Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation.

The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2 alpha phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2 alpha subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2 alpha kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2 alpha. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2 alpha 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2 alpha, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2 alpha impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2 alpha and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2 alpha inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.     

The association of eIF-2 with Met-tRNAi or eIF-2B alters the specificity of eIF-2 phosphatase

In unfractioned reticulocyte lysate, interaction of eukaryotic initiation factor 2 (eIF-2) with other components regulates the accessibility of phosphatases and kinases to phosphorylation sites on its alpha and beta subunits. Upon addition of eIF-2 phosphorylated on both alpha and beta subunits (eIF-2(alpha 32P, beta 32P) to lysate, the alpha subunit is rapidly dephosphorylated, but the beta subunit is not. In contrast, both sites are rapidly dephosphorylated by the purified phosphatase. The basis of this altered specificity appears to be the association of eIF-2 with other translational components rather than an alteration of the phosphatase. Formation of an eIF-2(alpha 32P,beta 32P) Met-tRNAi X GTP ternary complex prevents dephosphorylation of the beta subunit, but has no effect on the rate of alpha dephosphorylation. eIF-2B, a 280,000-dalton polypeptide complex required for GTP:GDP exchange, also protects the beta subunit phosphorylation site from the purified phosphatase. However, the dephosphorylation of eIF-2(alpha 32P) is inhibited by 75% while complexed with eIF-2B. The altered phosphatase specificity upon association of eIF-2 with eIF-2B also affects the access of protein kinases to these phosphorylation sites. In the eIF-2B X eIF-2 complex, the alpha subunit is phosphorylated at 30% the rate of free eIF-2. Under identical conditions, phosphorylation of eIF-2 beta can not be detected. These results illustrate the importance of substrate conformation and/or functional association with other components in determining the overall phosphorylation state allowed by alterations of kinase and phosphatase activities.     

Stimulation of protein synthesis in COS cells transfected with variants of the alpha-subunit of initiation factor eIF-2.

The role of eukaryotic initiation factor 2 (eIF-2) phosphorylation in translational control has been demonstrated in vivo by overexpressing variant forms of eIF-2 alpha that are not phosphorylated. COS-1 cells transiently transfected with expression vectors for human eIF-2 alpha contain 10-20-fold more eIF-2 alpha subunit than the endogenous COS cell eIF-2 trimeric complex. Expression of the variant form of eIF-2 alpha, Ser51Asp, where Asp replaces Ser51, causes inhibition of protein synthesis, whereas the Ser48Asp variant does not. When either Ser48 or Ser51 is replaced by Ala, the variants stimulate dihydrofolate reductase synthesis when the eIF-2 alpha kinase, DAI, is activated. In order to elucidate these mechanisms, we have separated eIF-2 trimeric complexes from free overexpressed eIF-2 alpha subunits by fast protein liquid chromatography Superose chromatography. Pulse-labeled cells transfected with wild-type or variant DNAs produced eIF-2 preparations with greater than 10-fold higher specific radioactivity in the alpha-subunit compared to the gamma-subunit, thus demonstrating that the human eIF-2 alpha produced from the plasmids readily exchanges into COS cell eIF-2 complexes. Both wild-type and Ser48Ala variant forms of the free 2 alpha-subunit, further purified by MonoQ chromatography, are poor substrates for the heme-regulated eIF-2 alpha kinase, HRI, but are good substrates for double-stranded RNA-activated inhibitor in vitro; the Ser51Ala variant subunit is not phosphorylated by either kinase. None of the purified free eIF-2 alpha subunits inhibits phosphorylation of eIF-2 in vitro, even at up to 8-fold molar excess. Examination of the extent of eIF-2 alpha phosphorylation in the COS cell eIF-2 complexes by two-dimensional polyacrylamide gel electrophoresis shows that the stimulation of dihydrofolate reductase synthesis by the Ser51Ala variant is most readily explained by failure of eIF-2 to be phosphorylated. Stimulation by the Ser48Ala variant appears to occur by mitigation of the effect of phosphorylation at Ser51 since the double variant, Ser48Ala-Ser51Asp, inhibits protein synthesis less than the single variant Ser51Asp. The evidence argues strongly against there being a second site of phosphorylation involved in translational repression.     

