Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon.

The double-stranded (ds) RNA-activated protein kinase from human cells is a 68 kd protein (p68 kinase) induced by interferon. On activation by dsRNA in the presence of ATP, the kinase becomes autophosphorylated and can catalyze the phosphorylation of the alpha subunit of eIF2, which leads to an inhibition of the initiation of protein synthesis. Here we report the molecular cloning and characterization of several related cDNAs from which can be deduced the full-length p68 kinase sequence. All of the cDNAs identify a 2.5 kb RNA that is strongly induced by interferon. The deduced amino acid sequence of the p68 kinase predicts a protein of 550 amino acids containing all of the conserved domains specific for members of the protein kinase family, including the catalytic domain characteristic of serine/threonine kinases. In vitro translation of a reconstructed full-length p68 kinase cDNA yields a protein of 68 kd that binds dsRNA, is recognized by a monoclonal antibody raised against the native p68 kinase, and is autophosphorylated.     

Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase

NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-gamma, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-kappaB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-kappaB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.     

Activation of double-stranded RNA-activated protein kinase by mild impairment of oxidative metabolism in neurons

Thiamine (vitamin B1) deficiency (TD) causes mild and chronic impairment of oxidative metabolism and induces neuronal death in specific brain regions. The mechanisms underlying TD-induced cell death, however, remain unclear. The double-stranded RNA-activated protein kinase (PKR), has been well known for its anti-viral function. Upon activation by viral infection or double-stranded RNA, PKR phosphorylates its substrate, the α-subunit of eukaryotic initiation factor-2 (eIF2α), leading to inhibition of translation. In response to various cellular stresses, PKR can also be stimulated by its protein activators, or its mouse homologue, PKR activator (RAX). We demonstrated that TD in mice induced phosphorylation of PKR at Thr446 and Thr451 and phosphorylation of eIF2α at Ser51 in the cerebellum and the thalamus. TD caused phosphorylation of PKR and eIF2α, as well as nuclear translocation of PKR in primary cultures of cerebellar granule neurons. PKR phosphorylation is necessary for its nuclear translocation because TD failed to induce nuclear translocation of a T446A/T451A PKR mutant. Both PKR inhibitor and dominant-negative PKR mutant protected cerebellar granule neurons against TD-induced cell death. TD promoted the association between RAX and PKR. Antioxidant vitamin E dramatically decreased the RAX/PKR association and ameliorated TD-induced cell death. Our results indicate that TD-induced neuronal death is at least partially mediated by the activation of PKR.     

Possible involvement of double-stranded RNA-activated protein kinase in cell death by influenza virus infection.

We reported previously that influenza virus infection induces the apoptotic death of HeLa cells associated with activation of the Fas gene. In this report, we show that transfection with a PKR having a point mutation in the catalytic domain of K at 296 to R suppressed both the augmented expression of Fas and cell death by influenza virus infection. These results suggested the involvement of PKR in influenza virus-induced cell death.     

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.     

Expression of human cardiac-specific homeobox protein in Escherichia coli

Human cardiac-specific homeobox protein cDNA (hCsx) was cloned into expression plasmid pET32a and fused with Escherichia coli thioredoxin (Trx). The Trx-Csx fusion protein was under the control of bacteriophage T7 promoter. When expressed in E. coli BL21(DE3), about half of the recombinant Trx-Csx products existed in the form of insoluble inclusion bodies. When coexpressed with human protein disulfide isomerase, more than 90% of Trx-Csx products accumulated in the soluble form in the cell lysate. The recombinant Csx fusion protein was purified by one-step metal-chelating affinity chromatography.     

Binding of Epstein-Barr virus small RNA EBER-1 to the double-stranded RNA-activated protein kinase DAI.

Epstein-Barr virus encodes two small RNAs, EBER-1 and -2, that are abundantly expressed in latently infected cells. Recent evidence suggests a role for EBER-1 in regulation of translation since this RNA is able to prevent the inhibition of protein synthesis by double-stranded RNA in rabbit reticulocyte lysates. We show here that EBER-1 that has been synthesized in vitro forms a complex with the dsRNA-activated inhibitor of protein synthesis DAI, a protein kinase that specifically phosphorylates polypeptide chain initiation factor eIF-2. Gel retardation assays and UV crosslinking experiments indicate that complex formation is specific for EBER-1 and requires the presence of some secondary structure in the molecule. RNA competition studies show that EBER-1-DAI complex formation is not inhibited in the presence of other small RNA species, heparin or the synthetic double-stranded RNA, poly(I).poly(C). SDS gel analysis reveals the existence of two forms of the crosslinked complex, of 64-68kDa and 46-53kDa, both of which are recognized by anti-DAI antibodies in immunoprecipitation experiments. These data suggest that EBER-1 regulates protein synthesis through its ability to interact with DAI.     

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.     

Expression in Escherichia coli and simple purification of human Fhit protein

The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5',5"'-P(1),P(3)-triphosphate (Ap(3)A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His(6)-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as "H6TV," fused to the N-terminus of Fhit. Expression of H6TV-Fhit in BL21(DE3) cells for 3 h at 37 degrees C produced 30 mg of H6TV-Fhit from 1 L of cell culture ( approximately 4 g of cells). The H6TV-Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV-Fhit with rTEV protease at 4 degrees C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4 degrees C without loss of activity. The pure protein was stable at -20 degrees C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K(m) value for Ap(3)A of 0.9 microM and a k(cat)(monomer) value of 7.2 +/- 1.6 s(-1) (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV-Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap(3)A.     

Semliki forest virus capsid protein inhibits the initiation of translation by upregulating the double-stranded RNA-activated protein kinase (PKR)

We investigated the possible translational role which elevated concentrations of highly purified Semliki Forest virus (SFV) capsid (C)-protein molecules may play in a cell-free translation system. Here we decomonstrate that in the absence of double-stranded RNA high concentrations of C protein triggered the phosphorylation of the interferon-induced, double-stranded RNA-activated protein kinase, PKR. Activated PKR in turn phosphorylated its natural substrate, the subunit of eukaryotic initiation factor 2 (eIF-2), thereby inhibiting initiation of host cell translation. These findings were further strengthened by experiments showing that during natural infection with SFV the maximum phosphorylation of PKR coincided with the maximum synthesis of C protein 4–9 hours post infection. Thus, our results demonstrate that high concentrations of C-protein molecules may act in a hitherto novel mechanism on PKR to inhibit host cell protein synthesis during viral infection.     

Expression of protein IIIa of human adenovirus type 2 in Escherichia coli

We have constructed a plasmid encoding the protein IIIa gene of human adenovirus type 2. The gene was expressed under the control of the hybrid tac (trp-lac) promoter; the protein was synthesized at levels up to 5% of newly synthesized protein after IPTG induction. The protein IIIa produced in Escherichia coli has an apparent Mr on sodium dodecyl sulfate-polyacrylamide gels of 67 kDa, and was revealed with anti-adenovirus serum in Western blotting. The protein IIIa produced in bacteria was phosphorylated in the presence of [gamma-32P]ATP.     

Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli

An expression vector has been constructed for the type I regulatory subunit of cAMP-dependent protein kinase. A cDNA clone for the bovine RI-subunit has been inserted into pUC7. When Escherichia coli JM105 was transformed with this plasmid, R-subunit was expressed in amounts that approached 4 mg/liter. The expressed protein was visualized in total cell extracts by photolabeling with 8-azidoadenosine 3':5'-mono[32P]phosphate following transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose. Expression of R-subunit was independent of isopropyl-beta-D-thiogalactopyranoside. R-subunit accumulated in large amounts only in the stationary phase of growth, and the addition of isopropyl-beta-D-thiogalactopyranoside during the log phase of growth actually blocked the accumulation of R-subunit. Maximum expression (20 mg/liter) was achieved when E. coli 222 was transformed with the RI-containing plasmid. E. coli 222 is a strain that contains two mutations; it is cya- and also has a mutation in the catabolite gene activator protein (crp) that enables the protein to bind to DNA in the absence of cAMP. The expressed RI-subunit was a soluble, dimeric protein, and no significant proteolysis was apparent in the cell extract. The purified RI-subunit bound 2 mol of cAMP/mol of R monomer, reassociated with C-subunit to form holoenzyme, and migrated as a dimer on sodium dodecyl sulfate-polyacrylamide gels in the absence of reducing agents. The expressed protein was also susceptible to limited proteolysis, yielding a monomeric cAMP-binding fragment having a molecular weight of 35,000. In all of these properties, the expressed protein was indistinguishable from RI purified from bovine tissue even though the R-subunit expressed in E. coli represents a fusion protein that contains 10 additional amino acids at the amino terminus that are provided by the lac Z' gene of the vector. This NH2-terminal sequence was confirmed by amino acid sequencing.     

A protein kinase C-like activity in Escherichia coli

The protein kinase C (PKC) family comprises calcium- and phospholipid-dependent kinases whose activity is stimulated by diacylglycerol and tumour-promoting phorbol esters such as 12-tetradecanoyl phorbol-13-acetate (TPA). In the Gram-negative bacterium Escherichia coli, functional similarity to PKC was demonstrated in crude extracts by calcium and phospholipid-dependent, TPA-stimulated phosphorylation of a small number of endogenous substrates. Activity was reduced by sphingosine, a known inhibitor of eukaryotic PKC. Structural similarity to PKC was demonstrated in crude and partially purified bacterial extracts by cross-reactivity with several monoclonal antibodies. This revealed isozyme-specific homology between a protein(s) of relative molecular mass 80-85,000 in E. coli and the alpha- and gamma-isozymes, but probably not the beta-isozyme, of eukaryotic PKC.