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.     

Structural requirements for porphyrin inhibition of the hemin-controlled protein kinase and maintenance of protein synthesis in reticulocytes

The role of hemin in the maintenance of protein synthesis in reticulocyte lysates was examined by comparing the effects of various porphyrins and metalloporphyrins on the protein kinase activity of the hemin-controlled repressor and on protein synthesis. The porphyrin requirements for maintenance of protein synthesis were relatively specific. Iron and cobalt metalloporphyrins sustained protein synthesis whereas other metalloporphyrins, metal-deficient porphyrins, and non-porphyrin precursor and degradation products of protoporphyrin IX were ineffective. These same compounds were examined for their effectiveness in inhibiting the protein kinase activity of the hemin-controlled repressor with initiation factor 2 (eIF-2). Most of the metalloporphyrins and porphyrins tested were inhibitory. The presence of the iron atom in the porphyrin was not essential for inhibition, but the maintenance of the integrity of the porphyrin ring was imperative. The porphyrins which inhibited the hemin-regulated protein kinase contained vinyl groups or ethyl groups, or were protonated in the 2- and 4-positions of the porphyrin ring, whereas those with bulky or acidic groups in these positions were ineffective. Precursor and degradation products of protoporphyrin IX and synthetic porphyrins modified at other positions had no effect on the enzyme. Both hemin and protoporphyrin IX inhibited phosphorylation of eIF-2 exogenously added to a reticulocyte lysate; however, hemin sustained protein synthesis in the lysate, whereas protoporphyrin IX did not. These results suggest that regulation of the protein kinase phosphorylating the alpha subunit of eIF-2 is not the only point at which hemin modulates protein synthesis in reticulocytes and reticulocyte lysates, since a correlation between inhibition of protein synthesis, inhibition of protein kinase activity, and phosphorylation of eIF-2 is not observed with all porphyrins.     

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.     

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.     

Possible involvement of the 90-kDa heat shock protein in the regulation of protein synthesis

The heme-sensitive eukaryotic initiation factor (eIF)-2 alpha kinase regulates translational activity in reticulocytes by phosphorylation of the smallest subunit of eukaryotic peptide initiation factor 2, eIF-2. Highly purified preparations of the kinase contain an abundant 90-kDa polypeptide which appears to modulate the activity of the enzyme. The physical properties and structural characteristics of the reticulocyte 90-kDa peptide are similar to those of the 90-kDa heat shock protein (hsp 90) from HeLa and other mammalian cells. The reticulocyte and HeLa cell proteins are shown to be immunologically cross-reactive. A direct comparison of the two proteins by one-dimensional peptide mapping of large peptides generated by limited proteolysis and by reversed-phase high performance liquid chromatography analysis of tryptic peptides indicates that they represent the same protein species. Like the 90-kDa reticulocyte protein, HeLa cell hsp 90 causes increased eIF-2 alpha phosphorylation by the heme-sensitive kinase and is a potent inhibitor of protein synthesis in the reticulocyte lysate system. A potential mechanism for the latter inhibition is inferred. These results implicate hsp 90 in the regulation of protein synthesis via its interaction with and perhaps regulation of the heme-sensitive kinase and phosphorylation of eIF-2 alpha.     

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.     

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.     

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.     

A novel role for aminoacyl-tRNA synthetases in the regulation of polypeptide chain initiation

Exposure of the temperature-sensitive leucyl-tRNA synthetase mutant of Chinese hamster ovary cells, tsH1, to the non-permissive temperature of 39.5 degrees C results in a rapid inhibition of polypeptide chain initiation. This inhibition is caused by a reduced ability of the eukaryotic initiation factor eIF-2 to participate in the formation of eIF-2.GTP.Met-tRNAf ternary complexes and thus in the formation of 43S ribosomal pre-initiation complexes. Associated with this decreased eIF-2 activity is an increased phosphorylation of the eIF-2 alpha subunit. It has previously been shown in other systems that phosphorylation of eIF-2 alpha slows the rate of recycling of eIF-2.GDP to eIF-2.GTP catalysed by the guanine nucleotide exchange factor eIF-2B. We show here that phosphorylation of eIF-2 alpha by the reticulocyte haem-controlled repressor also inhibits eIF-2B activity in cell-free extracts derived from tsH1 cells. Thus the observed increased phosphorylation of eIF-2 alpha at the non-permissive temperature in this system is consistent with impaired recycling of eIF-2 in vivo. Using a single-step temperature revertant of tsH1 cells, TR-3 (which has normal leucyl-tRNA synthetase activity at 39.5 degrees C), we demonstrate here that all inhibition of eIF-2 function reverts together with the synthetase mutation. This establishes the close link between synthetase function and eIF-2 activity. In contrast, recharging tRNALeu in vivo in tsH1 cells at 39.5 degrees C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF-2 function. This indicates that tRNA charging per se is not involved in the regulatory mechanism. Our data indicate a novel role for aminoacyl-tRNA synthetases in the regulation of eIF-2 function mediated through phosphorylation of the alpha subunit of this factor. However, in spite of the fact that cell-free extracts from Chinese hamster ovary cells contain protein kinase and phosphatase activities active against either exogenous or endogenous eIF-2 alpha, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5 degrees C.     

Translational stimulation by reovirus polypeptide sigma 3: substitution for VAI RNA and inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2.

COS cells transfected with plasmids that activate DAI depend on expression of virus-associated I (VAI) RNA to prevent the inhibitory effects of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) kinase (DAI) and restore the translation of vector-derived dihydrofolate reductase mRNA. This VAI RNA requirement could be completely replaced by reovirus polypeptide sigma 3, consistent with its double-stranded RNA (dsRNA)-binding activity. S4 gene transfection of 293 cells also partially restored adenovirus protein synthesis after infection with the VAI-negative dl331 mutant. In dl331-infected 293 cells, eIF-2 alpha was present mainly in the acidic, phosphorylated form, and trans complementation with polypeptide sigma 3 or VAI RNA decreased the proportion of eIF-2 alpha (P) from approximately 85 to approximately 30%. Activation of DAI by addition of dsRNA to extracts of S4 DNA-transfected COS cells required 10-fold-higher levels of dsRNA than extracts made from cells that were not producing polypeptide sigma 3. In extracts of reovirus-infected mouse L cells, the concentration of dsRNA needed to activate DAI was dependent on the viral serotype used for the infection. Although the proportion of eIF-2 alpha (P) was greater than that in uninfected cells, most of the factor remained in the unphosphorylated form, even at 16 h after infection, consistent with the partial inhibition of host protein synthesis observed with all three viral serotypes. The results indicate that reovirus polypeptide sigma 3 participates in the regulation of protein synthesis by modulating DAI and eIF-2 alpha phosphorylation.     

Mechanism of interferon action. Purification and substrate specificities of the double-stranded RNA-dependent protein kinase from untreated and interferon-treated mouse fibroblasts

The double-stranded RNA (dsRNA)-dependent protein kinase which catalyzes the phosphorylation of ribosome-associated protein P1 and the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2) was purified and characterized from mouse fibroblast L929 cells treated with either natural or recombinant interferon and from untreated cells. The dsRNA-dependent P1/eIF-2 alpha kinase was purified at least 1,500-fold from interferon-treated cells; the kinase activity that catalyzed the phosphorylation of eIF-2 alpha copurified with protein P1. The yield of P1/eIF-2 alpha protein kinase activity obtained following purification from cells treated with interferon was about 5-10 times greater than the yield from an equivalent number of untreated cells. The purified protein kinase remained dsRNA dependent. When P1 kinase was activated by dsRNA, a major phosphopeptide designated Xds was phosphorylated; Xds was not phosphorylated from P1 which had not been activated by dsRNA. The apparent native molecular weight of the purified mouse L929 dsRNA-dependent kinase as determined by sedimentation analysis was about 62,000, comparable to the molecular weight of 67,000 determined for denatured L929 phosphoprotein P1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase was highly selective for the alpha subunit of protein synthesis initiation factor eIF-2 and endogenous protein P1. Kinase activity was dependent upon Mg2+, and the Km for ATP was determined to be 5 X 10(-6) M. Histones (H1, H2A-B, H3, and H4) and protein synthesis initiation factors other than eIF-2 (eIF-3, eIF-4A, eIF-4B, and eIF-5) were not substrates or were very poor substrates for the purified dsRNA-dependent protein kinase. N-Ethylmaleimide, ethylenediaminetetraacetic acid, AMP, pyrophosphate, spermine, spermidine, and high concentrations of potassium inhibited both P1 and eIF-2 alpha phosphorylation by the purified kinase, whereas ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phenanthroline did not significantly affect the phosphorylation of either protein P1 or eIF-2 alpha.     

