Phospho-TP53 (Ser20) Antibody

What is the biological significance of p53 phosphorylation at Ser20?

Phosphorylation of p53 at Ser20 plays a crucial role in regulating p53 stability and activity. This modification occurs in response to various stresses and has several key functions:

• Disruption of p53-MDM2 interaction: Phosphorylation at Ser20 reduces the binding of p53 to its negative regulator MDM2, preventing p53 ubiquitination and proteasomal degradation .

• Enhanced p53 stability: By interfering with MDM2-mediated degradation, Ser20 phosphorylation leads to stabilization and accumulation of p53 protein .

• Increased transcriptional activity: Ser20 phosphorylation stabilizes the binding of the transcriptional co-activator p300 to p53 through interactions with multiple LxxLL peptide binding domains on p300 .

• Enhanced tetramerization: This modification promotes p53 tetramerization, which is essential for its DNA binding and transcriptional activities .

Studies with p53-Ala20 mutant (where Ser20 is replaced by Ala) demonstrate significantly impaired p53 apoptotic activity and increased susceptibility to negative regulation by MDM2, confirming the critical role of this phosphorylation site .

Which kinases phosphorylate p53 at Ser20 in response to different stress stimuli?

Different stress stimuli activate distinct kinase pathways that target p53 at Ser20:

Notably, ATM and ATR themselves cannot directly phosphorylate p53 at Ser20 (which lacks the SQ motif required for ATM substrates) but likely activate downstream kinases that perform this function .

How do different antibodies detect Phospho-p53 (Ser20) in experimental settings?

Several antibodies are available for detecting p53 phosphorylated at Ser20:

• Phospho-specific antibodies (e.g., AbS20p, AF3073, Cell Signaling #9287): These antibodies specifically recognize p53 only when Ser20 is phosphorylated .

• Conformation-sensitive antibodies (e.g., DO1 in high salt conditions): DO1 recognizes p53 only when Ser20 is NOT phosphorylated when used in high salt (800 mM NaCl) buffer conditions .

Validation methods:

• Peptide competition assays using p53 peptides either not phosphorylated or phosphorylated at specific sites (Ser15, Thr18, or Ser20) .

• Phosphatase treatment: Treatment of cell extracts with protein phosphatase reverses Ser20 phosphorylation and restores DO1 reactivity .

Applications include:

• Western blotting (WB) at 1:1000 dilution for detecting endogenous levels

• Immunohistochemistry (IHC) for tissue sections

• Immunofluorescence (IF/ICC) for cellular localization

• Flow cytometry for quantitative analysis

How is p53 Ser20 phosphorylation status altered in cancer and therapeutic settings?

p53 Ser20 phosphorylation levels change in response to cancer treatments:

• In squamous cell lung cancer: Patients treated with radiotherapy/cisplatin/vinorelbine show approximately 57% increase in p53 Ser20 phosphorylation levels, correlating with:

  • Increased total p53 expression (61% increase)

  • Elevated poly(ADP-ribose) levels (69% increase; p<0.01)

  • Changes in Ki-67 proliferation marker

These findings suggest that p53 Ser20 phosphorylation contributes to the antiproliferative and apoptotic effects of DNA-damaging cancer therapies .

What are the technical challenges in measuring p53 Ser20 phosphorylation in clinical samples?

Several methodological considerations are critical when measuring p53 Ser20 phosphorylation in clinical settings:

Challenges:

• Low abundance of wild-type p53: Wild-type p53 is expressed at lower levels than mutant p53, making detection technically difficult in non-transformed tissues .

• Antibody specificity: Ensuring antibodies don't cross-react with other phosphorylation sites (e.g., Ser15) is critical for accurate results .

• Timing of sample collection: Phosphorylation is dynamic and timing-dependent after stress exposure (optimal time points: ~2h post-IR, ~16h post-UV) .

• Heterogeneity of clinical samples: Mixed populations of cells with varying p53 status can complicate interpretation.

