Inhibition of protein synthesis in reticulocyte lysates by a double-stranded RNA component in HeLa mRNA

Double-stranded RNA (dsRNA) inhibits protein synthesis initiation in rabbit reticulocyte lysates by the activation of a latent dsRNA-dependent cAMP-independent protein kinase which phosphorylates the α-subunit of the eukaryotic initiation factor eIF-2. In this study, we describe a dsRNA-like component which is present in preparations of HeLa mRNA (poly A⁺) isolated from total cytoplasmic RNA. The inhibitory species in the HeLa cytoplasmic mRNA was detected by (a) its ability to inhibit protein synthesis with biphasic kinetics in reticulocyte lysates translating endogenous globin mRNA, and (b) by the inefficient translation of HeLa cytoplasmic mRNA in a nuclease-treated mRNA-dependent reticulocyte lysate. The inhibitory component was characterized as dsRNA by several criteria including (i) the ability to activate the lysate dsRNA-dependent eIF-2α kinase (dsI); (ii) the prevention of both dsI activation and inhibition of protein synthesis by high levels of dsRNA or cAMP; (iii) the reversal of inhibition by eIF-2; and (iv) the inability to inhibit protein synthesis in wheat germ extracts which lack latent dsI. By the same criteria, the putative dsRNA component(s) appears to be absent from preparations of HeLa mRNA isolated exclusively from polyribosomes.     

Purification and characterization of a latent precursor of a double-stranded RNA dependent protein kinase from reticulocyte lysates

Double-stranded RNA (dsRNA) activates a cyclic 3′: 5′-AMP independent protein kinase (dsI) in reticulocyte lysates which inhibits protein synthesis by phosphorylating the 38, 000 dalton (38K) subunit of the initiation factor eIF-2 (eIF-2α). A latent precursor form of dsI (latent dsI) has been partially purified (1000–2000 fold) from lysates. Activation of dsI at all stages in the purification of latent dsI requires ATP and low levels of dsRNA (1–20 ng/ml), and is accompanied by the phosphorylation of a broad 67,000 dalton (67K) band. However, as purification proceeds the 67K band is resolved into two phosphorylated polypeptides of 68,500 and 67,000 daltons ($\text{68.5K}\text{67K}$). Although latent dsI and activated dsI have distinctly different chromatographic properties, both forms have similar molecular weights (～120,000) and similar sedimentation coefficients (～3.8S) in glycerol gradients. The data support the view that one or both components of the $\text{68.5K}\text{67K}$ doublet are associated with the dsRNA-dependent protein kinase activity.     

Activation of the double-stranded RNA-dependent eIF-2 alpha kinase by cellular RNA from 3T3-F442A cells.

The interferon induced double-stranded-RNA-dependent eIF-2 alpha kinase has an established role in mediating part of interferons anti-viral effects. Several studies have suggested that it may have additional functions in cells not infected with virus. The mechanism of activation of the kinase and the consequences of its activity in uninfected cells remain to be determined. Our previous results have indicated that the activation (phosphorylation) of this kinase may be an important regulatory signal to the arrest of growth of mouse 3T3-F442A fibroblasts and their subsequent differentiation to adipocytes. We have found that the phosphorylation of the kinase occurred in vivo in the absence of viral infection and in vitro without the addition of dsRNA. We demonstrate here that total cytoplasmic RNA from 3T3-F442A cells contains a regulatory RNA(s) capable of activating dsRNA-dependent eIF-2 alpha kinase. Fractionation of the cytoplasmic RNA by oligo(dT)-cellulose indicated that the regulatory RNA eluted with the poly(A)-rich RNA fraction. It bound tightly to the dsRNA-dependent eIF-2 alpha kinase and was immune-precipitated with its antibodies as a complex of regulatory RNA and dsRNA-dependent eIF-2 alpha kinase. The regulatory RNA activity was further purified by phenol extraction of immune precipitates containing this complex. These findings indicated that the regulatory RNA forms a specific complex with the dsRNA-dependent eIF-2 alpha kinase. The activity of the regulatory RNA was sensitive to the dsRNA-specific RNase VI but not to proteinase K, DNase I or ssRNA-specific RNase T1. The activation of the dsRNA-dependent eIF-2 alpha kinase by regulatory RNA was prevented by addition of a high concentration of poly(I).poly(C). The regulatory RNA was also shown to activate partially purified dsRNA-dependent eIF-2 alpha kinase prepared from rabbit reticulocyte lysates and to inhibit protein synthesis in reticulocyte lysates. Our findings, that cellular RNAs can specifically activate the dsRNA-dependent eIF-2 alpha kinase, are consistent with a physiological role for the dsRNA-dependent eIF-2 alpha kinase and interferon during cell growth and differentiation. The relationship of the regulatory RNA activity to growth and differentiation of 3T3-F442A cells is discussed.     

