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Translational Control of Protein Synthesis in Reticulocyte Lysates by eIF-2α Kinases

  • R. L. Matts
  • D. H. Levin
  • R. Petryshyn
  • N. S. Thomas
  • I. M. London
Part of the Proceedings in Life Sciences book series (LIFE SCIENCES)

Abstract

The initiation of protein synthesis in reticulocyte lysates is inhibited by heme deficiency, double-stranded RNA, or oxidized glutathione (Zucker and Schulman 1968; Rabinovitz et al. 1969; Hunt et al. 1972; Ehrenfeld and Hunt 1971; Kosower et al. 1972). In lysates, the inhibitions are all characterized by a brief period of control linear synthesis, followed by an abrupt decline in this rate as protein synthesis shuts off. The inhibitions are due primarily to the activation of cAMP-independent protein kinases that specifically phosphorylate the 38000 mol. wt. α-subunit of the eukaryotic initiation factor-2 (eIF-2) (Levin et al. 1976; Kramer et al. 1976;Ranu and London 1976; Farrell et al. 1977; Gross and Mendelewski 1977; Levin and London 1978; Ernst et al. 1978a). Similar inhibitions of protein synthesis are produced by adding the purified inhibitors, the heme-regulated eIF-2α kinase (HRI), or the double-stranded RNA-activated eIF-2α kinase (dsI), to reticulocyte lysates. The inhibition of protein synthesis is a result of a block in the early steps of protein chain initiation and is immediately preceded by a decrease in the formation of the [eIF-2 Met-tRNAf·GTP] ternary complex and the [40S-eIF-2-Met-tRNAf·GTP] 43S initiation complex (de Haro et al. 1978; Ranu et al. 1978; de Haro and Ochoa 1978; Ranu and London 1979; de Haro and Ochoa 1979; Das et al. 1979). Studies by Cherbas and London (1976) suggested that the principal effect of HRI was the impairment of the recycling of eIF-2. In vitro studies have suggested that the recycling of eIF-2 requires the presence of an additional multipolypeptide factor, whose function is inhibited by the phosphorylation of eIF-2.

