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PubMed1979

The respective roles of the protein kinase and pppA2' p5' A2' p5 A-activated endonuclease in the inhibition of protein synthesis by double stranded RNA in rabbit reticulocyte lysates.

Double-stranded RNA (dsRNA) inhibits protein synthesis in rabbit reticulocyte lysates by activating the synthesis of the endonuclease effector pppA2' p5' A2' p5' A(2-5A) and a protein kinase which phosphorylates the protein synthesis initiation factor eIF-2. Under certain assay conditions, high concentrations of dsRNA are without inhibitory effect in many lysates (high dsRNA "reversible" lysates). In these lysates natural dsRNA at low concentrations stimulated protein kinase activity to a greater extent than did the synthetic dsRNA poly rI.rC. Synthesis of 2--5A was greater when poly rI.rC was used. However, a number of factors, including the salt concentration and messenger RNA used, combine to determine the overall effect of dsRNA on protein synthesis under any given set of experimental conditions.

Adenine Nucleotides↗
PubMed1978

Interferon action: two distinct pathways for inhibition of protein synthesis by double-stranded RNA.

Double-stranded RNA inhibits protein synthesis in at least two ways. It activates a protein kinase that blocks peptide chain initiation by phosphorylating the peptide chain initiation factor eIF-2 and also activates an endonuclease that inactivates different mRNAs at different rates. The protein kinase and the endonuclease have been partially purified from interferon-treated Ehrlich ascites tumor cells. The 2',5'-oligoadenylates [pppA(2'p5'A)n], found found earlier to be mediators in the activation of the endonuclease by double-stranded RNA, are not mediators in the activation of the protein kinase by double-stranded RNA.

Adenine Nucleotides↗
PubMed1978

Regulation of protein synthesis: activation by double-stranded RNA of a protein kinase that phosphorylates eukaryotic initiation factor 2.

Incubation of reticulocyte lysates or isolated crude ribosomes with low levels of double-stranded RNA (0.1-10 ng/ml) induces the formation of an inhibitor of protein synthesis initiation similar to that observed in heme deficiency. The inhibitor is associated with a cyclic AMP-independent protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) that phosphorylates the small polypeptide (38,000 daltons) of the eukaryotic initiation factor eIF-2. Activation of the inhibitor requires ATP in addition to double-stranded RNA and is accompanied by the phosphorylation of a 67,000-dalton polypeptide of unknown function. The inhibitor remains associated with the ribosomes during high-speed sedimentation. Once formed, the ribosome-associated inhibitor phosphorylates eIF-2 and inhibits protein synthesis in the absence of double-stranded RNA. Inhibition is prevented by exogenous eIF-2. The bound inhibitor can be solubilized by extraction with 0.5 M KCl. The soluble inhibitor preparation retains the ability to phosphorylate the small polypeptide of eIF-2 and to inhibit protein synthesis. Untreated crude ribosomes also contain cyclic AMP-independent protein kinase activities that phosphorylate the middle polypeptide (49,000 daltons) of eIF-2 and several polypeptide subunits of eIF-3 (160,000, 125,000, and 65,000 daltons); these kinase activities are not affected by double-stranded RNA and do not inhibit protein synthesis.

Animals↗
PubMed1977

Characterization of a rat liver factor that inhibits initiation of protein synthesis in rabbit reticulocyte lysates.

Protein synthesis in rabbit reticulocytes and their lysates is regulated by heme. In heme-deficient reticulocyte lysates, protein synthesis proceeds at the initial rate for several minutes and then declines abruptly. Inhibition of protein synthesis is due to the activation of a heme-regulated translational inhibitor (HRI) which blocks the initiation of protein synthesis. Addition of the isolated HRI to hemin-supplemented lysates causes inhibition of initiation similar to that observed in heme-deficiency. HRI has been shown to be a protein kinase that specifically phosphorylates the Met-tRNA(f) binding factor (eIF-2). We have isolated an inhibitor (LI) of protein chain initiation from rat liver which displays properties similar to those of HRI: (i) the chromatographic behavior of LI on DEAE-Sephadex, DEAE-cellulose, and phosphocellulose is similar to that of HRI; (ii) both LI and HRI inhibit protein chain initiation in rabbit reticulocyte lysates with the same kinetics of inhibition-i.e., an initial period of synthesis for several minutes at the control rate followed by an abrupt decline in the rate of initiation; (iii) both inhibitions are prevented or reversed by eIF-2; (iv) GTP (2 mM) prevents, and ATP (2 mM) potentiates, the inhibition of protein synthesis induced by either inhibitor; (v) LI is associated with a protein kinase that also phosphorylates the 38,000-dalton subunit of elF-2. These findings indicate that a mechanism for the regulation of protein synthesis similar to that found in rabbit reticulocytes may be present in rat liver.

