Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1989 Apr;9(4):1576–1586. doi: 10.1128/mcb.9.4.1576

Purification and activation of the double-stranded RNA-dependent eIF-2 kinase DAI.

M Kostura 1, M B Mathews 1
PMCID: PMC362574  PMID: 2725516

Abstract

The double-stranded RNA (dsRNA)-dependent protein kinase DAI (also termed dsI and P1) possesses two kinase activities; one is an autophosphorylation activity, and the other phosphorylates initiation factor eIF-2. We purified the enzyme, in a latent form, to near homogeneity from interferon-treated human 293 cells. The purified enzyme consisted of a single polypeptide subunit of approximately 70,000 daltons, retained its dependence on dsRNA for activation, and was sensitive to inhibition by adenovirus VA RNAI. Autophosphorylation required a suitable concentration of dsRNA and was second order with respect to DAI concentration, which suggests an intermolecular mechanism in which one DAI molecule phosphorylates a neighboring molecule. Once autophosphorylated, the enzyme could phosphorylate eIF-2 but seemed unable to phosphorylate other DAI molecules, which implies a change in substrate specificity upon activation. VA RNAI blocked autophosphorylation and activation but permitted the activated enzyme to phosphorylate eIF-2. VA RNAI also blocked the binding of dsRNA to the enzyme. The data are consistent with a model in which activation requires the interaction of two molecules of DAI with dsRNA, followed by intermolecular autophosphorylation of the latent enzyme. VA RNAI would block activation by preventing the interaction between DAI and dsRNA.

Full text

PDF
1586

Images in this article

1578Image
on p.1578
1579Image
on p.1579
1580Image
on p.1580
1582Image
on p.1582
1584Image
on p.1584

Selected References

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

  1. Baglioni C., Benvin S., Maroney P. A., Minks M. A., Nilsen T. W., West D. K. Interferon-induced enzymes: activation and role in te antiviral state. Ann N Y Acad Sci. 1980;350:497–509. doi: 10.1111/j.1749-6632.1980.tb20652.x. [DOI] [PubMed] [Google Scholar]
  2. Baglioni C. Interferon-induced enzymatic activities and their role in the antriviral state. Cell. 1979 Jun;17(2):255–264. doi: 10.1016/0092-8674(79)90151-x. [DOI] [PubMed] [Google Scholar]
  3. Berry M. J., Knutson G. S., Lasky S. R., Munemitsu S. M., Samuel C. E. Mechanism of interferon action. Purification and substrate specificities of the double-stranded RNA-dependent protein kinase from untreated and interferon-treated mouse fibroblasts. J Biol Chem. 1985 Sep 15;260(20):11240–11247. [PubMed] [Google Scholar]
  4. 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]
  5. Franklin R. M. Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proc Natl Acad Sci U S A. 1966 Jun;55(6):1504–1511. doi: 10.1073/pnas.55.6.1504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Galabru J., Hovanessian A. G. Two interferon-induced proteins are involved in the protein kinase complex dependent on double-stranded RNA. Cell. 1985 Dec;43(3 Pt 2):685–694. doi: 10.1016/0092-8674(85)90241-7. [DOI] [PubMed] [Google Scholar]
  7. Galabru J., Hovanessian A. Autophosphorylation of the protein kinase dependent on double-stranded RNA. J Biol Chem. 1987 Nov 15;262(32):15538–15544. [PubMed] [Google Scholar]
  8. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Katze M. G., DeCorato D., Safer B., Galabru J., Hovanessian A. G. Adenovirus VAI RNA complexes with the 68 000 Mr protein kinase to regulate its autophosphorylation and activity. EMBO J. 1987 Mar;6(3):689–697. doi: 10.1002/j.1460-2075.1987.tb04809.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Katze M. G., Detjen B. M., Safer B., Krug R. M. Translational control by influenza virus: suppression of the kinase that phosphorylates the alpha subunit of initiation factor eIF-2 and selective translation of influenza viral mRNAs. Mol Cell Biol. 1986 May;6(5):1741–1750. doi: 10.1128/mcb.6.5.1741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kimchi A., Zilberstein A., Schmidt A., Shulman L., Revel M. The interferon-induced protein kinase PK-i from mouse L cells. J Biol Chem. 1979 Oct 10;254(19):9846–9853. [PubMed] [Google Scholar]
  13. Kitajewski J., Schneider R. J., Safer B., Munemitsu S. M., Samuel C. E., Thimmappaya B., Shenk T. Adenovirus VAI RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced eIF-2 alpha kinase. Cell. 1986 Apr 25;45(2):195–200. doi: 10.1016/0092-8674(86)90383-1. [DOI] [PubMed] [Google Scholar]
  14. Kitajewski J., Schneider R. J., Safer B., Shenk T. An adenovirus mutant unable to express VAI RNA displays different growth responses and sensitivity to interferon in various host cell lines. Mol Cell Biol. 1986 Dec;6(12):4493–4498. doi: 10.1128/mcb.6.12.4493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Laurent A. G., Krust B., Galabru J., Svab J., Hovanessian A. G. Monoclonal antibodies to an interferon-induced Mr 68,000 protein and their use for the detection of double-stranded RNA-dependent protein kinase in human cells. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4341–4345. doi: 10.1073/pnas.82.13.4341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Lengyel P. Biochemistry of interferons and their actions. Annu Rev Biochem. 1982;51:251–282. doi: 10.1146/annurev.bi.51.070182.001343. [DOI] [PubMed] [Google Scholar]
  19. Levin D. H., Petryshyn R., London I. M. Characterization of purified double-stranded RNA-activated eIF-2 alpha kinase from rabbit reticulocytes. J Biol Chem. 1981 Jul 25;256(14):7638–7641. [PubMed] [Google Scholar]
  20. Mellits K. H., Mathews M. B. Effects of mutations in stem and loop regions on the structure and function of adenovirus VA RNAI. EMBO J. 1988 Sep;7(9):2849–2859. doi: 10.1002/j.1460-2075.1988.tb03141.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Monstein H. J., Philipson L. The conformation of adenovirus VAI-RNA in solution. Nucleic Acids Res. 1981 Sep 11;9(17):4239–4250. doi: 10.1093/nar/9.17.4239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. O'Malley R. P., Duncan R. F., Hershey J. W., Mathews M. B. Modification of protein synthesis initiation factors and the shut-off of host protein synthesis in adenovirus-infected cells. Virology. 1989 Jan;168(1):112–118. doi: 10.1016/0042-6822(89)90409-1. [DOI] [PubMed] [Google Scholar]
  23. O'Malley R. P., Mariano T. M., Siekierka J., Mathews M. B. A mechanism for the control of protein synthesis by adenovirus VA RNAI. Cell. 1986 Feb 14;44(3):391–400. doi: 10.1016/0092-8674(86)90460-5. [DOI] [PubMed] [Google Scholar]
  24. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  25. Ochoa S. Regulation of protein synthesis initiation in eucaryotes. Arch Biochem Biophys. 1983 Jun;223(2):325–349. doi: 10.1016/0003-9861(83)90598-2. [DOI] [PubMed] [Google Scholar]
  26. Paez E., Esteban M. Resistance of vaccinia virus to interferon is related to an interference phenomenon between the virus and the interferon system. Virology. 1984 Apr 15;134(1):12–28. doi: 10.1016/0042-6822(84)90268-x. [DOI] [PubMed] [Google Scholar]
  27. Pain V. M. Initiation of protein synthesis in mammalian cells. Biochem J. 1986 May 1;235(3):625–637. doi: 10.1042/bj2350625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Petryshyn R., Chen J. J., London I. M. Detection of activated double-stranded RNA-dependent protein kinase in 3T3-F442A cells. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1427–1431. doi: 10.1073/pnas.85.5.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Petryshyn R., Levin D. H., London I. M. Double-stranded RNA-dependent eIF-2alpha protein kinase. Methods Enzymol. 1983;99:346–362. doi: 10.1016/0076-6879(83)99070-5. [DOI] [PubMed] [Google Scholar]
  30. Petryshyn R., Levin D. H., London I. M. Purification and characterization of a latent precursor of a double-stranded RNA dependent protein kinase from reticulocyte lysates. Biochem Biophys Res Commun. 1980 Jun 30;94(4):1190–1198. doi: 10.1016/0006-291x(80)90545-8. [DOI] [PubMed] [Google Scholar]
  31. Ranu R. S. Regulation of protein synthesis in rabbit reticulocyte lysates: purification and initial characterization of the double stranded RNA activated protein kinase. Biochem Biophys Res Commun. 1980 Nov 17;97(1):252–262. doi: 10.1016/s0006-291x(80)80162-8. [DOI] [PubMed] [Google Scholar]
  32. Reichel P. A., Merrick W. C., Siekierka J., Mathews M. B. Regulation of a protein synthesis initiation factor by adenovirus virus-associated RNA. Nature. 1985 Jan 17;313(5999):196–200. doi: 10.1038/313196a0. [DOI] [PubMed] [Google Scholar]
  33. Rice A. P., Kerr I. M. Interferon-mediated, double-stranded RNA-dependent protein kinase is inhibited in extracts from vaccinia virus-infected cells. J Virol. 1984 Apr;50(1):229–236. doi: 10.1128/jvi.50.1.229-236.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Samuel C. E., Knutson G. S., Berry M. J., Atwater J. A., Lasky S. R. Purification of double-stranded RNA-dependent protein kinase from mouse fibroblasts. Methods Enzymol. 1986;119:499–516. doi: 10.1016/0076-6879(86)19070-7. [DOI] [PubMed] [Google Scholar]
  36. Schneider R. J., Safer B., Munemitsu S. M., Samuel C. E., Shenk T. Adenovirus VAI RNA prevents phosphorylation of the eukaryotic initiation factor 2 alpha subunit subsequent to infection. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4321–4325. doi: 10.1073/pnas.82.13.4321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schneider R. J., Shenk T. Impact of virus infection on host cell protein synthesis. Annu Rev Biochem. 1987;56:317–332. doi: 10.1146/annurev.bi.56.070187.001533. [DOI] [PubMed] [Google Scholar]
  38. Sen G. C., Taira H., Lengyel P. Interferon, double-stranded RNA, and protein phosphorylation. Characteristics of a double-stranded RNA-activated protein kinase system partially purified from interferon treated Ehrlich ascites tumor cells. J Biol Chem. 1978 Sep 10;253(17):5915–5921. [PubMed] [Google Scholar]
  39. Siekierka J., Mariano T. M., Reichel P. A., Mathews M. B. 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. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1959–1963. doi: 10.1073/pnas.82.7.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Whitaker-Dowling P., Youngner J. S. Characterization of a specific kinase inhibitory factor produced by vaccinia virus which inhibits the interferon-induced protein kinase. Virology. 1984 Aug;137(1):171–181. doi: 10.1016/0042-6822(84)90020-5. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Zilberstein A., Kimchi A., Schmidt A., Revel M. Isolation of two interferon-induced translational inhibitors: a protein kinase and an oligo-isoadenylate synthetase. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4734–4738. doi: 10.1073/pnas.75.10.4734. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES