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PubMed2005

Tyrosine phosphorylation acts as a molecular switch to full-scale activation of the eIF2alpha RNA-dependent protein kinase.

Phosphorylation of the alpha-subunit of translation eukaryotic initiation factor-2 (eIF2) leads to the inhibition of protein synthesis in response to diverse conditions of stress. Serine/threonine RNA-dependent protein kinase (PKR) is an eIF2alpha kinase family member induced by type I IFN and activated in response to dsRNA or virus infection. Herein, we demonstrate that human PKR is a dual specificity kinase phosphorylated at Y101, Y162 and Y293 in vitro and in vivo. Site-specific tyrosine phosphorylation is essential for efficient dsRNA-binding, dimerization, kinase activation and eIF2alpha phosphorylation of PKR. Biologically, tyrosine phosphorylation of PKR mediates the antiviral and antiproliferative properties of the kinase through its ability to control translation. Our data demonstrate an important role of tyrosine phosphorylation in biochemical and biological processes caused or mediated by the activation of the eIF2alpha kinase PKR.

Animals↗

PubMed2003

Stable and controllable RNA interference: Investigating the physiological function of glutathionylated actin.

RNA interference is an effective method to silence specific gene expression. Its application to mammalian cells, however, has been hampered by various shortcomings. Recently, it was reported that introduction of 22-bp double-stranded RNAs (dsRNAs) would specifically suppress expression of endogenous and heterogeneous genes in various mammalian cell lines. However, using this method, we failed to knock out proteins of interest effectively. Here we report the development of a stable and controllable method for generating dsRNA intracellularly. Tetracycline-responsive transactivator-containing cells were transfected with a vector capable of tetracycline-induced bidirectionally overexpressing sense and antisense RNA to form dsRNA in vivo. With this method, glutaredoxin, monitored by Western blot, was knocked out by overexpressing 290-base sense and antisense RNA in NIH 3T3 cells controlled by tetracycline or doxycycline. By using these glutaredoxin knocked-out cells, we have demonstrated that actin deglutathionylation plays a key role in growth factor-mediated actin polymerization, translocalization, and reorganization near the cell periphery.

3T3 Cells↗

PubMed1978

Electron microscopic heteroduplex analysis of "killer" double-stranded RNA species from yeast.

Wild-type and mutant double-stranded RNA (dsRNA) species from the yeast Saccharomyces cerevisiae were studied by electron microscopic heteroduplex mapping to determine the sequence relationships among the different RNA molecules. Three mutant dsRNAs, 1.5, 1.4, and 0.73 kilobase, were found to be derived by the same internal deletion of the wild-type (I83 kilobases) molecule. This deletion includes a wild-type (1.83 kilobases) molecule. This deletion includes a segment of about 200 base pairs that was estimated to be nearly 100% A+U. In addition, the sequences of the two larger mutant RNA species are tandem, direct duplications. One of the duplicated molecules appears to have a second internal deletion that occurred after the duplication. The mutant dsRNAs are functionally similar to the defective interfering virus particles of animal viruses--all of the mutant species prevent the propagation of the wild-type dsRNA when both are present in the same cell. The four dsRNAs share the same sequences at their termini, a finding that may suggest that these sequences are important for the replication of the dsRNAs.

Base Sequence↗

PubMed1979

In situ phosphorylation of the alpha subunit of eukaryotic initiation factor 2 in reticulocyte lysates inhibited by heme deficiency, double-stranded RNA, oxidized glutathione, or the heme-regulated protein kinase.

