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

Teresa Cesaro,Thomas Michiels,Yohei Hayashi,Fabian Borghese,Didier Vertommen,Fanny Wavreil

doi:10.1038/s41598-021-88610-z

Teresa Cesaro, Thomas Michiels + Show 4 more

Open Access

https://doi.org/10.1038/s41598-021-88610-z

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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 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 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 or dimerization, suggesting a model where negative charges occurring 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.

Highlights

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
A phosphorylatable residue is found at the same position (− 3) relative to the dsRBD in other human proteins such ADAR1 (DRBM2), TRBP (DRBM2), and DICER
Aberrant PKR activation may be detrimental to the host as PKR activity typically leads to mRNA translation blockade and to cell apoptosis

Summary

Introduction

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. We identified a novel PKR phosphorylation site, Ser[6], located 3 amino acids upstream of the first double-stranded RNA binding motif (DRBM1). Another Ser residue occurs in PKR at position 97, the very same position relative to the DRBM2. Phosphomimetic mutations (Ser-to-Asp) inhibited PKR activation following either poly (I:C) transfection or virus infection These mutations moderately affected dsRNA binding or dimerization, suggesting a model where negative charges occurring at position 6 and 97 tighten the interaction of DRBMs with the kinase domain, keeping PKR in an inactive closed conformation even in the presence of dsRNA. PKR stays in a so-called closed conformation, in which the second DRBM6 is tightly interacting with the kinase domain, hiding the kinase substrate-binding pocket and thereby preventing its random activation[7]

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