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Mol Cell Biol. 2005 July; 25(13): 5389–5395. doi: 10.1128/MCB.25.13.5389-5395.2005. | PMCID: PMC1157004 |
Copyright © 2005, American Society for Microbiology Functional Analysis of the Roles of Posttranslational Modifications at the p53 C Terminus in Regulating p53 Stability and Activity Lijin Feng,1 Tongxiang Lin,1 Hiroaki Uranishi,2 Wei Gu,2 and Yang Xu1* Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0322,1 Institute for Cancer Genetics, Columbia University, 1150 St. Nicholas Avenue, New York, New York 100322 Received December 13, 2004; Revised January 10, 2005; Accepted April 5, 2005. 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. p53 is one of the most frequently mutated tumor suppressor genes in human cancers ( 16). It is a transcriptional factor composed of four functional domains: the N-terminal transactivation domain, through which p53 interacts with coactivators or corepressors, the central sequence-specific DNA binding domain, the tetramerization domain, and the extreme C-terminal regulatory domain ( 21, 25). In unstressed cells, p53 is present in a latent form and is maintained at low levels through rapid protein degradation. Recent studies showed that Mdm2, Pirh2, and COP1 can all facilitate p53 degradation via the ubiquitin-proteasome pathway by functioning as ubiquitin ligase ( 11, 15, 17, 22, 24). In response to genotoxic and cellular stresses, the stability and activity of p53 are greatly induced, leading to cell cycle arrest, DNA repair, and/or apoptosis, depending on the cell types ( 1, 2, 21, 32, 39, 40). While the mechanism of activation of p53 responses after various genotoxic and cellular stresses remains to be established, accumulating evidence indicates that posttranslational modifications of p53 play important roles in regulating its stability and transcriptional activity ( 1). Specifically, the multiple lysine residues at the extreme carboxyl-terminal domain of p53 (the last 30 amino acids) can be posttranslationally modified by multiple mechanisms, including phosphorylation, acetylation, ubiquitination, neddylation, and methylation, in response to DNA damage and other cellular stresses ( 5, 10, 14, 36, 38, 41, 43). In this context, human p53 can potentially be acetylated by CBP/p300 at five lysine residues: Lys370, Lys372, Lys373, Lys381, and Lys382 ( 14, 30). Using an acetylation-specific antibody, it has been shown that the acetylation of p53 at Lys373 and Lys382 is significantly induced in response to DNA damage in vivo ( 19, 29, 36). In addition, human p53 can also be acetylated by P/CAF (p300/CBP-associated factor) at Lys320 in vitro and that this acetylation event is induced by DNA damage in vivo ( 29, 36). The roles of p53 acetylation have been studied extensively. Gu et al. suggested that CBP/p300 mediated acetylation of p53 can increase p53 sequence-specific DNA-binding activity in vitro by electrophoretic mobility shift assay using short oligonucleotides ( 14). Several subsequent studies also supported the idea that the acetylation of p53 can dramatically stimulate its sequence-specific DNA-binding activity both in vitro and in vivo, possibly by an acetylation-induced conformational change ( 29, 30, 36). However, recent studies showed that p53 binds to its cognate promoters constitutively ( 20). In addition, acetylation does not increase the p53 DNA-binding activity when the protein is assayed for binding to an artificially reconstituted chromatin ( 12). Instead, this and other studies showed that p53 acetylation is important for the recruitment of coactivators ( 3, 12). Recent studies also showed that the p53 C terminus is required for p53 linear diffusion on chromatin and its efficient DNA binding as well as transactivation of target promoters in vivo ( 31). However, acetylation and other modifications of the C terminus do not increase this p53 activity. Several studies also suggested that ubiquitination and acetylation at the C terminus of p53 can regulate p53 stability. In this context, one study showed that the p53 C terminus was required for Mdm2-mediated degradation of p53 but not Mdm2-p53 interaction ( 23). Since the lysine residues at the C terminus might be ubiquitinated by Mdm2, two studies tested the importance of the C-terminal lysine residues in p53 stabilization. In one study, all six lysine residues were changed to arginine (6KR mutant) to prevent ubiquitination but preserve the structure of p53 ( 34). While the 6KR mutant interacted with Mdm2 normally, it could not undergo Mdm2-mediated ubiquitination and degradation in transfected tumor cell lines, leading to p53 