Adenosine Triphosphate↗
PubMed1978

Inhibition of protein synthesis initiation by oxidized glutathione: activation of a protein kinase that phosphorylates the alpha subunit of eukaryotic initiation factor 2.

Oxidized glutathione (GSSG) (0.02-0.5 mM) inhibits reticulocyte lysates by a mechanism similar to that observed in heme deficiency. Incubation of hemin-supplemented postribosomal supernates with GSSG results in the activation of a translational inhibitor [I(GSSG)]. The activation of I(GSSG) is enhanced by the presence of an energy-regenerating system. The simultaneous addition of 1 mM dithiothreitol blocks the activation of the GSSG-induced inhibitor; however, once inhibitor is formed, its activity is not affected by 1 mM dithiothreitol. GSSG-treated postribosomal supernates and partially purified preparations of I(GSSG) inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. Inhibition by I(GSSG) is blocked by cyclic AMP (2-10 mM) and is potentiated by ATP (2 mM). The inhibition is also blocked or reversed by eukaryotic initiation factor eIF-2. The activation of I(GSSG) is accompanied by an increased cyclic AMP-independent protein kinase activity which phosphorylates the 38,000-dalton component (alpha subunit) of eIF-2; however, GSSG treatment of supernates does not alter the activity of the cyclic AMP-independent protein kinase activity that phosphorylates the 49,000-dalton polypeptide component (beta subunit) of eIF-2. These data indicate that GSSG treatment of reticulocyte lysates results in the activation of a protein kinase with inhibitory and phosphorylation properties similar to those of the heme-regulated cyclic AMP-independent protein kinase which is activated in heme deficiency.

Cell-Free System↗
PubMed1978

Mode of action of the hemin-controlled inhibitor of protein synthesis.

Despite the finding that the hemin-controlled translational inhibitor in reticulocyte lysates is a cyclic AMP-independent protein kinase that phosphorylates the small subunit of the initiation factor eIF-2, the mechanism of inhibition of translation remained unexplained. Whereas treatment of hemin-containing lysates with inhibitor in the presence of ATP inhibited translation, the same treatment of highly purified eIF-2 did not affect its ability to form a ternary complex with initiator Met-tRNA and GTP or a 40S initiation complex. We have isolated from ribosomal salt washes a protein (eIF-2 stimulating protein) that enhances the capacity of unphosphorylated eIF-2 to form ternary or 40S initiation complexes but has no effect on the phosphorylated factor. At low concentrations, eIF-2 is virtually inactive without this stimulating protein. Therefore, the translational inhibitor acts by converting eIF-2 to a form that is not stimulated by the stimulating protein.

Heme↗
PubMed1979

Regulation of protein synthesis.

Synthesis of globin in reticulocyte lysates depends on the presence of heme, the prosthetic group of hemoglobin. In the absence of heme, an inhibitor of polypeptide chain initiation is activated. The inhibitor is a cyclic AMP-independent protein kinase that catalyzes the phosphorylation by ATP of the small subunit of the initiation factor eIF-2. This blocks the interaction of eIF-2 with eIF-2-stimulating protein (ESP) that is essential for initiation. Our observations are consistent with the view that the inhibitor is activated by phosphorylation catalyzed by a cyclic AMP-dependent protein kinase. Heme inhibits this enzyme and, in this way, prevents activation of the inhibitor of chain initiation.

Animals↗
PubMed1979

Regulation of protein synthesis in rabbit reticulocyte lysates: additional initiation factor required for formation of ternary complex (eIF-2.GTP.Met-tRNAf) and demonstration of inhibitory effect of heme-regulated protein kinase.

