A novel function of eIF2alpha kinases as inducers of the phosphoinositide-3 kinase signaling pathway

Phosphoinositide-3 kinase (PI3K) plays an important role in signal transduction in response to a wide range of cellular stimuli involved in cellular processes that promote cell proliferation and survival. Phosphorylation of the alpha subunit of the eukaryotic translation initiation factor eIF2 at Ser51 takes place in response to various types of environmental stress and is essential for regulation of translation initiation. Herein, we show that a conditionally active form of the eIF2alpha kinase PKR acts upstream of PI3K and turns on the Akt/PKB-FRAP/mTOR pathway leading to S6 and 4E-BP1 phosphorylation. Also, induction of PI3K signaling antagonizes the apoptotic and protein synthesis inhibitory effects of the conditionally active PKR. Furthermore, induction of the PI3K pathway is impaired in PKR(-/-) or PERK(-/-) mouse embryonic fibroblasts (MEFs) in response to various stimuli that activate each eIF2alpha kinase. Mechanistically, PI3K signaling activation is indirect and requires the inhibition of protein synthesis by eIF2alpha phosphorylation as demonstrated by the inactivation of endogenous eIF2alpha by small interfering RNA or utilization of MEFs bearing the eIF2alpha Ser51Ala mutation. Our data reveal a novel property of eIF2alpha kinases as activators of PI3K signaling and cell survival.     

Resistance to vesicular stomatitis virus infection requires a functional cross talk between the eukaryotic translation initiation factor 2alpha kinases PERK and PKR

Phosphorylation of the alpha (alpha) subunit of the eukaryotic translation initiation factor 2 (eIF2) leads to the inhibition of protein synthesis in response to diverse stress conditions, including viral infection. The eIF2alpha kinase PKR has been shown to play an essential role against vesicular stomatitis virus (VSV) infection. We demonstrate here that another eIF2alpha kinase, the endoplasmic reticulum-resident protein kinase PERK, contributes to cellular resistance to VSV infection. We demonstrate that mouse embryonic fibroblasts (MEFs) from PERK(-/-) mice are more susceptible to VSV-mediated apoptosis than PERK(+/+) MEFs. The higher replication capacity of VSV in PERK(-/-) MEFs results from their inability to attenuate viral protein synthesis due to an impaired eIF2alpha phosphorylation. We also show that VSV-infected PERK(-/-) MEFs are unable to fully activate PKR, suggesting a cross talk between the two eIF2alpha kinases in virus-infected cells. These findings further implicate PERK in virus infection, and provide evidence that the antiviral and antiapoptotic roles of PERK are mediated, at least in part, via the activation of PKR.     

Suppression of viral replication by stress-inducible GADD34 protein via the mammalian serine/threonine protein kinase mTOR pathway

GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2alpha nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense.     

Importance of eIF2alpha phosphorylation and stress granule assembly in alphavirus translation regulation

Alphavirus infection results in the shutoff of host protein synthesis in favor of viral translation. Here, we show that during Semliki Forest virus (SFV) infection, the translation inhibition is largely due to the activation of the cellular stress response via phosphorylation of eukaryotic translation initiation factor 2alpha subunit (eIF2alpha). Infection of mouse embryo fibroblasts (MEFs) expressing a nonphosphorylatable mutant of eIF2alpha does not result in efficient shutoff, despite efficient viral protein production. Furthermore, we show that the SFV translation enhancer element counteracts the translation inhibition imposed by eIF2alpha phosphorylation. In wild-type MEFs, viral infection induces the transient formation of stress granules (SGs) containing the cellular TIA-1/R proteins. These SGs are disassembled in the vicinity of viral RNA replication, synchronously with the switch from cellular to viral gene expression. We propose that phosphorylation of eIF2alpha and the consequent SG assembly is important for shutoff to occur and that the localized SG disassembly and the presence of the enhancer aid the SFV mRNAs to elude general translational arrest.     