Tissue distribution and immunoreactivity of heme-regulated eIF-2 alpha kinase determined by monoclonal antibodies.

A highly purified preparation of heme-regulated inhibitor (HRI), an eIF-2 alpha kinase, from rabbit reticulocyte lysates has been used for generating monoclonal antibodies (mAB). Two hybridoma clones secreting HRI-specific antibodies (mAB A and mAB F) were obtained. Both antibodies immunoprecipitated biosynthetically labeled as well as phosphorylated HRI in reticulocyte lysates and also recognized denatured HRI in a Western blot. In in vitro protein kinase assays, preincubation of HRI with the antibodies significantly diminished both autokinase and eIF-2 alpha kinase activities. HRI from reticulocyte lysates could be quantitatively removed by immunoprecipitation with mAB F, and such HRI-depleted lysates were able to maintain protein synthesis under conditions of heme deficiency. With these monoclonal antibodies, HRI was detected only in the reticulocytes and bone marrow of anemic rabbits, among several rabbit tissues tested. The antibodies did not detect cross-reacting HRI in rat or human reticulocytes or in mouse erythroleukemic cells or human K562 cells even after induction of differentiation, although eIF-2 alpha kinase activity was detected in them. Polyclonal anti-rabbit HRI antibody detected HRI in rat reticulocytes. However, no cross-reacting HRI was detected by polyclonal antibody in human reticulocytes or other cell types tested. These findings suggest that HRI is not ubiquitous, and may be erythroid-specific, and that it is antigenically different in different species.     

Lack of inhibition of protein synthesis by double-stranded RNA in a cell-free system prepared from human reticulocytes

There are two inhibitors of protein synthesis which are related to the activity of interferon. One is a protein kinase which phosphorylates the α subunit of the eucaryotic initiation factor 2 (eIF-2). The other is an enzyme which synthesizes an unusual oligonucleotide that in turn activates a RNA endonuclease. In nucleated cells the synthesis of the inhibitors is induced by interferon but they must be activated in a subsequent lysate by double-stranded RNA (dsRNA). Rabbit reticulocytes, however, contain the inactive forms of the inhibitors in a constitutive manner and require only dsRNA activation. We report here the effect of dsRNA on protein synthesis and the generation of ribosomal eIF-2α kinase and heat-stable (oligonucleotide) inhibitory activity in human reticulocyte lysates. Our findings indicate that human reticulocytes, in contrast to rabbit reticulocytes, do not contain the interferon-related inhibitors of protein synthesis in a constitutive manner. Addition of dsRNA to the human reticulocyte cell-free system does not result in significant inhibition. Furthermore, no generation of ribosomal eIF-2α kinase or heatstable inhibitory activity could be detected. Direct addition of oligonucleotide or eIF-2α kinase (of rabbit origin), however, does result in inhibition of the human system. Thus, the ultimate inhibition mechanisms do appear operative in the human reticulocyte lysates. The differences between the rabbit and human systems may be due to either basic differences in the mechanism of interferon action or simply to variation in the history or maturity of the cells studied.     

Partial purification and characterization of heat stable protein phosphatase inhibitor-2 from rabbit reticulocytes

Previous studies indicated that the species of type 1 and type 2 protein phosphatases (PP-1, PP-2) in rabbit reticulocytes are similar to those of rabbit skeletal muscle and rabbit liver. Reticulocyte PP-1 was found to be selectively inhibited by the heat stable protein phosphatase inhibitor-2 (I-2) from rabbit skeletal muscle. Of interest was the observation that muscle I-2 appeared to regulate protein synthesis in reticulocyte lysates by inhibiting an eIF-2 alpha phosphatase with type 1 properties. In this study we have characterized reticulocyte inhibitor-2 (I-2) and find that its properties are similar to those of skeletal muscle I-2. (i) Both I-2 species are stable to boiling and to acid treatment, and have similar chromatographic profiles on DEAE-cellulose and on Blue Sepharose CL-6B. (ii) The two I-2 species migrate electrophoretically as 26-28,000 dalton polypeptides in SDS-acrylamide gels. (iii) Both skeletal muscle I-2 and reticulocyte I-2 selectively inhibit isolated reticulocyte PP-1 and endogenous PP-1 in the lysate. (iv) Reticulocyte I-2 co-chromatographs with PP-1 on DEAE-cellulose, and over 90% of lysate I-2 can be isolated from this partially purified PP-1. (v) Both inhibitor-2 species are active in the unphosphorylated state, but upon addition to lysates, both are phosphorylated by endogenous cAMP-independent protein kinase(s). In addition a preliminary analysis using a polyclonal antibody against muscle inhibitor-1 confirmed biochemical analyses which indicate that lysates are deficient in inhibitor-1.     

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.     

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.     

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.     

Small molecule modulators of eukaryotic initiation factor 2α kinases,the key regulators of protein synthesis

Eukaryotic initiation factor 2 alpha kinases (eIF-2α kinases) are key mediators of stress response in cells. In mammalian cells, there are four eIF-2α kinases, namely HRI (Heme-Regulated Inhibitor), PKR (RNA-dependent Protein Kinase), PERK (PKR-like ER Kinase) and GCN2 (General Control Non-derepressible 2). These kinases get activated during diverse cytoplasmic stress conditions and phosphorylate the alpha-subunit of eIF2, leading to global protein synthesis inhibition. Therefore, eIF-2α kinases play a vital role in various cellular processes such as proliferation, differentiation, apoptosis and cell signaling. Deregulation of eIF-2α kinases and protein synthesis has been linked to numerous pathological conditions such as certain cancers, anemia and neurodegenerative disorders. Thus, modulation of these kinases by small molecules holds a great therapeutic promise. In this review we have compiled the available information on inhibitors and activators of these four eIF-2α kinases. The review concludes with a note on the selectivity issue of currently available modulators and future perspectives for the design of specific small molecule probes.     

Growth-related expression of a double-stranded RNA-dependent protein kinase in 3T3 cells

Cultured mouse 3T3-F442A and 3T3-C2 fibroblasts exhibit a transient double-stranded RNA (dsRNA)-dependent phosphorylation of a 67,000-dalton protein (67K) without prior treatment with interferon (IFN). This phosphoprotein is similar but not identical to the dsRNA-dependent eukaryotic initiation factor-2 (eIF-2) alpha protein kinase (dsI), which regulates protein synthesis in rabbit reticulocytes. We have studied the relationship between cell growth and phosphorylation of the 67K protein (designated 3T3-dsRNA-dependent eIF-2 alpha kinase). A low level of dsRNA-dependent phosphorylation of 3T3-dsI was detectable in extracts prepared from cells not treated with IFN and grown at a low cell density. The phosphorylation of dsI and the phosphorylation of a 38K protein identified as the alpha-subunit (38K) of 3T3-eIF-2 (eIF-2 alpha) occurred concomitantly; the levels of these phosphorylations confluent and thereafter decreased markedly. Treatment of cells with IFN at all stages of growth resulted in an increase in phosphorylation of dsI. 3T3-F442A and 3T3-C2 fibroblasts were found to produce and secrete IFN at levels sufficient to induce an elevated dsI activity.     

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.     

