve cotranslational leader peptidase action. In contrast, the PHO5 leader is apparently removed from the fusion preprotoxin.

DNA↗

PubMed1988

Detection of activated double-stranded RNA-dependent protein kinase in 3T3-F442A cells.

We have previously reported that cultured mouse 3T3-F442A cells exhibit a transient, double-stranded RNA (dsRNA)-dependent phosphorylation of the dsRNA-dependent eIF-2 alpha kinase (eIF-2 alpha, alpha-subunit of the eukaryotic initiation factor 2) (dsI). When dsI is activated by low levels of dsRNA, it is a potent inhibitor of protein synthesis. The transient expression of dsI is due to an autocrine effect of interferon at specific stages of growth and differentiation, and it may represent a mechanism for regulating cell growth and differentiation in 3T3-F442A cells. In this report, the purification of dsI from 3T3-F442A cell cultures by a two-step procedure is described. A specific immune serum to dsI was prepared by immunizing a rabbit with highly pure preparations. Immune precipitation studies demonstrate that the serum reacts with phosphorylated dsI both in vitro and in vivo and with de novo synthesized dsI after induction with interferon. We find that dsI of 3T3 cells can undergo phosphorylation in vitro without the addition of dsRNA and in vivo in the absence of viral infection. These results are consistent with a physiologic role for dsI in the growth and differentiation of these cells.

Animals↗

PubMed1992

Identification of double-stranded RNA-binding domains in the interferon-induced double-stranded RNA-activated p68 kinase.

The double-stranded RNA (dsRNA)-binding domain of the human p68 kinase has been localized to the N-terminal half of the enzyme by using progressive deletion analysis and in vitro binding assays. To further define the domains responsible for binding to dsRNA, we cloned the mouse dsRNA-activated p65 kinase and used sequence alignment to identify conserved domains in the N-terminal region. Deletions in either of two 12-amino-acid-long and arginine- or lysine-rich regions abrogated binding to dsRNA. Moreover, in an in vivo growth inhibition assay in the yeast Saccharomyces cerevisiae, these mutants failed to exhibit a slow-growth phenotype.

Amino Acid Sequence↗

PubMed1992

A conserved double-stranded RNA-binding domain.

We have identified a double-stranded (ds)RNA-binding domain in each of two proteins: the product of the Drosophila gene staufen, which is required for the localization of maternal mRNAs, and a protein of unknown function, Xlrbpa, from Xenopus. The amino acid sequences of the binding domains are similar to each other and to additional domains in each protein. Database searches identified similar domains in several other proteins known or thought to bind dsRNA, including human dsRNA-activated inhibitor (DAI), human trans-activating region (TAR)-binding protein, and Escherichia coli RNase III. By analyzing in detail one domain in staufen and one in Xlrbpa, we delimited the minimal region that binds dsRNA. On the basis of the binding studies and computer analysis, we have derived a consensus sequence that defines a 65- to 68-amino acid dsRNA-binding domain.

Amino Acid Sequence↗

PubMed1995

Chicken double-stranded RNA adenosine deaminase has apparent specificity for Z-DNA.

A M(r) 140,000 protein has been purified from chicken lungs to apparent homogeneity. The protein binds with high affinity to a non-BNA conformation, which is most likely to the Z-DNA. The protein also has a binding site for double-stranded RNA (dsRNA). Peptide sequences from this protein show similarity to dsRNA adenosine deaminase, an enzyme that deaminates adenosine in dsRNA to form inosine. Assays for this enzyme confirm that dsRNA adenosine deaminase activity and Z-DNA binding are properties of the same molecule. The coupling of these two activities in a single molecule may indicate a distinctive mechanism of gene regulation that is, in part, dependent on DNA topology. As such, DNA topology, through its effects on the efficiency and extent of RNA editing may be important in the generation of new phenotypes during evolution.

Adenosine Deaminase↗

PubMed1995

Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity.

The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.

Base Sequence↗

PubMed1998

A mutant cell line defective in response to double-stranded RNA and in regulating basal expression of interferon-stimulated genes.

Although much progress has been made in identifying the signaling pathways that mediate the initial responses to interferons (IFNs), much less is known about how IFN-stimulated genes (ISGs) are kept quiescent in untreated cells, how the response is sustained after the initial induction, and how ISG expression is down-regulated, even in the continued presence of IFN. We have used the cell sorter to isolate mutant cells with constitutively high ISG expression. A recessive mutant, P2.1, has higher constitutive ISG levels than the parental U4C cells, which do not respond to any IFN. Unexpectedly, P2.1 cells also are deficient in the expression of ISGs in response to double-stranded RNA (dsRNA). Electrophoretic mobility-shift assays revealed that the defect is upstream of the activation of the transcription factors NFkappaB and IFN regulatory factor 1. Analysis of the pivotal dsRNA-dependent serine/threonine kinase PKR revealed that the wild-type kinase is present and is activated normally in response to dsRNA in P2.1 cells. Together, these data suggest that the defect in P2.1 cells is either downstream of PKR or in a component of a distinct pathway that is involved both in activating multiple transcription factors in response to dsRNA and in regulating the basal expression of ISGs.

