Inhibition of protein synthesis in reovirus-infected HeLa cells with elevated levels of interferon-induced protein kinase activity

Protein synthesis was inhibited in one line of interferon-treated HeLa cells (line 2) upon infection with reovirus, but not in different HeLa cells (line 1) treated in the same way. The inhibition resulted in polysome runoff, suggesting that it was due to an impairment of peptide chain initiation. Interferon induces the synthesis of a protein kinase, which is activated in cell-free systems by double-stranded RNA and phosphorylates the alpha subunit of eukaryotic initiation factor 2, thus inhibiting the initiation of protein synthesis. Therefore, we measured the level of this protein kinase in extracts prepared from the two HeLa cell lines. Cells of line 2 showed about 3-4 times more protein kinase activity than cells of line 1. The inhibition of protein synthesis upon infection with reovirus was correlated with an increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 in interferon-treated cells labeled with 32P. The kinase was presumably activated in intact cells by viral double-stranded RNA, but this activation resulted in inhibition of protein synthesis only in cells with elevated levels of the kinase.     

Regulation of protein synthesis in rabbit reticulocyte lysates by guanosine triphosphate

GTP (2 mM) promotes protein synthesis in rabbit reticulocyte lysates in which protein chain initiation is inhibited by the activation of specific adenosine 3′:5′ cyclic monophosphate independent protein kinases in: 1) heme deficiency; or 2) in hemin-supplemented lysates by the addition of the purified heme-regulated protein kinase (HRI); or 3) oxidized glutathione; or 4) by low levels of double stranded RNA. The molecular basis for the promotion of protein synthesis by GTP under these various conditions was investigated by examining the $\text{in}\text{,}\text{situ}$ state of eIF-2 phosphorylation. The results show that GTP (2 mM) blocks eIF-2 phosphorylation and also promotes the dephosphorylation of phosphorylated eIF-2. These findings suggest that GTP restores protein synthesis by a common mechanism that involves the relief of eIF-2 from phosphorylation. The nonphosphorylated eIF-2 is, therefore, available for the maintenance and the restoration of protin chain initiation cycle.     

Role of reversing factor in the inhibition of protein synthesis initiation by oxidized glutathione

The inhibitions of protein synthesis initiation in heme-deficient reticulocyte lysates and in GSSG-treated hemin-supplemented lysates are both characterized by the activation of heme-regulated eIF-2 alpha kinase, which phosphorylates the alpha-subunit of eukaryotic initiation factor (eIF-2). In both inhibitions, the accumulation of eIF phosphorylated in alpha-subunit (eIF-2(alpha P)) leads to the sequestration of reversing factor (RF) in a phosphorylated 15 S complex, RF.eIF-2(alpha P), in which RF is nonfunctional. A sensitive assay for the detection of endogenous RF activity in protein-synthesizing lysates indicates that, in GSSG-inhibited (1 mM GSSG) lysates, RF is more profoundly inhibited than in heme-deficient lysates. RF inactivation in GSSG-induced inhibition appears to be due to two separate but additive effects: (i) the formation of the phosphorylated 15 S RF complex, RF.eIF-2(alpha P), and (ii) the formation of disulfide complexes which inhibit RF activity. Both inhibitory effects are overcome by catalytic levels of exogenous RF which permits the resumption of protein synthesis. RF activity and protein synthesis in GSSG-inhibited lysates are efficiently restored by the delayed addition of glucose-6-P or 2-deoxyglucose-6-P (1 mM). The rescue of protein synthesis by hexose phosphate (1 mM) is proportional to the extent of RF recovery and is due in part to NADPH generation; even at levels of hexose phosphate (50 microM) too low to support protein synthesis, partial restoration of RF activity occurs due to increased NADPH/NADP+ ratios. The ability of dithiothreitol (1 mM) to restore RF activity in GSSG-treated but not heme-deficient lysates also provides evidence for a reducing mechanism which functions at the level of RF. The results suggest that NADPH plays a role in the maintenance of sulfhydryl groups essential for RF activity.     

A kinase able to phosphorylate exogenous protein synthesis initiation factor eIF-2 alpha is present in lysates of mengovirus-infected L cells.

Infection of mouse L929 cells by mengovirus resulted in the expression of a kinase activity that selectively phosphorylated the small, 38,000-molecular-weight subunit of eucaryotic initiation factor 2 and histone H2. This kinase activity was independent of host cell RNA synthesis and was located in the postribosomal supernatant (S-100 fraction) early after infection (up to 3 h). At later times after infection (5 h), kinase activity was also associated with the polysome fraction. The kinase present in the S-100 fraction bound strongly to DEAE-cellulose, its peak activity eluting at 0.5 M KCl. Kinase activity was independent of the presence of exogenous double-stranded RNA, and KCl at concentrations greater than 0.1 M inhibited eucaryotic initiation factor 2 phosphorylation. The 67,000-molecular-weight phosphoprotein activated in interferon-treated cells by double-stranded RNA was not detected by standard phosphorylation assays in lysates from mengovirus-infected cells. Labeling of this protein in vivo during 5 h of infection was also not detected. The DEAE-cellulose-purified mengovirus kinase inhibited protein synthesis in reticulocyte lysates, and the inhibition was not reversible by high concentrations of poly(I).poly(C).     

Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis

Protein synthesis, in particular peptide-chain elongation, consumes cellular energy. Anoxia activates AMP-activated protein kinase (AMPK, see ), resulting in the inhibition of biosynthetic pathways to conserve ATP. In anoxic rat hepatocytes or in hepatocytes treated with 5-aminoimidazole-4-carboxamide (AICA) riboside, AMPK was activated and protein synthesis was inhibited. The inhibition of protein synthesis could not be explained by changes in the phosphorylation states of initiation factor 4E binding protein-1 (4E-BP1) or eukaryotic initiation factor 2alpha (eIF2alpha). However, the phosphorylation state of eukaryotic elongation factor 2 (eEF2) was increased in anoxic and AICA riboside-treated hepatocytes and in AICA riboside-treated CHO-K1 cells, and eEF2 phosphorylation is known to inhibit its activity. Incubation of CHO-K1 cells with increasing concentrations of 2-deoxyglucose suggested that the mammalian target of the rapamycin (mTOR) signaling pathway did not play a major role in controlling the level of eEF2 phosphorylation in response to mild ATP depletion. In HEK293 cells, transfection of a dominant-negative AMPK construct abolished the oligomycin-induced inhibition of protein synthesis and eEF2 phosphorylation. Lastly, eEF2 kinase, the kinase that phosphorylates eEF2, was activated in anoxic or AICA riboside-treated hepatocytes. Therefore, the activation of eEF2 kinase by AMPK, resulting in the phosphorylation and inactivation of eEF2, provides a novel mechanism for the inhibition of protein synthesis.     

Phosphorylation of initiation factor elF-2 and the control of reticulocyte protein synthesis

When rabbit reticulocyte lysates are incubated in the absence of hemin or in the presence of low concentrations of double-stranded RNA, the rate of initiation of protein synthesis is severely reduced after a lag period in which control rates are observed. This reduced initiation rate is due to inhibition of the binding of Methionyl-tRNAf to native 40S ribosomal subunits and is caused by a macromolecular inhibitor which is activated under these conditions. This paper shows that the inhibitors activated in these two situations appear to be different entities, but that in both cases, the inhibitor has an associated protein kinase activity which is highly selective for the small subunit of elF-2, the initiation factor which catalyzes binding of Methionyl-tRNAf to 40S subunits. We present several lines of evidence in support of the hypothesis that the phosphorylation of elF-2 by these kinases is basis of the control of initiation in lysates incubated under these conditions.     

Glucosamine-induced endoplasmic reticulum stress attenuates apolipoprotein B100 synthesis via PERK signaling

Glucosamine impairs hepatic apolipoprotein B100 (apoB100) production by inducing endoplasmic reticulum (ER) stress and enhancing cotranslational and posttranslational apoB100 degradation (Qiu, W., R. K. Avramoglu, A. C. Rutledge, J. Tsai, and K. Adeli. Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins. J. Lipid Res. 2006. 47: 1749–1761). Here, we report that glucosamine also regulates apoB100 protein synthesis via ER-stress-induced PERK activation. Short-term (4 h) glucosamine treatment of HepG2 cells reduced both cellular (by 62%) and secreted apoB100 (by 43%) without altering apoB100 mRNA. Treatment with proteasomal inhibitors only partially prevented the suppressive effects of glucosamine, suggesting that mechanisms other than proteasomal degradation may also be involved. Glucosamine-induced ER stress was associated with a significantly reduced apoB100 synthesis with no significant change in posttranslational decay rates, suggesting that glucosamine exerted its effect early during apoB biosynthesis. The role of PERK and its substrate, α-subunit of eukaryotic initiation factor 2 (eIF2α), in the suppressive effects of glucosamine on apoB synthesis was then investigated. Coexpression of apoB15 (normally resistant to intracellular degradation) with wild-type double stranded (ds) RNA activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) in COS-7 cells resulted in a dramatic reduction in the levels of newly synthesized apoB15. Interestingly, cotransfection with apoB15 and a kinase inactive PERK mutant (K618A) increased apoB15 expression. In addition, short-term glucosamine treatment stimulated an increase in phosphorylation of PERK and eIF2α. Taken together, these data suggest that in addition to the induction of ER-associated degradation and other degradative pathways, ER stress is associated with suppression of apoB synthesis via a PERK-dependent mechanism.     

