MDM2-C Functions as an E3 Ubiquitin Ligase

BACKGROUND: Mouse double minute 2 (MDM2) is an E3 ubiquitin ligase that is over-expressed in many cancers and regulates target proteins through ubiquitination. Full-length MDM2 (MDM2-FL) is best known for targeting wild-type p53 for degradation by the proteasome, but the functions of the many splice variants of MDM2 are under-explored. The three well-studied alternative MDM2 isoforms are MDM2-A/ALT2, MDM2-B/ALT1, and MDM2-C/ALT3. MDM2-A and MDM2-B are capable of down-regulating MDM2-FL activity and have transforming activity in cancers with mutant p53. The MDM2 isoform MDM2-C is over-expressed in breast cancer and correlates with decreased survival in the context of mutant p53 expression. Therefore, MDM2-C requires further study to determine if it has biochemical activities similar to MDM2-FL. Hypothesis: We hypothesized that like MDM2-FL, the MDM2-C isoform (lacking exons 5–9 and containing a full C-terminal RING finger sequence) would maintain E3 ubiquitin ligase activity. MATERIALS AND METHODS: In order to explore the biochemical function of MDM2-C, we used an in vitro ubiquitination assay and a glutaraldehyde cross-linking assay. RESULTS: Here we report, for the first time, that MDM2-C has E3 auto-ubiquitin ligase activity, which can promote ubiquitination of wild-type p53 and mutant p53 R273H, and also can form a protein–protein interaction with p53 proteins. CONCLUSION: This information strongly positions MDM2-C as a protein with biochemical activities that may explain the varied outcomes observed in patients with high-level expression of MDM2-C in the presence of wild-type p53 versus mutant p53.