Cell Line↗

PubMed1995

Activation of the double-stranded RNA-regulated protein kinase by depletion of endoplasmic reticular calcium stores.

Perturbants of the endoplasmic reticulum (ER), including Ca(2+)-mobilizing agents, provoke a rapid suppression of translational initiation in conjunction with an increased phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF)-2. Depletion of ER Ca2+ stores was found to signal the activation of a specific eIF-2 alpha kinase. Analysis of extracts derived from cultured cells that had been pretreated with Ca2+ ionophore A23187 or thapsigargin revealed a 2-3-fold increase in eIF-2 alpha kinase activity without detectable changes in eIF-2 alpha phosphatase activity. A peptide of 65-68 kDa, which was phosphorylated concurrently with eIF-2 alpha in extracts of pretreated cells, was identified as the interferon-inducible, double-stranded RNA (dsRNA)-regulated protein kinase (PKR). Depletion of ER Ca2+ stores did not alter the PKR contents of extracts. When incubated with reovirus dsRNA, extracts derived from cells with depleted ER Ca2+ stores displayed greater degrees of phosphorylation of PKR and of eIF-2 alpha than did control extracts. The enhanced dsRNA-dependent phosphorylation of PKR was observed regardless of prior induction of the kinase with interferon. Lower concentrations of dsRNA were required for maximal phosphorylation of PKR in extracts of treated as compared to control preparations. These findings suggest that PKR mediates the translational suppression occurring in response to perturbation of ER Ca2+ homeostasis.

3T3 Cells↗

PubMed1995

Platelet-derived growth factor signal transduction through the interferon-inducible kinase PKR. Immediate early gene induction.

The interferon-inducible, double-stranded RNA (dsRNA)-dependent eukaryotic initiation factor-2 alpha kinase PKR has primarily been characterized as a component of the interferon-mediated cellular antiviral response. Several lines of evidence now exist that suggest that PKR plays a role in the regulation of growth in uninfected cells. The most direct examples are the finding of an oncogenic variant of PKR and the effects of activators and inhibitors of PKR phosphorylation on the expression of platelet-derived growth factor (PDGF)-inducible genes. Previous reports have shown that 1) dsRNA, a direct activator of PKR, induces the genes c-myc, c-fos, and JE; 2) 2-aminopurine, a chemical inhibitor of PKR, blocks the induction of these genes by serum; and 3) activated p21ras induces a cellular inhibitor of PKR. We report here that activation of PKR was correlated with the induction of the immediate early genes c-fos, c-myc, and JE by PDGF in the following situations: 1) PDGF induction of these genes, also inducible by dsRNA, was blocked by two inhibitors of PKR activation: 2-aminopurine and v-ras; 2) PDGF induction of another immediate early gene, egr-1, which could not be induced by dsRNA, was not blocked by 2-aminopurine or v-ras; 3) agents that reverse v-ras inhibition of PKR activation also reversed the v-ras block of PDGF induction of c-myc, c-fos, and JE; 4) down-regulation of PKR protein levels by antisense inhibition of translation blocked the induction of c-myc, c-fos, and JE by PDGF, but had no effect on egr-1 induction; and finally, 5) PKR was autophosphorylated in vivo in response to PDGF. These results provide direct evidence that PKR activation functions as a second messenger in a growth factor signal transduction pathway. Thus, PKR may serve as a common mediator of growth-promoting and growth inhibitory signals.

2-Aminopurine↗

PubMed1995

Activation of interferon-inducible 2'-5' oligoadenylate synthetase by adenoviral VAI RNA.

2'-5' oligoadenylate (2-5(A)) synthetase and protein kinase, RNA activated (PKR) are the only two known enzymes that bind double-stranded RNA (dsRNA) and get activated by it. We have previously identified their dsRNA binding domains, which do not have any sequence homology. Here, we report a profound difference between the two enzymes with respect to the structural features of the dsRNA that are required for their activation. The adenoviral virus-associated type I (VAI) RNA cannot activate PKR, although it binds to the protein and thereby prevents its activation by authentic dsRNA. In contrast, we observed that VAI RNA can both bind and activate 2-5(A) synthetase. Mutations in VAI RNA, which removed occasional mismatches present in its double-stranded stems, markedly enhanced its 2-5(A) synthetase-activating capacity. These mutants, however, are incapable of activating PKR. Other mutations, which disrupted the structure of the central stem-loop region of the VAI RNA, reduced its ability to activate 2-5(A) synthetase. These debilitated mutants could bind to the synthetase protein, although they fail to bind to PKR.