Inhibition of protein synthesis in reticulocyte lysates by a double-stranded RNA component in HeLa mRNA

Double-stranded RNA (dsRNA) inhibits protein synthesis initiation in rabbit reticulocyte lysates by the activation of a latent dsRNA-dependent cAMP-independent protein kinase which phosphorylates the α-subunit of the eukaryotic initiation factor eIF-2. In this study, we describe a dsRNA-like component which is present in preparations of HeLa mRNA (poly A⁺) isolated from total cytoplasmic RNA. The inhibitory species in the HeLa cytoplasmic mRNA was detected by (a) its ability to inhibit protein synthesis with biphasic kinetics in reticulocyte lysates translating endogenous globin mRNA, and (b) by the inefficient translation of HeLa cytoplasmic mRNA in a nuclease-treated mRNA-dependent reticulocyte lysate. The inhibitory component was characterized as dsRNA by several criteria including (i) the ability to activate the lysate dsRNA-dependent eIF-2α kinase (dsI); (ii) the prevention of both dsI activation and inhibition of protein synthesis by high levels of dsRNA or cAMP; (iii) the reversal of inhibition by eIF-2; and (iv) the inability to inhibit protein synthesis in wheat germ extracts which lack latent dsI. By the same criteria, the putative dsRNA component(s) appears to be absent from preparations of HeLa mRNA isolated exclusively from polyribosomes.     

The Atlantic salmon Z-DNA binding protein kinase phosphorylates translation initiation factor 2 alpha and constitutes a unique orthologue to the mammalian dsRNA-activated protein kinase R

The translation initiation factor 2 alpha (eIF2alpha)-kinase, dsRNA-activated protein kinase (PKR), constitutes one of the major antiviral proteins activated by viral infection of vertebrates. PKR is activated by viral double-stranded RNA and subsequently phosphorylates the alpha-subunit of translation initiation factor eIF2. This results in overall down regulation of protein synthesis in the cell and inhibition of viral replication. Fish appear to have a PKR-like protein that has Z-DNA binding domains instead of dsRNA binding domains in the regulatory domain, and has thus been termed Z-DNA binding protein kinase (PKZ). We present the cloning of the Atlantic salmon PKZ cDNA and show its upregulation by interferon in Atlantic salmon TO cells and poly inosinic poly cytodylic acid in head kidney. We also demonstrate that recombinant Atlantic salmon PKZ, expressed in Escherichia coli, phosphorylates eIF2alphain vitro. This is the first demonstration that PKZ is able to phosphorylate eIF2alpha. PKZ activity, as measured by phosphorylation of eIF2alpha, was increased after addition of Z-DNA, but not by dsRNA. In addition, we show that wild-type Atlantic salmon PKZ, but not the kinase defective variant K217R, has a direct inhibitory effect on protein synthesis after transient expression in Chinook salmon embryo cells. Overall, the results support a role for PKZ, like PKR, in host defense against virus infection.     

The effects of haem on translational control of protein synthesis in cell-free extracts from fed and lysine-derived Ehrlich ascites tumour cells

1. Addition of haem to cell-free extracts from Ehrlich ascites tumour cells stimulates protein synthesis only in extracts from cells previously incubated in nutritionally complete conditions. Extracts from amino-acid-deprived cells do not respond to haem. The stimulation of protein synthesis in fed cell extracts is due to increased initiation on endogenous mRNA mediated by an increase in the levels of 40-S-subunit X Met-tRNA initiation complexes. Extracts from starved cells exhibit a defect in 40-S initiation complex formation which cannot be overcome by haem. 2. Experiments to test for the presence of an inhibitor of initiation in Ehrlich cell extracts by monitoring effects on translation in haem-supplemented reticulocyte lysates have revealed that extracts from both fed and starved cells contain one or more inhibitory activities which shut off protein synthesis, dissagregate polysomes and reduce the level of 40-S initiation complexes in the lysate. Extracts from starved cells are more inhibitory for protein synthesis than those from fed cells. 3. Initiation factor eIF-2 is phosphorylated by an endogenous Ehrlich cell protein kinase in vitro, but this occurs to the same extent in extracts from fed and starved cells. 4. We propose a possible model for the role of eIF-2 in the control of protein synthesis by amino acid supply in Ehrlich cells.     

Interferon-induced dsRNA-activated protein kinase (PKR)*: antiproliferative,antiviral and antitumoral functions

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a serine/threonine kinase induced in mammalian cells by interferon. It is responsible for the phosphorylation of the protein synthesis initiation factor eIF-2, thus mediating inhibition of protein synthesis. Studies on the expression of the human protein kinase in the yeast Saccharomyces cerevisae and in murine NIH 3T3 cells, provided evidence for the antiproliferative, antiviral and antitumoral functions of this dsRNA activated protein kinase.     