Inhibition of host and viral translation during vesicular stomatitis virus infection. eIF2 is responsible for the inhibition of viral but not host translation

In cells that allow replication of vesicular stomatitis virus (VSV), there are two phases of translation inhibition: an early block of host translation and a later inhibition of viral translation. We investigated the phosphorylation of the alpha subunit of the eIF2 complex during these two phases of viral infection. In VSV-infected cells, the accumulation of phosphorylated (inactivated) eIF2alpha did not begin until well after host protein synthesis was inhibited, suggesting that it only plays a role in blocking viral translation later after infection. Consistent with this, cells expressing an unphosphorylatable eIF2alpha showed prolonged viral protein synthesis without an effect on host protein synthesis inhibition. Induction of eIF2alpha phosphorylation at early times of viral infection by treatment with thapsigargin showed that virus and host translation are similarly inhibited, demonstrating that viral and host messages are similarly sensitive to eIF2alpha phosphorylation. A recombinant virus that expresses a mutant matrix protein and is defective in the inhibition of host and virus protein synthesis showed an altered phosphorylation of eIF2alpha, demonstrating an involvement of viral protein function in inducing this antiviral response. This analysis of eIF2alpha phosphorylation, coupled with earlier findings that the eIF4F complex is modified earlier during VSV infection, supports a temporal/kinetic model of translation control, where at times soon after infection, changes in the eIF4F complex result in the inhibition of host protein synthesis; at later times, inactivation of the eIF2 complex blocks VSV protein synthesis.     

Activation of translation initiation factor eIF2B by insulin requires phosphatidyl inositol 3-kinase

Eukaryotic initiation factor eIF2B mediates a key regulatory step in peptide-chain initiation and is acutely activated by insulin, although it is not clear how. Inhibitors of phosphatidylinositide 3-kinase blocked activation of eIF2B, although rapamycin, which inhibits the p70 S6 kinase pathway, did not. Furthermore, a dominant negative mutant of PI 3-kinase also prevented activation of eIF2B, while a Sos-mutant, which blocks MAP kinase activation, did not. The data demonstrate that a pathway distinct from MAP and p70 S6 kinases regulates eIF2B. Glycogen synthase kinase-3 (GSK-3) phosphorylates and inactivates eIF2B. In all cases, eIF2B and GSK-3 were regulated reciprocally. Dominant negative PI 3-kinase abolished the insulin-induced inhibition of GSK-3. These data strongly support the hypothesis that insulin activates eIF2B through a signalling pathway involving PI 3-kinase and inhibition of GSK-3.     

IGF-I and insulin regulate eIF4F formation by different mechanisms in muscle and liver in the ovine fetus

The mechanisms by which insulin-like growth factor I (IGF-I) and insulin regulate eukaryotic initiation factor (eIF)4F formation were examined in the ovine fetus. Insulin infusion increased phosphorylation of eIF4E-binding protein (4E-BP1) in muscle and liver. IGF-I infusion did not alter 4E-BP1 phosphorylation in liver. In muscle, IGF-I increased 4E-BP1 phosphorylation by 27%; the percentage in the gamma-form in the IGF-I group was significantly lower than that in the insulin group. In liver, only IGF-I increased eIF4G. Both IGF-I and insulin increased eIF4E. eIF4G binding in muscle, but only insulin decreased the amount of 4E-BP1 associated with eIF4E. In liver, only IGF-I increased eIF4E. eIF4G binding. Insulin increased the phosphorylation of p70 S6 kinase (p70(S6k)) in both muscle and liver and protein kinase B (PKB/Akt) in muscle, two indicative signal proteins in the phosphatidylinositol (PI) 3-kinase pathway. IGF-I increased PKB/Akt phosphorylation in muscle but had no effect on p70(S6k) phosphorylation in muscle or liver. We conclude that insulin and IGF-I modulate eIF4F formation; however, the two hormones have different regulatory mechanisms. Insulin increases phosphorylation of 4E-BP1 and eIF4E. eIF4G binding in muscle, whereas IGF-I regulates eIF4F formation by increasing total eIF4G. Insulin, but not IGF-I, decreased 4E-BP1 content associated with eIF4E. Insulin regulates translation initiation via the PI 3-kinase-p70(S6k) pathway, whereas IGF-I does so mainly via mechanisms independent of the PI 3-kinase-p70(S6k) pathway.     

A potential role for extracellular signal-regulated kinases in prostaglandin F2alpha-induced protein synthesis in smooth muscle cells

To understand the mechanisms of prostaglandin F2alpha (PGF2alpha)-induced protein synthesis in vascular smooth muscle cells (VSMC), we have studied its effect on two major signal transduction pathways: mitogen-activated protein kinases and phosphatidylinositol 3-kinase (PI3-kinase) and their downstream targets ribosomal protein S6 kinase (p70(S6k)) and eukaryotic initiation factor eIF4E and its regulator 4E-BP1. PGF2alpha induced the activities of extracellular signal-regulated kinase 2 (ERK2) and Jun N-terminal kinase 1 (JNK1) groups of mitogen-activated protein kinases, PI3-kinase, and p70(S6k) in a time-dependent manner in growth-arrested VSMC. PGF2alpha also induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and basic fibroblast growth factor-2 (bFGF-2) expression in VSMC. Whereas inhibition of PI3-kinase by wortmannin completely blocked the p70(S6k) activation, it only partially decreased the ERK2 activity, and had no significant effect on global protein synthesis and bFGF-2 expression induced by PGF2alpha. Rapamycin, a potent inhibitor of p70(S6k), also failed to prevent PGF2alpha-induced global protein synthesis and bFGF-2 expression, although it partially decreased ERK2 activity. In contrast, inhibition of ERK2 activity by PD 098059 led to a significant loss of PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and bFGF-2 expression. PGF2alpha-induced phosphorylation of eIF4E and 4E-BP1 was also found to be sensitive to inhibition by both wortmannin and rapamycin. These findings demonstrate that 1) PI3-kinase-dependent and independent mechanisms appear to be involved in PGF2alpha-induced activation of ERK2; 2) PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation appear to be mediated by both ERK-dependent and PI3-kinase-dependent rapamycin-sensitive mechanisms; and 3) ERK-dependent eIF4E phosphorylation but not PI3-kinase-dependent p70(S6k) activation correlates with PGF2alpha-induced global protein synthesis and bFGF-2 expression in VSMC.     

The alpha subunit of eukaryotic initiation factor 2B (eIF2B) is required for eIF2-mediated translational suppression of vesicular stomatitis virus

Eukaryotic translation initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor that converts protein synthesis initiation factor 2 (eIF2) from a GDP-bound form to the active eIF2-GTP complex. Cellular stress can repress translation initiation by activating kinases capable of phosphorylating the alpha subunit of eIF2 (eIF2α), which sequesters eIF2B to prevent exchange activity. Previously, we demonstrated that tumor cells are sensitive to viral replication, possibly due to the occurrence of defects in eIF2B that overcome the inhibitory effects of eIF2α phosphorylation. To extend this analysis, we have investigated the importance of eIF2Bα function and report that this subunit can functionally substitute for its counterpart, GCN3, in yeast. In addition, a variant of mammalian eIF2Bα harboring a point mutation (T41A) was able overcome translational inhibition invoked by amino acid depravation, which activates Saccharomyces cerevisiae GCN2 to phosphorylate the yeast eIF2α homolog SUI2. Significantly, we also demonstrate that the loss of eIF2Bα, or the expression of the T41A variant in mammalian cells, is sufficient to neutralize the consequences of eIF2α phosphorylation and render normal cells susceptible to virus infection. Our data emphasize the importance of eIF2Bα in mediating the eIF2 kinase translation-inhibitory activity and may provide insight into the complex nature of viral oncolysis.     

Phosphorylation of serine 51 in initiation factor 2 alpha (eIF2 alpha) promotes complex formation between eIF2 alpha(P) and eIF2B and causes inhibition in the guanine nucleotide exchange activity of eIF2B