Effects of hemin and porphyrin compounds on intersubunit disulfide formation of heme-regulated eIF-2 alpha kinase and the regulation of protein synthesis in reticulocyte lysates.

To study the mechanism by which heme regulates the heme-regulated eIF-2 alpha kinase (HRI), the effects of various protoporphyrin IX (PP) compounds on the kinase activities and intersubunit disulfide formation of HRI and on protein synthesis in reticulocyte lysates were examined. Hemin and cobalt protoporphyrin (CoPP) are more effective than ZnPP, NiPP, SnPP, and metal-free PP in promoting intersubunit disulfide bond formation in HRI, in inhibiting the autokinase and eIF-2 alpha kinase activities of HRI, in inhibiting phosphorylation of eIF-2 alpha in rabbit reticulocytes, in maintaining protein synthesis, and in reversing the inhibition of protein synthesis in heme deficiency. There is an apparent correlation of in vitro intersubunit disulfide formation of HRI and the regulation of HRI kinase activities and protein synthesis by these porphyrin compounds. HRI in the reticulocyte lysate can be cross-linked by 1,6-bismaleimidohexane (bis-NEM). The formation of bis-NEM cross-linked dimers in lysates is prevented completely by N-ethylmaleimide (NEM) which alkylates free sulfhydryl groups and is diminished by hemin and CoPP. These results support the view that HRI in hemin-supplemented lysates is in equilibrium between the noncovalently linked dimer and the disulfide-linked dimer. The molecular size of HRI in control, hemin-supplemented, or NEM-treated hemin-supplemented lysates is identical to that of purified HRI; activation of HRI and changes in its thiol status do not significantly affect its molecular size.     

Partial purification and characterization of phorbol ester-regulated translational inhibitor(s) in human HL-60 leukemic cells

Addition of nanomolar concentration of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) to the human promyelocytic HL-60 leukemia cells is associated with a cessation of cellular proliferation and a subsequent differentiation into macrophage-like cells. Because the growth rate of mammalian cells is tightly coupled to the functions of the protein synthetic machinery, we have examined whether TPA induces a change in HL-60 translational functions. Addition of control HL-60 cell extracts to rabbit reticulocyte lysates results in a pronounced inhibition of protein synthesis, while TPA-treated HL-60 cell extracts are significantly less inhibitory. The reduction in TPA-induced translational inhibitory activity can be observed after a 3-6-h treatment and reaches a maximum after 24 h. Fractionation of control cell extracts on DEAE-cellulose columns reveals two inhibitory activities, eluting at 100 and 350 mM KCl, respectively. The DEAE-100 inhibitor(s) is further resolved into two activities by heparin-agarose column chromatography (HEP-100 and HEP-250). TPA treatment of HL-60 cells for 48 h completely eliminates the HEP-250 inhibitory activity and reduces the HEP-100 and the DEAE-350 inhibitory activities by 50 and 25%. Inhibition of protein synthesis in rabbit reticulocyte lysates by DEAE-100 inhibitory activities can be partially reversed by the addition of globin mRNA while translational inhibition by DEAE-350 inhibitor(s) can be reversed by addition of eukaryotic initiation factor (eIF) 2 or fructose 6-phosphate. The DEAE-100 inhibitor(s) causes extensive degradation of radioactive polynucleotides while the DEAE-350 inhibitor(s) is capable of phosphorylating both the alpha- and the beta-subunits of the highly purified rabbit reticulocyte initiation factor eIF-2. These data show that the DEAE-100 inhibitor(s) contains a nuclease while the DEAE-350 inhibitor(s) is associated with eIF-2 alpha and eIF-2 beta protein kinases.     

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.     

