Phospho-BRCA1 (Ser1524) Antibody

What is the functional significance of BRCA1 phosphorylation at Ser1524 in the DNA damage response pathway?

BRCA1 phosphorylation at Ser1524 is a critical post-translational modification that occurs in response to DNA damage. In Xenopus models, this phosphorylation is mediated through an ATR-dependent and Claspin-mediated recruitment mechanism following DNA damage . When cells experience genotoxic stress, BRCA1 becomes hyperphosphorylated at multiple serine residues, including Ser1524.

Research findings indicate that phosphorylation at this residue begins approximately 3-6 hours after DNA damage induction and correlates with ATM activation . This timing suggests a role in the intermediate response to DNA damage rather than the immediate early response. The phosphorylation persists for several hours and appears to be part of the cellular machinery that coordinates repair of damaged DNA.

Unlike phosphorylation at Ser1387, which is specifically required for the ATM-mediated S-phase checkpoint after ionizing radiation, Ser1524 phosphorylation appears to have different functional outcomes . When investigating DNA damage responses, monitoring phosphorylation at this residue provides insight into ATR-mediated events, particularly those involving processing of stalled replication forks.

How can I effectively validate the specificity of Phospho-BRCA1 (Ser1524) antibodies in experimental settings?

Validating phospho-specific antibodies requires comprehensive controls and multiple approaches:

Phosphatase Treatment Validation:

• Treat half of your protein sample with lambda phosphatase before Western blotting

• The phosphatase will remove phosphate groups, eliminating detection by phospho-specific antibodies

• Observe the disappearance of the phospho-BRCA1 band in treated samples compared to untreated controls

Stimulus-Response Validation:

• Compare untreated cells with those exposed to known inducers of BRCA1 phosphorylation (IR, UV, or chemotherapeutic agents)

• A properly functioning antibody will show increased signal in treated samples

• Example: U2OS cells exposed to 10 Gy IR for 1 hour show significantly increased Phospho-BRCA1 detection

Molecular Weight Verification:

• BRCA1 has a molecular weight of approximately 220 kDa

• Verify that your detected band appears at this position (note that hyperphosphorylation may cause a mobility shift)

• Antibody specifications indicate expected detection at 207-220 kDa

Dose-Response Relationship:

• Perform a titration of DNA damage-inducing agents

• A properly functioning antibody will show dose-dependent increases in phosphorylation

• As shown in studies with acetaldehyde, phosphorylation becomes detectable at 0.5 mM and increases at higher concentrations

What are the optimal experimental conditions for detecting Phospho-BRCA1 (Ser1524) in different sample types?

Western Blotting Protocol:

• Use Tris-Acetate gels (3-8%) for full-length BRCA1 detection

• Transfer to nitrocellulose membranes using standard transfer systems

• Block in TBST with 5% milk

• Use antibody dilutions of 1:1000 for most commercial Phospho-BRCA1 (Ser1524) antibodies

• For best results, load 15-50 μg protein per lane

Cell Culture Conditions:

• Timing is critical - peak phosphorylation typically occurs 3-6 hours post-DNA damage

• For UV-induced phosphorylation, collect samples 1-6 hours after exposure

• For IR-induced phosphorylation, collect samples 1-3 hours after exposure

Sample Preparation:

• Use phosphatase inhibitors in lysis buffers to preserve phosphorylation status

• Include protease inhibitors to prevent degradation of the high molecular weight BRCA1 protein

• Maintain cold temperatures throughout processing

Cell Types with Reliable Detection:

• Human osteosarcoma cell lines (U2OS) show robust phosphorylation

• Human breast cancer cell lines are appropriate model systems

• Primary fibroblasts can also be used but may show lower expression levels

How do different DNA damaging agents influence BRCA1 phosphorylation at Ser1524?

Different DNA damaging agents trigger distinct phosphorylation patterns at BRCA1 residues:

Research has demonstrated that while ATM predominantly phosphorylates BRCA1 following double-strand breaks from ionizing radiation, ATR is more involved in phosphorylation following UV damage or replication stress . This distinction is important when designing experiments to study specific DNA damage response pathways.

Notably, even low concentrations of DNA damaging agents can induce phosphorylation. For example, acetaldehyde concentrations as low as 0.1 mM show detectable effects, with maximal phosphorylation at 0.75 mM . This dose-response relationship can be used to calibrate experimental conditions.

How does the phosphorylation status at Ser1524 correlate with other BRCA1 phosphorylation sites in coordinating different DNA repair pathways?

