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

Kostura, M., Mathews, M. B. (April 1989) Purification and activation of the double-stranded RNA-dependent eIF-2 kinase DAI. Molecular & Cellular Biology, 9 (4). pp. 1576-86. ISSN 0270-7306

URL: http://www.ncbi.nlm.nih.gov/pubmed/2725516
DOI: 10.1128/MCB.9.4.1576

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.

Item Type: Paper
Uncontrolled Keywords: Binding Sites Binding, Competitive Enzyme Activation Humans Kinetics Molecular Weight Phosphorylation Protein Conformation Protein Kinases/*isolation & purification/metabolism RNA, Double-Stranded/metabolism RNA, Viral/metabolism Research Support, U.S. Gov't, P.H.S. eIF-2 Kinase
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > dsRNA
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > kinase
CSHL Authors:
Communities: CSHL labs
Depositing User: Gail Sherman
Date: April 1989
Date Deposited: 28 Jun 2017 19:36
Last Modified: 28 Jun 2017 19:36
PMCID: PMC362574
Related URLs:
URI: http://repository.cshl.edu/id/eprint/34867

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