Src phosphorylation converts Mdm2 from a ubiquitinating to a neddylating E3 ligase

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 6; pp. 1749 - 1754
• Main Authors: Batuello, Christopher N., Hauck, Paula M., Gendron, Jaimie M., Lehman, Jason A., Mayo, Lindsey D.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 10.02.2015; National Acad Sciences
• Subjects: Animals; Biological Sciences; Blotting, Western; Cell growth; Cell Line; Enzymes; Humans; Immunoprecipitation; Mass Spectrometry; Mice; NEDD8 Protein; Phosphorylation; Proteins; Proto-Oncogene Proteins c-mdm2 - metabolism; Signal Transduction - physiology; src-Family Kinases - metabolism; Tumors; Ubiquitination; Ubiquitins - metabolism; Mdm2; Src; Nedd8
• DOI: 10.1073/pnas.1416656112
• ISSN: 0027-8424

Murine double minute-2 protein (Mdm2) is a multifaceted phosphorylated protein that plays a role in regulating numerous proteins including the tumor suppressor protein p53. Mdm2 binds to and is involved in conjugating either ubiquitin or Nedd8 (Neural precursor cell expressed, developmentally down-regulated 8) to p53. Although regulation of the E3 ubiquitin activity of Mdm2 has been investigated, regulation of the neddylating activity of Mdm2 remains to be defined. Here we show that activated c-Src kinase phosphorylates Y281 and Y302 of Mdm2, resulting in an increase in Mdm2 stability and its association with Ubc12, the E2 enzyme of the neddylating complex. Mdm2-dependent Nedd8 conjugation of p53 results in transcriptionally inactive p53, a process that is reversed with a small molecule inhibitor to either Src or Ubc12. Thus, our studies reveal how Mdm2 may neutralize and elevate p53 in actively proliferating cells and also provides a rationale for using therapies that target the Nedd8 pathway in wild-type p53 tumors. Significance Conjugation of large polypeptides such as Neural precursor cell expressed, developmentally down-regulated 8 (Nedd8) and ubiquitin to proteins is a critical regulatory process for normal cell function. There are more than 1,000 E3 ligases that are involved in the ubiquitin pathway. The predominant activity of one E3, murine double minute-2 protein (Mdm2), has been characterized to mediate ubiquitination of the tumor suppressor p53 in response to genotoxic stress. We show that under growth conditions, active Src kinase binds to and phosphorylates Mdm2 at Y281 and Y302, which recruits the Nedd8 E2 enzyme, Ubc12. This recruitment converts Mdm2 to an E3 that neddylates p53. We provide the first evidence, to our knowledge, showing how phosphorylation may redirect ligase activity. This mechanism may be applicable to numerous E3 ligases and provides a basis for therapeutic intervention.