Effects of glucose 6-phosphate and hemin on activation of heme-regulated eIF-2 alpha kinase in gel-filtered reticulocyte lysates

In heme-deficient reticulocyte lysates, protein synthesis initiation is inhibited due to the activation of a heme-regulated protein kinase which blocks protein synthesis by the specific phosphorylation of the alpha-sub-unit of eukaryotic initiation factor 2 (eIF-2 alpha). The restoration of synthesis requires both hemin and glucose-6-P (Ernst, V., Levin, D. H., and London, I. M. (1978) J. Biol. Chem. 253, 7163-7172). The sugar phosphate fulfills two functions in initiation: (i) the generation of NADPH, and (ii) an effector function in some step in initiation. This latter effect is readily demonstrated in lysates depleted of low molecular weight components by filtration in dextran gels. In gel-filtered lysates, linear protein synthesis is sustained only by the addition of both hemin (20 microM) and glucose-6-P (or 2-deoxyglucose-6-P) (50-500 microM). The omission of either component gives rise to inhibitions which are characterized by the activation of heme-regulated eIF-2 alpha kinase and the concomitant phosphorylation of both endogenous heme-regulated eIF-2 alpha kinase and endogenous eIF-2 alpha, indicating that glucose-6-P is involved in the regulation of heme-regulated eIF-2 alpha kinase. In support of this, we find (a) that gel-filtered lysates incubated with hemin but depleted of glucose-6-P produce sufficient heme-regulated eIF-2 alpha kinase to inhibit protein synthesis when mixed with normal hemin-supplemented lysates; (b) the inhibitions of protein synthesis produced by heme-regulated eIF-2 alpha kinase generated either in glucose-6-P-depleted lysates or heme-deficient lysates are reversed by added eIF-2; and (c) the eIF-2 alpha kinase activities formed in the absence of either hemin or glucose-6-P are both neutralized by an anti-heme-regulated eIF-2 alpha kinase antiserum. We conclude that the physiological activation of heme-regulated eIF-2 alpha kinase is controlled by both hemin and glucose-6-P.     

The 60 S ribosomal subunit as a carrier of eukaryotic initiation factor 2 and the site of reversing factor activity during protein synthesis

Studies on the recycling of eukaryotic initiation factor 2 (eIF-2) during protein synthesis in normal and heme-deficient reticulocyte lysates indicate that eIF-2 binds physiologically to the 60 S ribosomal subunit. Several findings suggest that the 60 S subunit serves as a carrier for eIF-2 during protein synthesis. The addition of purified eIF-2 (beta-32P) to normal hemin-supplemented lysates results in its binding to polyribosomal 60 S subunits; the binding is temperature-dependent. In lysates inhibited by heme deficiency, phosphorylated eIF-2 alpha can be detected on polyribosomal 60 S subunits early in the initial linear phase of protein synthesis; after polyribosomal disaggregation and shut-off of protein synthesis, phosphorylated eIF-2 alpha accumulates on free 60 S ribosome subunits and on the 60 S subunits of 80 S ribosome couples. The phosphorylated eIF-2 alpha associated with the 60 S subunits in heme-deficient lysates appears to be present as the binary complex [eIF-2 (alpha P) X GDP]; the binding of this complex to the 60 S subunit is tight and is not affected by treatment with 25 mM EDTA or by sedimentation in sucrose gradients. Reversal of the inhibition of protein synthesis in heme-deficient lysates by the addition of reversing factor results in a rapid binding of reversing factor to the 60 S subunits and a concomitant dissociation of [eIF-2(alpha P) X GDP]. These findings suggest that the [eIF-2 X GDP] binary complex formed during the assembly of the 80 S initiation complex binds to the 60 S subunit of polyribosomes and is subsequently released by the action of reversing factor.     

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.     

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.     

Correlation between the distribution of the reversing factor and eukaryotic initiation factor 2 in heme-deficient or double-stranded RNA-inhibited reticulocyte lysates

The recycling of eukaryotic initiation factor eIF-2 requires the exchange of GDP for GTP, in a reaction catalyzed by the reversing factor (RF). Recent studies have suggested that a 60 S ribosomal subunit-bound eIF-2.GDP complex is an intermediate in protein chain initiation. We have monitored the distribution of RF in heme-deficient and dsRNA-inhibited lysates by immunoblot analysis of sucrose gradient fractions and have compared the distribution with that of eIF-2(alpha-32P). RF and eIF-2(alpha P) were both found to be tightly associated with 60 S and 80 S ribosomes, as their distribution did not change in gradients containing up to 0.1 M K+. The association of eIF-2(alpha-32P) and RF with 60 S and 80 S ribosomes was enhanced in the presence of F-, indicating the presence of an endogenous ribosome-associated phosphatase activity which is capable of dephosphorylating eIF-2(alpha P) in the absence of F-. These observations are consistent with the hypothesis that under physiologic conditions, RF interacts with the 60 S-bound eIF-2.GDP complex to promote the dissociation of GDP from eIF-2 and the release of eIF-2 from the 60 S subunit as a complex with RF.     

2,3-Bisphosphoglycerate inhibits hemoglobin synthesis and phosphorylation of initiation factor 2 by casein kinase II in reticulocyte lysates

2,3-Bisphosphoglycerate inhibited protein synthesis in reticulocyte lysates with 50% inhibition at 2 mM. Glycerate 2,3-P2 increased the Mg2+ optimum for protein synthesis by chelation of Mg2+, but Mg2+ addition did not completely reverse the inhibition, suggesting an additional site of action. eIF-2 has been used to examine the activity of casein kinase II in reticulocyte lysates in response to glycerate 2,3-P2. When glycerate 2,3-P2 was increased to 4mM, phosphorylation of eIF-2 beta was increasingly inhibited. Thus inhibition of phosphorylation of translational components by casein kinase II can be correlated with inhibition of globin synthesis at physiological concentrations of glycerate 2,3-P2.     

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.     

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.     

Mechanism of inhibition of peptide chain initiation by amino acid deprivation in perfused rat liver. Regulation involving inhibition of eukaryotic initiation factor 2 alpha phosphatase activity.

In previous studies, initiation of protein synthesis was shown to be inhibited in perfused rat livers deprived of single essential amino acids. In the present study, histidinol, a competitive inhibitor of histidinyl-tRNA synthetase, was used to amplify the effects of histidine deprivation on protein synthesis in perfused liver to facilitate investigation of mechanisms involved in the inhibition of peptide chain initiation. Protein synthesis was reduced to 77% of the control rate in livers deprived of histidine and to 13% of the control rate in livers deprived of histidine and exposed to 2.0 mM histidinol. The inhibition of protein synthesis caused by histidine deprivation alone was accompanied by a 2-fold increase in the number of free ribosomal particles, a 29% decrease in Met-tRNA(i) binding to 43 S preinitiation complexes, and a 31% reduction in activity of eukaryotic initiation factor 2B (eIF-2B). By comparison, histidine deprivation combined with histidinol addition resulted in a 3-fold increase in free ribosomal particles, a 66% decrease in Met-tRNAi binding, and a 78% reduction in eIF-2B activity. The proportion of the alpha-subunit of eukaryotic initiation factor two (eIF-2) in the phosphorylated form increased from 8.9 +/- 0.8% in control livers to 52.4 +/- 5.5% in response to histidinol. The increase in the amount of eIF-2 alpha in the phosphorylated form apparently was not due to an increase in kinase activity, because there was no change in eIF-2 alpha kinase activity in extracts of liver perfused with medium containing histidinol compared to controls. Instead, the increased phosphorylation of eIF-2 alpha was associated with an inhibition of eIF-2 alpha phosphatase activity. Thus, in contrast to other systems that have been examined, the mechanism involved in the increase in the phosphorylation state of eIF-2 alpha appears to involve an inhibition of eIF-2 alpha phosphatase activity rather than activation of an eIF-2 alpha kinase.     

The relationship between protein synthesis and heat shock proteins levels in rabbit reticulocyte lysates.

