MDM2 E3 ligase activity is essential for p53 regulation and cell cycle integrity

MDM2 and MDM4 are key regulators of p53 and function as oncogenes when aberrantly expressed. MDM2 and MDM4 partner to suppress p53 transcriptional transactivation and polyubiquitinate p53 for degradation. The importance of MDM2 E3-ligase-mediated p53 regulation remains controversial. To resolve this, we generated mice with an Mdm2 L466A mutation that specifically compromises E2 interaction, abolishing MDM2 E3 ligase activity while preserving its ability to bind MDM4 and suppress p53 transactivation. Mdm2(L466A/L466A) mice exhibit p53-dependent embryonic lethality, demonstrating MDM2 E3 ligase activity is essential for p53 regulation in vivo. Unexpectedly, cells expressing Mdm2(L466A) manifest cell cycle G2-M transition defects and increased aneuploidy even in the absence of p53, suggesting MDM2 E3 ligase plays a p53-independent role in cell cycle regulation and genome integrity. Furthermore, cells bearing the E3-dead MDM2 mutant show aberrant cell cycle regulation in response to DNA damage. This study uncovers an uncharacterized role for MDM2's E3 ligase activity in cell cycle beyond its essential role in regulating p53's stability in vivo. Author summaryThe most frequently mutated protein in human cancer, the p53 tumor suppressor protein, is negatively regulated by the potentially oncogenic proteins MDM2 and MDM4. MDM2/MDM4 regulates p53 through two mechanisms, MDM2 E3 ubiquitin ligase activity marks p53 for degradation while MDM2/MDM4 can bind p53 to inhibit its ability to promote RNA transcription. Whether these mechanisms contribute to normal p53 regulation in vivo remains controversial. Using a newly developed mouse model that genetically separates these two mechanisms, we find that mice expressing MDM2 deficient specifically for E3 ubiquitin ligase activity do not survive embryonic development because unregulated p53 is lethal. In contrast to prior reports, MDM2 E3 ubiquitin ligase activity is thus required for p53 regulation during embryonic development. In addition, cells lacking MDM2 E3 ubiquitin ligase activity have cell cycle defects regardless of p53 status, uncovering a p53-independent function for MDM2 in regulating the cell cycle. Activating p53 by blocking physical interaction with MDM2/MDM4 is one currently pursued approach for cancer therapy, but this approach does not account for cancer-promoting activities of MDM2/MDM4 independent of p53. Findings reported here suggest targeting MDM2 E3 ligase activity directly may be advantageous as it would inhibit both p53-dependent and p53-independent oncogenic mechanisms.