This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kostura, M Articles by Mathews, M B Search for Related Content PubMed PubMed Citation Articles by Kostura, M Articles by Mathews, M B

Purification and activation of the double-stranded RNA-dependent eIF-2 kinase DAI.

Cold Spring Harbor Laboratory, New York 11724.

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.

This article has been cited by other articles:

• Coventry, V. K., Conn, G. L. (2008). Analysis of adenovirus VA RNAI structure and stability using compensatory base pair modifications. Nucleic Acids Res 36: 1645-1653 [Abstract] [Full Text]  
• Ohoka, N., Hattori, T., Kitagawa, M., Onozaki, K., Hayashi, H. (2007). Critical and Functional Regulation of CHOP (C/EBP Homologous Protein) through the N-terminal Portion. J. Biol. Chem. 282: 35687-35694 [Abstract] [Full Text]  
• Schregel, V., Auerochs, S., Jochmann, R., Maurer, K., Stamminger, T., Marschall, M. (2007). Mapping of a self-interaction domain of the cytomegalovirus protein kinase pUL97. J. Gen. Virol. 88: 395-404 [Abstract] [Full Text]  
• Marciniak, S. J., Ron, D. (2006). Endoplasmic reticulum stress signaling in disease.. Physiol. Rev. 86: 1133-1149 [Abstract] [Full Text]  
• Obeng, E. A., Carlson, L. M., Gutman, D. M., Harrington, W. J. Jr, Lee, K. P., Boise, L. H. (2006). Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells. Blood 107: 4907-4916 [Abstract] [Full Text]  
• Ruf, I. K., Lackey, K. A., Warudkar, S., Sample, J. T. (2005). Protection from Interferon-Induced Apoptosis by Epstein-Barr Virus Small RNAs Is Not Mediated by Inhibition of PKR. J. Virol. 79: 14562-14569 [Abstract] [Full Text]  
• Lu, P. D., Harding, H. P., Ron, D. (2004). Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response. J. Cell Biol. 167: 27-33 [Abstract] [Full Text]  
• Perkins, D. J., Barber, G. N. (2004). Defects in Translational Regulation Mediated by the {alpha} Subunit of Eukaryotic Initiation Factor 2 Inhibit Antiviral Activity and Facilitate the Malignant Transformation of Human Fibroblasts. Mol. Cell. Biol. 24: 2025-2040 [Abstract] [Full Text]  
• Tan, S.-L., Tareen, S. U., Melville, M. W., Blakely, C. M., Katze, M. G. (2002). The Direct Binding of the Catalytic Subunit of Protein Phosphatase 1 to the PKR Protein Kinase Is Necessary but Not Sufficient for Inactivation and Disruption of Enzyme Dimer Formation. J. Biol. Chem. 277: 36109-36117 [Abstract] [Full Text]  
• Nussbaum, J. M., Gunnery, S., Mathews, M. B. (2002). The 3'-untranslated regions of cytoskeletal muscle mRNAs inhibit translation by activating the double-stranded RNA-dependent protein kinase PKR. Nucleic Acids Res 30: 1205-1212 [Abstract] [Full Text]  
• Samuel, C. E. (2001). Antiviral Actions of Interferons. Clin. Microbiol. Rev. 14: 778-809 [Abstract] [Full Text]  
• Pitha, P. M. (2001). Latently Expressed Human Herpesvirus 8-Encoded Interferon Regulatory Factor 2 Inhibits Double-Stranded RNA-Activated Protein Kinase. J. Virol. 75: 2345-2352 [Abstract] [Full Text]  
• Taylor, D. R., Tian, B., Romano, P. R., Hinnebusch, A. G., Lai, M. M. C., Mathews, M. B. (2001). Hepatitis C Virus Envelope Protein E2 Does Not Inhibit PKR by Simple Competition with Autophosphorylation Sites in the RNA-Binding Domain. J. Virol. 75: 1265-1273 [Abstract] [Full Text]  
• Abraham, N., Stojdl, D. F., Duncan, P. I., Methot, N., Ishii, T., Dube, M., Vanderhyden, B. C., Atkins, H. L., Gray, D. A., McBurney, M. W., Koromilas, A. E., Brown, E. G., Sonenberg, N., Bell, J. C. (1999). Characterization of Transgenic Mice with Targeted Disruption of the Catalytic Domain of the Double-stranded RNA-dependent Protein Kinase, PKR. J. Biol. Chem. 274: 5953-5962 [Abstract] [Full Text]  
• Kumar, K. U., Srivastava, S. P., Kaufman, R. J. (1999). Double-Stranded RNA-Activated Protein Kinase (PKR) Is Negatively Regulated by 60S Ribosomal Subunit Protein L18. Mol. Cell. Biol. 19: 1116-1125 [Abstract] [Full Text]  
• Romano, P. R., Zhang, F., Tan, S.-L., Garcia-Barrio, M. T., Katze, M. G., Dever, T. E., Hinnebusch, A. G. (1998). Inhibition of Double-Stranded RNA-Dependent Protein Kinase PKR by Vaccinia Virus E3: Role of Complex Formation and the E3 N-Terminal Domain. Mol. Cell. Biol. 18: 7304-7316 [Abstract] [Full Text]  
• Berlanga, J. J., Herrero, S., de Haro, C. (1998). Characterization of the Hemin-sensitive Eukaryotic Initiation Factor 2alpha Kinase from Mouse Nonerythroid Cells. J. Biol. Chem. 273: 32340-32346 [Abstract] [Full Text]  