Keywords

Ternary Complex Creatine Phosphokinase Binary Complex Translational Control Eukaryotic Initiation Factor 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Amesz H, Goumans H, Haubrich-Morre T, Voorma HO, Benne R (1979) Purification and characterization of a protein factor that reverses the inhibition of protein synthesis by the heme-regulated translational inhibitor in rabbit reticulocyte lysates. Eur J Biochem 98: 513–520PubMedCrossRefGoogle Scholar
  2. Cherbas L, London IM (1976) Mechanism of delayed inhibition of protein synthesis in heme-deficient rabbit reticulocyte lysates. Proc Natl Acad Sci USA 73: 3506–3510PubMedCrossRefGoogle Scholar
  3. Clemens MJ, Pain VM, Wong S, Henshaw EC (1982) Phosphorylation inhibits guanine nucleotide exchange on eukaryotic initiation factor 2. Nature (Lond) 296: 93–95CrossRefGoogle Scholar
  4. Clemens MJ, Safer B, Merrick WC, Anderson WF, London IM (1975) Inhibition of protein synthesis in rabbit reticulocyte lysates by double stranded RNA and oxidized glutathione: independent mode of action on polypeptide chain initiation. Proc Natl Acad Sci USA 72: 1286–1290PubMedCrossRefGoogle Scholar
  5. Das A, Ralston RO, Grace M et al. (1979) Protein synthesis in rabbit reticulocytes-mechanism of protein synthesis inhibition by heme-regulated inhibitor 24. Proc Natl Acad Sci USA 76: 5076–5080PubMedCrossRefGoogle Scholar
  6. deHaro C, Datta A, Ochoa S (1978) Mode of action of the hemin controlled inhibitor of protein synthesis. Proc Natl Acad Sci USA 75: 243–247CrossRefGoogle Scholar
  7. deHaro C, Ochoa S (1978) Mode of action of hemin-controlled inhibitor of protein synthesis-studies with factors from rabbit reticulocytes 2. Proc Natl Acad Sci USA 75: 2713–2716CrossRefGoogle Scholar
  8. deHaro C, Ochoa S (1979) Further studies on the mode of action of heme controlled translational inhibitor. Proc Natl Acad Sci USA 76: 1741–1745CrossRefGoogle Scholar
  9. Ehrenfeld E, Hunt T (1971) Double-stranded poliovirus RNA inhibits initiation of protein synthesis by reticulocyte lysates. Proc Natl Acad Sci USA 68: 1075–1078PubMedCrossRefGoogle Scholar
  10. Ernst V, Levin DH, London IM (1978a) Inhibition of protein synthesis initiation by oxidized glut- athione-activation of a protein kinase that phosphorylates alpha-subunit of eukaryotic initiation factor 2. Proc Natl Acad Sci USA 75: 4110–4114PubMedCrossRefGoogle Scholar
  11. Ernst V, Levin DH, London IM (1978b) Evidence that glucose 6-phosphate regulates protein synthesis initiation in reticulocyte lysates. J Biol Chem 253: 7163–7162PubMedGoogle Scholar
  12. Ernst V, Levin DH, Ranu RS, London IM (1976) Control of protein synthesis in reticulocyte lysates- effects of 3’-5’-cyclic AMP, ATP and GTP upon inhibitions induced by heme deficiency, double- stranded RNA, and a reticulocyte translational inhibitor. Proc Natl Acad Sci USA 73: 1112–1116PubMedCrossRefGoogle Scholar
  13. Farrell P, Balkow J, Hunt T, Jackson RJ, Trachsel H (1977) Phosphorylation of initiation factor F-2 and the control of reticulocyte protein synthesis. Cell 11: 187–200PubMedCrossRefGoogle Scholar
  14. Farrell PJ, Hunt T, Jackson RJ (1978) Analysis of phosphorylation of protein synthesis initiation factor F-2 by two-dimensional gel electrophoresis. Eur J Biochem 89: 517–521PubMedCrossRefGoogle Scholar
  15. Grace M, Ralston RO, Banerjee AC, Gupta NK (1982) Protein synthesis in rabbit reticulocytes. Characteristics of the protein factor RF that reverses inhibition in heme-deficient reticulocyte lysates. 30. Proc Natl Acad Sci USA 79: 6517–6521PubMedCrossRefGoogle Scholar
  16. Gross M, Mendelewski J (1977) Additional evidence that the hemin controlled translational repressor from rabbit reticulocytes is a protein kinase. Biochem Biophys Res Commun 74: 559–569PubMedCrossRefGoogle Scholar
  17. Hunt T, Vanderhoff G, London IM (1972) Control of globin synthesis, the role of heme. J Mol Biol 66: 471–481PubMedCrossRefGoogle Scholar
  18. Kaempfer R (1974) Identification and NA-binding properties of an initiation factor capable of relieving translational inhibition induced by heme deprivation or double-stranded RNA. Biochem Biophys Res Commun 61: 591–597CrossRefGoogle Scholar
  19. Konieczny A, Safer B (1983) Purification of the eukaryotic initiation factor 2, eukaryotic initiation factor 2B complex and characterization of its guanine nucleotide exchange activity during protein synthesis initiation. J Biol Chem 258: 3402–3408PubMedGoogle Scholar
  20. Kosower NS, Vanderhoff GA, Kosower EM (1972) Glutathione VIII. The effects of glutathione disulfide on initiation of protein synthesis. Biochim Biophys Acta 272: 623–627PubMedGoogle Scholar
  21. Kramer G, Cimadivella M, Hardesty B (1976) Specificity of the protein kinase activity associated with the hemin controlled repressor of rabbit reticulocyte. Proc Natl Acad Sci USA 73: 3078–3082PubMedCrossRefGoogle Scholar
