Regulation of Interferon-Induced Protein Kinase PKR: Modulation of P58 IPK Inhibitory Function by a Novel Protein, P52 rIPK

Abstract

ABSTRACT 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-induced 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 P52 rIPK , 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 P52 rIPK 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.