RNF8-dependent and RNF8-independent regulation of 53BP1 in response to DNA damage

The DNA damage surveillance network orchestrates cellular responses to DNA damage through the recruitment of DNA damage-signaling molecules to DNA damage sites and the concomitant activation of protein phosphorylation cascades controlled by the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases. Activation of ATM/ATR triggers cell cycle checkpoint activation and adaptive responses to DNA damage. Recent studies suggest that protein ubiquitylation or degradation plays an important role in the DNA damage response. In this study, we examined the potential role of the proteasome in checkpoint activation and ATM/ATR signaling in response to UV light-induced DNA damage. HeLa cells treated with the proteasome inhibitor MG-132 showed delayed phosphorylation of ATM substrates in response to UV light. UV light-induced phosphorylation of 53BP1, as well as its recruitment to DNA damage foci, was strongly suppressed by proteasome inhibition, whereas the recruitment of upstream regulators of 53BP1, including MDC1 and H2AX, was unaffected. The ubiquitin-protein isopeptide ligase RNF8 was critical for 53BP1 focus targeting and phosphorylation in ionizing radiation-damaged cells, whereas UV light-induced 53BP1 phosphorylation and targeting exhibited partial dependence on RNF8 and the ubiquitin-conjugating enzyme UBC13. Suppression of RNF8 or UBC13 also led to subtle defects in UV light-induced G2/M checkpoint activation. These findings are consistent with a model in which RNF8 ubiquitylation pathways are essential for 53BP1 regulation in response to ionizing radiation, whereas RNF8-independent pathways contribute to 53BP1 targeting and phosphorylation in response to UV light and potentially other forms of DNA replication stress.     

Proapoptotic p53-interacting protein 53BP2 is induced by UV irradiation but suppressed by p53

p53 is an important mediator of the cellular stress response with roles in cell cycle control, DNA repair, and apoptosis. 53BP2, a p53-interacting protein, enhances p53 transactivation, impedes cell cycle progression, and promotes apoptosis through unknown mechanisms. We now demonstrate that endogenous 53BP2 levels increase following UV irradiation induced DNA damage in a p53-independent manner. In contrast, we found that the presence of a wild-type (but not mutant) p53 gene suppressed 53BP2 steady-state levels in cell lines with defined p53 genotypes. Likewise, expression of a tetracycline-regulated wild-type p53 cDNA in p53-null fibroblasts caused a reduction in 53BP2 protein levels. However, 53BP2 levels were not reduced if the tetracycline-regulated p53 cDNA was expressed after UV damage in these cells. This suggests that UV damage activates cellular factors that can relieve the p53-mediated suppression of 53BP2 protein. To address the physiologic significance of 53BP2 induction, we utilized stable cell lines with a ponasterone A-regulated 53BP2 cDNA. Conditional expression of 53BP2 cDNA lowered the apoptotic threshold and decreased clonogenic survival following UV irradiation. Conversely, attenuation of endogenous 53BP2 induction with an antisense oligonucleotide resulted in enhanced clonogenic survival following UV irradiation. These results demonstrate that 53BP2 is a DNA damage-inducible protein that promotes DNA damage-induced apoptosis. Furthermore, 53BP2 expression is highly regulated and involves both p53-dependent and p53-independent mechanisms. Our data provide new insight into 53BP2 function and open new avenues for investigation into the cellular response to genotoxic stress.     

UV irradiation triggers ubiquitin-dependent degradation of p21(WAF1) to promote DNA repair

p53-mediated increase in cyclin-dependent kinase inhibitor p21(WAF1) protein is thought to be the major mediator of cell cycle arrest after DNA damage. Previously p21 protein levels have been reported to increase or to decrease after UV irradiation. We show that p21 protein is degraded after irradiation of a variety of cell types with low but not high doses of UV. Cell cycle arrest occurs despite p21 degradation via Tyr(15) inhibitory phosphorylation of cdk2 and differs from the classical p21-dependent checkpoint elicited by ionizing radiation. In contrast to the basal turnover of p21, degradation of p21 switches to ubiquitin/Skp2-dependent proteasome pathway following UV irradiation. ATR activation after UV irradiation is essential for signaling p21 degradation. Finally, UV-induced p21 degradation is essential for optimal DNA repair. These results provide novel insight into regulation of p21 protein and its role in the cellular response to DNA damage.     

