Regulation of interferon-induced protein kinase PKR: Modulation of P58(IPK) inhibitory function by a novel protein, p52(rIPK)

The cellular response to environmental signals is largely dependent upon the induction of responsive protein kinase signaling pathways. Within these pathways, distinct protein-protein interactions play a role in determining the specificity of the response through regulation of kinase function. The interferon-lnduced serine/ threonine protein kinase, PKR, is activated in response to various environmental stimuli. Like many protein kinases, PKR is regulated through direct interactions with activator and inhibitory molecules, including P58(IPK), a cellular PKR inhibitor. P58(IPK) functions to represses PKR-mediated phosphorylation of the eukaryotic initiation factor 2α subunit (eIF-2α) through a direct interaction, thereby relieving the PKR-imposed block on mRNA translation and cell growth. To further define the molecular mechanism underlying regulation of PKR, we have utilized an interaction cloning strategy to identify a novel cDNA encoding a P58(IPK)- interacting protein. This protein, designated P52r(IPK), possesses limited homology to the charged domain of Hsp90 and is expressed in a wide range of cell lines. P52(rIPK) and P58(IPK) interacted in a yeast two-hybrid assay and were recovered as a complex from mammalian cell extracts. When coexpressed with PKR in yeast, P58(IPK) repressed PKR-mediated eIF-2α phosphorylation, inhibiting the normally toxic and growth-suppressive effects associated with PKR function. Conversely, introduction of P52(rIPK) into these strains resulted in restoration of both PKR activity and eIF-2α phosphorylation, concomitant with growth suppression due to inhibition of P58(IPK) function. Furthermore, P52(rIPK) inhibited P58(IPK) function in a reconstituted in vitro PKR-regulatory assay. Our results demonstrate that P58(IPK) is inhibited through a direct interaction with P52r(IPK) which, in turn, results in upregulation of PKR activity. Taken together, our data describe a novel protein kinase-regulatory system which encompasses an intersection of interferon-, stress-, and growth-regulatory pathways.