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PKR activity modulation by phosphomimetic mutations of serine residues located three aminoacids upstream of double-stranded RNA binding motifs

ABSTRACT: Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), better known as PKR plays a key role in the response to viral infections and cellular homeostasis by regulating mRNA translation. Upon binding dsRNA, PKR is activated through homodimerization and subsequent autophosphorylation on Thr446 and Thr451 residues. In this study, we identified a novel PKR phosphorylation site, Ser6, located 3 aminoacids upstream the first double-stranded RNA binding domain (DRBM1). Another Ser residue occurs in PKR at position 97, the very same position relative to the DRBM2. Ser or Thr residues also occur 3 amino acids upstream DRBMs of other proteins such as ADAR1 or DICER. Phosphoinhibiting mutations (Ser-to-Ala) introduced at Ser6 and Ser97 spontaneously activated PKR. In contrast, phosphomimetic mutations (Ser-to-Asp) inhibited PKR activation following either poly (I:C) transfection or virus infection. These mutations moderately affected dsRNA binding, suggesting a model where negative charges occuring at position 6 and 97 tighten the interaction of DRBMs with the kinase domain, thus keeping PKR in an inactive, closed conformation even in the presence of dsRNA. This study provides new insights on PKR regulation mechanisms and identifies Ser6 and Ser97 as potential targets to modulate PKR activity for therapeutic purposes

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Didier Vertommen

LAB HEAD: Thomas Michiels

PROVIDER: PXD022564 | Pride | 2021-05-10

REPOSITORIES: Pride

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PKR activity modulation by phosphomimetic mutations of serine residues located three aminoacids upstream of double-stranded RNA binding motifs.

Cesaro Teresa T Hayashi Yohei Y Borghese Fabian F Vertommen Didier D Wavreil Fanny F Michiels Thomas T

Scientific reports 20210428 1

Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), better known as PKR, plays a key role in the response to viral infections and cellular homeostasis by regulating mRNA translation. Upon binding dsRNA, PKR is activated through homodimerization and subsequent autophosphorylation on residues Thr446 and Thr451. In this study, we identified a novel PKR phosphorylation site, Ser6, located 3 amino acids upstream of the first double-stranded RNA binding motif (DRBM1). Another Ser resi ...[more]

PMID: 33911136

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Project description:Sensing of double-stranded RNA (dsRNA) is an important component of innate immunity. Proteins like PKR and MDA5 recognize dsRNA and activate various pathways to fight viral infection. In addition to viral dsRNA, many endogenous RNAs containing double-stranded regions can be misrecognized as immunogenic. The RNA editing enzyme ADAR1, specifically its p150 isoform, has been shown to suppress activation of PKR and MDA5 through its A-to-I editing activity. While the cytoplasmic ADAR1-p150 isoform has been well established within this role, the functions of the nuclear ADAR1-p110 isoform are less understood. To address this knowledge gap, we utilized proximity labeling by APEX2 to identify putative ADAR1-p110 interacting proteins. We identified 110 proteins across three breast cancer cell lines that either interact with p110 or are within close proximity. Many of these proteins have known roles in RNA metabolism. Nine of the identified proteins belong to the DEAD or DEAH box families of RNA helicases, including DHX9. DHX9 is overexpressed in breast cancer, with expression closely correlated with that of p110. By co-immunoprecipitation we confirmed that p110 interacts with DHX9. Knockdown of DHX9 in several triple-negative breast cancer cell lines caused cell death and activation of the dsRNA sensor PKR. In two cell lines that are refractory to knockdown of ADAR1, the combined knockdown of DHX9 and ADAR1 caused a substantial increase in PKR activity, where knockdown of either alone had no effect. Knockdown of DHX9 and ADAR1 caused activation of the type I IFN pathway, RNase L and NF-KB signaling. Activation of these proteins and pathways was not seen with individual knockdown of ADAR1 or DHX9. Activation of PKR following combined knockdown of ADAR1 and DHX9 could be rescued by expression of p110, p150, DHX9, and catalytically inactive DHX9. Additionally PKR activation could be rescued by the dsRBM of DHX9, revealing an important role for dsRBMs in suppressing PKR activation. Together these results reveal an important role for DHX9 in suppressing dsRNA sensing by multiple pathways.

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