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PMID: 9528799 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2alpha kinases PKR and GCN2.

Molecular and cellular biology ·Vol. 18 ·No. 4 ·1998-04-00 ·Pages 2282-97

Romano PR, Garcia-Barrio MT, Zhang X, Wang Q, Taylor DR, Zhang F, Herring C, Mathews MB, Qin J, Hinnebusch AG

Abstract

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 alpha (eIF2alpha) 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 eIF2alpha 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.

MeSH Terms
3T3 Cells Amino Acid Sequence Amino Acid Substitution Animals Binding Sites COS Cells Conserved Sequence DNA-Binding Proteins Enzyme Activation Fungal Proteins/genetics,metabolism Gene Expression Regulation, Enzymologic Humans Mass Spectrometry Mice Mice, Nude Molecular Sequence Data Mutagenesis, Site-Directed Neoplasms, Experimental/etiology Peptide Initiation Factors/genetics,metabolism Peptides/chemical synthesis,metabolism Phosphorylation Protein Biosynthesis Protein Kinases/genetics,metabolism Saccharomyces cerevisiae/enzymology,genetics Saccharomyces cerevisiae Proteins Substrate Specificity Threonine/metabolism eIF-2 Kinase/genetics,metabolism
Chemicals
DNA-Binding Proteins Fungal Proteins Peptide Initiation Factors Peptides Saccharomyces cerevisiae Proteins Threonine Protein Kinases eIF-2 Kinase
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Romano P R
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.
Garcia-Barrio M T
Zhang X
Wang Q
Taylor D R
Zhang F
Herring C
Mathews M B
Qin J
Hinnebusch A G
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.18.4.2282
Published
1998-04-00
Pages
2282-97
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC121479
Subset
IM
Grants
NIAID NIH HHS · AI 34552 · United States
External Links
PubMed source DOI: 10.1128/MCB.18.4.2282 PMC Full Text View journal details
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