Phospho-TP53 (Ser315) Antibody

What is the significance of p53 phosphorylation at Ser315?

Phosphorylation at Ser315 is a post-translational modification of the p53 tumor suppressor protein that plays a key role in regulating its function as a transcription factor. This specific modification enhances p53's transcriptional activity across multiple target genes. Studies have demonstrated that near-stoichiometric Ser315 phosphorylation of endogenous p53 protein occurs after UV irradiation in MCF7 and A375 cells, coinciding with elevated p53-dependent transcription . When this serine residue is mutated to alanine (preventing phosphorylation), p53 shows substantially reduced specific activity as a transcription factor . This indicates that phosphorylation at Ser315 is an activating modification that contributes to p53's role in cellular stress responses including DNA damage, cell cycle regulation, and apoptosis.

How does Ser315 phosphorylation differ from other p53 phosphorylation sites?

Unlike some other phosphorylation sites like Ser15 (which is directly involved in DNA damage response signaling), Ser315 is located in the C-terminal regulatory domain of p53. While Ser15 phosphorylation appears to be a nucleating event for other modifications following DNA damage , Ser315 phosphorylation affects p53 through a different mechanism. Research indicates that antibodies targeting the C-terminal negative regulatory domain can activate p53's transcription factor activity, suggesting that modifications in this region (including Ser315 phosphorylation) play a crucial role in stimulating p53-dependent gene expression . Unlike some phosphorylation sites that may have promoter-selective effects, Ser315 phosphorylation appears to have a more general enhancing effect on p53 transcriptional activity. Interestingly, substitution of another major p53 phosphorylation site, Ser392, had no detectable effect on p53 activity under conditions where other modifications were crucial .

Which enzymes are responsible for phosphorylating p53 at Ser315?

Research indicates that cyclin-dependent protein kinases (CDKs) are primarily responsible for phosphorylating p53 at Ser315. This is supported by studies showing that treatment of cells with Roscovitine, a cyclin-dependent protein kinase inhibitor, promotes a reduction in the specific activity of both endogenous and ectopically expressed p53 . In vitro phosphorylation assays have successfully used CDKs to phosphorylate p53 at Ser315 . This kinase-substrate relationship places p53 Ser315 phosphorylation in the context of cell cycle regulation, as CDKs are key regulators of cell cycle progression, suggesting a possible link between cell cycle status and p53 activation through this specific modification. This differs from Ser15 phosphorylation, which is primarily mediated by DNA damage response kinases like ATM and ATR .

What are the optimal methods for detecting p53 phosphorylation at Ser315?

Several complementary methods can be used to detect p53 phosphorylation at Ser315, each with specific advantages for different research applications:

• Phospho-specific antibodies: The most direct approach involves using antibodies that specifically recognize p53 phosphorylated at Ser315. These are available in formats suitable for western blotting, immunoprecipitation, and immunofluorescence microscopy. Western blotting with phospho-specific antibodies can be quantitative when properly normalized to total p53 levels .

• Cell-based ELISA: This method allows for qualitative determination of p53 (phospho Ser315) concentration through an indirect ELISA format where the phosphorylated protein is captured by anti-p53 (phospho Ser315) antibodies and detected using HRP-conjugated secondary antibodies . The method includes multiple normalization approaches: anti-GAPDH antibody as an internal positive control, Crystal Violet whole-cell staining for cell density normalization, and anti-p53 antibody to normalize phosphorylated target to total p53 levels .

• Native phospho-specific IgG binding assay: As described in research, this can be used for quantitating the extent of p53 phosphorylation at Ser315, allowing researchers to define when one, two, three, or four phosphates per tetramer are present after in vitro phosphorylation by cyclin-dependent protein kinases . This method provides valuable information about stoichiometry that other approaches cannot easily determine.

• Functional assays with phospho-mimetic and phospho-deficient mutants: Comparing the activities of wild-type p53, S315A (phospho-deficient), and S315D (phospho-mimetic) mutants in transcriptional assays provides indirect evidence of the functional significance of phosphorylation at this site .

What cell lines are most suitable for studying Ser315 phosphorylation of p53?

Based on the research literature, several cell lines have been successfully used to study p53 Ser315 phosphorylation:

• MCF7 (human breast cancer cells): These cells retain wild-type p53 and show detectable Ser315 phosphorylation that increases after UV irradiation . Their robust p53-dependent transcriptional responses make them ideal for studying the relationship between phosphorylation and downstream gene expression.

• A375 (human melanoma cells): Similar to MCF7, these cells display increased Ser315 phosphorylation after UV exposure . Their melanoma origin makes them particularly relevant for studying UV response pathways.

• Saos-2 (human osteosarcoma cells): These p53-null cells have been used for transfection studies with wild-type or mutant (S315A) p53 to assess the functional consequences of Ser315 phosphorylation . The absence of endogenous p53 makes them ideal for ectopic expression studies without interference.

• HCT116, U2OS and H1299 cells: While not specifically mentioned for Ser315 studies in the search results, these cell lines have been used extensively for studying other p53 phosphorylation sites . HCT116 cells (human colon carcinoma) with wild-type p53 are particularly valuable as isogenic p53-null variants are available for comparison studies.

For studying endogenous regulation, cells with wild-type p53 like MCF7 or HCT116 are preferable. For ectopic expression studies, p53-null cells like Saos-2 or H1299 allow expression of p53 mutants without interference from endogenous p53.

How can researchers induce and measure changes in p53 Ser315 phosphorylation?

Researchers can induce changes in p53 Ser315 phosphorylation through several complementary approaches:

• UV irradiation: Studies have shown that UV exposure leads to near-stoichiometric phosphorylation of p53 at Ser315 in certain cell lines (MCF7, A375) . This provides a physiologically relevant stimulus that activates multiple stress response pathways.

• CDK modulation: Since cyclin-dependent kinases phosphorylate Ser315, researchers can manipulate CDK activity using:

  • CDK inhibitors like Roscovitine (which has been shown to reduce p53 activity)

  • Cell cycle synchronization to enrich for specific CDK activities

  • CDK overexpression systems

• DNA damage agents: While the search results focus specifically on UV irradiation, other DNA damaging agents that activate p53 might similarly affect Ser315 phosphorylation. Etoposide, ionizing radiation, and other genotoxic agents could be tested .

To measure changes, researchers should employ multiple complementary methods:

• Western blotting with phospho-specific antibodies, comparing to total p53 levels detected with antibodies like DO-1, CM1, or PAb 1801

• Phospho-p53 (Ser315) cell-based ELISA assays which allow for detection of changes under different stimulation conditions

• Functional readouts such as p53-dependent reporter gene assays to correlate phosphorylation with transcriptional activity

• Chromatin immunoprecipitation (ChIP) assays to determine if Ser315 phosphorylation affects p53 binding to target gene promoters like p21/CDKN1A

A time-course experiment is essential to determine the kinetics of phosphorylation following stimulation, as modifications can be transient and dynamic.

How does Ser315 phosphorylation impact p53's interaction with other proteins?

Phosphorylation of p53 at Ser315 likely alters its interaction with various protein partners, although the specific mechanisms deserve further investigation. Based on research with other phosphorylation sites, we can infer several potential interaction changes:

Ser315 phosphorylation may influence p53's ability to recruit transcriptional co-activators. For example, phosphorylation at Ser15 stimulates association with important histone/lysine acetyltransferases (HATs) such as p300 and CBP . These interactions promote acetylation of multiple lysine residues in p53 and contribute to its stabilization by blocking ubiquitylation . Given that Ser315 phosphorylation enhances transcriptional activity , it may similarly facilitate recruitment of chromatin modifiers to p53-responsive promoters.

The location of Ser315 in the C-terminal domain suggests it may regulate tetramerization dynamics or DNA binding properties. The native phospho-specific IgG binding assay described in the research can quantitate "one, two, three, or four phosphates/tetramer" , indicating that not all subunits in a p53 tetramer are necessarily phosphorylated equally. This variable stoichiometry might create preferential interactions with specific cofactors.

To directly study these interactions, immunoprecipitation experiments similar to those described in the research could be employed . Comparing wild-type p53, p53-S315A, and p53-S315D would reveal phosphorylation-dependent protein interactions.

