Eukaryotic Initiation Factor eIF2B Research Articles

EP198: EIF3F compound heterozygous genotype-phenotype association

Phenotypes for newly described gene-disease associations often undergo rapid evolution as additional cases are described. The expansion of exome and genome sequencing has contributed to this phenomenon by allowing ascertainment of diseases based on genotype rather than phenotype. We describe an example of phenotype expansion for the EIF3F gene, which encodes an essential subunit of the mammalian eukaryotic initiation factor (eIF2B) complex. EIF2F is important for translation regulation and is involved in cell proliferation and growth, cell cycle control, differentiation, and apoptosis.

EIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition.

RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αβγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2α. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2α phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2α binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2α.IMPORTANCE RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.

Expression, purification, and crystallization of Schizosaccharomyces pombe eIF2B.

Tight control of protein synthesis is necessary for cells to respond and adapt to environmental changes rapidly. Eukaryotic translation initiation factor (eIF) 2B, the guanine nucleotide exchange factor for eIF2, is a key target of translation control at the initiation step. The nucleotide exchange activity of eIF2B is inhibited by the stress-induced phosphorylation of eIF2. As a result, the level of active GTP-bound eIF2 is lowered, and protein synthesis is attenuated. eIF2B is a large multi-subunit complex composed of five different subunits, and all five of the subunits are the gene products responsible for the neurodegenerative disease, leukoencephalopathy with vanishing white matter. However, the overall structure of eIF2B has remained unresolved, due to the difficulty in preparing a sufficient amount of the eIF2B complex. To overcome this problem, we established the recombinant expression and purification method for eIF2B from the fission yeast Schizosaccharomyces pombe. All five of the eIF2B subunits were co-expressed and reconstructed into the complex in Escherichia coli cells. The complex was successfully purified with a high yield. This recombinant eIF2B complex contains each subunit in an equimolar ratio, and the size exclusion chromatography analysis suggests it forms a heterodecamer, consistent with recent reports. This eIF2B increased protein synthesis in the reconstituted in vitro human translation system. In addition, disease-linked mutations led to subunit dissociation. Furthermore, we crystallized this functional recombinant eIF2B, and the crystals diffracted to 3.0 Å resolution.

Biochemical effects of mutations in the gene encoding the alpha subunit of eukaryotic initiation factor (eIF) 2B associated with Vanishing White Matter disease.

BackgroundLeukoencephalopathy with Vanishing White Matter (VWM) is an autosomal recessive disorder caused by germline mutations in the genes EIF2B1-5, which encode the 5 subunits of the eukaryotic translation initiation factor eIF2B. To date, analysis of the biochemical effects of mutations in the EIF2B2-5 genes has been carried out, but no study has been performed on mutations in the EIF2B1 gene. This gene encodes eIF2Bα, the smallest subunit in eIF2B which has an important role in both the structure and regulation of the eIF2B complex.MethodseIF2B subunits were overexpressed in HEK293 cells and isolated from the resulting cell lysates by affinity chromatography. Formation of the eIF2B complex and binding of its substrate, eIF2, was assessed by western blot. Assays of the guanine nucleotide exchange (GEF) activity were also carried out.ResultsOf the 5 eIF2Bα mutations studied, we found 3 that showed loss or reduction of binding of eIF2Bα to the rest of the complex, one with increased GEF activity, and one where no effects on activity or complex formation were observed.ConclusionsThis is the first study on eIF2Bα VWM mutations. We show that some mutations cause expected decreases in GEF activity or complex formation, similar to a majority of observed VWM mutations. However, we also observe some unexpected changes which hint at other effects of these mutations on as yet undescribed functions of eIF2B.

Use of comparative proteomics to identify the effects of creatine pyruvate on lipid and protein metabolism in broiler chickens

Four hundred male chickens were selected to study the effects of pyruvate (Pyr), creatine pyruvate (CrPyr) and creatine (Cr) on the expression of hepatic mitochondrial and cytoplasm proteins associated with lipid and protein metabolism. Mitochondrial purification was accomplished using the two-step differential centrifugation and density gradient method, and the activities of organelle-specific marker enzymes were determined to assess the purity of the mitochondria. Proteins were extracted and fractionated by two-dimensional electrophoresis and the differential protein spots were assessed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. CrPyr reduced fatty acid accumulation by down-regulating adipose differentiation-related protein, inhibited ATP synthase expression, and reduced cholesteryl ester transfer protein (CETP) expression, thus reducing the levels of high density lipoprotein and triglycerol (TG) levels (thereby lowering fat and cholesterol deposition). CrPyr increased the expression of eukaryotic translation initiation factor (eIF) 2B, calreticulin (CRT) and eIF3a, thus promoting protein synthesis. CrPyr up-regulated the expression of fatty acid-binding proteins, CETP and apolipoprotein A-IV in cytoplasmic extracts, and these proteins accelerated the decomposition of fatty acids and TG, thus reducing fat deposition. In conclusion, CrPyr plays an important role in lipolysis and protein synthesis, and this effect was more pronounced than was the effect of Pyr and Cr.

