Serine-15 phosphorylated p53 in complex with Mdm2 is inhibited from activating p53 effector pathways.

摘要

We have studied a previously unidentified pathway for the inactivation of wild type p53 by comparing the p53 response of two different cell lines; one that undergoes the expected tumor suppressor mediated response (ML-1) and one that does not (MANCA). Both the ML-1 and MANCA cell lines contain wild type p53. The signal transduction pathway causing accumulation of nuclear p53 protein is intact in both cell lines as treatment with camptothecin, etoposide, and mitomycin C cause p53 levels to increase. While the ML-1 p53 growth arrest and apoptosis pathways are functional, the same pathways in the MANCA cell line are impaired. The MANCA p53 protein is phosphorylated at Serine-15, however the p53 dependent induction of waf1, gadd45, mdm2, fas, and noxa are strikingly compromised. Surprisingly, there is extensive p53 DNA binding activity in the MANCA cells and this binding remains unchanged after DNA damage. We have found that MANCA cells have an abundance of the Mdm2 protein, and this same protein was barley detectable in the ML-1 cell line. Coimmunoprecipitation experiments with p53 and Mdm2 antibodies have shown a specific interaction with the MANCA p53 and Mdm2 proteins, even in the presence of DNA damage. Interestingly, there was no such interaction detected in the ML-1 cell line upon DNA damage. In the presence of DNA damage there was also a change in the phosphorylation state of the Mdm2 protein in the ML-1 cell line, and this same change was not seen in the MANCA cell line. Our data suggests a model in which the p53-Mdm2 protein-protein interaction in the MANCA cell line may render the p53 protein inactive. This complex may not allow for the transcriptional activation of p53 target genes mediating growth arrest and apoptosis.