Phospho-ESR1 (Ser106) Antibody

What is the biological significance of Ser106 phosphorylation in estrogen receptor alpha signaling?

Phosphorylation of estrogen receptor-α (ERα) at Ser106 in transcription activation function 1 (AF-1) plays a critical role in stimulating ERα activity through ligand-independent mechanisms. This post-translational modification is primarily mediated by extracellular signal-regulated kinases 1 and 2 (Erk1/2) mitogen-activated protein kinase (MAPK) both in vitro and upon stimulation of MAPK activity in vivo . Research has demonstrated that Ser106 phosphorylation, often co-occurring with Ser104 phosphorylation, significantly enhances ERα transcriptional activity . Interestingly, acidic amino acid substitution experiments have shown that modifications at Ser104 or Ser106 stimulate ERα activity to a greater extent than equivalent substitutions at the more commonly studied Ser118 site . This enhanced activation suggests a particularly important role for Ser106 phosphorylation in modulating estrogen receptor function.

The MAPK-mediated phosphorylation of Ser106 enables ERα to respond to growth factor signaling pathways, creating a mechanism for cross-talk between steroid hormone and growth factor signaling networks . Furthermore, Ser106 phosphorylation contributes to the agonist activity of selective estrogen receptor modulators (SERMs) such as 4-hydroxytamoxifen (OHT), potentially contributing to resistance mechanisms in breast cancer treatment .

How does Ser106 phosphorylation compare with other phosphorylation sites on estrogen receptor alpha?

ESR1 undergoes complex regulation through multiple phosphorylation events at different sites, each with distinct functional implications. The table below compares key phosphorylation sites identified in breast cancer tissues:

Multiple studies have demonstrated that Ser104/106 phosphorylation works synergistically with other modifications to regulate ERα activity. Importantly, studies using specific antibodies have enabled detection of these multiple phosphorylated ERα forms in breast cancer tissue, suggesting potential clinical relevance for therapy selection .

What are the optimal applications and dilution ranges for Phospho-ESR1 (Ser106) antibodies?

Phospho-ESR1 (Ser106) antibodies can be utilized across various experimental platforms, each requiring specific optimization. The following table outlines recommended applications and dilution ranges based on validated antibody performance:

For optimal results in each application, consider the following methodological recommendations:

For Western blotting: Include phosphatase inhibitors in sample preparation buffers to preserve phosphorylation status. Fresh sample preparation is critical as phosphorylated epitopes can be labile. When performing quantitative analysis, normalize phospho-specific signal to total ERα expression levels .

For immunohistochemistry: Heat-induced antigen retrieval using citrate buffer is recommended . Due to the critical importance of tissue collection and processing time on phospho-epitope preservation, samples should be fixed as quickly as possible after collection. Semi-quantitative scoring systems combining staining intensity (0-3) and percentage of positive cells (0-100%) are commonly employed, generating a final score range of 0-300 .

How can researchers validate the specificity of Phospho-ESR1 (Ser106) antibodies?

Demonstrating antibody specificity is crucial for accurate interpretation of phosphorylation data. The following methodological approach is recommended for validating Phospho-ESR1 (Ser106) antibodies:

• Site-directed mutagenesis validation: Generate ERα mutants where Ser106 is substituted by alanine (S106A). These mutants should not be recognized by the phospho-specific antibody. Similarly, mutation of adjacent sites (such as S104A) should not eliminate antibody binding if the antibody is truly specific for pSer106 .

• Peptide competition assays: Pre-incubate the antibody with phosphorylated peptides containing the Ser106 site. This competition should abolish specific signal in subsequent applications. Non-phosphorylated peptides of the same sequence should not significantly affect antibody binding .

• Kinase modulation: Treat samples with MAPK pathway activators (such as PMA) to enhance Ser106 phosphorylation or inhibitors (such as U0126) to reduce phosphorylation. The signal intensity should correlate with the expected phosphorylation status changes .

• Phosphatase treatment: Exposing samples to lambda phosphatase before antibody application should eliminate specific binding of phospho-antibodies.

• Control for cross-reactivity: Test antibodies against related phosphorylation sites (e.g., Ser104, Ser118) to ensure specificity for the Ser106 epitope.

Research has shown that Ser106 phosphorylation can be stimulated following treatment with estradiol (E2) and phorbol 12-myristate 13-acetate (PMA) . These treatments provide positive controls for antibody validation experiments.

How can Phospho-ESR1 (Ser106) antibodies be used to study tamoxifen resistance mechanisms in breast cancer?

Phosphorylation of ERα at Ser106 has significant implications for tamoxifen resistance mechanisms in breast cancer therapy. Researchers can utilize Phospho-ESR1 (Ser106) antibodies to investigate these mechanisms through several methodological approaches:

• Tissue microarray analysis: Using validated phospho-specific antibodies to examine Ser106 phosphorylation status in breast cancer patient cohorts with known responses to tamoxifen therapy. The relationship between phosphorylation levels and clinical outcomes can be assessed through semi-quantitative scoring methods as described in previous studies . This approach may help identify patient subgroups more likely to develop resistance.

