Phospho-ESR1 (S305) Antibody

What is the significance of ESR1 S305 phosphorylation in breast cancer research?

Phosphorylation at serine 305 of ESR1 represents a critical post-translational modification with substantial implications for breast cancer pathophysiology. This modification:

• Modulates ER conformational dynamics, altering interactions with coregulators

• Contributes to ligand-independent activation of ESR1

• Converts tamoxifen from an antagonist to an agonist, promoting endocrine resistance

• Redirects ER to new transcriptional start sites, establishing a distinct gene expression profile associated with poor clinical outcomes

• Enhances receptor dimerization and DNA binding capacity

The S305 residue is located in the receptor's hinge region, which coordinates functional synergy between activation function domains (AF-1 and AF-2) in response to estrogen and antiestrogens .

Which kinases are known to phosphorylate ESR1 at S305?

Multiple kinases target the S305 residue of ESR1, each with distinct regulatory mechanisms and downstream implications:

Understanding which kinase is active in a specific research context is crucial for experimental design and data interpretation.

How should I validate the specificity of a phospho-ESR1 (S305) antibody?

Rigorous validation of phospho-specific antibodies is essential for experimental reproducibility. A comprehensive validation approach should include:

• Western blot with control lysates:

  • Use MCF-7 cells (ER-positive breast cancer cells) as positive control

  • Include samples with/without treatments that induce S305 phosphorylation (e.g., PKA activators)

  • Perform peptide competition assays using the immunizing phosphopeptide to confirm specificity

• Site-directed mutagenesis controls:

  • Generate S305A mutant (phospho-null) to confirm antibody specificity

  • Test K303R/S305A double mutant to evaluate interplay between mutations

• Phosphatase treatment:

  • Treat positive control samples with phosphatase to demonstrate phospho-dependence of signal

• Cross-reactivity assessment:

  • Test against closely related phosphorylation sites on ESR1 (e.g., S294, S236) to confirm site specificity

Document the validation process meticulously to ensure experimental rigor and reproducibility.

How does S305 phosphorylation interact with the K303R mutation in ESR1?

The K303R mutation (lysine to arginine substitution at residue 303) has significant interactions with S305 phosphorylation, creating complex regulatory dynamics:

• K303R mutant ESR1 is a more efficient substrate for phosphorylation by PKA at S305 compared to wild-type receptor

• The S305 residue shows constitutively higher phosphorylation in K303R mutant ESR1

• Mutation of S305 to alanine (S305A) in the K303R background completely abrogates this phosphorylation

• Phosphorylation of S305 in K303R mutants enhances interaction with insulin-like growth factor 1 (IGF-1) signaling

• This interaction contributes to estrogen hypersensitivity, aromatase inhibitor resistance, and tamoxifen resistance

The K303R mutation introduces a significant alteration in a region of major post-translational modifications (acetylation, ubiquitination, sumoylation, and methylation) adjacent to the S305 phosphorylation site . This creates a complex interplay that affects receptor function and therapeutic response.

What methods can detect changes in ESR1 conformation following S305 phosphorylation?

Phosphorylation at S305 induces conformational changes in ESR1 that affect ligand response and coregulator interactions. Advanced methods to detect these changes include:

• FRET (Fluorescence Resonance Energy Transfer):

  • Allows real-time monitoring of ER conformational changes upon binding of different ligands

  • Can detect differential responses to SERMs and SERDs in the context of S305 phosphorylation

  • Reveals how different antiestrogens respond to various phospho-modifications in ESR1

• Coregulator binding assays:

  • Peptide arrays allow assessment of multiple coregulators simultaneously

  • Can detect how S305 phosphorylation enhances binding to coregulators in a ligand-independent manner

  • Demonstrate how phosphorylation alters the orientation between ER and coactivators like SRC-1

• In-cell protein fragment complementation assays:

  • Enables assessment of protein-protein interactions in living cells

  • Can evaluate dimerization efficiency of wild-type versus S305 phosphorylated receptor

• Hydrogen/deuterium exchange mass spectrometry:

  • Analyzes solvent accessibility changes to detect conformational alterations

  • Provides detailed structural insights into how phosphorylation affects receptor dynamics

These techniques offer complementary information about the structural and functional consequences of S305 phosphorylation.

How can I distinguish between different kinase-mediated phosphorylation events at S305?