• Tan, S.-L., Gale, M. J. Jr., Katze, M. G. (1998). Double-Stranded RNA-Independent Dimerization of Interferon-Induced Protein Kinase PKR and Inhibition of Dimerization by the Cellular P58IPK Inhibitor. Mol. Cell. Biol. 18: 2431-2443 [Abstract] [Full Text]  
• Dever, T. E., Sripriya, R., McLachlin, J. R., Lu, J., Fabian, J. R., Kimball, S. R., Miller, L. K. (1998). Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2alpha kinase homolog. Proc. Natl. Acad. Sci. USA 95: 4164-4169 [Abstract] [Full Text]  
• Romano, P. R., Garcia-Barrio, M. T., Zhang, X., Wang, Q., Taylor, D. R., Zhang, F., Herring, C., Mathews, M. B., Qin, J., Hinnebusch, A. G. (1998). Autophosphorylation in the Activation Loop Is Required for Full Kinase Activity In Vivo of Human and Yeast Eukaryotic Initiation Factor 2alpha Kinases PKR and GCN2. Mol. Cell. Biol. 18: 2282-2297 [Abstract] [Full Text]  
• Zhu, S., Romano, P. R., Wek, R. C. (1997). Ribosome Targeting of PKR Is Mediated by Two Double-stranded RNA-binding Domains and Facilitates in Vivo Phosphorylation of Eukaryotic Initiation Factor-2. J. Biol. Chem. 272: 14434-14441 [Abstract] [Full Text]  
• Brand, S. R., Kobayashi, R., Mathews, M. B. (1997). The Tat Protein of Human Immunodeficiency Virus Type 1Is a Substrate and Inhibitor of the Interferon-induced, Virally Activated Protein Kinase, PKR. J. Biol. Chem. 272: 8388-8395 [Abstract] [Full Text]  
• Wu, S., Kaufman, R. J. (1997). A Model for the Double-stranded RNA (dsRNA)-dependent Dimerization and Activation of the dsRNA-activated Protein Kinase PKR. J. Biol. Chem. 272: 1291-1296 [Abstract] [Full Text]  
• Raveh, T., Hovanessian, A. G., Meurs, E. F., Sonenberg, N., Kimchi, A. (1996). Double-stranded RNA-dependent Protein Kinase Mediates c-Myc Suppression Induced by Type I Interferons. J. Biol. Chem. 271: 25479-25484 [Abstract] [Full Text]  
• Patel, R. C., Stanton, P., Sen, G. C. (1996). Specific Mutations Near the Amino Terminus of Double-stranded RNA-dependent Protein Kinase (PKR) Differentially Affect Its Double-stranded RNA Binding and Dimerization Properties. J. Biol. Chem. 271: 25657-25663 [Abstract] [Full Text]  
• Zhu, S., Sobolev, A. Y., Wek, R. C. (1996). Histidyl-tRNA Synthetase-related Sequences in GCN2 Protein Kinase Regulate in Vitro Phosphorylation of eIF-2. J. Biol. Chem. 271: 24989-24994 [Abstract] [Full Text]  
• Wu, S., Kaufman, R. J. (1996). Double-stranded (ds) RNA Binding and Not Dimerization Correlates with the Activation of the dsRNA-dependent Protein Kinase (PKR). J. Biol. Chem. 271: 1756-1763 [Abstract] [Full Text]  
• Prostko, C. R., Dholakia, J. N., Brostrom, M. A., Brostrom, C. O. (1995). Activation of the Double-stranded RNA-regulated Protein Kinase by Depletion of Endoplasmic Reticular Calcium Stores. J. Biol. Chem. 270: 6211-6215 [Abstract] [Full Text]  
• Mundschau, L. J., Faller, D. V. (1995). Platelet-derived Growth Factor Signal Transduction through the Interferon-inducible Kinase PKR. J. Biol. Chem. 270: 3100-3106 [Abstract] [Full Text]  
• Jimenez-Garcia, L., Green, S., Mathews, M., Spector, D. (1993). Organization of the double-stranded RNA-activated protein kinase DAI and virus-associated VA RNAI in adenovirus-2-infected HeLa cells. J. Cell Sci. 106: 11-22 [Abstract]  
• Green, S R, Mathews, M B (1992). Two RNA-binding motifs in the double-stranded RNA-activated protein kinase, DAI.. Genes Dev. 6: 2478-2490 [Abstract]  
• Koromilas, A., Roy, S, Barber, G., Katze, M., Sonenberg, N (1992). Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase. Science 257: 1685-1689 [Abstract]  
• Tian, B., Mathews, M. B. (2001). Functional Characterization of and Cooperation between the Double-stranded RNA-binding Motifs of the Protein Kinase PKR. J. Biol. Chem. 276: 9936-9944 [Abstract] [Full Text]  
• Zhang, F., Romano, P. R., Nagamura-Inoue, T., Tian, B., Dever, T. E., Mathews, M. B., Ozato, K., Hinnebusch, A. G. (2001). Binding of Double-stranded RNA to Protein Kinase PKR Is Required for Dimerization and Promotes Critical Autophosphorylation Events in the Activation Loop. J. Biol. Chem. 276: 24946-24958 [Abstract] [Full Text]  
• Parker, L. M., Fierro-Monti, I., Mathews, M. B. (2001). Nuclear Factor 90 Is a Substrate and Regulator of the Eukaryotic Initiation Factor 2 Kinase Double-stranded RNA-activated Protein Kinase. J. Biol. Chem. 276: 32522-32530 [Abstract] [Full Text]