A phosphorylation-dependent switch in the disordered p53 transactivation domain regulates DNA binding

The tumor-suppressor p53 is a critical regulator of the cellular response to DNA damage and is tightly regulated by posttranslational modifications. Thr55 in the AD2 interaction motif of the N-terminal transactivation domain functions as a phosphorylation-dependent regulatory switch that modulates p53 activity. Thr55 is constitutively phosphorylated, becomes dephosphorylated upon DNA damage, and is subsequently rephosphorylated to facilitate dissociation of p53 from promoters and inactivate p53-mediated transcription. Using NMR and fluorescence spectroscopy, we show that Thr55 phosphorylation inhibits DNA-binding by enhancing competitive interactions between the disordered AD2 motif and the structured DNA-binding domain (DBD). Nonphosphorylated p53 exhibits positive cooperativity in binding DNA as a tetramer. Upon phosphorylation of Thr55, cooperativity is abolished and p53 binds initially to cognate DNA sites as a dimer. As the concentration of phosphorylated p53 is further increased, a second dimer binds and causes p53 to dissociate from the DNA, resulting in a bell-shaped binding curve. This autoinhibition is driven by favorable interactions between the DNA-binding surface of the DBD and the multiple phosphorylated AD2 motifs within the tetramer. These interactions are augmented by additional phosphorylation of Ser46 and are fine-tuned by the proline-rich domain (PRD). Removal of the PRD strengthens the AD2–DBD interaction and leads to autoinhibition of DNA binding even in the absence of Thr55 phosphorylation. This study reveals the molecular mechanism by which the phosphorylation status of Thr55 modulates DNA binding and controls both activation and termination of p53-mediated transcriptional programs at different stages of the cellular DNA damage response.

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Interaction between p53 N terminus and core domain regulates specific and nonspecific DNA binding

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903077116 ◽

2019 ◽

Vol 116 (18) ◽

pp. 8859-8868 ◽

Cited By ~ 10

Author(s):

Fan He ◽

Wade Borcherds ◽

Tanjing Song ◽

Xi Wei ◽

Mousumi Das ◽

...

Keyword(s):

Dna Binding ◽

Posttranslational Modifications ◽

Dynamic Control ◽

Transactivation Domains ◽

Binding Surface ◽

N Terminus ◽

Response To Stress ◽

Stress Signals

The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. Dynamic control of p53 degradation and DNA binding in response to stress signals are critical for tumor suppression. The p53 N terminus (NT) contains two transactivation domains (TAD1 and TAD2), a proline-rich region (PRR), and multiple phosphorylation sites. Previous work revealed the p53 NT reduced DNA binding in vitro. Here, we show that TAD2 and the PRR inhibit DNA binding by directly interacting with the sequence-specific DNA binding domain (DBD). NMR spectroscopy revealed that TAD2 and the PRR interact with the DBD at or near the DNA binding surface, possibly acting as a nucleic acid mimetic to competitively block DNA binding. In vitro and in vivo DNA binding analyses showed that the NT reduced p53 DNA binding affinity but improved the ability of p53 to distinguish between specific and nonspecific sequences. MDMX inhibits p53 binding to specific target promoters but stimulates binding to nonspecific chromatin sites. The results suggest that the p53 NT regulates the affinity and specificity of DNA binding by the DBD. The p53 NT-interacting proteins and posttranslational modifications may regulate DNA binding, partly by modulating the NT–DBD interaction.

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Differential Contribution of N- and C-Terminal Regions of HIF1α and HIF2α to Their Target Gene Selectivity

International Journal of Molecular Sciences ◽

10.3390/ijms21249401 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9401

Author(s):

Antonio Bouthelier ◽

Florinda Meléndez-Rodríguez ◽

Andrés A. Urrutia ◽

Julián Aragonés

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Cellular Response ◽

Target Gene ◽

Target Genes ◽

Transactivation Domain ◽

Transactivation Domains ◽

Relative Contribution

Cellular response to hypoxia is controlled by the hypoxia-inducible transcription factors HIF1α and HIF2α. Some genes are preferentially induced by HIF1α or HIF2α, as has been explored in some cell models and for particular sets of genes. Here we have extended this analysis to other HIF-dependent genes using in vitro WT8 renal carcinoma cells and in vivo conditional Vhl-deficient mice models. Moreover, we generated chimeric HIF1/2 transcription factors to study the contribution of the HIF1α and HIF2α DNA binding/heterodimerization and transactivation domains to HIF target specificity. We show that the induction of HIF1α-dependent genes in WT8 cells, such as CAIX (CAR9) and BNIP3, requires both halves of HIF, whereas the HIF2α transactivation domain is more relevant for the induction of HIF2 target genes like the amino acid carrier SLC7A5. The HIF selectivity for some genes in WT8 cells is conserved in Vhl-deficient lung and liver tissue, whereas other genes like Glut1 (Slc2a1) behave distinctly in these tissues. Therefore the relative contribution of the DNA binding/heterodimerization and transactivation domains for HIF target selectivity can be different when comparing HIF1α or HIF2α isoforms, and that HIF target gene specificity is conserved in human and mouse cells for some of the genes analyzed.

