THE TUMOR-SUPPRESSOR P53 AND THE ONCOPROTEIN SIMIAN VIRUS-40 T-ANTIGEN BIND TO OVERLAPPING DOMAINS ON THE MDM2 PROTEIN

The oncogene mdm2 has been found to be amplified in human sarcomas, an d the gene product binds to the tumor suppressor p53. In this report, we describe the dissection of the MDM2-binding domain on p53 as well a s the p53-binding domain on MDM2. We also demonstrate that the oncopro tein simian virus 40 T antigen binds to the product of cellular oncoge ne mdm2. We have constructed several N- and C-terminal deletion mutant s of p53 and MDM2, expressed them in vitro, and assayed their in vitro association capability. The N-terminal boundary of the p53-binding do main on MDM2 is between amino acids 1 and 58, while the C-terminal bou ndary is between amino acids 221 and 155. T antigen binds to an overla pping domain on the MDM2 protein. On the other hand, the MDM2-binding domain of p53 is defined by amino acids 1 and 159 at the N terminus. A t the C terminus, binding is progressively reduced as amino acids 327 to 145 are deleted. We determined the effect of human MDM2 on the tran sactivation ability of wild-type human p53 in the Saos-2 osteosarcoma cell line, which does not have any endogenous p53. Human MDM2 inhibite d the ability of human p53 to transactivate the promoter with p53-bind ing sites. Thus, human MDM2 protein, like the murine protein, can inac tivate the transactivation ability of human p53. Interestingly, both t he transactivation domain and the MDM2-binding domain of p53 are situa ted near the N terminus. We further show that deletion of the N-termin al 58 amino acids of MDM2, which eliminates p53 binding, also abolishe s the capability of inactivating p53-mediated transactivation. This fi nding suggests a correlation of in vitro p53-MDM2 binding with MDM2's ability in vivo to interfere with p53-mediated transactivation.