Technical approaches to overcome challenges:

• Use high-sensitivity detection methods with proper controls

• Employ multiple antibody approach (using both phospho-specific and conformation-sensitive antibodies)

• Include phosphatase treatment controls to confirm specificity

• Normalize results to total p53 expression levels

• Consider analysis of specific cellular fractions (e.g., nuclear extracts)

How can researchers distinguish between direct and indirect effects on p53 Ser20 phosphorylation when studying novel compounds?

To determine whether novel compounds directly or indirectly affect p53 Ser20 phosphorylation:

Methodological approach:

• Perform kinase inhibitor studies: Use specific inhibitors for known Ser20 kinases (ATM inhibitor KU-55933, CK1 inhibitor D4476, AMPK inhibitor Compound C) to identify the pathway involved .

• Conduct in vitro kinase assays: Test if purified kinases can directly phosphorylate p53 at Ser20 in the presence of the compound .

• Utilize p53 mutants: Compare effects on wild-type p53 versus p53-Ala20 mutant to confirm Ser20-specific effects .

• Employ phospho-peptide competition assays: Use peptides with phosphorylated Ser20 vs. non-phosphorylated peptides to evaluate direct binding events .

• Perform cell-free systems analyses: Study the direct effect of compounds on purified proteins in reconstituted systems.

Example experimental design from research:
In studies identifying CK1 as the HHV-6B-induced p53 Ser20 kinase, researchers:

• Treated infected cells with specific CK1 inhibitor D4476

• Observed dose-dependent attenuation of Ser20 phosphorylation

• Confirmed specificity by showing the CK1 inhibitor had no effect on X-ray-induced phosphorylation (which is ATM-dependent)

What are the experimental approaches to study Ser20 phosphorylation kinetics in living cells?

Advanced techniques for studying phosphorylation dynamics:

• Live-cell phospho-specific antibody-based biosensors:

  • Fluorescently labeled antibody fragments that recognize phosphorylated Ser20

  • Enables real-time monitoring of phosphorylation status

• Pulse-chase experiments with synchronized cells:

  • Synchronize cells at specific cell cycle stages

  • Apply stress stimulus

  • Sample at multiple time points (e.g., 0, 15, 30, 60, 120, 240 min)

  • Analyze Ser20 phosphorylation by Western blot or flow cytometry

• Phosphatase inhibition time-course:

  • Apply stress stimulus followed by phosphatase inhibitors at various intervals

  • Determines both phosphorylation and dephosphorylation kinetics

• Validation approach:

  • Combine multiple detection methods (Western blot, immunofluorescence, flow cytometry)

  • Use multiple antibodies with different recognition properties

  • Include proper controls (phosphatase treatment, kinase inhibitors)

The MOLT-3 cell line (human acute lymphoblastic leukemia T-cell line) has been validated as a model system for studying stress-induced p53 Ser20 phosphorylation dynamics with these approaches .

How do contradictory findings regarding the role of Chk1/Chk2 in p53 Ser20 phosphorylation complicate research interpretation?

The literature contains several contradictory findings regarding Chk1/Chk2 and p53 Ser20 phosphorylation:

Contradictory findings:

• Some studies identify Chk1 and Chk2 as direct kinases for p53 Ser20, enhancing p53 tetramerization, stability, and activity .

• Other research shows that CHK2 inhibitor does not prevent X-ray-induced Ser20 phosphorylation, suggesting Chk2 is not the primary kinase in this context .

• CHK1 inhibitor (SB218078) not only fails to prevent X-ray-induced Ser20 phosphorylation but actually elevates it and stabilizes basal p53 levels .

Methodological approaches to resolve contradictions:

• Experimental context standardization:

  • Use consistent cell types, stress conditions, and time points

  • The MOLT-3 cell line provides a validated model system

• Multiple kinase inhibitors and genetic approaches:

  • Combine pharmacological (inhibitors) and genetic (siRNA, CRISPR) approaches

  • Test both gain-of-function and loss-of-function approaches

• In-depth pathway analysis:

  • Consider ATM → Chk2 → Ser20 as potential pathway

  • Investigate whether Chk1 inhibition activates compensatory mechanisms

  • Examine whether Chk1/Chk2 might phosphorylate Ser20 in specific contexts or cell types

Research interpretation guideline:
The findings suggest that while Chk1/Chk2 may phosphorylate p53 at Ser20 in certain contexts, they are not universally required for Ser20 phosphorylation across all stress conditions, highlighting the complexity and context-dependency of p53 regulation.