Identification of Intersubunit Domain Interactions within Eukaryotic Initiation Factor (eIF) 2B, the Nucleotide Exchange Factor for Translation Initiation

In eukaryotic translation initiation, eIF2B is the guanine nucleotide exchange factor (GEF) required for reactivation of the G protein eIF2 between rounds of protein synthesis initiation. eIF2B is unusually complex with five subunits (α-ε) necessary for GEF activity and its control by phosphorylation of eIF2α. In addition, inherited mutations in eIF2B cause a fatal leukoencephalopathy. Here we describe experiments examining domains of eIF2Bγ and ε that both share sequence and predicted tertiary structure similarity with a family of phospho-hexose sugar nucleotide pyrophosphorylases. Firstly, using a genetic approach, we find no evidence to support a significant role for a potential nucleotide-binding region within the pyrophosphorylase-like domain (PLD) of eIF2Bε for nucleotide exchange. These findings are at odds with one mechanism for nucleotide exchange proposed previously. By using a series of constructs and a co-expression and precipitation strategy, we find that the eIF2Bε and -γ PLDs and a shared second domain predicted to form a left-handed β helix are all critical for interprotein interactions between eIF2B subunits necessary for eIF2B complex formation. We have identified extensive interactions between the PLDs and left-handed β helix domains that form the eIF2Bγε subcomplex and propose a model for domain interactions between eIF2B subunits.

Inhibition of Glycogen Synthase Kinase 3β Activity with Lithium In Vitro Attenuates Sepsis-Induced Changes in Muscle Protein Turnover

Loss of lean body mass is a characteristic feature of the septic response, and the mechanisms responsible for this decrease and means of prevention have not been fully elucidated. The present study tested the hypothesis that in vitro treatment of skeletal muscle with lithium chloride (LiCl), a glycogen synthase kinase (GSK) 3 inhibitor, would reverse both the sepsis-induced increase in muscle protein degradation and inhibition of protein synthesis. Sepsis decreased GSK-3[beta] phosphorylation and increased GSK-3[beta] activity, under basal conditions. Sepsis increased muscle protein degradation, with a concomitant increase in atrogin 1 and MuRF1 mRNA and 26S proteosome activity. Incubation of septic muscle with LiCl completely reversed the increased GSK-3[beta] activity and decreased proteolysis to basal nonseptic values, but only partially reduced proteosome activity and did not diminish atrogene expression. Lithium chloride also did not ameliorate the sepsis-induced increase in LC3-II, a marker for activated autophagy. In contrast, LiCl increased protein synthesis only in nonseptic control muscle. The inability of septic muscle to respond to LiCl was independent of its ability to reverse the sepsis-induced increase in eukaryotic initiation factor (eIF) 2B[varepsilon] phosphorylation, decreased eIF2B activity, or the reduced phosphorylation of FOXO3, but instead was more closely associated with the continued suppression of mTOR (mammalian target of rapamycin) kinase activity (e.g., reduced phosphorylation of 4E-BP1 and S6). These data suggest that in vitro lithium treatment, which inhibited GSK-3[beta] activity, (a) effectively reversed the sepsis-induced increase in proteolysis, but only in part by a reduction in the ubiquitin-proteosome pathway and not by a reduction in autophagy; and (b) was ineffective at reversing the sepsis-induced decrease in muscle protein synthesis. This lithium-resistant state seems mediated at the level of mTOR and not eIF2/eIF2B. Hence, use of GSK-3[beta] inhibitors in the treatment of sepsis may not be expected to fully correct the imbalance in muscle protein turnover.

HIGHLIGHTS FROM THE LITERATURE

HIGHLIGHTS FROM THE LITERATURE

Exon Deletion in the Non-catalytic Domain of eIF2Bɛ Due to a Splice Site Mutation Leads to Infantile Forms of CACH/VWM with Severe Decrease of eIF2B GEF Activity

The CACH/VWM syndrome is an autosomal recessive leukodystrophy characterized by a broad spectrum of clinical presentations and by diffuse cavitary degeneration of the white matter on MRI. Mutations responsible for this disorder are missense or frameshift mutations occurring in the five genes (EIF2B1-5) that encode the translation eukaryotic initiation factor eIF2B. We found that a patient with infantile CACH/VWM carries a mutation in the acceptor splice site of EIF2B5 exon 6. In lymphoblastoid cells of the patient, we detected an abnormal EIF2B5 transcript in which exon 6 was absent, however, the predicted protein product lacking part of the non-catalytic domain encoded by exon 6 was not detected. The eIF2B GEF activity was severely decreased. These data support the importance of the non-catalytic domain of the eIF2Bepsilon subunit in the eIF2B complex formation and activity.

The Unfolded Protein Response in Vanishing White Matter Disease

Leukoencephalopathy with vanishing white matter (VWM) is an autosomal-recessive disorder in which febrile infections may provoke major neurologic deterioration. Characteristic pathologic findings include cystic white matter degeneration, foamy oligodendrocytes, dysmorphic astrocytes and oligodendrocytes, oligodendrocytosis, and apoptotic losses of oligodendrocytes. VWM is caused by mutations in eukaryotic initiation factor (eIF) 2B (eIF2B). eIF2B plays an important role in the regulation of protein synthesis. Mutant eIF2B may impair the ability of cells to regulate protein synthesis in response to stress and perhaps even under normal conditions. An overload of misfolded proteins in the endoplasmic reticulum activates the unfolded protein response (UPR), a compensatory mechanism that inhibits synthesis of new proteins and induces both prosurvival and proapoptotic signals. We have studied the activation of the UPR in VWM through the immunohistochemical expression of its upstream components PERK and phosphorylated eIF2alpha (eIF2alphaP) and combined immunohistochemical and Western blot analysis of the downstream effector proteins activating transcription factor-4 (ATF4) and C/EBP homologous protein (CHOP) in 4 VWM brains and 3 age-matched controls. We demonstrate activation of the UPR in glia of patients with VWM. Our findings may point to a possible explanation for the dysmorphic glia, the increased numbers of oligodendrocytes, and the apoptotic loss of oligodendrocytes in VWM.

Structure of the catalytic fragment of translation initiation factor 2B and identification of a critically important catalytic residue.

Eukaryotic initiation factor (eIF) 2B catalyzes the nucleotide activation of eIF2 to its active GTP-bound state. The exchange activity has been mapped to the C terminus of the eIF2Bepsilon subunit. We have determined the crystal structure of residues 544-704 from yeast eIF2Bepsilon at 2.3-A resolution, and this fragment is an all-helical protein built around the conserved aromatic acidic (AA) boxes also found in eIF4G and eIF5. The eight helices are organized in a manner similar to HEAT repeats. The molecule is highly asymmetric with respect to surface charge and conservation. One area in the N terminus is proposed to be directly involved in catalysis. In agreement with this hypothesis, mutation of glutamate 569 is shown to be lethal. An acidic belt and a second area in the C terminus containing residues from the AA boxes are important for binding to eIF2. Two mutations causing the fatal human genetic disease leukoencephalopathy with vanishing white matter are buried and appear to disrupt the structural integrity of the catalytic domain rather than interfering directly with catalysis or binding of eIF2.

EIF2B-Related Disorders: Antenatal Onset and Involvement of Multiple Organs

Leukoencephalopathy with vanishing white matter, also called "childhood ataxia with central nervous system hypomyelination," is the first human disease related to mutations in any of the five genes encoding subunits of eukaryotic initiation factor eIF2B or any translation factor at all. eIF2B is essential in all cells of the body for protein synthesis and the regulation of this protein synthesis under different stress conditions. It is surprising that mutations in the eIF2B genes have been reported to lead to abnormalities of the white matter of the brain only, although it has been shown recently that ovarian failure may accompany the leukoencephalopathy. Another surprising observation is that the onset of the disease varies from early childhood to adulthood, with the exception of Cree leukoencephalopathy, a disease related to a particular mutation in one of the eIF2B genes, which invariably has its onset within the first year of life. We analyzed the eIF2B genes of nine patients with an antenatal- or early-infantile-onset encephalopathy and an early demise and found mutations in eight of the patients. In addition to signs of a serious encephalopathy, we found oligohydramnios, intrauterine growth retardation, cataracts, pancreatitis, hepatosplenomegaly, hypoplasia of the kidneys, and ovarian dysgenesis. Until now, no evidence had been found for a genotype-phenotype correlation, but the consistently severe phenotype in affected siblings among our patients and in Cree encephalopathy patients suggests an influence of the genotype on the phenotype.

Alterations in the expression of mRNAs and proteins that code for species relevant to eIF2B activity after an acute bout of resistance exercise.

The focus of the study described herein was to examine the relative expression levels of mRNAs and proteins relevant to the regulation of translational initiation, and hence protein synthesis, in the time course after an acute bout of resistance exercise in male Sprague-Dawley rats. Significant increases in the relative abundance of the mRNAs coding for the epsilon (33%) and gamma (26%) subunits of eukaryotic initiation factor (eIF) 2B were observed 48 h after the exercise bout. Furthermore, the mRNA coding for the delta subunit of eIF2B was also significantly increased, both 24 h (46%) and 48 h (44%) postexercise. There was a relative decrease in three eIF2Bepsilon kinase mRNAs, namely sequences coding for glycogen synthase kinase 3beta (49%), casein kinase I (48%), and casein kinase II (42%) 48 h into the recovery period. Additionally, there was a significant decrease in expression of the mRNAs coding for eIF2alpha (28% 24 h postexercise) and one of its regulatory kinases, double-stranded RNA-activated protein kinase (33% 48 h postexercise). Finally, an increase in eIF2B total protein (124%) was observed within 3 h postexercise. These results suggest that there may be rapid translational regulation of mRNAs coding for species relevant to translational initiation after an acute bout of resistance exercise. Furthermore, transcription of these mRNAs is altered further into the recovery period, and this might play a role in protein synthetic capacity on subsequent bouts of resistance exercise.

Endotoxin induces differential regulation of mTOR-dependent signaling in skeletal muscle and liver of neonatal pigs.

In the present study, differential responses of regulatory proteins involved in translation initiation in skeletal muscle and liver during sepsis were studied in neonatal pigs treated with lipopolysaccharide (LPS). LPS did not alter eukaryotic initiation factor (eIF) 2B activity in either tissue. In contrast, binding of eIF4G to eIF4E to form the active mRNA-binding complex was repressed in muscle and enhanced in liver. Phosphorylation of eIF4E-binding protein, 4E-BP1, and ribosomal protein S6 kinase, S6K1, was reduced in muscle during sepsis but increased in liver. Finally, changes in 4E-BP1 and S6K1 phosphorylation were associated with altered phosphorylation of the protein kinase mammalian target of rapamycin (mTOR). Overall, the results suggest that translation initiation in both skeletal muscle and liver is altered during neonatal sepsis by modulation of the mRNA-binding step through changes in mTOR activation. Moreover, the LPS-induced changes in factors that regulate translation initiation are more profound than previously reported changes in global rates of protein synthesis in the neonate. This finding suggests that the initiator methionyl-tRNA-rather than the mRNA-binding step in translation initiation may play a more critical role in maintaining protein synthesis rates in the neonate during sepsis.

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.

Reduced amino acid availability inhibits muscle protein synthesis and decreases activity of initiation factor eIF2B.

We have examined the effect of a hemodialysis-induced 40% reduction in plasma amino acid concentrations on rates of muscle protein synthesis and breakdown in normal swine. Muscle protein kinetics were measured by tracer methodology using [(2)H(5)]phenylalanine and [1-(13)C]leucine and analysis of femoral arterial and venous samples and tissue biopsies. Net amino acid release by muscle was accelerated during dialysis. Phenylalanine utilization for muscle protein synthesis was reduced from the basal value of 45 +/- 8 to 25 +/- 6 nmol x min(-1) x 100 ml leg(-1) between 30 and 60 min after start of dialysis and was stimulated when amino acids were replaced while dialysis continued. Muscle protein breakdown was unchanged. The signal for changes in synthesis appeared to be changes in plasma amino acid concentrations, as intramuscular concentrations remained constant throughout. The changes in muscle protein synthesis were accompanied by a reduction or stimulation, respectively, in the guanine nucleotide exchange activity of eukaryotic initiation factor (eIF)2B following hypoaminoacidemia vs. amino acid replacement. We conclude that a reduction in plasma amino acid concentrations below the normal basal value signals an inhibition of muscle protein synthesis and that corresponding changes in eIF2B activity suggest a possible role in mediating the response.

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.

Evidence that the dephosphorylation of Ser(535) in the epsilon-subunit of eukaryotic initiation factor (eIF) 2B is insufficient for the activation of eIF2B by insulin.

