The 58-kDa cellular inhibitor of the double stranded RNA-dependent protein kinase requires the tetratricopeptide repeat 6 and DnaJ motifs to stimulate protein synthesis in vivo.
Double stranded RNA-dependent protein kinase (PKR) is a double stranded RNA-activated, interferon-induced serine-threonine kinase that participates in both the antiviral and antiproliferative properties of interferon. We previously found that influenza virus inhibited PKR function by recruiting or activating a cellular inhibitor termed P58(IPK). The present study was undertaken to complement our earlier analyses, which demonstrated that P58(IPK) efficiently inhibited PKR autophosphorylation and activity in vitro. We now report that P58(IPK) down-regulates PKR and, in turn, stimulates protein synthetic rates inside the cell. Using transfection analysis, we show that P58(IPK) stimulates translation of secreted embryonic alkaline phosphatase reporter gene mRNA. Furthermore, we found that at least two regions of the P58(IPK) molecule were required for PKR inhibitory activity in COS-1 cells: (i) the DnaJ similarity region at the carboxyl terminus (amino acids 391-504); and (ii) the tetratricopeptide repeat 6 (TPR6) domain (amino acids 222-255) located in the middle of the P58(IPK) protein and within the eukaryotic protein synthesis initiation factor 2alpha homology region. P58(IPK) variants lacking either one of these regions were unable to stimulate secreted embryonic alkaline phosphatase protein synthetic rates. Consistent with this data is the observation that the DeltaTPR6 mutant (the P58(IPK) variant lacking the TPR6 motif) failed to block PKR activity in vitro. Based on these data and our earlier in vitro functional and PKR-P58(IPK) binding analyses, a revised model of PKR regulation by P58(IPK) is presented.
Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195, USA. honey@u.washington.edu
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D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D008954:Models, Biological; D014176:Protein Biosynthesis; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D012097:Repressor Proteins / Q000494:pharmacology*; D019892:eIF-2 Kinase
D004791:Enzyme Inhibitors; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D047428:Protein Kinase Inhibitors; D012097:Repressor Proteins; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D000469:Alkaline Phosphatase