Phosphorylation of protein synthesis initiation factor-2. Identification of the site in the alpha-subunit phosphorylated in reticulocyte lysates.

The data presented here show that serine-51 of the alpha-subunit of eukaryotic initiation factor eIF-2 is the only residue phosphorylated by the eIF-2 alpha-specific kinases HCR (haem-controlled repressor) and dsI (double-stranded RNA-activated inhibitor) in vitro. This confirms our earlier finding that serine-48 is not labelled by either kinase. Methodology appropriate for the examination of phosphorylation sites in eIF-2 alpha in whole cells and their extracts has been developed, and used to study the site(s) in eIF-2 alpha labelled in reticulocyte lysates. Only serine-51 became phosphorylated under conditions of haem-deficiency or in the presence of double-stranded RNA. No evidence for a second phosphorylation site on the alpha-subunit was obtained with the lysates and conditions used here.     

Two phosphorylation sites on eIF-2 alpha

Protein synthesis in mammalian cells can be regulated through phosphorylation/dephosphorylation of the alpha subunit of initiation factor 2, eIF-2. Two specific kinases have been identified that apparently phosphorylate the same site(s). Controversy exists as to whether serine-48 is a phosphorylation site in addition to serine-51. A recent publication is discussed that, in this author's view, answers the question of the phosphorylation sites. It is suggested that phosphorylation proceeds sequentially with serine-51 being the first and serine-48 the second phosphorylation site. Phosphorylation of both sites is required for inhibition of protein synthesis.     

Phosphorylation of initiation factor eIF-2 alpha, binding of mRNA to 48 S complexes, and its reutilization in initiation of protein synthesis

The formation of 80 S initiation complexes containing labeled viral mRNA was drastically inhibited when mRNA binding assays were carried out with reticulocyte lysate preincubated with double-stranded RNA (dsRNA). When the assays were analyzed by centrifugation on sucrose gradients, the mRNA incubated with lysate pretreated with dsRNA sedimented as a 48 S complex. Met-tRNA, GDP, and phosphorylated initiation factor eIF-2(alpha P) were shown to co-sediment with the 48 S complex. Therefore, the formation of this complex was attributed to the phosphorylation of eIF-2 alpha by a dsRNA-activated protein kinase. These observations suggested that mRNA could bind to a 40 S ribosomal subunit containing Met-tRNAf, GDP, and eIF-2(alpha P), but the joining of a 60 S ribosomal subunit was inhibited. When the 48 S complex was isolated and incubated with lysate without added dsRNA, the mRNA could form 80 S initiation complexes. The shift of mRNA from 48 S to 80 S complexes was also observed when the eIF-2 alpha kinase activity was inhibited by the addition of 2-aminopurine. This shift was quite slow, however, when compared to the rate of binding of free mRNA to 80 S initiation complexes. The 2-aminopurine was effective in reversing the inhibition of protein synthesis by dsRNA and in maintaining a linear rate of protein synthesis for 3 h in lysates. Without added 2-aminopurine, protein synthesis was inhibited after 90 min even in lysates supplemented with hemin and eIF-2(alpha P) was detected in these lysates. This finding indicated that eIF-2 alpha phosphorylation could be in part responsible for limiting the duration of protein synthesis in mammalian cell-free systems.     

