Phospho-TP53 (T55) Recombinant Monoclonal Antibody

What is TP53 and why is the T55 phosphorylation site significant?

TP53 (tumor protein p53) is a master tumor suppressor that controls cellular responses to genotoxic stress. This multifunctional transcription factor induces cell cycle arrest, DNA repair, or apoptosis upon binding to target DNA sequences . T55 is located in the AD2 interaction motif of the N-terminal transactivation domain (NTAD) and functions as a phosphorylation-dependent regulatory switch that modulates p53 activity . This site is particularly important because its phosphorylation status changes dynamically during the cellular stress response cycle - it is constitutively phosphorylated in unstressed cells, becomes dephosphorylated upon DNA damage, and is subsequently rephosphorylated to facilitate dissociation of p53 from promoters and inactivate p53-mediated transcription .

How does T55 phosphorylation affect TP53 function at the molecular level?

T55 phosphorylation modulates synergistic intramolecular interactions between the disordered transactivation domain and the structured DNA-binding domain (DBD) . This phosphorylation enhances competitive interactions between the AD2 motif and the DBD, inhibiting DNA binding. Specifically:

• Non-phosphorylated p53 exhibits positive cooperativity in binding DNA as a tetramer

• Upon T55 phosphorylation, cooperativity is abolished and p53 binds initially to cognate DNA sites as a dimer

• As phosphorylated p53 concentration increases, a second dimer binds, causing 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.

What are the recommended applications for Phospho-TP53 (T55) antibodies?

Phospho-TP53 (T55) antibodies have been validated for multiple experimental techniques, with specific dilution recommendations:

When designing experiments, it is crucial to optimize antibody concentration for your specific cell type or tissue sample, as background signal and optimal conditions may vary .

How can I validate the specificity of Phospho-TP53 (T55) antibodies in my experiments?

To ensure the specificity of Phospho-TP53 (T55) antibodies, implement these validation strategies:

• Use phosphatase treatment controls: Treat half of your sample with lambda phosphatase to remove phosphorylation and confirm antibody specificity for the phosphorylated form

• Include T55A mutant controls: Express a T55A mutant of TP53 that cannot be phosphorylated at this position as a negative control

• Perform CK2 kinase treatment: CK2 kinase specifically phosphorylates T55 in TP53, and can be used to generate positive controls

• Employ blocking peptides: Use phosphorylated and non-phosphorylated peptides containing the T55 region to confirm specificity

• Validate with multiple antibody clones: Cross-validate results using different antibody clones that recognize the same phospho-epitope

Research by Kruse et al. demonstrates that T55-specific phosphorylation can be confirmed using NMR spectroscopy, where phosphorylated residues produce characteristic, non-overlapped cross-peaks in [1H, 15N]-TROSY spectra .

How can Phospho-TP53 (T55) antibodies be used to study the DNA damage response?

Phospho-TP53 (T55) antibodies are powerful tools for studying the temporal dynamics of p53 regulation during DNA damage response:

• Time-course experiments: Monitor T55 phosphorylation status at different time points after DNA damage induction to track the dephosphorylation and subsequent rephosphorylation cycle

• Co-immunoprecipitation studies: Identify proteins that interact specifically with phosphorylated or non-phosphorylated T55-TP53

• Chromatin immunoprecipitation (ChIP): Determine how T55 phosphorylation affects p53 occupancy at specific promoters

• Fluorescence microscopy: Track subcellular localization changes associated with T55 phosphorylation status

Research has shown that T55 is constitutively phosphorylated in unstressed cells, facilitating association with the nuclear export factor CRM1, export from the nucleus, and cytoplasmic degradation. Upon DNA damage, T55 becomes dephosphorylated, allowing p53 to bind DNA and activate target genes. The subsequent rephosphorylation of T55 promotes dissociation from DNA and terminates the p53 transcriptional response .

What is the relationship between T55 phosphorylation and other post-translational modifications of TP53?

