Inhibitory sequences in the N-terminus of the double-stranded-RNA-dependent protein kinase, PKR, are important for regulating phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha).
During viral infection, phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) by the interferon-induced RNA-dependent protein kinase, PKR, leads to inhibition of translation initiation and viral proliferation. Activation of PKR is mediated by association of virally encoded double-stranded RNAs (dsRNAs) with two dsRNA binding domains (dsRBDs) located in the N-terminus of PKR. To better understand the molecular mechanisms regulating PKR, we characterized the activities of wild-type and mutant versions of human PKR expressed and purified from yeast. The catalytic rate of eIF2alpha phosphorylation by our purified PKR was increased in response to dsRNA, but not single-stranded RNA or DNA, consistent with the properties previously described for PKR purified from mammalian sources. While both dsRBD1 and dsRBD2 were required for activation of PKR by dsRNA, only deletion of dsRBD1 severely reduced the basal eIF2alpha kinase activity. Removal of as few as 25 residues at the C-terminal junction of dsRBD2 dramatically increased eIF2alpha kinase activity and characterization of larger deletions that included dsRBD1 demonstrated that removal of these negative-acting sequences could bypass the dsRBD1 requirement for in vitro phosphorylation of eIF2alpha. Heparin, a known in vitro activator of PKR, enhanced eIF2alpha phosphorylation by PKR mutants lacking their entire N-terminal sequences, including the dsRBDs. The results indicate that induction of PKR activity is mediated by multiple mechanisms, one of which involves release of inhibition by negative-acting sequences in PKR.
Vattem
K M
KM
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Staschke
K A
KA
Zhu
S
S
Wek
R C
RC
D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D012330:RNA, Double-Stranded; D006493:Heparin; D019892:eIF-2 Kinase