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Mol Cell Biol, May 1998, p. 2431-2443, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Double-Stranded RNA-Independent Dimerization of Interferon-Induced Protein Kinase PKR and Inhibition of Dimerization by the Cellular P58IPK Inhibitor

Seng-Lai Tan,1 Michael J. Gale Jr.,1 and Michael G. Katze1,2,*

Department of Microbiology1 and Regional Primate Research Center,2 University of Washington, Seattle, Washington 98195

Received 21 August 1997/Returned for modification 16 October 1997/Accepted 22 January 1998

The interferon (IFN)-induced, double-stranded RNA-activated protein kinase (PKR) mediates the antiviral and antiproliferative actions of IFN, in part, via its translational inhibitory properties. Previous studies have demonstrated that PKR forms dimers and that dimerization is likely to be required for activation and/or function. In the present study we used multiple approaches to examine the modulation of PKR dimerization. Deletion analysis with the lambda  repressor fusion system identified a previously unrecognized site involved in PKR dimerization. This site comprised amino acids (aa) 244 to 296, which span part of the third basic region of PKR and the catalytic subdomains I and II. Using the yeast two-hybrid system and far-Western analysis, we verified the importance of this region for dimerization. Furthermore, coexpression of the 52-aa region alone inhibited the formation of full-length PKR dimers in the lambda  repressor fusion and two-hybrid systems. Importantly, coexpression of aa 244 to 296 exerted a dominant-negative effect on wild-type kinase activity in a functional assay. Due to its role as a mediator of IFN-induced antiviral resistance, PKR is a target of viral and cellular inhibitors. Curiously, PKR aa 244 to 296 contain the binding site for a select group of specific inhibitors, including the cellular protein P58IPK. We demonstrated, utilizing both the yeast and lambda  systems, that P58IPK, a member of the tetratricopeptide repeat protein family, can block kinase activity by preventing PKR dimerization. In contrast, a nonfunctional form of P58IPK lacking a TPR motif did not inhibit kinase activity or perturb PKR dimers. These results highlight a potential mechanism of PKR inhibition and define a novel class of PKR inhibitors. Finally, the data document the first known example of inhibition of protein kinase dimerization by a cellular protein inhibitor. On the basis of these results we propose a model for the regulation of PKR dimerization.

* Corresponding author. Mailing address: University of Washington, Health Sciences Bldg., Rm. I-321, 1705 Pacific St., NE, Seattle, WA 98195. Phone: (206) 543-8837. Fax: (206) 685-0305. E-mail: honey{at}u.washington.edu.

Mol Cell Biol, May 1998, p. 2431-2443, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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