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An ATP/ADP-Dependent Molecular Switch Regulates the Stability of p53-DNA Complexes

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7501 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7501-7510 ◽

Cited By ~ 24

Author(s):

Andrei L. Okorokov ◽

Jo Milner

Keyword(s):

Dna Damage ◽

Dna Binding ◽

Cellular Response ◽

Atp Hydrolysis ◽

P53 Protein ◽

Molecular Switch ◽

Dna Complexes ◽

Switch Mechanism ◽

The Stability ◽

First Time

ABSTRACT Interaction with DNA is essential for the tumor suppressor functions of p53. We now show, for the first time, that the interaction of p53 with DNA can be stabilized by small molecules, such as ADP and dADP. Our results also indicate an ATP/ADP molecular switch mechanism which determines the off-on states for p53-DNA binding. This ATP/ADP molecular switch requires dimer-dimer interaction of the p53 tetramer. Dissociation of p53-DNA complexes by ATP is independent of ATP hydrolysis. Low-level ATPase activity is nonetheless associated with ATP-p53 interaction and may serve to regenerate ADP-p53, thus recycling the high-affinity DNA binding form of p53. The ATP/ADP regulatory mechanism applies to two distinct types of p53 interaction with DNA, namely, sequence-specific DNA binding (via the core domain of the p53 protein) and binding to sites of DNA damage (via the C-terminal domain). Further studies indicate that ADP not only stabilizes p53-DNA complexes but also renders the complexes susceptible to dissociation by specific p53 binding proteins. We propose a model in which the DNA binding functions of p53 are regulated by an ATP/ADP molecular switch, and we suggest that this mechanism may function during the cellular response to DNA damage.

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Cellular DNA Damage Response to Zinc Oxide Nanoparticles Hinges on the Tumor Suppressor p53 Pathway

Proceedings of the 14th Asia Pacific Confederation of Chemical Engineering Congress ◽

10.3850/978-981-07-1445-1_805 ◽

2012 ◽

Author(s):

David Leong

Keyword(s):

Dna Damage ◽

Zinc Oxide ◽

Tumor Suppressor ◽

Dna Damage Response ◽

Zinc Oxide Nanoparticles ◽

Oxide Nanoparticles ◽

Tumor Suppressor P53 ◽

P53 Pathway ◽

Damage Response ◽

Cellular Dna

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Biophysical investigation of the dual binding surfaces of human transcription factors FOXO4 and p53

10.1101/2021.01.11.425814 ◽

2021 ◽

Author(s):

Jinwoo Kim ◽

Dabin Ahn ◽

Chin-Ju Park

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Cellular Senescence ◽

Structural Information ◽

Titration Calorimetry ◽

Transactivation Domain ◽

Forkhead Box ◽

Binding Surface ◽

Forkhead Domain ◽

Oncogenic Stress

AbstractCellular senescence is protective against external oncogenic stress, but its accumulation causes aging-related diseases. Forkhead box O4 (FOXO4) and p53 are human transcription factors known to promote senescence by interacting in the promyelocytic leukemia bodies. Inhibiting their binding is a strategy for inducing apoptosis of senescent cells, but the binding surfaces that mediate the interaction of FOXO4 and p53 remain elusive. Here, we investigated two binding sites involved in the interaction between FOXO4 and p53 by using NMR spectroscopy. NMR chemical shift perturbation analysis showed that the binding between FOXO4’s forkhead domain (FHD) and p53’s transactivation domain (TAD), and between FOXO4’s C-terminal transactivation domain (CR3) and p53’s DNA binding domain (DBD), mediate the FOXO4-p53 interaction. Also, we showed that the CR3-binding surface of FOXO4 FHD interacts with p53 TAD2, and four residues of FOXO4 CR3 interact with the DNA-binding surface of p53 DBD. Further isothermal titration calorimetry experiments showed that the FOXO4 FHD-p53 TAD interaction takes precedence with high affinity and that the FOXO4 CR3-p53 DBD interaction follows. This work provides structural information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence.

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p53 protein at the hub of cellular DNA damage response pathways through sequence-specific and non-sequence-specific DNA binding

Carcinogenesis ◽

10.1093/carcin/22.6.851 ◽

2001 ◽

Vol 22 (6) ◽

pp. 851-860 ◽

Cited By ~ 112

Author(s):

Y. Liu

Keyword(s):

Dna Damage ◽

Dna Binding ◽

Dna Damage Response ◽

P53 Protein ◽

Damage Response ◽

Cellular Dna

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RAX Activates Tumor Suppressor p53.