While multiple kinases can phosphorylate S305, distinguishing between them requires sophisticated experimental approaches:

• Kinase-specific inhibitors:

  • Use selective inhibitors for PKA (e.g., H-89), Pak1 (e.g., IPA-3), or Akt (e.g., MK-2206)

  • Monitor S305 phosphorylation reduction following specific inhibitor treatment

  • Combine with kinase activity assays to confirm target engagement

• Kinase-specific activators:

  • PKA activation with forskolin or cAMP analogs

  • Cytokine treatment to activate IKKβ

  • Growth factor stimulation for Pak1 or Akt activation

• Immunoprecipitation with kinase-specific antibodies:

  • Pull down specific kinases and perform in vitro kinase assays

  • Use recombinant kinases to phosphorylate wild-type and mutant ESR1 in vitro

• Phospho-proteomics:

  • Mass spectrometry-based approaches can identify kinase-specific phosphorylation patterns

  • Combine with kinase prediction algorithms to identify consensus motifs

  • Analysis of co-occurring phosphorylation events can provide insight into active kinase networks

Documentation of activation conditions and timing is crucial, as different kinases may operate under specific cellular contexts.

What are the optimal fixation and immunostaining protocols for phospho-ESR1 (S305) detection in tissue samples?

Detecting phospho-epitopes in fixed tissues requires specialized protocols to preserve phosphorylation status:

• Fixation considerations:

  • Formalin fixation time should be optimized (12-24 hours) to prevent epitope masking

  • Phosphate-buffered formaldehyde is preferred to preserve phospho-epitopes

  • Samples should be processed promptly to minimize phosphatase activity

• Antigen retrieval:

  • Heat-induced epitope retrieval in 10 mM citrate buffer (pH 6.0) for 10 minutes

  • Cool at room temperature for 20 minutes before proceeding

  • Addition of phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) to all buffers

• Antibody dilution and incubation:

  • Optimal dilution range for immunofluorescence: 1:200-1:1000

  • Include phosphatase inhibitors in antibody diluent

  • Consider overnight incubation at 4°C to maximize signal

• Controls:

  • Include phosphatase-treated sections as negative controls

  • Use breast cancer tissues with known S305 phosphorylation status as positive controls

  • Consider dual staining with total ESR1 antibody to normalize phospho-signal

This methodology has been validated in human breast cancer tissue sections using appropriate controls .

How should I integrate phospho-ESR1 (S305) analysis into studies of endocrine resistance?

Endocrine resistance is multifactorial, and integrating phospho-ESR1 (S305) analysis requires a comprehensive approach:

• Clinical sample considerations:

  • Analyze paired samples (pre- and post-treatment) from patients receiving endocrine therapy

  • Correlate S305 phosphorylation with treatment response and clinical outcomes

  • Consider analyzing circulating tumor cells or cell-free DNA for ESR1 mutations affecting S305 phosphorylation

• In vitro models:

  • Develop resistant cell lines through long-term exposure to endocrine therapies

  • Compare S305 phosphorylation levels between parental and resistant lines

  • Use site-directed mutagenesis (S305A or S305E) to evaluate causality in resistance

• Pathway analysis:

  • Examine cross-talk between S305 phosphorylation and other signaling pathways (IGF-1R/IRS-1/Akt)

  • Investigate how S305 phosphorylation affects chromatin binding patterns and gene expression

  • Analyze how S305 affects response to different antiestrogens (SERMs vs. SERDs)

• Therapeutic targeting:

  • Evaluate S305 phosphorylation as a biomarker for selecting optimal endocrine therapy

  • Test blocking peptides that inhibit S305 phosphorylation to restore drug sensitivity

  • Consider combination approaches targeting both ESR1 and upstream kinases

This integrated approach provides mechanistic insights and identifies potential therapeutic strategies to overcome resistance.

How do I interpret contradictory results between phospho-ESR1 (S305) levels and endocrine therapy response?

