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
. 1995 Aug 29;92(18):8283–8287. doi: 10.1073/pnas.92.18.8283

The interferon-inducible double-stranded RNA-activated protein kinase self-associates in vitro and in vivo.

R C Patel 1, P Stanton 1, N M McMillan 1, B R Williams 1, G C Sen 1
PMCID: PMC41141  PMID: 7545299

Abstract

The interferon-inducible double-stranded (ds) RNA-activated protein kinase (PKR) exhibits antiviral, anticellular, and antitumor activities. The mechanisms of its enzymatic activation by autophosphorylation and of the observed transdominant inhibitory phenotype of enzymatically inactive mutants have invoked PKR dimerization. Here we present direct evidence in support of PKR-PKR interaction. We show that radiolabeled PKR can specifically interact with matrix-bound unlabeled PKR in the absence of dsRNA. The self-association activity resides, in part, in the N-terminal region of 170 residues, which also constitutes the dsRNA-binding domain (DRBD). DRBD can bind to matrix-bound PKR or to matrix-bound DRBD. Dimerization of DRBD was directly demonstrated by chemical crosslinking. Affinity chromatography and electrophoretic mobility supershift assays demonstrated that mutants that fail to bind dsRNA can still exhibit protein-protein interaction. The PKR-PKR interaction could also be observed in a two-hybrid transcriptional activation assay in mammalian cells and consequently is likely to be an important feature of PKR activity in vivo.

Full text

PDF
8283

Images in this article

8284Fig. 1
on p.8284
8285Fig. 2
on p.8285
8285Fig. 3
on p.8285
8286Fig. 4
on p.8286

Selected References

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

  1. Barber G. N., Tomita J., Garfinkel M. S., Meurs E., Hovanessian A., Katze M. G. Detection of protein kinase homologues and viral RNA-binding domains utilizing polyclonal antiserum prepared against a baculovirus-expressed ds RNA-activated 68,000-Da protein kinase. Virology. 1992 Dec;191(2):670–679. doi: 10.1016/0042-6822(92)90242-h. [DOI] [PubMed] [Google Scholar]
  2. Black T. L., Safer B., Hovanessian A., Katze M. G. The cellular 68,000-Mr protein kinase is highly autophosphorylated and activated yet significantly degraded during poliovirus infection: implications for translational regulation. J Virol. 1989 May;63(5):2244–2251. doi: 10.1128/jvi.63.5.2244-2251.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Colthurst D. R., Campbell D. G., Proud C. G. Structure and regulation of eukaryotic initiation factor eIF-2. Sequence of the site in the alpha subunit phosphorylated by the haem-controlled repressor and by the double-stranded RNA-activated inhibitor. Eur J Biochem. 1987 Jul 15;166(2):357–363. doi: 10.1111/j.1432-1033.1987.tb13523.x. [DOI] [PubMed] [Google Scholar]
  5. Davies G. E., Stark G. R. Use of dimethyl suberimidate, a cross-linking reagent, in studying the subunit structure of oligomeric proteins. Proc Natl Acad Sci U S A. 1970 Jul;66(3):651–656. doi: 10.1073/pnas.66.3.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davies M. V., Elroy-Stein O., Jagus R., Moss B., Kaufman R. J. The vaccinia virus K3L gene product potentiates translation by inhibiting double-stranded-RNA-activated protein kinase and phosphorylation of the alpha subunit of eukaryotic initiation factor 2. J Virol. 1992 Apr;66(4):1943–1950. doi: 10.1128/jvi.66.4.1943-1950.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Desai S. Y., Patel R. C., Sen G. C., Malhotra P., Ghadge G. D., Thimmapaya B. Activation of interferon-inducible 2'-5' oligoadenylate synthetase by adenoviral VAI RNA. J Biol Chem. 1995 Feb 17;270(7):3454–3461. doi: 10.1074/jbc.270.7.3454. [DOI] [PubMed] [Google Scholar]
  8. Dubois M. F., Hovanessian A. G. Modified subcellular localization of interferon-induced p68 kinase during encephalomyocarditis virus infection. Virology. 1990 Dec;179(2):591–598. doi: 10.1016/0042-6822(90)90126-c. [DOI] [PubMed] [Google Scholar]
  9. Feng G. S., Chong K., Kumar A., Williams B. R. Identification of double-stranded RNA-binding domains in the interferon-induced double-stranded RNA-activated p68 kinase. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5447–5451. doi: 10.1073/pnas.89.12.5447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Green S. R., Mathews M. B. Two RNA-binding motifs in the double-stranded RNA-activated protein kinase, DAI. Genes Dev. 1992 Dec;6(12B):2478–2490. doi: 10.1101/gad.6.12b.2478. [DOI] [PubMed] [Google Scholar]
  11. Hershey J. W. Translational control in mammalian cells. Annu Rev Biochem. 1991;60:717–755. doi: 10.1146/annurev.bi.60.070191.003441. [DOI] [PubMed] [Google Scholar]
  12. Hovanessian A. G., Galabru J. The double-stranded RNA-dependent protein kinase is also activated by heparin. Eur J Biochem. 1987 Sep 15;167(3):467–473. doi: 10.1111/j.1432-1033.1987.tb13360.x. [DOI] [PubMed] [Google Scholar]
  13. Hovanessian A. G. The double stranded RNA-activated protein kinase induced by interferon: dsRNA-PK. J Interferon Res. 1989 Dec;9(6):641–647. doi: 10.1089/jir.1989.9.641. [DOI] [PubMed] [Google Scholar]
  14. Imani F., Jacobs B. L. Inhibitory activity for the interferon-induced protein kinase is associated with the reovirus serotype 1 sigma 3 protein. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7887–7891. doi: 10.1073/pnas.85.21.7887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Judware R., Petryshyn R. Partial characterization of a cellular factor that regulates the double-stranded RNA-dependent eIF-2 alpha kinase in 3T3-F442A fibroblasts. Mol Cell Biol. 1991 Jun;11(6):3259–3267. doi: 10.1128/mcb.11.6.3259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Katze M. G. The war against the interferon-induced dsRNA-activated protein kinase: can viruses win? J Interferon Res. 1992 Aug;12(4):241–248. doi: 10.1089/jir.1992.12.241. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Koromilas A. E., Roy S., Barber G. N., Katze M. G., Sonenberg N. Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase. Science. 1992 Sep 18;257(5077):1685–1689. doi: 10.1126/science.1382315. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Kumar A., Haque J., Lacoste J., Hiscott J., Williams B. R. Double-stranded RNA-dependent protein kinase activates transcription factor NF-kappa B by phosphorylating I kappa B. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6288–6292. doi: 10.1073/pnas.91.14.6288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Langland J. O., Jacobs B. L. Cytosolic double-stranded RNA-dependent protein kinase is likely a dimer of partially phosphorylated Mr = 66,000 subunits. J Biol Chem. 1992 May 25;267(15):10729–10736. [PubMed] [Google Scholar]
  23. Lee S. B., Green S. R., Mathews M. B., Esteban M. Activation of the double-stranded RNA (dsRNA)-activated human protein kinase in vivo in the absence of its dsRNA binding domain. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10551–10555. doi: 10.1073/pnas.91.22.10551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee T. G., Tomita J., Hovanessian A. G., Katze M. G. Purification and partial characterization of a cellular inhibitor of the interferon-induced protein kinase of Mr 68,000 from influenza virus-infected cells. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6208–6212. doi: 10.1073/pnas.87.16.6208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lengyel P. Tumor-suppressor genes: news about the interferon connection. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5893–5895. doi: 10.1073/pnas.90.13.5893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Manche L., Green S. R., Schmedt C., Mathews M. B. Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol Cell Biol. 1992 Nov;12(11):5238–5248. doi: 10.1128/mcb.12.11.5238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maran A., Maitra R. K., Kumar A., Dong B., Xiao W., Li G., Williams B. R., Torrence P. F., Silverman R. H. Blockage of NF-kappa B signaling by selective ablation of an mRNA target by 2-5A antisense chimeras. Science. 1994 Aug 5;265(5173):789–792. doi: 10.1126/science.7914032. [DOI] [PubMed] [Google Scholar]
  28. Marcus P. I., Sekellick M. J. Interferon induction by viruses. XVI. 2-Aminopurine blocks selectively and reversibly an early stage in interferon induction. J Gen Virol. 1988 Jul;69(Pt 7):1637–1645. doi: 10.1099/0022-1317-69-7-1637. [DOI] [PubMed] [Google Scholar]
  29. McCormack S. J., Thomis D. C., Samuel C. E. Mechanism of interferon action: identification of a RNA binding domain within the N-terminal region of the human RNA-dependent P1/eIF-2 alpha protein kinase. Virology. 1992 May;188(1):47–56. doi: 10.1016/0042-6822(92)90733-6. [DOI] [PubMed] [Google Scholar]
  30. McMillan N. A., Carpick B. W., Hollis B., Toone W. M., Zamanian-Daryoush M., Williams B. R. Mutational analysis of the double-stranded RNA (dsRNA) binding domain of the dsRNA-activated protein kinase, PKR. J Biol Chem. 1995 Feb 10;270(6):2601–2606. doi: 10.1074/jbc.270.6.2601. [DOI] [PubMed] [Google Scholar]
  31. Meurs E. F., Galabru J., Barber G. N., Katze M. G., Hovanessian A. G. Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):232–236. doi: 10.1073/pnas.90.1.232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Meurs E. F., Watanabe Y., Kadereit S., Barber G. N., Katze M. G., Chong K., Williams B. R., Hovanessian A. G. Constitutive expression of human double-stranded RNA-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth. J Virol. 1992 Oct;66(10):5805–5814. doi: 10.1128/jvi.66.10.5805-5814.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Olsen H. S., Cochrane A. W., Dillon P. J., Nalin C. M., Rosen C. A. Interaction of the human immunodeficiency virus type 1 Rev protein with a structured region in env mRNA is dependent on multimer formation mediated through a basic stretch of amino acids. Genes Dev. 1990 Aug;4(8):1357–1364. doi: 10.1101/gad.4.8.1357. [DOI] [PubMed] [Google Scholar]
  36. Patel R. C., Sen G. C. Characterization of the interactions between double-stranded RNA and the double-stranded RNA binding domain of the interferon induced protein kinase. Cell Mol Biol Res. 1994;40(7-8):671–682. [PubMed] [Google Scholar]
  37. Patel R. C., Sen G. C. Identification of the double-stranded RNA-binding domain of the human interferon-inducible protein kinase. J Biol Chem. 1992 Apr 15;267(11):7671–7676. [PubMed] [Google Scholar]
  38. Patel R. C., Stanton P., Sen G. C. Role of the amino-terminal residues of the interferon-induced protein kinase in its activation by double-stranded RNA and heparin. J Biol Chem. 1994 Jul 15;269(28):18593–18598. [PubMed] [Google Scholar]
  39. Petryshyn R., Chen J. J., London I. M. Growth-related expression of a double-stranded RNA-dependent protein kinase in 3T3 cells. J Biol Chem. 1984 Dec 10;259(23):14736–14742. [PubMed] [Google Scholar]
  40. Romano P. R., Green S. R., Barber G. N., Mathews M. B., Hinnebusch A. G. Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae. Mol Cell Biol. 1995 Jan;15(1):365–378. doi: 10.1128/mcb.15.1.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Samuel C. E. Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology. 1991 Jul;183(1):1–11. doi: 10.1016/0042-6822(91)90112-o. [DOI] [PubMed] [Google Scholar]
  42. Sharp T. V., Xiao Q., Jeffrey I., Gewert D. R., Clemens M. J. Reversal of the double-stranded-RNA-induced inhibition of protein synthesis by a catalytically inactive mutant of the protein kinase PKR. Eur J Biochem. 1993 Jun 15;214(3):945–948. doi: 10.1111/j.1432-1033.1993.tb17998.x. [DOI] [PubMed] [Google Scholar]
  43. Sonenberg N. Measures and countermeasures in the modulation of initiation factor activities by viruses. New Biol. 1990 May;2(5):402–409. [PubMed] [Google Scholar]
  44. Takacs A. M., Das T., Banerjee A. K. Mapping of interacting domains between the nucleocapsid protein and the phosphoprotein of vesicular stomatitis virus by using a two-hybrid system. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10375–10379. doi: 10.1073/pnas.90.21.10375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Tiwari R. K., Kusari J., Kumar R., Sen G. C. Gene induction by interferons and double-stranded RNA: selective inhibition by 2-aminopurine. Mol Cell Biol. 1988 Oct;8(10):4289–4294. doi: 10.1128/mcb.8.10.4289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Zapp M. L., Hope T. J., Parslow T. G., Green M. R. Oligomerization and RNA binding domains of the type 1 human immunodeficiency virus Rev protein: a dual function for an arginine-rich binding motif. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7734–7738. doi: 10.1073/pnas.88.17.7734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zinn K., Keller A., Whittemore L. A., Maniatis T. 2-Aminopurine selectively inhibits the induction of beta-interferon, c-fos, and c-myc gene expression. Science. 1988 Apr 8;240(4849):210–213. doi: 10.1126/science.3281258. [DOI] [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