Functional expression and characterization of the interferon-induced double-stranded RNA activated P68 protein kinase from Escherichia coli

Abstract

The P68 protein (referred to as P68 on the basis of its molecular weight of 68,000 in human cells) is a serine/threonine kinase induced by interferon treatment and activated by double-stranded (ds) RNAs. Although extensively studied, little is currently known about the regulation of kinase function at the molecular level. What is known is that activation of this enzyme triggers a series of events which lead to an inhibition of protein synthesis initiation and may, in turn, play an integral role in the antiviral response to interferon. To begin to understand P68 and its biological functions in the eukaryotic cell, we have expressed the protein kinase in Escherichia coli under control of the bacteriophage T7 promoter. In rifampicin-treated cells, metabolically labeled with [35S]methionine and induced by IPTG, the P68 kinase was the predominant labeled product. Further, P68 was recovered from extracts as a fully functional enzyme, shown by its ability to become activated and phosphorylate its natural substrate, the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2). Moreover, P68 was phosphorylated in vivo in E. coli, providing conclusive evidence that the kinase has the capacity to phosphorylate and activate itself in the absence of other eukaryotic proteins. In contrast, a mutant P68 protein, containing a single amino acid substitution in the invariant lysine in catalytic domain II, was completely inactive. Interestingly, both the mutant and wild-type protein kinases efficiently bound activator dsRNAs despite the fact that only the latter was activated by these RNAs. Finally, the expressed kinase could be isolated from contaminating E. coli proteins in an active form by immunoaffinity chromatography with a monoclonal antibody specific for P68.

PACT, a Stress-modulated Cellular Activator of Interferon-induced Double-stranded RNA-activated Protein Kinase, PKR

“…These results differ from our previous results that bacterially expressed affinity-purified PACT can activate PKR in vitro. The discrepancy between in vitro and in vivo results may be explained by the fact that many recombinant proteins including PKR, are known to be constitutively phosphorylated in bacteria (46). It remains to be seen if PACT phosphorylation is essential either for its association with PKR or its activation in vivo.…”

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

“…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.…”

Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity

“…Wild-type human PKR is isolated from E.coli or Saccharomyces cerevisiae overexpression systems as a highly phosphorylated protein (6,21,22,27). Since E.coli lacks eukaryotic kinases and a kinase-inactive mutant of PKR (K296R) is not phosphorylated during overexpression in bacteria or yeast, the phosphorylation observed is attributed to PKR autophosphorylation (22,27).…”

“…Since E.coli lacks eukaryotic kinases and a kinase-inactive mutant of PKR (K296R) is not phosphorylated during overexpression in bacteria or yeast, the phosphorylation observed is attributed to PKR autophosphorylation (22,27). This autophosphorylation during overexpression is believed to arise from the binding of endogenous RNAs that serve to activate the enzyme (27).…”

“…These results suggested that autophosphorylation may have an effect on the RNAbinding properties of the enzyme (26). Interestingly, although wild-type PKR and several site-directed mutants have been overexpressed using a variety of different expression systems in various organisms, the extent to which kinase active recombinant enzyme can be stimulated by RNA in vitro has been disputed (6,27). Thus, convenient, efficient sources of recombinant PKR in its dephosphorylated form that responds to RNA activation in vitro are needed.…”