Impact of post-translational modifications of p53 on its mitochondrial localization

The tumor suppressor p53 is able to translocate to the mitochondria in stressed cells, where it interacts with BCL-2 family members to induce outer mitochondrial membrane (OMM) permeabilization and cytochrome c release. p53 transcriptional activity is regulated by a set of post-translational modifications (PTMs). How these modifications impact on p53 pro-apoptotic mitochondrial activity remains poorly understood. We determined the kinetic of p53 mitochondrial translocation in cancer cell lines exposed to genotoxic stresses. We observed that p53 mitochondrial localization was characterized by a bell-shaped kinetic curve, showing an early peak followed by a marked decrease of p53 level at later time points. Serine 392 phosphorylation of p53 influenced its mitochondrial translocation and transcription-independent apoptotic function. We used CRISPR/Cas9 genome editing to substitute serine 392 by a non-phosphorylatable alanine in HCT-116 carcinoma cells. The S392A mutant displayed normal transcriptional activity following a genotoxic stress but was markedly impaired for mitochondrial translocation. The lower ability of p53 S392A to localize to the mitochondria was correlated with reduced apoptosis. These observations were also confirmed using enforced expression of the S392A p53 mutant or a phospho-mimetic S392E mutant in H1299 carcinoma cells. During late apoptosis, the decrease of p53 mitochondrial level was correlated to a decreased of Serine 20 phosphorylation. Moreover, a p53 S20A mutant translocated well to mitochondria after a stress. However, its abundance at the mitochondria decreased rapidly over time by comparison to wt p53. The p53 S20A mutant was compromised for interaction with BAK, a major anchor for p53wt at the OMM. In conclusion, our data evidence that serine 392 and 20 regulate p53 pro-apoptotic activity at the mitochondria. More generally, our work highlights that PTMs modulate both p53 transcription-independent function and transcriptional activity.