2',5'-Oligoadenylate Synthetase↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed2002

Double-stranded RNA signaling by Toll-like receptor 3 requires specific tyrosine residues in its cytoplasmic domain.

Double-stranded (ds) RNA, a common product of viral infection, can induce transcription of many cellular genes, including the 561 gene that encodes P56, a regulator of protein synthesis. Here, we report that induction of the 561 mRNA by exogenous dsRNA is mediated by Toll-like receptor 3 (TLR3), and it requires no new protein synthesis. Because gene induction by dsRNA is blocked by inhibitors of tyrosine kinases, we investigated the potential roles of the five tyrosine residues present in the cytoplasmic domain of TLR3 by their individual and combinatorial mutations. Transfection assays, using a reporter gene driven by the 561 promoter, identified specific tyrosine residues to be essential for TLR3 signaling. This conclusion was further validated in permanent cell lines expressing tyrosine-mutant TLR3 proteins; in some of these cell lines dsRNA failed to induce the 561 mRNA. Our results provide the first demonstration of the importance of TLR3 cytoplasmic tyrosine residues in dsRNA signaling.

Cell Line↗

PubMed2000

IRF3 and IRF7 phosphorylation in virus-infected cells does not require double-stranded RNA-dependent protein kinase R or Ikappa B kinase but is blocked by Vaccinia virus E3L protein.

Induction of interferon-alpha (IFNalpha) gene expression in virus-infected cells requires phosphorylation-induced activation of the transcription factors IRF3 and IRF7. However, the kinase(s) that targets these proteins has not been identified. Using a combined pharmacological and genetic approach, we found that none of the kinases tested was responsible for IRF phosphorylation in cells infected with Newcastle disease virus (NDV). Although the broad-spectrum kinase inhibitor staurosporine potently blocked IRF3 and -7 phosphorylation, inhibitors for protein kinase C, protein kinase A, MEK, SAPK, IKK, and protein kinase R (PKR) were without effect. Both IkappaB kinase and PKR have been implicated in IFN induction, but cells genetically deficient in IkappaB kinase, PKR, or the PKR-related genes PERK, IRE1, or GCN2 retained the ability to phosphorylate IRF7 and induce IFNalpha. Interestingly, PKR mutant cells were defective for response to double-stranded (ds) RNA but not to virus infection, suggesting that dsRNA is not the only activating viral component. Consistent with this notion, protein synthesis was required for IRF7 phosphorylation in virus-infected cells, and the kinetics of phosphorylation and viral protein production were similar. Despite evidence for a lack of involvement of dsRNA and PKR, vaccinia virus E3L protein, a dsRNA-binding protein capable of inhibiting PKR, was an effective IRF3 and -7 phosphorylation inhibitor. These results suggest that a novel cellular protein that is activated by viral products in addition to dsRNA and is sensitive to E3L inhibition is responsible for IRF activation and reveal a novel mechanism for the anti-IFN effect of E3L distinct from its inhibition of PKR.

Animals↗

PubMed2003

Poly(I-C)-induced Toll-like receptor 3 (TLR3)-mediated activation of NFkappa B and MAP kinase is through an interleukin-1 receptor-associated kinase (IRAK)-independent pathway employing the signaling components TLR3-TRAF6-TAK1-TAB2-PKR .

Recent studies show that a member of the interleukin-1 (IL-1)/Toll receptor superfamily, Toll-like receptor 3 (TLR3), recognizes double-stranded RNA (dsRNA). Because of the similarity in their cytoplasmic domains, IL-1/Toll receptors share signaling components that associate with the IL-1 receptor, including IL-1 receptor-associated kinase (IRAK), MyD88, and TRAF6. However, we find that, in response to dsRNA, TLR3 can mediate the activation of both NFkappaB and mitogen-activated protein (MAP) kinases in IL-1-unresponsive mutant cell lines, including IRAK-deficient I1A and I3A cells, which are defective in a component that is downstream of IL-1R but upstream of IRAK. These results clearly indicate that TLR3 does not simply share the signaling components employed by the IL-1 receptor. Through biochemical analyses we have identified an IRAK-independent TLR3-mediated pathway. Upon binding of dsRNA to TLR3, TRAF6, TAK1, and TAB2 are recruited to the receptor to form a complex, which then translocates to the cytosol where TAK1 is phosphorylated and activated. The dsRNA-dependent protein kinase (PKR) is also detected in this signal-induced TAK1 complex. Kinase inactive mutants of TAK1 (TAK1DN) and PKR (PKRDN) inhibit poly(dI.dC)-induced TLR3-mediated NFkappaB activation, suggesting that both of these kinases play important roles in this pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Protein kinase R (PKR) interacts with and activates mitogen-activated protein kinase kinase 6 (MKK6) in response to double-stranded RNA stimulation.