stabilization and activation ( 34). In addition, mutation of four lysine residues (Lys372, 373, 381, and 382) to alanine (A4 mutant) also abrogated p53 ubiquitination and degradation ( 33). Since acetylation of the same lysine residues might prevent ubiquitination at these sites, it has been suggested that p53 acetylation can stabilize p53. In support of this notion, increased levels of p53 acetylation by deacetylase inhibitors could inhibit p53 degradation in vivo, and the p53 degradation requires deacetylation ( 19, 26). In addition to acetylation and ubiquitination, the C-terminal lysine residues can also be modified by other mechanisms. Lys386 of p53 can be modified by conjugation to a small ubiquitin-like protein (SUMO-1) in vitro and in vivo ( 13, 35), although the role of this modification remains controversial. Recent studies also showed that the ubiquitin-like protein Nedd8 can be covalently linked to p53 at Lys370, -372, and -373, and Mdm2-dependent neddylation of p53 negatively regulates its transcriptional activity ( 43). Additionally, methylation of p53 at Lys372 by Set9 methyltransferase has been identified and suggested to restrict p53 in the nucleus and stabilize p53 ( 10). To further study the physiological roles of the posttranslational modifications at the C-terminal lysine residues in regulating p53 stability and activity, we employed homologous recombination and LoxP/Cre-mediated deletion to introduce six lysine-to-arginine mutations (Lys367, -369, -370, -378, -379, and -383 to Arg; K6R) into the endogenous mouse p53 gene in embryonic stem (ES) cells. Analysis of p53 stability and activity before and after DNA damage indicated that ubiquitination of the six C-terminal lysine residues is not required for p53 degradation. However, the net effects of these modifications increase p53 transcriptional activities in a cell type-dependent manner. Construction of targeting vector and generation of p53K6R mutant ES cells. The six lysine residues, Lys367, Lys369, Lys370, Lys378, Lys379, and Lys383, are encoded by exon 11 of the murine p53 gene. A fragment of mouse p53 genomic sequence extending from exon 2 through exon 11 was cloned into the pBS vector. Site-directed mutagenesis was performed to introduce Lys-to-Arg mutations into cloned exon 11. To facilitate screening of ES cell clones, an EcoRI restriction site was introduced into intron 1, and a neomycin resistance gene driven by the phosphoglycerate kinase promoter (PGK-Neor gene) flanked by loxP sites was inserted into intron 7. This targeting vector was then linearized with NotI and electroporated into AY ES cells. To excise the PGK-Neor gene flanked by loxP sites, 20 μg of a circular plasmid expressing the Cre enzyme was transiently transfected into the mutant ES cells, and the LoxP/Cre-mediated deletion was screened by PCR using primers shown in Fig. . The PGK-Neor-deleted ES cell clones identified by PCR were then subcloned, and the Cre deletion was further confirmed by Southern blotting using the same probe. Derivation, culture, and treatment of p53K6R MEFs. We employed the Hprt-deficient blastocyst complementation approach to generate mouse embryonic fibroblasts (MEFs) from the mutant ES cells as previously described ( 42). MEFs recovered from the embryos at E12.5 were cultured and selected in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, glutamine, antibiotics, 50 μM β-mercaptoethanol, and HAT (0.016 mg/ml hypoxanthine, 0.01 mM aminopterin, and 0.0048 mg/ml thymidine). After we confirmed that all the live MEFs were derived from mutant ES cells, the HAT selection was removed and MEFs were cultured in normal medium. MEFs were treated with 60 J/m 2 UV-C light or 0.25 μM doxorubicin (Sigma) and harvested at different time points for the analysis of protein levels or gene expression. Culture and treatment of ES cells. ES cells were cultured on feeder layers in DMEM supplemented with 15% fetal bovine serum, glutamine, nonessential amino acids, sodium pyruvate, antibiotics, 100 μM 2-mercaptoethanol, and recombinant leukemia inhibitory factor (LIF). Before experiments, ES cells were split and plated on gelatin-coated plates in the presence of LIF but without feeder layer cells. ES cells were exposed to 60 J/m2 UV-C light and harvested at different time points for protein levels or at 8 h for gene expression analysis. Ubiquitination assay. AY and K6R ES cells were cultured in 10-cm plates. The cells were treated for 6 h with proteasome inhibitors, 25 μM N-acetyl-L-leucyl-L-leucyl-L-norleucine (LLnL) and 25 μM MG132, before being harvested and were then lysed with RIPA buffer (1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], Tris-HCl [pH 7.8], 150 mM NaCl, 1 mM dithiothreitol, 0.5 mM EDTA, 25 μM LLnL, 25 μM MG132, 5 mM N-ethylmaleimide, and fresh proteinase inhibitors) with mild sonication. Protein extracts of samples containing 2 × 106 cells were incubated with 1 μg p53 antibody against the full-length protein (Santa Cruz Biotechnology) for 1 h at 4°C. Protein A/G-agarose beads (30 μl) were added, and the reaction mixtures were further incubated overnight at 4°C. The immunoprecipitates were subsequently resolved by 8% SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed by Western blotting with antiubiquitin antibody (P4D1; Santa Cruz Biotechnology) or anti-p53 antibody (Pab240; Santa Cruz Biotechnology). IR-induced apoptosis in thymocytes. Thymocytes were recovered from 4- to 6-week-old mice and cultured in DMEM supplemented with 5% FCS and 25 mM HEPES (pH 7.4) before treatment. Thymocytes were treated with 5 Gy of ionizing radiation (IR) and harvested at different time points for analysis of p53 protein levels. For the apoptosis assay, thymocytes were exposed to 2.5, 5, 10, or 20 Gy of IR, and apoptotic cells were identified 10 h after treatment by staining with annexin V. For real-time PCR, thymocytes were irradiated with 5 Gy of IR, and RNA was harvested 8 h after treatment. Western blot analysis. Protein extract from 2 × 104 ES cells and MEFs or 2 × 105 thymocytes were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% dry milk and probed with CM5, a polyclonal antibody against p53 (Novo Laboratories, Inc.), or polyclonal antibodies specifically against mouse p53 phosphorylated at Ser18 or Ser389 (Cell Signaling Technology). The membranes were subsequently incubated with horseradish peroxide-conjugated secondary antibody, developed with ECL Plus (Amersham Biosciences), and exposed to X-ray film. To determine whether the total amount of proteins loaded in each lane was comparable, the membranes were probed with a goat polyclonal antibody against β-actin (Santa Cruz Biotechnology). RNA preparation and quantitative real-time PCR analysis. Total RNA was prepared from the frozen cell pellets by following RNeasy RNA cleanup protocol (QIAGEN). Up to 1 μg RNA from individual samples was reverse transcribed to cDNA using a SuperscriptII first-strand synthesis kit (Invitrogen). Real-time PCR was performed on an ABI PRISM 7000 sequence detection system with SYBR green PCR MasterMix (ABI). The average threshold cycle ( Ct) for each gene was determined from triplicate reactions, and the levels of gene expression were determined relative to the average Ct value of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as previously described ( 4). The primer sequences used in real-time PCR were described previously ( 7). Statistic analysis. To determine statistical significance, quantitative RT-PCR data and apoptosis data were subjected to a two-tailed Student's t-test analysis. Significance was noted for P values less than 0.05. Generation of K6R mutant murine ES cells. A DNA fragment harboring murine p53 exon 11 was cloned into pBluescript, and the nucleotides encoding the six lysine residues (Lys367, -369, -370, -378, -379, and -383) were mutated to those encoding arginine by site-directed mutagenesis. The knock-in vector was constructed by replacing the germ line exon 11 of the cloned mouse p53 genomic DNA with the K6R exon, followed by inserting the PGK-Neo r gene flanked by LoxP sites into intron 7 (Fig. ). To facilitate the mutagenesis process, the knock-in vector was electroporated into AY ES cells, which contain one wild-type p53 allele and the AY allele with exons 2 to 4 replaced with a LoxP site (Fig. ) ( 42). The AY allele does not produce any truncated p53 protein; therefore, the genotype of the AY ES cell line is p53 +/−. Homologous recombination between the wild-type allele of AY ES cells and the targeting vector was screened by Southern blotting analysis of genomic DNA with EcoRI digestion and hybridization to probe 1 (Fig. ). The PGK-Neo r gene in the mutant ES cells was deleted through transient expression of the Cre enzyme as previously described ( 8). The LoxP/Cre-mediated deletion was confirmed by Southern blot analysis with EcoRI digestion and hybridization to probe 1 (Fig. ). The final knock-in ES cells were designated p53K6R. Both the genomic DNA and cDNA from p53K6R ES cells were sequenced to confirm that the p53 gene expressed in p53K6R ES cells harbored the six lysine-to-arginine mutations but no other mutations. p53 responses to DNA damage in p53K6R ES cells. Since previous cell line transfection studies indicated that the C-terminal lysine residues of p53 