Interpreting seemingly contradictory results requires consideration of multiple factors:

• Technical considerations:

  • Different antibodies may have varying specificities and sensitivities

  • Phosphorylation can be lost during sample processing if phosphatase inhibitors are inadequate

  • Heterogeneity within tumor samples may yield inconsistent results

• Biological complexity:

  • S305 phosphorylation effects depend on ESR1 mutation status (particularly K303R)

  • Different kinases phosphorylating S305 may have distinct downstream effects

  • Cross-talk with other phosphorylation sites (e.g., S118) can modify outcomes

  • Response may vary by antiestrogen class (SERMs vs. SERDs)

• Methodological approach:

  • Analyze multiple phosphorylation sites simultaneously to detect compensatory mechanisms

  • Consider dynamic temporal changes in phosphorylation status

  • Evaluate both nuclear and cytoplasmic phospho-ESR1 localization

• Research-backed interpretation framework:

  • S305 phosphorylation converts tamoxifen from antagonist to agonist but may not affect SERD response

  • Phosphorylation at S305 can lead to phosphorylation at S118 through a positive feedback loop

  • The phospho-S305 establishes a 26-gene expression classifier that identifies patients with poor outcomes after tamoxifen treatment

This multi-dimensional analysis helps resolve apparent contradictions in experimental results.

What are the implications of cross-talk between S305 and other phosphorylation sites on ESR1?

ESR1 contains multiple phosphorylation sites that interact in complex regulatory networks:

• S305-S118 cross-talk:

  • S305 phosphorylation triggers subsequent phosphorylation of S118

  • This effect is potentiated by tamoxifen treatment

  • S305-induced ER transactivation requires functional S118

  • Creates a positive feedback loop: estrogen activates Pak1, which potentiates PKA activity for maximal S305 phosphorylation, which enhances S118 phosphorylation

• S305-K303 interplay:

  • Negative cross-talk exists between S305 phosphorylation and K303 acetylation

  • The K303R mutation enhances S305 phosphorylation efficiency

  • This cross-talk mediates estrogen hypersensitivity and resistance to aromatase inhibitors

• Integration with global phosphorylation patterns:

  • Phosphoproteomics approaches reveal phosphorylation dynamics affected by ESR1 mutations

  • Different combinations of phospho-modifications create distinct responses to various antiestrogens

• Structural consequences:

  • Phosphorylated S305 forms a charge-linked bridge with the AF-2 domain of ER

  • This enables inter-domain communication and constitutive activity from the coactivator-binding site

  • Altered orientation between ER and coactivators renders ER transcriptionally active in the presence of tamoxifen

Understanding these intricate cross-talk mechanisms is essential for developing targeted therapeutic strategies that address the complexity of endocrine resistance.

How might phospho-ESR1 (S305) analysis inform personalized medicine approaches for breast cancer?

Phospho-ESR1 (S305) analysis holds significant promise for personalizing breast cancer treatment:

• Predictive biomarker development:

  • S305 phosphorylation status may predict response to specific endocrine therapies

  • The phospho-S305-dependent 26-gene expression classifier identifies patients with poor outcomes after tamoxifen treatment

  • Combined assessment of S305 phosphorylation and ESR1 mutations could guide therapy selection

• Therapeutic resistance mechanisms:

  • Patients with tumors showing high S305 phosphorylation might benefit from SERD therapy rather than SERMs

  • S305 phosphorylation in K303R mutant tumors suggests potential benefit from combined targeting of IGF-1R/IRS-1/Akt pathways

  • Monitoring S305 phosphorylation during treatment could identify emerging resistance mechanisms

• Novel therapeutic approaches:

  • Development of S305 phosphorylation blocking peptides to restore endocrine therapy sensitivity

  • Investigation of specific kinase inhibitors (PKA, Pak1, TBK1, IKKβ) as combination therapies

  • Consideration of next-generation ESR1 degraders for patients with phospho-S305-mediated resistance

  • Exploration of drugs like H3B-6545 that covalently inactivate ESR1 by targeting different sites (e.g., S530)

• Clinical trial stratification:

  • Incorporating S305 phosphorylation status into inclusion criteria or stratification variables

  • Evaluating emerging agents like lasofoxifene or bazedoxifene specifically in patients with phospho-S305-positive tumors

This approach integrates molecular profiling with therapeutic decision-making to optimize patient outcomes.

What role might phospho-ESR1 (S305) play in understanding the impact of inflammatory signaling on breast cancer progression?

