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Cyclin a-CDK phosphorylation regulates MDM2 protein interactions
1Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Abstract:
The product of the MDM2 gene interacts with and regulates a number of proteins, in particular the tumor suppressor p53. The MDM2 protein is likely to be extensively modified in vivo, and such modification may regulate its functions in cells. We identified a potential cyclin-dependent kinase (CDK) site in murine MDM2, and found the protein to be efficiently phosphorylated in vitro by cyclin A-containing complexes (cyclin A-CDK2 and cyclin A-CDK1), but MDM2 was either weakly or not phosphorylated by other cyclin-containing complexes. Moreover, a peptide containing a putative MDM2 cyclin recognition motif specifically inhibited phosphorylation by cyclin A-CDK2. The site of cyclin A-CDK2 phosphorylation was identified as Thr-216 by two-dimensional phosphopeptide mapping and mutational analysis. Phosphorylation of MDM2 at Thr-216 both weakens its interaction with p53 and modestly augments its binding to p19(ARF). Interestingly, an MDM2-specific monoclonal antibody, SMP14, cannot recognize MDM2 phosphorylated at Thr-216. Changes in SMP14 reactivity of MDM2 in staged cell extracts indicate that phosphorylation of MDM2 at Thr-216 in vivo is most prevalent at the onset of S phase when cyclin A first becomes detectable.
Insights
MDM2 protein phosphorylation by cyclin A-CDK2 at Thr-216 weakens its interaction with p53 and alters its binding to p19ARF. This modification, prevalent during S phase, affects MDM2 antibody recognition.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- Oncogenesis
Background:
- MDM2 protein regulates the tumor suppressor p53.
- Post-translational modifications of MDM2 influence its cellular functions.
- Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle.
Purpose of the Study:
- To investigate the role of MDM2 phosphorylation in regulating its interaction with p53 and p19ARF.
- To identify the specific kinase responsible for MDM2 phosphorylation and the site of modification.
- To correlate MDM2 phosphorylation with cell cycle progression.
Main Methods:
- In vitro kinase assays using murine MDM2 and various cyclin-CDK complexes.
- Peptide inhibition assays to identify cyclin recognition motifs.
- Two-dimensional phosphopeptide mapping and mutational analysis to pinpoint phosphorylation sites.
- Western blot analysis using a specific monoclonal antibody (SMP14) to detect phosphorylated MDM2 in cell extracts.
Main Results:
- MDM2 is efficiently phosphorylated by cyclin A-CDK2 and cyclin A-CDK1, but not by other cyclin-containing complexes.
- Phosphorylation occurs at Thr-216, weakening MDM2 binding to p53 and enhancing binding to p19ARF.
- Phosphorylation at Thr-216 prevents recognition by the MDM2-specific antibody SMP14.
- In vivo, Thr-216 phosphorylation is most prominent at the start of S phase, coinciding with cyclin A expression.
Conclusions:
- Cyclin A-CDK2-mediated phosphorylation of MDM2 at Thr-216 is a key regulatory mechanism.
- This phosphorylation event modulates the interaction of MDM2 with its binding partners p53 and p19ARF.
- MDM2 phosphorylation at Thr-216 serves as a cell cycle-dependent marker, detectable during S phase.