Impaired myocardial protein synthesis induced by acute alcohol intoxication is associated with changes in eIF4F

The purpose of the present study was to examine potential mechanisms for the known inhibitory effect of acute alcohol exposure on myocardial protein synthesis. Rats were injected intraperitoneally with either ethanol (75 mmol/kg) or saline, and protein synthesis was measured in vivo 2.5 h thereafter by use of the flooding-dose L-[(3)H]phenylalanine technique. Rates of myocardial protein synthesis and translational efficiency in alcohol-treated rats were decreased compared with control values. Free (nonpolysome bound) 40S and 60S ribosomal subunits were increased 50% after alcohol treatment, indicating an impaired peptide-chain initiation. To identify mechanisms responsible for this impairment, several eukaryotic initiation factors (eIF) were analyzed. Acute alcohol intoxication did not significantly alter the myocardial content of eIF2 alpha or eIF2B epsilon, the extent of eIF2 alpha phosphorylation, or the activity of eIF2B. Acute alcohol exposure increased the binding of 4E-binding protein 1 (4E-BP1) to eIF4E (55%), diminished the amount of eIF4E bound to eIF4G (70%), reduced the amount of 4E-BP1 in the phosphorylated gamma-form (40%), and decreased the phosphorylation of p70S6 kinase and the ribosomal protein S6. There was no significant difference in either the plasma insulin-like growth factor (IGF) I concentration (total or free) or expression of IGF-I or IGF-II mRNA in heart between the two groups. These data suggest that the acute alcohol-induced impairment in myocardial protein synthesis results, in part, from an inhibition in peptide-chain initiation, which is associated with marked changes in eIF4E availability and p70S6 kinase phosphorylation but is independent of changes in the eIF2/2B system and IGFs.     

Skeletal muscle protein synthesis and degradation exhibit sexual dimorphism after chronic alcohol consumption but not acute intoxication

Epidemiological evidence suggests alcoholic myopathy is more severe in females than males, but comparable animal studies are lacking that make elucidating the biochemical locus for this defect problematic. The present study determined whether skeletal muscle protein synthesis and markers of degradation exhibit a sexual dimorphic response to either chronic alcohol consumption or acute intoxication. Male and female rats were fed an alcohol-containing diet, pair-fed for 26 wk (chronic), or received an intraperitoneal injection of alcohol (acute). In males, chronic alcohol decreased gastrocnemius protein synthesis by 20%. This reduction was associated with a twofold increase in the inactive eukaryotic initiation factor (eIF) 4E.4E-binding protein 1 (4E-BP1) complex and a 60% reduction in the active eIF4E.eIF4G complex. This redistribution of eIF4E was associated with decreased phosphorylation of both 4E-BP1 and eIF4G (50-55%). The phosphorylation of ribosomal protein S6 was also reduced 60% in alcohol-consuming male rats. In contrast, neither rates of protein synthesis nor indexes of translation initiation in muscle were altered in alcohol-fed female rats despite blood alcohol levels comparable to males. Chronic alcohol ingestion did not alter atrogin-1 or muscle RING finger-1 mRNA content (biomarkers of muscle proteolysis) in males but increased their expression in females 50-100%. Acute alcohol intoxication produced a comparable decrease in muscle protein synthesis and translation initiation in both male and female rats. Our data demonstrate a sexual dimorphism for muscle protein synthesis, translation initiation, and proteolysis in response to chronic, but not acute, alcohol intoxication; however, they do not support evidence indicating females are more sensitive toward the development of alcoholic skeletal muscle myopathy.     

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.     