Inflammatory signaling intersects with ESR1 signaling through S305 phosphorylation in complex ways:

• Inflammatory cytokine-induced phosphorylation:

  • Inflammatory cytokines induce S305 phosphorylation via IKKβ

  • This phosphorylation establishes an ER cistrome that substantially overlaps with the estrogen-dependent ER cistrome

  • Results in cytokine-induced constitutive activation of ER, bypassing the need for estrogen

• Invasion and metastasis promotion:

  • Cytokine treatment of MCF-7 cells increases extravasation and invasion

  • Cytokines prevent the effects of tamoxifen in cells with wild-type ER but not in cells expressing S305A mutant ER

  • IKKβ-mediated S305 phosphorylation promotes the expansion of stem/basal-like cells and a dormant, metastatic phenotype

• Tumor microenvironment considerations:

  • Inflammatory cells in the tumor microenvironment may contribute to S305 phosphorylation

  • TBK1, which regulates S305 phosphorylation, connects innate immune response with ER signaling

  • Patients with tumors highly expressing TBK1 respond poorly to tamoxifen treatment and show high risk for relapse

• Therapeutic implications:

  • Anti-inflammatory approaches might reduce S305 phosphorylation and enhance endocrine therapy response

  • IKKβ inhibitors could be explored as combination therapy for patients with inflammatory signatures

  • Monitoring inflammatory markers alongside S305 phosphorylation could improve treatment selection

This research direction connects tumor microenvironment, inflammatory signaling, and endocrine response through S305 phosphorylation.

What are the optimal conditions for using phospho-ESR1 (S305) antibodies in different experimental applications?

Each experimental application requires specific optimization for phospho-ESR1 (S305) antibody use:

• Western blotting:

  • Recommended dilution: Typically 1:500

  • Sample preparation: Include phosphatase inhibitors in lysis buffer

  • Controls: Include peptide competition with immunizing phosphopeptide

  • Cell lines: MCF-7 cells serve as positive control

• Immunofluorescence:

  • Recommended dilution: 1:200-1:1000

  • Fixation: 4% paraformaldehyde for cultured cells; formalin for tissue sections

  • Antigen retrieval: 10 mM citrate buffer, pH 6.0, 10 min boiling followed by 20 min cooling

  • Controls: Include S305A mutant cells as negative control

• ELISA:

  • Recommended dilution: 1:20000

  • Coating conditions: Optimize antigen concentration and buffer pH

  • Detection system: HRP-conjugated secondary antibody with appropriate substrate

  • Standard curve: Include phosphorylated and non-phosphorylated peptide controls

• Chromatin immunoprecipitation (ChIP):

  • Cross-linking: 1% formaldehyde, 10 minutes at room temperature

  • Sonication: Optimize conditions to generate 200-500 bp fragments

  • Immunoprecipitation: 2-5 μg antibody per ChIP reaction

  • Controls: Include IgG control and total ESR1 antibody

These conditions should be further optimized for specific experimental systems and antibody lots to ensure reproducible results.

How can phospho-ESR1 (S305) analysis be integrated into multi-omics approaches for comprehensive breast cancer research?

Integration of phospho-ESR1 (S305) analysis into multi-omics frameworks provides comprehensive insights:

• Proteogenomic integration:

  • Combine ESR1 mutation analysis with phosphoproteomics to correlate genetic alterations with phosphorylation patterns

  • Integrate transcriptomics to identify genes regulated by phospho-S305 ER, such as the 26-gene classifier

  • Compare protein expression levels with phosphorylation status to understand regulatory networks

• Kinome profiling:

  • Map active kinase networks that converge on S305 phosphorylation

  • Identify additional substrates of S305-targeting kinases to understand broader pathway effects

  • Use kinase inhibitor screens to determine functional dependencies

• Epigenomic correlations:

  • Perform ChIP-seq to map phospho-S305 ER binding sites genome-wide

  • Compare with ATAC-seq data to correlate with chromatin accessibility

  • Integrate with histone modification data to understand transcriptional regulation

• Single-cell approaches:

  • Apply single-cell phosphoproteomics to capture heterogeneity in S305 phosphorylation

  • Correlate with single-cell transcriptomics to understand cell-specific responses

  • Map cellular trajectories during treatment response and resistance development

• Clinical translation:

  • Develop multiplexed immunohistochemistry panels including phospho-S305 ER

  • Integrate with genomic profiling for comprehensive biomarker analysis

  • Design clinical trials with integrated biomarker analyses to guide treatment decisions

This multi-omics approach provides a systems-level understanding of how S305 phosphorylation influences breast cancer biology and therapeutic response.