Post-translational modifications of p53 tumor suppressor: determinants of its functional targets

Tumor suppressor p53 functions as a "guardian of the genome" to prevent cells from transformation. p53 is constitutively ubiquitinated and degradated in unstressed conditions, thereby suppressing the expression. However, cellular stimuli enable p53 to escape from the negative regulation, and then stably expressed p53 transactivates its target genes to induce cell cycle arrest, DNA repair, or apoptosis. Promoter preference of target genes is determined by modification status of p53. Because p53 has two critical roles in the decision of cell fate, stopping cell cycle to repair damaged DNA or induction of apoptotic cell death in response to DNA damage, elucidation of switching mechanisms on p53 functions is of particular importance. Here we review recent evidence how several post-translational modifications of p53 including methylation, phosphorylation, acetylation, and ubiquitination, affect the functions of p53 in response to cellular stress.     

Hzf Determines cell survival upon genotoxic stress by modulating p53 transactivation

A critical unresolved issue about the genotoxic stress response is how the resulting activation of the p53 tumor suppressor can lead either to cell-cycle arrest and DNA repair or to apoptosis. We show here that hematopoietic zinc finger (Hzf), a zinc-finger-containing p53 target gene, modulates p53 transactivation functions in an autoregulatory feedback loop. Hzf is induced by p53 and binds to its DNA-binding domain, resulting in preferential transactivation of proarrest p53 target genes over its proapoptotic target genes. Thus, p53 activation results in cell-cycle arrest in Hzf wild-type MEFs, while in Hzf(-/-) MEFs, apoptosis is induced. Exposure of Hzf null mice to ionizing radiation resulted in enhanced apoptosis in several organs, as compared to in wild-type mice. These findings provide novel insights into the regulation of p53 transactivation function and suggest that Hzf functions as a key player in regulating cell fate decisions in response to genotoxic stress.     

ATM and MET kinases are synthetic lethal with nongenotoxic activation of p53

The p53 tumor suppressor orchestrates alternative stress responses including cell cycle arrest and apoptosis, but the mechanisms defining cell fate upon p53 activation are poorly understood. Several small-molecule activators of p53 have been developed, including Nutlin-3, but their therapeutic potential is limited by the fact that they induce reversible cell cycle arrest in most cancer cell types. We report here the results of a genome-wide short hairpin RNA screen for genes that are lethal in combination with p53 activation by Nutlin-3, which showed that the ATM and MET kinases govern cell fate choice upon p53 activation. Genetic or pharmacological interference with ATM or MET activity converts the cellular response from cell cycle arrest into apoptosis in diverse cancer cell types without affecting expression of key p53 target genes. ATM and MET inhibitors also enable Nutlin-3 to kill tumor spheroids. These results identify new pathways controlling the cellular response to p53 activation and aid in the design of p53-based therapies.     

Stra6, a retinoic acid-responsive gene,participates in p53-induced apoptosis after DNA damage

Stra6 is the retinoic acid (RA)-inducible gene encoding the cellular receptor for holo-retinol binding protein. This transmembrane protein mediates the internalization of retinol, which then upregulates RA-responsive genes in target cells. Here, we show that Stra6 can be upregulated by DNA damage in a p53-dependent manner, and it has an important role in cell death responses. Stra6 expression induced significant amounts of apoptosis in normal and cancer cells, and it was also able to influence p53-mediated cell fate decisions by turning an initial arrest response into cell death. Moreover, inhibition of Stra6 severely compromised p53-induced apoptosis. We also found that Stra6 induced mitochondria depolarization and accumulation of reactive oxygen species, and that it was present not only at the cellular membrane but also in the cytosol. Finally, we show that these novel functions of Stra6 did not require downstream activation of RA signalling. Our results present a previously unknown link between the RA and p53 pathways and provide a rationale to use retinoids to upregulate Stra6, and thus enhance the tumour suppressor functions of p53. This may have implications for the role of vitamin A metabolites in cancer prevention and treatment.     

Coordination between cell proliferation and apoptosis after DNA damage in Drosophila

Exposure to genotoxic stress promotes cell cycle arrest and DNA repair or apoptosis. These “life” or “death” cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, the precise regulation of p53 is essential to maintain tissue homeostasis and to prevent cancer development. However, how cell cycle progression has an impact on p53 cell fate decision-making is mostly unknown. In this work, we demonstrate that Drosophila p53 proapoptotic activity can be impacted by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation-induced apoptosis. We show that p53 binding to the regulatory elements of the proapoptotic genes and its ability to activate their expression is compromised in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 proapoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 proapoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.Subject terms: Cell biology, Development, Gene regulation, Molecular biology     

Structural basis of DNA recognition by p53 tetramers

The tumor-suppressor protein p53 is among the most effective of the cell's natural defenses against cancer. In response to cellular stress, p53 binds as a tetramer to diverse DNA targets containing two decameric half-sites, thereby activating the expression of genes involved in cell-cycle arrest or apoptosis. Here we present high-resolution crystal structures of sequence-specific complexes between the core domain of human p53 and different DNA half-sites. In all structures, four p53 molecules self-assemble on two DNA half-sites to form a tetramer that is a dimer of dimers, stabilized by protein-protein and base-stacking interactions. The protein-DNA interface varies as a function of the specific base sequence in correlation with the measured binding affinities of the complexes. The new data establish a structural framework for understanding the mechanisms of specificity, affinity, and cooperativity of DNA binding by p53 and suggest a model for its regulation by regions outside the sequence-specific DNA binding domain.     

