c-Abl regulates p53 levels under normal and stress conditions by preventing its nuclear export and ubiquitination

The p53 protein is subject to Mdm2-mediated degradation by the ubiquitin-proteasome pathway. This degradation requires interaction between p53 and Mdm2 and the subsequent ubiquitination and nuclear export of p53. Exposure of cells to DNA damage results in the stabilization of the p53 protein in the nucleus. However, the underlying mechanism of this effect is poorly defined. Here we demonstrate a key role for c-Abl in the nuclear accumulation of endogenous p53 in cells exposed to DNA damage. This effect of c-Abl is achieved by preventing the ubiquitination and nuclear export of p53 by Mdm2, or by human papillomavirus E6. c-Abl null cells fail to accumulate p53 efficiently following DNA damage. Reconstitution of these cells with physiological levels of c-Abl is sufficient to promote the normal response of p53 to DNA damage via nuclear retention. Our results help to explain how p53 is accumulated in the nucleus in response to DNA damage.     

Oligomerization of p53 Precedes its Association with Dynein and Nuclear Accumulation

Previous studies have identified several proteins that associate with microtubules and the dynein motor complex including p53, the glucocorticoid and the vitamin D receptors, and the APC (adenomatous polyposis coli) protein; but neither the residues important for this interaction nor the physical state of the proteins involved have been clarified. We observed in SN12C cells harboring a mutant p53 truncated at amino acid 336, impaired nuclear localization and impaired association with dynein. This finding was confirmed and extended by examining a series of truncated p53 proteins that identified residues 336 to 348 as crucial for association with dynein and nuclear transport. Point mutations identified the importance of residues involved in p53 oligomerization in this process, establishing a p53 oligomer as the cargo for dynein transport. The association of cytosolic p53 oligomers with dynein occurs independent of microtubules indicating that following this association, the p53/dynein complex then associates with microtubules and is transported to the peri-nuclear region. These studies suggest that mutations or modifications that affect p53 oligomerization not only interfere with DNA binding but also with its intracellular distribution. They also highlight the importance of an intact microtubule network in the trafficking of crucial cellular proteins.     

hGTSE-1 expression stimulates cytoplasmic localization of p53

hGTSE-1 (human G(2) and S phase-expressed-1) is a cell cycle-regulated protein mainly localized in the cytoplasm and apparently associated with the microtubules. hGTSE-1 is able to down-regulate levels and activity of the p53 tumor suppressor protein: it binds the C-terminal region of p53 and represses its ability to induce apoptosis after DNA damage. Here we report that, after DNA damage, hGTSE-1 becomes stabilized in a p53-independent way and accumulated in the nucleus. Further characterization of hGTSE-1 localization revealed increased nuclear staining in unstressed cells after treatment with the nuclear export inhibitor leptomycin B, or when a nuclear export signal (NES) located in its C-terminal region was mutated. Finally, we provide evidence that hGTSE-1 ectopic expression, in addition to p53 protein levels down-regulation, is able to enhance cytoplasmic localization of p53. Interestingly, NES-mutated hGTSE-1 accumulates in the nucleus, binds p53 but looses its ability to enhance cytoplasmic redistribution of p53 and to regulate p53 protein levels. Similarly, when wild type hGTSE-1 functions on p53 were analyzed in cells lacking Mdm2, it failed in regulating both p53 localization and protein levels, thus indicating that hGTSE-1 requires an intact NES and functional Mdm2 for the regulation of p53. Our results provide new insights into the mechanism of hGTSE-1 function, whereby its characterized nucleo-cytoplasmic shuttling ability is required to regulate p53.     

ATM and Chk2 kinase target the p53 cofactor Strap

The p53 cofactor Strap (stress responsive activator of p300) is directly targeted by the DNA damage signalling pathway where phosphorylation by ATM (ataxia telangiectasia mutated) kinase facilitates nuclear accumulation. Here, we show that Strap regulation reflects the coordinated interplay between different DNA damage-activated protein kinases, ATM and Chk2 (Checkpoint kinase 2), where phosphorylation by each kinase provides a distinct functional consequence on the activity of Strap. ATM phosphorylation prompts nuclear accumulation, which we show occurs by impeding nuclear export, whereas Chk2 phosphorylation augments protein stability once Strap has attained a nuclear location. These results highlight the various functional roles undertaken by the DNA damage signalling kinases in Strap control and, more generally, shed light on the pathways that contribute to the regulation of the p53 response.     

p53 is required for nuclear but not mitochondrial DNA damage-induced degeneration

