Double-Stranded-RNA-Activated Protein Kinase PKR Enhances Transcriptional Activation by Tumor Suppressor p53

Abstract

The tumor suppressor p53 plays a key role in inducing G₁ arrest and apoptosis following DNA damage. The double-stranded-RNA-activated protein PKR is a serine/threonine interferon (IFN)-inducible kinase which plays an important role in regulation of gene expression at both transcriptional and translational levels. Since a cross talk between IFN-inducible proteins and p53 had already been established, we investigated whether and how p53 function was modulated by PKR. We analyzed p53 function in several cell lines derived from PKR^+/+ and PKR^−/− mouse embryonic fibroblasts (MEFs) after transfection with the temperature-sensitive (ts) mutant of mouse p53 [p53(Val135)]. Here we report that transactivation of transcription by p53 and G₀/G₁ arrest were impaired in PKR^−/− cells upon conditions that ts p53 acquired a wild-type conformation. Phosphorylation of mouse p53 on Ser¹⁸ was defective in PKR^−/− cells, consistent with an impaired transcriptional induction of the p53-inducible genes encoding p21^WAF/Cip1 and Mdm2. In addition, Ser¹⁸ phosphorylation and transcriptional activation by mouse p53 were diminished in PKR^−/− cells after DNA damage induced by the anticancer drug adriamycin or γ radiation but not by UV radiation. Furthermore, the specific phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 inhibited the induction of phosphorylation of Ser¹⁸ of p53 by adriamycin to a higher degree in PKR^+/+ cells than in PKR^−/− cells. These novel findings suggest that PKR enhances p53 transcriptional function and implicate PKR in cell signaling elicited by a specific type of DNA damage that leads to p53 phosphorylation, possibly through a PI-3 kinase pathway.

ACKNOWLEDGMENTS

We thank Y.-L. Yang and C. Weissmann for PKR^+/+ and PKR^−/− MEFs; B. Vogelstein for the tsmutant p53(Val135) and mouse p21^WAF1/Cip1 cDNAs; A. Levine for BALB/c (10)1 cells, mdm2 cDNA, and anti-Mdm2 MAbs 2A10 and 4B11; C. Midgley and D. Lane for wild-type mouse p53 and mouse p53 Ser¹⁸-Ala cDNAs; K. McDonald for flow cytometry analysis; S. Lehnert for assistance with the γ-irradiation experiments; S. Kirchhoff and H. Hauser for communicating results before publication; and J. Th’ng and C. Couture for helpful discussions and suggestions.

This work was supported by research grants from the National Cancer Institute of Canada (NCIC), Medical Research Council (MRC) of Canada, and The Cancer Research Society (CRS) Inc. to A.E.K. A.R.C. is a recipient of a CRS studentship, and A.H.-T.W. is a recipient of a NCIC Terry Fox research award. A.E.K. is a member of the Terry Fox Group in Molecular Oncology and an MRC Scientist awardee.