How does phosphorylation at Ser20 coordinate with other p53 post-translational modifications in the DNA damage response?

Phosphorylation of p53 at Ser20 functions within a complex network of post-translational modifications:

Coordination with other phosphorylation sites:

• Ser15 phosphorylation (by ATM, ATR, DNA-PK): Often occurs in concert with Ser20 phosphorylation after DNA damage

• Ser37 phosphorylation (by ATM, ATR, DNA-PK): Works together with Ser15 to impair MDM2 binding

• Ser46 phosphorylation: Specifically regulates the apoptotic function of p53

• Ser6/Ser9 phosphorylation (by CK1δ and CK1ε): May coordinate with Ser20 in specific stress responses

• Ser392 phosphorylation: Influences DNA binding and transcriptional activation, and is increased in human tumors

Integration with other modifications:

• Acetylation: Ser20 phosphorylation stabilizes p300/CBP binding, promoting subsequent acetylation of p53 at multiple lysine residues

• Ubiquitination: Ser20 phosphorylation interferes with MDM2-mediated ubiquitination, preventing proteasomal degradation

Experimental approaches to study modification crosstalk:

• Sequential immunoprecipitation: First IP with anti-phospho-Ser20, then probe for other modifications

• Mass spectrometry: Analyze the complete modification pattern of p53 molecules

• Site-directed mutagenesis: Create combinatorial mutations (e.g., S15A/S20A double mutant)

• Kinetic studies: Determine the temporal order of modifications after stress

Understanding this coordination is critical for developing therapeutic strategies targeting the p53 pathway in cancer and other diseases.

What are the optimal experimental conditions for detecting p53 Ser20 phosphorylation after different stresses?

Based on published research, the following experimental conditions optimize detection of p53 Ser20 phosphorylation:

For ionizing radiation (IR)-induced phosphorylation:

• Dose: 9 Gy

• Cell harvest time: 2 hours post-irradiation

• Detection method: Immunoprecipitation with AbS20p antibody or DO1 (in high salt buffer)

• Positive control: ATM inhibitor KU-55933 should attenuate signal

• Cell model: MOLT-3 cell line (validated model)

For UV light-induced phosphorylation:

• Dose: 50 J/m²

• Cell harvest time: 16 hours post-exposure

• Detection method: Same as for IR

• Cell model: U2-OS cells or MOLT-3

For metabolic stress (AMPK pathway):

• Inducer: AICAR (elevates intracellular AMP levels)

• Control: AMPK inhibitor Compound C should attenuate signal

• Cell model: MOLT-3 cell line

For virus-induced phosphorylation:

• Virus: Human herpesvirus 6B (HHV-6B)

• Control: CK1 inhibitor D4476 should attenuate signal

• Cell model: Infected cell lines

Buffer conditions for Western blot/IP:

• High salt (800 mM NaCl): Required for DO1 antibody to show phospho-specificity

• Phosphatase inhibitors: Critical to preserve phosphorylation status

• Lysis buffer: 20 mM Tris-HCl (pH 8), 1 mM EDTA, 0.05% Tween-20

What controls should be included when validating the specificity of a Phospho-p53 (Ser20) antibody?

