The B56α Regulatory Subunit of Protein Phosphatase 2A Is a Target for Regulation by Double-Stranded RNA-Dependent Protein Kinase PKR

Abstract

PKR is a cellular serine/threonine kinase that phosphorylates eukaryotic translation initiation factor 2α (eIF2α) to regulate protein synthesis. PKR also plays a role in the regulation of transcription, programmed cell death and the cell cycle, processes which likely involve other substrates. In a yeast two-hybrid screen, we isolated human protein phosphatase 2A (PP2A) regulatory subunit B56α as a PKR-interacting protein. The interaction between B56α and PKR was confirmed by in vitro binding assays as well as by in vivo coimmunoprecipitation, and this interaction is dependent on the catalytic activity of PKR. Moreover, recombinant B56α was efficiently phosphorylated by PKR in vitro and an isoelectric point shift in B56α was detected in extracts from cells induced with the PKR activator pIC. An in vitro dephosphorylation assay showed that when B56α was phosphorylated by PKR, the activity of PP2A trimeric holoenzyme was increased. A functional interaction between B56α and PKR was observed in cotransfection assays, where a B56α-mediated increase in luciferase expression was inhibited by cotransfection with wild-type PKR. This is likely due to a decreased level of eIF4E phosphorylation caused by an increase in PP2A activity following PKR phosphorylation of B56α. Taken together, our data indicate that PKR can modulate PP2A activity by phosphorylating B56α to regulate cellular activities.

ACKNOWLEDGMENTS

We thank Bruce Carpick for preparing the recombinant human wild-type PKR proteins, Ruorong Cai for the mutant PKR L362Q construct, and Kee-Chuan Goh for help with RNA assays.

This study is made possible by a grant from the National Institutes of Health (AI34039-02).