What is the relationship between Ser315 phosphorylation and other post-translational modifications of p53?

The relationship between Ser315 phosphorylation and other p53 post-translational modifications represents a complex regulatory network that requires integrated analysis:

Phosphorylation events on p53 can be hierarchical, with certain modifications creating conditions favorable for subsequent ones. For example, Ser15 phosphorylation appears to nucleate other modifications . Research shows that phosphorylation can promote acetylation of multiple lysine residues in p53's DNA binding and carboxy-terminal domains . This suggests that Ser315 phosphorylation might similarly influence other modifications, creating a specific "modification signature" that determines p53's ultimate activity.

Different modifications show varying functional importance. The research indicates that while Ser15 and Ser315 phosphorylation significantly impact p53 function, substitution of Ser392 had no detectable effect under the same experimental conditions . This highlights the non-equivalence of different modification sites and the importance of determining their individual and combined contributions.

The sequence of modifications matters. In murine cells, Ser18 (the ortholog of human Ser15) phosphorylation contributes to protection against late-onset tumor development . Understanding whether Ser315 phosphorylation works in parallel, upstream, or downstream of such protective modifications would provide insight into the complete p53 regulatory network.

Methodologically, mass spectrometry approaches coupled with site-specific mutations and domain-specific antibodies would allow mapping of the complete modification landscape under different conditions, revealing how Ser315 phosphorylation fits into the broader regulatory picture.

How does p53 Ser315 phosphorylation contribute to cell fate decisions following DNA damage?

The role of Ser315 phosphorylation in determining cell fate following DNA damage appears to be connected to its enhancement of p53's transcriptional activity:

Research demonstrates that p53 in which Ser315 is substituted by alanine (S315A) fails to mediate p53-dependent transcription from multiple promoters, including the key cell cycle regulator p21/CDKN1A . Since p21 is critical for implementing cell cycle arrest, this suggests that Ser315 phosphorylation is necessary for this cell fate option. The phospho-mimetic S315D mutation restores this function, confirming the significance of phosphorylation at this position .

The research indicates that UV irradiation, which can lead to both cell cycle arrest and apoptosis depending on damage severity, induces near-stoichiometric Ser315 phosphorylation . This suggests this modification is integral to UV damage response pathways. Since CDKs mediate Ser315 phosphorylation , there may be crosstalk between cell cycle status and p53 activation, potentially influencing whether cells arrest or undergo apoptosis.

Interestingly, different p53-responsive promoters show varying sensitivity to Ser315 phosphorylation. The research indicates that while S315A mutation completely abolished activation of the p21 promoter, it only reduced activity at the BAX and MDM2 promoters . This differential effect might contribute to selective gene expression patterns that influence cell fate decisions following damage.

To fully understand this role, researchers should examine how Ser315 phosphorylation affects the complete p53 transcriptional program using genome-wide approaches like ChIP-seq and RNA-seq, correlating phosphorylation status with specific cell fate outcomes.

What controls should be included when using phospho-TP53 (Ser315) antibodies?

When using phospho-TP53 (Ser315) antibodies, researchers should include several types of controls to ensure reliable and interpretable results:

• Positive controls:

  • Lysates from cells treated with known inducers of Ser315 phosphorylation (e.g., UV-irradiated MCF7 or A375 cells)

  • In vitro phosphorylated p53 (using purified p53 and CDKs)

  • Cells expressing phospho-mimetic S315D mutant

• Negative controls:

  • Lysates from cells treated with CDK inhibitors like Roscovitine

  • Extracts from cells expressing p53-S315A mutant protein

  • p53-null cells (e.g., H1299) to confirm antibody specificity

  • Phosphatase-treated samples to confirm phospho-specificity

• Normalization controls:

  • Total p53 detection (using antibodies like DO-1, CM1, or PAb 1801)

  • GAPDH as a loading control

  • Crystal Violet staining for cell density normalization in cell-based assays

• Experimental controls:

  • Time-course sampling to capture dynamic changes in phosphorylation

  • Dose-response analysis for treatments

  • Parallel analysis of other p53 phosphorylation sites for comparison

The cell-based ELISA kit mentioned in the research includes multiple normalization methods, demonstrating the importance of proper controls for quantitative assessment of phosphorylation . These include anti-GAPDH antibody as an internal positive control, Crystal Violet whole-cell staining for cell density normalization, and anti-p53 antibody to normalize phosphorylated p53 to total p53 levels .

