The p53-CerS6 interaction on the ER integrates p53 and sphingolipid signaling pathways in metabolic stress response

Ceramides, a diverse class of bioactive sphingolipids, play a pivotal role in cellular stress response. We have previously reported a novel mechanism by which a particular member of this class, C₁₆-ceramide, activates death pathways in cancer cells. In this mechanism, C₁₆-ceramide, generated by ceramide synthase 6 (CerS6), binds directly to the tumor suppressor p53 and protects it from MDM2-mediated degradation, thereby promoting a stress response. In the present study, we investigated the mechanism by which p53 acquires ceramide, a highly hydrophobic molecule. Using bimolecular fluorescence complementation, we show that in cells under metabolic stress, p53 is recruited to the cytoplasmic surface of the ER by CerS6, the ER resident enzyme generating C₁₆-ceramide. The direct contact between the two proteins enables the transfer of C₁₆-ceramide from CerS6 to the DNA-binding domain of p53. Domain deletion experiments and pulldown assays with purified recombinant cytoplasmic fragments of CerS6 showed that the p53 physically interacts with the two large cytoplasmic loops of CerS6, forming a complex on the surface of the ER membrane. The interaction between p53 and CerS6 strictly requires the presence of C₁₆-ceramide in the catalytic site of the enzyme. Substitution of the CerS6 lumenal loop, which defines the enzyme's acyl chain specificity, with the corresponding loop of CerS2, which generates very-long-chain ceramides, abolished the interaction with p53. Our results uncover a previously unknown process, the direct C₁₆-ceramide transfer to the p53 DNA-binding domain at the ER surface, which is a mechanism regulating activation of this tumor suppressor. The findings that this mechanism is triggered upon metabolic stress induction by specific chemotherapeutics, as well as by nutrient and growth factor deprivation, highlight it as a promising target for cancer treatment.