Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system.

Abstract

Eukaryotic viruses have devised numerous strategies to downregulate activity of the interferon-induced, double-stranded (dsRNA)-activated protein kinase (referred to as p68 on the basis of its Mr of 68,000 in human cells). Viruses must exert this control to avoid extensive phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) by p68 and the resultant negative effects on protein synthesis initiation. To begin to define the molecular mechanisms underlying this regulation, we optimized expression of p68 in an in vitro transcription-translation system utilizing the full-length cDNA clone. The in vitro-expressed kinase was autophosphorylated in response to dsRNAs and heparin in a manner similar to that for the native p68 provided that the kinase inhibitor, 2-aminopurine, was present during the in vitro translation reaction. Further, the activated kinase efficiently phosphorylated its natural substrate, the alpha subunit of eIF-2. Binding experiments revealed that the expressed kinase complexed with the dsRNA activator, reovirus dsRNA, as well as the adenovirus-encoded inhibitor, VAI RNA. Interestingly, both the reovirus RNAs and VAI RNA also complexed with protein kinase molecules that lacked the carboxyl terminus and all catalytic domains. Deletion analysis confirmed that the p68 amino terminus contained critical determinants for reovirus dsRNA and VAI RNA binding. Further, reovirus dsRNA efficiently bound to, but failed to activate, p68 kinase molecules containing a single amino acid substitution in the invariant lysine 295 present in catalytic domain H. Taken together, these data demonstrate that this expression system permits a detailed mutagenic analysis of the regions of p68 required for interaction with virus-encoded activators and repressors.

Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation

“…This finding is in accordance with our previous results showing that a full-length mutant PKR, deficient in dsRNA binding, could still interact with P58 IPK (66). This finding contrasts dramatically with observations regarding adenovirus-encoded VAI RNA, which interacts with the PKR amino-terminal regulatory domains (39,41,58) and functions to inhibit PKR through competitive inhibition of dsRNA binding. Our results localize the minimal P58 IPK interactive site(s) on PKR to the 52-aa region comprising aa 244 to 296 (Fig.…”

“…However, we cannot formally exclude the possibility that the (39,41,58). The P58 IPK and K3L interactive domains both localize to nonoverlapping regions within the PKR protein kinase catalytic domain (shaded).…”

“…Construction of the PKR mutants PKR ⌬362-367 in pCDNA1neo (Invitrogen) and of PKR K296R, PKR 1-296, and PKR 244-551 in pET11a (Novagen) and pCDNA1neo was previously described (4,17,41,43). For construction of GAL4 transcriptional activation domain (AD) and GAL4 DNA-binding domain (BD) fusions, the NdeI linker sequence, ccatatgg, was ligated into the SmaI site of pGAD424 (AD) and pGBT9 (BD; Clontech) to generate pGAD425 and pGBT10, respectively.…”

“…Two micrograms of each in vitro transcription product was used to program a rabbit reticulocyte lysate in vitro translation system (Promega) in the presence of [ 35 S]methionine as described previously (41). To verify that the translation products were of the expected sizes, an aliquot of each was analyzed by sodium dodecyl (SDS)-sulfate polyacrylamide gel electrophoresis (PAGE) and fluorography.…”

“…Transcripts encoding full-length and mutant PKR and P58 IPK were generated essentially as described previously (41), using the T7 promoter of pCDNA1neo or pET15b (Novagen). Linear transcripts encoding full-length PKR (K296R), PKR aa 1 to 296 or 244 to 551, or the PKR ⌬362-367 mutant (aa 362 to 367 deleted) were generated from the corresponding EcoRV-digested pCDNA1neo construct.…”