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. 1996 Nov;16(11):6295–6302. doi: 10.1128/mcb.16.11.6295

Autophosphorylation sites participate in the activation of the double-stranded-RNA-activated protein kinase PKR.

D R Taylor 1, S B Lee 1, P R Romano 1, D R Marshak 1, A G Hinnebusch 1, M Esteban 1, M B Mathews 1
PMCID: PMC231632  PMID: 8887659

Abstract

The interferon-induced RNA-dependent protein kinase PKR is found in cells in a latent state. In response to the binding of double-stranded RNA, the enzyme becomes activated and autophosphorylated on several serine and threonine residues. Consequently, it has been postulated that autophosphorylation is a prerequisite for activation of the kinase. We report the identification of PKR sites that are autophosphorylated in vitro concomitantly with activation and examine their roles in the activation of PKR. Mutation of one site, threonine 258, results in a kinase that is less efficient in autophosphorylation and in phosphorylating its substrate, the initiation factor eIF2, in vitro. The mutant kinase is also impaired in vivo, displaying reduced ability to inhibit protein synthesis in yeast and mammalian cells and to induce a slow-growth phenotype in Saccharomyces cerevisiae. Mutations at two neighboring sites, serine 242 and threonine 255, exacerbated the effect. Taken together with earlier results (S. B. Lee, S. R. Green, M. B. Mathews, and M. Esteban, Proc. Natl. Acad. Sci. USA 91:10551-10555, 1994), these data suggest that the central part of the PKR molecule, lying between its RNA-binding and catalytic domains, regulates kinase activity via autophosphorylation.

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Selected References

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  1. Barber G. N., Edelhoff S., Katze M. G., Disteche C. M. Chromosomal assignment of the interferon-inducible double-stranded RNA-dependent protein kinase (PRKR) to human chromosome 2p21-p22 and mouse chromosome 17 E2. Genomics. 1993 Jun;16(3):765–767. doi: 10.1006/geno.1993.1262. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  4. Brasier A. R., Tate J. E., Habener J. F. Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. Biotechniques. 1989 Nov-Dec;7(10):1116–1122. [PubMed] [Google Scholar]
  5. Bycroft M., Grünert S., Murzin A. G., Proctor M., St Johnston D. NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5. EMBO J. 1995 Jul 17;14(14):3563–3571. doi: 10.1002/j.1460-2075.1995.tb07362.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen J. J., Throop M. S., Gehrke L., Kuo I., Pal J. K., Brodsky M., London I. M. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase of rabbit reticulocytes: homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2 alpha kinase. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7729–7733. doi: 10.1073/pnas.88.17.7729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Dever T. E., Chen J. J., Barber G. N., Cigan A. M., Feng L., Donahue T. F., London I. M., Katze M. G., Hinnebusch A. G. Mammalian eukaryotic initiation factor 2 alpha kinases functionally substitute for GCN2 protein kinase in the GCN4 translational control mechanism of yeast. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4616–4620. doi: 10.1073/pnas.90.10.4616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dever T. E., Feng L., Wek R. C., Cigan A. M., Donahue T. F., Hinnebusch A. G. Phosphorylation of initiation factor 2 alpha by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast. Cell. 1992 Feb 7;68(3):585–596. doi: 10.1016/0092-8674(92)90193-g. [DOI] [PubMed] [Google Scholar]
  10. Ellis L., Clauser E., Morgan D. O., Edery M., Roth R. A., Rutter W. J. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell. 1986 Jun 6;45(5):721–732. doi: 10.1016/0092-8674(86)90786-5. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Flint A. J., Paladini R. D., Koshland D. E., Jr Autophosphorylation of protein kinase C at three separated regions of its primary sequence. Science. 1990 Jul 27;249(4967):408–411. doi: 10.1126/science.2377895. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Galabru J., Krust B., Hovanessian A. G. Interferon-mediated protein kinase activity in different fractions of mouse L-929 cells. J Interferon Res. 1984 Fall;4(4):469–480. doi: 10.1089/jir.1984.4.469. [DOI] [PubMed] [Google Scholar]
  17. Green S. R., Manche L., Mathews M. B. Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI. Mol Cell Biol. 1995 Jan;15(1):358–364. doi: 10.1128/mcb.15.1.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Hanks S. K., Quinn A. M., Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. doi: 10.1126/science.3291115. [DOI] [PubMed] [Google Scholar]
  20. Herrera R., Rosen O. M. Autophosphorylation of the insulin receptor in vitro. Designation of phosphorylation sites and correlation with receptor kinase activation. J Biol Chem. 1986 Sep 15;261(26):11980–11985. [PubMed] [Google Scholar]
  21. Hinnebusch A. G. Involvement of an initiation factor and protein phosphorylation in translational control of GCN4 mRNA. Trends Biochem Sci. 1990 Apr;15(4):148–152. doi: 10.1016/0968-0004(90)90215-w. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Kmiecik T. E., Shalloway D. Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell. 1987 Apr 10;49(1):65–73. doi: 10.1016/0092-8674(87)90756-2. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. Lee S. B., Esteban M. The interferon-induced double-stranded RNA-activated protein kinase induces apoptosis. Virology. 1994 Mar;199(2):491–496. doi: 10.1006/viro.1994.1151. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Lee S. B., Melkova Z., Yan W., Williams B. R., Hovanessian A. G., Esteban M. The interferon-induced double-stranded RNA-activated human p68 protein kinase potently inhibits protein synthesis in cultured cells. Virology. 1993 Jan;192(1):380–385. doi: 10.1006/viro.1993.1048. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Maran A., Mathews M. B. Characterization of the double-stranded RNA implicated in the inhibition of protein synthesis in cells infected with a mutant adenovirus defective for VA RNA. Virology. 1988 May;164(1):106–113. doi: 10.1016/0042-6822(88)90625-3. [DOI] [PubMed] [Google Scholar]
  35. Mathews M. B., Shenk T. Adenovirus virus-associated RNA and translation control. J Virol. 1991 Nov;65(11):5657–5662. doi: 10.1128/jvi.65.11.5657-5662.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McMillan N. A., Chun R. F., Siderovski D. P., Galabru J., Toone W. M., Samuel C. E., Mak T. W., Hovanessian A. G., Jeang K. T., Williams B. R. HIV-1 Tat directly interacts with the interferon-induced, double-stranded RNA-dependent kinase, PKR. Virology. 1995 Nov 10;213(2):413–424. doi: 10.1006/viro.1995.0014. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Mochly-Rosen D., Koshland D. E., Jr Domain structure and phosphorylation of protein kinase C. J Biol Chem. 1987 Feb 15;262(5):2291–2297. [PubMed] [Google Scholar]
  40. Mohammadi M., Dikic I., Sorokin A., Burgess W. H., Jaye M., Schlessinger J. Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction. Mol Cell Biol. 1996 Mar;16(3):977–989. doi: 10.1128/mcb.16.3.977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mundschau L. J., Faller D. V. Platelet-derived growth factor signal transduction through the interferon-inducible kinase PKR. Immediate early gene induction. J Biol Chem. 1995 Feb 17;270(7):3100–3106. doi: 10.1074/jbc.270.7.3100. [DOI] [PubMed] [Google Scholar]
  42. Offermann M. K., Zimring J., Mellits K. H., Hagan M. K., Shaw R., Medford R. M., Mathews M. B., Goodbourn S., Jagus R. Activation of the double-stranded-RNA-activated protein kinase and induction of vascular cell adhesion molecule-1 by poly (I).poly (C) in endothelial cells. Eur J Biochem. 1995 Aug 15;232(1):28–36. doi: 10.1111/j.1432-1033.1995.tb20777.x. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Piwnica-Worms H., Saunders K. B., Roberts T. M., Smith A. E., Cheng S. H. Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src. Cell. 1987 Apr 10;49(1):75–82. doi: 10.1016/0092-8674(87)90757-4. [DOI] [PubMed] [Google Scholar]
  46. Proud C. G., Colthurst D. R., Ferrari S., Pinna L. A. The substrate specificity of protein kinases which phosphorylate the alpha subunit of eukaryotic initiation factor 2. Eur J Biochem. 1991 Feb 14;195(3):771–779. doi: 10.1111/j.1432-1033.1991.tb15765.x. [DOI] [PubMed] [Google Scholar]
  47. Proud C. G. PKR: a new name and new roles. Trends Biochem Sci. 1995 Jun;20(6):241–246. doi: 10.1016/s0968-0004(00)89025-8. [DOI] [PubMed] [Google Scholar]
  48. Rice A. P., Kostura M., Mathews M. B. Identification of a 90-kDa polypeptide which associates with adenovirus VA RNAI and is phosphorylated by the double-stranded RNA-dependent protein kinase. J Biol Chem. 1989 Dec 5;264(34):20632–20637. [PubMed] [Google Scholar]
  49. Rodriguez J. F., Rodriguez D., Rodriguez J. R., McGowan E. B., Esteban M. Expression of the firefly luciferase gene in vaccinia virus: a highly sensitive gene marker to follow virus dissemination in tissues of infected animals. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1667–1671. doi: 10.1073/pnas.85.5.1667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rodriguez J. F., Smith G. L. Inducible gene expression from vaccinia virus vectors. Virology. 1990 Jul;177(1):239–250. doi: 10.1016/0042-6822(90)90477-9. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Russo G. L., Vandenberg M. T., Yu I. J., Bae Y. S., Franza B. R., Jr, Marshak D. R. Casein kinase II phosphorylates p34cdc2 kinase in G1 phase of the HeLa cell division cycle. J Biol Chem. 1992 Oct 5;267(28):20317–20325. [PubMed] [Google Scholar]
  53. 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]
  54. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  55. St Johnston D., Brown N. H., Gall J. G., Jantsch M. A conserved double-stranded RNA-binding domain. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10979–10983. doi: 10.1073/pnas.89.22.10979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. 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]
  57. Thomis D. C., Samuel C. E. Mechanism of interferon action: characterization of the intermolecular autophosphorylation of PKR, the interferon-inducible, RNA-dependent protein kinase. J Virol. 1995 Aug;69(8):5195–5198. doi: 10.1128/jvi.69.8.5195-5198.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. 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]
  59. Weinmaster G., Pawson T. Protein kinase activity of FSV (Fujinami sarcoma virus) P130gag-fps shows a strict specificity for tyrosine residues. J Biol Chem. 1986 Jan 5;261(1):328–333. [PubMed] [Google Scholar]
  60. Wettenhall R. E., Aebersold R. H., Hood L. E. Solid-phase sequencing of 32P-labeled phosphopeptides at picomole and subpicomole levels. Methods Enzymol. 1991;201:186–199. doi: 10.1016/0076-6879(91)01017-v. [DOI] [PubMed] [Google Scholar]
  61. Yang Y. L., Reis L. F., Pavlovic J., Aguzzi A., Schäfer R., Kumar A., Williams B. R., Aguet M., Weissmann C. Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase. EMBO J. 1995 Dec 15;14(24):6095–6106. doi: 10.1002/j.1460-2075.1995.tb00300.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

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