Abstract 1090: The RAX-PKR signaling axis enhances p53-mediated cell cycle arrest by a SUMOylation-dependent mechanism
Authors: Bennett Richard L. , Pan Yu , Hui Teng , Kerney Krystal R. , May W. Stratford 
Published: February 2011

Cellular stresses including IL-3 withdrawal from factor-dependent hematopoietic cells, inflammatory cytokine treatment or viral infection promote RAX-dependent activation of the double-stranded RNA dependent protein kinase PKR. Previously, our laboratory and others have demonstrated that upon activation PKR phosphorylates the alpha subunit of eIF2 to inhibit protein synthesis and initiate apoptosis. In addition, recent findings also indicate that PKR may regulate the transcription factors p53, STAT1 and NF-κB. Now, we report that stable or transient expression of RAX in a variety of cell lines induces increased protein stability and transcriptional activity of p53 to promote cell cycle arrest. Significantly, reporter assays demonstrate a RAX-dosage dependent increase in p53 activity by as much as 5 fold compared to control cells. Furthermore, western blotting and qPCR indicate that exogenous RAX expression promotes increased levels of the p53 downstream target p21 in normal but not in p53-/- MEF cells. Significantly, genotoxic damage by cisplatin or gamma irradiation is additive to RAX for upregulation of p53 and p21. Consistent with p53's effect on the cell cycle, expression of exogenous RAX promotes an almost 2-fold increase in the G1 population of MEF cells compared to control cells. Furthermore, while the expression of RAX, PKR or p53 alone has little effect on the cell cycle of H1299 cells, co-expression of p53 with either RAX or PKR promotes a 25 − 35% increase in the population of cells in G1. Importantly, RAX-induced p53 activation is PKR dependent since exogenous RAX expression fails to promote upregulation of p53 or p21 in pkr-/- cells even following gamma irradiation. Furthermore, RAX-dependent p53 activation can be inhibited by expression of the dominant negative RAX(S18A) mutant, and siRNA knockdown of endogenous RAX reduces p21 expression by 5 fold following stress. RAX expression does not affect p53-MDM2 interaction, p53 ubiquitination, or the level of p53 mRNA. Mechanistically, RAX interacts and co-localizes with the SUMO E2 conjugating enzyme, Ubc9, to promote p53 SUMOylation on lysine 386 in a RAX-dosage dependent manner. This is followed by G1 cell cycle arrest which can be reversed by the desumoylase SENP1. In addition, co-expression of RAX with the SUMOylation deficient p53(K386R) mutant inhibits RAX-induced, p53-dependent G1 growth arrest. Significantly, phosphorylation of p53(K386R) on serine 392 is also reduced compared to wt p53, indicating that SUMOylation of p53 may be necessary for p53 phosphorylation on serine 392. Taken together, our findings indicate that RAX-PKR stress signaling is a modulator of the p53 response, RAX-induced p53 SUMOylation may facilitate p53 tumor suppression activity, and functional crosstalk between RAX-PKR dependent translation inhibition and p53-dependent cell cycle arrest may occur in response to stress.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1090.