Eukaryotic initiation factor (eIF) 2B is a guanine-nucleotide exchange factor that plays a key role in the regulation of protein synthesis. It is activated by insulin, serum and other agents that stimulate general protein synthesis. The largest (epsilon) subunit of eIF2B is a substrate for glycogen synthase kinase (GSK)-3 in vitro, and phosphorylation by GSK3 inhibits the activity of eIF2B. The site of phosphorylation has previously been identified as Ser(535). GSK3 is inactivated by phosphorylation in response to insulin or serum. In Chinese-hamster ovary cells, insulin and serum bring about the dephosphorylation of Ser(535) in vivo, concomitantly with the phosphorylation of GSK3, and these effects are mediated through signalling via phosphoinositide 3-kinase. We have made use of inhibitors of GSK3 to determine whether GSK3 is responsible for phosphorylation of Ser(535) in vivo and to explore the role of phosphorylation of Ser(535) in the regulation of eIF2B. Treatment of cells with LiCl or with either of two recently developed GSK3 inhibitors, SB-415286 and SB-216763, brought about the dephosphorylation of Ser(535), which strongly indicates that this site is indeed a target for GSK3 in vivo. However, these compounds did not elicit significant activation of eIF2B, indicating, consistent with conclusions from one of our previous studies, that additional inputs are required for the activation of eIF2B. Our results also show that each of the inhibitors used affects overall protein synthesis and have additional effects on translation factors or signalling pathways apparently unrelated to their effects on GSK3, indicating that caution must be exercised when interpreting data obtained using these compounds.

Effect of CCK and intracellular calcium to regulate eIF2B and protein synthesis in rat pancreatic acinar cells.

Pancreatic secretagogues enhance acinar protein synthesis at physiological concentrations and inhibit protein synthesis at high concentrations. We investigated the potential role in this process of the eukaryotic translation initiation factor (eIF)2B. Cholecystokinin (CCK) at 10-100 pM did not significantly affect eIF2B activity, which averaged 35.4 nmol guanosine 5'-diphosphate exchanged per minute per milligram protein under control conditions; higher CCK concentrations reduced eIF2B activity to 38.2% of control. Carbamylcholine chloride (Carbachol, CCh), A-23187, and thapsigargin also inhibited eIF2B and protein synthesis, whereas bombesin and the CCK analog JMV-180 were without effect. Previous studies have shown that eIF2B can be negatively regulated by glycogen synthase kinase-3 (GSK-3). However, GSK-3 activity, as assessed by phosphorylation state, was inhibited at high concentrations of CCK, an effect that should have stimulated, rather than repressed, eIF2B activity. An alternative mechanism for regulating eIF2B is through phosphorylation of the alpha-subunit of eIF2, which converts it into an inhibitor of eIF2B. CCK, CCh, A-23187, and thapsigargin all enhanced eIF2alpha phosphorylation, suggesting that eIF2B activity is regulated by eIF2alpha phosphorylation under these conditions. Removal of Ca(2+) from the medium enhanced the inhibitory action of CCK on both protein synthesis and eIF2B activity as well as further increasing eIF2alpha phosphorylation. Although it is likely that other mechanisms account for the stimulation of acinar protein synthesis, these results suggest that the inhibition of acinar protein synthesis by CCK occurs as a result of depletion of Ca(2+) from the endoplasmic reticulum lumen leading to phosphorylation of eIF2alpha and inhibition of eIF2B.

The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes

BackgroundActivation of human resting T lymphocytes results in an immediate increase in protein synthesis. The increase in protein synthesis after 16–24 h has been linked to the increased protein levels of translation initiation factors. However, the regulation of protein synthesis during the early onset of T cell activation has not been studied in great detail. We studied the regulation of protein synthesis after 1 h of activation using αCD3 antibody to stimulate the T cell receptor and αCD28 antibody to provide the co-stimulus.ResultsActivation of the T cells with both antibodies led to a sustained increase in the rate of protein synthesis. The activities and/or phosphorylation states of several translation factors were studied during the first hour of stimulation with αCD3 and αCD28 to explore the mechanism underlying the activation of protein synthesis. The initial increase in protein synthesis was accompanied by activation of the guanine nucleotide exchange factor, eukaryotic initiation factor (eIF) 2B, and of p70 S6 kinase and by dephosphorylation of eukaryotic elongation factor (eEF) 2. Similar signal transduction pathways, as assessed using signal transduction inhibitors, are involved in the regulation of protein synthesis, eIF2B activity and p70 S6 kinase activity. A new finding was that the p38 MAPK α/β pathway was involved in the regulation of overall protein synthesis in primary T cells. Unexpectedly, no changes were detected in the phosphorylation state of the cap-binding protein eIF4E and the eIF4E-binding protein 4E-BP1, or the formation of the cap-binding complex eIF4F.ConclusionsBoth eIF2B and p70 S6 kinase play important roles in the regulation of protein synthesis during the early onset of T cell activation.