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Mol Cell Biol, April 1998, p. 2282-2297, Vol. 18, No. 4
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Autophosphorylation in the Activation Loop Is Required for Full Kinase Activity In Vivo of Human and Yeast Eukaryotic Initiation Factor 2 Kinases PKR and GCN2

Patrick R. Romano,¹ Minerva T. Garcia-Barrio,¹ Xiaolong Zhang,² Qizhi Wang,^3, Deborah R. Taylor,^3, Fan Zhang,¹ Christopher Herring,² Michael B. Mathews,^3,§ Jun Qin,² and Alan G. Hinnebusch^1,*

Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development,¹ and Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute,² Bethesda, Maryland 20892, and Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724³

Received 24 October 1997/Returned for modification 11 December 1997/Accepted 22 December 1997

The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit  (eIF2) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2 kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.

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^* Corresponding author. Mailing address: Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Building 6A, Room B1A-13A, Bethesda, MD 20892-2716. Phone: (301) 496-4480. Fax: (301) 496-6828. E-mail: ahinnebusch{at}nih.gov.

Present address: Department of Oncology, Novartis Pharmaceuticals, East Hanover, NJ 07936.

Present address: Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA 90033.

^§ Present address: Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103.

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