BRCA1 contains multiple phosphorylation sites that work in concert to orchestrate DNA repair:

Research indicates that mutation of both Ser1423 and Ser1524 abolishes the ability of BRCA1 to mediate the G2/M checkpoint, while mutation at Ser1387 specifically disrupts the S-phase checkpoint . This suggests that different phosphorylation combinations direct BRCA1 toward specific DNA repair mechanisms or cell cycle checkpoints.

Studies investigating the temporal sequence of BRCA1 phosphorylation events reveal that initial phosphorylation at certain sites can prime the protein for subsequent modifications. For instance, ATM-mediated phosphorylation often precedes and facilitates ATR-dependent phosphorylation events. The sequence and combination of these phosphorylation events likely determine which repair pathway (homologous recombination vs. non-homologous end joining) will be preferentially activated .

What techniques can be employed to study the dynamic interplay between Phospho-BRCA1 (Ser1524) and its binding partners in live cells?

Investigating the dynamic protein interactions of phosphorylated BRCA1 requires sophisticated methodologies:

Proximity Ligation Assays (PLA):

• Enables visualization of protein-protein interactions in fixed cells

• Can specifically detect interactions involving phosphorylated BRCA1

• Use antibodies against Phospho-BRCA1 (Ser1524) and potential binding partners

• Quantify interaction signals in response to DNA damage or other stimuli

FRET-Based Biosensors:

• Design FRET pairs with BRCA1 and interaction partners

• Monitor real-time changes in protein proximity following DNA damage

• Can reveal transient interactions that might be missed in co-immunoprecipitation studies

• Combine with phospho-specific antibodies for validation

Live-Cell Phosphorylation Tracking:

• Utilize genetically encoded biosensors that change conformation upon phosphorylation

• Monitor kinetics of BRCA1 phosphorylation/dephosphorylation cycles

• Correlate with recruitment of repair factors to damage sites

ChIP-Seq and Re-ChIP Approaches:

• Identify genomic loci where phosphorylated BRCA1 is recruited

• Compare binding patterns of different phosphorylated forms

• Explore co-occupancy with other DNA repair factors

• Example protocol: First immunoprecipitate with general BRCA1 antibody, then re-ChIP with Phospho-BRCA1 (Ser1524) specific antibody

Mass Spectrometry-Based Interactomics:

• Perform immunoprecipitation with phospho-specific antibodies

• Identify interaction partners unique to phosphorylated vs. non-phosphorylated BRCA1

• Quantify changes in the interactome following DNA damage

• Can reveal novel binding partners specific to the phosphorylated state

These approaches provide complementary information about the functional consequences of BRCA1 phosphorylation at Ser1524 in the context of DNA damage response pathways.

How can I develop experimental models to investigate the functional consequences of disrupted BRCA1 Ser1524 phosphorylation in cancer progression?

Creating experimental models to study phosphorylation site-specific functions requires sophisticated approaches:

CRISPR-Based Phospho-Site Mutagenesis:

• Generate cell lines with S1524A mutation (phospho-dead) or S1524E mutation (phospho-mimetic)

• Compare DNA repair efficiency, cell cycle progression, and genomic stability

• Measure sensitivity to DNA damaging agents and PARP inhibitors

• Assess changes in transcriptional programs regulated by BRCA1

Phospho-Specific Rescue Experiments:

• In BRCA1-deficient cell lines (like HCC1937), introduce:

  • Wild-type BRCA1

  • S1524A mutant

  • S1524E mutant

• Compare restoration of DNA repair capabilities and checkpoint functions

• Previous studies showed that S1423/S1524 mutations affected G2/M checkpoint function

3D Organoid Models:

• Develop organoids from normal breast or ovarian tissue

• Introduce phospho-site mutations using CRISPR

• Analyze effects on tissue architecture and response to DNA damage

• Monitor for spontaneous transformation events

Patient-Derived Xenograft Models:

• Sequence patient tumors for mutations affecting Ser1524 or kinases that phosphorylate this site

• Create PDX models to study response to therapeutic agents

• Compare with PDX models having intact phosphorylation sites

Integrated Multi-Omics Analysis:

• Combine phosphoproteomics, transcriptomics, and genomic instability assays

• Identify cellular pathways specifically affected by Ser1524 phosphorylation status

• Correlate with patient outcomes and therapeutic responses in existing cancer datasets

When designing these experiments, consider that the functional impact of S1524 phosphorylation may be context-dependent, varying across tissue types, cancer subtypes, and in response to different cellular stresses. The experimental approach should reflect the specific research question being addressed.

What are the most reliable methods for quantifying changes in BRCA1 Ser1524 phosphorylation levels in patient-derived samples?

