Skip to main content
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
. 1979 May;76(5):2118–2122. doi: 10.1073/pnas.76.5.2118

In situ phosphorylation of the α subunit of eukaryotic initiation factor 2 in reticulocyte lysates inhibited by heme deficiency, double-stranded RNA, oxidized glutathione, or the heme-regulated protein kinase

Vivian Ernst *, Daniel H Levin *, Irving M London *,
PMCID: PMC383548  PMID: 287050

Abstract

Protein synthesis initiation in reticulocyte lysates is inhibited by heme deficiency, low levels of double-stranded RNA (dsRNA), oxidized glutathione (GSSG), or the purified kinase (HRI) that acts on the α polypeptide of eukaryotic initiation factor 2 (eIF-2α). The phosphoprotein profiles produced in lysates in response to these various conditions have been monitored directly in lysates after labeling for brief periods with pulses of [γ-32P]ATP. The [32P]phosphoprotein profiles were analyzed by electrophoresis in sodium dodecyl sulfate/polyacrylamide slab gels under conditions in which the HRI and eIF-2α polypeptides were clearly distinguished. All four modes of inhibition produced a rapid phosphorylation of eIF-2α compared to control lysates, which displayed little or no phosphorylation of eIF-2α. In heme-deficient lysates, phosphorylation of eIF-2α occurred rapidly both before and after the shut-off of protein synthesis; the delayed addition of hemin to these lysates resulted in a decrease in the phosphorylation of eIF-2α and the subsequent restoration of protein synthesis. These data suggest that rapid turnover of phosphate occurs at the site(s) of eIF-2α phosphorylation. In lysates inhibited by heme deficiency, GSSG, or added HRI, the phosphorylation of eIF-2α was accompanied by the rapid in situ phosphorylation of HRI. The inhibition of initiation induced by dsRNA was accompanied by the phosphorylation of eIF-2α and a 67,000-dalton polypeptide but not HRI. These observations in situ indicate that (i) the phosphorylation of eIF-2α is the critical event in these inhibitions of protein chain initiation, and (ii) the phosphorylation of HRI is associated with its activation in heme deficiency.

Keywords: protein synthesis regulation, Met-tRNAfMet binding factor, inhibition of initiation, [32P]phosphoprotein profiles

Full text

PDF
2118

Images in this article

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., Godchaux W. Factors affecting the rate of protein synthesis in lysate systems from reticulocytes. Arch Biochem Biophys. 1968 May;125(2):671–683. doi: 10.1016/0003-9861(68)90625-5. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Benne R., Edman J., Traut R. R., Hershey J. W. Phosphorylation of eukaryotic protein synthesis initiation factors. Proc Natl Acad Sci U S A. 1978 Jan;75(1):108–112. doi: 10.1073/pnas.75.1.108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Ernst V., Levin D. H., London I. M. Evidence that glucose 6-phosphate regulates protein synthesis initiation in reticulocyte lysates. J Biol Chem. 1978 Oct 25;253(20):7163–7172. [PubMed] [Google Scholar]
  7. Ernst V., Levin D. H., London I. M. Inhibition of protein synthesis initiation by oxidized glutathione: activation of a protein kinase that phosphorylates the alpha subunit of eukaryotic initiation factor 2. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4110–4114. doi: 10.1073/pnas.75.9.4110. [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. Farrell P. J., Hunt T., Jackson R. J. Analysis of phosphorylation of protein synthesis initiation factor eIF-2 by two-dimensional gel electrophoresis. Eur J Biochem. 1978 Sep 1;89(2):517–521. doi: 10.1111/j.1432-1033.1978.tb12556.x. [DOI] [PubMed] [Google Scholar]
  10. Floyd G. A., Traugh J. A. Phosphorylation of ribosomal-associated proteins in reticulocyte lysates. Methods Enzymol. 1979;60:511–521. doi: 10.1016/s0076-6879(79)60048-4. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Gross M., Mendelewski J. Control of protein synthesis by hemin. An association between the formation of the hemin-controlled translational repressor and the phosphorylation of a 100 000 molecular weight protein. Biochim Biophys Acta. 1978 Oct 24;520(3):650–663. doi: 10.1016/0005-2787(78)90150-8. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Issinger O. G., Benne R., Hershey J. W., Traut R. R. Phosphorylation in vitro of eukaryotic initiation factors IF-E2 and IF-E3 by protein kinases. J Biol Chem. 1976 Oct 25;251(20):6471–6474. [PubMed] [Google Scholar]
  16. Kosower N. S., Vanderhoff G. A., Kosower E. M. Glutathione. 8. The effects of glutathione disulfide on initiation of protein synthesis. Biochim Biophys Acta. 1972 Jul 31;272(4):623–637. [PubMed] [Google Scholar]
  17. 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]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Levin D., London I. M. Regulation of protein synthesis: activation by double-stranded RNA of a protein kinase that phosphorylates eukaryotic initiation factor 2. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1121–1125. doi: 10.1073/pnas.75.3.1121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Rabinovitz M., Freedman M. L., Fisher J. M., Maxwell C. R. Translational control in hemoglobin syntheskis. Cold Spring Harb Symp Quant Biol. 1969;34:567–578. doi: 10.1101/sqb.1969.034.01.064. [DOI] [PubMed] [Google Scholar]
  22. Ranu R. S., London I. M., Das A., Dasgupta A., Majumdar A., Ralston R., Roy R., Gupta N. K. Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase: inhibition of interaction of Met-tRNAfMet binding factor with another initiation factor in formation of Met-tRNAfMet.40S ribosomal subunit complexes. Proc Natl Acad Sci U S A. 1978 Feb;75(2):745–749. doi: 10.1073/pnas.75.2.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Tahara S. M., Traugh J. A., Sharp S. B., Lundak T. S., Safer B., Merrick W. C. Effect of hemin on site-specific phosphorylation of eukaryotic initiation factor 2. Proc Natl Acad Sci U S A. 1978 Feb;75(2):789–793. doi: 10.1073/pnas.75.2.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Trachsel H., Ranu R. S., London I. M. Regulation of protein synthesis in rabbit reticulocyte lysates: purification and characterization of heme-reversible translational inhibitor. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3654–3658. doi: 10.1073/pnas.75.8.3654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Traugh J. A., Lundak T. S. Phosphorylation of translational initiation factor 3 (eIF-3) by cyclic AMP-regulated protein kinase. Biochem Biophys Res Commun. 1978 Jul 28;83(2):379–384. doi: 10.1016/0006-291x(78)91001-x. [DOI] [PubMed] [Google Scholar]
  27. Traugh J. A., Tahara S. M., Sharp S. B., Safer B., Merrick W. C. Factors involved in initiation of haemoglobin synthesis can be phosphorylated in vitro. Nature. 1976 Sep 9;263(5573):163–165. doi: 10.1038/263163a0. [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