Double-stranded RNA-activated protein kinase (PKR) is negatively regulated by 60S ribosomal subunit protein L18

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2alpha in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2alpha phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.     

Calcium mobilization by ryanodine promotes the phosphorylation of initiation factor 2alpha subunit and inhibits protein synthesis in cultured neurons

Protein synthesis plays an important role in the viability and function of the cell. There is evidence indicating that Ca2+ may be a physiological regulator of the translational process. In the present study, the effect of agents that increase intracellular calcium levels by different mechanisms, as well as repercussion on the rate of protein synthesis, including phosphorylation of initiation factor 2alpha subunit, and double-stranded RNA-dependent eIF-2alpha kinase (PKR) activity were analyzed. Glutamate (100 microM) and K+ (60 mM), which increase intracellular calcium levels (the former mostly by the influx of extracellular calcium via voltage-sensitive calcium channels, and the latter by receptor-operated calcium channels), and carbachol (1 mM), as well as glutamate, which mobilizes intracellular calcium from the endoplasmic reticulum via activation of inositol 1,4,5-trisphosphate receptor, did not modify any of the analyzed parameters. Nevertheless, 100 nM ryanodine, which increases intracellular calcium concentration by activating the ryanodine receptor, promoted a significant decrease in the rate of protein synthesis and increased both initiation factor 2alpha subunit phosphorylation and PKR activity. From our results, we can conclude that inhibition of protein synthesis is dependent on the mobilization of intracellular calcium from internal stores. Moreover, they strongly suggest that this inhibition is only promoted when calcium is increased via ryanodine receptor, and possibly by activation of PKR activity.     

A eukaryotic translation initiation factor 2-associated 67 kDa glycoprotein partially reverses protein synthesis inhibition by activated double-stranded RNA-dependent protein kinase in intact cells

A eukaryotic translation initiation factor 2 (eIF-2)-associated 67 kDa glycoprotein (p67) protects the eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, and this promotes protein synthesis in the presence of active eIF-2 alpha kinases in vitro [Ray, M. K., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539-543]. We have now examined the effect of overexpression of this cellular eIF-2 kinase inhibitor in an in vivo system using transiently transfected COS-l cells. In this system, coexpression of genes that inhibit PKR activity restores translation of plasmid-derived mRNA. We now report the following. (1) Transient transfection of COS-1 cells with a p67 expression vector increased p67 synthesis by 20-fold over endogenous levels in the isolated subpopulation of transfected cells. (2) Cotransfection of p67 cDNA increased translation of plasmid-derived mRNAs. (3) Overexpression of p67 reduced phosphorylation of coexpressed eIF-2 alpha. (4) p67 synthesis was inhibited by cotransfection with an eIF-2 alpha mutant S51D, a mutant that mimics phosphorylated eIF-2 alpha, indicating that p67 cannot bypass translational inhibition mediated by phosphorylation of the eIF-2 alpha-subunit. These results show that the cellular protein p67 can reverse PKR-mediated translational inhibition in intact cells.     

Ribosome targeting of PKR is mediated by two double-stranded RNA-binding domains and facilitates in vivo phosphorylation of eukaryotic initiation factor-2

Protein kinase PKR is activated in mammalian cells during viral infection, leading to phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha) and inhibition of protein synthesis. This antiviral response is thought to be mediated by association of double-stranded RNA (ds-RNA), a by-product of viral replication, with two ds-RNA-binding domains (DRBDs) located in the amino terminus of PKR. Recent studies have observed that expression of mammalian PKR in yeast leads to a slow growth phenotype due to hyperphosphorylation of eIF-2alpha. In this report, we observed that while DRBD sequences are required for PKR to function in the yeast model system, these sequences are not required for in vitro phosphorylation of eIF-2alpha. To explain this apparent contradiction, we proposed that these sequences are required to target the kinase to the translation machinery. Using sucrose gradient sedimentation, we found that wild-type PKR was associated with ribosomes, specifically with 40 S particles. Deletions or residue substitutions in the DRBD sequences blocked kinase interaction with ribosomes. These results indicate that in addition to mediating ds-RNA control of PKR, the DRBD sequences facilitate PKR association with ribosomes. Targeting to ribosomes may enhance in vivo phosphorylation of eIF-2alpha, by providing PKR access to its substrate.     

Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control

In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). In mammals, the phosphorylation was shown to be carried out by eIF-2alpha kinases PKR and HRI. We report the identification and characterization of a cDNA from rat pancreatic islet cells that encodes a new related kinase, which we term pancreatic eIF-2alpha kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2alpha kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced in Escherichia coli or Sf-9 insect cells, we demonstrate that PEK is autophosphorylated on both serine and threonine residues and that the recombinant enzyme can specifically phosphorylate eIF-2alpha on serine-51. Northern blot analyses indicate that PEK mRNA is expressed in all tissues examined, with highest levels in pancreas cells. Consistent with our mRNA assays, PEK activity was predominantly detected in pancreas and pancreatic islet cells. The regulatory role of PEK in protein synthesis was demonstrated both in vitro and in vivo. The addition of recombinant PEK to reticulocyte lysates caused a dose-dependent inhibition of translation. In the Saccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2alpha kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2alpha. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes, suggesting that this eIF-2alpha kinase plays an important role in translational control from nematodes to mammals.     

Regulation of T cell activation in vitro and in vivo by targeting the OX40-OX40 ligand interaction: amelioration of ongoing inflammatory bowel disease with an OX40-IgG fusion protein, but not with an OX40 ligand-IgG fusion protein

The role of GRP78/BiP in coordinating endoplasmic reticular (ER) protein processing with mRNA translation was examined in GH3 pituitary cells. ADP-ribosylation of GRP78 and eukaryotic initiation factor (eIF)-2alpha phosphorylation were assessed, respectively, as indices of chaperone inactivation and the inhibition of translational initiation. Inhibition of protein processing by ER stress (ionomycin and dithiothreitol) resulted in GRP78 deribosylation and eIF-2 phosphorylation. Suppression of translation relative to ER protein processing (cycloheximide) produced approximately 50% ADP-ribosylation of GRP78 within 90 min without eIF-2 phosphorylation. ADP-ribosylation was reversed in 90 min by cycloheximide removal in a manner accelerated by ER stressors. Cycloheximide sharply reduced eIF-2 phosphorylation in response to ER stressors for about 30 min; sensitivity returned as GRP78 became increasingly ADP-ribosylated. Reduced sensitivity of eIF-2 to phosphorylation appeared to derive from the accumulation of free, unmodified chaperone as proteins completed processing without replacements. Prolonged (24 h) incubations with cycloheximide resulted in the selective loss of the ADP-ribosylated form of GRP78 and increased sensitivity of eIF-2 phosphorylation in response to ER stressors. Brefeldin A decreased ADP-ribosylation of GRP78 in parallel with increased eIF-2 phosphorylation. The cytoplasmic stressor, arsenite, which inhibits translational initiation through eIF-2 phosphorylation without affecting the ER, also produced ADP-ribosylation of GRP78.     

Regulation of protein synthesis in ventricular myocytes by vasopressin. The role of sarcoplasmic/endoplasmic reticulum Ca2+ stores

