Pseudosubstrate Inhibition of Protein Kinase PKR by Swine Pox Virus C8L Gene Product

Self Cite

As part of the mammalian cell innate immune response, the doublestranded RNA activated protein kinase PKR phosphorylates the translation initiation factor eIF2␣ to inhibit protein synthesis and thus block viral replication. Poxviruses including vaccinia and smallpox viruses express PKR inhibitors such as the vaccinia virus K3L protein that resembles the N-terminal substrate-targeting domain of eIF2␣. Whereas high-level expression of human PKR was toxic in yeast, this growth inhibition was suppressed by coexpression of the K3L protein.We used this yeast assay to screen for PKR mutants that are resistant to K3L inhibition, and we identified 12 mutations mapping to the C-terminal lobe of the PKR kinase domain. The PKR mutations specifically conferred resistance to the K3L protein both in yeast and in vitro. Consistently, the PKR-D486V mutation led to nearly a 15-fold decrease in K3L binding affinity yet did not impair eIF2␣ phosphorylation. Our results support the identification of the eIF2␣-binding site on an extensive face of the C-terminal lobe of the kinase domain, and they indicate that subtle changes to the PKR kinase domain can drastically impact pseudosubstrate inhibition while leaving substrate phosphorylation intact. We propose that these paradoxical effects of the PKR mutations on pseudosubstrate vs. substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2␣ around the Ser-51 phosphorylation site.eIF2 ͉ translational control

mentioning

confidence: 69%

Section: Screen To Identify Pkr Mutants Resistant To Inhibition By Thmentioning

confidence: 99%

Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein

Seo

Liu

Kawagishi-Kobayashi

et al. 2008

Self Cite

“…Elucidating the mechanism by which PKR function is subverted by viral products has enhanced our understanding of how PKR and other eIF2α family kinases naturally function. In particular, viral mimicry appeared as a recurring theme: analysis of the pseudosubstrate inhibitor K3L from vaccinia virus revealed key molecular determinants required for PKR recognition of its natural substrate eIF2α (10)(11)(12); analysis of the RNA binding protein E3L from vaccinia virus revealed how PKR itself senses dsRNA through comparable infrastructure (13)(14)(15); and analysis of the RNA inhibitor VAI from adenovirus showed how a structured RNA decoy can competitively engage the dsRNA-binding domains of PKR in a manner that circumvents kinase domain activation (16)(17)(18).…”

Section: Significancementioning

confidence: 99%

Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe

Cao

Fixsen

et al. 2015

Self Cite

Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by eIF2α family kinases is a conserved mechanism to limit protein synthesis under specific stress conditions. The baculovirus-encoded protein PK2 inhibits eIF2α family kinases in vivo, thereby increasing viral fitness. However, the precise mechanism by which PK2 inhibits eIF2α kinase function remains an enigma. Here, we probed the mechanism by which PK2 inhibits the model eIF2α kinase human RNA-dependent protein kinase (PKR) as well as native insect eIF2α kinases. Although PK2 structurally mimics the C-lobe of a protein kinase domain and possesses the required docking infrastructure to bind eIF2α, we show that PK2 directly binds the kinase domain of PKR (PKR KD ) but not eIF2α. The PKR KD -PK2 interaction requires a 22-residue N-terminal extension preceding the globular PK2 body that we term the "eIF2α kinase C-lobe mimic" (EKCM) domain. The functional insufficiency of the N-terminal extension of PK2 implicates a role for the adjacent EKCM domain in binding and inhibiting PKR. Using a genetic screen in yeast, we isolated PK2-activating mutations that cluster to a surface of the EKCM domain that in bona fide protein kinases forms the catalytic cleft through sandwiching interactions with a kinase N-lobe. Interaction assays revealed that PK2 associates with the N-but not the C-lobe of PKR KD . We propose an inhibitory model whereby PK2 engages the N-lobe of an eIF2α kinase domain to create a nonfunctional pseudokinase domain complex, possibly through a lobe-swapping mechanism. Finally, we show that PK2 enhances baculovirus fitness in insect hosts by targeting the endogenous insect heme-regulated inhibitor (HRI)-like eIF2α kinase. viral mimicry | lobe-swap | HRI | eIF2α kinase inhibition | PKR E ukaryotic cells possess diverse mechanisms for molecular adaptation to stress conditions. Eukaryotic translation initiation factor 2α (eIF2α) kinases are evolutionarily conserved enzymes that detect specific stress signals and induce changes in translation to produce an adaptive response. The four eIF2α kinases in humans possess divergent regulatory domains that enable response to different stress signals: (i) the RNA-dependent protein kinase (PKR) senses viral infection to mediate antiviral immunity; (ii) the PKR-like endoplasmic reticulum kinase (PERK) detects unfolded proteins in the endoplasmic reticulum to regulate the unfolded protein response; (iii) the heme-regulated inhibitor kinase (HRI) senses free heme to coordinate globin synthesis in red blood cells; and (iv) the general control nonrepressible-2 kinase (GCN2) detects amino acid insufficiencies to regulate metabolite homeostasis (1). Although eIF2α kinases respond to different stress stimuli, their protein kinase domains are highly similar and allow transmission of an overlapping signal that potently inhibits cellular translation. Upon stress-induced activation, eIF2α kinases phosphorylate a common cellular substrate, eIF2α, at a conserved site corresponding to Ser51 in human eIF2α. eIF2 is a...

“…Vaccinia virus uses two strategies to evade PKR. First is expression of E3L, which binds and masks dsRNAs (26). The second is expression of K3L, which binds and inhibits PKR via its ability to mimic the natural substrate of this enzyme, eIF2␣ (26).…”

Section: Rnai In Embryonic Stem Cellsmentioning

confidence: 99%

Stable suppression of gene expression by RNAi in mammalian cells

Paddison

Caudy²,

Hannon³

2002

491

290

In a diverse group of organisms including plants, Caenorhabditis elegans, Drosophila, and trypanosomes, double-stranded RNA (dsRNA) is a potent trigger of gene silencing. In several model systems, this natural response has been developed into a powerful tool for the investigation of gene function. Use of RNA interference (RNAi) as a genetic tool has recently been extended to mammalian cells, being inducible by treatment with small, Ϸ22-nt RNAs that mimic those produced in the first step of the silencing process. Here, we show that some cultured murine cells specifically silence gene expression upon treatment with long dsRNAs (Ϸ500 nt). This response shows hallmarks of conventional RNAi including silencing at the posttranscriptional level and the endogenous production of Ϸ22-nt small RNAs. Furthermore, enforced expression of long, hairpin dsRNAs induced stable gene silencing. The ability to create stable ''knock-down'' cell lines expands the utility of RNAi in mammalian cells by enabling examination of phenotypes that develop over long time periods and lays the groundwork for by using RNAi in phenotype-based, forward genetic selections.