Phosphorothioated binary complex of eukaryotic initiation factor eIF-2 and GDP inhibits protein synthesis in hemin-supplemented reticulocyte lysates

The rabbit reticulocyte heme-regulated eIF-2 alpha kinase (HRI) utilizes adenosine-5'-0-(3-thiotriphosphate) (ATP-gamma-S) as a substrate for its autophosphorylation and activation, and for the phosphorylation of eIF-2. The phosphorothioated binary complex [eIF-2(alpha-[35S]P) . GDP], interacted with the reticulocyte reversing factor (RF) in in vitro assays, and inhibited the ability of RF to catalyze GDP exchange from (eIF-2 . [3H]GDP) complexes. The phosphorothioate residue in the binary complex was resistant to phosphatase action under protein synthesis conditions. eIF-2(alpha-[35S]P) . GDP inhibited protein synthesis in hemin-supplemented lysates with biphasic kinetics, but had no effect on protein synthesis in heme-deficient lysates. The data reported here indicate that phosphorylation of eIF-2 . GDP alone, through the ability of eIF-2(alpha-P) . GDP to bind and sequester RF, is sufficient to inhibit protein chain initiation in the reticulocyte lysate.     

Purification and properties of eIF-2 phosphatase

Eukaryotic initiation factor 2 (eIF-2) phosphatase has been purified 840-fold to apparent homogeneity from rabbit reticulocyte lysate. Native eIF-2 phosphatase has a Mr = 98,000, pI = 6.1, s20,w = 5.1, and a Stokes radius = 38 A. A subunit composition of one 60,000-dalton polypeptide and one 38,000-dalton polypeptide is indicated. The Km for [32P]eIF-2 is 30 microM and the Vmax = 1.1 nmol of phosphate released/min/microgram. The 38,000-dalton subunit of eIF-2 phosphatase does not co-electrophorese with the catalytic subunit of liver phosphorylase phosphatase, a type 1 protein phosphatase. The specificity of eIF-2 phosphatase for phosphorylation sites on th alpha- and beta-subunits of eIF-2 appears to be determined by the environment of the phosphatase and substrate. Both the alpha- and beta-subunits of [32P]eIF-2 are rapidly dephosphorylated by the purified phosphatase. In unfractionated lysate and in unfractionated lysate supplemented with an equivalent activity of the purified phosphatase, only the alpha-subunit of eIF-2 is dephosphorylated. This indicates other factors are present in the lysate which govern the dephosphorylation of eIF-2.     

Evidence for a second phosphorylation site on eIF-2 alpha from rabbit reticulocytes

Ser 51 in the NH2-terminal sequence of the alpha-subunit of eukaryotic peptide initiation factor 2 (eIF-2) has been identified as a second phosphorylation site for the heme-controlled eIF-2 alpha kinase from rabbit reticulocytes. Increased phosphorylation of this serine relative to the previously described phosphorylation site (Ser 48) is observed when the kinase reaction is carried out in the presence of the alpha-subunit of spectrin. A synthetic peptide corresponding to eIF-2 alpha (41-54) is phosphorylated only in Ser 51 by the eIF-2 alpha kinase.     

Regulation of protein synthesis in eukaryotes. Eukaryotic initiation factor eIF-2 and eukaryotic recycling factor eRF from neuroblastoma cells

eIF-2 purified from neuroblastoma cells consists of three subunits, which appear to be of molecular weight identical to those of the subunits of rabbit reticulocyte eIF-2. A protein fraction has been isolated from neuroblastoma cells with characteristics similar to eRF from reticulocytes: stimulation of amino acid incorporation in a hemin-deprived reticulocyte lysate, the removal of GDP from eIF-2-GDP complexes, a 4-5-fold stimulatory effect in a two-step reaction measuring 40 S preinitiation complex formation and a 3-3.5-fold stimulation in the methionyl-puromycin synthesis. In the methionyl-puromycin-synthesizing system phosphorylated eIF-2 is not responsive to the addition of this fraction from neuroblastoma cells. The protein fraction contains eRF which seems to be similar to the eRF isolated from Ehrlich ascites tumor cells and somewhat distinct from the reticulocyte factor. Incubation of neuroblastoma cell lysate in the presence of [gamma-32P]ATP results in the phosphorylation of a protein of Mr 36 000, migrating on SDS-polyacrylamide gels to the position of eIF-2 alpha. This protein is also phosphorylated in vitro by HRI from reticulocytes. These results may reflect a common underlying principle for the quantitative regulation of protein synthesis in eukaryotic cells.     

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.     

Regulation of protein synthesis in rabbit reticulocyte lysate. Glucose 6-phosphate is required to maintain the activity of eukaryotic initiation factor (eIF)-2B by a mechanism that is independent of the phosphorylation of eIF-2 alpha

Previous studies from other laboratories, using rabbit reticulocyte lysate filtered through Sephadex G-25 or G-50, have demonstrated that glucose 6-phosphate is required to maintain active rates of translation, but its mechanism of action is currently unsettled. We have tested whether glucose 6-phosphate is required to prevent activation of the hemin-controlled translational repressor and the phosphorylation of the smallest or alpha subunit of eukaryotic initiation factor 2 (eIF-2). We have found that antibody to the hemin-controlled translational repressor can completely restore protein synthesis in reticulocyte lysate, filtered through Sephadex G-25, that is incubated in the absence of hemin and presence of glucose 6-phosphate, but cannot restore protein synthesis in such lysate incubated in the presence of hemin and absence of glucose 6-phosphate. We have also found, using a modification of the method of Matts and London [1984) J. Biol. Chem. 259, 6708-6711) to measure the ability of gel-filtered lysate to dissociate and exchange GDP from eIF-2.GDP, that this endogenous eIF-2B activity is reduced to the same low level in the presence of hemin and absence of glucose 6-phosphate as it is in the absence of hemin and presence of glucose 6-phosphate. Although there is a low level of phosphorylation of eIF-2 alpha in gel-filtered lysate given hemin but no glucose 6-phosphate, it cannot account for the loss of eIF-2B activity, since this phosphorylation is removed by antibody to the hemin-controlled translational repressor or isocitrate, which do not restore protein synthesis or eIF-2B activity, and not by fructose 1,6-diphosphate, which does partially restore protein synthesis and eIF-2B activity. These findings suggest that sugar phosphates may exert a direct effect on eIF-2B and may be required for its proper function. Additional support for this conclusion is our finding that protein synthesis and eIF-2B activity in partially hemin-deficient lysate can be restored by high levels of glucose 6-phosphate or fructose 1,6-diphosphate without a reduction in the level of phosphorylated eIF-2 alpha, suggesting that such levels of sugar phosphate may permit restoration of normal function with a limiting amount of eIF-2B.     

The use of [14C]eukaryotic initiation factor 2 to measure the endogenous pool size of eukaryotic initiation factor 2 in rabbit reticulocyte lysate.

[14C]Eukaryotic initiation factor 2 (eIF-2), obtained by reductive methylation of the purified initiation factor, was shown to be active in the unfractionated reticulocyte lysate. This allowed a direct measurement of the endogenous pool size of eIF-2 in rabbit reticulocyte lysate according to the principle of isotope dilution. A value of 20 to 30 pmol/ml of lysate was obtained. Although translational inhibition resulting from hemin deficiency appears to be characterized by a change from catalytic to stoichiometric utilization of eIF-2, the pool size of eIF-2 is too small to account for the normal period of protein synthesis before the onset of translation inhibition. This suggests, therefore, that additional events to eIF-2 alpha phosphorylation may be required for translational inhibition.     

Control of protein synthesis in human reticulocytes by heme-regulated and double-stranded RNA dependent eIF-2 alpha kinases

Heme-deficiency and double-stranded RNA (dsRNA) activate distinct cyclic 3':5'-AMP independent protein kinases (HRI and dsI, respectively) in rabbit reticulocyte lysates. These kinases inhibit protein synthesis by phosphorylating the 38,000 daltons (38K) subunit of the initiation factor eIF-2 (eIF-2 alpha). Using separation techniques to obtain a reticulocyte enriched fraction and reticulocyte-free erythrocytes, we have prepared lysates of these fractions from normal human whole blood. Human reticulocyte-enriched lysates contain the hemin-regulated and dsRNA-dependent protein kinases which inhibit protein synthesis and which phosphorylate rabbit eIF-2 alpha. An endogenous 38K polypeptide which co-migrates with rabbit eIF-2 alpha is also phosphorylated. In contrast, human mature erythrocytes contain little or no heme-regulated or dsRNA-dependent eIF-2 alpha kinase activities which are inhibitory of protein synthesis.     

Evidence that phosphorylation of eIF-2(alpha) prevents the eIF-2B-mediated dissociation of eIF-2 X GDP from the 60 S subunit of complete initiation complexes

Recent observations have indicated that eukaryotic initiation factor (eIF)-2 and GTP or GDP normally bind to 60 S ribosomal subunits in rabbit reticulocyte lysate and that when eIF-2 alpha is phosphorylated and polypeptide chain initiation is inhibited, eIF-2 X GDP accumulates on 60 S subunits due to impaired dissociation that is normally mediated by the reversing factor (eIF-2B). Current findings now indicate that inhibition due to phosphorylation of eIF-2 alpha is mediated, at least in part, by the inability to dissociate eIF-2 X GDP from the 60 S subunit of complete initiation complexes. At the onset of inhibition, there is an accumulation of Met-tRNA(f) and eIF-2 on the polysomes, despite a marked reduction in Met-tRNA(f) bound to 40 S subunits and Met-peptidyl-tRNA bound to the polysomes. This initial effect is not associated with the formation of "half-mers" (polysomes containing an extra unpaired 40 S subunit), and the 40 S X Met-tRNA(f) complexes, though reduced, still sediment at 43 S. When inhibition is maximal and the polysomes are largely disaggregated, there is an accumulation of 48 S complexes consisting of a 40 S subunit and Met-tRNA(f) bound to globin mRNA as well as small polysomal half-mers, such that residual protein synthesis occurs to about the same degree on "1 1/2"s and "2 1/2"s as on mono-, di-, and triribosomes. Exogenous eIF-2B increases protein synthesis on mono-, di-, and triribosomes and decreases that on half-mers. This is associated with reduced binding of Met-tRNA(f) and eIF-2 to ribosomal particles sedimenting at 80 S and greater and a shift from 48 S to 43 S complexes. These results suggest that eIF-2B must normally promote dissociation of eIF-2 X GDP from the 60 S subunit of complete initiation complexes before they can elongate but cannot when eIF-2 alpha is phosphorylated, resulting in the accumulation of these complexes, some of which dissociate into Met-tRNA(f) X 40 S X mRNA and 60 S X eIF-2 X GDP.     

Inhibition of rabbit reticulocyte lysate protein synthesis by heavy metal ions involves the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2

The effect of heavy metal ions (in particular Cd2+, Hg2+, and Pb2+) on protein synthesis in hemin-supplemented reticulocyte lysates was investigated. Heavy metal ions were found to inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. The shut off of protein synthesis occurred in conjunction with the phosphorylation of the alpha-subunit of the eukaryotic initiation factor (eIF) 2, the loss of reversing factor (RF) activity, and the disaggregation of polyribosomes. Addition of eIF-2 or RF to heavy metal ion-inhibited lysates restored protein synthesis to levels observed in hemin-supplemented controls. The stimulation of protein synthesis observed upon the addition of cAMP to heavy metal ion-inhibited lysates correlated with the inhibition of eIF-2 alpha phosphorylation and the restoration of RF activity. The partial restoration of protein synthesis observed upon the addition of MgGTP to heavy metal ion-inhibited lysates correlated with a partial inhibition of eIF-2 alpha phosphorylation. Addition of glucose 6-phosphate was found to have no effect on protein synthesis of eIF-2 alpha phosphorylation under these conditions. Antiserum raised to the reticulocyte heme-regulated eIF-2 alpha kinase inhibited the phosphorylation of eIF-2 alpha catalyzed by Hg2+-inhibited lysate. The inhibition of protein synthesis observed in the presence of heavy metal ions correlated with the relative biological toxicity of the ions. Highly toxic ions (AsO-2, Cd2+, Hg2+, Pb2+) inhibited protein synthesis by 50% at concentrations of 2.5-10 microM. Cu2+, Fe3+, and Zn2+, which are moderately to slightly toxic ions, inhibited protein synthesis by 50% at concentrations of 40, 250, and 300 microM, respectively. The data presented here indicate that heavy metal ions inhibit protein chain initiation in hemin-supplemented lysates by stimulating the phosphorylation of eIF-2 alpha apparently through the activation of the heme-regulated eIF-2 alpha kinase rather than through inhibition of the rate of eIF-2 alpha dephosphorylation.     

The conversion of eIF-2.GDP to eIF-2.GTP by eIF-2B requires Met-tRNA(fMet).

We have investigated why the recycling of eIF-2.GDP to eIF-2.GTP, mediated by the guanine nucleotide exchange factor eIF-2B, is rapid in rabbit reticulocyte lysate, reconstituted for optimal protein synthesis, but slow in an isolated reaction with purified eIF-2B. We have found that purified eIF-2B dissociates eIF-2.[3H]GDP as efficiently in the presence of GTP as it does in the presence of GDP provided Met-tRNA(fMet) is added. tRNA(fMet) is ineffective, and there is no Met-tRNA(fMet) requirement for exchange with GDP. Exchange of eIF-2 bound GDP for GTP is completely dependent upon Met-tRNA(fMet) in the presence of ATP, suggesting that under physiological conditions efficient recycling of eIF-2.GDP to eIF-2.GTP requires conversion of the latter, a relatively unstable complex, to a more stable Met-tRNA(fMet).eIF-2.GTP complex.     

Regulation of eukaryotic initiation factor-2B activity by polyamines and amino acid starvation in rabbit reticulocyte lysate

We recently reported that the translational control of protein synthesis by glucose 6-phosphate in gel-filtered, rabbit reticulocyte lysate is exerted on the activity of eukaryotic initiation factor (eIF)-2B, the factor that catalyzes the exchange of GTP for GDP bound to eIF-2, by a mechanism that is independent of the phosphorylation of eIF-2 (alpha subunit). We now demonstrate that two other conditions regulate the activity of eIF-2B in rabbit reticulocyte lysate: polyamines (spermidine and spermine) and amino acid deficiency. In the absence of added polyamines, protein synthesis in gel-filtered lysate is reduced to about 70% and eIF-2B activity to about 35% of optimal. The former is likely a result of the latter, since we find that reticulocyte lysate has about twice the eIF-2B necessary to recycle the eIF-2.GDP generated under conditions of optimal protein synthesis. In contrast, the reduction in eIF-2B activity (to about 50% of optimal) occurring in the absence of added amino acids in unfractionated or gel-filtered lysate is insufficient, by itself, to slow the rate of protein synthesis, and the inhibition of protein synthesis that does occur with amino acid deficiency is exerted on polypeptide chain elongation, not initiation. The reduction in eIF-2B activity occurring with amino acid deficiency cannot be reversed by adding more glucose 6-phosphate or polyamines nor can the reduced eIF-2B activity seen with polyamine deficiency be overcome by increasing the glucose 6-phosphate, suggesting that these three components regulate eIF-2B activity by different mechanisms.     

Abrogation of translation initiation factor eIF-2 phosphorylation causes malignant transformation of NIH 3T3 cells.

The interferon induced double-stranded RNA-activated kinase, PKR, has been suggested to act as a tumor suppressor since expression of a dominant negative mutant of PKR causes malignant transformation. However, the mechanism of transformation has not been elucidated. PKR phosphorylates translation initiation factor eIF-2 alpha on Ser51, resulting in inhibition of protein synthesis and cell growth arrest. Consequently, it is possible that cell transformation by dominant negative PKR mutants is caused by inhibition of eIF-2 alpha phosphorylation. Here, we demonstrate that in NIH 3T3 cells transformed by the dominant negative PKR mutant (PKR delta 6), eIF-2 alpha phosphorylation is dramatically reduced. Furthermore, expression of a mutant form of eIF-2 alpha, which cannot be phosphorylated on Ser51 also caused malignant transformation of NIH 3T3 cells. These results are consistent with a critical role of phosphorylation of eIF-2 alpha in control of cell proliferation, and indicate that dominant negative PKR mutants transform cells by inhibition of eIF-2 alpha phosphorylation.