The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been invoked in different signaling pathways. In cells pre-exposed to the PKR inhibitor 2-aminopurine or in PKR-null cells, the activation of p38 mitogen-activated protein kinase (MAPK) following dsRNA stimulation is attenuated. We found that the p38 MAPK activator MKK6, but not its close relatives MKK3 or MKK4, exhibited an increased affinity for PKR following the exposure of cells to poly(rI:rC), a dsRNA analog. In vitro kinase assays revealed that MKK6 was efficiently phosphorylated by PKR, and this could be inhibited by 2-aminopurine. Expression of kinase-inactive PKR (K296R) in cells inhibited the poly(IC)-induced phosphorylation of MKK3/6 detected by phosphospecific antiserum but did not affect the poly(IC)-induced gel migration retardation of MKK3. This suggests that poly(IC)-mediated in vivo activation of MKK6, but not MKK3, is through PKR. Consistent with this observation, PKR was capable of activating MKK6 as assessed in a coupled kinase assay containing the components of the p38 MAPK pathway. Our results indicate that the interaction of MKK6 and PKR provides a mechanism for regulating p38 MAPK activation in response to dsRNA stimulation.

Cell Line↗

PubMed2006

Critical role for Cryopyrin/Nalp3 in activation of caspase-1 in response to viral infection and double-stranded RNA.

Viral infection induces the production of interleukin (IL)-1beta and IL-18 in macrophages through the activation of caspase-1, but the mechanism by which host cells sense viruses to induce caspase-1 activation is unknown. In this report, we have identified a signaling pathway leading to caspase-1 activation that is induced by double-stranded RNA (dsRNA) and viral infection that is mediated by Cryopyrin/Nalp3. Stimulation of macrophages with dsRNA, viral RNA, or its analog poly(I:C) induced the secretion of IL-1beta and IL-18 in a cryopyrin-dependent manner. Consistently, caspase-1 activation triggered by poly(I:C), dsRNA, and viral RNA was abrogated in macrophages lacking cryopyrin or the adaptor ASC (apoptosis-associated speck-like protein containing a caspase-activating and recruitment domain) but proceeded normally in macrophages deficient in Toll-like receptor 3 or 7. We have also shown that infection with Sendai and influenza viruses activates the cryopyrin inflammasome. Finally, cryopyrin was required for IL-1beta production in response to poly(I:C) in vivo. These results identify a mechanism mediated by cryopyrin and ASC that links dsRNA and viral infection to caspase-1 activation resulting in IL-1beta and IL-18 production.

Animals↗

PubMed2004

The roles of two IkappaB kinase-related kinases in lipopolysaccharide and double stranded RNA signaling and viral infection.

Viral infection and stimulation with lipopolysaccharide (LPS) or double stranded RNA (dsRNA) induce phosphorylation of interferon (IFN) regulatory factor (IRF)-3 and its translocation to the nucleus, thereby leading to the IFN-beta gene induction. Recently, two IkappaB kinase (IKK)-related kinases, inducible IkappaB kinase (IKK-i) and TANK-binding kinase 1 (TBK1), were suggested to act as IRF-3 kinases and be involved in IFN-beta production in Toll-like receptor (TLR) signaling and viral infection. In this work, we investigated the physiological roles of these kinases by gene targeting. TBK1-deficient embryonic fibroblasts (EFs) showed dramatic decrease in induction of IFN-beta and IFN-inducible genes in response to LPS or dsRNA as well as after viral infection. However, dsRNA-induced expression of these genes was residually detected in TBK1-deficient cells and intact in IKK-i-deficient cells, but completely abolished in IKK-i/TBK1 doubly deficient cells. IRF-3 activation, in response not only to dsRNA but also to viral infection, was impaired in TBK1-deficient cells. Together, these results demonstrate that TBK1 as well as, albeit to a lesser extent, IKK-i play a crucial role in the induction of IFN-beta and IFN-inducible genes in both TLR-stimulated and virus-infected EFs.

Animals↗