The Mdm-2 amino terminus is required for Mdm2 binding and SUMO-1 conjugation by the E2 SUMO-1 conjugating enzyme Ubc9.

Thomas Buschmann,Dimitri Lerner,Chee-Gun Lee,Ze'Ev Ronai

doi:10.1074/jbc.m103786200

Thomas Buschmann, Dimitri Lerner + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m103786200

Copy DOI

Abstract

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.

Highlights

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)
Conjugation of ubiquitin or ubiquitin-like proteins is mediated by multiple enzymatic reactions catalyzed by a single ubiquitin-activating enzyme (E1),1 a few ubiquitin carrier proteins (E2s), and when applicable, by a large variety of ubiquitin-protein isopeptide ligases (E3s)
Ubc9 Binding Is Mapped to the Mdm2 Amino-terminal Domain—As noted, binding of Ubc9, which serves as an E2conjugating enzyme for SUMO-1 [19], is prerequisite to Mdm2 sumoylation

Summary

Introduction

Covalent attachment of SUMO-1 to Mdm requires the activation of a heterodimeric Aos1-Uba enzyme (ubiquitin-activating enzyme (E1)) followed by the conjugation of Sumo-1 to Mdm by Ubc, a protein with a strong sequence similarity to ubiquitin carrier proteins (E2s). Within the amino-terminal helix both structural and sequence alignments do not match to ubiquitin-conjugating enzymes due to one mismatched amino acid, which confers a different recognition surface on Ubc9 [12, 13] This together with a profound change in the electrostatic surface are likely to contribute to the recognition of selective substrates in the SUMO-1 conjugation pathways [12, 13]. A growing list of proteins was shown to exhibit Ubc binding followed by SUMO-1 conjugation, the implication of this modification to the function of the proteins is not yet clear. Among such proteins are Fas (Apo-1/CD95; Ref. 33), Werner syndrome protein [34] ATF2 [35], c-Jun [36], poly(ADPribose) polymerase [37], and viral proteins including bovine papillomavirus E1 [38], the cytomegalovirus 1E2-p86 [39], and the adenovirus E1A [40]

Methods

Results

Conclusion