Protein synthesis initiation in reticulocyte lysates is inhibited by heme deficiency, low levels of double-stranded RNA (dsRNA), oxidized glutathione (GSSG), or the purified kinase (HRI) that acts on the alpha polypeptide of eukaryotic initiation factor 2 (eIF-2alpha). The phosphoprotein profiles produced in lysates in response to these various conditions have been monitored directly in lysates after labeling for brief periods with pulses of [gamma-(32)P]ATP. The [(32)P]phosphoprotein profiles were analyzed by electrophoresis in sodium dodecyl sulfate/polyacrylamide slab gels under conditions in which the HRI and eIF-2alpha polypeptides were clearly distinguished. All four modes of inhibition produced a rapid phosphorylation of eIF-2alpha compared to control lysates, which displayed little or no phosphorylation of eIF-2alpha. In heme-deficient lysates, phosphorylation of eIF-2alpha occurred rapidly both before and after the shut-off of protein synthesis; the delayed addition of hemin to these lysates resulted in a decrease in the phosphorylation of eIF-2alpha and the subsequent restoration of protein synthesis. These data suggest that rapid turnover of phosphate occurs at the site(s) of eIF-2alpha phosphorylation. In lysates inhibited by heme deficiency, GSSG, or added HRI, the phosphorylation of eIF-2alpha was accompanied by the rapid in situ phosphorylation of HRI. The inhibition of initiation induced by dsRNA was accompanied by the phosphorylation of eIF-2alpha and a 67,000-dalton polypeptide but not HRI. These observations in situ indicate that (i) the phosphorylation of eIF-2alpha is the critical event in these inhibitions of protein chain initiation, and (ii) the phosphorylation of HRI is associated with its activation in heme deficiency.

Adenosine Triphosphate↗

PubMed1991

Tat-dependent adenosine-to-inosine modification of wild-type transactivation response RNA.

Tat is a potent activator of gene expression in human immunodeficiency virus type 1 (HIV-1). Activation by Tat requires a cis-acting element, the transactivation response (TAR) site, located in the viral long terminal repeat and the 5' end of all viral mRNAs. Sequences in TAR RNA can fold into a specific stem-loop structure, and certain features of the stem-loop are essential for Tat-mediated transactivation. In Xenopus oocytes, TAR sequences can inhibit the translation of 3' cis-linked mRNAs. However, coinjection of Tat and the TAR-containing RNA into oocyte nuclei relieves this translational inhibition [Braddock, M., Chambers, A., Wilson, W., Esnout, M. A., Adams, S.E. & Kingsman, S.M. (1989) Cell 58, 269-279]. We report here that the intramolecular TAR stem-loop structure is a substrate for the double-stranded RNA (dsRNA)-modifying activity, which converts adenosines to inosines. This activity is located in the nuclei of Xenopus oocytes. The specificity and extent of modification of adenosines in TAR is dependent on Tat. We propose that the dsRNA-modifying activity may be one of the cellular proteins that interacts with TAR in the nucleus. The possible role of TAR RNA modification in the expression of HIV-1 is discussed.

Adenosine↗

PubMed1994

TAR RNA-binding protein is an inhibitor of the interferon-induced protein kinase PKR.

A cDNA encoding a double-stranded-RNA (dsRNA)-binding protein was isolated by screening a HeLa cell cDNA expression library for proteins that bind the HIV-1 Rev-responsive-element RNA. The cDNA encoded a protein that was identical to TRBP, the previously reported cellular protein that binds the transactivation response element (TAR) RNA of human immunodeficiency virus type 1. TRBP inhibited phosphorylation of the interferon-induced ribosome-associated protein kinase PKR and of the eukaryotic translation initiation factor eIF-2 alpha in a transient-expression system in which the translation of a reporter gene was inhibited by the localized activation of PKR. TRBP expression in HeLa cells complemented the growth and protein-synthesis defect of a vaccinia virus mutant lacking the expression of the dsRNA-binding protein E3L. These results implicate TRBP as a cellular regulatory protein that binds RNAs containing specific secondary structure(s) to mediate the inhibition of PKR activation and stimulate translation in a localized manner.

Animals↗

PubMed1995

Mutagenic analysis of double-stranded RNA adenosine deaminase, a candidate enzyme for RNA editing of glutamate-gated ion channel transcripts.

Mutagenic analysis of the substrate binding and catalytic domains of double-stranded RNA (dsRNA) adenosine deaminase (DRADA) was carried out. This nuclear enzyme is likely to be involved in the RNA editing of glutamate-gated ion channels that are essential for fast excitatory neurotransmission in mammalian brain. The deletion of the first or the third of the three dsRNA binding motifs within the substrate binding domain dramatically decreases enzyme activity, whereas the second motif seems to be dispensable. The results indicate that the three motifs are not functionally equivalent in the catalytic action of DRADA. Mutation of the putative zinc-coordinating residues, His910, Cys966, and Cys1036, abolished the DRADA activity. Similarly, the Glu912 residue, predicted to be involved in the proton transfer functions of the enzyme, was found to be indispensable. Our results reinforce the previous proposal that the hydrolytic deamination mechanism of DRADA may be more similar to that of the cytidine deaminases than of adenosine deaminases.

