Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1978 Mar;75(3):1121–1125. doi: 10.1073/pnas.75.3.1121

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

Daniel Levin 1,2,3, Irving M London 1,2,3
PMCID: PMC411420  PMID: 274704

Abstract

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.

Keywords: initiation in reticulocyte lysates, translational control, Met-tRNAfMet

Full text

PDF
1121

Images in this article

1124Image
on p.1124

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adamson S. D., Herbert E., Kemp S. F. Effects of hemin and other porphyrins on protein synthesis in a reticulocyte lysate cell-free system. J Mol Biol. 1969 Jun 14;42(2):247–258. doi: 10.1016/0022-2836(69)90041-2. [DOI] [PubMed] [Google Scholar]
  2. Anderson W. F., Bosch L., Cohn W. E., Lodish H., Merrick W. C., Weissbach H., Wittmann H. G., Wool I. G. International symposium on protein synthesis. Summary of Fogarty Center-NIH Workshop held in Bethesda, Maryland on 18-20 October, 1976. FEBS Lett. 1977 Apr 1;76(1):1–10. doi: 10.1016/0014-5793(77)80109-9. [DOI] [PubMed] [Google Scholar]
  3. Clemens M. J., Henshaw E. C., Rahamimoff H., London I. M. Met-tRNAfMet binding to 40S ribosomal subunits: a site for the regulation of initiation of protein synthesis by hemin. Proc Natl Acad Sci U S A. 1974 Aug;71(8):2946–2950. doi: 10.1073/pnas.71.8.2946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clemens M. J., Safer B., Merrick W. C., Anderson W. F., London I. M. Inhibition of protein synthesis in rabbit reticulocyte lysates by double-stranded RNA and oxidized glutathione: indirect mode of action on polypeptide chain initiation. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1286–1290. doi: 10.1073/pnas.72.4.1286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Darnbrough C., Legon S., Hunt T., Jackson R. J. Initiation of protein synthesis: evidence for messenger RNA-independent binding of methionyl-transfer RNA to the 40 S ribosomal subunit. J Mol Biol. 1973 May 25;76(3):379–403. doi: 10.1016/0022-2836(73)90511-1. [DOI] [PubMed] [Google Scholar]
  6. Ehrenfeld E., Hunt T. Double-stranded poliovirus RNA inhibits initiation of protein synthesis by reticulocyte lysates. Proc Natl Acad Sci U S A. 1971 May;68(5):1075–1078. doi: 10.1073/pnas.68.5.1075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ernst V., Levin D. H., Ranu R. S., London I. M. Control of protein synthesis in reticulocyte lysates: effects of 3':5'-cyclic AMP, ATP, and GTP on inhibitions induced by hemedeficiency, double-stranded RNA, and a reticulocyte translationa inhibitor. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1112–1116. doi: 10.1073/pnas.73.4.1112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Farrell P. J., Balkow K., Hunt T., Jackson R. J., Trachsel H. Phosphorylation of initiation factor elF-2 and the control of reticulocyte protein synthesis. Cell. 1977 May;11(1):187–200. doi: 10.1016/0092-8674(77)90330-0. [DOI] [PubMed] [Google Scholar]
  9. Gross M., Mendelewski J. Additional evidence that the hemin-controlled translational repressor from rabbit reticulocytes is a protein kinase. Biochem Biophys Res Commun. 1977 Jan 24;74(2):559–569. doi: 10.1016/0006-291x(77)90340-0. [DOI] [PubMed] [Google Scholar]
  10. Gross M., Rabinovitz M. Control of globin synthesis by hemin: factors influencing formation of an inhibitor of globin chain initiation in reticulocyte lysates. Biochim Biophys Acta. 1972 Dec 6;287(2):340–352. doi: 10.1016/0005-2787(72)90383-8. [DOI] [PubMed] [Google Scholar]
  11. Gross M., Rabinovitz M. Partial purification of a translational repressor mediating hemin control of globin synthesis and implication of results on the site of inhibition. Biochem Biophys Res Commun. 1973 Feb 5;50(3):832–838. doi: 10.1016/0006-291x(73)91320-x. [DOI] [PubMed] [Google Scholar]
  12. Hovanessian A. G., Brown R. E., Kerr I. M. Synthesis of low molecular weight inhibitor of protein synthesis with enzyme from interferon-treated cells. Nature. 1977 Aug 11;268(5620):537–540. doi: 10.1038/268537a0. [DOI] [PubMed] [Google Scholar]