Lack of inhibition of protein synthesis by double-stranded RNA in a cell-free system prepared from human reticulocytes

There are two inhibitors of protein synthesis which are related to the activity of interferon. One is a protein kinase which phosphorylates the α subunit of the eucaryotic initiation factor 2 (eIF-2). The other is an enzyme which synthesizes an unusual oligonucleotide that in turn activates a RNA endonuclease. In nucleated cells the synthesis of the inhibitors is induced by interferon but they must be activated in a subsequent lysate by double-stranded RNA (dsRNA). Rabbit reticulocytes, however, contain the inactive forms of the inhibitors in a constitutive manner and require only dsRNA activation. We report here the effect of dsRNA on protein synthesis and the generation of ribosomal eIF-2α kinase and heat-stable (oligonucleotide) inhibitory activity in human reticulocyte lysates. Our findings indicate that human reticulocytes, in contrast to rabbit reticulocytes, do not contain the interferon-related inhibitors of protein synthesis in a constitutive manner. Addition of dsRNA to the human reticulocyte cell-free system does not result in significant inhibition. Furthermore, no generation of ribosomal eIF-2α kinase or heatstable inhibitory activity could be detected. Direct addition of oligonucleotide or eIF-2α kinase (of rabbit origin), however, does result in inhibition of the human system. Thus, the ultimate inhibition mechanisms do appear operative in the human reticulocyte lysates. The differences between the rabbit and human systems may be due to either basic differences in the mechanism of interferon action or simply to variation in the history or maturity of the cells studied.     

Partial purification of a ribonucleic acid cAMP-independent protein kinase from embryonic chicken muscle

A cAMP-indepedent protein kinase (P38 kinase) from embryonic chicken muscle with ability to phosphorylate a 38,000 molecular weight polypeptide and to bind to RNAs has been further characterized. An approximately 2000-fold purification of this enzyme was achieved by a combination of affinity and ion-exchange chromatography. Our studies indicate that this protein kinase can not phosphorylate the small subunit of rabbit reticulocyte initiation factor eIF-2 in the presence of its normal endogenous substrate, nor is it activated over a wide range of concentrations of double-stranded RNA. This P38 kinase is, therefore, distinct from the hemin-regulated translational inhibitor of protein synthesis in rabbit reticulocytes and from the interferon-induced protein kinase identified In several systems.     

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA

Translation of the hepatitis C genome is mediated by internal ribosome entry on the structurally complex 5' untranslated region of the large viral RNA. Initiation of protein synthesis by this mechanism is independent of the cap-binding factor eIF4E, but activity of the initiator Met-tRNA(f)-binding factor eIF2 is still required. HCV protein synthesis is thus potentially sensitive to the inhibition of eIF2 activity that can result from the phosphorylation of the latter by the interferon-inducible, double-stranded RNA-activated protein kinase PKR. Two virally encoded proteins, NS5A and E2, have been shown to reduce this inhibitory effect of PKR by impairing the activation of the kinase. Here we present evidence for a third viral strategy for PKR inhibition. A region of the viral RNA comprising part of the internal ribosome entry site (IRES) is able to bind to PKR in competition with double-stranded RNA and can prevent autophosphorylation and activation of the kinase in vitro. The HCV IRES itself has no PKR-activating ability. Consistent with these findings, cotransfection experiments employing a bicistronic reporter construct and wild-type PKR indicate that expression of the protein kinase is less inhibitory towards HCV IRES-driven protein synthesis than towards cap-dependent protein synthesis. These data suggest a dual function for the viral IRES, with both a structural role in promoting initiation complex formation and a regulatory role in preventing inhibition of initiation by PKR.     

Purification and characterization of a protein synthesis inhibitor associated with vaccinia virus

A protein synthesis inhibitor, solubilized from vaccinia virus (Ben-Hamida, F., Person, A., and Beaud, G. (1983) J. Virol. 45, 452-455), has been purified to homogeneity, yielding a basic protein with molecular mass of 11 kDa. This purified protein migrates as a single spot in two-dimensional gel analysis (isoelectric point above 8.6). It is phosphorylated by the vaccinia-associated protein kinase, and it aggregates in the absence of reducing agents. This 11-kDa protein inhibits protein synthesis when added to a reticulocyte lysate at a stoichiometric ratio of approximately one protein molecule/ribosome, and it associates with the ribosome fraction after incubation in reticulocyte lysates or in Ehrlich ascites tumor cell lysates. As previously described for the inhibitor associated with vaccinia cores, the purified inhibitor inhibits the formation of the 40 S ribosomal subunit X Met-tRNAi ribosomal initiation complex. It has no detectable effect on the formation of the ternary complex (Met-tRNAi X GTP X eucaryotic initiation factor 2). This inhibitor associated with vaccinia virus particles may be involved in the shutoff of host protein synthesis and may also be responsible for the absence of virus replication in some cell-virus systems.