by Katherine Wood, Ph.D.
The development of cancer involves changes in gene expression. The tumor suppressor protein p53 is probably the most studied regulator of transcription activators. The p53 protein is a sequence-specific transcription factor that responds to stress by inducing or repressing many different genes, thereby regulating such key cellular processes as cell cycle control, cell death, and DNA repair. DNA damaging agents including ionizing radiation, UV light, anti-cancer drugs such as topoisomerase inhibitors, inhibitors of transcription, DNA cross linking agents, and environmental agents such as cadmium have all been shown to alter p53 activity. Importantly, non-genotoxic stresses such as hypoxia, microtubule disruption, oncogene activation, and replicative senescence are also known to modify p53..
The crystal structure of p53 has been determined and functional motifs have been mapped: the amino terminus interacts with transcriptional machinery and regulatory proteins (residues 1-101 in human p53), the carboxyl terminus carries a regulatory and a tetramerization domain (residues 293-393), and the central highly structured region (residues 102-292) is a sequence-specific DNA binding domain.(2)
Mutation in p53Since the identification, cloning and determination of the tumor suppressor function of p53, a remarkable number of mutations in p53 have been identified. Li-Fraumeni cancer-prone families carry germ line mutations in p53 and approximately 50% of all solid tumors in humans are associated with p53 mutation. The majority of mutations interfere in tetramerization or act to disrupt the transcriptional activation function of p53. More than 95% of the mutations in p53 occur in the central part of the gene and are missense mutations. These missense mutations not only disrupt the normal biological function of the p53 protein but also cause a change in p53 stability, increasing the half-life from minutes to hours. Mutant p53 protein usually exhibits a dominant inhibitory effect since the mutant form will oligomerize with the wild type p53 and reduce or prevent DNA binding.
In healthy individuals, the development of antibodies against p53 is rare, however, anti-p53 antibodies in patients with neoplasias is common. The majority of individuals with anti-p53 antibodies carry tumor-specific mutant p53. Less commonly, some patients exhibit detectable p53 but have no obvious mutation in tumor tissue. The accumulation of the mutant p53 allows for immunological detection and thus provides the opportunity for the generation of diagnostic tools to detect the oncogenic p53 changes in patient samples.
p53 and post-translational modificationsWild type p53 is a short lived protein and in the absence of stress is present at only low levels in the cell. Stress leads to nuclear accumulation of the protein and subsequent binding to specific DNA sequences. This p53 DNA-binding may induce or inhibit expression of at least 150 different genes. The outcome of this p53 stabilization and activation may be cell cycle arrest, senescence, or apoptosis depending upon the cell status and the nature and strength of the stress.
The diverse cellular response to stress, both genotoxic and non-genotoxic, is determined at least in part by post-translational modifications to p53 (see Figure 1). It is now known that there are at least 18 sites within p53, concentrated within the amino and carboxy termini, that can be post-translationally modified, and the majority of these modifications are phosphorylations.(1) During G0 of the cell cycle, p53 is phosphorylated on Ser 9, amino acids 9, 15, and 20 are phosphorylated during G1, and amino acids 37 and 392 are phosphorylated during G2/M.(3) Significant changes in the pattern of phosphorylation have been identified in response to DNA damaging agents and all the N-terminal serines and threonines may be phosphorylated or dephosphorylated in response to DNA damage. Rapid degradation of p53 by the 26S proteosome maintains only a low level of the protein in healthy cells. In resting cells, p53 is rapidly degraded through ubiquitination at lysines that target the protein to the 26S proteosome. The multiple ubiquitination of p53 is controlled in part by Jun N-terminal kinase that binds to p53 and targets it for ubiquitination. Ubiquitination is also promoted by phosphorylation at Thr 155, and other nearby residues, by the COP9 signalsome. In dividing cells, the MDM2 E3 ligase ubiquitinates p53 at multiple C-terminal lysines. The binding of MDM2 to the transactivation domain of p53 in turn leads to ubiquitination at C-terminal lysines.(4) Thus, post-translational modifications of p53 contribute to maintaining only low levels of p53 in healthy resting and dividing cells.
In response to stress, p53 is stabilized. Jun N-terminal kinase (JNK) is activated by DNA damage and phosphorylates p53 at Thr 81; this releases JNK and enhances p53 transcriptional activity. Although mutation of Thr 155 and inhibition of the COP9 signalsome kinase both lead to an increased p53 half-life, the role of the signalsome in contribution to p53 stability in response to stress is unknown. Phosphorylation at the N-terminus of p53 by kinases, and competition for phosphorylation sites activated by different stress pathways, interfere with MDM2 complex formation. MDM2 is itself a target for post-translational modifications in response to stress, which can alter interaction with p53. The carboxyl terminus is also known to be important for stability although the mechanism is unclear.
Transcriptional activation of p53 requires both stabilization and activation.(5). The mechanisms for activation of transcriptional activity remain unresolved because chromatin binding does not seem to be rate limiting in vivo. Recently, several deacetylases have been shown to act on p53 and thereby inhibit p53-mediated transcription. Sites known to be acetylated in human p53 are Lys 320, Lys 373 and Lys 382. One model proposes that p53 targets histone acetyl transferases to the promoters of p53 activated target genes, and similarly, p53 targets the histone deacetylases to promoters of p53-repressed genes. N-terminal phosphorylation of p53 promotes C-terminal acetylation and the association of p300/CREB binding protein with the C-terminus of p53. Acetylation may interfere with C-terminal ubiquitination and alter interactions of p53 with other proteins and DNA. Acetylation may play a key role in stabilization of the p300:p53 complex and recent evidence suggests that DNA acts as an allosteric ligand to activate acetylation.(6) Recently, it has been proposed that the Human Immunodeficiency Virus Tat protein, in association with other proteins, may promote neoplasia during Acquired Immune-Deficiency Syndrome through inhibition of acetylation of Lys 320.(7)
In the course of understanding normal functioning of p53, the complexity of post-translational modifications to p53 has become apparent. The profile of p53 modifications differs depending not only upon the stress applied to the cell but is also cell type specific. Each of the target modifications, be it phosphorylation, acetylation, ubiquitination or sumoylation, are associated with one or more regulators, which themselves may be modified in response to stress. Although many cancers are associated with mutations in p53 that directly disrupt function, alterations to the post-translational state of p53 may also be significant in neoplastic disease: a mutant p53 has been described that may be stabilized by altered phosphorylation at Ser 392.(8) Monitoring the impact of mutations in upstream regulators of p53 through their impact on the post-translational profile of p53 will be of increasing importance.
The key tools available to researchers for the study of post-translational profiles of p53 are antibodies specific for each modified site within p53. These antibodies are particularly useful in Western blotting of immunoprecipitated p53, allowing the in vivo phosphorylation or acetylation status of p53 to be determined. The generation of new polyclonal and monoclonal antibodies with the high affinity needed to allow specific determination of the full profile of p53 modification at every target site will be an important development for p53 and cancer researchers.
About the authorKatherine Wood, Ph.D. is Director of Research and Development at Trevigen Inc. Dr. Wood studied p53 in neurological models of apoptosis prior to joining Trevigen, a research products company with focus on DNA damage and downstream consequences, including apoptosis. Trevigen has recently added anti-acetylated human and mouse p53 antibodies to their comprehensive DNA damage detection product line.
More information about p53 transcription and related antibodies is available from: