Ser18 and Ser23 Phosphorylation Plays Synergistic Roles in Activating p53-Dependent Neuronal Apoptosis

Tumor suppressor p53 is required for the neuronal apoptosis in response to DNA double-stranded break (DSB) damage. Posttranslational modifications such as phosphorylation play important roles in activating p53-dependent apoptosis after DNA damage. In support of this notion, our recent studies indicate that Ser18 and Ser23 phosphorylation together plays critical roles in activating p53 apoptotic activities in vivo. Thymocytes derived from p53S18/23A mice are essentially resistant to p53-dependent apoptosis after DNA DSB damage. In addition, identical to p53-deficiency, p53S18/23A knock-in mutation completely rescues the embryonic lethality of XRCC4-/- mice, which die of the massive p53-dependent apoptosis of embryonic neurons likely as a result of accumulated endogenous DNA damage. To dissect the contribution of Ser18 and Ser23 phosphorylation to p53-dependent neuronal apoptosis, we report here that neither p53S18A nor p53S23A mutation alone can rescue the embryonic lethality of XRCC4-/- mice. Therefore, Ser18 and Ser23 phosphorylation plays synergistic and critical roles in activating p53-dependent neuronal apoptosis.     

Phosphorylation of serine 18 regulates distinct p53 functions in mice

The p53 protein acts a tumor suppressor by inducing cell cycle arrest and apoptosis in response to DNA damage or oncogene activation. Recently, it has been proposed that phosphorylation of serine 15 in human p53 by ATM (mutated in ataxia telangiectasia) kinase induces p53 activity by interfering with the Mdm2-p53 complex formation and inhibiting Mdm2-mediated destabilization of p53. Serine 18 in murine p53 has been implicated in mediating an ATM- and ataxia telangiectasia-related kinase-dependent growth arrest. To explore further the physiological significance of phosphorylation of p53 on Ser18, we generated mice bearing a serine-to-alanine mutation in p53. Analysis of apoptosis in thymocytes and splenocytes following DNA damage revealed that phosphorylation of serine 18 was required for robust p53-mediated apoptosis. Surprisingly, p53Ser18 phosphorylation did not alter the proliferation rate of embryonic fibroblasts or the p53-mediated G(1) arrest induced by DNA damage. In addition, endogenous basal levels and DNA damage-induced levels of p53 were not affected by p53Ser18 phosphorylation. p53Ala18 mice developed normally and were not susceptible to spontaneous tumorigenesis, and the reduced apoptotic function of p53Ala18 did not rescue the embryo-lethal phenotype of Mdm2-null mice. These results indicate that phosphorylation of the ATM target site on p53 specifically regulates p53 apoptotic function and further reveal that phosphorylation of p53 serine 18 is not required for p53-mediated tumor suppression.     

Ser18 and 23 phosphorylation is required for p53-dependent apoptosis and tumor suppression

Mouse p53 is phosphorylated at Ser18 and Ser23 after DNA damage. To determine whether these two phosphorylation events have synergistic functions in activating p53 responses, we simultaneously introduced Ser18/23 to Ala mutations into the endogenous p53 locus in mice. While partial defects in apoptosis are observed in p53S18A and p53S23A thymocytes exposed to IR, p53-dependent apoptosis is essentially abolished in p53S18/23A thymocytes, indicating that these two events have critical and synergistic roles in activating p53-dependent apoptosis. In addition, p53S18/23A, but not p53S18A, could completely rescue embryonic lethality of Xrcc4(-/-) mice that is caused by massive p53-dependent neuronal apoptosis. However, certain p53-dependent functions, including G1/S checkpoint and cellular senescence, are partially retained in p53(S18/23A) cells. While p53(S18A) mice are not cancer prone, p53S18/23A mice developed a spectrum of malignancies distinct from p53S23A and p53(-/-) mice. Interestingly, Xrcc4(-/-)p53S18/23A mice fail to develop tumors like the pro-B cell lymphomas uniformly developed in Xrcc4(-/-) p53(-/-) animals, but exhibit developmental defects typical of accelerated ageing. Therefore, Ser18 and Ser23 phosphorylation is important for p53-dependent suppression of tumorigenesis in certain physiological context.     

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.     

