Cesar de Haro

Antiviral effect of the mammalian translation initiation factor 2α kinase GCN2 against RNA viruses

In mammals, four different protein kinases, heme‐regulated inhibitor, double‐stranded RNA‐dependent protein kinase (PKR), general control non‐derepressible‐2 (GCN2) and PKR‐like endoplasmic reticulum kinase, regulate protein synthesis in response to environmental stresses by phosphorylating the α‐subunit of the initiation factor 2 (eIF2α). We now report that mammalian GCN2 is specifically activated in vitro upon binding of two nonadjacent regions of the Sindbis virus (SV) genomic RNA to its histidyl‐tRNA synthetase‐related domain. Moreover, endogenous GCN2 is activated in cells upon SV infection. Strikingly, fibroblasts derived from GCN2−/− mice possess an increased permissiveness to SV or vesicular stomatitis virus infection. We further show that mice lacking GCN2 are extremely susceptible to intranasal SV infection, demonstrating high virus titers in the brain compared to similarly infected control animals. The overexpression of wild‐type GCN2, but not the catalytically inactive GCN2‐K618R variant, in NIH 3T3 cells impaired the replication of a number of RNA viruses. We determined that GCN2 inhibits SV replication by blocking early viral translation of genomic SV RNA. These findings point to a hitherto unrecognized role of GCN2 as an early mediator in the cellular response to RNA viruses.

CHARACTERIZATION OF THE HEMIN-SENSITIVE EUKARYOTIC INITIATION FACTOR 2ALPHA KINASE FROM MOUSE NONERYTHROID CELLS

The heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase (heme-regulated inhibitor (HRI)) is activated by heme deficiency in reticulocytes and plays an important role in translational control in these cells. Previously, HRI was cloned from rabbit reticulocytes and rat brain, but a heme-regulated eIF2α kinase activity has only been purified from erythroid cells. In this study, we report the purification of a heme-sensitive eIF2α kinase activity from both mouse liver and NIH 3T3 cell extracts. Furthermore, we have cloned and characterized this mouse liver eIF2α kinase (mHRI), which exhibits 83 and 94% identities to rabbit and rat HRIs, respectively. Both the purified enzyme and recombinant mHRI exhibited an autokinase and an eIF2α kinase activity, and both activities were inhibitedin vitro by hemin. In addition, wild-type mHRI, but not the inactive mHRI-K196R mutant, was autophosphorylated in vivowhen it was expressed in 293 cells. Quantitation of mHRI mRNA expression in various mouse tissues by reverse transcription-polymerase chain reaction revealed relatively high levels in liver, kidney, and testis. These results provide strong evidence that mHRI is a ubiquitous eIF2α kinase of mammalian cells, suggesting that it could play important roles in the translational regulation of nonerythroid tissues.

GCN2 Has Inhibitory Effect on Human Immunodeficiency Virus-1 Protein Synthesis and Is Cleaved upon Viral Infection

The reversible phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2alpha) is a well-characterized mechanism of translational control in response to a wide variety of cellular stresses, including viral infection. Beside PKR, the eIF2alpha kinase GCN2 participates in the cellular response against viral infection by RNA viruses with central nervous system tropism. PKR has also been involved in the antiviral response against HIV-1, although this antiviral effect is very limited due to the distinct mechanisms evolved by the virus to counteract PKR action. Here we report that infection of human cells with HIV-1 conveys the proteolytic cleavage of GCN2 and that purified HIV-1 and HIV-2 proteases produce direct proteolysis of GCN2 in vitro, abrogating the activation of GCN2 by HIV-1 RNA. Transfection of distinct cell lines with a plasmid encoding an HIV-1 cDNA clone competent for a single round of replication resulted in the activation of GCN2 and the subsequent eIF2alpha phosphorylation. Moreover, transfection of GCN2 knockout cells or cells with low levels of phosphorylated eIF2alpha with the same HIV-1 cDNA clone resulted in a marked increase of HIV-1 protein synthesis. Also, the over-expression of GCN2 in cells led to a diminished viral protein synthesis. These findings suggest that viral RNA produced during HIV-1 infection activates GCN2 leading to inhibition of viral RNA translation, and that HIV-1 protease cleaves GCN2 to overcome its antiviral effect.

Role of Mitogen-Activated Protein Kinase Sty1 in Regulation of Eukaryotic Initiation Factor 2α Kinases in Response to Environmental Stress in Schizosaccharomyces pombe

ABSTRACT The mitogen-activated protein kinase (MAPK) Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. In fission yeast, three distinct eukaryotic initiation factor 2α (eIF2α) kinases, two mammalian HRI-related protein kinases (Hri1 and Hri2) and the Gcn2 ortholog, regulate protein synthesis in response to cellular stress conditions. In this study, we demonstrate that both Hri1 and Hri2 exhibited an autokinase activity, specifically phosphorylated eIF2α, and functionally replaced the endogenous Saccharomyces cerevisiae Gcn2. We further show that Gcn2, but not Hri1 or Hri2, is activated early after exposure to hydrogen peroxide and methyl methanesulfonate (MMS). Cells lacking Gcn2 exhibit a later activation of Hri2. The activated MAPK Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress but not in response to MMS. In contrast, Hri2 is the primary activated eIF2α kinase in response to heat shock. In this case, the activation of Sty1 appears to be transitory and does not contribute to the modulation of the eIF2α kinase stress pathway. In strains lacking Hri2, a type 2A protein phosphatase is activated soon after heat shock to reduce eIF2α phosphorylation. Finally, the MAPK Sty1, but not the eIF2α kinases, is essential for survival upon oxidative stress or heat shock, but not upon MMS treatment. These findings point to a regulatory coordination between the Sty1 MAPK and eIF2α kinase pathways for a particular range of stress responses.

New roles of the fission yeast eIF2α kinases Hri1 and Gcn2 in response to nutritional stress

Summary In fission yeast, three distinct eukaryotic initiation factor 2&agr; (eIF2&agr;) kinases (Hri1, Hri2 and Gcn2), regulate protein synthesis in response to various environmental stresses. Thus, Gcn2 is activated early after exposure to hydrogen peroxide (H2O2) and methyl methanesulfonate (MMS), whereas Hri2 is the primary activated eIF2&agr; kinase in response to heat shock. The function of Hri1 is still not completely understood. It is also known that the mitogen-activated protein kinase Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress. In this study, we demonstrate that Hri1 is mainly activated, and its expression upregulated, during transition from exponential growth to the stationary phase in response to nutritional limitation. Accordingly, both Hri1 and Gcn2, but not Hri2, are activated upon nitrogen source deprivation. In contrast, Hri2 is stimulated early during glucose starvation. We also found that Gcn2 is implicated in nitrogen starvation-induced growth arrest in the cell cycle G1 phase as well as in the non-selective protein degradation process caused upon this particular cellular stress. Moreover, Gcn2, but not Hri1 or Hri2, is essential for survival of cells growing in minimal medium, upon oxidative stress or glucose limitation. We further show that eIF2&agr; phosphorylation at serine 52 by the eIF2&agr; kinases is necessary for efficient cell cycle arrest in the G1 phase, for the consequent protein degradation and for sexual differentiation, under nitrogen starvation. Therefore, the eIF2&agr; kinase signalling pathway modulates G1 phase cell cycle arrest, cell survival and mating under nutritional stress in the fission yeast Schizosaccharomyces pombe.

