Expression of p53KRKKK enhances mitochondrial translocation of p38 MAPK and phosphorylation of HtrA2/Omi in Ras-transformed cells. (A) NIH3T3 cells and p53^−/− MEFs were infected with a control or Ha-RasV12–expressing retrovirus. After subcellular fractionation of the cytosol (Cyto) and mitochondria (Mito), the distribution of p53 was evaluated by immunoblotting. Black arrowhead indicates nonspecific band (N.S.). COX IV and α-tubulin were used as mitochondrial and cytosolic markers, respectively. (B–F) p53^−/− MEFs were infected with a Ha-RasV12–expressing retrovirus together with a control or p53KRKKK-expressing retrovirus. (B, top) DIC images of cells. Cell was stained for nucleus with DAPI (middle) or for p53 (bottom). Bar, 20 µm. (C) After subcellular fractionation of the cytosol and mitochondria, the distribution of p38 MAPK was evaluated by immunoblotting. (D) The level of phosphorylated HtrA2/Omi in the mitochondrial fraction was evaluated by immunoblotting. (E) Inverted invasion assays were performed. Images of serial Z-sections (10-µm intervals). Bars, 100 µm. (F) Cells invaded 30 µm or more were quantified from image in E. Data represent the mean ± SD; n = 3. *, P < 0.01. (G) Schematic of p53-mediated suppression of Ras-driven invasion via HtrA2/Omi. After Ras transformation, concurrent events of mitochondria fission and p53-mediated activation of HtrA2/Omi induced by p38 MAPK in mitochondria leads to the release of activated HtrA2/Omi. Consequentially, actin polymerization and p130Cas phosphorylation are inhibited, resulting in the suppression of invasion.