While the consequences of nuclear DNA damage have been well studied, the exact consequences of acute and selective mitochondrial DNA (mtDNA) damage are less understood. DNA damaging chemotherapeutic drugs are known to activate p53-dependent apoptosis in response to sustained nuclear DNA damage. While it is recognized that whole-cell exposure to these drugs also damages mtDNA, the specific contribution of mtDNA damage to cellular degeneration is less clear. To examine this, we induced selective mtDNA damage in neuronal axons using microfluidic chambers that allow for the spatial and fluidic isolation of neuronal cell bodies (containing nucleus and mitochondria) from the axons (containing mitochondria). Exposure of the DNA damaging drug cisplatin selectively to only the axons induced mtDNA damage in axonal mitochondria, without nuclear damage. We found that this resulted in the selective degeneration of only the targeted axons that were exposed to DNA damage, where ROS was induced but mitochondria were not permeabilized. mtDNA damage-induced axon degeneration was not mediated by any of the three known axon degeneration pathways: apoptosis, axon pruning, and Wallerian degeneration, as Bax-deficiency, or Casp3-deficiency, or Sarm1-deficiency failed to protect the degenerating axons. Strikingly, p53, which is essential for degeneration after nuclear DNA damage, was also not required for degeneration induced with mtDNA damage. This was most evident when the p53-deficient neurons were globally exposed to cisplatin. While the cell bodies of p53-deficient neurons were protected from degeneration in this context, the axons farthest from the cell bodies still underwent degeneration. These results highlight how whole cell exposure to DNA damage activates two pathways of degeneration; a faster, p53-dependent apoptotic degeneration that is triggered in the cell bodies with nuclear DNA damage, and a slower, p53-independent degeneration that is induced with mtDNA damage.Subject terms: Cell biology, Neuroscience     

Differential roles of ATM- and Chk2-mediated phosphorylations of Hdmx in response to DNA damage

The p53 tumor suppressor plays a major role in maintaining genomic stability. Its activation and stabilization in response to double strand breaks (DSBs) in DNA are regulated primarily by the ATM protein kinase. ATM mediates several posttranslational modifications on p53 itself, as well as phosphorylation of p53's essential inhibitors, Hdm2 and Hdmx. Recently we showed that ATM- and Hdm2-dependent ubiquitination and subsequent degradation of Hdmx following DSB induction are mediated by phosphorylation of Hdmx on S403, S367, and S342, with S403 being targeted directly by ATM. Here we show that S367 phosphorylation is mediated by the Chk2 protein kinase, a downstream kinase of ATM. This phosphorylation, which is important for subsequent Hdmx ubiquitination and degradation, creates a binding site for 14-3-3 proteins which controls nuclear accumulation of Hdmx following DSBs. Phosphorylation of S342 also contributed to optimal 14-3-3 interaction and nuclear accumulation of Hdmx, but phosphorylation of S403 did not. Our data indicate that binding of a 14-3-3 dimer and subsequent nuclear accumulation are essential steps toward degradation of p53's inhibitor, Hdmx, in response to DNA damage. These results demonstrate a sophisticated control by ATM of a target protein, Hdmx, which itself is one of several ATM targets in the ATM-p53 axis of the DNA damage response.     

53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer

53BP1 is a conserved nuclear protein that is implicated in the DNA damage response. After irradiation, 53BP1 localizes rapidly to nuclear foci, which represent sites of DNA double strand breaks, but its precise function is unclear. Using small interference RNA (siRNA), we demonstrate that 53BP1 functions as a DNA damage checkpoint protein. 53BP1 is required for at least a subset of ataxia telangiectasia-mutated (ATM)-dependent phosphorylation events at sites of DNA breaks and for cell cycle arrest at the G2-M interphase after exposure to irradiation. Interestingly, in cancer cell lines expressing mutant p53, 53BP1 was localized to distinct nuclear foci and ATM-dependent phosphorylation of Chk2 at Thr 68 was detected, even in the absence of irradiation. In addition, Chk2 was phosphorylated at Thr 68 in more than 50% of surgically resected lung and breast tumour specimens from otherwise untreated patients [corrected]. We conclude that the constitutive activation of the DNA damage checkpoint pathway may be linked to the high frequency of p53 mutations in human cancer, as p53 is a downstream target of Chk2 and ATM.     

Actin Polymerization Negatively Regulates p53 Function by Impairing Its Nuclear Import in Response to DNA Damage

Actin, one of the most evolutionarily conservative proteins in eukaryotes, is distributed both in the cytoplasm and the nucleus, and its dynamics plays important roles in numerous cellular processes. Previous evidence has shown that actin interacts with p53 and this interaction increases in the process of p53 responding to DNA damage, but the physiological significance of their interaction remains elusive. Here, we show that DNA damage induces both actin polymerization and p53 accumulation. To further understand the implication of actin polymerization in p53 function, cells were treated with actin aggregation agent. We find that the protein level of p53 decrease. The change in p53 is a consequence of the polymeric actin anchoring p53 in the cytoplasm, thus impairing p53 nuclear import. Analysis of phosphorylation and ubiquitination of p53 reveals that actin polymerization promotes the p53 phosphorylation at Ser315 and reduces the stabilization of p53 by recruiting Aurora kinase A. Taken together, our results suggest that the actin polymerization serves as a negative modulator leading to the impairment of nuclear import and destabilization of p53. On the basis of our results, we propose that actin polymerization might be a factor participating in the process of orchestrating p53 function in response to DNA damage.     

Hypoxia induces accumulation of p53 protein, but activation of a G1-phase checkpoint by low-oxygen conditions is independent of p53 status.

