Mechanism of activation of the double‐stranded‐RNA‐dependent protein kinase, PKR

Abstract

An important defense against viral infection involves inhibition of translation by PKR phosphorylation of the a subunit of eIF2. Binding of viral dsRNAs to two dsRNAbinding domains (dsRBDs) in PKR leads to relief of an inhibitory region and activation of eIF2 kinase activity. Interestingly, while deletion of the regulatory region of PKR significantly induces activity in vitro, the truncated kinase does not inhibit translation in vivo, suggesting that these sequences carry out additional functions required for PKR control. To delineate these functions and determine the order of events leading to activation of PKR, we fused truncated PKR to domains of known function and assayed the chimeras for in vivo activity. We found that fusion of a heterologous dimerization domain with the PKR catalytic domain enhanced autophosphorylation and eIF2 kinase function in vivo. The dsRBDs also mediate ribosome association and we proposed that such targeting increases the localized concentration of PKR, enhancing interaction between PKR molecules. We addressed this premise by linking the truncated PKR to RAS sequences mediating farnesylation and membrane localization and found that the fusion protein was functional in vivo. These results indicate that cellular localization along with oligomerization enhances interaction between PKR molecules. Alanine substitution for the phosphorylation site, threonine 446, impeded in vivo and in vitro activity of the PKR fusion proteins, while aspartate or glutamate substitutions partially restored the function of the truncated kinase. These results indicate that both dimerization and cellular localization play a role in transient protein±protein interactions and that transautophosphorylation is the final step in the mechanism of activation of PKR.

Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells

“…Values are a measure of a ratio of firefly vs. Renilla luciferase units (relative light units, RLU) and represent the mean values of three independent transfections. Immunoblot analysis of phosphorylated and total levels of eIF2␣ were carried out as described (24).…”

Dimerization and Release of Molecular Chaperone Inhibition Facilitate Activation of Eukaryotic Initiation Factor-2 Kinase in Response to Endoplasmic Reticulum Stress

“…Filters were washed three times in TBS-T solution, and the PEK-antibody complex was detected by enhanced chemiluminescence. Immunoblots measuring eIF2␣ phosphorylation were carried out with antibody that specifically recognizes phosphorylated eIF2 at Ser-51 (Research Genetics) (37). Total eIF2␣ in lysates was detected by immunoblot using monoclonal antibody generously provided by Dr. Scot Kimball (Pennsylvania State University, College of Medicine, Hershey, PA) that recognizes either phosphorylated or nonphosphorylated forms of this initiation factor.…”

“…Such oligomerization is facilitated by the cooperative binding of dsRNA between the dsRBDs, although protein-protein contacts may also participate. Additionally, we have proposed that association of PKR to ribosomes enhances the localized concentration of PKR required to facilitate dimerization (37,52). Binding of dsRNA to the dsRBDs also contributes to an activated conformational change in PKR involving the release of a proposed inhibitory interaction between a region flanking dsRBD-2 and the kinase catalytic domain (39,53).…”

“…In the example of PKR, phosphorylation occurs between polypeptides at multiple serine and threonine residues. Notably, Thr-446 and Thr-451 in the activation loop of the catalytic domain of PKR are a prerequisite for induced eIF2 kinase activity (37,54). Mass spectrometric analysis of PEK phosphorylated in vitro has identified 10 serine, threonine, and tyrosine residues in the kinase domain (35), including the activation loop residue Thr-980 that was specifically recognized by our phospho-PEK antibody (Fig.…”

Novel Therapeutic Approach for Breast Cancer

“…PKR is recognized as a mediator of antiviral and antitumor responses in target cells. Two dsRNAbinding domains reside in the NH 2 terminus, and interaction with dsRNA or other activators modifies the conformation of PKR allowing it to undergo autophosphorylation and activation [87][88][89]. Once activated, PKR is able to phosphorylate a variety of target substrates, the most well characterized being eIF-2α, which can lead to the inhibition of protein synthesis, growth suppression and induction of apoptosis [90][91][92].…”