Defective apoptosis and B-cell lymphomas in mice with p53 point mutation at Ser 23

Phosphorylation of the p53 tumor suppressor at Ser20 (murine Ser23) has been proposed to be critical for disrupting p53 interaction with its negative regulator, MDM2, and allowing p53 stabilization. To determine the importance of Ser23 for the function of p53 in vivo, we generated a mouse in which the endogenous p53 locus was targeted to replace Ser23 with alanine. We show that, in mouse embryonic fibroblasts generated from Ser23 mutant mice, Ser23 mutation did not dramatically reduce IR-induced p53 protein stabilization or p53-dependent cell cycle arrest. However, in Ser23 mutant thymocytes and in the developing cerebellum, p53 stabilization following IR was decreased and resistance to apoptosis was observed. Homozygous Ser23 mutant animals had a reduced lifespan, but did not develop thymic lymphomas or sarcomas that are characteristic of p53-/- mice. Instead, Ser23 mutant animals died between 1 and 2 years with tumors that were most commonly of B-cell lineage. These data support an important role for Ser20/23 phosphorylation in p53 stabilization, apoptosis and tumor suppression.     

ATR is not required for p53 activation but synergizes with p53 in the replication checkpoint.

ATR (ataxia telangiectasia and Rad-3-related) is a protein kinase required for survival after DNA damage. A critical role for ATR has been hypothesized to be the regulation of p53 and other cell cycle checkpoints. ATR has been shown to phosphorylate p53 at Ser(15), and this damage-induced phosphorylation is diminished by expression of a catalytically inactive (ATR-kd) mutant. p53 function could not be examined directly in prior studies of ATR, however, because p53 was mutant or because cells expressed the SV40 large T antigen that blocks p53 function. To test the interactions of ATR and p53 directly we generated human U2OS cell lines inducible for either wild-type or kinase-dead ATR that also have an intact p53 pathway. Indeed, ATR-kd expression sensitized these cells to DNA damage and caused a transient decrease in damage-induced serine 15 phosphorylation of p53. However, we found that the effects of ATR-kd expression do not result in blocking the response of p53 to DNA damage. Specifically, prior ATR-kd expression had no effect on DNA damage-induced p53 protein up-regulation, p53-DNA binding, p21 mRNA up-regulation, or G(1) arrest. Instead of promoting survival via p53 regulation, we found that ATR protects cells by delaying the generation of mitotic phosphoproteins and inhibiting premature chromatin condensation after DNA damage or hydroxyurea. Although p53 inhibition (by E6 or MDM2 expression) had little effect on premature chromatin condensation, when combined with ATR-kd expression there was a marked loss of the replication checkpoint. We conclude that ATR and p53 can function independently but that loss of both leads to synergistic disruption of the replication checkpoint.     

AMP-Activated Protein Kinase Induces p53 by Phosphorylating MDMX and Inhibiting Its Activity

AMP-activated protein kinase (AMPK) has been shown to activate p53 in response to metabolic stress. However, the underlying mechanisms remain unclear. Here we show that metabolic stresses induce AMPK-mediated phosphorylation of human MDMX on Ser342 in vitro and in cells, leading to enhanced association between MDMX and 14-3-3. This markedly inhibits p53 ubiquitylation and significantly stabilizes and activates p53. By striking contrast, no phosphorylation of MDM2 by AMPK was noted. AMPK-mediated MDMX phosphorylation, MDMX–14-3-3 binding, and p53 activation were drastically reduced in mouse embryo fibroblasts harboring endogenous MDMX with S341A (mouse homologue of human serine 342), S367A, and S402A (mouse homologue of human serine 403) mutations. Moreover, deficiency of AMPK prevented MDMX–14-3-3 interaction and p53 activation. The activation of p53 through AMPK-mediated MDMX phosphorylation and inactivation was further confirmed by using cell and animal model systems with two AMPK activators, metformin and salicylate (the active form of aspirin). Together, the results unveil a mechanism by which metabolic stresses activate AMPK, which, in turn, phosphorylates and inactivates MDMX, resulting in p53 stabilization and activation.     

