RAX Activates Tumor Suppressor p53.

Recently, our laboratory identified RAX as a unique cellular activator for the interferon-induced double-stranded RNA-dependent protein kinase, PKR. Cellular stresses such as IL-3 withdrawal from factor dependent hematopoietic cells, inflammatory cytokine and chemotherapy treatment or viral infection promote RAX phosphorylation with activation of PKR leading to inhibition of new protein synthesis. In addition, PKR can also regulate the transcription factors p53, STAT1 and NF-kB. Now we report a novel activity for RAX in regulating the expression and activity of the tumor suppressor, p53. Results indicate that increased RAX expression in p53 expressing cells, such as HEK293, mouse embryo fibroblast (MEF) or human osteosarcoma U2OS promotes an increase in the steady-state level of endogenous p53 and its activation of the p53 target gene, p21. Furthermore, when RAX is expressed in U2OS cells or coexpressed with p53 in p53 null H1299 cells, enhanced, dose-dependent p53 transcriptional activity is observed as assessed using a p53 luciferase reporter. Consistent with these findings, flow cytometry demonstrates that either RAX or PKR can stimulate p53-dependent G1 arrest that precedes apoptosis. Since p53 is a major ‘sensor’ for DNA damage leading to cell cycle arrest and apoptosis, we also examined whether RAX enhances p53’s response to DNA damage. Following gamma irradiation or Cisplatin treatment of p53 expressing cells, expression of exogenous RAX augments while ‘knock down’ of endogenous RAX by siRNA or expression of the dominant-negative, RAX (S18A) mutant, potently blocks any increase in p53 and p21 expression compared to vector-only control cells. While we find no evidence that RAX directly interacts with p53, results indicate that RAX promotes p53 SUMOylation but does not affect p53 ubiquitination or interaction with MDM2. We now propose that RAX augments the p53 stress-dependent cellular response potentially by a PKR-dependent mechanism involving SUMOylation. Since p53 is only mutated in apparently 15% of hematologic malignancies versus more than 50% in solid tumors, it is envisioned that a more detailed understanding of the mechanism by which RAX activates p53 may serve as a basis for novel antileukemic strategies.