Protein synthesis in H9c2 ventricular myocytes was subject to rapid inhibition by agents that release Ca2+ from the sarcoplasmic/endoplasmic reticulum, including thapsigargin, ionomycin, caffeine, and arginine vasopressin. Inhibitions were attributable to the suppression of translational initiation and were coupled to the mobilization of cell-associated Ca2+ and the phosphorylation of eIF2alpha. Ionomycin and thapsigargin produced relatively stringent degrees of Ca2+ mobilization that produced an endoplasmic reticulum (ER) stress response. Translational recovery was associated with the induction of ER chaperones and resistance to translational inhibition by Ca2+-mobilizing agents. Vasopressin at physiologic concentrations mobilized 60% of cell-associated Ca2+ and decreased protein synthesis by 50% within 20-30 min. The inhibition of protein synthesis was exerted through an interaction at the V1 vascular receptor, was imposed at physiologic extracellular Ca2+ concentrations, and became refractory to hormonal washout within 10 min of treatment. Inhibition was found to attenuate after 30 min, with full recovery occurring in 2 h. Translational recovery did not involve an ER stress response but rather was derived from the partial repletion of intracellular Ca2+ stores. Longer exposures to vasopressin were invariably accompanied by increased rates of protein synthesis. Translational inhibition by vasopressin, but not by Ca2+-mobilizing drugs, was both preventable and reversible by treatment with phorbol ester, which reduced the extent of Ca2+ mobilization occurring in response to the hormone. Larger and more prolonged translational inhibitions occurred after down-regulation of protein kinase C. This report provides the first compelling evidence that hormonally induced mobilization of sarcoplasmic/endoplasmic reticulum Ca2+ stores is regulatory upon mRNA translation.     

Phosphorylation of plant eukaryotic initiation factor-2 by the plant-encoded double-stranded RNA-dependent protein kinase, pPKR, and inhibition of protein synthesis in vitro

Regulation of protein synthesis by eukaryotic initiation factor-2alpha (eIF-2alpha) phosphorylation is a highly conserved phenomenon in eukaryotes that occurs in response to various stress conditions. Protein kinases capable of phosphorylating eIF-2alpha have been characterized from mammals and yeast. However, the phenomenon of eIF2-alpha-mediated regulation of protein synthesis and the presence of an eIF-2alpha kinase has not been demonstrated in higher plants. We show that plant eIF-2alpha (peIF-2alpha) and mammalian eIF-2alpha (meIF-2alpha) are phosphorylated similarly by both the double-stranded RNA-binding kinase, pPKR, present in plant ribosome salt wash fractions and the meIF-2alpha kinase, PKR. By several criteria, phosphorylation of peIF-2alpha is directly correlated with pPKR protein and autophosphorylation levels. Significantly, pPKR is capable of specifically phosphorylating Ser51 in a synthetic eIF-2alpha peptide, a key characteristic of the eIF-2alpha kinase family. Taken together, these data support the concept that pPKR is a member of the eIF-2alpha kinase family. In addition, the inhibition of brome mosaic virus RNA in vitro translation in wheat germ lysates by the addition of double-stranded RNA, phosphorylated peIF-2alpha, meIF-2alpha, or activated human PKR suggests that plant protein synthesis may be regulated via phosphorylation of eIF-2alpha.     

Double-stranded (ds) RNA binding and not dimerization correlates with the activation of the dsRNA-dependent protein kinase (PKR)

Upon binding to double-stranded (ds) RNA, the dsRNA-dependent protein kinase (PKR) sequentially undergoes autophosphorylation and activation. Activated PKR may exist as a dimer and phosphorylates the eukaryotic translation initiation factor 2 alpha subunit (cIF-2 alpha) to inhibit polypeptide chain initiation. Transfection of COS-1 cells with a plasmid cDNA expression vector encoding a marker gene, activates endogenous PKR, and selectively inhibits translation of the marker mRNA, dihydrofolate reductase (DHFR). This system was used to study the dsRNA binding and dimerization requirements for over-expressed PKR mutants and subdomains to affect DHFR translation. DHFR translation was rescued by expression of either an ATP hydrolysis defective mutant PKR K296P, the amino-terminal 1-243 fragment containing two dsRNA binding motifs, or the isolated first RNA binding motif (amino acids 1-123). Mutation of K64E within the dsRNA binding motif 1 destroyed dsRNA binding and the ability to rescue DHFR translation. Immunoprecipitation of T7 epitope-tagged PKR derivatives from cell lysates detected interaction between intact PKR and the amino-terminal 1-243 fragment as well as a 1-243 fragment harboring the K64E mutation. Expression of adenovirus VAI RNA, a potent inhibitor of PKR activity, did not disrupt this interaction. In contrast, intact PKR did not interact with fragments containing the first dsRNA binding motif (1-123), the second dsRNA binding motif (98-243), or the isolated PKR kinase catalytic domain (228-551). These results demonstrate that the translational stimulation mediated by the dominant negative PKR mutant does not require dimerization, but requires the ability to bind dsRNA and indicate these mutants act by competition for binding to activators.     

Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and lambda repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.     

The herpes simplex virus US11 protein effectively compensates for the gamma1(34.5) gene if present before activation of protein kinase R by precluding its phosphorylation and that of the alpha subunit of eukaryotic translation initiation factor 2

In herpes simplex virus-infected cells, viral gamma134.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1alpha to dephosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). The amino acid sequence of the gamma134.5 protein which interacts with the phosphatase has high homology to a domain of the eukaryotic protein GADD34. A class of compensatory mutants characterized by a deletion which results in the juxtaposition of the alpha47 promoter next to US11, a gamma2 (late) gene in wild-type virus-infected cells, has been described. In cells infected with these mutants, protein synthesis continues even in the absence of the gamma134.5 gene. In these cells, PKR is activated but eIF-2alpha is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2alpha. We report the following: (i) in cells infected with these mutants, US11 protein was made early in infection; (ii) US11 protein bound PKR and was phosphorylated; (iii) in in vitro assays, US11 blocked the phosphorylation of eIF-2alpha by PKR activated by poly(I-C); and (iv) US11 was more effective if present in the reaction mixture during the activation of PKR than if added after PKR had been activated by poly(I-C). We conclude the following: (i) in cells infected with the compensatory mutants, US11 made early in infection binds to PKR and precludes the phosphorylation of eIF-2alpha, whereas US11 driven by its natural promoter and expressed late in infection is ineffective; and (ii) activation of PKR by double-stranded RNA is a common impediment countered by most viruses by different mechanisms. The gamma134.5 gene is not highly conserved among herpesviruses. A likely scenario is that acquisition by a progenitor of herpes simplex virus of a portion of the cellular GADD34 gene resulted in a more potent and reliable means of curbing the effects of activated PKR. US11 was retained as a gamma2 gene because, like many viral proteins, it has multiple functions.     

The 58-kilodalton inhibitor of the interferon-induced double-stranded RNA-activated protein kinase is a tetratricopeptide repeat protein with oncogenic properties

The interferon-induced RNA-dependent protein kinase (PKR) is considered to play an important role in the cellular defense against viral infection and, in addition, has been suggested to be a tumor suppressor gene because of its growth-suppressive properties. Activation of PKR by double-stranded RNAs leads to the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) and a resultant block to protein synthesis initiation. To avoid the consequences of kinase activation, many viruses have developed strategies to down-regulate PKR. Recently, we reported on the purification and characterization of a cellular inhibitor of PKR (referred to as p58), which is activated during influenza virus infection. Subsequent cloning and sequencing has revealed that p58 is a member of the tetratricopeptide repeat (TPR) family of proteins. To further examine the physiological role of this PKR inhibitor, we stably transfected NIH 3T3 cells with a eukaryotic expression plasmid containing p58 cDNA under control of the cytomegalovirus early promoter. By taking advantage of a recently characterized p58 species-specific monoclonal antibody, we isolated cell lines that overexpressed p58. These cells exhibited a transformed phenotype, growing at faster rates and higher saturation densities and exhibiting anchorage-independent growth. Most importantly, inoculation of nude mice with p58-overexpressing cells gave rise to the production of tumors. Finally, murine PKR activity and endogenous levels of eIF-2 alpha phosphorylation were reduced in the p58-expressing cell lines compared with control cells. These data, taken together, suggest that p58 functions as an oncogene and that one mechanism by which the protein induces malignant transformation is through the down-regulation of PKR and subsequent deregulation of protein synthesis.     

