Reversal of the double‐stranded‐RNA‐induced inhibition of protein synthesis by a catalytically inactive mutant of the protein kinase PKR

Abstract

The interferon-inducible double-stranded-RNA(dsRNA)-dependent protein kinase PKR has been implicated in both the antiviral and cell growth-regulatory effects of the interferons. Over-expression of the wild-type form of this protein inhibits cell proliferation, whereas over-expression of inactive mutant forms transforms cells to a tumourigenic phenotype. It has been suggested that mutant PKR exerts a dominant negative effect on the activity of the wild-type protein lunase. We have investigated this possibility using the rabbit reticulocyte cell-free translation system in which protein synthesis is inhibited by dsRNA due to activation of PKR and phosphorylation of initiation factor eIF-2. Addition of a highly purified inactive PKR mutant, synthesised in a baculovirus-infected insect cell system, rescues protein synthesis from inhibition by low concentrations of dsRNA in a dose-dependent manner. The PKR mutant has no effect on protein synthesis in the absence of dsRNA or in the presence of another inhibitory protein kinase, the haem-controlled repressor. Inhibition of translation can be re-established in the presence of the mutant PKR by adding a higher concentration of dsRNA. These results suggest that inactive mutant PKR does exert a dominant negative effect on wild-type PKR and that this may be due to competition for dsRNA binding.The interferon-inducible double-stranded-(dsRNA)-activated protein kinase PKR, previously called DAI or p68, is believed to play a major role in the antiviral and antiproliferative effects of the interferons (IFN) [l]. Human PKR is a single polypeptide chain of M , approximately 62000 [21. The molecule consists of a protein kinase catalytic domain located in the C-terminal half, which contains conserved subdomains common to all protein kinases [3], and two dsRNAbinding domains situated in the N-terminal portion of the protein [4-61. PKR has two separate and well-characterized activities: in the presence of dsRNA the protein undergoes autophosphorylation and activation [7], and following such activation PKR phosphorylates the a subunit of polypeptide chain initiation factor eIF-2. Phosphorylation of eIF-2a leads to inhibition of protein synthesis by a well-established mechanism [8, 91.As well as being a mediator of the antiviral affects of IFN, PKR has now been identified as a potential tumoursuppressor-gene product (reviewed in [lo] nude mice [ l l , 121. The specific mutations used to inactivate the kinase were either a change of Lys to Arg at position 296 or deletion of amino-acid residues 361-366 between protein-kinase sub-domains V and VI of PKR. Cells transfected with wild-type kinase or with plasmid vector alone were nontumourigenic. Indeed those cells expressing the wild-type kinase exhibited inhibition of cell growth [12], behaviour which is consistent with recent evidence that the expression of human PKR in yeast cells results in poor growth [13]. These results suggest that over-expression of mutant forms of PKR leads to tumourigenesis due to interference with the active form ...

The catalytic activity of dsRNA-dependent protein kinase, PKR, is required for NF-κB activation

“…We have found that K296R acts as a dominant negative mutant of NF-kB activation triggered by PKR. These results con®rm previous results using in vitro expression systems in which the PKR-dependent protein synthesis inhibition was reverted by a PKR mutant (Sharp et al, 1993). However, it should be emphasized that high amounts of K296R expression were needed to signi®cantly inhibit NF-kB translocation, thus suggesting that WT mutant PKR heterodimers could still retain catalytic activity.…”

“…Although the precise mechanism of action is controversial (Proud, 1995), it has been ®rmly established that expression of PKR (K296R) mutant interferes with WT PKR catalytic activity in a dominant negative fashion (Sharp et al, 1993). Thus, we reasoned that if PKR (K296R) mutant is not able to activate NF-kB, it should also interfere with WT PKR-triggered NF-kB activation.…”

RNase L Mediates Transient Control of the Interferon Response through Modulation of the Double-stranded RNA-dependent Protein Kinase PKR

“…Very high levels of dsRNA (e.g. Ͼ10 g/ml) inhibit PKR (35), but these levels do not appear to be obtained in vivo. Conversely, at low m.o.i., antiviral effects of elevated PKR may not be observed due to insufficient dsRNA.…”

“…The RNA-PKR interaction is complex and is dependent on concentration, length, and structure of dsRNA molecules. PKR levels and optimum interactions are necessary for dimerization of PKR for full activity (32)(33)(34)(35)(36). Thus, at high m.o.i., the combination of elevated levels of both viral dsRNA intermediates and IFNinduced PKR accounts for enhanced translational arrest leading to superior antiviral action of IFN-␣ in RNase L-null cells.…”

Double-stranded RNA-dependent Protein Kinase Mediates c-Myc Suppression Induced by Type I Interferons

“…First, we generated M1-S6 cells that expressed the catalytically inactive mutant form of human PKR, K296R (15), previously shown to act in a transdominant mode (29,30). The kinase inactivation was achieved by the conversion of the conserved lysine at position 296 to arginine.…”