Adenosine Deaminase↗

PubMed1995

Characterization of specific DNA-binding factors activated by double-stranded RNA as positive regulators of interferon alpha/beta-stimulated genes.

Viral infection results in transcriptional activation of the cellular interferon alpha/beta-stimulated genes (ISGs) independent of the autocrine action of interferon alpha/beta (IFN-alpha/beta). Induction of ISG expression by virus appears to be mediated through production of viral double-stranded RNA (dsRNA). Previously, we identified two novel dsRNA-activated factors (DRAFs) that bind to the interferon-stimulated response element (ISRE), the DNA sequence that mediates transcriptional activation by IFN-alpha/beta. In this report we define sequences that flank the classical ISRE to be necessary for DRAF1 binding. More significantly, it is shown that the sequences required to bind DRAF1 correlate with the ability to mediate ISG induction by virus. These results strongly suggest that DRAF1 is a positive regulator of ISG transcription. DRAF1 is shown to bind selectively to the promoters of those ISGs which are strongly induced by viral infection, again suggesting the functional significance of this factor. UV cross-linking experiments indicate that DRAF1 and DRAF2 share a common DNA-binding subunit of approximately 70 kDa which is referred to as the DRAF binding component (DRAFB). DRAFB is shown to preexist in the cytoplasm of unstimulated cells. Consistent with this observation, both DRAF1 and DRAF2 are activated in the cytoplasm prior to nuclear translocation.

Base Sequence↗

PubMed1997

The Tat protein of human immunodeficiency virus type 1 is a substrate and inhibitor of the interferon-induced, virally activated protein kinase, PKR.

We demonstrate that the interferon-induced, double-stranded (ds) RNA-activated kinase, PKR, is able to bind to and phosphorylate the human immunodeficiency virus type 1 (HIV-1) trans-activating protein, Tat. Furthermore, Tat can inhibit the activation and activity of the kinase. Phosphorylation of Tat by PKR is dependent on the prior activation of PKR by dsRNA and occurs on serine and threonine residues adjacent to the basic region important for TAR RNA binding and Tat function. Activated PKR efficiently phosphorylates both the two-exon form of Tat (Tat-86) and the single exon form (Tat-72). Mutagenesis indicates that the interaction between PKR and Tat requires the RNA-binding region of Tat. Tat competes with eukaryotic initiation factor 2, a well-characterized substrate of PKR, for phosphorylation by activated PKR. Tat also inhibits the autophosphorylation of PKR by dsRNA. This biochemical evidence of an intimate relationship between Tat, an important regulator of HIV transcription, and PKR, a pleiotropic cellular regulator, may provide insights into HIV-1 pathogenesis and, more generally, virus/host interactions.

Amino Acid Sequence↗

PubMed1998

Localization of the active site of HIV-1 reverse transcriptase-associated RNase H domain on a DNA template using site-specific generated hydroxyl radicals.

Reverse transcriptase (RT)-associated ribonuclease H (RNase H) can cleave both the RNA template of DNA/RNA hybrids as well as double-stranded (ds) RNA. This report shows that human immunodeficiency virus (HIV)-RT can also cleave the template strand of dsDNA when Mg2+ is replaced by Fe2+ in the RNase H active site of HIV-RT. The cleavage mechanisms as well as the positions of the cut vary depending on whether RNA or DNA is used. While DNA is cleaved 17 base positions upstream of the primer 3'-end, RNA is cleaved 18 base positions upstream. Competition experiments show that Fe2+ replaces the catalytically active Mg2+ of RT-associated RNase H. The bound Fe2+ is the source of locally generated OH-radicals that cleave the most proximate base in the DNA. Electrophoretic mobility studies of the cleaved fragments suggest that DNA is cleaved by an oxidative mechanism, while RNA is cleaved by an enzymatic mechanism which is indistinguishable from the Mg2+-dependent cleavage. The Fe2+-dependent cuts can be used to trace the active site of RT-associated RNase H on dsDNA as well as on dsRNA and DNA/RNA hybrids. The observed 1 base difference in the cleavage positions on DNA and RNA templates can be attributed to conformational differences of the bound nucleic acids. We suggest that the lower pitch of dsRNA and DNA/RNA hybrids compared with dsDNA permits accommodation of an additional base pair in the region between the primer 3'-end and the Fe2+-dependent cleavage position at the RNase H active site.

