Acetylation of mouse p53 at lysine 317 negatively regulates p53 apoptotic activities after DNA damage

Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxyl terminus by CBP/p300 in response to genotoxic and some nongenotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317-to-Arg (K317R) missense mutation into the endogenous p53 gene of mice. p53 protein accumulates to normal levels in p53(K317R) mouse embryonic fibroblasts (MEFs) and thymocytes after DNA damage. While p53-dependent gene expression is largely normal in p53(K317R) MEFs after various types of DNA damage, increased p53-dependent apoptosis was observed in p53(K317R) thymocytes, epithelial cells from the small intestine, and cells from the retina after ionizing radiation (IR) as well as in E1A/Ras-expressing MEFs after doxorubicin treatment. Consistent with these findings, p53-dependent expression of several proapoptotic genes was significantly increased in p53(K317R) thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.     

Acetylation of p53 inhibits its ubiquitination by Mdm2

In response to DNA damage, the activity of the p53 tumor suppressor is modulated by protein stabilization and post-translational modifications including acetylation. Interestingly, both acetylation and ubiquitination can modify the same lysine residues at the C terminus of p53, implicating a role of acetylation in the regulation of p53 stability. However, the direct effect of acetylation on Mdm2-mediated ubiquitination of p53 is still lacking because of technical difficulties. Here, we have developed a method to obtain pure acetylated p53 proteins from cells, and by using an in vitro purified system, we provide the direct evidence that acetylation of the C-terminal domain is sufficient to abrogate its ubiquitination by Mdm2. Importantly, even in the absence of DNA damage, acetylation of the p53 protein is capable of reducing the ubiquitination levels and extending its half-life in vivo. Moreover, we also show that acetylation of p53 can affect its ubiquitination through other mechanisms in addition to the site competition. This study has significant implications regarding a general mechanism by which protein acetylation modulates ubiquitination-dependent proteasome proteolysis.     

Functional analysis of the acetylation of human p53 in DNA damage responses

As a critical tumor suppressor, p53 is inactivated in human cancer cells by somatic gene mutation or disruption of pathways required for its activation. Therefore, it is critical to elucidate the mechanism underlying p53 activation after genotoxic and cellular stresses. Accumulating evidence has indicated the importance of posttranslational modifications such as acetylation in regulating p53 stability and activity. However, the physiological roles of the eight identified acetylation events in regulating p53 responses remain to be fully understood. By employing homologous recombination, we introduced various combinations of missense mutations (lysine to arginine) into eight acetylation sites of the endogenous p53 gene in human embryonic stem cells (hESCs). By determining the p53 responses to DNA damage in the p53 knock-in mutant hESCs and their derivatives, we demonstrate physiological importance of the acetylation events within the core domain (K120 and K164) and at the C-terminus (K370/372/373/381/382/ 386) in regulating human p53 responses to DNA damage.     

Distinct p53 acetylation cassettes differentially influence gene-expression patterns and cell fate

The activity of the p53 gene product is regulated by a plethora of posttranslational modifications. An open question is whether such posttranslational changes act redundantly or dependently upon one another. We show that a functional interference between specific acetylated and phosphorylated residues of p53 influences cell fate. Acetylation of lysine 320 (K320) prevents phosphorylation of crucial serines in the NH(2)-terminal region of p53; only allows activation of genes containing high-affinity p53 binding sites, such as p21/WAF; and promotes cell survival after DNA damage. In contrast, acetylation of K373 leads to hyperphosphorylation of p53 NH(2)-terminal residues and enhances the interaction with promoters for which p53 possesses low DNA binding affinity, such as those contained in proapoptotic genes, leading to cell death. Further, acetylation of each of these two lysine clusters differentially regulates the interaction of p53 with coactivators and corepressors and produces distinct gene-expression profiles. By analogy with the "histone code" hypothesis, we propose that the multiple biological activities of p53 are orchestrated and deciphered by different "p53 cassettes," each containing combination patterns of posttranslational modifications and protein-protein interactions.     

