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.
Kinases PKR and GCN2


Laboratory of Eukaryotic Gene Regulation,
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.
*
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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