Mutations in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) that overcome the inhibitory effect of eIF-2 alpha phosphorylation on translation initiation

Phosphorylation of eIF-2 alpha in Saccharomyces cerevisiae by the protein kinase GCN2 leads to inhibition of general translation initiation and a specific increase in translation of GCN4 mRNA. We isolated mutations in the eIF-2 alpha structural gene that do not affect the growth rate of wild-type yeast but which suppress the toxic effects of eIF-2 alpha hyperphosphorylation catalyzed by mutationally activated forms of GCN2. These eIF-2 alpha mutations also impair translational derepression of GCN4 in strains expressing wild-type GCN2 protein. All four mutations alter single amino acids within 40 residues of the phosphorylation site in eIF-2 alpha; however, three alleles do not decrease the level of eIF-2 alpha phosphorylation. We propose that these mutations alter the interaction between eIF-2 and its recycling factor eukaryotic translation initiation factor 2B (eIF-2B) in a way that diminishes the inhibitory effect of phosphorylated eIF-2 on the essential function of eIF-2B in translation initiation. These mutations may identify a region in eIF-2 alpha that participates directly in a physical interaction with the GCN3 subunit of eIF-2B.     

Depletion of intracellular Ca2+ stores, phosphorylation of eIF2alpha, and sustained inhibition of translation initiation mediate the anticancer effects of clotrimazole

Regulation of translation initiation plays a critical role in the control of cell growth and division in eukaryotic cells. Translation of many growth regulatory proteins including cyclins depends critically on translation initiation factors because their mRNAs are translated inefficiently. We report that clotrimazole, a potent antiproliferative agent both in vitro and in vivo, inhibits cell growth by interfering with translation initiation. In particular, clotrimazole causes a sustained depletion of intracellular Ca2+ stores, which results in activation of PKR, phosphorylation of eIF2alpha, and thereby in inhibition of protein synthesis at the level of translation initiation. Consequently, clotrimazole preferentially decreases the expression of the growth promoting proteins cyclin A, E and D1, resulting in inhibition of cyclin-dependent kinase activity and blockage of cell cycle in G1.     

Cultivation of cell-polymer cartilage implants in bioreactors

Primary T-cells are metabolically quiescent, with little DNA, RNA or protein synthesis. Upon mitogenic stimulation the rate of protein synthesis increases 10-fold. We have studied the role of eIF-2 and eIF-4 alpha (eIF-4E) expression in the mechanism of translational activation. During this period, the levels of eIF-2 alpha and eIF-4 alpha mRNA increase some 50-fold. Similar to the increase in ribosomes and mRNA, the number of eIF-2 alpha, eIF-2 beta, and eIF-4 alpha molecules per cell also increase 2-3-fold. This suggests that in addition to an increase in the pool size of translational components, an additional mechanism exists which results in an increased efficiency of factor utilization. We have looked at initiation factor phosphorylation. We find that eIF-2 alpha does not undergo significant changes in its phosphorylation state nor is there a change in the efficiency of eIF-2 utilization. However, there is a rapid increase in the phosphorylation state of eIF-4 alpha which correlates with the rapid increase in translational activity. It thus appears there are 2 distinct components responsible for the translational activation of quiescent T-cells during mitogenic stimulation. The first is the phosphorylation of eIF-4 alpha, with a concomitant increase in the efficiency of eIF-4 alpha utilization. The second is an increase in the pool sizes of eIF-2 and eIF-4 alpha.     

Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2alpha kinase homolog

Phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) is a common cellular mechanism to limit protein synthesis in stress conditions. Baculovirus PK2, which resembles the C-terminal half of a protein kinase domain, was found to inhibit both human and yeast eIF2alpha kinases. Insect cells infected with wild-type, but not pk2-deleted, baculovirus exhibited reduced eIF2alpha phosphorylation and increased translational activity. The negative regulatory effect of human protein kinase RNA-regulated (PKR), an eIF2alpha kinase, on virus production was counteracted by PK2, indicating that baculoviruses have evolved a unique strategy for disrupting a host stress response. PK2 was found in complex with PKR and blocked kinase autophosphorylation in vivo, suggesting a mechanism of kinase inhibition mediated by interaction between truncated and intact kinase domains.     

Ribosome-binding domain of eukaryotic initiation factor-2 kinase GCN2 facilitates translation control

A family of protein kinases regulate translation initiation in response to cellular stresses by phosphorylation of eukaryotic initiation factor-2 (eIF-2). One family member from yeast, GCN2, contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic domain. It is thought that uncharged tRNA accumulating during amino acid starvation binds to the synthetase-related sequences and stimulates phosphorylation of the alpha subunit of eIF-2. In this report, we define another domain in GCN2 that functions to target the kinase to ribosomes. A truncated version of GCN2 containing only amino acid residues 1467 to 1590 can independently associate with the translational machinery. Interestingly, this region of GCN2 shares sequence similarities with the core of the double-stranded RNA-binding domain (DRBD). Substitutions of the lysine residues conserved among DRBD sequences block association of GCN2 with ribosomes and impaired the ability of the kinase to stimulate translational control in response to amino acid limitation. Additionally, as found for other DRBD sequences, recombinant protein containing GCN2 residues 1467-1590 can bind double-stranded RNA in vitro, suggesting that interaction with rRNA mediates ribosome targeting. These results indicate that appropriate ribosome localization of the kinase is an obligate step in the mechanism leading to translational control by GCN2.     

Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2alpha kinases PKR and GCN2

The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit alpha (eIF2alpha) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2alpha kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.     

Mutant influenza viruses with a defective NS1 protein cannot block the activation of PKR in infected cells

A short model genome RNA and also the genome RNA of influenza A virus bearing both 5'- and 3'-terminal common sequences activated the interferon-induced double-stranded-RNA-dependent protein kinase, PKR, by stimulating autophosphorylation in vitro. The activated PKR catalyzed phosphorylation of the alpha subunit of eucaryotic translation initiation factor 2 (eIF2alpha). The NS1 protein efficiently eliminated the PKR-activating activity of these RNAs by binding to them. Two mutant NS1 proteins, each harboring a single amino acid substitution at different regions, exhibited temperature sensitivity in their RNA binding activity in the mutant virus-infected cell lysates as well as when they were prepared as fusion proteins expressed in bacteria. The virus strains carrying these mutant NS1 proteins exhibited temperature sensitivity in virus protein synthesis at the translational level, as reported previously, and could not repress the autophosphorylation of PKR developing during the virus growth, which is normally suppressed by a viral function(s). As a result, the level of eIF2alpha phosphorylation was elevated 2.5- to 3-fold. The defect in virus protein synthesis was well correlated with the level of phosphorylation of PKR and eIF2alpha.     

Antibodies to the E2 protein of GB virus C/hepatitis G virus: low prevalence in Asian countries

The alpha-subunit of eukaryotic initiation factor eIF2 (eIF2alpha) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulated in vivo by phosphorylation of eIF2alpha at Ser51. In the present study, rat eIF2alpha was expressed in Sf21 cells using the baculovirus expression system. The recombinant protein was purified to >90% homogeneity in a single immunoaffinity chromatographic step. The protein was free of endogenous eIF2alpha kinase activity and was rapidly phosphorylated by the eIF2alpha kinases HCR and PKR. A variant of eIF2alpha in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2alpha was not phosphorylated by either HCR or PKR, demonstrating that the kinases specifically phosphorylate the correct site in the recombinant protein even in the absence of the other two subunits of the protein. In summary, a rapid and inexpensive method for obtaining eIF2alpha has been developed. Use of the wildtype and variant forms of eIF2alpha to measure eIF2alpha kinase activity in cell and tissue extracts should greatly facilitate examination of the regulation of mRNA translation under a variety of conditions.