Heme deficiency in rabbit reticulocytes and their lysates leads to the activation of a heme-regulated translational inhibitor (HRI) which causes the cessation of polypeptide initiation. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 binds Met-tRNA(f) and GTP in ternary complex. As a continuation of the studies on the molecular basis of the inhibition of the formation of 40S ribosomal subunit-Met-tRNA(f) complexes by HRI [Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., Roy, R. & Gupta, N. K. (1978) Proc. Natl. Acad. Sci. USA 75, 745-749], we describe here the isolation and some characteristics of a factor that is required for the HRI-catalyzed inhibition of eIF-2-promoted ternary complex formation. In the presence of 1 mM Mg(2+), ternary complex formation by eIF-2 is dependent on the presence of this stabilization factor (SF). Under these conditions, SF increases the rate and the extent of ternary complex formation. This finding suggests that the interaction of SF with eIF-2 causes a conformational change that stabilizes eIF-2 and promotes efficient ternary complex formation by increasing the affinity of eIF-2 for GTP and Met-tRNA(f). In the absence of Mg(2+), however, eIF-2 efficiently forms the ternary complex and SF has little effect on its ternary complex formation capacity-hence, the name eIF-2 stabilization factor (SF). In the presence of SF, HRI markedly inhibits (70-80%) the ternary complex formation capacity of eIF-2. The inhibitory effect requires both HRI and ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of eIF-2. Both the rate and the extent of the SF-dependent ternary complex formation are inhibited. These findings are consistent with the idea that phosphorylation causes a conformational change in eIF-2 such that its interactions with other initiation factors in the formation and the binding of ternary complex to 40S ribosomal subunits are inhibited.

Animals↗
PubMed1978

Effect of hemin on site-specific phosphorylation of eukaryotic initiation factor 2.

Initiation factor 2 (eIF-2) is phosphorylated in vitro by two different cyclic nucleotide-independent protein kinases. As previously shown, a protein kinase activity that comigrates with the major casein kinase activity from rabbit reticulocytes phosphorylates eIF-2beta. In addition, a second protein kinase that specifically phosphorylates eIF-2alpha has been identified. Both protein kinase activities demonstrate cyclic nucleotide-independent activity and are not inhibited by the inhibitor protein diagnostic for cyclic AMP-regulated protein kinase activities. Phosphorylation of eIF-2alpha is almost completely inhibited by 20--35 muM hemin, whereas phosphorylation of eIF-2beta is only partially inhibited. Hemin acts by decreasing the rate of incorporation of phosphate into eIF-2alpha. The protein kinase activity that modifies eIF-2alpha has been shown to have inhibitory activity in the cell-free protein-synthesizing system, whereas the protein kinase for eIF-2beta has no effect. The identity of the former enzyme with the hemin-controlled repressor and role of hemin in the control of initiation are discussed.

Adenosine Triphosphate↗
PubMed1979

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.

Animals↗
PubMed1979

Protein synthesis in rabbit reticulocytes: characteristics of a postribosomal supernatant factor that reverses inhibition of protein synthesis in heme-deficient lysates and inhibition of ternary complex (Met-tRNAfMet.eIF-2.GTP) formation by heme-regulated inhibitor.

During heme deficiency in reticulocyte lysates, a translational inhibitor (heme-regulated inhibitor, HRI) that blocks polypeptide chain initiation is activated. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNAfMet binding factor, eIF-2. Phosphorylation of eIF-2 by HRI prevents its interaction with at least two additional factors, resulting in a net reduction in formation of ternary complex (Met-tRNAfMet.eIF-2.GTP) and AUG-dependent transfer of Met-tRNAfMet to 40S ribosomal subunits. A factor (sRF) that reverses protein synthesis inhibition in heme-deficient lysates has been purified from reticulocyte postribosomal supernatant. sRF also reverses the inhibition of ternary complex formation by HRI in a fractionated system. The ternary complex inhibition reversal activity and the protein synthesis inhibition reversal activity cosediment at 12.5 S upon glycerol density gradient centrifugation, and both activities are sensitive to heat or N-ethylmaleimide. Purified sRF does not dephosphorylate eIF-2 whose phosphorylation has been catalyzed by HRI, nor does the sRF prevent the phosphorylation of eIF-2 by HRI in a fractionated system. sRF stimulates ternary complex formation by both phosphorylated and nonphosphorylated eIF-2. These observations suggest that the sensitivity of protein synthesis to phosphorylation of eIF-2 by HRI may be modulated by the concentration and activity of sRF.

