Purification and Activation of the Double-stranded RNA-dependent EIF-2 Kinase DAI

Overview

Journal Mol Cell Biol

Specialty Cell Biology

Date 1989 Apr 1

PMID 2725516

Citations 81

Authors

M Kostura

M B Mathews

Affiliations

Soon will be listed here.

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.

Citing Articles

Activation of PKR by a short-hairpin RNA.

Cottrell K, Ryu S, Donelick H, Mai H, Young A, Pierce J Sci Rep. 2024; 14(1):23533.

PMID: 39384561 PMC: 11464672. DOI: 10.1038/s41598-024-74477-3.

Activation of PKR by a short-hairpin RNA.

Cottrell K, Ryu S, Donelick H, Mai H, Pierce J, Bass B bioRxiv. 2024; .

PMID: 38766230 PMC: 11100704. DOI: 10.1101/2024.05.08.592371.

RNA recognition by PKR during DNA virus infection.

Zhang R, Karijolich J J Med Virol. 2024; 96(2):e29424.

PMID: 38285432 PMC: 10832991. DOI: 10.1002/jmv.29424.

The competitive landscape of the dsRNA world.

Cottrell K, Andrews R, Bass B Mol Cell. 2023; 84(1):107-119.

PMID: 38118451 PMC: 10843539. DOI: 10.1016/j.molcel.2023.11.033.

The integrated stress response in cancer progression: a force for plasticity and resistance.

Lines C, McGrath M, Dorwart T, Conn C Front Oncol. 2023; 13:1206561.

PMID: 37601686 PMC: 10435748. DOI: 10.3389/fonc.2023.1206561.

References

Franklin R . Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proc Natl Acad Sci U S A. 1966; 55(6):1504-11. PMC: 224351. DOI: 10.1073/pnas.55.6.1504. View

OMalley R, Duncan R, Hershey J, Mathews M . Modification of protein synthesis initiation factors and the shut-off of host protein synthesis in adenovirus-infected cells. Virology. 1989; 168(1):112-8. DOI: 10.1016/0042-6822(89)90409-1. View

Hunter T, Hunt T, Jackson R, Robertson H . The characteristics of inhibition of protein synthesis by double-stranded ribonucleic acid in reticulocyte lysates. J Biol Chem. 1975; 250(2):409-17. View

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; 68(1):119-24. DOI: 10.1016/0014-5793(76)80418-8. View

Lebleu B, Sen G, Shaila S, Cabrer B, Lengyel P . Interferon, double-stranded RNA, and protein phosphorylation. Proc Natl Acad Sci U S A. 1976; 73(9):3107-11. PMC: 430945. DOI: 10.1073/pnas.73.9.3107. View

Roberts W, Hovanessian A, Brown R, Clemens M, Kerr I . Interferon-mediated protein kinase and low-molecular-weight inhibitor of protein synthesis. Nature. 1976; 264(5585):477-80. DOI: 10.1038/264477a0. View

Farrell P, Balkow K, Hunt T, Jackson R, Trachsel H . Phosphorylation of initiation factor elF-2 and the control of reticulocyte protein synthesis. Cell. 1977; 11(1):187-200. DOI: 10.1016/0092-8674(77)90330-0. View

Graham F, Smiley J, Russell W, Nairn R . Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977; 36(1):59-74. DOI: 10.1099/0022-1317-36-1-59. View

Sen G, 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; 253(17):5915-21. View

10.

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; 75(10):4734-8. PMC: 336194. DOI: 10.1073/pnas.75.10.4734. View

11.

Baglioni C . Interferon-induced enzymatic activities and their role in the antriviral state. Cell. 1979; 17(2):255-64. DOI: 10.1016/0092-8674(79)90151-x. View

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; 254(19):9846-53. View

13.

Petryshyn R, LEVIN D, LONDON I . Purification and characterization of a latent precursor of a double-stranded RNA dependent protein kinase from reticulocyte lysates. Biochem Biophys Res Commun. 1980; 94(4):1190-8. DOI: 10.1016/0006-291x(80)90545-8. View

14.

Oakley B, Kirsch D, Morris N . A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980; 105(2):361-3. DOI: 10.1016/0003-2697(80)90470-4. View

15.

Ranu R . 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; 97(1):252-62. DOI: 10.1016/s0006-291x(80)80162-8. View

16.

Baglioni C, Benvin S, Maroney P, Minks M, Nilsen T, West D . 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. View

17.

LEVIN D, Petryshyn R, LONDON I . Characterization of purified double-stranded RNA-activated eIF-2 alpha kinase from rabbit reticulocytes. J Biol Chem. 1981; 256(14):7638-41. View

18.

Monstein H, Philipson L . The conformation of adenovirus VAI-RNA in solution. Nucleic Acids Res. 1981; 9(17):4239-50. PMC: 327432. DOI: 10.1093/nar/9.17.4239. View

19.

Lengyel P . Biochemistry of interferons and their actions. Annu Rev Biochem. 1982; 51:251-82. DOI: 10.1146/annurev.bi.51.070182.001343. View

20.

Ochoa S . Regulation of protein synthesis initiation in eucaryotes. Arch Biochem Biophys. 1983; 223(2):325-49. DOI: 10.1016/0003-9861(83)90598-2. View