Mutational Analysis of the Double-stranded RNA (dsRNA) Binding Domain of the dsRNA-activated Protein Kinase, PKR

doi:10.1074/jbc.270.6.2601

Mutational Analysis of the Double-stranded RNA (dsRNA) Binding Domain of the dsRNA-activated Protein Kinase, PKR (*)

From the Department of Cancer Biology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

^§ To whom correspondence and reprint requests should be addressed:
Dept. of Cancer Biology, Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195.
Tel.: 216-445-9652; Fax: 216-445-6269.

Abstract

The interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase, PKR, is an inhibitor of translation and has antiviral, antiproliferative, and antitumor properties. Previously, the dsRNA binding domain had been located within the N-terminal region of PKR and subsequently shown to include two nearly identical domains comprising residues 55-75 and 145-166. We have undertaken both random and site-directed, alanine-scanning mutagenesis in order to investigate the contribution of individual amino acids within these domains to dsRNA binding. Here we identify 2 residues that were absolutely required for dsRNA binding, glycine 57 and lysine 60. Mutation of 2 other residues within the domain (lysine 64 and leucine 75) resulted in less than 10% binding (compared to wild type). We have also identified a number of other residues that influence dsRNA binding to varying degrees. Mutants that were unable to bind dsRNA were not active in vitro and possessed no antiproliferative activity in vivo. However, dsRNA binding mutants were partially transdominant over wild type PKR in mammalian cells, suggesting that binding of dsRNA activator is not the mechanism responsible for the phenotype of PKR mutants.

Footnotes

↵* This work was supported in part by NIAID Grant R01 A1 34039-02 from the National Institutes of Health and a grant from the Human Frontiers Science Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PKR

protein kinase, RNA-activated

CAT

chloramphenicol acetyltransferase

ds

double-stranded

eIF

eukaryotic initiation factor

poly(I):poly(C)

polyinosonic acid:polycytidylic acid

FPLC

fast protein liquid chromatography

PAGE

polyacrylamide gel electrophoresis.
↵² N. A. J. McMillan, D. P. Siderovski, J. Galabru, W. M. Toone, T. W. Mak, A. G. Hovanessian, and B. R. G. Williams, submitted for publication.
↵³ N. A. J. McMillan, B. W. Carpick, B. Hollis, W. M. Toone, M. Zamanian-Daryoush, and B. R. G. Williams, unpublished observation.
↵⁴ A. Kumar and B. R. G. Williams, unpublished observation.
- Received September 15, 1994.