Tip60-dependent acetylation of p53 modulates the decision between cell-cycle arrest and apoptosis

Upon DNA damage and other types of stress, p53 induces either cell-cycle arrest or apoptosis depending on the cellular context. However, the molecular mechanisms that govern the choice between cell-cycle arrest and apoptosis are not well understood. Here, we show that Tip60 is required for both cell growth arrest and apoptosis mediated by p53 and also induces its acetylation specifically at lysine 120 (K120) within the DNA-binding domain. Interestingly, this modification is crucial for p53-dependent apoptosis but is dispensable for its mediated growth arrest. K120 is a recurrent site for p53 mutation in human cancer, and the corresponding acetylation-defective tumor mutant (K120R) abrogates p53-mediated apoptosis, but not growth arrest. Thus, our study demonstrates that Tip60-dependent acetylation of p53 at K120 modulates the decision between cell-cycle arrest and apoptosis, and it reveals that the DNA-binding core domain is an important target for p53 regulation by posttranslational modifications.     

Ubiquitination of p53 at multiple sites in the DNA-binding domain

The tumor suppressor p53 is negatively regulated by the ubiquitin ligase MDM2. The MDM2 recognition site is at the NH2-terminal region of p53, but the positions of the actual ubiquitination acceptor sites are less well defined. Lysine residues at the COOH-terminal region of p53 are implicated as sites for ubiquitination and other post-translational modifications. Unexpectedly, we found that substitution of the COOH-terminal lysine residues did not diminish MDM2-mediated ubiquitination. Ubiquitination was not abolished even after the entire COOH-terminal regulatory region was removed. Using a method involving in vitro proteolytic cleavage at specific sites after ubiquitination, we found that p53 was ubiquitinated at the NH2-terminal portion of the protein. The lysine residue within the transactivation domain is probably not essential for ubiquitination, as substitution with an arginine did not affect MDM2 binding or ubiquitination. In contrast, several conserved lysine residues in the DNA-binding domain are critical for p53 ubiquitination. Removal of the DNA-binding domain reduced ubiquitination and increased the stability of p53. These data provide evidence that in addition to the COOH-terminal residues, p53 may also be ubiquitinated at sites in the DNA-binding domain.     

Deacetylation of the DNA-binding domain regulates p53-mediated apoptosis

In unstressed cells, the p53 tumor suppressor is highly unstable. DNA damage and other forms of cellular stress rapidly stabilize and activate p53. This process is regulated by a complex array of post-translational modifications that are dynamically deposited onto p53. Recent studies show that these modifications orchestrate p53-mediated processes such as cell cycle arrest and apoptosis. Cancer cells carry inherent genetic damage, but avoid arrest and apoptosis by inactivating p53. Defining the enzymatic machinery that regulates the stress-induced modification of p53 at single-residue resolution is critical to our understanding of the biochemical mechanisms that control this critical tumor suppressor. Specifically, acetylation of p53 at lysine 120, a DNA-binding domain residue mutated in human cancer, is essential for triggering apoptosis. Given the oncogenic properties of deacetylases and the success of deacetylase inhibitors as anticancer agents, we investigated the regulation of Lys(120) deacetylation using pharmacologic and genetic approaches. This analysis revealed that histone deacetylase 1 is predominantly responsible for the deacetylation of Lys(120). Furthermore, treatment with the clinical-grade histone deacetylase inhibitor entinostat enhances Lys(120) acetylation, an event that is mechanistically linked to its apoptotic effect. These data expand our understanding of the mechanisms controlling p53 function and suggest that regulation of p53 modification status at single-residue resolution by targeted therapeutics can selectively alter p53 pathway function. This knowledge may impact the rational application of deacetylase inhibitors in the treatment of human cancer.     

