Determinants of C16-Ceramide Binding to p53

The tumor suppressor p53 coordinates cellular stress responses, but underlying mechanisms remain incompletely understood. We previously demonstrated that, in response to metabolic stress, C16-ceramide produced by ceramide synthase 6 (CerS6) directly binds to p53, thus preventing its MDM2-mediated degradation and promoting p53 activation. Here, we investigated the structural requirements and functional consequences of ceramide binding to p53. Using a panel of p53 mutants, including naturally occurring oncogenic variants, we characterized the ceramide-binding interface of p53 and the role of amino acid substitutions within this region in metabolic stress signaling. We found that disruption of ceramide binding impaired stress-induced p53–CerS6 interaction at the endoplasmic reticulum (ER), attenuated induction of p53 target genes, and reduced cellular sensitivity to stress. Of note, certain cancer-associated p53 mutants retained ceramide binding and the ability to activate stress responses. These studies were further extended to monitoring the p53–CerS6 interaction on the ER and associated membrane aggregation using fluorescence techniques. We showed that metabolic stress-induced ER remodeling was distinct from the canonical UPR. Overall, our study defines the ceramide-binding surface within the p53 DNA-binding domain and provides novel insight into the functional role of the ceramide–p53 interaction.