Acute alcohol intoxication enhances myocardial eIF4G phosphorylation despite reducing mTOR signaling

Acute alcohol intoxication impairs myocardial protein synthesis in rats, secondary to a diminished mRNA translational efficiency. Decreased mRNA translational efficiency occurs through altered regulation of peptide chain initiation. The purpose of the present set of experiments was to determine whether acute alcohol intoxication alters the phosphorylation state of eukaryotic initiation factor (eIF) 4G, eIF4G.eIF4E complex formation, and the mammalian target of rapamycin (mTOR) signaling pathway in the heart. Acute alcohol intoxication was induced by injection of alcohol (75 mmol/kg body wt ip). Control animals received an equal volume of saline. Alcohol administration enhanced phosphorylation of eIF4G (Ser(1108)) approximately threefold. Alcohol administration lowered formation of the active eIF4G.eIF4E complex by >90%, whereas it increased the abundance of the inactive 4E-binding protein 1 (4E-BP1).eIF4E complex by approximately 160%. Phosphorylation of mTOR on Ser(2448) and Ser(2481) was decreased by 50%. Reduced mTOR phosphorylation did not result from decreased phosphorylation of PKB. Phosphorylation of 4E-BP1 and S6 kinase 1 (Thr(389)), downstream targets of mTOR, were also reduced after acute alcohol administration. These data suggest that acute alcohol-induced impairments in myocardial mRNA translation initiation result, in part, from marked decreases in eIF4G.eIF4E complex formation, which appear to be independent of changes in phosphorylation of eIF4G but dependent on mTOR.     

Hyperoxia alters the expression and phosphorylation of multiple factors regulating translation initiation

Hyperoxia is cytotoxic and depresses many cellular metabolic functions including protein synthesis. Translational control is exerted primarily during initiation by two mechanisms: 1) through inhibition of translation initiation complex formation via sequestration of the cap-binding protein, eukaryotic initiation factor (eIF) 4E, with inhibitory 4E-binding proteins (4E-BP); and 2) by prevention of eIF2-GTP-tRNA(i)(Met) formation and eIF2B activity by phosphorylated eIF2alpha. In this report, exposure of human lung fibroblasts to 95% O2 decreased the incorporation of thymidine into DNA at 6 h and the incorporation of leucine into protein beginning at 12 h. The reductions in DNA and protein synthesis were accompanied by increased phosphorylation of eIF4E protein and reduced phosphorylation of 4E-BP1. At 24 h, hyperoxia shifted 4E-BP1 phosphorylation to lesser-phosphorylated isoforms, increased eIF4E expression, and increased the association of eIF4E with 4E-BP1. Although hyperoxia did not change eIF2alpha expression, it increased its phosphorylation at Ser51, but not until 48 h. In addition, the activation of eIF2alpha was not accompanied by the formation of stress granules. These findings suggest that hyperoxia diminishes protein synthesis by increasing eIF4E phosphorylation and enhancing the affinity of 4E-BP1 for eIF4E.     

Interaction between RAX and PKR modulates the effect of ethanol on protein synthesis and survival of neurons

Ethanol exposure inhibits protein synthesis and causes cell death in the developing central nervous system. The double-stranded RNA (dsRNA)-activated protein kinase (PKR), a serine/threonine protein kinase, plays an important role in translational regulation and cell survival. PKR has been well known for its anti-viral response. Upon activation by viral infection or dsRNA, PKR phosphorylates its substrate, the alpha-subunit of eukaryotic translation initiation factor-2 (eIF2alpha) leading to inhibition of translation initiation. It has recently been shown that, in the absence of a virus or dsRNA, PKR can be activated by direct interactions with its protein activators, PACT, or its mouse homologue, RAX. We have demonstrated that exposure to ethanol increased the phosphorylation of PKR and eIF2alpha in the developing cerebellum. The effect of ethanol on PKR/eIF2alpha phosphorylation positively correlated to the expression of PACT/RAX in cultured neuronal cells. Using PKR inhibitors and PKR null mouse fibroblasts, we verified that ethanol-induced eIF2alpha phosphorylation was mediated by PKR. Overexpression of a wild-type RAX dramatically enhanced sensitivity to ethanol-induced PKR/eIF2alpha phosphorylation, as well as translational inhibition and cell death. In contrast, overexpression of a mutant (S18A) RAX inhibited ethanol-mediated PKR/eIF2alpha activation. Ethanol promoted PKR and RAX association in cells expressing wild-type RAX but not in cells expressing S18A RAX. S18A RAX functioned as a dominant negative protein and blocked ethanol-induced inhibition of protein synthesis and cell death. Our results suggest that the interactions between PKR and PACT/RAX modulate the effect of ethanol on protein synthesis and cell survival in the central nervous system.     

