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. 1978 Jan;75(1):108–112. doi: 10.1073/pnas.75.1.108

Phosphorylation of eukaryotic protein synthesis initiation factors.

R Benne, J Edman, R R Traut, J W Hershey
PMCID: PMC411193  PMID: 272626

Abstract

Phosphorylation of eukaryotic initiation factors was examined both in intact cells and in vitro with purified components. Intact rabbit reticulocytes were incubated in a medium containing[32P]phosphate, and eight initiation factors were isolated and partially purified. The purified factors were analyzed on dodecyl sulfate/polyacrylamide gels and compared with highly purified nonradioactive factors. Significant amounts of radioactivity were found associated with initiation factors eIF-2, polypeptide 2 (molecular weight 53,000); eIF-3, polypeptides 2 and 4 (molecular weights 110,000 and 67,000); and eIF-4B. Purfied initiation factors from rabbit reticulocytes were also treated in vitro with [gamma-32P]ATP and a cyclic AMP-independent protein kinase isolated from rabbit erythrocytes. Only the factor polypeptides phosphorylated intracellularly were phosphorylated in vitro. The results suggest that the cyclic AMP-independent protein kinase is responsible for the phosphorylation of specific initiation factors in cells active in protein synthesis and that it may play a role in regulating translation.

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Selected References

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

  1. 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]
  2. Benne R., Hershey J. W. Purification and characterization of initiation factor IF-E3 from rabbit reticulocytes. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3005–3009. doi: 10.1073/pnas.73.9.3005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benne R., Luedi M., Hershey J. W. Purification and characterization of initiation factors IF-E4 and IF-E6 from rabbit reticulocytes. J Biol Chem. 1977 Aug 25;252(16):5798–5803. [PubMed] [Google Scholar]
  4. Benne R., Wong C., Luedi M., Hershey J. W. Purification and characterization of initiation factor IF-E2 from rabbit reticulocytes. J Biol Chem. 1976 Dec 10;251(23):7675–7681. [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Datta A., de Haro C., Sierra J. M., Ochoa S. Role of 3':5'-cyclic-AMP-dependent protein kinase in regulation of protein synthesis in reticulocyte lysates. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1463–1467. doi: 10.1073/pnas.74.4.1463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Glynn I. M., Chappell J. B. A simple method for the preparation of 32-P-labelled adenosine triphosphate of high specific activity. Biochem J. 1964 Jan;90(1):147–149. doi: 10.1042/bj0900147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Golini F., Thach S. S., Birge C. H., Safer B., Merrick W. C., Thach R. E. Competition between cellular and viral mRNAs in vitro is regulated by a messenger discriminatory initiation factor. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3040–3044. doi: 10.1073/pnas.73.9.3040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gressner A. M., Wool I. G. Effect of experimental diabetes and insulin on phosphorylation of rat liver ribosomal protein S6. Nature. 1976 Jan 15;259(5539):148–150. doi: 10.1038/259148a0. [DOI] [PubMed] [Google Scholar]
  11. Gressner A. M., Wool I. G. The phosphorylation of liver ribosomal proteins in vivo. Evidence that only a single small subunit protein (S6) is phosphorylated. J Biol Chem. 1974 Nov 10;249(21):6917–6925. [PubMed] [Google Scholar]
  12. 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]
  13. Kabat D., Chappell M. R. Competition between globin messenger ribonucleic acids for a discriminating initiation factor. J Biol Chem. 1977 Apr 25;252(8):2684–2690. [PubMed] [Google Scholar]
  14. Kabat D. Phosphorylation of ribosomal proteins in rabbit reticulocytes. Characterization and regulatory aspects. Biochemistry. 1970 Oct 13;9(21):4160–4175. doi: 10.1021/bi00823a019. [DOI] [PubMed] [Google Scholar]
  15. Kramer G., Henderson A. B., Pinphanichakarn P., Wallis M. H., Hardesty B. Partial reaction of peptide initiation inhibited by phosphorylation of either initiation factor eIF-2 or 40S ribosomal proteins. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1445–1449. doi: 10.1073/pnas.74.4.1445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kumar R., Tao M. Multiple forms of casein kinase from rabbit erythrocytes. Biochim Biophys Acta. 1975 Nov 20;410(1):87–98. doi: 10.1016/0005-2744(75)90209-0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Schreier M. H., Staehelin T. Initiation of mammalian protein synthesis: the importance of ribosome and initiation factor quality for the efficiency of in vitro systems. J Mol Biol. 1973 Feb 19;73(3):329–349. doi: 10.1016/0022-2836(73)90346-x. [DOI] [PubMed] [Google Scholar]
  20. Stahl J., Böhm H., Bielka H. Enzymatic phosphorylation of eukaryotic ribosomal proteins and factors of protein biosynthesis. Acta Biol Med Ger. 1974;33(5-6):667–676. [PubMed] [Google Scholar]
  21. Traugh J. A., Porter G. G. A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro. Biochemistry. 1976 Feb 10;15(3):610–616. doi: 10.1021/bi00648a025. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Traugh J. A., Traut R. R. Characterization of protein kinases from rabbit reticulocytes. J Biol Chem. 1974 Feb 25;249(4):1207–1212. [PubMed] [Google Scholar]
  24. Traugh J. A., Traut R. R. Recent advances in the preparation of mammalian ribosomes and analysis of their protein composition. Methods Cell Biol. 1973;7:67–103. doi: 10.1016/s0091-679x(08)61772-0. [DOI] [PubMed] [Google Scholar]
  25. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

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