Phosphorylation of serine 51 residue on the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) inhibits the guanine nucleotide exchange (GNE) activity of eIF2B, presumably, by forming a tight complex with eIF2B. Inhibition of the GNE activity of eIF2B leads to impairment in eIF2 recycling and protein synthesis. We have partially purified the wild-type (wt) and mutants of eIF2alpha in which the serine 51 residue was replaced with alanine (51A mutant) or aspartic acid (51D mutant) in the baculovirus system. Analysis of these mutants has provided novel insight into the role of 51 serine in the interaction between eIF2 and eIF2B. Neither mutant was phosphorylated in vitro. Both mutants decreased eIF2alpha phosphorylation occurring in hemin and poly(IC)-treated reticulocyte lysates due to the activation of double-stranded RNA-dependent protein kinase (PKR). However, addition of 51D, but not 51A mutant eIF2alpha protein promoted inhibition of the GNE activity of eIF2B in hemin-supplemented rabbit reticulocyte lysates in which relatively little or no endogenous eIF2alpha phosphorylation occurred. The 51D mutant enhanced the inhibition in GNE activity of eIF2B that occurred in hemin and poly(IC)-treated reticulocyte lysates where PKR is active. Our results show that the increased interaction between eIF2 and eIF2B protein, occurring in reticulocyte lysates due to increased eIF2alpha phosphorylation, is decreased significantly by the addition of mutant 51A protein but not 51D. Consistent with the idea that mutant 51D protein behaves like a phosphorylated eIF2alpha, addition of this partially purified recombinant subunit, but not 51A or wt eIF2alpha, increases the interaction between eIF2 and 2B proteins in actively translating hemin-supplemented lysates. These findings support the idea that phosphorylation of the serine 51 residue in eIF2alpha promotes complex formation between eIF2alpha(P) and eIF2B and thereby inhibits the GNE activity of eIF2B.     

Caerulein-induced acute pancreatitis inhibits protein synthesis through effects on eIF2B and eIF4F

Acute pancreatitis (AP) has been shown in some studies to inhibit total protein synthesis in the pancreas, whereas in other studies, protein synthesis was not affected. Previous in vitro work has shown that high concentrations of cholecystokinin both inhibit protein synthesis and inhibit the activity of the guanine nucleotide exchange factor eukaryotic initiation factor (eIF)2B by increasing the phosphorylation of eIF2alpha. We therefore evaluated in C57BL/6 mice the effects of caerulein-induced AP on pancreatic protein synthesis, eIF2B activity and other protein translation regulatory mechanisms. Repetitive hourly injections of caerulein were administered at 50 microg/kg ip. Pancreatic protein synthesis was reduced 10 min after the initial caerulein administration and was further inhibited after three and five hourly injections. Caerulein inhibited the two major regulatory points of translation initiation: the activity of the guanine nucleotide exchange factor eIF2B (with an increase of eIF2alpha phosphorylation) and the formation of the eIF4F complex due, in part, to degradation of eIF4G. This inhibition was not accounted for by changes in the upstream stimulatory pathway, because caerulein activated Akt as well as phosphorylating the downstream effectors of mTOR, 4E-BP1, and ribosomal protein S6. Caerulein also decreased the phosphorylation of the eukaryotic elongation factor 2, implying that this translation factor was not inhibited in AP. Thus the inhibition of pancreatic protein synthesis in this model of AP most likely results from the inhibition of translation initiation as a result of increased eIF2alpha phosphorylation, reduction of eIF2B activity, and the inhibition of eIF4F complex formation.     

Phosphorylation of eukaryotic initiation factor eIF2Bepsilon in skeletal muscle during sepsis

We reported that the inhibition of protein synthesis in skeletal muscle during sepsis correlated with reduced eukaryotic initiation factor eIF2B activity. The present studies define changes in eIF2Bepsilon phosphorylation in gastrocnemius of septic animals. eIF2B kinase activity was significantly elevated 175% by sepsis compared with sterile inflammation, whereas eIF2B phosphatase activity was unaffected. Phosphorylation of eIF2Bepsilon-Ser(535) was significantly augmented over 2-fold and 2.5-fold after 3 and 5 days and returned to control values after 10 days of sepsis. Phosphorylation of glycogen synthase kinase-3 (GSK-3), a potential upstream kinase responsible for the elevated phosphorylation of eIF2Bepsilon, was significantly reduced over 36 and 41% after 3 and 5 days and returned to control values after 10 days of sepsis. The phosphorylation of PKB, a kinase thought to directly phosphorylate and inactivate GSK-3, was significantly reduced approximately 50% on day 3, but not on days 5 or 10, postinfection compared with controls. Treatment of septic rats with TNF-binding protein prevented the sepsis-induced changes in eIF2Bepsilon and GSK-3 phosphorylation, implicating TNF in mediating the effects of sepsis. Thus increased phosphorylation of eIF2Bepsilon via activation of GSK-3 is an important mechanism to account for the inhibition of skeletal muscle protein synthesis during sepsis. Furthermore, the study presents the first demonstration of changes in eIF2Bepsilon phosphorylation in vivo.     

Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1

Vesicular stomatitis virus (VSV) modulates protein synthesis in infected cells in a way that allows the translation of its own 5'-capped mRNA but inhibits the translation of host mRNA. Previous data have shown that inactivation of eIF2alpha is important for VSV-induced inhibition of host protein synthesis. We tested whether there is a role for eIF4F in this inhibition. The multisubunit eIF4F complex is involved in the regulation of protein synthesis via phosphorylation of cap-binding protein eIF4E, a subunit of eIF4F. Translation of host mRNA is significantly reduced under conditions in which eIF4E is dephosphorylated. To determine whether VSV infection alters the eIF4F complex, we analyzed eIF4E phosphorylation and the association of eIF4E with other translation initiation factors, such as eIF4G and the translation inhibitor 4E-BP1. VSV infection of HeLa cells resulted in the dephosphorylation of eIF4E at serine 209 between 3 and 6 h postinfection. This time course corresponded well to that of the inhibition of host protein synthesis induced by VSV infection. Cells infected with a VSV mutant that is delayed in the ability to inhibit host protein synthesis were also delayed in dephosphorylation of eIF4E. In addition to decreasing eIF4E phosphorylation, VSV infection also resulted in the dephosphorylation and activation of eIF4E-binding protein 4E-BP1 between 3 and 6 h postinfection. Analysis of cap-binding complexes showed that VSV infection reduced the association of eIF4E with the eIF4G scaffolding subunit at the same time as its association with 4E-BP1 increased and that these time courses correlated with the dephosphorylation of eIF4E. These changes in the eIF4F complex occurred over the same time period as the onset of viral protein synthesis, suggesting that activation of 4E-BP1 does not inhibit translation of viral mRNAs. In support of this idea, VSV protein synthesis was not affected by the presence of rapamycin, a drug that blocks 4E-BP1 phosphorylation. These data show that VSV infection results in modifications of the eIF4F complex that are correlated with the inhibition of host protein synthesis and that translation of VSV mRNAs occurs despite lowered concentrations of the active cap-binding eIF4F complex. This is the first noted modification of both eIF4E and 4E-BP1 phosphorylation levels among viruses that produce capped mRNA for protein translation.     

Serine 48 in initiation factor 2 alpha (eIF2 alpha) is required for high-affinity interaction between eIF2 alpha(P) and eIF2B

Phosphorylation of the serine 51 residue in the alpha-subunit of translational initiation factor 2 in eukaryotes (eIF2 alpha) impairs protein synthesis presumably by sequestering eIF2B, a rate-limiting pentameric guanine nucleotide exchange protein which catalyzes the exchange of GTP for GDP in the eIF2-GDP binary complex. To further understand the importance of eIF2 alpha phosphorylation in the interaction between eIF2 alpha(P) and eIF2B proteins and thereby the regulation of eIF2B activity, we expressed the wild type (wt) and a mutant eIF2 alpha in which the serine 48 residue was replaced with alanine (48A mutant) in the baculovirus system. The findings reveal that the expression of both of these recombinant subunits was very efficient (15-20% of the total protein) and both proteins were recognized by an eIF2 alpha monoclonal antibody and were phosphorylated to the same extent by reticulocyte eIF2 alpha kinases. However, partially purified recombinant subunits (wt or 48A mutant) were not phosphorylated as efficiently as the eIF2 alpha subunit present in the purified reticulocyte trimeric eIF2 complex and were also found to inhibit the phosphorylation of eIF2 alpha of the trimeric complex. Furthermore, the extents of inhibition of eIF2B activity and formation of the eIF2 alpha(P)-eIF2B complex that occurs due to eIF2 alpha phosphorylation in poly(IC)-treated rabbit reticulocyte lysates were decreased significantly in the presence of insect cell extracts expressing the 48A mutant eIF2 alpha compared to those for wt. These findings support the hypothesis that the serine 48 residue is required for high-affinity interaction between eIF2 alpha(P) and eIF2B.