Cyclin a-CDK phosphorylation regulates MDM2 protein interactions.
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.
Zhang
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Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Prives
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D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D019925:Cyclin A / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000737:chemistry* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005982:Glutathione Transferase / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins; D010449:Peptide Mapping; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D025803:Tumor Suppressor Protein p14ARF
D000911:Antibodies, Monoclonal; D019925:Cyclin A; D009687:Nuclear Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D025803:Tumor Suppressor Protein p14ARF; D013912:Threonine; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D005982:Glutathione Transferase; D018844:Cyclin-Dependent Kinases