are important for ubiquitin-mediated degradation of p53 ( 33, 34), we determined the p53 stability in the p53K6R ES cells before and after DNA damage induced by UV radiation. p53 protein levels were similarly low before DNA damage but greatly induced in both AY and p53K6R ES cells after UV radiation (Fig. ). While the protein levels of p53 were slightly reduced at 4 h after UV radiation, the peak levels of p53 induction were similar between AY and p53K6R ES cells at 8 h after UV radiation (Fig. ). Therefore, the K6R mutation had no significant impact on p53 stabilization in ES cells with or without DNA damage. In addition, the K6R mutation had no significant impact on p53 phosphorylation at the N terminus (Ser18) or C terminus (Ser389) (Fig. ). The normal stability of p53 in p53K6R ES cells in the absence of DNA damage indicates that other lysine residues of p53 can also be ubiquitinated and contribute to the destabilization of p53. In support of this notion, p53 was ubiquitinated in p53K6R ES cells (Fig. ). However, the levels of p53 ubiquitination appeared to be slightly reduced in p53K6R ES cells, indicating that the lysine residues at the C terminus are ubiquitination targets. Activation of p53 transcriptional activities by DNA damage is important to maintain genetic stability in ES cells ( 9, 28). To determine whether K6R affects p53 transcriptional activities, we analyzed the p53-dependent gene expression in p53K6R and control AY ES cells after UV radiation. While there was no significant difference in the p53-dependent induction of perp mRNA in p53K6R and AY ES cells after UV radiation, the expression of several other p53 target genes, including p21, Mdm2, Noxa, Pidd, and PUMA, was significantly reduced in p53K6R ES cells 8 h after UV radiation compared with that in AY ES cells (Fig. ). As internal controls, no significant difference in the mRNA levels of p53 or actin was detected in p53K6R and AY ES cells after UV radiation (Fig. ). Since the protein levels of p53 were similar in K6R and AY ES cells 8 h after UV radiation, these findings indicated that K6R mutation impaired p53-dependent transcriptional activity in a promoter-specific manner in ES cells after DNA damage. p53 responses to DNA damage in p53K6R MEFs. MEFs undergo p53-dependent cell cycle arrest after DNA damage. Therefore, we examined the effects of K6R mutation on p53 stability and activity in p53K6R MEFs after DNA damage. We employed the Hprt-deficient blastocyst complementation to derive p53K6R MEFs from ES cells as previously described ( 42). Since the genotype of AY ES cells is p53 +/−, p53 protein levels in p53K6R and the control p53 +/− MEFs before and after DNA damage were analyzed. Consistent with the findings in ES cells, the protein levels of p53 were very low before UV treatment and similarly induced in both p53K6R and p53 +/− MEFs after UV radiation (Fig. ). p53 stabilization also appeared to be normal in p53K6R MEFs after DNA damage induced by doxorubicin (Fig. ). Consistent with the findings in ES cells, K6R mutation did not affect the phosphorylation of p53 at Ser18 or Ser389 after UV radiation and doxorubicin treatment (data not shown). To test whether the K6R mutation affects p53-dependent transcriptional activities, we analyzed the expression levels of several p53 target genes, including p21, Mdm2, Bax, and Pidd, after UV radiation and doxorubicin treatment (Fig. ). There is no significant difference in the p53-dependent gene expression between p53K6R and control MEFs after these types of DNA damage. In addition, there was no significant difference in the mRNA levels of p53 and actin between p53K6R and p53 +/− MEFs after DNA damage (Fig. ). Therefore, the K6R mutation has no apparent impact on p53 stability and activity in MEFs after DNA damage. p53 responses to IR in p53K6R thymocytes. Mouse thymocytes undergo p53-dependent apoptosis in response to IR. To determine the effects of K6R mutation on p53-dependent apoptotic activities in thymocytes, we derived the p53K6R and control AY thymocytes by Rag2-deficient blastocyst complementation assay as described previously ( 18, 42). In addition, p53 +/− thymocytes were also used as controls because the genotype of AY cells is p53 +/− and previous studies indicated that p53 responses to IR are the same in AY thymocytes and p53 +/− thymocytes. Consistent with the notion that the K6R mutation does not affect p53 stabilization, p53 protein levels were similar in p53K6R and control thymocytes before and after IR (Fig. ). p53K6R thymocytes were slightly more resistant to p53-dependent apoptosis after IR than control AY thymocytes (Fig. ). However, while