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, and 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 as the p53-binding domain on MDM2. We also demonstrate that the oncoprotein simian virus 40 T antigen binds to the product of cellular oncogene mdm2. We have constructed several N- and C-terminal deletion mutants of p53 and MDM2, expressed them in vitro, and assayed their in vitro association capability. The N-terminal boundary of the p53-binding domain on MDM2 is between amino acids 1 and 58, while the C-terminal boundary is between amino acids 221 and 155. T antigen binds to an overlapping 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. At the C terminus, binding is progressively reduced as amino acids 327 to 145 are deleted. We determined the effect of human MDM2 on the transactivation ability of wild-type human p53 in the Saos-2 osteosarcoma cell line, which does not have any endogenous p53. Human MDM2 inhibited the ability of human p53 to transactivate the promoter with p53-binding sites. Thus, human MDM2 protein, like the murine protein, can inactivate the transactivation ability of human p53. Interestingly, both the transactivation domain and the MDM2-binding domain of p53 are situated near the N terminus. We further show that deletion of the N-terminal 58 amino acids of MDM2, which eliminates p53 binding, also abolishes the capability of inactivating p53-mediated transactivation. This finding suggests a correlation of in vitro p53-MDM2 binding with MDM2's ability in vivo to interfere with p53-mediated transactivation.
Brown
D R
DR
Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.
Deb
S
S
Muñoz
R M
RM
Subler
M A
MA
Deb
S P
SP
D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017931:DNA Primers; D017353:Gene Deletion; D015870:Gene Expression; D016158:Genes, p53; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000378:metabolism*; D009687:Nuclear Proteins; D009857:Oncogenes; D012516:Osteosarcoma; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins; D051736:Proto-Oncogene Proteins c-mdm2; D013539:Simian virus 40 / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D017931:DNA Primers; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D015500:Chloramphenicol O-Acetyltransferase; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2