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 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.
Buschmann
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T
Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029, USA.
Lerner
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D
Lee
C G
CG
Ronai
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D016475:3T3 Cells; D000818:Animals; D002461:Cell Line, Transformed; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D051379:Mice; D009687:Nuclear Proteins; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D025842:SUMO-1 Protein / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D014466:Ultraviolet Rays; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D025842:SUMO-1 Protein; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; C497206:MDM2 protein, human; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9