How can phospho-TP53 (Ser315) antibodies be validated for specificity?

Validating phospho-TP53 (Ser315) antibodies for specificity is crucial to ensure reliable research results. A comprehensive validation approach should include:

• Genetic validation:

  • Compare antibody reactivity between wild-type p53 and p53-S315A mutant under identical conditions

  • Test reactivity in p53-null cells (e.g., H1299) with and without reconstituted wild-type or mutant p53

  • Perform siRNA or CRISPR-mediated knockdown of p53 to confirm signal reduction correlates with p53 levels

• Biochemical validation:

  • In vitro phosphorylation: Compare reactivity with unphosphorylated p53 versus p53 phosphorylated in vitro by CDKs

  • Phosphatase treatment: Signal should be eliminated after treatment with lambda phosphatase

  • Peptide competition assays: Pre-incubation with phospho-Ser315 peptide should block antibody binding, while unphosphorylated peptide should not

• Physiological validation:

  • Correlation with known stimuli: Antibody should show increased reactivity after treatments known to enhance Ser315 phosphorylation (e.g., UV irradiation)

  • Reduction with inhibitors: Signal should decrease when cells are treated with CDK inhibitors like Roscovitine

  • Correlation with functional outcomes: Changes in signal should correlate with altered p53 transcriptional activity

• Multiple detection methods:

  • Compare results between different techniques (Western blot, ELISA, immunofluorescence)

  • Use mass spectrometry as an antibody-independent method to confirm phosphorylation status

The native phospho-specific IgG binding assay described in the research provides an additional validation method by allowing quantitation of phosphorylation stoichiometry , which can be used to confirm that antibody signal strength correlates with phosphorylation levels.

What are common pitfalls when interpreting results from phospho-TP53 (Ser315) antibody experiments?

Researchers should be aware of several potential pitfalls when interpreting results from phospho-TP53 (Ser315) antibody experiments:

• Stoichiometry considerations:

  • Phosphorylation may occur on only a fraction of total p53 molecules, making detection challenging without sufficient sensitivity

  • The research mentions a native phospho-specific IgG binding assay for quantitating the extent of phosphorylation (one to four phosphates per tetramer) , highlighting that p53 tetramers may have variable phosphorylation states

  • Bulk measurements may mask significant cell-to-cell heterogeneity in phosphorylation levels

• Context dependency:

  • Phosphorylation patterns vary significantly between cell types and treatments

  • The research demonstrates that UV irradiation induces Ser315 phosphorylation in specific cell lines , but this may not generalize to all cell types or stress conditions

  • Results from one experimental system (e.g., specific cell line or treatment) require validation in multiple systems

• Temporal dynamics:

  • Phosphorylation is a dynamic modification; timing of analysis after stimulation is critical

  • Without appropriate time-course experiments, peak phosphorylation events may be missed

  • The kinetics of phosphorylation may differ between cell types and treatments

• Total p53 fluctuations:

  • p53 protein levels often change dramatically in response to stress, complicating interpretation

  • Proper normalization to total p53 is essential, as mentioned in the research on cell-based ELISA methods

  • Changes in apparent phosphorylation could reflect altered total protein rather than modified phosphorylation status

• Antibody limitations:

  • Cross-reactivity with other phosphorylation sites, particularly those with similar surrounding sequences

  • Potential epitope masking due to protein-protein interactions or other post-translational modifications

  • Batch-to-batch variability in antibody specificity and sensitivity

To address these pitfalls, researchers should employ multiple complementary approaches, including phospho-mimetic and phospho-deficient mutants, alongside appropriate controls and normalization strategies.

How can phospho-TP53 (Ser315) antibodies be used to study cancer mechanisms?

