Signaling by the integrated stress response kinase PKR is fine-tuned by dynamic clustering

Abstract

The double-stranded RNA sensor kinase PKR is one of four integrated stress response (ISR) sensor kinases that phosphorylate the alpha subunit of the eukaryotic initiation factor 2 (eIF2α) in response to stress. The current model of PKR activation considers the formation of back-to-back PKR dimers as a prerequisite for signal propagation. Here we show that PKR signaling involves the assembly of dynamic PKR clusters. PKR clustering is driven by ligand binding to PKR’s sensor domain and by front-to-front interfaces between PKR’s kinase domains. PKR clusters are discrete, heterogeneous, autonomous coalescences that share some protein components with processing bodies. Strikingly, eIF2α is not recruited to PKR clusters, and PKR cluster disruption enhances eIF2α phosphorylation. Together, these results support a model in which PKR clustering buffers downstream signaling, which may enable proofreading the ISR.

APOBEC3B drives PKR-mediated translation shutdown and protects stress granules in response to viral infection

“…5H) Thus, A3B has a critical function in regulating PKR during infection with different types of RNA viruses. Finally, we asked whether A3B impacts PKR-dsRNA condensates that are formed when dsRNAs are present in the cytoplasm 80,81 . We transfected cells with poly(I:C)-FITC to monitor PKR colocalization with dsRNAs and we confirmed that PKR formed large foci that are associated with poly(I:C) (Supplementary Fig.…”

Optogenetic control of the integrated stress response reveals proportional encoding and the stress memory landscape

“…We chose to engineer optogenetic control over PKR for two reasons. First, PKR's activation mechanism involves high-order self-association (Corbet et al, 2022;Mayo et al, 2019;Zappa et al, 2021), which can be mimicked using optogenetic clustering tools. Second, PKR is cytoplasmically localized and its sensor domain is isolated to a contiguous stretch of amino acids on the polypeptide chain, making it easier to engineer in comparison to the other ISR kinases (Cole, 2007;Zhu et al, 1997).…”

The integrated stress response induces a common cell-autonomous death receptor 5-dependent apoptosis switch

“…ISRIB (Sigma-Aldrich), 50 µM Z-VAD-FMK (SelleckChem), or 1 µg/ml FcDR5 (R&D systems) as indicated. FKBP-PKR was activated with 100 nM of the homodimerization ligand AP20187 (Takara), as previously described (Zappa et al, 2022).…”

“…Pseudoclonal cell populations were obtained by fluorescence-activated cell sorting using a narrow gate placed over the mean of the signal distribution as previously described (Zappa et al, 2022).…”

“…To dissect the molecular circuitry exclusive to the terminal ISR and avoid the pleiotropic effects of stress-inducing agents, we employed a chemical-genetics approach consisting of an engineered ISR sensor kinase, FKBP-PKR, which can be activated with a small molecule ligand to induce the ISR (Zappa et al, 2022). Unlike the classical ISR stress-inducing agents mentioned above, FKBP-PKR allows a precise, stress-free activation of a "pure" ISR.…”