Destabilizing missense mutations in the tumour suppressor protein p53 enhance its ubiquitination in vitro and in vivo.
p53 ubiquitination catalysed by MDM2 (murine double minute clone 2 oncoprotein) provides a biochemical assay to dissect stages in E3-ubiquitin-ligase-catalysed ubiquitination of a conformationally flexible protein. A mutant form of p53 (p53(F270A)) containing a mutation in the second MDM2-docking site in the DNA-binding domain of p53 (F270A) is susceptible to modification of long-lived and high-molecular-mass covalent adducts in vivo. Mutant F270A is hyperubiquitinated in cells as defined by immunoprecipitation and immunoblotting with an anti-ubiquitin antibody. Transfection of His-tagged ubiquitin along with p53(R175H) or p53(F270A) also results in selective hyperubiquitination in cells under conditions where wild-type p53 is refractory to covalent modification. The extent of mutant p53(R175H) or p53(F270A) unfolding in cells as defined by exposure of the DO-12 epitope correlates with the extent of hyperubiquitination, suggesting a link between substrate conformation and E3 ligase function. The p53(F270A:6KR) chimaeric mutant (where 6KR refers to the simultaneous mutation of lysine residues at positions 370, 372, 373, 381, 382 and 386 to arginine) maintains the high-molecular-mass covalent adducts and is modified in an MDM2-dependent manner. Using an in vitro ubiquitination system, mutant p53(F270A) and the p53(F270A:6KR) chimaeric mutant is also subject to hyperubiquitination outwith the C-terminal domain, indicating direct recognition of the mutant p53 conformation by (a) factor(s) in the cell-free ubiquitination system. These data identify an in vitro and in vivo assay with which to dissect how oligomeric protein conformational alterations are linked to substrate ubiquitination in cells. This has implications for understanding the recognition of misfolded proteins during aging and in human diseases such as cancer.
Cancer Research UK Cell Signalling Unit, p53 Signal Transduction Group, University of Edinburgh, South Crewe Road, Edinburgh EH4 2XR, Scotland, UK.
Harumi
H
Shimizu
David
D
Saliba
Maura
M
Wallace
Lee
L
Finlan
Patrick R R
PR
Langridge-Smith
Ted R
TR
Hupp
D020816:Amino Acid Motifs; D045744:Cell Line, Tumor; D002474:Cell-Free System; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D009154:Mutation; D020125:Mutation, Missense; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D025801:Ubiquitin / Q000378:metabolism*
D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D051736:Proto-Oncogene Proteins c-mdm2