The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation, leading to G₁ arrest.
Cellular stresses, including growth factor deprivation, inflammatory cytokines or viral infection promote RAX/PACT-dependent activation of the double-stranded RNA-dependent protein kinase, PKR, to phosphorylate eIF2α, resulting in translation inhibition and apoptosis. In addition, PKR has been reported to regulate p53, STAT1 and NFκB. Here, we report that RAX/PACT interacts with the SUMO E2 ligase Ubc9 to stimulate p53-Ubc9 association and reversible p53 sumoylation on lysine 386. In addition, expression of RAX/PACT in a variety of cell lines promotes p53 stability and activity to increase p53 target gene expression. Significantly, while the 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 the desumoylase SENP1 fails to induce such a G₁ arrest. Furthermore, co-expression of p53, RAX/PACT and the dominantnegative PKR(K296R) mutant inhibits RAX/PACT-induced, p53-dependent G₁ growth arrest and expression of RAX/PACT in pkr(+/+) but not pkr(-/-) MEF cells promotes p53 and p21 expression following gamma irradiation. Significantly, p53 stability is decreased in cells with reduced RAX/PACT or PKR following doxorubicin treatment, and expression of exogenous RAX/ PACT promotes phosphorylation of wild-type but not p53(K386R) on serine 392. Collectively, results indicate that, in response to stress, the RAX/PACT-PKR signaling pathway may inhibit p53 protein turnover by a sumoylation-dependent mechanism with promotion of p53 phosphorylation and translational activation leading to G₁ cell cycle arrest.
Department of Medicine, Division of Hematology and Oncology, University of Florida, Gainesville, FL, USA.
Richard L
RL
Bennett
Yu
Y
Pan
Jaime
J
Christian
Teng
T
Hui
W Stratford
WS
May
D045744:Cell Line, Tumor; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism*; D059585:G1 Phase Cell Cycle Checkpoints; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D016601:RNA-Binding Proteins / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D013312:Stress, Physiological; D058207:Sumoylation; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D019892:eIF-2 Kinase / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D015852:Eukaryotic Initiation Factor-2; D005136:Eye Proteins; D018398:Homeodomain Proteins; C104647:PRKRA protein, human; C486393:RAX protein, human; D016601:RNA-Binding Proteins; C495901:TP53 protein, human; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D044763:Ubiquitin-Conjugating Enzymes; D019892:eIF-2 Kinase; D000098843:Ubiquitin-Conjugating Enzyme UBC9