TP53 is regulated by a complex network of post-translational modifications that interact to fine-tune its activity:

• Synergy with S46 phosphorylation: Research indicates that T55 phosphorylation-mediated autoinhibition is augmented by additional phosphorylation of Ser46, suggesting these modifications work together to regulate p53 activity

• Proline-rich domain (PRD) interactions: The PRD fine-tunes the strength of interactions between phosphorylated T55 and the DNA binding domain. Removal of the PRD strengthens the AD2-DBD interaction and leads to autoinhibition of DNA binding even without T55 phosphorylation

• Interplay with other phosphorylation sites: Experimental data suggests T55 phosphorylation exists within a network including phosphorylation at S6, S46, and S392, with CK2 kinase able to phosphorylate all these sites

The table below summarizes how different p53 constructs with various phosphorylation states affect DNA binding:

These data demonstrate how phosphorylation at T55 reduces DNA binding affinity and introduces autoinhibition .

How does T55 phosphorylation influence p53 interaction with DNA at physiological concentrations?

At physiological concentrations (estimated basal p53 concentration of ~350 nM in vivo), T55 phosphorylation significantly impacts DNA binding:

• Complete dissociation from weak binding sites: Upon T55 phosphorylation, p53 completely dissociates from DINP1 DNA recognition elements

• Partial dissociation from strong binding sites: T55 phosphorylation causes partial dissociation from p21 consensus sites

• Abrogation of DNA binding cooperativity: Non-phosphorylated p53 exhibits positive cooperativity in binding DNA as a tetramer, but T55 phosphorylation abolishes this cooperativity

• Dimeric binding followed by autoinhibition: Phosphorylated p53 initially binds to DNA as a dimer with reduced affinity, but as concentration increases, a second dimer binds, causing dissociation from DNA

Experiments using fluorescence anisotropy demonstrate that when preformed p53:DNA complexes (with 10 nM DNA and 300 nM p53) are incubated with CK2 kinase to phosphorylate T55, significant dissociation occurs, confirming the regulatory role of T55 phosphorylation at near-physiological concentrations .

What are the optimal storage and handling conditions for Phospho-TP53 (T55) antibodies?

Proper storage and handling are crucial for maintaining antibody activity:

• Long-term storage: Store at -20°C for up to one year

• Short-term/frequent use: Store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles: Aliquot antibodies before freezing to minimize degradation

• Buffer composition: Most commercial Phospho-TP53 (T55) antibodies are supplied in PBS with stabilizers such as 0.02% sodium azide and 50% glycerol at pH 7.2

• Working dilution preparation: Dilute in fresh buffer immediately before use and avoid storing diluted antibody

Following these guidelines will help maintain antibody specificity and sensitivity in your experiments.

What controls should be included when using Phospho-TP53 (T55) antibodies?

Include these essential controls to ensure reliable results:

• Positive controls:

  • CK2-treated samples (CK2 specifically phosphorylates T55)

  • Cell lines with known constitutive T55 phosphorylation (unstressed cells)

• Negative controls:

  • DNA damage-induced samples (when T55 is dephosphorylated)

  • T55A mutant p53 samples (cannot be phosphorylated at position 55)

  • Total p53 detection (using non-phospho-specific antibodies)

  • Secondary antibody-only controls

• Validation controls:

  • Phosphatase-treated samples to confirm phospho-specificity

  • Blocking peptide competition assays

  • Multiple antibody clones to verify results

For the most rigorous validation, consider employing mass spectrometry to directly confirm phosphorylation status at T55.

How can I optimize Western blot protocols specifically for Phospho-TP53 (T55) detection?

For optimal Western blot detection of phospho-T55 TP53:

• Sample preparation:

  • Add phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate) to lysis buffers

  • Process samples quickly and keep them cold to prevent dephosphorylation

  • Consider using phospho-protein enrichment techniques for low abundance samples

• SDS-PAGE conditions:

  • Use fresh gels with appropriate percentage (10-12% typically works well for p53)

  • Include phosphorylated protein markers

• Transfer conditions:

  • Use PVDF membranes for better protein retention

  • Wet transfer often works better than semi-dry for phospho-proteins

• Blocking and antibody incubation:

  • For primary antibody, use recommended dilutions (1:500-1:5000 for WB)

  • Consider using 5% BSA instead of milk for blocking and antibody dilution

  • Incubate primary antibody overnight at 4°C for optimal sensitivity

• Detection systems:

  • Use high-sensitivity ECL or fluorescent secondary antibodies

  • Consider signal enhancement systems for low abundance targets

These optimizations will help ensure specific detection of phospho-T55 p53, particularly in samples with low expression levels.

How can Phospho-TP53 (T55) antibodies contribute to cancer research?

Phospho-TP53 (T55) antibodies offer valuable insights for cancer research:

• Diagnostic biomarker development: Assess if T55 phosphorylation status correlates with cancer progression or treatment response

• Drug discovery: Screen compounds that modulate T55 phosphorylation to potentially restore normal p53 function

• Resistance mechanisms: Investigate whether altered T55 phosphorylation contributes to therapy resistance

• Cancer subtype characterization: Determine if T55 phosphorylation patterns differ between cancer types or subtypes

• Cell stress response analysis: Examine how various stressors alter T55 phosphorylation in different cancer cell models

Research shows that T55 is constitutively phosphorylated in unstressed cells, which facilitates nuclear export and degradation of p53 . This suggests that dysregulation of T55 phosphorylation could contribute to aberrant p53 activity in cancer cells, making it an important target for investigation.

What is the functional significance of the T55 phosphorylation switch in the context of tumor suppression?

The T55 phosphorylation switch plays a crucial role in p53-mediated tumor suppression through several mechanisms:

• Regulation of p53 stability: In unstressed cells, T55 phosphorylation facilitates association with the nuclear export factor CRM1, promoting nuclear export and cytoplasmic degradation of p53

• Modulation of DNA binding: T55 phosphorylation inhibits p53 binding to DNA by enhancing competitive interactions between the AD2 motif and the DNA-binding domain

• Cell cycle checkpoint control: The phosphorylation status of T55 regulates p53's ability to bind promoters that control cell cycle arrest genes

• Apoptotic response regulation: T55 phosphorylation status affects p53's interaction with apoptotic gene promoters

• Termination of stress response: Rephosphorylation of T55 after DNA damage promotes dissociation of p53 from promoters and inactivates p53-mediated transcription

The dynamic regulation of T55 phosphorylation ensures that p53's tumor suppressor functions are activated appropriately in response to cellular stress and then properly terminated once the stress has been addressed, preventing prolonged activation that could be detrimental to normal cells.

What emerging research areas involve Phospho-TP53 (T55) antibodies?

Several promising research directions involve Phospho-TP53 (T55) antibodies:

• Single-cell analysis: Investigating T55 phosphorylation heterogeneity within tumors using single-cell techniques

• Cancer stem cell regulation: Examining how T55 phosphorylation affects p53 function in cancer stem cells

• Immune response modulation: Studying the impact of T55 phosphorylation on p53's role in immune signaling

• Combination therapy development: Testing how modulators of T55 phosphorylation might synergize with established cancer therapies

• Liquid biopsy applications: Developing assays to detect phospho-T55 p53 in circulating tumor cells or exosomes

• Structural biology approaches: Using cryo-EM or other advanced techniques to visualize how T55 phosphorylation alters p53 tetramer conformation and interaction with DNA

Current research has revealed the importance of the proline-rich domain in fine-tuning interactions between phosphorylated T55 and the DNA binding domain , suggesting that targeting these domain interactions could be a novel therapeutic approach.

How might advances in antibody technology improve Phospho-TP53 (T55) detection and research?

Emerging antibody technologies could enhance Phospho-TP53 (T55) research:

• Recombinant antibody engineering: Development of higher-specificity recombinant antibodies with reduced batch-to-batch variability

• Bispecific antibodies: Creating antibodies that simultaneously detect T55 phosphorylation and other post-translational modifications

• Intrabodies: Engineered antibodies for live-cell imaging of T55 phosphorylation dynamics

• Nanobodies: Smaller antibody fragments for improved tissue penetration and spatial resolution in imaging

• Proximity labeling technologies: Combining phospho-T55 antibodies with proximity labeling to identify proteins that interact specifically with phosphorylated T55

• Multiplexed detection systems: Developing methods to simultaneously visualize multiple phosphorylation sites on p53