Translation initiation factor modifications and the regulation of protein synthesis in apoptotic cells

The rate of protein synthesis is rapidly down-regulated in mammalian cells following the induction of apoptosis. Inhibition occurs at the level of polypeptide chain initiation and is accompanied by the phosphorylation of the alpha subunit of initiation factor eIF2 and the caspase-dependent cleavage of initiation factors eIF4G, eIF4B, eIF2alpha and the p35 subunit of eIF3. Proteolytic cleavage of these proteins yields characteristic products which may exert regulatory effects on the translational machinery. Inhibition of caspase activity protects protein synthesis from long-term inhibition in cells treated with some, but not all, inducers of apoptosis. This review describes the initiation factor modifications and the possible signalling pathways by which translation may be regulated during apoptosis. We discuss the significance of the initiation factor cleavages and other changes for protein synthesis, and the implications of these events for our understanding of the cellular changes associated with apoptosis.     

Alcohol impairs insulin and IGF-I stimulation of S6K1 but not 4E-BP1 in skeletal muscle

The present study determined whether acute alcohol (ethanol; EtOH) intoxication in rats impaired components of the insulin- and IGF-I-signaling pathway in skeletal muscle. Rats were administered EtOH, and 2.5 h thereafter either insulin, IGF-I, or saline was injected and the gastrocnemius removed. EtOH did not alter the total amount or tyrosine phosphorylation of the insulin receptor, IGF-I receptor, insulin receptor substrate (IRS)-1, or protein kinase B (PKB)/Akt under basal or hormone-stimulated conditions. In contrast, the ability of insulin or IGF-I to phosphorylate T389 and T421/S424 on S6K-1 was markedly diminished by EtOH, and these changes were associated with a reduction in the phosphorylation of the ribosomal protein S6. Under basal conditions, EtOH altered the distribution of eukaryotic initiation factor (eIF)4E, as evidenced by a decreased amount of active eIF4E. eIF4G complex, an increased amount of inactive eIF4E. 4E-binding protein (BP)1 complex, and decreased 4E-BP1 phosphorylation. In contrast, EtOH did not impair the ability of either hormone to reverse the changes in eIF4E distribution or 4E-BP1 phosphorylation. Pretreatment with a glucocorticoid receptor antagonist was unable to attenuate either the basal EtOH-induced changes in eIF4E distribution or the impaired ability of IGF-I to stimulate S6K1 and S6 phosphorylation. Hence, acute alcohol intoxication alters selected aspects of translational control under both basal and anabolic hormone-stimulated conditions in skeletal muscle in a glucocorticoid-independent manner.     

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.     

The double-stranded RNA-activated protein kinase PKR is dispensable for regulation of translation initiation in response to either calcium mobilization from the endoplasmic reticulum or essential amino acid starvation