JimMY on the stage

Cellular DNA undergoes constant assault from a wide range of genotoxic stress. In order to maintain genome integrity, cells develop a repertoire of sophisticated systems to detect DNA damage and mediate cellular responses to DNA damage. Defects in the DNA damage response have been implicated in a variety of disorders including aging and cancer. Tumor suppressor p53 is a key intermediate in DNA damage response by inducing cell-cycle arrest to allow repair or promoting apoptosis to eliminate irreparably damaged cells. A recent study described a novel layer of p53-mediated cellular response to DNA damage, i.e. modulation of cell adhesion and motility. JMY, a p53 co-factor, was demonstrated to be a multifunctional protein that coordinates cell adhesion and motility with nuclear p53 response. These results suggest that abnormal JMY activity and/or localization could contribute to tumor invasion and reveal JMY as a potential therapeutic target.     

The absence of Ser389 phosphorylation in p53 affects the basal gene expression level of many p53-dependent genes and alters the biphasic response to UV exposure in mouse embryonic fibroblasts

Phosphorylation is important in p53-mediated DNA damage responses. After UV irradiation, p53 is phosphorylated specifically at murine residue Ser389. Phosphorylation mutant p53.S389A cells and mice show reduced apoptosis and compromised tumor suppression after UV irradiation. We investigated the underlying cellular processes by time-series analysis of UV-induced gene expression responses in wild-type, p53.S389A, and p53^−/− mouse embryonic fibroblasts. The absence of p53.S389 phosphorylation already causes small endogenous gene expression changes for 2,253, mostly p53-dependent, genes. These genes showed basal gene expression levels intermediate to the wild type and p53^−/−, possibly to readjust the p53 network. Overall, the p53.S389A mutation lifts p53-dependent gene repression to a level similar to that of p53^−/− but has lesser effect on p53-dependently induced genes. In the wild type, the response of 6,058 genes to UV irradiation was strictly biphasic. The early stress response, from 0 to 3 h, results in the activation of processes to prevent the accumulation of DNA damage in cells, whereas the late response, from 12 to 24 h, relates more to reentering the cell cycle. Although the p53.S389A UV gene response was only subtly changed, many cellular processes were significantly affected. The early response was affected the most, and many cellular processes were phase-specifically lost, gained, or altered, e.g., induction of apoptosis, cell division, and DNA repair, respectively. Altogether, p53.S389 phosphorylation seems essential for many p53 target genes and p53-dependent processes.     

Two faces of p53: aging and tumor suppression

The p53 tumor suppressor protein, often termed guardian of the genome, integrates diverse physiological signals in mammalian cells. In response to stress signals, perhaps the best studied of which is the response to DNA damage, p53 becomes functionally active and triggers either a transient cell cycle arrest, cell death (apoptosis) or permanent cell cycle arrest (cellular senescence). Both apoptosis and cellular senescence are potent tumor suppressor mechanisms that irreversibly prevent damaged cells from undergoing neoplastic transformation. However, both processes can also deplete renewable tissues of proliferation-competent progenitor or stem cells. Such depletion, in turn, can compromise the structure and function of tissues, which is a hallmark of aging. Moreover, whereas apoptotic cells are by definition eliminated from tissues, senescent cells can persist, acquire altered functions, and thus alter tissue microenvironments in ways that can promote both cancer and aging phenotypes. Recent evidence suggests that increased p53 activity can, at least under some circumstances, promote organismal aging. Here, we discuss the role of p53 as a key regulator of the DNA damage responses, and discuss how p53 integrates the outcome of the DNA damage response to optimally balance tumor suppression and longevity.     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation, and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.     

Mutation of mouse p53 Ser23 and the response to DNA damage

Recent studies have suggested that phosphorylation of human p53 at Ser20 is important for stabilizing p53 in response to DNA damage through disruption of the interaction between MDM2 and p53. To examine the requirement for this DNA damage-induced phosphorylation event in a more physiological setting, we introduced a missense mutation into the endogenous p53 gene of mouse embryonic stem (ES) cells that changes serine 23 (S23), the murine equivalent of human serine 20, to alanine (A). Murine embryonic fibroblasts harboring the p53(S23A) mutation accumulate p53 as well as p21 and Mdm2 proteins to normal levels after DNA damage. Furthermore, ES cells and thymocytes harboring the p53(S23A) mutation also accumulate p53 protein to wild-type levels and undergo p53-dependent apoptosis similarly to wild-type cells after DNA damage. Therefore, phosphorylation of murine p53 at Ser23 is not required for p53 responses to DNA damage induced by UV and ionizing radiation treatment.     

Characterisation of the sites of DNA damage-induced 53BP1 phosphorylation catalysed by ATM and ATR

The 53BP1 tumour suppressor, an important regulator of genome stability, is phosphorylated in response to ionising radiation (IR) by the ATM protein kinase, itself an important regulator of cellular responses to DNA damage. The only known sites of phosphorylation in 53BP1 are Ser25 and/or Ser29 but 53BP1 lacking these residues is still phosphorylated after DNA damage. In this study, we use mass spectrometry-based together with bioinformatic analysis to identify novel DNA damage-regulated sites of 53BP1 phosphorylation. Several new sites were identified that conform to the consensus Ser/Thr-Gln motif phosphorylated by ATM and related kinases. Phospho-specific antibodies were raised, and were used to demonstrate ATM-dependent phosphorylation of these residues in 53BP1 after exposure of cells to IR. Surprisingly, 53BP1 was also phosphorylated on these residues after exposure of cells to UV light. In this case, 53BP1 phosphorylation did not require ATM but required ATR instead. These data reveal that 53BP1 is phosphorylated on multiple residues in response to different types of DNA damage, and that 53BP1 is regulated by ATR in response to UV-induced DNA damage.     

Effects of hyperthermia on p53 protein expression and activity

Although p53 responses after DNA damage have been investigated extensively, p53 responses after heat shock, which exerts cytotoxic action by mechanisms other than direct induction of DNA damage, are less well characterized. We investigated, therefore, the effect of hyperthermic exposures on the levels and DNA-binding activity of p53. Experiments were carried out with U2OS and ML-1 cells, known to express wild-type p53 protein. Although heating at 41 degrees C for up to 6 h had only a small effect on p53 levels or DNA binding activity, exposure to temperatures between 42.5 and 45.5 degrees C caused an immediate decrease in protein levels that was associated with a reduction in DNA binding activity. This observation is compatible with a high lability of p53 to heat shock, or heat sensitivity of the pathway regulating p53 levels in non-stressed cells. When cells were heated to 42.5 degrees C and returned to normal temperatures, a strong p53 response associated with an increase in protein levels and DNA binding activity was observed, suggesting the production of p53-inducing cellular damage. At higher temperatures, however, this response was compromised in an exposure-time-dependent manner. The increase in DNA binding activity was more heat sensitive than the increase in p53 levels and was inhibited at lower temperatures and shorter exposure times. Thus, the pathway of p53 activation is itself heat sensitive and compromised at high levels of exposure. Compared to p53 activation after exposure to ionizing radiation, heat-induced activation is rapid and short lived. When cells were exposed to combined heat and radiation, the response observed approximated that of cells exposed to heat alone. Wortmannin at 10 microM inhibited p53 activation for up to 2 h after heat shock suggesting the involvement of wortmannin-sensitive kinases, such as DNA-PK and ATM. Heat shock causes phosphorylation of p53 at Serine-15, but there is no correlation between phosphorylation at this site and activation of the protein. The results in aggregate indicate p53 activation in the absence of DNA damage by a heat-sensitive mechanism operating with faster kinetics than radiation-induced p53 activation. The former response may induce pathways preventing other stimuli from activating p53, as heat-induced activation of p53 is dominant over activation of p53 by DNA damage in combined-treatment experiments. These observations suggest means for abrogating p53 induction after DNA damage with the purpose of potentiating response and enhancing cell killing.