Functional analysis of the roles of posttranslational modifications at the p53 C terminus in regulating p53 stability and activity.
Posttranslational modification of the tumor suppressor p53 plays important roles in regulating its stability and activity. Six lysine residues at the p53 C terminus can be posttranslationally modified by various mechanisms, including acetylation, ubiquitination, neddylation, methylation, and sumoylation. Previous cell line transfection studies show that ubiquitination of these lysine residues is required for ubiquitin-dependent degradation of p53. In addition, biochemical and cell line studies suggested that p53 acetylation at the C terminus might stabilize p53 and activate its transcriptional activities. To investigate the physiological functional outcome of these C-terminal modifications in regulating p53 stability and activity, we introduced missense mutations (lysine to arginine) at the six lysine residues (K6R) into the endogenous p53 gene in mouse embryonic stem (ES) cells. The K6R mutation prevents all posttranslational modifications at these sites but conserves the structure of p53. In contrast to conclusions of previous studies, analysis of p53 stability in K6R ES cells, mouse embryonic fibroblasts, and thymocytes showed normal p53 stabilization in K6R cells both before and after DNA damage, indicating that ubiquitination of these lysine residues is not required for efficient p53 degradation. However, p53-dependent gene expression was impaired in K6R ES cells and thymocytes in a promoter-specific manner after DNA damage, indicating that the net outcome of the posttranslational modifications at the C terminus is to activate p53 transcriptional activities after DNA damage.
Feng
Lijin
L
Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322, USA.
Lin
Tongxiang
T
Uranishi
Hiroaki
H
Gu
Wei
W
Xu
Yang
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D004249:DNA Damage / Q000528:radiation effects; D005786:Gene Expression Regulation; D016158:Genes, p53; D051379:Mice; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D013950:Thymus Gland / Q000166:cytology / Q000378:metabolism; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D001120:Arginine