Purification and properties of protein kinase C from rabbit reticulocyte lysates

We have previously reported that addition of Ca2+ and phospholipid (PL) inhibits translation in hemin-containing reticulocyte lysates through activation of a eukaryotic protein synthesis initiation factor (eIF-2) kinase. The possibility that this activation was mediated by a Ca2+-PL-dependent protein kinase (protein kinase C, PKC) appeared unlikely by the observation that it was prevented or reversed by NADPH-generating systems. Nevertheless, reticulocyte lysates contain a potent PKC activity and we deemed it desirable to isolate this enzyme to answer unequivocally the question whether it does or does not activate eIF-2 alpha kinase. We have purified reticulocyte PKC to near homogeneity with Mr 95,500 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme absolutely depended upon both Ca2+ and phosphatidylserine for activity on histone H1 or the beta-subunit of initiation factor eIF-2 and underwent autophosphorylation in a Ca2+- and PL-dependent manner. Mild treatment with trypsin yielded an Mr 82,000 polypeptide that still required Ca2+ and PL for activity. This Mr agrees with that reported for other PKCs, suggesting that these enzymes may undergo limited degradation during isolation. Further proteolytic treatment converted the reticulocyte enzyme into a Ca2+- and PL-dependent form, as is known for PKCs from other sources. The highly purified PKC had no effect on translation in hemin-supplemented reticulocyte lysates.

Protein Phosphorylation and Translational Control in Reticulocytes: Activation of the Heme-Controlled Translational Inhibitor by Calcium Ions and Phospholipid

The synthesis of globin, the major protein synthesized by reticulocytes, requires the presence of heme, the prosthetic group of hemoglobin. The absence of heme leads to the activation of a nucleotide-independent protein kinase that phosphorylates the alpha subunit of the chain initiation factor eIF-2. This modification interferes with the catalytic function of eIF-2 in protein synthesis initiation. Recent progress in our understanding of the molecular mechanism of this inhibition is briefly reviewed. The same phosphorylation is catalyzed by a different enzyme (DAI) which, while constitutive in reticulocytes, is induced by interferon in other cells. This enzyme is activated by low concentrations of double-stranded RNA in conjunction with ATP. The mechanisms of activation of these enzymes are still poorly understood. HCI is believed to form an inactive complex with heme and become active when the heme is removed by hemoglobin formation. The proinhibitor form of HCI (proHCI) is unstable in vitro and, even in the presence of heme, is irreversibly inactivated by SH-binding reagents, alkaline pH, slightly elevated temperatures, or high hydrostatic pressure. In hemin-supplemented reticulocyte lysates proHCI can also be reversibly activated by oxidized glutathione (GSSG) or NADPH depletion as well as by polyunsaturated fatty acids and by Ca2+-phospholipid. The mechanism of activation of HCI by GSSG has not been clarified although it appears to involve oxidation of proHCI SH groups to disulfides. Like activation by GSSG, the activation of HCI by polyunsaturated fatty acids and by Ca2+-phospholipid also appears to be largely due to oxidation of some of the enzymes SH groups. There thus appear to be two fully independent mechanisms of HCI activation in reticulocyte lysates, one involving heme deficiency, the other involving oxidation of proHCI SH groups. The latter, but not the former, can be prevented or reversed by NADPH generators or dithiols. ProHCI appears to be maintained in the reduced, inactive state by a system involving NADPH, thioredoxin, and thioredoxin reductase.

Translational inhibition by eIF-2-phospholipid complex in mammalian cell-free systems

The polypeptide chain initiation factor 2 (eIF‐2) binds phospholipid (PL) and becomes a potent inhibitor of translation in hemin‐supplemented reticulocyte lysates [De Haro et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6711–6715]. This binding is independent of calcium ions and seems to be specific for phosphatidylinositol or phosphatidylserine; phosphatidic and arachidonic acids are inactive. Like α‐subunit‐phosphorylated eIF‐2, eIF‐2·PL traps GEF in a non‐dissociable eIF‐2·PL·GEF complex whereby GEF is no longer able to recycle. Initiation is inhibited when no free GEF is available. Translational inhibition by eIF‐2·PL is rescued by equimolar amounts of eIF‐2·GEF. On the basis of this stoichiometry, we have estimated that reticulocyte lysates contain about 60 pmol of GEF/ml (60 nM) eIF‐2·PL also inhibits translation in cell‐free mouse liver extracts and this inhibition is prevented by reticulocyte eIF‐2·GEF suggesting that GEF also functions in liver. However, the eIF‐2·PL complex does not affect translation in such non‐mammalian eukaryotic systems as wheat germ and Drosophila embryos.