A comprehensive validation of Phospho-p53 (Ser20) antibodies should include:

Essential controls:

• Peptide competition assays:

  • Incubate antibody with biotinylated peptides corresponding to p53 residues 7-29:

    • Non-phosphorylated

    • Phosphorylated at Ser15

    • Phosphorylated at Thr18

    • Phosphorylated at Ser20

  • A specific antibody should bind only to Ser20-phosphorylated peptide

• Phosphatase treatment:

  • Treat cell extracts containing phosphorylated p53 with protein phosphatase

  • Should abolish reactivity with phospho-specific antibodies (AbS20p)

  • Should restore reactivity with antibodies like DO1 in high salt conditions

• p53-null cells or p53 knockdown:

  • Include p53-null or knockdown samples to confirm absence of signal

• Mutant p53 (Ser20Ala):

  • Express p53 with Ser20 mutated to Ala

  • Should not be recognized by phospho-specific antibody

• Specific kinase inhibitors:

  • Treat cells with specific inhibitors before stress induction:

    • ATM inhibitor KU-55933 for IR-induced phosphorylation

    • CK1 inhibitor D4476 for virus-induced phosphorylation

    • AMPK inhibitor Compound C for metabolic stress-induced phosphorylation

  • Should see dose-dependent attenuation of signal

Advanced validation approaches:

• Multiple antibody cross-validation: Compare results from different commercial antibodies targeting the same site

• Mass spectrometry confirmation: Verify phosphorylation state by mass spectrometry analysis

How can researchers accurately quantify changes in p53 Ser20 phosphorylation levels across experimental conditions?

Accurate quantification of p53 Ser20 phosphorylation requires:

Methodological considerations:

• Normalization strategy:

  • Always normalize phospho-p53 (Ser20) signal to total p53 protein levels

  • Account for potential changes in total p53 expression due to stabilization

  • Consider additional normalization to housekeeping proteins (β-actin, GAPDH)

• Quantification techniques:

  • Western blot: Use digital imaging and analysis software (e.g., ImageJ)

  • Flow cytometry: Provides single-cell resolution and statistical robustness

  • ELISA: Offers quantitative measurement with standard curves

  • Mass spectrometry: Provides absolute quantification of phosphorylation stoichiometry

• Statistical analysis:

  • Perform at least three independent biological replicates

  • Apply appropriate statistical tests (t-test, ANOVA) with multiple testing correction

  • Report both magnitude of change (fold change) and statistical significance

Example quantification approach from literature:
In studies of squamous cell lung cancer patients treated with radiotherapy/cisplatin/vinorelbine:

• Researchers reported a ~57% increase in p53 Ser20 phosphorylation

• This was correlated with a 61% increase in total p53 expression

• Changes were analyzed in relation to proliferation marker Ki-67 and poly(ADP-ribose) levels (69% increase; p<0.01)

• Patient-matched samples (before and after treatment) were used to control for inter-individual variation

What are the considerations when developing research models to study the biological consequences of altered p53 Ser20 phosphorylation?

When developing models to study p53 Ser20 phosphorylation:

Model system options:

• Cell line models:

  • MOLT-3 cell line: Validated model for studying stress-induced Ser20 phosphorylation

  • U2-OS cells: Used for studying UV-induced phosphorylation

  • H1299 cells: p53-null cells useful for expressing p53 mutants

  • Consider p53 status: Wild-type, mutant, or null depending on research question

• Genetic manipulation approaches:

  • Ser20 to Ala mutation (S20A): Prevents phosphorylation, shows increased MDM2 binding and reduced stability

  • Ser20 to Asp/Glu mutation (S20D/E): Phosphomimetic mutation

  • CRISPR/Cas9 genome editing: For endogenous modification of Ser20

• In vivo models:

  • Mouse models: Transgenic mice with p53-Ala20 mutation show spontaneous B-cell lymphoma development

  • Patient samples: As demonstrated in studies of squamous cell lung cancer patients

Biological readouts to assess:

• p53 stability: Half-life determination using cycloheximide chase

• p53-MDM2 interaction: Co-immunoprecipitation assays

• Transcriptional activity: Reporter assays, qRT-PCR of p53 target genes

• Cell fate decisions: Apoptosis (flow cytometry), cell cycle arrest (BrdU incorporation)

• Long-term consequences: Cellular transformation, tumorigenesis

Experimental design considerations:

• Include both short-term (minutes to hours) and long-term (days to weeks) analyses

• Examine effects under basal and stressed conditions

• Consider tissue/cell type-specific effects

• Compare effects of different stress stimuli that utilize distinct kinase pathways