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The contribution of the RING finger domain of MDM2 to cell cycle progression
M Argentini1, N Barboule, B Wasylyk
1Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Illkirch, France.
Abstract:
The MDM2 oncoprotein binds to p53 and abrogates p53-mediated G1 arrest and apoptosis. We show that MDM2 over-expression accelerates cell cycle progression of RPM12650 cells by overcoming the negative effect of endogenous wild type p53 at the G1/S checkpoint. The interaction with p53 and transcription inhibition are necessary but not sufficient. The RING finger domain of MDM2 is also required for the positive effect of MDM2 on the cell cycle. Surprisingly, several point mutants in the zinc binding sites of the RING finger are fully competent for cell cycle stimulation even though they abolish MDM2-directed degradation of p53 and MDM2 E3-ligase activity. In contrast, alterations in and around the cryptic nucleolar localization sequence (KR motif) inhibit MDM2-mediated cell cycle progression as well as p53 degradation and MDM2 E3 ligase activity. We found that all the RING mutants decrease inhibition of both p53 dependent reporters and endogenous p21CIP1/WAF1/SDI1. These results indicate that the RING finger of MDM2 has a role in the regulation of the cell cycle that is independent of p53 degradation and endogenous p21CIP1/WAF1/SDI1 regulation.
Insights
MDM2 oncoprotein accelerates cell cycle progression by interacting with p53. Its RING finger domain is crucial for this effect, independent of p53 degradation or p21 regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- MDM2 oncoprotein inhibits p53 tumor suppressor activity.
- MDM2 binds to p53, preventing G1 arrest and apoptosis.
- Overexpression of MDM2 is implicated in various cancers.
Purpose of the Study:
- To investigate the role of MDM2 in cell cycle regulation.
- To determine the specific domains of MDM2 required for cell cycle progression.
- To elucidate the relationship between MDM2's E3 ligase activity, p53 degradation, and cell cycle effects.
Main Methods:
- Overexpression of wild-type and mutant MDM2 in RPM12650 cells.
- Cell cycle analysis using flow cytometry.
- Western blotting to assess p53 and p21 levels.
- Reporter assays to measure p53 transcriptional activity.
- Analysis of MDM2 E3 ligase activity and p53 degradation.
Main Results:
- MDM2 overexpression accelerates cell cycle progression at the G1/S checkpoint.
- The RING finger domain of MDM2 is essential for promoting cell cycle progression.
- Mutants in the RING finger's zinc-binding sites retain cell cycle-stimulating activity despite lacking p53 degradation ability.
- Alterations in the cryptic nucleolar localization sequence (KR motif) impair MDM2's cell cycle effects, p53 degradation, and E3 ligase activity.
- RING mutants reduce the inhibition of p53-dependent reporters and endogenous p21CIP1/WAF1/SDI1.
Conclusions:
- MDM2 promotes cell cycle progression through mechanisms independent of its E3 ligase activity and p53 degradation.
- The RING finger domain plays a critical role in MDM2-mediated cell cycle regulation.
- The KR motif is important for both MDM2's E3 ligase function and its cell cycle-promoting activity.