• Cell line models of acquired resistance: Developing tamoxifen-resistant cell lines through long-term culture with tamoxifen and analyzing changes in Ser106 phosphorylation status compared to parental cells. Phospho-ESR1 (Ser106) antibodies can be used in Western blot and immunofluorescence applications to track these changes .

• MAPK pathway inhibition studies: Since Ser106 is phosphorylated by MAPK, combining tamoxifen with MAPK pathway inhibitors (such as MEK inhibitors) may prevent or reverse resistance. Phospho-ESR1 (Ser106) antibodies can monitor the efficacy of such combination treatments in modulating ERα phosphorylation .

• Site-directed mutagenesis experiments: Generating S106A mutants (preventing phosphorylation) or S106E mutants (mimicking constitutive phosphorylation) to directly assess the contribution of this specific site to tamoxifen response in cellular models.

Importantly, research has shown that Ser104 and Ser106 are required for the agonist activity of the selective ER modulator 4-hydroxytamoxifen , supporting their potential role in resistance mechanisms.

What experimental approaches can elucidate the interplay between Ser106 phosphorylation and other post-translational modifications of ERα?

Estrogen receptor alpha undergoes numerous post-translational modifications that collectively regulate its function. Investigating the interplay between Ser106 phosphorylation and other modifications requires sophisticated experimental designs:

• Sequential chromatin immunoprecipitation (ChIP): Using Phospho-ESR1 (Ser106) antibodies followed by antibodies targeting other modifications to determine if multiple modifications co-occur at specific genomic loci.

• Mass spectrometry-based approaches: Immunoprecipitating ERα and analyzing the modification landscape through mass spectrometry to identify patterns of co-occurring modifications with Ser106 phosphorylation. This approach can reveal previously unknown relationships between different modifications.

• Proximity ligation assays: Detecting spatial relationships between Ser106 phosphorylation and other modifications in situ at the single-molecule level.

• Pulse-chase experiments: Tracking the temporal sequence of modifications to determine if Ser106 phosphorylation precedes or follows other modifications such as ubiquitination.

• Combinatorial mutagenesis: Generating ERα mutants with mutations at multiple modification sites to assess functional consequences.

The extensive post-translational modification profile of ERα includes phosphorylation by various kinases (cyclin A/CDK2, CK1, MAPK), glycosylation (containing N-acetylglucosamine), ubiquitination (regulated by LATS1 via DCAF1, deubiquitinated by OTUB1, ubiquitinated by STUB1/CHIP and UBR5), dimethylation (by PRMT1 at Arg-260), demethylation (by JMJD6), and palmitoylation (by ZDHHC7 and ZDHHC21) . Each of these modifications may interact with Ser106 phosphorylation to fine-tune receptor function in different cellular contexts.

What are the critical factors for successful detection of Phospho-ESR1 (Ser106) in tissue samples?

Detecting phosphorylated proteins in tissue samples presents unique challenges compared to cell culture systems. For optimal detection of Phospho-ESR1 (Ser106) in tissues, several critical factors must be addressed:

• Tissue collection and fixation protocols: Phospho-epitopes are extremely labile and can be rapidly lost during tissue procurement. Research has demonstrated that the time from tissue collection to fixation significantly impacts phospho-epitope preservation. Ideally, tissues should be fixed or flash-frozen within 20 minutes of collection .

• Antigen retrieval optimization: Heat-induced antigen retrieval using citrate buffer (pH 6.0) has been successfully employed for Phospho-ESR1 detection in tissue microarrays . The optimal duration and temperature may require empirical determination for each tissue type.

• Signal amplification strategies: Since phosphorylation represents a substoichiometric modification, signal amplification methods such as tyramide signal amplification may improve detection sensitivity, especially in tissues with low ERα expression levels.

• Quantification methodology: Semi-quantitative scoring systems that assess both staining intensity (scale 0–3) and percentage of positive cells (0–100%) are recommended, generating a composite score range of 0–300 . Multiple independent evaluators should assess staining to ensure reliability.

• Validation with multiple antibodies: When possible, confirm findings using multiple antibodies against the same phosphorylation site or complementary approaches such as Phos-tag gel electrophoresis to detect mobility shifts associated with phosphorylation.

Researchers should be aware that no relevant clinical cut-off points have been definitively established for Phospho-ESR1 (Ser106) in the literature. Studies have used the 25th percentile of IHC scores as a threshold for positivity , but optimal thresholds may depend on the specific research question and cohort characteristics.

How can researchers address challenges in multiplexed detection of different phosphorylated forms of ESR1?

Simultaneous detection of multiple phosphorylated forms of ESR1 provides valuable insights into the complex regulation of this receptor but presents significant technical challenges. Researchers can employ the following methodological approaches:

• Sequential immunostaining protocols: Performing serial staining with different phospho-specific antibodies on the same tissue section, using methods to strip or quench previous antibodies between rounds. This approach requires careful validation to ensure complete removal of previous antibodies and preservation of epitopes.

