Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58^IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation

Abstract

Expression of the double-stranded RNA-activated protein kinase (PKR) is induced by interferons, with PKR activity playing a pivotal role in establishing the interferon-induced antiviral and antiproliferative states. PKR is directly regulated by physical association with the specific inhibitor, P58 IPK , a cellular protein of the tetratricopeptide repeat (TPR) family, and K3L, the product of the corresponding vaccinia virus gene. P58 IPK and K3L repress PKR activation and activity. To investigate the mechanism of P58 IPK -and K3L-mediated PKR inhibition, we have used a combination of in vitro and in vivo binding assays to identify the interactive regions of these proteins. The P58 IPK -interacting site of PKR was mapped to a 52-amino-acid aa segment (aa 244 to 296) spanning the ATP-binding region of the protein kinase catalytic domain. The interaction with PKR did not require the C-terminal DNA-J homology region of P58 IPK but was dependent on the presence of the eukaryotic initiation factor 2-␣ homology region, mapping to the 34 aa within the sixth P58 IPK TPR motif. Consistent with other TPR proteins, P58 IPK formed multimers in vivo: the N-terminal 166 aa were both necessary and sufficient for complex formation. A parallel in vivo analysis to map the K3L-binding region of PKR revealed that like P58 IPK , K3L interacted exclusively with the PKR protein kinase catalytic domain. In contrast, however, the K3L-binding region of PKR was localized to within aa 367 to 551, demonstrating that each inhibitor bound PKR in unique, nonoverlapping domains. These data, taken together, suggest that P58 IPK and K3L may mediate PKR inhibition by distinct mechanisms. Finally, we will propose a model of PKR inhibition in which P58 IPK or a P58 IPK complex binds PKR and interferes with nucleotide binding and autoregulation, while formation of a PKR-K3L complex interferes with active-site function and/or substrate association.

Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

“…As wild-type PKR is toxic to bacteria (3), we used PKR containing the K296R mutation, which is catalytically inactive yet retains the dimerization and protein-protein interaction characteristics of wt PKR (11,21,33,34,51,54,64). In vitro-translated, radiolabeled pTRS1 was incubated with GST alone or GST-PKRK296R, and protein binding was analyzed by SDS-PAGE and autoradiography.…”

Acute hepatitis C virus infection: A chronic problem

“…65 The cause of dysfunctional CD4 ϩ T cells in chronic HCV infection is not clear. However, exhaustion from continuous T cell stimulation, 83 the induction of T-cell anergy, 84 the induction of T regulatory cells that inhibit the immune responses, 85,86 and the sup-pression of immune responses by HCV proteins 48,[87][88][89][90][91][92][93] represent several intriguing hypotheses. Without sufficient HCV-specific CD4 help, HCV-specific CD8 ϩ T cell and heterologous neutralizing antibody responses may develop but fail to clear viremia.…”

Regulation of Interferon-Induced Protein Kinase PKR: Modulation of P58IPK Inhibitory Function by a Novel Protein, P52rIPK

“…Segregated pGADGH library plasmids were isolated from yeast colonies which failed to grow in the absence of tryptophan, transformed into Escherichia coli, and purified. Specificity of the yeast two-hybrid protein interaction was determined by introducing the recovered library plasmids into S. cerevisiae Hf7c harboring pBD-P58 IPK or control plasmids encoding the BD vector alone (pGBT9; Clontech), BD-simian virus 40 (SV40) T antigen (pTD1), or BD-lamin (pLAM5Ј) (31). BD-P58…”