Animals↗
PubMed1979

The interferon-induced protein kinase PK-i from mouse L cells.

Interferon-treated L cells are characterized by an increased protein kinase activity that can selectively phosphorylate the small subunit of eukaryotic initiation factor 2. This protein kinase, PK-i, has been extensively purified and shown to be a potent inhibitor of mRNA translation. The purified PK-i contains the endogenously phosphorylated 67,000 Mr protein characteristic of interferon-treated cell extracts. PK-i can also phosphorylate arginine-rich histones. Purified PK-i can be activated by preincubation with ATP (but not adenylyl imidodiphosphate) and low concentrations of double-stranded RNA. The activation results in an increase in the first rate of eIF-2 phosphorylation. Activated PK-i becomes resistant to high concentrations of double-stranded RNA and more thermostable. A stimulator of PK-i activity, factor A, was isolated, as well as a specific phosphoprotein phosphatase that dephosphorylates the 67,000 Mr protein and eIF-2. These two factors, which are present in untreated L cells, may regulate the translation inhibitory activity of the interferon-induced and double-stranded RNA-activated protein kinase PK-i.

Animals↗
PubMed1979

Regulation of protein synthesis in reticulocyte lysates: immune serum inhibits heme-regulated protein kinase activity and differentiates heme-regulated protein kinase from double-stranded RNA-induced protein kinase.

A specific immune serum to the heme-regulated inhibitor (HRI) has been prepared by immunizing chickens with highly purified reversible HRI prepared from rabbit reticulocyte lysates. Studies with this immune serum demonstrate that the behavior of purified reversible HRI is similar to that of the inhibitor activated in rabbit reticulocyte lysates: the immune serum (i) inhibits the phosphorylation of the small subunit (38,000 daltons) of the eukaryotic initiation factor eIF-2 by both crude and purified inhibitor preparations; (ii) prevents the concomitant inhibition of protein synthesis by both crude and purified inhibitor preparations; and (iii) prevents the autophosphorylation of the 95,000-dalton polypeptide in purified and crude HRI preparations. The protein kinase and inhibitory activities of crude and partially purified preparations of the double-stranded RNA-induced inhibitor of protein synthesis are not affected by the immune serum prepared to reversible HRI. These results indicate that the inhibitor induced by double-stranded RNA is antigenically distinct from the reversible HRI.

Animals↗
PubMed1978

Regulation of protein synthesis in rabbit reticulocyte lysates: purification and characterization of heme-reversible translational inhibitor.

To define the mechanism of regulation of the protein kinase that is activated in heme deficiency and that inhibits initiation of protein synthesis, we have isolated and purified the heme-reversible form of the protein kinase from rabbit reticulocytes. The inhibitory activity is found in a single band after polyacrylamide gel electrophoresis under nondenaturing conditions. It migrates as a 95,000-dalton polypeptide in 15% sodium dodecyl sulfate/polyacrylamide gels. This purified inhibitor becomes self-phosphorylated in the presence of ATP; the phosphorylated protein and the inhibitory activity copurify. The inhibitor produces characteristic biphasic kinetics of inhibition in reticulocyte lysates and phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2); the inhibition is reversed by added eIF-2. In contrast to the heme-irreversible inhibitor, this heme-reversible inhibitor is no longer inhibitory after incubation with 20 micron hemin. Incubation with hemin also inhibits self-phosphorylation. Preincubation of the heme-reversible inhibitor in the presence of ATP potentiates the inhibition of protein synthesis in the subsequent incubation, as does treatment with N-ethylmaleimide. Phosphorylation of the heme-reversible inhibitor and inhibition of protein synthesis in the lysate due to phosphorylation of eIF-2 appear to be related. These findings suggest that hemin acts directly on the heme-reversible inhibitor.