The alpha-subunit of eukaryotic initiation factor eIF2 is a preferred substrate for the double-stranded RNA-activated protein kinase, PKR. Phosphorylation of eIF2alpha converts the factor from a substrate into a competitive inhibitor of the guanine nucleotide exchange factor, eIF2B, leading to a decline in mRNA translation. Early studies provided evidence implicating PKR as the kinase that phosphorylates eIF2alpha under conditions of cell stress such as the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum, i.e., the unfolded protein response (UPR). However, the recent identification of a trans-microsomal membrane eIF2alpha kinase, termed PEK or PERK, suggests that this kinase, and not PKR, might be the kinase that is activated by misfolded protein accumulation. Similarly, genetic studies in yeast provide compelling evidence that a kinase termed GCN2 phosphorylates eIF2alpha in response to amino acid deprivation. However, no direct evidence showing activation of the mammalian homologue of GCN2 by amino acid deprivation has been reported. In the present study, we find that in fibroblasts treated with agents that promote the UPR, protein synthesis is inhibited as a result of a decrease in eIF2B activity. Furthermore, the reduction in eIF2B activity is associated with enhanced phosphorylation of eIF2alpha. Importantly, the magnitude of the change in each parameter is identical in wildtype cells and in fibroblasts containing a chromosomal deletion in the PKR gene (PKR-KO cells). In a similar manner, we find that during amino acid deprivation the inhibition of protein synthesis and extent of increase in eIF2alpha phosphorylation are identical in wildtype and PKR-KO cells. Overall, the results show that PKR is not required for increased eIF2alpha phosphorylation or inhibition of protein synthesis under conditions promoting the UPR or in response to amino acid deprivation.     

Effects of chronic alcohol consumption on regulation of myocardial protein synthesis

Heart disease represents an important etiology of mortality in chronic alcoholics. The purpose of the present study was to examine potential mechanisms for the inhibitory effect of chronic alcohol exposure (16 wk) on the regulation of myocardial protein metabolism. Chronic alcohol feeding resulted in a lower heart weight and 25% loss of cardiac protein per heart compared with pair-fed controls. The loss of protein mass resulted in part from a diminished (30%) rate of protein synthesis. Ethanol exerted its inhibition of protein synthesis through diminished translational efficiency rather than lower RNA content. Chronic ethanol administration decreased the abundance of eukaryotic initiation factor (eIF)4G associated with eIF4E in the myocardium by 36% and increased the abundance of the translation response protein (4E-BP1) associated with eIF4E. In addition, chronic alcohol feeding significantly reduced the extent of p70S6 kinase (p70(S6K)) phosphorylation. The decreases in the phosphorylation of 4E-BP1 and p70(S6K) did not result from a reduced abundance of mammalian target of rapamycin (mTOR). These data suggest that a chronic alcohol-induced impairment in myocardial protein synthesis results in part from inhibition in peptide chain initiation secondary to marked changes in eIF4E availability and p70(S6K) phosphorylation.     

Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIF2alpha

Hypoxia profoundly influences tumor development and response to therapy. While progress has been made in identifying individual gene products whose synthesis is altered under hypoxia, little is known about the mechanism by which hypoxia induces a global downregulation of protein synthesis. A critical step in the regulation of protein synthesis in response to stress is the phosphorylation of translation initiation factor eIF2alpha on Ser51, which leads to inhibition of new protein synthesis. Here we report that exposure of human diploid fibroblasts and transformed cells to hypoxia led to phosphorylation of eIF2alpha, a modification that was readily reversed upon reoxygenation. Expression of a transdominant, nonphosphorylatable mutant allele of eIF2alpha attenuated the repression of protein synthesis under hypoxia. The endoplasmic reticulum (ER)-resident eIF2alpha kinase PERK was hyperphosphorylated upon hypoxic stress, and overexpression of wild-type PERK increased the levels of hypoxia-induced phosphorylation of eIF2alpha. Cells stably expressing a dominant-negative PERK allele and mouse embryonic fibroblasts with a homozygous deletion of PERK exhibited attenuated phosphorylation of eIF2alpha and reduced inhibition of protein synthesis in response to hypoxia. PERK(-/-) mouse embryo fibroblasts failed to phosphorylate eIF2alpha and exhibited lower survival after prolonged exposure to hypoxia than did wild-type fibroblasts. These results indicate that adaptation of cells to hypoxic stress requires activation of PERK and phosphorylation of eIF2alpha and suggest that the mechanism of hypoxia-induced translational attenuation may be linked to ER stress and the unfolded-protein response.     