Loss of p53 Ser18 and Atm results in embryonic lethality without cooperation in tumorigenesis

Phosphorylation at murine Serine 18 (human Serine 15) is a critical regulatory process for the tumor suppressor function of p53. p53Ser18 residue is a substrate for ataxia-telangiectasia mutated (ATM) and ATM-related (ATR) protein kinases. Studies of mice with a germ-line mutation that replaces Ser18 with Ala (p53(S18A) mice) have demonstrated that loss of phosphorylation of p53Ser18 leads to the development of tumors, including lymphomas, fibrosarcomas, leukemia and leiomyosarcomas. The predominant lymphoma is B-cell lymphoma, which is in contrast to the lymphomas observed in Atm(-/-) animals. This observation and the fact that multiple kinases phosphorylate p53Ser18 suggest Atm-independent tumor suppressive functions of p53Ser18. Therefore, in order to examine p53Ser18 function in relationship to ATM, we analyzed the lifespan and tumorigenesis of mice with combined mutations in p53Ser18 and Atm. Surprisingly, we observed no cooperation in survival and tumorigenesis in compound p53(S18A) and Atm(-/-) animals. However, we observed embryonic lethality in the compound mutant animals. In addition, the homozygous p53Ser18 mutant allele impacted the weight of Atm(-/-) animals. These studies examine the genetic interaction of p53Ser18 and Atm in vivo. Furthermore, these studies demonstrate a role of p53Ser18 in regulating embryonic survival and motor coordination.     

Treatment of Chronic Myeloid Leukemia Cells with Imatinib (STI571) Impairs p53 Accumulation in Response to DNA Damage

Chronic myelogenous leukaemia (CML) is induced by the Bcr-Abl fusion protein. Inhibition of Bcr-Abl by STI571 is widely used to treat CML patients. Unlike in most cancer types, the frequency of p53 mutations in CML is low. Here, we investigated the effect of STI571 treatment of CML cells on p53 regulation. Exposure of CML cells, including established cell lines and freshly isolated cells from patients, to STI571 reduced p53 protein levels, and severely impaired its accumulation in response to DNA damage. This may be explained by the status of p53 serine 20 phosphorylation. In non-stressed CML cells, serine 20 of p53 is constitutively phosphorylated by Chk1, and is inhibited by STI571. In response to DNA damage, however, this phosphorylation is mediated by Chk1 and Chk2, and is only partially inhibited by STI571. CML cells expressing wild-type p53 are more resistant to treatment with STI571, but moderately more sensitive to DNA damage, than CML cells lacking p53. An enhanced induction of apoptosis by STI571 and DNA damage is observed in CML cells bearing wild-type p53, but not in cells lacking functional p53. This implies that the status of p53 may affect the response of CML cells to this combined treatment.     

Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells

This protocol describes a rapid, precise method for generating sets of embryonic stem (ES) cells or mouse embryonic fibroblasts (MEFs) harboring point mutations in the p53 tumor suppressor gene (officially known as Trp53). The strategy uses cells from the Trp53 (p53-null) 'platform' mouse, which allows site-specific integration of plasmid DNA into the Trp53 locus. Simple PCR protocols identify correctly targeted clones and immunoblots verify re-expression of the protein. We also present protocol modifications needed for efficient recovery of MEF clones expressing p53 constructs that retain wild-type function, including growth at low (3%) oxygen and transient downregulation of p53 regulators to forestall cell senescence of primary MEFs. A library of cell lines expressing various p53 mutants derived from the same population of primary fibroblasts or platform ES cells can be acquired and screened in less than 1 month.     

ATM mediates interdependent activation of p53 and ERK through formation of a ternary complex with p-p53 and p-ERK in response to DNA damage

DNA damage in eukaryotic cells induces signaling pathways mediated by the ATM, p53 and ERK proteins, but the interactions between these pathways are not completely known. To address this issue, we performed a time course analysis in human embryonic fibroblast cells treated with DNA-damaging agents. DNA damage induced the phosphorylation of p53 at Ser 15 (p-p53) and the phosphorylation of ERK (p-ERK). Inhibition of p53 by a dominant negative mutant or in p53(-/-) fibroblast cells abolished ERK phosphorylation. ERK inhibitor prevented p53 phosphorylation, indicating that phosphorylations of p53 and p-ERK are interdependent each other. A time course analysis showed that ATM interacted with p-p53 and p-ERK in early time (0.5 h) and interaction between ATM-bound p-p53 and p-ERK or ATM-bound p-ERK and p-p53 occurred in late time (3 h) of DNA damage. These results indicate that ATM mediates interdependent activation of p53 and ERK through formation of a ternary complex between p-p53 and p-ERK in response to DNA damage to cause growth arrest.