p53-dependent induction of Mdm2, Bax, and Pidd was similar in p53K6R and control thymocytes, induction of p53-targeted apoptotic genes, including Killer/DR5 and PUMA, was significantly impaired in p53K6R thymocytes after IR (Fig. ). Therefore, we concluded that the K6R mutation impaired p53 transcription activities in a promoter-specific manner in thymocytes after IR. Multiple lysine residues in the extreme C-terminal domain of p53 can be posttranslationally modified by several mechanisms. To investigate the physiological importance of these posttranslational modifications, we introduced the six lysine-to-arginine missense mutations into the endogenous p53 gene in ES cells, which can be differentiated in vivo into MEFs and thymocytes. Previous cell line transfection studies reached the conclusion that the C-terminal lysine residues are ubiquitination sites by Mdm2 and are required for ubiquitin-dependent p53 degradation ( 33, 34). In contrast to these previous findings, our studies indicated that the K6R mutation has no significant impact on p53 stability either before or after DNA damage in mouse ES cells, MEFs, and thymocytes. While our findings do not argue against the notion that these C-terminal lysine residues could be ubiquitinated and involved in regulating p53 stability, the finding that p53 was ubiquitinated in p53K6R ES cells clearly indicates that ubiquitination of lysine residues residing in other regions of p53 could play redundant roles in ubiquitin-dependent p53 degradation. One likely explanation for the apparent discrepancy between previous findings and our findings is that the p53 −/− tumor cell lines or mouse cells used in previous studies might harbor additional mutations that disrupt the redundant pathways involved in ubiquitination of p53 at other lysine residues. In addition, previous studies only focused on the involvement of Mdm2 in the ubiquitination of p53 by overexpressing Mdm2 with p53K6R mutant and drew the conclusion that the six C-terminal lysine residues are the primary targets for Mdm2-mediated ubiquitination ( 33, 34). However, recently identified additional E3 ligases for p53, including the p53 targets Pirh2 and COP1, might ubiquitinate p53 at alternative lysine residues and thus destabilize p53K6R ( 11, 24). In this context, overexpression of Pirh2/COP1/Mdm2 with p53K6R might lead to the ubiquitination and degradation of p53K6R. Consistent with this notion, while p53K6R is as unstable as wild-type p53, p53 Gln25,Ser26, which cannot interact with Mdm2 and is essentially abolished in its p53-dependent expression of genes such as Pirh2 and COP1, is constitutively stable ( 9). p53 acetylation is correlated with p53 stabilization after DNA damage ( 44). Previous studies have suggested that acetylation of lysine residues, particularly the ones at the C terminus, might prevent ubiquitination of the same residues, thus leading to p53 stabilization ( 19, 27). Since our findings indicate that ubiquitination at the C-terminal lysine residues is not required for efficient p53 degradation, the importance of the competition of acetylation and ubiquitination at the p53 C-terminal lysine residues remains questionable. However, our findings do not argue against the notion that p53 acetylation at multiple lysine residues, including those within the extreme C terminus, can prevent ubiquitination, leading to p53 stabilization. In addition to their impact on p53 stability, the functions of the posttranslational modifications at C-terminal lysine residues in regulating p53 activities have been extensively studied in biochemical and cell line transfection studies. In this context, various posttranslational modifications of p53 at the C terminus have been suggested for different roles in regulating p53 activities. Acetylation of p53 at the C terminus has been thought to recruit coactivators to the p53-dependent promoters and thus to activate p53-dependent transcription ( 3, 12). However, neddylation of p53 at the C-terminal lysine residues might inhibit p53 transcriptional activities ( 43). Our analysis of p53-dependent transcriptional activities in K6R mutant ES cells, MEFs, and thymocytes indicated that K6R mutation impaired p53 activities after DNA damage in ES cells and thymocytes. The lack of defects in MEFs might be due to the activation of functionally redundant posttranslational modification pathways that are induced by stresses of in vitro culturing, as indicated by the findings that p53 activities are normally increased during the continuous passage of MEFs, eventually leading to cellular senescence ( 6, 37). 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