Phosphorylation Of Initiation Factor eIF-2 Research Articles

Phosphorylation of Initiation Factor eIF2 in Response to Stress Conditions Is Mediated by Acidic Ribosomal P1/P2 Proteins in Saccharomyces cerevisiae

Eukaryotic cells contain an unusually large cytoplasmic pool of P1/P2 phosphoproteins, which form the highly flexible 60S subunit stalk that is required to interact with and activate soluble translation factors. In cells, cytoplasmic P1/P2 proteins are exchanged for ribosome-bound proteins in a process that can modulate ribosome function and translation. Here, we analysed different S. cerevisiae stalk mutants grown under stress conditions that result in eIF2α phosphorylation. These mutants either lack a cytoplasmic pool of stalk proteins or contain free but not ribosome-bound proteins. Only cells that contain free P1/P2 proteins induce eIF2 phosphorylation in vivo in response to glucose starvation or osmotic stress. Moreover, we show that free S. cerevisiae P1/P2 proteins can induce in vitro phosphorylation of the initiation factor eIF2 by stimulating the autophosphorylation and activation of GCN2 kinase. Indeed, these ribosomal proteins do not stimulate other eIF2α kinases, such as PKR and HRI. P1/P2 and the known GCN2 activator deacylated tRNA compete for stimulating the eIF2α kinase activity of GCN2, although the P1/P2 proteins are considerably more active. These findings reveal a capacity of free cytoplasmic ribosomal stalk components to stimulate eIF2α phosphorylation, which in turn would modulate translation in response to specific forms of stress that may be linked with the previously described regulatory function of the ribosomal stalk.

Reversal of the double-stranded-RNA-induced inhibition of protein synthesis by a catalytically inactive mutant of the protein kinase PKR.

The interferon-inducible double-stranded-RNA(dsRNA)-dependent protein kinase PKR has been implicated in both the antiviral and cell growth-regulatory effects of the interferons. Over-expression of the wild-type form of this protein inhibits cell proliferation, whereas over-expression of inactive mutant forms transforms cells to a tumourigenic phenotype. It has been suggested that mutant PKR exerts a dominant negative effect on the activity of the wild-type protein kinase. We have investigated this possibility using the rabbit reticulocyte cell-free translation system in which protein synthesis is inhibited by dsRNA due to activation of PKR and phosphorylation of initiation factor eIF-2. Addition of a highly purified inactive PKR mutant, synthesised in a baculovirus-infected insect cell system, rescues protein synthesis from inhibition by low concentrations of dsRNA in a dose-dependent manner. The PKR mutant has no effect on protein synthesis in the absence of dsRNA or in the presence of another inhibitory protein kinase, the haem-controlled repressor. Inhibition of translation can be re-established in the presence of the mutant PKR by adding a higher concentration of dsRNA. These results suggest that inactive mutant PKR does exert a dominant negative effect on wild-type PKR and that this may be due to competition for dsRNA binding.

Epstein-Barr virus gene expression in interferon-treated cells: Implications for the regulation of protein synthesis and the antiviral state

This paper presents data on the effects of interferon treatment on Epstein-Barr virus (EBV) gene expression in latently infected Daudi Burkitt's lymphoma cells, and reviews the possible role of viral gene products in the regulation of translation. In Daudi cells the main virally coded RNAs are the small untranslated RNAs EBER-1 and EBER-2, two mRNAs for the DNA binding protein EBNA-1, and a number of small RNAs containing sequences from the BamHI W repeat region of the viral genome. Interferon treatment does not change the qualitative pattern of EBV gene expression but decreases the levels of the EBNA-1 mRNAs. The chromatographic behaviour of EBV-encoded RNAs on CF11-cellulose indicates that many contain double-stranded regions; these RNAs co-purify with RNA that activates the interferon-induced, dsRNA-sensitive protein kinase DAI. Computer analysis indicates that the exons transcribed from the BamHI W repeats have the potential for formation of very stable secondary structures. Many viruses can counteract the inhibition of protein synthesis mediated by the DAI-catalysed phosphorylation of initiation factor eIF-2 and our data suggest that the small RNA EBER-1 may fulfil this function in the EBV system. During the infection and immortalization of B lymphocytes by EBV the synthesis of large amounts of EBER-1 RNA might thus allow the virus to circumvent one of the interferon-mediated mechanisms of host cell defence.

Control of Translation in Adenovirus-Infected Cells

The initiation of protein synthesis in adenovirus-infected cells is regulated during the late phase in two ways, which may be related. The overall translation rate is maintained by a small viral RNA, VA RNAI, which prevents the phosphorylation of initiation factor eIF-2 by a double-stranded RNA-activated protein kinase, DAI. In addition, the relative efficiency of translation of host cell and viral mRNA populations is regulated in the infected cell during the late phase such that viral mRNAs are selectively utilized. Three viral elements have been implicated in this process: the 5' leader present on most late viral mRNAs; the late protein, 100K; and VA RNA. This article reviews the mechanisms underlying these translational control phenomena.

Regulation of polypeptide chain initiation in Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase. Changes in phosphorylation of initiation factor eIF-2 and in the activity of the guanine nucleotide exchange factor GEF.

When cultures of the temperature-sensitive Chinese hamster ovary cell mutant tsH1 are shifted from 34 degrees C (permissive temperature) to 39.5 degrees C (nonpermissive temperature), protein synthesis is inhibited by more than 80%. This is due principally to a block in activity of polypeptide chain initiation factor eIF-2. In this paper we show that there is impairment of the ability of the guanine nucleotide exchange factor (GEF) to displace GDP from eIF-2 X GDP complexes in extracts from cells incubated at the nonpermissive temperature. Addition of GEF or of high concentrations of eIF-2 stimulates protein synthesis to the level observed in control cell extracts, suggesting that GEF is rate-limiting for eIF-2 activity and overall protein synthesis at the nonpermissive temperature. Analysis of eIF-2 by two-dimensional gel electrophoresis and immunoblotting reveals an increase in the proportion of the alpha subunit in the phosphorylated form from 5.5 +/- 2.4% to 17.2 +/- 3.9% on shifting tsH1 cells from 34 to 39.5 degrees C. No such effect is seen in wild-type cells, which do not exhibit temperature-sensitive protein synthetic activity. Since the primary lesion in tsH1 cells is in their leucyl-tRNA synthetase, these results suggest a role for eIF-2 phosphorylation and GEF activity in coupling the rate of polypeptide chain initiation to the activity of the chain elongation machinery.

Inhibition of HeLa cell protein synthesis under heat shock conditions in the absence of initiation factor eIF-2α phosphorylation

Subjecting a HeLa cell suspension culture to an increase in incubation temperature (from 37 degrees to 42 degrees C) results in the rapid cessation of polypeptide chain synthesis followed by a gradual increase in the synthesis of a class of polypeptides referred to as the heat-shock proteins. It has been proposed that the initial, rapid shutoff of protein synthesis (less than 20 min) is due to the phosphorylation of initiation factor eIF-2 in its alpha subunit, a modification known to result in the inhibition of polypeptide synthesis. Using an in vitro translation system derived from heat-shocked HeLa cells grown in suspension culture, we were unable to find any evidence implicating eIF-2 alpha phosphorylation in the initial shutoff of translation during the heat shock response. These results suggest that the rapid inhibition of protein synthesis observed under heat shock conditions is mediated by a mechanism(s) other than eIF-2 alpha phosphorylation.

Translational control by adenovirus: lack of virus-associated RNAI during adenovirus infection results in phosphorylation of initiation factor eIF-2 and inhibition of protein synthesis.

The dl331 mutant of adenovirus serotype 5 fails to produce virus-associated (VA) RNAI, and cells infected with this mutant do not synthesize proteins efficiently at late times in infection. The translational defect occurs at the level of polypeptide chain initiation, and cell-free extracts prepared from dl331-infected cells exhibit the defect observed in vivo. Addition of either eukaryotic initiation factor 2 (eIF-2) or guanine nucleotide exchange factor (GEF) to these cell-free extracts restores translational activity, with GEF functioning more efficiently in this regard. These results suggest that cells infected with the dl331 mutant develop a translational block at the level of GEF-catalyzed guanine nucleotide exchange and that this block is most likely established through phosphorylation of the alpha subunit of eIF-2. In the present investigation we show that endogenous HeLa cell GEF activity is significantly reduced in cells infected with the dl331 mutant. Further, in contrast to cells infected with wild-type serotype 2 adenovirus, dl331-infected cells contain increased eIF-2 alpha kinase activity. These results indicate that VA RNAI plays a role in suppressing eIF-2 alpha kinase activity during adenovirus infection of HeLa cells.

Assembly and breakdown of mammalian protein synthesis initiation complexes: regulation by guanine nucleotides and by phosphorylation of initiation factor eIF-2.

Eukaryotic cell polypeptide chain initiation factor eIF-2 forms ternary complexes with GTP and initiator Met-tRNAf. These complexes can be destabilized in vitro by the addition of salt-washed 40S ribosomal subunits. Our evidence suggests that this destabilization is mediated by GDP generated by premature hydrolysis of the GTP molecule present in the ternary complex. With complexes formed by using a partially purified preparation of eIF-2 from Ehrlich ascites tumor cells, it is possible to reverse the 40S subunit induced inhibition by creating conditions which eliminate free GDP from the system. This reversal probably occurs due to exchange of GTP for the GDP bound to the initiation factor, in a reaction catalyzed by another factor present in the eIF-2 preparation. However, if the eIF-2 has previously been phosphorylated by the reticulocyte heme-controlled repressor, the 40S subunit induced inhibition cannot be reversed by elimination of free GDP. The instability of initiation complexes containing eIF-2, together with the impairment of guanine nucleotide exchange after phosphorylation of eIF-2 [Clemens, M.J., Pain, V.M., Wong, S.-T., & Henshaw, E. C. (1982) Nature (London) 296, 93-95], may be an important aspect of the mechanism of the inhibition of translation by the heme-controlled repressor.

Purification and phosphorylation of initiation factor eIF-2 from rabbit skeletal muscle

FEBS LettersVolume 143, Issue 1 p. 55-59 Full-length articleFree Access Purification and phosphorylation of initiation factor eIF-2 from rabbit skeletal muscle Christopher G. Proud, Christopher G. Proud School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, England Preset address, for correspondence: The Biological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ, England Search for more papers by this authorVirginia M. Pain, Virginia M. Pain School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, EnglandSearch for more papers by this author Christopher G. Proud, Christopher G. Proud School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, England Preset address, for correspondence: The Biological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ, England Search for more papers by this authorVirginia M. Pain, Virginia M. Pain School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, EnglandSearch for more papers by this author First published: June 21, 1982 https://doi.org/10.1016/0014-5793(82)80273-1Citations: 28 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 P. Borst, J.H.J. Hoeijmakers, Plasmid, 2, (1979), 20– 40. 2 F.R. Opperdoes, P. Borst, FEBS Lett., 80, (1977), 360– 369. 3 M.B. Taylor, H. Berghausen, P. Heyworth, N. Messenger, L.J. Rees, W.E. Gutteridge, Int. J. Biochem., 11, (1980), 117– 120. 4 G.H. Coombs, J.A. Craft, D.T. Hart, Mol. Biochem. Parasitol., (1982), in press 5 D.J. Hammond, W.E. Gutteridge, F.R. Opperdoes, FEBS Lett., 128, (1981), 27– 29. 6 F.R. Opperdoes, A. Markoš, R.F. Steiger, Mol. Biochem. Parasitol., 4, (1981), 291– 309. 7 R.A. Klein, D.J. Linstead, M.V. Wheeler, Parasitology, 71, (1975), 93– 107. 8 I.B.R. Bowman, Trypanosomiases and Leishmaniases with Special Reference to Chagas' Disease. Ciba Found. Symp 20, (1974), Elsevier Biomedical Amsterdam, New York 255– 271. (new ser.) 9 R. Brun, M. Schönenberger, Acta Trop., 36, (1979), 289– 292. 10 R.F. Steiger, F.R. Opperdoes, J. Bontemps, Eur. J. Biochem., 105, (1980), 163– 175. 11 H. Beaufay, A. Amar-Costesec, E.D. Korn Methods in Membrane Biology 6, (1976), Plenum New York 1– 100. 12 H.U. Bergmeyer 2nd edn. Methods of Enzymatic Analysis (1974), Academic Press New York 13 G.H. Dixon, H.L. Kornberg, Biochem. J., 72, (1960), 3P– 14 R.G. Gibbs, J.G. Morris, Methods Enzymol., 17A, (1970), 981– 992. 15 K.E. Richardson, J.S. Thompson, Methods Enzymol., 17A, (1970), 163– 166. 16 T.G. Cooper, H. Beevers, J. Biol. Chem., 244, (1969), 3507– 3513. 17 S. Stein, P. Böhlen, J. Stone, W. Dairman, S. Udenfriend, Arch. Biochem. Biophys., 155, (1973), 203– 212. 18 E. Orellano, J.J. Cazzulo, Mol. Biochem. Parasitol., 3, (1981), 1– 11. 19 F.R. Opperdoes, P. Borst, H. Spits, Eur. J. Biochem., 76, (1977), 21– 28. 20 F.R. Opperdoes, Mol. Biochem. Parasitol., 3, (1981), 181– 186. 21 J.F. Ryley, Biochem. J., 85, (1962), 211– 223. 22 M.A. Cataldi de Flombaum, J.J.B. Cannata, J.J. Cazzulo, E.I. Segura, Comp. Biochem. Physiol., 58B, (1977), 67– 69. Citing Literature Volume143, Issue1June 21, 1982Pages 55-59 ReferencesRelatedInformation

Phosphorylation inhibits guanine nucleotide exchange on eukaryotic initiation factor 2.