Blood ◽

10.1182/blood.v108.11.1449.1449 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1449-1449

Author(s):

Yu Pan ◽

Richard L. Bennett ◽

W. Stratford May

Keyword(s):

Dna Damage ◽

Tumor Suppressor ◽

Cellular Response ◽

State Level ◽

Hematologic Malignancies ◽

Dominant Negative ◽

Luciferase Reporter ◽

G1 Arrest ◽

Tumor Suppressor P53 ◽

Stress Dependent

Abstract Recently, our laboratory identified RAX as a unique cellular activator for the interferon-induced double-stranded RNA-dependent protein kinase, PKR. Cellular stresses such as IL-3 withdrawal from factor dependent hematopoietic cells, inflammatory cytokine and chemotherapy treatment or viral infection promote RAX phosphorylation with activation of PKR leading to inhibition of new protein synthesis. In addition, PKR can also regulate the transcription factors p53, STAT1 and NF-kB. Now we report a novel activity for RAX in regulating the expression and activity of the tumor suppressor, p53. Results indicate that increased RAX expression in p53 expressing cells, such as HEK293, mouse embryo fibroblast (MEF) or human osteosarcoma U2OS promotes an increase in the steady-state level of endogenous p53 and its activation of the p53 target gene, p21. Furthermore, when RAX is expressed in U2OS cells or coexpressed with p53 in p53 null H1299 cells, enhanced, dose-dependent p53 transcriptional activity is observed as assessed using a p53 luciferase reporter. Consistent with these findings, flow cytometry demonstrates that either RAX or PKR can stimulate p53-dependent G1 arrest that precedes apoptosis. Since p53 is a major ‘sensor’ for DNA damage leading to cell cycle arrest and apoptosis, we also examined whether RAX enhances p53’s response to DNA damage. Following gamma irradiation or Cisplatin treatment of p53 expressing cells, expression of exogenous RAX augments while ‘knock down’ of endogenous RAX by siRNA or expression of the dominant-negative, RAX (S18A) mutant, potently blocks any increase in p53 and p21 expression compared to vector-only control cells. While we find no evidence that RAX directly interacts with p53, results indicate that RAX promotes p53 SUMOylation but does not affect p53 ubiquitination or interaction with MDM2. We now propose that RAX augments the p53 stress-dependent cellular response potentially by a PKR-dependent mechanism involving SUMOylation. Since p53 is only mutated in apparently 15% of hematologic malignancies versus more than 50% in solid tumors, it is envisioned that a more detailed understanding of the mechanism by which RAX activates p53 may serve as a basis for novel antileukemic strategies.

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Viral Transport of DNA Damage That Mimics a Stalled Replication Fork

Journal of Virology ◽

10.1128/jvi.79.1.569-580.2005 ◽

2005 ◽

Vol 79 (1) ◽

pp. 569-580 ◽

Cited By ~ 45

Author(s):

Jaana Jurvansuu ◽

Kenneth Raj ◽

Andrzej Stasiak ◽

Peter Beard

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Cellular Response ◽

Replication Fork ◽

Dna Polymerase Delta ◽

Viral Dna ◽

Focus Formation ◽

Viral Dna Replication ◽

Cross Links ◽

Cellular Dna

ABSTRACT Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.

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Cytokine Storm as a Cellular Response to dsDNA Breaks: A New Proposal

Frontiers in Immunology ◽

10.3389/fimmu.2021.622738 ◽

2021 ◽

Vol 12 ◽

Author(s):

Snehal Shabrish ◽

Indraneel Mittra

Keyword(s):

Dna Damage ◽

Cellular Response ◽

The Body ◽

Host Cells ◽

Preclinical Studies ◽

Cytokine Storm ◽

Vicious Cycle ◽

Cascading Effect ◽

Microbial Invasion ◽

Cellular Dna

Pathogenesis of cytokine storm is poorly understood. In this article we propose a new mechanism and suggest innovative therapeutic avenues for its prevention. We have reported that particles of cell-free chromatin (cfCh) that are released from the billions of cells that die in the body everyday can illegitimately integrate into genomes of healthy cells to trigger dsDNA breaks. The latter leads to apoptosis and/or intense activation of inflammatory cytokines in the affected cells. We hypothesise that a similar phenomenon of dsDNA breaks and inflammation is involved in cytokine storm. The abundant cfCh particles that are released from dying host cells following viral/microbial invasion initiate a cascading effect of more cell death resulting in a vicious cycle of further DNA damage, apoptosis and hyper-inflammation which culminate in cytokine storm. We propose that this unrelenting vicious cycle of cellular DNA damage and cytokine storm may be the underlying cause of high mortality from severe COVID-19. We discuss results of our preclinical studies wherein we have shown that endotoxin induced cytokine storm in mice can be reversed by three different agents that have the ability to inactivate cfCh. These agents may be worthy of investigation in clinical trials to reduce mortality from COVID-19.

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