Abstract 2042: The RAX/PACT-PKR stress-signaling pathway promotes p53 sumoylation and stability to induce G1 arrest

Abstract

Abstract Cellular stresses including IL-3 withdrawal from factor-dependent hematopoietic cells, inflammatory cytokine treatment or viral infection promote RAX/PACT-dependent activation of the interferon-induced, double-stranded RNA activated protein kinase PKR. Previously, we and others demonstrated that activated PKR phosphorylates the alpha subunit of eIF2 to inhibit protein synthesis and initiate apoptosis. In addition, recent reports indicate that PKR may regulate the transcription factors p53, STAT1 and NF-κB. Now we find that RAX/PACT interacts with the SUMO E2 ligase Ubc9 to stimulate p53-Ubc9 association and reversible p53 sumoylation on lysine 386. In addition, stable or transient expression of RAX/PACT in a variety of cell lines promotes p53 stability and activity to increase p53 target gene expression. Significantly, while expression of RAX/PACT, PKR or p53 alone has little effect on the cell cycle of p53-null H1299 cells, co-expression of p53 with either RAX/PACT or PKR promotes a 25 - 35% increase of cells in G1. In contrast, co-expression of RAX/PACT with the sumoylation-deficient p53 (K386R) mutant or with p53 and the desumoylase SENP1 fails to induce such a G1 arrest. Furthermore, RAX/PACT-induced, p53-dependent G1 growth arrest is inhibited by expressing the dominant-negative PKR (K296R) mutant, and expression of RAX/PACT in pkr+/+ but not pkr-/- MEF cells promotes p53 and p21 expression following gamma irradiation. Significantly, expression of exogenous RAX/PACT promotes phosphorylation of wild-type but not p53 (K386R) on serine 392, and p53 stability is decreased in cells with reduced RAX/PACT or PKR following doxorubicin treatment. Collectively, results indicate that in response to stress the RAX/PACT-PKR signaling pathway may promote p53 translational activation leading to G1 cell cycle arrest by a mechanism dependent on p53 sumoylation and phosphorylation that regulates protein turnover. Importantly, these novel findings may indicate a wider role for the RAX/PACT-PKR signaling pathway to direct sumoylation-dependent subcellular localization, protein stability or protein-protein interactions of downstream targets in response to cellular stress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2042. doi:1538-7445.AM2012-2042