  22. Leroux A, London IM (1982) Regulation of protein synthesis by phosphorylation of eukaryotic initiation factor 2-alpha in intact reticulocytes and reticulocyte lysates. Proc Natl Acad Sci USA 79: 2147–2151PubMedCrossRefGoogle Scholar
  23. Levin DH, London JM (1978) Regulation of protein synthesis-activation by double-stranded RNA of a protein kinase that phosphorylates eukaryotic initiation factor 2. Proc Natl Acad Sci USA 75: 1121–1125PubMedCrossRefGoogle Scholar
  24. Levin DH, Ranu RS, Ernst V, London IM (1976) Regulation of protein synthesis in reticulocyte lysates: Phosphorylation of methionyl-NA binding factor by protein kinase activity of translational inhibitor isolated from heme-deficient lysate. Proc Natl Acad Sci USA 73: 3112–3116PubMedCrossRefGoogle Scholar
  25. London IM, Fagard R, Leroux A, Levin DH, Matts R, Petryshyn R (1983) Goldwasser E (ed) Regulation of hemoglobin biosynthesis. Elsevier Biomedical, New York, pp 165–183Google Scholar
  26. Matts RL, Levin DH, London IM (1983) Effect of phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 on the function of reversing factor in the initiation of protein synthesis. Proc Natl Acad Sci USA 80: 2559–2563PubMedCrossRefGoogle Scholar
  27. Pain VM, Clemens MJ (1983) Assembly and breakdown of mammalian protein synthesis initiation complexes: regulation by guanine nucleotides and by phosphorylation of initiation factor eIF-2. Biochemistry 22: 726–733PubMedCrossRefGoogle Scholar
  28. Panniers R, Henshaw EC (1983) A GDP/GTP exchange factor essential for eukaryotic initiation factor 2 cycling in Ehrlich ascites tumor cells and its regulation by eukaryotic initiation factor 2 phosphorylation. J Biol Chem 258: 7928–7934PubMedGoogle Scholar
  29. Pelham HRB, Jackson RJ (1976) An efficient NA-dependent translation system from reticulocyte lysates. Eur J Biochem 67: 247–256PubMedCrossRefGoogle Scholar
  30. Rabinovitz M, Freedman ML, Fisher JM, Maxwell CR (1969) Translational control in hemoglobin synthesis. Cold Spring Harbor Symp Quant Biol 34: 567–578PubMedGoogle Scholar
  31. Ranu RS, London IM (1976) Regulation of protein synthesis in rabbit reticulocyte lysates-purification and initial characterization of cyclic 3’-5 ‘-AMP independent protein kinase of heme-regulated translational inhibitor (Phosphorylation of Met-NAf binding factor). Proc Natl Acad Sci USA 73: 4349–4353PubMedCrossRefGoogle Scholar
  32. Ranu RS, London IM (1979) 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. Proc Natl Acad Sci USA 76: 1079–1083PubMedCrossRefGoogle Scholar
  33. Ranu RS, London IM, Das A et al. (1978) Regulation of protein synthesis in rabbit reticulocyte lysates by heme-regulated protein kinase - inhibition of interaction of met-tRNAf met binding factor with another initation factor in formation of met-tRNAf met. 40S ribosomal subunit complexes. Proc Natl Acad Sci USA 75: 745–749PubMedCrossRefGoogle Scholar
  34. Siekierka J, Manne V, Mauser L, Ochoa S (1983) Polypeptide chain initiation in eukaryotes. Reversibility of the ternary complex-forming reaction. Proc Natl Acad Sci USA 80: 1232–1235PubMedCrossRefGoogle Scholar
  35. Siekierka J, Mauser L, Ochoa S (1982) Mechanism of polypeptide chain initiation in eukaryotes and its control by phosphorylation of the alpha-subunit of initiation factor 2. Proc Natl Acad Sci USA 79: 2537–2540PubMedCrossRefGoogle Scholar
  36. Siekierka J, Mitsui K, Ochoa S (1981) Mode of action of the heme-controlled translational inhibitor: relationship of eukaryotic initiation factor 2-stimulating protein to translation restoring factor. Proc Natl Acad Sci USA 78: 220–223PubMedCrossRefGoogle Scholar
  37. Walton GM, Gill GN (1975) Nucleotide regulation of eukaryotic protein synthesis initiation complex. Biochim Biophys Acta 390: 231–245PubMedGoogle Scholar
  38. Walton GM, Gill GN (1976) Regulation of ternary (Met-NAf.GTP.eukaryotic initiation factor 2) protein synthesis initiation complex formation by the adenylate energy charge. Biochim Biophys Acta 418: 195–203PubMedGoogle Scholar
  39. Voorma HO, Amesz H (1982) Grunberg-Manago M, Safer B (eds) Developments in biochemistry, vol 24. Elsevier Biomedical, New York, pp 297–309Google Scholar
  40. Zucker WV, Schulman HM (1968) Stimulation of globin-chain initiation by hemin in the reticulocyte cell-free system. Proc Natl Acad Sci USA 59: 582–589PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag, Berlin Heidelberg 1984

Authors and Affiliations

  • R. L. Matts
    • 1
  • D. H. Levin
    • 1
  • R. Petryshyn
    • 1
  • N. S. Thomas
    • 1
  • I. M. London
    • 1
  1. 1.Division of Health Sciences and Technology and The Department of BiologyThe Harvard Massachusetts Institute of TechnologyCambridgeUSA

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