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Updated: Aug 14, 2026
Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle
Published on: March 5, 2019
Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity
Abstract:
The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha), inhibiting the function of the eIF2 complex and continued initiation of translation. When bound to an activating RNA and ATP, PKR undergoes autophosphorylation reactions at multiple serine and threonine residues. This autophosphorylation reaction stimulates the eIF2alpha kinase activity of PKR. The binding of certain viral RNAs inhibits the activation of PKR. Wild-type PKR is obtained as a highly phosphorylated protein when overexpressed in Escherichia coli. We report here that treatment of the isolated phosphoprotein with the catalytic subunit of protein phosphatase 1 dephosphorylates the enzyme. The in vitro autophosphorylation and eIF2alpha kinase activities of the dephosphorylated enzyme are stimulated by addition of RNA. Thus, inactivation by phosphatase treatment of autophosphorylated PKR obtained from overexpression in bacteria generates PKR in a form suitable for in vitro analysis of the RNA-induced activation mechanism. Furthermore, we used gel mobility shift assays, methidiumpropyl-EDTA.Fe footprinting and affinity chromatography to demonstrate differences in the RNA-binding properties of phospho- and dephosphoPKR. We found that dephosphorylation of PKR increases binding affinity of the enzyme for both kinase activating and inhibiting RNAs. These results are consistent with an activation mechanism that includes release of the activating RNA upon autophosphorylation of PKR prior to phosphorylation of eIF2alpha.
Insights
Dephosphorylating RNA-dependent protein kinase (PKR) enhances its RNA binding affinity, facilitating studies on RNA-induced activation mechanisms crucial for translation control.
Area of Science:
- Biochemistry
- Molecular Biology
- Virology
Background:
- RNA-dependent protein kinase (PKR) is an interferon-induced enzyme regulating translation initiation.
- PKR activation involves autophosphorylation upon binding RNA and ATP, leading to eIF2alpha phosphorylation and translation inhibition.
- Viral RNAs can inhibit PKR activation, highlighting its role in antiviral defense.
Purpose of the Study:
- To investigate the role of phosphorylation in PKR's RNA-binding properties and activation mechanism.
- To develop a method for generating active PKR from overexpressed bacterial sources for in vitro studies.
- To elucidate the differences in RNA binding between phosphorylated and dephosphorylated PKR.
Main Methods:
- Overexpression of wild-type PKR in E. coli and subsequent purification of the phosphoprotein.
- Dephosphorylation of PKR using the catalytic subunit of protein phosphatase 1.
- In vitro assays for autophosphorylation and eIF2alpha kinase activity.
- Gel mobility shift assays, methidiumpropyl-EDTA.Fe footprinting, and affinity chromatography to assess RNA binding.
Main Results:
- Dephosphorylation of overexpressed PKR by protein phosphatase 1 generated an active enzyme.
- Dephosphorylated PKR showed stimulated autophosphorylation and eIF2alpha kinase activity upon RNA addition.
- Dephosphorylation significantly increased PKR's binding affinity for both activating and inhibiting RNAs.
- These findings suggest a model where RNA release follows PKR autophosphorylation before eIF2alpha phosphorylation.
Conclusions:
- Phosphatase treatment provides a method to generate active PKR from bacterial overexpression systems for mechanistic studies.
- Dephosphorylation enhances PKR's affinity for RNA, suggesting a conformational change regulating its activity.
- The results support a model where autophosphorylation-induced conformational changes modulate RNA binding and subsequent kinase activity, impacting translational control.
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