Protein kinase C delta regulates Ser46 phosphorylation of p53 tumor suppressor in the apoptotic response to DNA damage

The p53 tumor suppressor is activated in the cellular response to genotoxic stress. Transactivation of p53 target genes dictates cell cycle arrest and DNA repair or induction of apoptosis; however, a molecular mechanism responsible for these distinct functions remains unclear. Recent studies revealed that phosphorylation of p53 on Ser(46) was associated with induction of p53AIP1 expression, resulting in the commitment of the cell fate into apoptotic cell death. Moreover, upon exposure to genotoxic stress, p53DINP1 was expressed and recruited a kinase(s) to p53 that specifically phosphorylated Ser(46). Here, we show that the pro-apoptotic kinase, protein kinase C delta (PKCdelta), is involved in phosphorylation of p53 on Ser(46). PKCdelta-mediated phosphorylation is required for the interaction of PKCdelta with p53. The results also demonstrate that p53DINP1 associates with PKCdelta upon exposure to genotoxic agents. Consistent with these results, PKCdelta potentiates p53-dependent apoptosis by Ser(46) phosphorylation in response to genotoxic stress. These findings indicate that PKCdelta regulates p53 to induce apoptotic cell death in the cellular response to DNA damage.     

DAZAP2 acts as specifier of the p53 response to DNA damage

The DNA damage-responsive tumor suppressors p53 and HIPK2 are well established regulators of cell fate decision-making and regulate the cellular sensitivity to DNA-damaging drugs. Here, we identify Deleted in Azoospermia-associated protein 2 (DAZAP2), a small adaptor protein, as a novel regulator of HIPK2 and specifier of the DNA damage-induced p53 response. Knock-down or genetic deletion of DAZAP2 strongly potentiates cancer cell chemosensitivity both in cells and in vivo using a mouse tumour xenograft model. In unstressed cells, DAZAP2 stimulates HIPK2 polyubiquitination and degradation through interplay with the ubiquitin ligase SIAH1. Upon DNA damage, HIPK2 site-specifically phosphorylates DAZAP2, which terminates its HIPK2-degrading function and triggers its re-localization to the cell nucleus. Interestingly, nuclear DAZAP2 interacts with p53 and specifies target gene expression through modulating a defined subset of p53 target genes. Furthermore, our results suggest that DAZAP2 co-occupies p53 response elements to specify target gene expression. Collectively, our findings propose DAZAP2 as novel regulator of the DNA damage-induced p53 response that controls cancer cell chemosensitivity.     

p53-mediated transcriptional regulation and activation of the actin cytoskeleton regulatory RhoC to LIMK2 signaling pathway promotes cell survival

The central arbiter of cell fate in response to DNA damage is p53, which regulates the expression of genes involved in cell cycle arrest, survival and apoptosis. Although many responses initiated by DNA damage have been characterized, the role of actin cytoskeleton regulators is largely unknown. We now show that RhoC and LIM kinase 2 (LIMK2) are direct p53 target genes induced by genotoxic agents. Although RhoC and LIMK2 have well-established roles in actin cytoskeleton regulation, our results indicate that activation of LIMK2 also has a pro-survival function following DNA damage. LIMK inhibition by siRNA-mediated knockdown or selective pharmacological blockade sensitized cells to radio- or chemotherapy, such that treatments that were sub-lethal when administered singly resulted in cell death when combined with LIMK inhibition. Our findings suggest that combining LIMK inhibitors with genotoxic therapies could be more efficacious than single-agent administration, and highlight a novel connection between actin cytoskeleton regulators and DNA damage-induced cell survival mechanisms.     

p63 and p73: roles in development and tumor formation

The tumor suppressor p53 is critically important in the cellular damage response and is the founding member of a family of proteins. All three genes regulate cell cycle and apoptosis after DNA damage. However, despite a remarkable structural and partly functional similarity among p53, p63, and p73, mouse knockout studies revealed an unexpected functional diversity among them. p63 and p73 knockouts exhibit severe developmental abnormalities but no increased cancer susceptibility, whereas this picture is reversed for p53 knockouts. Neither p63 nor p73 is the target of inactivating mutations in human cancers. Genomic organization is more complex in p63 and p73, largely the result of an alternative internal promoter generating NH2-terminally deleted dominant-negative proteins that engage in inhibitory circuits within the family. Deregulated dominant-negative p73 isoforms might play an active oncogenic role in some human cancers. Moreover, COOH-terminal extensions specific for p63 and p73 enable further unique protein-protein interactions with regulatory pathways involved in development, differentiation, proliferation, and damage response. Thus, p53 family proteins take on functions within a wide biological spectrum stretching from development (p63 and p73), DNA damage response via apoptosis and cell cycle arrest (p53, TAp63, and TAp73), chemosensitivity of tumors (p53 and TAp73), and immortalization and oncogenesis (DeltaNp73).     

Decision making of the p53 network: Death by integration

The tumor suppressor protein p53 plays a central role in the multiple response pathways activated by DNA damage. In particular, p53 is involved in both the pro-survival response of cell cycle arrest and DNA repair, and the pro-death response of apoptosis. How does the p53 network coordinate the different pathways that lead to the opposite cell fates and what is its strategy in making the life-death decisions? To address these questions, we develop an integrated mathematical model that embraces three key modules of the p53 network: p53 core regulation, p53-induced cell cycle arrest and p53-dependent apoptosis initiation. Our analyses reveal that different aspects of the nuclear p53 dynamic profile are being used to differentially regulate the pro-survival and the pro-death modules. While the activation of the pro-survival module is dependent on the current or recent status of the DNA damage, the activation of the pro-death module relies on the accumulation or integration of the damage level over time. Thus, the cell will take the death fate if it cannot recover from the damage within a time period that is inversely proportional to the damage level. This “adaptive timer” strategy is likely to be adopted in other stress response systems.