Protein-protein interactions of the p53 family with the Bcl-2 family in hepatocellular carcinoma

Bcl-2 family proteins serve as critical regulators in apoptosis, either promoting or inhibiting cell death via the mitochondrial apoptosis signaling pathway. Wildtype p53 promotes apoptosis via transcription-dependent and transcription-independent mechanisms. The latter may be mediated by direct translocation of p53 to the mitochondria where it leads to outer mitochondrial membrane permeabilization (OMMP) by forming an inhibitory complex with the anti-apoptotic Bclxl protein.

We investigated direct protein-protein interactions (PPI) of the members of the p53 family (p53, p63 and p73) with different members of the Bcl-2 family to further characterize p53 family-mediated transcription-independent mitochondrial apoptosis signalling pathways.

We therefore adapted LUMIER technology (LUminescence-based Mammalian IntERactome mapping), an automated HTP technology for the systemic mapping of PPI networks in mammalian cells for the use in liver cells (Hep3B). The LUMIER-assay is based on the co-purification of two tagged proteins from transiently transfected cells using GATEWAY-compatible ORF clones. We selected a set of apoptosis-relevant proteins including anti-apoptotic members of the Bcl-2 family (Bcl2, Bclxl, Bag1 and Mcl1) but also pro-apoptotic members like Bax, Bid and Bak. Positive interactions with members of the p53 family were verified by co-immunoprecipitations.

We identified several new interactions of the p53 family with the Bcl-2 family. We demonstrated that Bclxl does not only interact with p53 but also with p63 and p73. Furthermore, another Bcl2 family member, Mcl1, revealed – so far undescribed – interactions with p53, p63 and p73. Such interactions of pro-apoptotic p53 family members with anti-apoptotic Bcl-2 family members may lead to OMPP. These data imply that p63 and p73– like p53– partly achieve their pro-apoptotic function at the mitochondria by facilitating interaction between Bclxl and BH3-only proteins.

In conclusion, we adapted LUMIER technology to directly map protein-protein interactions networks in a hepatocellular carcinoma cell line. We identified new and direct protein-protein interactions of p53, p63 and p73 with several members of the Bcl-2 family which may regulate the mitochondrial membrane.