There is extensive evidence that protein synthesis in reticulocyte lysates is inhibited during haem deficiency because of phosphorylation of initiation factor eIF-2 by a protein kinase (the haem-controlled represser, HCR)1–5. Other protein kinases, which are activated by low concentrations of double-stranded RNA, have also been identified in reticulocytes1,6 and in extracts from interferon-treated cells7–10, and these phosphorylate an identical site on eIF-2 11–12. Despite the extensive documentation of such enzymes, however, the precise mechanism of inhibition has remained unclear, mainly because phosphorylation fails to affect many partial reactions of polypeptide chain initiation13,14. We present here evidence that the exchange of GTP for GDP bound to eIF-2 is inhibited following phosphorylation of the factor. Such an effect may inactivate eIF-2 by impairing the ability to recycle between successive rounds of protein synthesis.

Regulation of protein synthesis in Ehrlich ascites tumour cells in culture.

Regulation of polypeptide-chain synthesis initiation appears to be an important control mechanism in eukaryotic tissues in response to many forms of stress. In the Ehrlich ascites-tumour cell, formation of the Met-tRNA,MCt-eIF-2.GTP ternary complex and/or the binding of this complex to the native 40s ribosomal subunit is inhibited during nutrient deprivation. In investigating the basis for this inhibition we have examined factors affecting ternary-complex formation in the Ehrlich cell and have compared this system with the reticulocyte, where phosphorylation of initiation factor eIF-2 by the haem-regulated inhibitor (HRI, or haem-controlled repressor, HCR) appears to

Interferon action: Isolation of Nuclease F, A translation inhibitor activated by interferon-induced (2′–5′) oligo-isoadenylate

Cell cultures treated by interferon become unable to support viral replication. At least one of interferon’s antiviral effects is to inhibit viral protein synthesis [l-3]. Interferon induces several biochemical mechanisms which could mediate this inhibition: (i) A double-stranded RNA (dsRNA) and ATP-dependent phosphorylation of initiation factor eIF2 and possibly other ribosome-associated proteins [4-71; (ii) The dsRNA ATP-dependent synthesis of an unusual oligo-adenylate isomer with 2’-5 phosphodiester bonds [7-91; (iii) A mechanism which affects polypeptide chain elongation, that does not require dsRNA and is reversed by tRNAs [lo-l 21. We have reported [3,9] the separation and isolation, from extracts of interferon-treated L cells, of the dsRNA ATP-activated protein kinase PKi, and of the (2’-5’) oligo-isoadenylate synthetase E. Here, we demonstrate that the oligonucleotide produced by E requires, to inhibit mRNA translation, an additional protein F already present constitutively in untreated cells. The purified F is shown to be an oligo-isoadenylate-dependent ribonuclease which degrades the mRNA template. Our data show that the dsRNA ATP-dependent nuclease activity reported [ 13,141 to be increased in extracts of interferon-treated cells, can be explained as the activation of a constitutive ribonuclease, F, by the product of the interferon-induced and dsRNA-dependent oligo-isoadenylate synthetase E. This is in

Analysis of phosphorylation of protein synthesis initiation factor eIF-2 by two-dimensional gel electrophoresis.

We describe a method for examining the state of phosphorylation of initiation factor eIF-2 in reticulocyte lysates. The procedure involves incubation of the lysate with iodo[1-14C]acetate in 8.5 M urea, and fractionation of the labelled proteins by two-dimensional acrylamide gel electrophoresis. This approach has been used to show that haem deficiency, double-stranded RNA, and oxidised glutathione, which all inhibit the initiation of protein synthesis in an analogous manner, all cause a net increase in the level of phosphorylated eIF-2 in the complete lysate protein synthesis system.

Phosphorylation of initiation factor elF-2 and the control of reticulocyte protein synthesis.

When rabbit reticulocyte lysates are incubated in the absence of hemin or in the presence of low concentrations of double-stranded RNA, the rate of initiation of protein synthesis is severely reduced after a lag period in which control rates are observed. This reduced initiation rate is due to inhibition of the binding of Methionyl-tRNAf to native 40S ribosomal subunits and is caused by a macromolecular inhibitor which is activated under these conditions. This paper shows that the inhibitors activated in these two situations appear to be different entities, but that in both cases, the inhibitor has an associated protein kinase activity which is highly selective for the small subunit of elF-2, the initiation factor which catalyzes binding of Methionyl-tRNAf to 40S subunits. We present several lines of evidence in support of the hypothesis that the phosphorylation of elF-2 by these kinases is basis of the control of initiation in lysates incubated under these conditions.