Binding Sites↗

PubMed1998

Phosphorylation of eukaryotic translation initiation factor 2 mediates apoptosis in response to activation of the double-stranded RNA-dependent protein kinase.

The interferon-inducible, double-stranded (ds) RNA-dependent serine/threonine protein kinase (PKR) plays a role in viral pathogenesis, cell growth, and differentiation and is implicated as a tumor suppressor gene. Expression of a trans-dominant negative, catalytically inactive mutant PKR protected NIH3T3 cells from apoptosis in response to either treatment with tumor necrosis factor alpha (TNF alpha), serum deprivation. In cells expressing mutant PKR, TNF alpha, but not dsRNA induced transcription from a nuclear factor kappa B-dependent promoter, demonstrating specificity for dsRNA in signaling through the PKR pathway. Serum or platelet-derived growth factor addition to serum-deprived mutant PKR-expressing cells induced transcription of the early response genes c-fos and c-jun, indicating that the immediate early response signaling was intact. Overexpression of wild-type PKR in a transient DNA transfection system was sufficient to induce apoptosis. TNF alpha-induced apoptosis correlated with increased phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha), the primary physiological substrate of the PKR. Furthermore, forced expression of a nonphosphorylatable S51A mutant eIF-2 alpha partially protected cells from TNF alpha-induced apoptosis, and expression of a S51D mutant eIF-2 alpha, a mutant that mimics phosphorylated eIF-2 alpha, was sufficient to induce apoptosis. Taken together, these studies identify a novel requirement for PKR in stress-induced apoptosis that is mediated through eIF-2 alpha phosphorylation.

3T3 Cells↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

Serotonin-2C receptor pre-mRNA editing in rat brain and in vitro by splice site variants of the interferon-inducible double-stranded RNA-specific adenosine deaminase ADAR1.

The interferon-inducible RNA-specific adenosine deaminase (ADAR1) is an RNA editing enzyme implicated in the site-selective deamination of adenosine to inosine in cellular pre-mRNAs. The pre-mRNA for the rat serotonin-2C receptor (5-HT2CR) possesses four editing sites (A, B, C, and D), which undergo A-to-I nucleotide conversions that alter the signaling function of the encoded G-protein-coupled receptor. Measurements of 5-HT2CR pre-mRNA editing in vitro revealed site-specific deamination catalyzed by ADAR1. Three splice site variants, ADAR1-a, -b, and -c, all efficiently edited the A site of 5-HT2CR pre-mRNA, but the D site did not serve as an efficient substrate for any of the ADAR1 variants. Mutational analysis of the three double-stranded (ds) RNA binding motifs present in ADAR1 revealed a different relative importance of the individual dsRNA binding motifs for deamination of the A site of 5-HT2CR and synthetic dsRNA substrates. Quantitative reverse transcription-polymerase chain reaction analyses demonstrated that the 5-HT2CR pre-mRNA was most highly expressed in the choroid plexus of rat brain. However, ADAR1 and the related deaminase ADAR2 showed significant expression in all regions of the brain examined, including cortex, hippocampus, olfactory bulb, and striatum, where the 5-HT2CR pre-mRNA was extensively edited.

Adenosine Deaminase↗

PubMed1999

Dimerization of the interferon type I receptor IFNaR2-2 is sufficient for induction of interferon effector genes but not for full antiviral activity.

We constructed chimeric receptors wherein the extracellular domain of the erythropoietin receptor (EpoR) was fused to the transmembrane and intracellular domains of the interferon (IFN) type I receptor subunits, IFNaR1 or IFNaR2-2. Transfection into 2fTGH and Tyk2-deficient 11,1 cells showed that EpoR/IFNaR2-2 alone was able to transduce a signal upon stimulation with erythropoietin (Epo), as judged by induction of the interferon type I-inducible 6-16 promoter. In contrast, protection against infection with encephalomyocarditis virus or vesicular stomatitis virus was reduced or absent, respectively. To further investigate the role of IFNaR1 in the induction of an antiviral state, we analyzed the Epo- versus IFNalpha-induced transcription of a set of genes, involved in antiviral protection. Up to 24 h after stimulation with Epo or IFNalpha, comparable transcription of the p56, dsRNA-dependent protein kinase, 2'-5'A synthetase, and MxA genes was seen. However, at later time points, only in the case of Epo induction, a sharp decrease of mRNA levels was observed. Western blotting analysis of dsRNA-dependent protein kinase showed a similar pattern at the protein level. Taken together, our results imply a role for IFNaR1 in the induction of sustained mRNA and protein levels that are likely required for optimal antiviral activity.