It has been convincingly demonstrated that genotoxic stresses cause the accumulation of the tumor suppressor gene p53. One important consequence of increased p53 protein levels in response to DNA damage is the activation of a G1-phase cell cycle checkpoint. It has also been shown that G1-phase cell cycle checkpoints are activated in response to other stresses, such as lack of oxygen. Here we show that hypoxia and heat, agents that induce cellular stress primarily by inhibiting oxygen-dependent metabolism and denaturing proteins, respectively, also cause an increase in p53 protein levels. The p53 protein induced by heat is localized in the cytoplasm and forms a complex with the heat shock protein hsc70. The increase in nuclear p53 protein levels and DNA-binding activity and the induction of reporter gene constructs containing p53 binding sites following hypoxia occur in cells that are wild type for p53 but not in cells that possess mutant p53. However, unlike ionizing radiation, the accumulation of cells in G1 phase by hypoxia is not strictly dependent on wild-type p53 function. In addition, cells expressing the human papillomavirus E6 gene, which show increased degradation of p53 by ubiquitination and fail to accumulate p53 in response to DNA-damaging agents, do increase their p53 levels following heat and hypoxia. These results suggest that hypoxia is an example of a "nongenotoxic" stress which induces p53 activity by a different pathway than DNA-damaging agents.     

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.     

The presence of a p53-like protein during pea seed maturation and germination

ABSTRACT

The mechanisms that allow monitoring of DNA damage and the activation of repair systems in plants are poorly known. In mammalian cells the tumor suppressor protein p53 plays an important role in the checkpoint pathway induced by DNA damage. In this work, we investigated the presence and distribution of the p53-like protein in pea root tip nuclei and its role during early germination in relation to DNA damage. In pea seed, PFGE and TdT assays show that DNA fragmentation occurs during maturation and dry seed storage, and that this DNA fragmentation is repaired at the beginning of germination before the onset of proliferation. In the same seeds, the p53-like protein was found during maturation and germination. Immunoblotting characterization of this protein led to the identification of a single specific protein of about 94 kDa, more abundant at the beginning of the hydration process than in actively cycling cells. Furthermore, the p53-like protein revealed different nuclear distribution patterns, probably in relation to the formation of DNA fragments in dry seeds, and to the reactivation of repair mechanisms during early germination. These data suggest that the presence of a p53-like protein in quiescent or proliferating pea embryo cells is related to DNA damage, and serves for the maintenance of genetic information and the development of normal seedlings.     

Downregulation of VRK1 by p53 in response to DNA damage is mediated by the autophagic pathway

Human VRK1 induces a stabilization and accumulation of p53 by specific phosphorylation in Thr18. This p53 accumulation is reversed by its downregulation mediated by Hdm2, requiring a dephosphorylated p53 and therefore also needs the removal of VRK1 as stabilizer. This process requires export of VRK1 to the cytosol and is inhibited by leptomycin B. We have identified that downregulation of VRK1 protein levels requires DRAM expression, a p53-induced gene. DRAM is located in the endosomal-lysosomal compartment. Induction of DNA damage by UV, IR, etoposide and doxorubicin stabilizes p53 and induces DRAM expression, followed by VRK1 downregulation and a reduction in p53 Thr18 phosphorylation. DRAM expression is induced by wild-type p53, but not by common human p53 mutants, R175H, R248W and R273H. Overexpression of DRAM induces VRK1 downregulation and the opposite effect was observed by its knockdown. LC3 and p62 were also downregulated, like VRK1, in response to UV-induced DNA damage. The implication of the autophagic pathway was confirmed by its requirement for Beclin1. We propose a model with a double regulatory loop in response to DNA damage, the accumulated p53 is removed by induction of Hdm2 and degradation in the proteasome, and the p53-stabilizer VRK1 is eliminated by the induction of DRAM that leads to its lysosomal degradation in the autophagic pathway, and thus permitting p53 degradation by Hdm2. This VRK1 downregulation is necessary to modulate the block in cell cycle progression induced by p53 as part of its DNA damage response.     

Septins regulate actin organization and cell-cycle arrest through nuclear accumulation of NCK mediated by SOCS7

Mammalian septins are GTP-binding proteins the functions of which are not well understood. Knockdown of SEPT2, 6, and 7 causes stress fibers to disintegrate and cells to lose polarity. We now show that this phenotype is induced by nuclear accumulation of the adaptor protein NCK, as the effects can be reversed or induced by cytoplasmic or nuclear NCK, respectively. NCK is carried into the nucleus by SOCS7 (suppressor of cytokine signaling 7), which possesses nuclear import/export signals. SOCS7 interacts with septins and NCK through distinct domains. DNA damage induces actin and septin rearrangement and rapid nuclear accumulation of NCK and SOCS7. Moreover, NCK expression is essential for cell-cycle arrest. The septin-SOCS7-NCK axis intersects with the canonical DNA damage cascade downstream of ATM/ATR and is essential for p53 Ser15 phosphorylation. These data illuminate an unanticipated connection between septins, SOCS7, NCK signaling, and the DNA damage response.