The Mdm-2 Amino Terminus Is Required for Mdm2 Binding and SUMO-1 Conjugation by the E2 SUMO-1 Conjugating Enzyme Ubc9

Covalent attachment of SUMO-1 to Mdm2 requires the activation of a heterodimeric Aos1-Uba2 enzyme (ubiquitin-activating enzyme (E1)) followed by the conjugation of Sumo-1 to Mdm2 by Ubc9, a protein with a strong sequence similarity to ubiquitin carrier proteins (E2s). Upon Sumo-1 conjugation, Mdm2 i...

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Bibliographic Details
Published in:The Journal of biological chemistry 2001-11, Vol.276 (44), p.40389-40395
Main Authors: Buschmann, Thomas, Lerner, Dimitri, Lee, Chee-Gun, Ronai, Ze'ev
Format: Article
Language:English
Subjects:
3T3 Cells
Animals
Cell Line, Transformed
Humans
Ligases - metabolism
Mice
Nuclear Proteins
Protein Binding
Proto-Oncogene Proteins - chemistry
Proto-Oncogene Proteins - metabolism
Proto-Oncogene Proteins c-mdm2
SUMO-1 Protein - metabolism
Tumor Suppressor Protein p53 - metabolism
Ubiquitin - metabolism
Ubiquitin-Conjugating Enzymes
Ultraviolet Rays
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Summary:Covalent attachment of SUMO-1 to Mdm2 requires the activation of a heterodimeric Aos1-Uba2 enzyme (ubiquitin-activating enzyme (E1)) followed by the conjugation of Sumo-1 to Mdm2 by Ubc9, a protein with a strong sequence similarity to ubiquitin carrier proteins (E2s). Upon Sumo-1 conjugation, Mdm2 is protected from self-ubiquitination and elicits greater ubiquitin-protein isopeptide ligase (E3) activity toward p53, thereby increasing its oncogenic potential. Because of the biological implication of Mdm2 sumoylation, we mapped Ubc9 binding on Mdm2. Here we demonstrate that Ubc9 can associate with Mdm2 only if amino acids 40–59 within the N terminus of Mdm2 are present. Mdm2 from which amino acids 40–59 have been deleted can no longer be sumoylated. Furthermore, addition of a peptide that corresponds to amino acids 40–59 on Mdm2 to a sumoylation reaction efficiently inhibits Mdm2 sumoylation in vitro and in vivo. In UV-treated cells Mdm2 exhibits reduced association with Ubc9, which coincides with decreased Mdm2 sumoylation. Our findings regarding the association of Ubc9 with Mdm2, and the effect of UV-irradiation on Ubc9 binding, point to an additional level in the regulation of Mdm2 sumoylation under normal growth conditions as well as in response to stress conditions.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M103786200