Alkaline Phosphatase↗

PubMed2002

Manganese(II) induces apoptotic cell death in NIH3T3 cells via a caspase-12-dependent pathway.

Under physiological conditions, manganese(II) exhibits catalase-like activity. However, at elevated concentrations, it induces apoptosis via a non-mitochondria-mediated mechanism (Oubrahim, H., Stadtman, E. R., and Chock, P. B. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9505-9510). In this study, we show that the Mn(II)-induced apoptosis, as monitored by caspase-3-like activity, in NIH3T3 cells was inhibited by calpain inhibitors I and II or the p38 MAP kinase inhibitor, SB202190. The control experiments showed that each of these inhibitors in the concentration ranges used exerted no effect on activated caspase-3-like activity. Furthermore, caspase-12 was cleaved in Mn(II)-treated cells, suggesting that the Mn(II)-induced apoptosis is mediated by caspase-12. This notion is confirmed by the observations that pretreatment of NIH3T3 cells with either caspase-12 antisense RNA or dsRNA corresponding to the full-length caspase-12 led to a dramatic decrease in caspase-3-like activity induced by Mn(II). The precise mechanism by which Mn(II) induced the apoptosis is not clear. Nevertheless, Mn(II), in part, exerts its effect via its ability to replace Ca(II) in the activation of m-calpain, which in turn activates caspase-12 and degrades Bcl-xL. In addition, the dsRNA(i) method serves as an effective technique for knocking out caspase-12 in NIH3T3 cells without causing apoptosis.

3T3 Cells↗

PubMed2006

Two tyrosine residues of Toll-like receptor 3 trigger different steps of NF-kappa B activation.

Innate immune response to viral infection is often triggered by Toll-like receptor 3 (TLR3)-mediated signaling by double-stranded (ds) RNA, which culminates in the activation of the transcription factor NF-kappaB and induction of NF-kappaB-driven genes. We demonstrated that dsRNA-induced phosphorylation of two specific tyrosine residues, 759 and 858, of TLR3 was necessary and sufficient for complete activation of the NF-kappaB pathway. When Tyr-759 of TLR3 was mutated, gene induction was inhibited, although NF-kappaB was partially activated. It was released from IkappaB and translocated to the nucleus but failed to bind to the kappaB site of the target A20 gene promoter. This defect could be attributed to incomplete phosphorylation of the RelA (p65) subunit of NF-kappaB, as revealed by two-dimensional gel analyses of p65, isolated from dsRNA-treated cells expressing either wild type TLR3 or the Tyr-759 --> Phe mutant TLR3. Thus, two phosphotyrosine residues of TLR3 activate two distinct pathways, one leading to NF-kappaB release and the other leading to its phosphorylation.

Cell Line↗

PubMed2000

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.

3T3 Cells↗

PubMed2001

Nuclear factor 90 is a substrate and regulator of the eukaryotic initiation factor 2 kinase double-stranded RNA-activated protein kinase.

Nuclear factor 90 (NF90) is a member of an expanding family of double-stranded (ds) RNA-binding proteins thought to be involved in gene expression. Originally identified in complex with nuclear factor 45 (NF45) as a sequence-specific DNA-binding protein, NF90 contains two double stranded RNA-binding motifs (dsRBMs) and interacts with highly structured RNAs as well as the dsRNA-activated protein kinase, PKR. In this report, we characterize the biochemical interactions between these two dsRBM containing proteins. NF90 binds to PKR through two independent mechanisms: an RNA-independent interaction occurs between the N terminus of NF90 and the C-terminal region of PKR, and an RNA-dependent interaction is mediated by the dsRBMs of the two proteins. Co-immunoprecipitation analysis demonstrates that NF90, NF45, and PKR form a complex in both nuclear and cytosolic extracts, and both proteins serve as substrates for PKR in vitro. NF90 is phosphorylated by PKR in its RNA-binding domain, and this reaction is partially blocked by the NF90 N-terminal region. The C-terminal region also inhibits PKR function, probably through competitive binding to dsRNA. A model for NF90-PKR interactions is proposed.

Animals↗