Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1995 Aug;69(8):5195–5198. doi: 10.1128/jvi.69.8.5195-5198.1995

Mechanism of interferon action: characterization of the intermolecular autophosphorylation of PKR, the interferon-inducible, RNA-dependent protein kinase.

D C Thomis 1, C E Samuel 1
PMCID: PMC189345  PMID: 7541849

Abstract

The interferon-inducible, RNA-dependent protein kinase (PKR) is activated by autophosphorylation, a process mediated by double-stranded RNA. A catalytically deficient, histidine-tagged mutant PKR protein [His-PKR(K296R)] was used as the substrate for characterization of the intermolecular phosphorylation catalyzed by purified wild-type PKR [PKR(Wt)]. The intermolecular autophosphorylation of His-PKR(K296R) by PKR(Wt) was RNA dependent. Excess His-PKR(K296R) substrate inhibited both the auto- and the trans-phosphorylation activities of PKR(Wt). Inhibition of PKR(Wt) by His-PKR(K296R) was relieved by higher concentrations of activator double-stranded RNA. Phosphopeptide analysis revealed that the sites of intermolecular autophosphorylation in His-PKR(K296R) were very similar, if not identical, to the sites that were autophosphorylated in PKR(Wt) and suggest a multiple of four major phosphorylation sites per PKR molecule.

Full Text

The Full Text of this article is available as a PDF (259.5 KB).

Selected References

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

  1. Barber G. N., Wambach M., Wong M. L., Dever T. E., Hinnebusch A. G., Katze M. G. Translational regulation by the interferon-induced double-stranded-RNA-activated 68-kDa protein kinase. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4621–4625. doi: 10.1073/pnas.90.10.4621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beattie E., Denzler K. L., Tartaglia J., Perkus M. E., Paoletti E., Jacobs B. L. Reversal of the interferon-sensitive phenotype of a vaccinia virus lacking E3L by expression of the reovirus S4 gene. J Virol. 1995 Jan;69(1):499–505. doi: 10.1128/jvi.69.1.499-505.1995. [DOI] [PMC free article] [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. Chong K. L., Feng L., Schappert K., Meurs E., Donahue T. F., Friesen J. D., Hovanessian A. G., Williams B. R. Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2. EMBO J. 1992 Apr;11(4):1553–1562. doi: 10.1002/j.1460-2075.1992.tb05200.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Benedetti A., Baglioni C. Inhibition of mRNA binding to ribosomes by localized activation of dsRNA-dependent protein kinase. Nature. 1984 Sep 6;311(5981):79–81. doi: 10.1038/311079a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Galabru J., Katze M. G., Robert N., Hovanessian A. G. The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase. Eur J Biochem. 1989 Jan 2;178(3):581–589. doi: 10.1111/j.1432-1033.1989.tb14485.x. [DOI] [PubMed] [Google Scholar]
  8. Henry G. L., McCormack S. J., Thomis D. C., Samuel C. E. Mechanism of interferon action. Translational control and the RNA-dependent protein kinase (PKR): antagonists of PKR enhance the translational activity of mRNAs that include a 161 nucleotide region from reovirus S1 mRNA. J Biol Regul Homeost Agents. 1994 Jan-Mar;8(1):15–24. [PubMed] [Google Scholar]
  9. Hershey J. W. Protein phosphorylation controls translation rates. J Biol Chem. 1989 Dec 15;264(35):20823–20826. [PubMed] [Google Scholar]
  10. Katze M. G., Wambach M., Wong M. L., Garfinkel M., Meurs E., Chong K., Williams B. R., Hovanessian A. G., Barber G. N. Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system. Mol Cell Biol. 1991 Nov;11(11):5497–5505. doi: 10.1128/mcb.11.11.5497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kostura M., Mathews M. B. Purification and activation of the double-stranded RNA-dependent eIF-2 kinase DAI. Mol Cell Biol. 1989 Apr;9(4):1576–1586. doi: 10.1128/mcb.9.4.1576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Krust B., Galabru J., Hovanessian A. G. Further characterization of the protein kinase activity mediated by interferon in mouse and human cells. J Biol Chem. 1984 Jul 10;259(13):8494–8498. [PubMed] [Google Scholar]
  13. Lasky S. R., Jacobs B. L., Samuel C. E. Mechanism of interferon action. Characterization of sites of phosphorylation in the interferon-induced phosphoprotein P1 from mouse fibroblasts: evidence for two forms of P1. J Biol Chem. 1982 Sep 25;257(18):11087–11093. [PubMed] [Google Scholar]
  14. McCormack S. J., Ortega L. G., Doohan J. P., Samuel C. E. Mechanism of interferon action motif I of the interferon-induced, RNA-dependent protein kinase (PKR) is sufficient to mediate RNA-binding activity. Virology. 1994 Jan;198(1):92–99. doi: 10.1006/viro.1994.1011. [DOI] [PubMed] [Google Scholar]
  15. Meurs E., Chong K., Galabru J., Thomas N. S., Kerr I. M., Williams B. R., Hovanessian A. G. Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell. 1990 Jul 27;62(2):379–390. doi: 10.1016/0092-8674(90)90374-n. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Samuel C. E. Mechanism of interferon action: phosphorylation of protein synthesis initiation factor eIF-2 in interferon-treated human cells by a ribosome-associated kinase processing site specificity similar to hemin-regulated rabbit reticulocyte kinase. Proc Natl Acad Sci U S A. 1979 Feb;76(2):600–604. doi: 10.1073/pnas.76.2.600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Samuel C. E. The eIF-2 alpha protein kinases, regulators of translation in eukaryotes from yeasts to humans. J Biol Chem. 1993 Apr 15;268(11):7603–7606. [PubMed] [Google Scholar]
  19. Sen G. C., Lengyel P. The interferon system. A bird's eye view of its biochemistry. J Biol Chem. 1992 Mar 15;267(8):5017–5020. [PubMed] [Google Scholar]
  20. Tanaka H., Samuel C. E. Mechanism of interferon action: structure of the mouse PKR gene encoding the interferon-inducible RNA-dependent protein kinase. Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):7995–7999. doi: 10.1073/pnas.91.17.7995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Thomis D. C., Doohan J. P., Samuel C. E. Mechanism of interferon action: cDNA structure, expression, and regulation of the interferon-induced, RNA-dependent P1/eIF-2 alpha protein kinase from human cells. Virology. 1992 May;188(1):33–46. doi: 10.1016/0042-6822(92)90732-5. [DOI] [PubMed] [Google Scholar]
  22. Thomis D. C., Samuel C. E. Mechanism of interferon action: autoregulation of RNA-dependent P1/eIF-2 alpha protein kinase (PKR) expression in transfected mammalian cells. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10837–10841. doi: 10.1073/pnas.89.22.10837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Thomis D. C., Samuel C. E. Mechanism of interferon action: evidence for intermolecular autophosphorylation and autoactivation of the interferon-induced, RNA-dependent protein kinase PKR. J Virol. 1993 Dec;67(12):7695–7700. doi: 10.1128/jvi.67.12.7695-7700.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES