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Mol Cell Biol. Jun 1995; 15(6): 3138–3146.
PMCID: PMC230545

Mutants of the RNA-dependent protein kinase (PKR) lacking double-stranded RNA binding domain I can act as transdominant inhibitors and induce malignant transformation.


Recently we reported that introduction of catalytically inactive PKR molecules into NIH 3T3 cells causes malignant transformation and the development of tumors in nude mice. We have proposed that PKR may be a tumor suppressor gene possibly because of its translational inhibitory properties. We have now designed and characterized a number of PKR mutants encoding proteins that retain their catalytic competence but are mutated in their regulatory double-stranded RNA (dsRNA) binding domains (RBDs). RNA binding analysis revealed that PKR proteins either lacking or with point mutations in the first RBD (RBD-1) bound negligible amounts of dsRNA activator or adenovirus VAI RNA inhibitor. Despite the lack of binding, such variants remained functionally competent but were much less active than wild-type PKR. PKR variants completely lacking RBD-1 were largely unresponsive to dsRNA in activation assays but could be activated by heparin. To complement these studies, we evaluated the effects of point mutations in RBD-1 or the removal of either RBD-1 or RBD-2 on the proliferation rate of mouse 3T3 cells. We were unsuccessful at isolating stably transformed cells expressing RBD-1 point mutants or RBD-2-minus mutants. In contrast, NIH 3T3 cells, which constitutively expressed PKR proteins that lacked RBD-1, were selected. These cells displayed a transformed phenotype and caused tumors after inoculation in nude mice. Further, levels of endogenous eIF-2 alpha phosphorylation in RBD-1-minus cell lines were reduced, suggesting that such mutants act in a dominant negative manner to inhibit the function of endogenous PKR. These results emphasize the importance of RBD-1 in PKR control of cell growth and provide additional evidence for the critical role played by PKR in the regulation of malignant transformation.

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Selected References

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  • Barber GN, Edelhoff S, Katze MG, Disteche CM. 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. [PubMed]
  • Barber GN, Thompson S, Lee TG, Strom T, Jagus R, Darveau A, Katze MG. The 58-kilodalton inhibitor of the interferon-induced double-stranded RNA-activated protein kinase is a tetratricopeptide repeat protein with oncogenic properties. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4278–4282. [PMC free article] [PubMed]
  • Barber GN, Tomita J, Garfinkel MS, Meurs E, Hovanessian A, Katze MG. 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. [PubMed]
  • Barber GN, Tomita J, Hovanessian AG, Meurs E, Katze MG. Functional expression and characterization of the interferon-induced double-stranded RNA activated P68 protein kinase from Escherichia coli. Biochemistry. 1991 Oct 22;30(42):10356–10361. [PubMed]
  • Barber GN, Wambach M, Wong ML, Dever TE, Hinnebusch AG, Katze MG. 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. [PMC free article] [PubMed]
  • Bigler J, Hokanson W, Eisenman RN. Thyroid hormone receptor transcriptional activity is potentially autoregulated by truncated forms of the receptor. Mol Cell Biol. 1992 May;12(5):2406–2417. [PMC free article] [PubMed]
  • Black TL, Safer B, Hovanessian A, Katze MG. 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. [PMC free article] [PubMed]
  • Carroll K, Elroy-Stein O, Moss B, Jagus R. Recombinant vaccinia virus K3L gene product prevents activation of double-stranded RNA-dependent, initiation factor 2 alpha-specific protein kinase. J Biol Chem. 1993 Jun 15;268(17):12837–12842. [PubMed]
  • Chong KL, Feng L, Schappert K, Meurs E, Donahue TF, Friesen JD, Hovanessian AG, Williams BR. Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2. EMBO J. 1992 Apr;11(4):1553–1562. [PMC free article] [PubMed]
  • Clarke PA, Schwemmle M, Schickinger J, Hilse K, Clemens MJ. Binding of Epstein-Barr virus small RNA EBER-1 to the double-stranded RNA-activated protein kinase DAI. Nucleic Acids Res. 1991 Jan 25;19(2):243–248. [PMC free article] [PubMed]
  • Delmas V, Laoide BM, Masquilier D, de Groot RP, Foulkes NS, Sassone-Corsi P. Alternative usage of initiation codons in mRNA encoding the cAMP-responsive-element modulator generates regulators with opposite functions. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4226–4230. [PMC free article] [PubMed]
  • Dever TE, Chen JJ, Barber GN, Cigan AM, Feng L, Donahue TF, London IM, Katze MG, Hinnebusch AG. 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. [PMC free article] [PubMed]
  • Dever TE, Feng L, Wek RC, Cigan AM, Donahue TF, Hinnebusch AG. 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. [PubMed]
  • Feng GS, Chong K, Kumar A, Williams BR. 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. [PMC free article] [PubMed]
  • 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]
  • Green SR, Mathews MB. Two RNA-binding motifs in the double-stranded RNA-activated protein kinase, DAI. Genes Dev. 1992 Dec;6(12B):2478–2490. [PubMed]
  • Hershey JW. Translational control in mammalian cells. Annu Rev Biochem. 1991;60:717–755. [PubMed]
  • Hovanessian AG. Interferon-induced and double-stranded RNA-activated enzymes: a specific protein kinase and 2',5'-oligoadenylate synthetases. J Interferon Res. 1991 Aug;11(4):199–205. [PubMed]
  • Hovanessian AG, Galabru J. The double-stranded RNA-dependent protein kinase is also activated by heparin. Eur J Biochem. 1987 Sep 15;167(3):467–473. [PubMed]
  • Katze MG. Regulation of the interferon-induced PKR: can viruses cope? Trends Microbiol. 1995 Feb;3(2):75–78. [PubMed]
  • Katze MG, DeCorato D, Safer B, Galabru J, Hovanessian AG. 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. [PMC free article] [PubMed]
  • Katze MG, Wambach M, Wong ML, Garfinkel M, Meurs E, Chong K, Williams BR, Hovanessian AG, Barber GN. 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. [PMC free article] [PubMed]
  • Konieczny A, Safer B. Purification of the eukaryotic initiation factor 2-eukaryotic initiation factor 2B complex and characterization of its guanine nucleotide exchange activity during protein synthesis initiation. J Biol Chem. 1983 Mar 10;258(5):3402–3408. [PubMed]
  • Koromilas AE, Roy S, Barber GN, Katze MG, Sonenberg N. Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase. Science. 1992 Sep 18;257(5077):1685–1689. [PubMed]
  • Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 1991 Nov;115(4):887–903. [PMC free article] [PubMed]
  • Kumar A, Haque J, Lacoste J, Hiscott J, Williams BR. 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. [PMC free article] [PubMed]
  • Langland JO, Jacobs BL. 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]
  • Laurent AG, Krust B, Galabru J, Svab J, Hovanessian AG. 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. [PMC free article] [PubMed]
  • Lee SB, Green SR, Mathews MB, 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. [PMC free article] [PubMed]
  • Lee TG, Tang N, Thompson S, Miller J, Katze MG. The 58,000-dalton cellular inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (PKR) is a member of the tetratricopeptide repeat family of proteins. Mol Cell Biol. 1994 Apr;14(4):2331–2342. [PMC free article] [PubMed]
  • Manche L, Green SR, Schmedt C, Mathews MB. Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol Cell Biol. 1992 Nov;12(11):5238–5248. [PMC free article] [PubMed]
  • Maran A, Maitra RK, Kumar A, Dong B, Xiao W, Li G, Williams BR, Torrence PF, Silverman RH. 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. [PubMed]
  • Maran A, Mathews MB. 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. [PubMed]
  • McCormack SJ, Ortega LG, Doohan JP, Samuel CE. 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. [PubMed]
  • McCormack SJ, Thomis DC, Samuel CE. 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. [PubMed]
  • Meurs E, Chong K, Galabru J, Thomas NS, Kerr IM, Williams BR, Hovanessian AG. Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell. 1990 Jul 27;62(2):379–390. [PubMed]
  • Meurs EF, Galabru J, Barber GN, Katze MG, Hovanessian AG. 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. [PMC free article] [PubMed]
  • Meurs EF, Watanabe Y, Kadereit S, Barber GN, Katze MG, Chong K, Williams BR, Hovanessian AG. 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. [PMC free article] [PubMed]
  • Sancho J, Ledbetter JA, Choi MS, Kanner SB, Deans JP, Terhorst C. CD3-zeta surface expression is required for CD4-p56lck-mediated upregulation of T cell antigen receptor-CD3 signaling in T cells. J Biol Chem. 1992 Apr 15;267(11):7871–7879. [PubMed]
  • Patel RC, Stanton P, Sen GC. 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]
  • Romano PR, Green SR, Barber GN, Mathews MB, Hinnebusch AG. 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. [PMC free article] [PubMed]
  • Roy S, Agy M, Hovanessian AG, Sonenberg N, Katze MG. The integrity of the stem structure of human immunodeficiency virus type 1 Tat-responsive sequence of RNA is required for interaction with the interferon-induced 68,000-Mr protein kinase. J Virol. 1991 Feb;65(2):632–640. [PMC free article] [PubMed]
  • Samuel CE. 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]
  • Sen GC, Lengyel P. The interferon system. A bird's eye view of its biochemistry. J Biol Chem. 1992 Mar 15;267(8):5017–5020. [PubMed]
  • St Johnston D, Brown NH, Gall JG, Jantsch M. A conserved double-stranded RNA-binding domain. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10979–10983. [PMC free article] [PubMed]
  • Tanaka H, Samuel CE. 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. [PMC free article] [PubMed]
  • Thomis DC, Samuel CE. 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. [PMC free article] [PubMed]
  • Thomis DC, Samuel CE. 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. [PMC free article] [PubMed]

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