Besides heme deficiency, protein synthesis in rabbit reticulocyte lysates becomes inhibited upon exposure to a variety of agents that mimic conditions which induce the heat shock response in cells. This inhibition has been demonstrated to be due primarily to the activation of the heme-regulated eIF-2 alpha kinase (HRI) which causes an arrest in the initiation of translation. In this report, the sensitivity of protein synthesis in hemin-supplemented lysates to inhibition by Hg2+, GSSG, methylene blue, and heat shock was examined in six different reticulocyte lysate preparations. The extent to which translation was inhibited in response to Hg2+, GSSG, methylene blue, and heat shock correlated inversely with the relative levels of the 70-kDa heat shock proteins (hsp 70) and a 56-kDa protein (p56) present in the lysates determined by Western blotting. The ability of hemin to restore protein synthesis upon addition to heme-deficient lysates was also examined. While the restoration of protein synthesis correlated roughly with the levels of hsp 90 present, the results also suggest that the heme regulation of HRI probably involves the interaction of HRI with several factors present in the lysate besides hsp 90. A comparison of two lysate preparations, which had a 2-fold difference in their protein synthesis rates, indicated that the slower translational rate of the one lysate could be accounted for by its low level of constitutive eIF-2 alpha phosphorylation, with its accompanying decrease in the eIF-2B activity and lower level of polyribosome loading. The present study supports the notion that the previously demonstrated interaction of HRI with hsp 90, hsp 70, and p56 in reticulocyte lysates may play a direct role in regulating HRI activation or activity. We hypothesize that the competition of denatured protein and HRI for the binding of hsp 70 may be a molecular signal that triggers the activation of HRI in reticulocyte lysates in response to stress. Possible functions for p56 in the regulation of HRI activity are also discussed.     

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.     

Evidence for the association of the heme-regulated eIF-2 alpha kinase with the 90-kDa heat shock protein in rabbit reticulocyte lysate in situ

Inhibition of protein synthesis initiation in rabbit reticulocyte lysates occurs in response to a variety of conditions including heme deficiency, addition of oxidants, and heat stress. The inhibition of translation occurs due to the activation of a heme-regulated protein kinase (HRI), which specifically phosphorylates the alpha-subunit of the eukaryotic initiation factor eIF-2. How the activation of HRI in hemin-supplemented lysate occurs in response to oxidants and heat stress is not well understood. Recently, the 90-kDa heat shock protein (hsp 90) has been reported to co-purify with HRI activity. In this report, we have used monoclonal antibodies directed against hsp 90 to determine whether HRI and hsp 90 are functionally associated in the reticulocyte lysate in situ. The AC88 antibody recognizes only free hsp 90 and only bound significant amounts of hsp 90 upon prolonged incubation in the absence of heme or upon N-ethylmaleimide treatment of hemin-supplemented lysates. HRI activity is not absorbed by the AC88 antibody. The 8D3 monoclonal antibody, which binds to both free hsp 90 and hsp 90 complexed to steroid hormone receptors, absorbed the hsp 90 present in hemin-supplemented lysates and reduced the HRI activity by 70-95%. Progressively more HRI activity is not adsorbed by the 8D3 antibody the longer the reticulocyte lysate is incubated in the absence of hemin. The HRI that is adsorbed from heme-deficient lysates by the 8D3 antibody is also more active. The sedimentation rate of HRI was analyzed by glycerol gradient centrifugation. HRI present in hemin-supplemented lysate was found to have a sedimentation coefficient of approximately 7.5-8 S and was adsorbed from fractions by the 8D3 antibody in association with hsp 90. A second peak of HRI activity with a sedimentation coefficient of approximately 4.5-5 S was detected upon glycerol gradient centrifugation of heme-deficient lysates. Upon Western blot analysis, heme-deficient lysates were found to have less hsp 90 in the 7.5-8 S region of glycerol gradients than hemin-supplemented lysates. The data suggest that HRI is associated with hsp 90 in an inactive form in hemin-supplemented lysates and dissociates from hsp 90 upon activation. There also appears to be an intermediate of active HRI which is associated with hsp 90 or which can reversibly associate with hsp 90. Similarities between the stages of HRI activation and steroid hormone receptor activation and transformation are discussed.     

Regulation of protein synthesis in rabbit reticulocyte lysates by guanosine triphosphate

GTP (2 mM) promotes protein synthesis in rabbit reticulocyte lysates in which protein chain initiation is inhibited by the activation of specific adenosine 3′:5′ cyclic monophosphate independent protein kinases in: 1) heme deficiency; or 2) in hemin-supplemented lysates by the addition of the purified heme-regulated protein kinase (HRI); or 3) oxidized glutathione; or 4) by low levels of double stranded RNA. The molecular basis for the promotion of protein synthesis by GTP under these various conditions was investigated by examining the $\text{in}\text{,}\text{situ}$ state of eIF-2 phosphorylation. The results show that GTP (2 mM) blocks eIF-2 phosphorylation and also promotes the dephosphorylation of phosphorylated eIF-2. These findings suggest that GTP restores protein synthesis by a common mechanism that involves the relief of eIF-2 from phosphorylation. The nonphosphorylated eIF-2 is, therefore, available for the maintenance and the restoration of protin chain initiation cycle.     

Synthesis of human initiation factor-2 alpha in Saccharomyces cerevisiae.

A human eIF-2 alpha cDNA (encoding alpha-subunit of the eukaryotic initiation factor-2) was expressed under the control of the galactose-regulated GAL1, 10 promoter, in Saccharomyces cerevisiae, in order to study the possible interactions of human eIF-2 alpha with the yeast protein synthesis apparatus. Isoelectric focusing coupled with Western-blot analysis demonstrated that the human eIF-2 alpha subunit synthesized in yeast under a variety of growth conditions was detected as two bands which co-migrated with the phosphorylated and unphosphorylated forms of rabbit eIF-2 alpha, suggesting covalent modification in vivo. Cell fractionation studies further demonstrated that the synthesised human eIF-2 alpha protein, though present in the cytoplasm, was largely associated with the yeast ribosomes, but could be removed from these by washing with 0.3 M KCl. This possible association of the synthesised human subunit into a three-subunit (alpha, beta and gamma) eIF-2 complex was further examined by partial purification of the yeast eIF-2 complex and estimation of the molecular mass of this complex. Immunoreactive eIF-2 alpha was found in fractions with eIF-2 activity and the estimated molecular mass (130 kDa) corresponded to that predicted for the eIF-2 trimer. These analyses suggest that human eIF-2 alpha subunit synthesised in yeast can become involved with the yeast protein synthetic apparatus, though whether this is a functional incorporation requires further genetic studies.     

The alpha subunit of initiation factor 2 is phosphorylated in vivo in the yeast Saccharomyces cerevisiae.

The phosphorylation state of the alpha subunit of initiation factor 2 (eIF-2 alpha) in Saccharomyces cerevisiae has been determined by two-dimensional gel electrophoresis and autoradiography of lysates from cultures grown under a variety of conditions. The alpha subunit was maintained in a phosphorylated state during logarithmic growth on fermentable and nonfermentable carbon sources, during starvation for an essential amino acid, during heat shock, during stationary phase, and during sporulation. Only when cells were starved for a carbon source for 2 h in 1 M sorbitol was eIF-2 alpha isolated in the nonphosphorylated state. This is in contrast with the studies in rabbit reticulocyte lysates, in which arrested protein synthesis was correlated with a relative increase in the extent of phosphorylation of eIF-2 alpha.     

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.     

Mechanism of stimulation of globin synthesis by adenosine-3',5'-monophosphate and guanosine 5'-triphosphate in a rabbit reticulocyte lysate system