• Multiplex immunofluorescence: Utilizing phospho-specific antibodies raised in different host species or of different isotypes that can be detected with spectrally distinct fluorophores. Advanced imaging systems allow detection of 4-7 distinct markers on a single tissue section.

• Mass cytometry (CyTOF): For cellular suspensions, metal-tagged antibodies against different phosphorylation sites can enable highly multiplexed analysis at the single-cell level.

• NanoString Digital Spatial Profiling: Combining morphological context with multiplexed protein analysis using oligonucleotide-tagged antibodies, enabling detection of numerous phosphorylation sites in spatially resolved regions of interest.

• Parallel serial sections: While not true multiplexing, analysis of adjacent tissue sections with different phospho-specific antibodies can provide insights into co-expression patterns.

Previous research has successfully detected multiple phosphorylated ERα forms in breast cancer tissue, including P-S104/106-ERα, P-S118-ERα, P-S167-ERα, P-S282-ERα, P-S294-ERα, P-T311-ERα, and P-S559-ERα using immunohistochemistry on tissue microarrays . This approach demonstrated the feasibility of profiling phosphorylated ERα isoforms as a potential strategy for selecting breast cancer patients who might benefit from specific endocrine therapy approaches.

How can Phospho-ESR1 (Ser106) antibodies be integrated into studies of MAPK-driven endocrine resistance?

MAPK pathway hyperactivation has been implicated in endocrine resistance mechanisms, making Phospho-ESR1 (Ser106) an important biomarker for investigating these processes. Researchers can integrate these antibodies into mechanistic studies through several approaches:

• Pharmacological inhibitor studies: Combining endocrine therapies with MAPK pathway inhibitors and monitoring changes in Ser106 phosphorylation. Research has shown that phosphorylation of S104 and S106 can be inhibited by the MEK1/2 inhibitor U0126 and by expression of kinase-dead Raf1 . Phospho-ESR1 (Ser106) antibodies can serve as pharmacodynamic markers for the efficacy of pathway inhibition.

• Patient-derived xenografts (PDX): Establishing PDX models from endocrine-resistant breast cancers and analyzing Ser106 phosphorylation status in response to different therapeutic interventions. This approach bridges preclinical and clinical research.

• Reverse phase protein arrays (RPPA): High-throughput analysis of phosphorylation networks, including Ser106, across large panels of cell lines or patient samples to identify patterns associated with resistance.

• Single-cell analysis: Examining heterogeneity in Ser106 phosphorylation at the single-cell level within tumors, potentially revealing resistant subpopulations that may not be detected in bulk analyses.

• Transcriptomic correlation studies: Integrating phosphorylation data with RNA-seq to identify gene expression signatures associated with high Ser106 phosphorylation states that might predict resistance.

Research has established that MAPK-mediated hyperphosphorylation of ERα at sites including Ser106 may contribute to resistance to tamoxifen in breast cancer . This provides a strong rationale for incorporating Phospho-ESR1 (Ser106) antibodies in studies targeting the MAPK-ERα signaling axis.

What novel methodologies are being developed for quantitative analysis of Ser106 phosphorylation in liquid biopsies?

Liquid biopsy approaches represent an exciting frontier for monitoring phosphorylation events in a minimally invasive manner. Emerging technologies for detecting Phospho-ESR1 (Ser106) in liquid biopsies include:

• Extracellular vesicle (EV) isolation and analysis: Analyzing phosphorylated ERα in tumor-derived EVs using sensitive detection methods such as proximity extension assays or digital ELISA platforms. This approach requires optimization of EV isolation protocols and validation of phospho-epitope stability during processing.

• Circulating tumor cell (CTC) phosphoprotein analysis: Capturing CTCs and performing immunofluorescence analysis of Ser106 phosphorylation status. This can be combined with other phenotypic markers to characterize CTC heterogeneity.

• Plasma phosphoprotein measurements: Developing ultrasensitive immunoassays capable of directly detecting phosphorylated ERα fragments in plasma, potentially using technologies like Simoa (single molecule array) or immuno-PCR.

• Phosphoproteomic analysis of cfDNA-associated proteins: Emerging evidence suggests cell-free DNA (cfDNA) may be associated with proteins from their cell of origin, offering a potential avenue for phosphoprotein detection.

• In vitro diagnostic multivariate index assays (IVDMIAs): Developing integrated assays that combine multiple biomarkers, including Phospho-ESR1 (Ser106), to create predictive signatures for therapy response.

These approaches are still in developmental stages, and researchers should focus on rigorous analytical validation, including establishing limits of detection, reproducibility, and correlation with tissue-based measurements. The preservation of phosphorylation status during sample collection and processing remains a significant challenge that requires standardized protocols with appropriate phosphatase inhibitors.