Tight Binding of the Phosphorylated α Subunit of Initiation Factor 2 (eIF2α) to the Regulatory Subunits of Guanine Nucleotide Exchange Factor eIF2B Is Required for Inhibition of Translation Initiation

Translation initiation factor 2 (eIF2) is a heterotrimeric protein that transfers methionyl-initiator tRNA(Met) to the small ribosomal subunit in a ternary complex with GTP. The eIF2 phosphorylated on serine 51 of its alpha subunit [eIF2(alphaP)] acts as competitive inhibitor of its guanine nucleotide exchange factor, eIF2B, impairing formation of the ternary complex and thereby inhibiting translation initiation. eIF2B is comprised of catalytic and regulatory subcomplexes harboring independent eIF2 binding sites; however, it was unknown whether the alpha subunit of eIF2 directly contacts any eIF2B subunits or whether this interaction is modulated by phosphorylation. We found that recombinant eIF2alpha (glutathione S-transferase [GST]-SUI2) bound to the eIF2B regulatory subcomplex in vitro, in a manner stimulated by Ser-51 phosphorylation. Genetic data suggest that this direct interaction also occurred in vivo, allowing overexpressed SUI2 to compete with eIF2(alphaP) holoprotein for binding to the eIF2B regulatory subcomplex. Mutations in SUI2 and in the eIF2B regulatory subunit GCD7 that eliminated inhibition of eIF2B by eIF2(alphaP) also impaired binding of phosphorylated GST-SUI2 to the eIF2B regulatory subunits. These findings provide strong evidence that tight binding of phosphorylated SUI2 to the eIF2B regulatory subcomplex is crucial for the inhibition of eIF2B and attendant downregulation of protein synthesis exerted by eIF2(alphaP). We propose that this regulatory interaction prevents association of the eIF2B catalytic subcomplex with the beta and gamma subunits of eIF2 in the manner required for GDP-GTP exchange.     

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.     

Alcohol myopathy: impairment of protein synthesis and translation initiation

Alcohol consumption leads to numerous morphological, biochemical and functional changes in skeletal and cardiac muscle. One such change observed in both tissues after either acute alcohol intoxication or chronic alcohol consumption is a characteristic decrease in the rate of protein synthesis. A decrease in translation efficiency appears to be responsible for at least part of the reduction. This review highlights advances in determining the molecular mechanisms by which alcohol impairs protein synthesis and places these observations in context of earlier studies on alcoholic myopathy. Both acute and chronic alcohol administration impairs translational control by modulating various aspects of peptide-chain initiation. Moreover, this alcohol-induced impairment in initiation is associated with a decreased availability of eukaryotic initiation factor (eIF) 4E in striated muscle, as evidenced by an increase in the amount of the inactive eIF4E.4E-BP1 complex and decrease in the active eIF4E.eIF4G complex. In contrast, alcohol does not produce consistent alterations in the control of translation initiation by the eIF2 system. The etiology of these changes remain unresolved. However, defects in the availability or effectiveness of various anabolic hormones, particularly insulin-like growth factor-I, are consistent with the alcohol-induced decrease in protein synthesis and translation initiation.     

The role of de novo ceramide synthesis in the mechanism of action of the tricyclic xanthate D609

The cytotoxic effects of several chemotherapeutic drugs have been linked to elevated de novo ceramide biosynthesis. However, the relationship between the intracellular site(s) of ceramide accumulation and cytotoxicity is poorly understood. Here we examined the relationship between the site of ceramide deposition and inhibition of protein translation and induction of apoptosis by the antitumor/antiviral xanthate, D609. In Chinese hamster ovary (CHO)-K1, HEK-293, and NIH-3T3 cells, D609 caused rapid (1-5 min) and sustained eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation followed by apoptosis after 24 h. Concurrently, D609 stimulated de novo ceramide synthesis and increased ceramide mass 2-fold by 2 h in CHO-K1 cells. In D609-treated CHO-K1 cells, sphingomyelin synthesis was stimulated by brefeldin A, and C5-DMB-ceramide transport to the Golgi apparatus was blocked, indicating ceramide accumulation in the endoplasmic reticulum (ER). However, D609-mediated eIF2alpha phosphorylation, inhibition of protein synthesis, and apoptosis in CHO-K1 cells were not attenuated by fumonisin B1 or l-cycloserine. Interestingly, short-chain ceramide promoted eIF2alpha phosphorylation and inhibited protein synthesis in CHO-K1 cells, indicating that the effectiveness of endogenous ceramide could be limited by access to signaling pathways. Thus, expansion of the ER ceramide pool by D609 was not implicated in early (eIF2alpha phosphorylation) or late (apoptotic) cytotoxic events.     