Vaccinia specific kinase inhibitory factor prevents translational inhibition by double-stranded RNA in rabbit reticulocyte lysate

Mouse L-cells infected with vaccinia virus produce a specific kinase inhibitory factor (SKIF) which inhibits the activation of the interferon-induced, double-stranded (ds)RNA-dependent, eukaryotic initiation factor (eIF)-2 alpha-specific protein kinase in L-cell extracts (Whitaker-Dowling, P., and Younger, J. S., (1984) Virology 137, 171). The effects of a partially purified preparation of SKIF have been examined in cell-free extracts of rabbit reticulocytes. Both the phosphorylation state of eIF-2 and protein synthetic activity have been determined. SKIF inhibits the phosphorylation of the alpha subunit of eIF-2 by dsRNA-dependent eIF-2 alpha-kinase in reticulocyte lysate, but does not affect phosphorylation of eIF-2 by the heme-sensitive kinase. In addition to its effects on eIF-2 alpha-PKds activity, SKIF prevents dsRNA-induced inhibition of protein synthesis in reticulocyte lysate. In contrast, SKIF does not prevent the translational inhibition caused by hemin depletion. These data provide a direct correlation between the effects of SKIF on eIF-2 alpha phosphorylation and on protein synthetic activity and demonstrate the specificity of SKIF. The results also show that SKIF does not abolish dsRNA sensitivity, but increases the concentration of dsRNA required to activate the kinase and phosphorylate eIF-2.     

Role of reversing factor in the inhibition of protein synthesis initiation by oxidized glutathione

The inhibitions of protein synthesis initiation in heme-deficient reticulocyte lysates and in GSSG-treated hemin-supplemented lysates are both characterized by the activation of heme-regulated eIF-2 alpha kinase, which phosphorylates the alpha-subunit of eukaryotic initiation factor (eIF-2). In both inhibitions, the accumulation of eIF phosphorylated in alpha-subunit (eIF-2(alpha P)) leads to the sequestration of reversing factor (RF) in a phosphorylated 15 S complex, RF.eIF-2(alpha P), in which RF is nonfunctional. A sensitive assay for the detection of endogenous RF activity in protein-synthesizing lysates indicates that, in GSSG-inhibited (1 mM GSSG) lysates, RF is more profoundly inhibited than in heme-deficient lysates. RF inactivation in GSSG-induced inhibition appears to be due to two separate but additive effects: (i) the formation of the phosphorylated 15 S RF complex, RF.eIF-2(alpha P), and (ii) the formation of disulfide complexes which inhibit RF activity. Both inhibitory effects are overcome by catalytic levels of exogenous RF which permits the resumption of protein synthesis. RF activity and protein synthesis in GSSG-inhibited lysates are efficiently restored by the delayed addition of glucose-6-P or 2-deoxyglucose-6-P (1 mM). The rescue of protein synthesis by hexose phosphate (1 mM) is proportional to the extent of RF recovery and is due in part to NADPH generation; even at levels of hexose phosphate (50 microM) too low to support protein synthesis, partial restoration of RF activity occurs due to increased NADPH/NADP+ ratios. The ability of dithiothreitol (1 mM) to restore RF activity in GSSG-treated but not heme-deficient lysates also provides evidence for a reducing mechanism which functions at the level of RF. The results suggest that NADPH plays a role in the maintenance of sulfhydryl groups essential for RF activity.     

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.     

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.     

Differing effects of the protein phosphatase inhibitors okadaic acid and microcystin on translation in reticulocyte lysates.

The effects of the cyanobacterial toxin and protein phosphatase inhibitor, microcystin, on translation in rabbit reticulocyte lysates have been studied. Microcystin inhibited translation with similar potency to the protein phosphatase inhibitor okadaic acid. Unlike low concentrations of okadaic acid, however, it inhibited both the initiation and elongation stages. This was demonstrated using EGTA to inhibit the phosphorylation and inactivation of elongation factor eEF-2. A method for detecting changes in eEF-2 phosphorylation was developed. eEF-2 was found to exist as three different species: eEF-2 was largely monophosphorylated in reticulocyte lysates under control conditions, the remainder being unphosphorylated. Okadaic acid and microcystin increased the level of the bisphosphorylated species. The implications of multiple phosphorylation of eEF-2 for the control of translation is discussed. Microcystin was also found to increase the phosphorylation of eIF-2 alpha (and therefore to inhibit initiation) at lower concentrations than okadaic acid, suggesting that the major eIF-2 alpha phosphatase in the reticulocyte lysate is phosphatase-1.