Molecular and Cellular Biology, July 2000, p. 5285-5299, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Regulatory Subunit of Protein
Phosphatase 2A Is a Target for Regulation by Double-Stranded
RNA-Dependent Protein Kinase PKR
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195,1 and Department of Genetics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 441062
Received 5 October 1999/Returned for modification 16 November 1999/Accepted 14 April 2000
PKR is a cellular serine/threonine kinase that phosphorylates
eukaryotic translation initiation factor 2
(eIF2
) 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 B56
as a PKR-interacting protein. The interaction between B56
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 B56
was efficiently
phosphorylated by PKR in vitro and an isoelectric point shift in B56
was detected in extracts from cells induced with the PKR activator pIC.
An in vitro dephosphorylation assay showed that when B56
was
phosphorylated by PKR, the activity of PP2A trimeric holoenzyme was
increased. A functional interaction between B56
and PKR was observed
in cotransfection assays, where a B56
-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
B56
. Taken together, our data indicate that PKR can modulate PP2A
activity by phosphorylating B56
to regulate cellular activities.
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