Adenosine Triphosphate↗
PubMed1978

Mode of action of the hemin-controlled inhibitor of protein synthesis: studies with factors from rabbit reticulocytes.

Previously [de Haro, C., Datta, A & Ochoa, S. (1978) Proc. Natl. Acad. Sci. USA 75, 243--247] it was shown with initiation factors from Artemia salina embryos that the activity of the initiator methionyl-tRNA binding factor eIF-2 is stimulated by another factor (ESP, for eIF-2 stimulating protein) present, like eIF-2, in ribosomal salt washes. Incubation of eIF-2 with translational inhibitor from rabit reticulocytes, in the presence of ATP, abolished the ESP effect. At physiological concentrations eIF-2 was virtually inactive without ESP. These observations indicated that the translational inhibitor acts by converting eIF-2 to a form that is not stimulated by ESP. The same observations have now been made with eIF-2 and ESP from rabbit reticulocytes but, in this case, the dependence of eIF-2 activity on ESP is much more pronounced than with the A. salina factors. eIF-2 from reticulocytes interacts with ESP from A. salina and conversely.

Animals↗
PubMed1979

The activity of eukaryotic initiation factor eIF-2 in ternary complex formation with GTP and Met-tRNA.

Ternary complex formation between [3H]Met-tRNAf, [14C]H3-eIF-2, and GTP was measured on nitrocellulose filters. It is shown that [3H]Met-tRNAf and [14C]H3-eIF-2 are present on the filter in equimolar amounts when ATP, creatine phosphate, and creatine kinase are included in the reaction mixture. Under these conditions the factor is 100% active. With small amounts of factor significant losses occur due to adsorption to the wall of the reaction vessels, resulting in seemingly low activities of eIF-2. These losses can be prevented by the presence of "stimulatory" proteins, which enhance the recovery of both [3H]Met-tRNAf and [14C]H3-eIF-2 on the filter but do not alter their ratio.

Animals↗
PubMed1979

Stimulation of protein synthesis and Met-tRNAf binding by phosphorylated sugars: studies on their mechanism of action.

It has been previously reported by J. R. Lenz et al. [(1978) Biochemistry 17, 80--87] that certain phosphorylated sugars stimulate protein synthesis in extracts of mammalian cells. This effect was found to be due to a stimulation of Met-tRNAf binding to 40S ribosomal subunits, both in whole extracts and with isolated ribosomes. However, formation of a ternary complex of Met-tRNAf, initiation factor eIF-2, and GTP was not stimulated. It was also shown that the stimulation is not due solely to metabolism of the sugars. The present communication further characterizes the stimulatory effect of the sugars. They were found to prevent the inactivation of ribosomes that occurs during protein synthesis incubations. The sugars were also found to inhibit cAMP-dependent protein kinases noncompetitively. However, they stimulate Met-tRNAf binding to 40S ribosomal subunits even under conditions in which an inhibition of protein kinase has no effect. Although it has bot been possible to demonstrate a direct association of the sugars with the 40S initiation complex, the evidence suggests that their effect is mediated by an interaction with one of the components involved in the formation of this complex.

Animals↗
PubMed1979

Initiation of mammalian protein synthesis. The multiple functions of the initiation factor eIF-3.

The protein synthesis initiation factor eIF-3 (a multicomponent protein complex) was labelled with 32P by phosphorylation with a protein kinase present in a partially purified 'hemin-controlled repressor' preparation. The interaction of the labelled factor with the 40 S ribosomal subunit during the course of initiation was followed. It binds to the 40 S subunit in the absence of other initiation factors and inhibits the Mg2+-dependent reassociation of the 40 S with the 60 S ribosomal subunit. It stimulates the binding of the ternary complex (eIF-2, GTP, Met-tRNAf) to the 40 S subunit, and earlier work (Trachsel, H., Schreier, M.H., Erni, B. and Staehelin, T. (1977) J. Mol. Biol. 116, 745-767) also showed it to be essential for the subsequent binding of mRNA. The factor is released from the 40 S initiation complex during the 60 S subunit joining reaction.

Animals↗