Association of p19(ARF) with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor suppressor p53.
We have demonstrated previously that the oncoprotein Mdm2 has a ubiquitin ligase activity for the tumor suppressor p53 protein. In the present study, we characterize this ubiquitin ligase activity of Mdm2. We first demonstrate the ubiquitination of several p53 point mutants and deletion mutants by Mdm2. The point mutants, which cannot bind to Mdm2, are not ubiquitinated by Mdm2. The ubiquitination of the C-terminal deletion mutants, which contain so-called Mdm2-binding sites, is markedly decreased, compared with that of wild-type p53. The binding of Mdm2 to p53 is essential for ubiquitination, but p53's tertiary structure and/or C-terminal region may also be important for this reaction. DNA-dependent protein kinase is known to phosphorylate p53 on Mdm2-binding sites, where DNA damage induces phosphorylation, and p53 phosphorylated by this kinase is not a good substrate for Mdm2. This suggests that DNA damage-induced phosphorylation stabilizes p53 by inhibiting its ubiquitination by Mdm2. We further investigated whether the tumor suppressor p19(ARF) affects the ubiquitin ligase activity of Mdm2 for p53. The activity of p19(ARF)-bound Mdm2 was found to be lower than that of free Mdm2, suggesting that p19(ARF) promotes the stabilization of p53 by inactivating Mdm2.
Honda
R
R
School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Yasuda
H
H
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008025:Ligases / Q000037:antagonists & inhibitors*; D009154:Mutation; D009687:Nuclear Proteins; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D014452:Ubiquitins; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D008025:Ligases