Phospho-TP53 (Ser315) antibodies offer valuable tools for investigating cancer mechanisms through multiple research approaches:

Cancer development studies can benefit from analyzing Ser315 phosphorylation across disease progression. The research notes that in human cancer, Ser15 phosphorylation occurs during the earliest stages of tumor development and may be an important element in activating p53 tumor suppressor function . Similarly, examining Ser315 phosphorylation in pre-malignant lesions compared to advanced tumors could reveal its role in cancer evolution. Since mutation at the Ser315 site (to alanine) reduces p53's activity as a transcription factor , understanding whether this phosphorylation site is functionally compromised in tumors could provide insights into mechanisms of p53 inactivation beyond coding mutations.

Therapeutic response monitoring represents another application. The research demonstrates that different stimuli lead to distinct patterns of p53 phosphorylation – DNA damage inducers like etoposide, UV, and ionizing radiation induce Ser15 phosphorylation, while Nutlin (which blocks MDM2-mediated degradation) activates p53 without significant Ser15 phosphorylation . Investigating how cancer therapeutics affect Ser315 phosphorylation could reveal mechanism-specific biomarkers of drug action.

Combination therapy development might be informed by understanding the relationship between CDK inhibition and p53 function. The research shows that Roscovitine (a CDK inhibitor) reduces p53 activity , suggesting that combination therapies involving CDK inhibitors might have complex effects on p53-dependent tumor suppression pathways that could be monitored using phospho-specific antibodies.

What is the relationship between TP53 mutations and Ser315 phosphorylation in cancer?

The relationship between TP53 mutations and Ser315 phosphorylation represents an important area for cancer research that requires systematic investigation:

First, researchers should consider whether common TP53 mutations affect the ability of p53 to be phosphorylated at Ser315. While the research shows that substituting Ser315 with alanine reduces p53's transcriptional activity , many cancer-associated mutations occur in the DNA-binding domain rather than at phosphorylation sites directly. These mutations might indirectly affect phosphorylation by altering protein conformation or kinase recognition motifs. Studies comparing Ser315 phosphorylation between wild-type and mutant p53 expressed in isogenic cell lines would address this question.

Second, the functional consequences of Ser315 phosphorylation might differ between wild-type and mutant p53. The research demonstrates that Ser315 phosphorylation enhances transcriptional activity of wild-type p53 , but some mutant p53 proteins exhibit gain-of-function activities unrelated to canonical transcription. Whether Ser315 phosphorylation modulates these alternative functions remains to be determined.

Third, the kinases responsible for Ser315 phosphorylation might be differentially regulated in cancer cells. The research identifies CDKs as the kinases that phosphorylate Ser315 , and since cell cycle dysregulation is a hallmark of cancer, altered CDK activity could affect the pattern of p53 phosphorylation. Comparing the effects of CDK inhibitors like Roscovitine on p53 phosphorylation in normal versus cancer cells would provide insight into this relationship.

How might multi-parametric analysis of p53 modifications improve cancer diagnostics?

Multi-parametric analysis of p53 modifications, including Ser315 phosphorylation, represents a promising approach for improving cancer diagnostics and treatment stratification:

The research demonstrates that different p53 phosphorylation sites have distinct functional impacts – Ser15 and Ser315 phosphorylation enhance transcriptional activity , while Ser392 substitution had no detectable effect under the same conditions . This suggests that the specific pattern or "barcode" of p53 modifications might provide more information than examining any single modification. By simultaneously analyzing multiple modifications (phosphorylation, acetylation, methylation, etc.), researchers could potentially identify signature patterns associated with specific cancer types, stages, or treatment responses.

Methodologically, this could be achieved through multiplex immunoassays using antibodies targeting different modifications, phospho-proteomics approaches, or through targeted mass spectrometry. The cell-based ELISA approach described in the research could potentially be adapted for multiplexed detection of different modifications from the same sample.

From a diagnostic perspective, such multi-parametric analysis could help distinguish between functionally active versus inactive p53 in tumors with wild-type TP53. The research shows that even without DNA damage, a basal level of p53 phosphorylation occurs in unstimulated cells , suggesting that the absolute presence or absence of phosphorylation may be less informative than the specific pattern and stoichiometry across multiple sites.

For treatment selection, understanding the complete modification status of p53 could potentially predict response to specific therapies that depend on p53 function, such as radiation, DNA-damaging chemotherapeutics, or newer agents that reactivate mutant p53.