  13. Howard G. A., Adamson S. D., Herbert E. Studies on cessation of protein synthesis in a reticulocyte lysate cell-free system. Biochim Biophys Acta. 1970 Jul 16;213(1):237–240. doi: 10.1016/0005-2787(70)90028-6. [DOI] [PubMed] [Google Scholar]
  14. Hunt T., Ehrenfeld E. Cytoplasm from poliovirus-infected HeLa cells inhibits cell-free haemoglobin synthesis. Nat New Biol. 1971 Mar 17;230(11):91–94. doi: 10.1038/newbio230091a0. [DOI] [PubMed] [Google Scholar]
  15. Hunt T., Vanderhoff G., London I. M. Control of globin synthesis: the role of heme. J Mol Biol. 1972 May 28;66(3):471–481. doi: 10.1016/0022-2836(72)90427-5. [DOI] [PubMed] [Google Scholar]
  16. Hunter T., Hunt T., Jackson R. J., Robertson H. D. The characteristics of inhibition of protein synthesis by double-stranded ribonucleic acid in reticulocyte lysates. J Biol Chem. 1975 Jan 25;250(2):409–417. [PubMed] [Google Scholar]
  17. Kerr I. M., Brown R. E., Hovanessian A. G. Nature of inhibitor of cell-free protein synthesis formed in response to interferon and double-stranded RNA. Nature. 1977 Aug 11;268(5620):540–542. doi: 10.1038/268540a0. [DOI] [PubMed] [Google Scholar]
  18. Kramer G., Cimadevilla J. M., Hardesty B. Specificity of the protein kinase activity associated with the hemin-controlled repressor of rabbit reticulocyte. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3078–3082. doi: 10.1073/pnas.73.9.3078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lebleu B., Sen G. C., Shaila S., Cabrer B., Lengyel P. Interferon, double-stranded RNA, and protein phosphorylation. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3107–3111. doi: 10.1073/pnas.73.9.3107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Legon S., Brayley A., Hunt T., Jackson R. J. The effect of cyclic AMP and related compounds on the control of protein synthesis in reticulocyte lysates. Biochem Biophys Res Commun. 1974 Feb 4;56(3):745–752. doi: 10.1016/0006-291x(74)90668-8. [DOI] [PubMed] [Google Scholar]
  21. Legon S., Jackson R. J., Hunt T. Control of protein synthesis in reticulocyte lysates by haemin. Nat New Biol. 1973 Jan 31;241(109):150–152. doi: 10.1038/newbio241150a0. [DOI] [PubMed] [Google Scholar]
  22. Levin D., Ranu R. S., Ernst V., London I. M. Regulation of protein synthesis in reticulocyte lysates: phosphorylation of methionyl-tRNAf binding factor by protein kinase activity of translational inhibitor isolated from hemedeficient lysates. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3112–3116. doi: 10.1073/pnas.73.9.3112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maxwell C. R., Kamper C. S., Rabinovitz M. Hemin control of globin synthesis: an assay for the inhibitor formed in the absence of hemin and some characteristics of its formation. J Mol Biol. 1971 May 28;58(1):317–327. doi: 10.1016/0022-2836(71)90249-x. [DOI] [PubMed] [Google Scholar]
  24. Ranu R. S., London I. M. Regulation of protein synthesis in rabbit reticulocyte lysates: purification and initial characterization of the cyclic 3':5'-AMP independent protein kinase of the heme-regulated translational inhibitor. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4349–4353. doi: 10.1073/pnas.73.12.4349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Roberts W. K., Hovanessian A., Brown R. E., Clemens M. J., Kerr I. M. Interferon-mediated protein kinase and low-molecular-weight inhibitor of protein synthesis. Nature. 1976 Dec 2;264(5585):477–480. doi: 10.1038/264477a0. [DOI] [PubMed] [Google Scholar]
  26. Weber L. A., Hickey E. D., Maroney P. A., Baglioni C. Inhibition of protein synthesis by Cl-. J Biol Chem. 1977 Jun 10;252(11):4007–4010. [PubMed] [Google Scholar]
  27. Zilberstein A., Federman P., Shulman L., Revel M. Specific phosphorylation in vitro of a protein associated with ribosomes of interferon-treated mouse L cells. FEBS Lett. 1976 Sep 15;68(1):119–124. doi: 10.1016/0014-5793(76)80418-8. [DOI] [PubMed] [Google Scholar]
  28. Zucker W. V., Schulman H. M. Stimulation of globin-chain initiation by hemin in the reticulocyte cell-free system. Proc Natl Acad Sci U S A. 1968 Feb;59(2):582–589. doi: 10.1073/pnas.59.2.582. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES