Inactivation of the PKR Protein Kinase and Stimulation of mRNA Translation by the Cellular Co-Chaperone P58IPK Does Not Require J Domain Function

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Abstract
P58IPK was discovered as an inhibitor of the interferon-induced, protein kinase, PKR. Upon virus infection, PKR can, as part of the host defense system, inhibit mRNA translation by phosphorylating the α subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2α). We previously found that influenza virus recruits the cellular P58IPK co-chaperone to inhibit PKR activity and thus facilitate viral protein synthesis. P58IPK contains nine tetratricopeptide repeat (TPR) motifs in addition to the highly conserved J domain found in all DnaJ chaperone family members. To define the role of molecular chaperones in regulating cell growth in addition to PKR regulation, we performed a detailed analysis of the P58IPK J domain. Using growth rescue assays, we found that the P58IPK J domain substituted for the J domains of other DnaJ proteins, including DnaJ in Escherichia coli and Ydj1 in Saccharomyces cerevisiae. This is the first time a cellular J domain from a mammalian DnaJ family member was shown to be functional in both prokaryotic DnaJ and eukaryotic Ydj1 constructs. Furthermore, point mutations within the conserved HPD residue cluster of the P58IPK J domain disrupted P58IPK J function including stimulation of ATPase activity of Hsp70. However, the P58IPK HPD mutants still inhibited PKR activity and thus supported cell growth in a yeast rescue assay. Overexpression of the HPD mutants of P58IPK, similar to their wild-type counterpart, also stimulated mRNA translation in a mammalian cell system. Taken together, our data necessitate a model of P58IPK inhibition of PKR kinase activity and stimulation of mRNA translation, which does not require classical J domain function found in the DnaJ molecular chaperone family.