N-terminal 130 amino acids of MDM2 are sufficient to inhibit p53-mediated transcriptional activation.
The human oncoprotein MDM2 binds with the tumor suppressor p53 and inhibits p53-directed transactivation. In this report we show that deletion of 336 amino acids from the C-terminus of human MDM2 does not decrease its efficiency to bind p53 in vivo and inhibit p53-directed transactivation. Even further deletion of MDM2 from the C-terminus up to amino acid 131 does not reduce its ability to inhibit p53-mediated transactivation. Since deletion up to amino acid 131 also deletes many antigenic sites of MDM2 and the truncated protein cannot be immunoprecipitated by the antibodies available to us, two internal deletions were made to define the p53-interaction domain. Internal deletion of four amino acids beginning at 110 residue (amino acids 110 to 113) did not reduce p53-binding or inhibition of p53-directed transactivation whereas internal deletion of amino acids 60 to 65 reduces but does not abolish these activities. Sequential deletion of amino acids from the N-terminus leads to sequential destruction of p53-binding and inhibition of transactivation capability of MDM2. Fourteen amino acids can be deleted from this end without any reduction of these activities. Deletion of 28 N-terminal amino acids residues drastically reduces, but does not abolish the p53-binding ability of the protein, as well as inhibition of p53-directed transactivation. Deletion of 58 amino acids from the N-terminus of the oncoprotein abolishes its ability to bind p53 in vivo and to inhibit p53-directed transactivation. These results locate the p53-binding domain of MDM2 within amino acids 14 to 154 and inhibition of transactivation domains of MDM2 within amino acid residues 14 to 130 suggesting possible p53-independent biological functions of the 491 amino acid long oncoprotein.
Leng
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Department of Microbiology, University of Texas Health Science Center at San Antonio 78284-7758, USA.
Brown
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Shivakumar
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D006801:Humans; D066298:In Vitro Techniques; D009687:Nuclear Proteins; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D051736:Proto-Oncogene Proteins c-mdm2; D011994:Recombinant Proteins; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*
D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2