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Double-stranded-RNA-activated protein kinase PKR enhances transcriptional activation by tumor suppressor p53
1Departments of Oncology and Medicine, McGill University, Montreal, Quebec.
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 gamma 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.
Insights
The double-stranded-RNA-activated protein PKR enhances tumor suppressor p53
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The tumor suppressor p53 is crucial for cell cycle arrest and apoptosis after DNA damage.
- PKR (double-stranded-RNA-activated protein kinase) is an interferon-inducible kinase regulating gene expression.
- Existing evidence suggests crosstalk between interferon-inducible proteins and p53.
Purpose of the Study:
- To investigate if and how PKR modulates p53 function.
- To analyze the impact of PKR deficiency on p53-mediated transcriptional activity and cell cycle arrest.
Main Methods:
- Utilized PKR+/+ and PKR-/- mouse embryonic fibroblasts (MEFs).
- Transfected cells with a temperature-sensitive mutant of mouse p53 [p53(Val135)].
- Assessed p53 transactivation, G0/G1 arrest, and p53 phosphorylation at Ser18 following DNA damage (adriamycin, gamma radiation, UV radiation) and PI-3 kinase inhibition.
Main Results:
- p53-mediated transactivation and G0/G1 arrest were impaired in PKR-/- cells.
- PKR deficiency led to defective p53 phosphorylation at Ser18 and reduced induction of p53-target genes (p21WAF/Cip1, Mdm2).
- DNA damage-induced Ser18 phosphorylation and transcriptional activation by p53 were diminished in PKR-/- cells, particularly after adriamycin or gamma radiation, but not UV radiation. PI-3 kinase inhibition affected p53 phosphorylation differently in PKR+/+ versus PKR-/- cells.
Conclusions:
- PKR enhances the transcriptional function of p53.
- PKR is implicated in the cell signaling pathways activated by specific types of DNA damage leading to p53 phosphorylation, potentially involving the PI-3 kinase pathway.