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

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^IPKrepressed 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^rIPKinhibited P58^IPK function in a reconstituted in vitro PKR-regulatory assay. Our results demonstrate that P58^IPKis inhibited through a direct interaction with P52^rIPKwhich, 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.

ACKNOWLEDGMENTS

We thank Pat Mcgiffort and University of Washington photography for data photos. We are grateful to Thomas Dever for advice on isoelectric focusing analyses, as well as for providing the J110 yeast strain and antibody to yeast eIF-2α. We also thank Marcus Korth and Gary Geiss for critical review of the manuscript. We are grateful to Seng-Lai Tan for sharing results prior to publication.

This work was supported by National Institutes of Health grants AI22646 and RR00166 to M.G.K. C.M.B. was supported in part by an undergraduate research fellowship award from the Howard Hughes Medical Institute and by a University of Washington Mary Gates research fellowship. M.W.M. was supported by a Public Health Service grant, National Research Service Award 32 GM07270, from the National Institute of General Medical Sciences. M.G. was supported by a fellowship from the Helen Hay Whitney Foundation.