Regulation of PKR by RNA: formation of active and inactive dimers.
PKR is a member of the eIF2α family of protein kinases that inhibit translational initiation in response to stress stimuli and functions as a key mediator of the interferon-induced antiviral response. PKR contains a dsRNA binding domain that binds to duplex regions present in viral RNAs, resulting in kinase activation and autophosphorylation. An emerging theme in the regulation of protein kinases is the allosteric linkage of dimerization and activation. The PKR kinase domain forms a back-to-back parallel dimer that is implicated in activation. We have developed a sensitive homo-Förster resonance energy transfer assay for kinase domain dimerization to directly probe the relationship among RNA binding, activation, and dimerization. In the case of perfect duplex RNAs, dimerization is correlated with activation and dsRNAs containing 30 bp or more efficiently induce kinase domain dimerization and activation. However, more complex duplex RNAs containing a 10-15 bp 2'-O-methyl RNA barrier produce kinase dimers but do not activate. Similarly, inactivating mutations within the PKR dimer interface that disrupt key electrostatic and hydrogen binding interactions fail to abolish dimerization. Our data support a model in which activating RNAs induce formation of a back-to-back parallel PKR kinase dimer whereas nonactivating RNAs either fail to induce dimerization or produce an alternative, inactive dimer configuration, providing an additional mechanism for distinguishing between host and pathogen RNA.
Department of Molecular and Cell Biology and ‡Department of Chemistry, University of Connecticut , Storrs, Connecticut 06269, United States.
Bushra
B
Husain
Department of Molecular and Cell Biology and ‡Department of Chemistry, University of Connecticut , Storrs, Connecticut 06269, United States.
Stephen
S
Hesler
Department of Molecular and Cell Biology and ‡Department of Chemistry, University of Connecticut , Storrs, Connecticut 06269, United States.
James L
JL
Cole
D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008745:Methylation; D008958:Models, Molecular; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D019892:eIF-2 Kinase / Q000737:chemistry / Q000378:metabolism*
D012330:RNA, Double-Stranded; D012313:RNA; D019892:eIF-2 Kinase