The inhibition of globin synthesis in hemin-deficient rabbit reticulocyte lysates is due to the activation of a hemin-controlled translational inhibitor (HCI) that specifically phosphorylates eIF-2 alpha. High concentrations of cAMP (5-10 mM) and GTP (1-2 mM) stimulated the globin synthesis in hemin-deficient lysates when these compounds were added at the initial stage of incubation. The mechanism of the stimulation by cAMP and GTP was studied using hemin-deficient lysates, the N-ethylmaleimide (NEM)-treated HCI-supplemented lysates and a partially purified initiation factor, eIF-2. As the stimulation of globin synthesis by these compounds must be due to the prevention of the inhibition of globin synthesis, or due to the restoration of globin synthesis, or both, the preventive and restorative effects of these compounds were examined. As for the preventive effect, it was observed that a) the activation of HCI in the postribosomal supernatant of reticulocytes was prevented by GTP, but not by cAMP, and b) cAMP and GTP inhibited the phosphorylation of eIF-2 alpha in hemin-deficient lysates. As for the restorative effect of cAMP and GTP, it was observed that c) these compounds restored the globin synthesis and the binding of [35S]Met-tRNAf to the 40S ribosomal subunits, and promoted the dephosphorylation of eIF-2(alpha P), d) the rates of the restored synthesis of globin were lower than the control, and e) cAMP promoted the release of [3H]GDP from the eIF-2(alpha P) X [3H]GDP complex and the formation of eIF-2(alpha P) X eIF-2B complex. Finding (d) indicates that steps involved in the restorative effect of these compounds may not contribute to the stimulation of the globin synthesis in hemin-deficient lysates. The data on the preventive and restorative effects of cAMP and GTP showed that these compounds affected multiple steps. That is, cAMP inhibited the phosphorylation of eIF-2 alpha and promoted both the release of GDP from eIF-2 and the formation of eIF-2(alpha P) X eIF-2B complex, and GTP prevented both the activation of HCI and the phosphorylation of eIF-2 alpha. Though cAMP and GTP affected multiple steps, it is suggested that cAMP stimulates the globin synthesis by inhibiting the phosphorylation of eIF-2 alpha and that GTP stimulates the globin synthesis chiefly by preventing the activation of HCI in hemin-deficient lysates.     

Phosphorylation of only serine-51 in protein synthesis initiation factor-2 is associated with inhibition of peptide-chain initiation in reticulocyte lysates

We have examined the phosphorylation of the alpha-subunit of initiation factor-2 (eIF-2 alpha) in reticulocyte lysates in which translational shut-off was induced by haem-deficiency or by double-stranded RNA. To maximise the phosphorylation of eIF-2 alpha, lysates were supplemented with the broad spectrum phosphatase inhibitor microcystin. Under all conditions tested, serine-51 was the only residue to become labelled. This is consistent with the observation of only two species of eIF-2 alpha in isoelectric focusing/immunoblotting analyses of lysates treated as described above.     

Inhibition of microsomal calcium sequestration causes an impairment of initiation of protein synthesis in perfused rat liver

The present study examined the effect of 2,5-di-(tert-butyl)-hydroquinone (tBuHQ), an inhibitor of liver microsomal calcium sequestration, on initiation of protein synthesis in perfused rat liver. Perfusion of livers with a concentration of tBuHQ previously shown to completely inhibit microsomal calcium sequestration in isolated hepatocytes caused a 50% inhibition of protein synthesis. The inhibition was characterized by an increase in liver content of free ribosomal particles and a decrease in polysomes indicating that peptide-chain initiation was slowed relative to elongation. Furthermore, the inhibition was associated with a 7.5-fold increase in the proportion of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2) present in the phosphorylated form and a reduction in the activity of eukaryotic initiation factor 2B (eIF-2B) to 37% of the control value. The results suggest that protein synthesis in rat liver is regulated directly by changes in intracellular calcium concentration through a mechanism involving modulation of the phosphorylation state of eIF-2 alpha.     

Regulation of protein synthesis in rabbit reticulocyte lysates: the hemeregulated protein kinase (HRI) and double stranded RNA induced protein kinase (dRI) phosphorylate the same site(s) on initiation factor eIF-2

Double stranded RNA (dsRNA) induced inhibitor (dRI) has been partially purified (80–100 fold). The dRI inhibits protein synthesis in rabbit reticulocyte lysates; the inhibition is overcome by the initiation factor eIF-2. The dRI preparations phosphorylate the 38,000-dalton subunit of eIF-2. Heme-deficiency in rabbit reticulocyte lysates also induces a translational inhibitor (HRI) which inhibits protein chain initiation by specifically phosphorylating the 38,000-dalton subunit of eIF-2. To establish correlation of the mechanism of inhibition of protein synthesis by dRI and HRI, the phosphopeptide patterns of eIF-2 phosphorylated by using HRI or dRI are compared. Treatment with various proteases of eIF-2 phosphorylated by HRI or dRI yield identical phosphopeptide patterns. This finding suggests that HRI and dRI phosphorylate the same site(s) of the 38,000-dalton subunit of eIF-2 and raises the possibility that dRI may also inhibit protein chain initiation by the mechanism similar to that of HRI.