Heat shock-induced translational alterations in HeLa cells. Initiation factor modifications and the inhibition of translation

Heat shock at 45 degrees C virtually abolishes protein synthesis in HeLa cells, but return to 37 degrees C effects a complete recovery and the concomitant synthesis of heat shock-induced proteins. Heat shock induces polysome disaggregation, indicating initiation is principally inhibited. In vitro assays for initiation factor activities reveal heat shock inhibits eukaryotic initiation factor 2 (eIF-2), eIF-(3 + 4F), and eIF-4B. Immunoblot analyses show that eIF-2 alpha and eIF-2 beta become modified during heat shock, and eIF-4B variants disappear. Upon return to 37 degrees C, these alterations reverse. The modifications of eIF-2 alpha and eIF-4B are due to phosphorylation and dephosphorylation, respectively. Enzymatic activities induced by heat shock inhibit protein synthesis and modify initiation factors in a rabbit reticulocyte lysate. Initiation factor modifications may contribute to, or cause, protein synthesis inhibition.     

Induction of interferon in HeLa cells of a protein kinase activated by double-stranded RNA

Treatment of HeLa cells with human fibroblast interferon results in increased levels of latent protein kinase activity that can be activated by double-stranded RNA (dsRNA). The protein kinase activity in extracts of interferon-treated cells is assayed by two methods: (a) inhibition of protein synthesis in rabbit reticulocyte lysates and (b) phosphorylation of two polypeptides of Mr 72000 (P1) and 38000 (the eIF-2 alpha subunit of initiation factor 2). When extracts of interferon-treated cells are fractionated by centrifugation at 150000 x g, the protein kinase activity is found in the pellet fraction. The kinase is maximally activated by 0.1 micrograms/ml poly(I) . poly(C). An increase in protein kinase activity is detected after 8 h of treatment with 100 units interferon/ml or after a 17-h treatment with 12.5 units/ml or greater interferon concentrations. Therefore, the kinase activity increases as a function of both time of treatment and interferon concentration. Addition of actinomycin D to cells during interferon treatment prevents this increase. The protein kinase activity decreases gradually over three days when interferon-treated cells are subsequently grown in the absence of interferon.     

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.     

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.     

The La antigen inhibits the activation of the interferon-inducible protein kinase PKR by sequestering and unwinding double-stranded RNA.

The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both nucleus and cytoplasm of eukaryotic cells. The spectrum of RNAs that interact with the La antigen includes species which also bind to the interferon-inducible protein kinase PKR. We have investigated whether the La antigen can regulate the activity of PKR and have observed that both the autophosphorylation of the protein kinase that accompanies its activation by dsRNA and the dsRNA-dependent phosphorylation of the alpha subunit of polypeptide chain initiation factor eIF-2 by PKR are inhibited in the presence of recombinant La antigen. This inhibition is partially relieved at higher concentrations of dsRNA. Once activated by dsRNA the protein kinase activity of PKR is insensitive to the La antigen. We have demonstrated by a filter binding assay that La is a dsRNA binding protein. Furthermore, when recombinant La is incubated with a 900 bp synthetic dsRNA or with naturally occurring reovirus dsRNA it converts these substrates to single-stranded forms. We conclude that the La antigen inhibits the dsRNA-dependent activation of PKR by binding and unwinding dsRNA and that it may therefore play a role in the regulation of this protein kinase in interferon-treated or virus-infected cells.     

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.     

Resistance of initiation factor 2 (eIF-2alpha) kinases to staurosporine: an approach for assaying the kinases in crude extracts.

We studied the effect of staurosporine on two well characterised mammalian eIF-2alpha kinases, the heme-regulated translational inhibitor (HRI), and interferon-inducible double-stranded RNA-activated protein kinase (PKR). Both pure eIF-2 and a synthetic peptide used to measure the activity of purified or immunoprecipitated enzymes (sequence ILLSELSRRRIRAI) were phosphorylated with purified enzymes and crude preparations of tissues or cells in the presence of the inhibitor. In the presence of 0.25 microM staurosporine (a concentration which completely inhibits a wide range of Ser/Thr protein kinases), the phosphorylation of eIF-2alpha by HRI and PKR was not inhibited. The lack of response of eIF-2alpha kinases to staurosporine allowed us to measure PKR activity in salt washed postmicrosomal supernatants without previous purification of the enzyme. In the presence of poly(I):poly(C), the PKR activator, we detected both an increased phosphorylation of eIF-2alpha and an increment in the autophosphorylation of PKR. We also confirmed an induction of PKR in cultured neuronal cells after treatment with interferon. The results obtained following phosphorylation of the synthetic peptide with crude extracts are less conclusive. Although its phosphorylation is specific because it displaces eIF-2 phosphorylation, and the presence of staurosporine prevents its phosphorylation by other serine/threonine kinases, it is a rather poor substrate for PKR.     

Double-Stranded RNA-Activated Protein Kinase (PKR) Is Negatively Regulated by 60S Ribosomal Subunit Protein L18

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2α), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2α in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2α phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.     

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.