P53蛋白赖氨酸甲基化与去甲基化

P53作为肿瘤抑制因子和转录调节因子在控制细胞周期、凋亡和DNA修复方面发挥重要作用。P53蛋白的稳定性和转录激活活性的调节主要依赖磷酸化、乙酰化、泛素化等多种翻译后修饰。最近研究发现一些组蛋白赖氨酸甲基转移酶和去甲基化酶可使P53蛋白C-端赖氨酸残基发生甲基化或去甲基化,调节P53蛋白的稳定性和转录激活活性。甲基化和去甲基化与其它翻译后修饰相互作用构成“P53密码”调节P53蛋白功能。     

Acetylation of Lysine 382 and Phosphorylation of Serine 392 in p53 Modulate the Interaction between p53 and MDC1 In Vitro

Occurrence of DNA damage in a cell activates the DNA damage response, a survival mechanism that ensures genomics stability. Two key members of the DNA damage response are the tumor suppressor p53, which is the most frequently mutated gene in cancers, and MDC1, which is a central adaptor that recruits many proteins to sites of DNA damage. Here we characterize the in vitro interaction between p53 and MDC1 and demonstrate that p53 and MDC1 directly interact. The p53-MDC1 interaction is mediated by the tandem BRCT domain of MDC1 and the C-terminal domain of p53. We further show that both acetylation of lysine 382 and phosphorylation of serine 392 in p53 enhance the interaction between p53 and MDC1. Additionally, we demonstrate that the p53-MDC1 interaction is augmented upon the induction of DNA damage in human cells. Our data suggests a new role for acetylation of lysine 382 and phosphorylation of serine 392 in p53 in the cellular stress response and offers the first evidence for an interaction involving MDC1 that is modulated by acetylation.     

The ARF/Oncogene Pathway Activates p53 Acetylation within the DNA Binding Domain

Stabilization of the p53 tumor suppressor is a critical event in the response to various forms of cellular stress. Two distinct signaling pathways are thought to lead to this stabilization, depending on the type of cellular stress encountered. Genotoxic stress, such as chromosomal breaks or lesions induced by chemotherapeutic agents, result in the activation of the well-characterized DNA damage response pathway. Conversely, cellular stress that results from the aberrant activation of oncogenes triggers p53 stabilization via the induction of the p19ARF pathway. While activation of the DNA damage pathway ultimately causes a complex array of post-translational modifications on p53, activation few if any modifications have been demonstrated to occur following activation of the p19ARF pathway. We and others have recently identified a novel modification on p53, acetylation of lysine 120 within the DNA binding domain. This acetylation event is eliminated by tumor-derived mutations in p53 and its presence is required for the tumor suppressor apoptotic function of p53. We demonstrate here that both the DNA damage response pathway and the p19ARF/oncogene stress pathway induce the acetylation of p53 at lysine 120.     

Multiple lysine mutations in the C-terminal domain of p53 interfere with MDM2-dependent protein degradation and ubiquitination

To investigate the effect of mutations in the p53 C-terminal domain on MDM2-mediated degradation, we introduced single and multiple point mutations into a human p53 cDNA at four putative acetylation sites (amino acid residues 372, 373, 381, and 382). Substitution of all four lysine residues by alanines (the A4 mutant) and single lysine-to-alanine substitutions were functional in sequence-specific DNA binding and transactivation; however, the A4 mutant protein was resistant to MDM2-mediated degradation, whereas the single lysine substitutions were not. Although the A4 mutant protein and the single lysine substitutions both bound MDM2 reasonably well, the single lysine substitutions underwent normal MDM2-dependent ubiquitination, whereas the A4 protein was inefficiently ubiquitinated. In addition, the A4 mutant protein was found in the cytoplasm as well as in the nucleus of a subpopulation of cells, unlike wild-type p53, which is mostly nuclear. The partially cytoplasmic distribution of A4 mutant protein was not due to a defect in nuclear import because inhibition of nuclear export by leptomycin B resulted in nuclear accumulation of the protein. Taken together, the data suggest that mutations in the putative acetylation sites of the p53 C-terminal domain interfere with ubiquitination, thereby regulating p53 degradation.