Functional analysis of the roles of posttranslational modifications at the p53 C terminus in regulating p53 stability and activity

Abstract

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.

FIG. 1.

Generation of p53K6R ES cells. (A) Genomic configuration of the endogenous p53 genes in AY ES cells. The AY cell line has one wild-type p53 allele and one mutant p53 allele (AY allele) with exons 2 to 4 replaced with a LoxP site. The filled boxes represent p53 exons, and the filled bar represents the probe for Southern blot analysis. The 14-kb germ line EcoRI fragment and 6-kb EcoRI fragment of AY allele are indicated. (B) Targeting construct. The PGK-Neo^r gene flanked by LoxP sites was inserted into intron 7. The K6R mutation within exon 11 is indicated by an asterisk. (C) Targeted configuration after homologous recombination between the wild-type p53 allele and the targeting vector. The 9.8-kb mutant EcoRI fragment is shown. The positions of the primer sets used to screen for LoxP/Cre-mediated deletion are shown by arrowheads. (D) p53K6R knock-in allele. The size of the mutant EcoRI fragment is indicated. (E) Southern blotting analysis of the genomic DNA derived from targeted AY ES cells before PGK-Neo^r deletion. Genomic DNA was digested with EcoRI and hybridized to the probe. The positions of EcoRI fragments derived from the germ line, AY allele, and targeted allele are indicated by arrows. Lane 1, AY ES cells; lane 2, targeted AY ES cells. (F) Southern blot analysis of genomic DNA from wild-type ES cells (lane 1), AY ES cells (lane 2), targeted AY ES cells before LoxP/Cre deletion (lane 3), and p53K6R knock-in ES cells after deletion of the PGK-Neo^r gene (lane 4). Genomic DNA was digested with EcoRI and hybridized to the probe. The positions of the 14-kb germ line, 12.5-kb PGK-Neo^r-deleted, 9.8-kb PGK-Neo^r-inserted, and 6-kb AY EcoRI fragments are indicated.

FIG. 2.

p53 responses to DNA damage in p53K6R ES cells. (A) Induction of p53 in AY and p53K6R ES cells. ES cells were cultured on gelatinized plates in the presence of LIF but without feeder layer cells before UV radiation. Cell extracts were prepared from AY and p53K6R ES cells at the indicated time points after 60 J/m² UV-C irradiation and analyzed for p53 protein levels or p53 phosphorylation levels at Ser18 and Ser389. Genotypes of ES cells are shown on top, and p53 and actin are indicated on the right. (B) Ubiquitination levels of p53 in p53K6R ES cells. Western blot analysis of the ubiquitination levels of p53 immunoprecipitated from AY and p53K6R ES cells either not treated (lanes 3 and 5) or treated with proteasome inhibitors (25 μM MG132 + 25 μM LLnL) for 6 h (lanes 4 and 6) as indicated. An unsaturated amount of anti-full-length p53 antibody (1 μg) was used to ensure similar total amounts of immunoprecipitated p53. p53^−/− samples were also analyzed to control for the specificity of p53 antibody. Ubiquitinated p53 and total p53 are indicated. (C) p53-dependent transcriptional activity in p53K6R ES cells after UV radiation. The mRNA levels of several p53 target genes in AY and p53K6R ES cells 8 h after 60 J/m² UV radiation were analyzed by quantitative real-time PCR. The mRNA levels of each gene were standardized by the mRNA level of GAPDH. p53 and actin mRNAlevels were also analyzed as non-p53-dependent controls. The ratio of mRNA levels in p53K6R cells after UV radiation versus those in AY cells is presented. Mean values from three independent experiments are presented, with standard deviations. The P value representing the statistical significance of the difference between each mean value and 1 is shown on top of each ratio bar. The approximate levels of induction for each p53 target gene in AY ES cells after UV treatment are as follows: p21, 10-fold; Mdm2, 3.5-fold; Noxa, 7-fold; Perp, 7.5-fold; Pidd, 3-fold; PUMA, 5-fold.

FIG. 3.

p53 responses to DNA damage in p53K6R MEFs. Induction of p53 protein levels in p53^+/− and p53K6R MEFs after 60 J/m² UV treatment (A) or 0.25 μM doxorubicin treatment (B). The genotype and time points after DNA damage are indicated on the top. p53 and actin are indicated on the right. p53-dependent transcriptional activity in p53K6R and control MEFs 18 h after treatment with 60 J/m² UV radiation (C) or 24 h after treatment with 0.25 μM doxorubicin (D). The mRNA levels of p53 target genes were analyzed by quantitative real-time PCR. In addition, p53 and actin mRNA levels were analyzed as internal controls. The ratio of mRNA levels in treated p53K6R MEFs to those in treated p53^+/− MEFs is indicated. Mean values from at least three independent experiments are presented, with standard deviations. The P values are shown on top of each bar. The levels of induction for each p53 target gene in p53^+/− MEFs after UV radiation are as follows: p21, 5-fold; Mdm2, 5-fold; Bax, 1.5-fold; Pidd, 7-fold. The levels of induction after doxorubicin treatment are as follows: p21, 11-fold; Mdm2, 7-fold; Bax, 2.5-fold; Pidd, 8.5-fold.

FIG. 4.

p53 responses to DNA damage in p53K6R thymocytes. (A) Induction of p53 protein levels in p53^+/− and p53K6R thymocytes after IR. The genotype and time points after IR are indicated on the top. p53 and actin are indicated on the right. (B) p53-dependent apoptosis in p53K6R and control AY thymocytes 10 h after increasing dosages of IR. The P values representing the statistical significance of the difference in apoptosis between p53K6R and AY thymocytes are also indicated. (C) p53-dependent transcriptional activity in p53K6R and control p53^+/− thymocytes 8 h after 5 Gy of IR. The ratio of mRNA levels in irradiated p53K6R thymocytes to those in irradiated p53^+/− thymocytes is shown. Mean values from three independent experiments are shown, with standard deviations. P values are indicated. The levels of induction of p53 target genes in p53^+/− thymocytes after IR are as follows: p21, 17-fold; Mdm2, 3-fold; Bax, 15-fold; Killer/RD5: 4-fold; Pidd, 5-fold; PUMA, 28-fold.