In vivo regulation of protein synthesis by phosphorylation of the alpha subunit of wheat eukaryotic initiation factor 2

The regulation of protein synthesis is a critical component in the maintenance of cellular homeostasis. A major mechanism of translational control in response to diverse abiotic and biotic stress signals involves the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). The pathway has been demonstrated in all eukaryotes except plants, although components of a putative plant pathway have been characterized. To evaluate the in vivo capability of plant eIF2alpha to participate in the translation pathway, we have used vaccinia virus recombinants that constitutively express wheat eIF2alpha and inducibly express the eIF2alpha dsRNA-stimulated protein kinase, PKR, in BSC-40 cells. Activation of PKR in cells expressing wild-type wheat eIF2alpha resulted in an inhibition of cellular and viral protein synthesis and an induction of cellular apoptosis correlating with phosphorylation of eIF2alpha on serine 51. Expression of a nonphosphorylatable mutant (51A) of plant eIF2alpha reversed the PKR-mediated translational block as well as the PKR-induced apoptosis. A direct interaction of the plant proteins with the mammalian translational initiation apparatus is supported by coimmunoprecipitation of wild-type plant eIF2alpha and the 51A mutant with mammalian eIF2gamma and the localization of the plant proteins in ribosome fractions. These findings suggest that plant eIF2alpha is capable of interacting with the guanine nucleotide exchange factor eIF2B within the context of the eIF2 holoenzyme and provide direct evidence for its ability to participate in phosphorylation-mediated translational control in vivo.     

Calcineurin is required for translational control of protein synthesis in rat pancreatic acini

CCK increases the rate of net protein synthesis in rat pancreatic acini by activating initiation and elongation factors required for translation. The immunosuppressant FK506 inhibits the Ca²⁺-calmodulin-dependent phosphatase calcineurin in pancreatic acinar cells and blocks pancreatic growth induced by chronic CCK treatment. To test a requirement for calcineurin in the activation of the translational machinery stimulated by CCK, we evaluated the effects of FK506 on protein synthesis and on regulatory initiation and elongation factors in rat pancreatic acini in vitro. CCK acutely increased protein synthesis in acini from normal rats with a maximum increase at 100 pM CCK to 170 ± 11% of control. The immunosuppressant FK506 dose-dependently inhibited CCK-stimulated protein synthesis over the same concentration range that blocked calcineurin activity, as assessed by dephosphorylation of the calcineurin substrate calcium-regulated heat-stable protein of 24 kDa. Another immunosuppressant, cyclosporin A, inhibited protein synthesis, but its effects appeared more complex. FK506 also inhibited protein synthesis stimulated by bombesin and carbachol. FK506 did not significantly affect the activity of the initiation factor-2B, or the phosphorylation of the initiation factor-2, ribosomal protein protein S6, or the mRNA cap binding protein eukaryotic initiation factor (eIF) 4E. Instead, blockade of calcineurin with FK506 reduced the phosphorylation of the eIF4E binding protein, reduced the formation of the eIF4F complex, and increased the phosphorylation of eukaryotic elongation factor 2. From these results, we conclude that calcineurin activity is required for protein synthesis, and this action may be related to an effect on the formation of the mRNA cap binding complex and the elongation processes. exocrine pancreas; cholecystokinin; translation initiation factors; protein phosphatase 2B; immunosuppressants