Inhibitory sequences in the N-terminus of the double-stranded-RNA-dependent protein kinase, PKR, are important for regulating phosphorylation of eukaryotic initiation factor 2 alpha (elF2 alpha)

During viral infection, phosphorylation of the a subunit of eukaryotic init iation factor 2 (eIF2 alpha) by the interferon-induced RNA-dependent protei n kinase, PKR, leads to inhibition of translation initiation and viral prol iferation. Activation of PKR is mediated by association of virally encoded double-stranded RNAs (dsRNAs) with two dsRNA binding domains (dsRBDs) locat ed in the N-terminus of PKR. To better understand the molecular mechanisms regulating PKR, we characterized the activities of wild-type and mutant ver sions of human PKR expressed and purified from yeast. The catalytic rate of eIF2a phosphorylation by our purified PKR was increased in response to dsR NA, but not single-stranded RNA or DNA, consistent with the properties prev iously 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 eIF2 alpha kinase activity. Removal of as few as 25 residues at the C-terminal junction of dsRBD2 dramatically incre ased eIF2 alpha kinase activity and characterization of larger deletions th at included dsRBD1 demonstrated that removal of these negative-acting seque nces could bypass the dsRBD1 requirement for in vitro phosphorylation of eI F2a. Heparin, a known in vitro activator of PKR, enhanced eIF2 alpha phosph orylation by PKR mutants lacking their entire N-terminal sequences, includi ng the dsRBDs. The results indicate that induction of PKR activity is media ted by multiple mechanisms, one of which involves release of inhibition by negative-acting sequences in PKR.