The conformationally flexible S9-S10 linker region in the core domain of p53 contains a novel MDM2 binding site whose mutation increases ubiquitination of p53 in vivo.
Although the N-terminal BOX-I domain of the tumor suppressor protein p53 contains the primary docking site for MDM2, previous studies demonstrated that RNA stabilizes the MDM2.p53 complex using a p53 mutant lacking the BOX-I motif. In vitro assays measuring the specific activity of MDM2 in the ligand-free and RNA-bound state identified a novel MDM2 interaction site in the core domain of p53. As defined using phage-peptide display, the RNA.MDM2 isoform exhibited a notable switch in peptide binding specificity, with enhanced affinity for novel peptide sequences in either p53 or small nuclear ribonucleoprotein-U (snRNP-U) and substantially reduced affinity for the primary p53 binding site in the BOX-I domain. The consensus binding site for the RNA.MDM2 complex within p53 is SGXLLGESXF, which links the S9-S10 beta-sheets flanking the BOX-IV and BOX-V motifs in the core domain and which is a site of reversible conformational flexibility in p53. Mutation of conserved amino acids in the linker at Ser(261) and Leu(264), which bridges the S9-S10 beta-sheets, stimulated p53 activity from reporter templates and increased MDM2-dependent ubiquitination of p53. Furthermore, mutation of the conserved Phe(270) within the S10 beta-sheet resulted in a mutant p53, which binds more stably to RNA.MDM2 complexes in vitro and which is strikingly hyper-ubiquitinated in vivo. Introducing an Ala(19) mutation into the p53(F270A) protein abolished both RNA.MDM2 complex binding and hyper-ubiquitination in vivo, thus indicating that p53(F270A) protein hyper-ubiquitination depends upon MDM2 binding to its primary site in the BOX-I domain. Together, these data identify a novel MDM2 binding interface within the S9-S10 beta-sheet region of p53 that plays a regulatory role in modulating the rate of MDM2-dependent ubiquitination of p53 in cells.
Shimizu
Harumi
H
Department of Molecular and Cellular Pathology, The Cancer Research UK Laboratories, The University of Dundee, Dundee DD1 9SY, Scotland.
Burch
Lindsay R
LR
Smith
Amanda J
AJ
Dornan
David
D
Wallace
Maura
M
Ball
Kathryn L
KL
Hupp
Ted R
TR
D000483:Alleles; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D016158:Genes, p53; D006801:Humans; D007150:Immunohistochemistry; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins; D019151:Peptide Library; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*; D025801:Ubiquitin / Q000378:metabolism*
D008024:Ligands; D009687:Nuclear Proteins; D019151:Peptide Library; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D012694:Serine; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D007930:Leucine