Quantifying phosphorylation in clinical samples presents unique challenges that require specialized approaches:

Optimized Immunohistochemistry (IHC) Protocols:

• Use phospho-specific antibodies validated for IHC applications

• Implement antigen retrieval methods optimized for phospho-epitopes

• Include positive controls (irradiated cell pellets) and negative controls (phosphatase-treated sections)

• Use digital pathology tools for standardized quantification

• Develop a scoring system based on staining intensity and percentage of positive cells

Reverse Phase Protein Array (RPPA):

• High-throughput method for analyzing multiple samples simultaneously

• Requires minimal amounts of protein (advantageous for limited clinical material)

• Can detect phosphorylation changes with high sensitivity

• Allows for normalization to total BRCA1 protein levels

Targeted Mass Spectrometry:

• Develop Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM) assays

• Directly quantify phosphorylated and non-phosphorylated peptides containing Ser1524

• Calculate stoichiometry of phosphorylation

• Sample preparation protocol:

  • Extract proteins from flash-frozen tissue

  • Enrich for BRCA1 by immunoprecipitation

  • Perform tryptic digestion

  • Enrich for phosphopeptides using TiO₂ or immobilized metal affinity chromatography

  • Analyze by LC-MS/MS with heavy-labeled synthetic phosphopeptide standards

Proximity Ligation Assay (PLA) for Tissue Sections:

• Combine antibodies against total BRCA1 and phospho-Ser1524

• Generates signal only when both epitopes are present and proximal

• Provides spatial information about phosphorylation within tissue architecture

• Allows correlation with other markers in the tumor microenvironment

Preprocessing Requirements for Clinical Samples:

• Samples must be collected and preserved rapidly to maintain phosphorylation status

• Use phosphatase inhibitors during sample collection and processing

• Consider the effects of tissue ischemia time on phosphorylation levels

• Document preanalytical variables for proper interpretation of results

When implementing these methods for clinical research, establish clear cutoff values and validation procedures to ensure reproducible quantification across different patient cohorts.

How does the loss of BRCA1 Ser1524 phosphorylation impact the recruitment and function of DNA repair complexes at sites of DNA damage?

The loss of BRCA1 Ser1524 phosphorylation has multi-faceted effects on DNA repair complex dynamics:

Impact on Protein-Protein Interactions:
BRCA1 phosphorylation at Ser1524 changes its interaction profile with key DNA repair components. When this phosphorylation is absent, research shows altered binding to:

• BARD1, BRCA1's primary binding partner

• DNA end resection machinery including CtIP

• RAD51 loading factors essential for homologous recombination

Spatiotemporal Recruitment Dynamics:
Studies investigating deregulation of BRCA1 have found that proper phosphorylation is critical for:

• Timely recruitment to DNA damage sites

• Appropriate retention duration at damage locations

• Formation of distinct nuclear foci patterns

• Coordination with γ-H2AX and other damage markers

Impact on End Resection:
BRCA1-BARD1 directly promotes double-strand break repair by stimulating long-range DNA end resection pathways . Loss of Ser1524 phosphorylation appears to compromise this function by:

• Reducing activation of WRN-DNA2-RPA resection machinery

• Impairing the processing of complex DNA structures at break sites

• Decreasing the efficiency of homologous recombination

Cell Cycle-Specific Effects:
The consequences of impaired Ser1524 phosphorylation vary across the cell cycle:

• In S/G2 phases: More severe impairment of homologous recombination

• In G1 phase: Altered regulation of non-homologous end joining pathways

• These differences highlight the context-dependent roles of BRCA1 phosphorylation

Repair Pathway Choice:
Loss of Ser1524 phosphorylation shifts the balance between competing DNA repair pathways:

• Decreased homologous recombination efficiency

• Potential increase in error-prone non-homologous end joining

• Accumulation of chromosomal aberrations, as demonstrated in cells with compromised BRCA1 function

These findings suggest that therapeutic strategies targeting ATR or other kinases responsible for Ser1524 phosphorylation may be effective in cancers relying on functional BRCA1-mediated repair.

What are the emerging techniques for studying the relationship between BRCA1 Ser1524 phosphorylation and chromatin modifications in the DNA damage response?

Advanced methodologies are revealing intricate connections between BRCA1 phosphorylation and chromatin dynamics:

CUT&RUN and CUT&Tag with Phospho-Specific Antibodies:

• Provides high-resolution mapping of phosphorylated BRCA1 binding sites on chromatin

• Can be performed with limited cell numbers (advantage over traditional ChIP)

• Allows correlation with histone modifications at the same genomic locations

• Implementation protocol:

  • Immobilize intact cells on ConA beads

  • Permeabilize cell membrane

  • Introduce phospho-BRCA1 (Ser1524) antibody

  • Add pA-MNase for targeted chromatin cleavage

  • Release and sequence DNA fragments

Sequential ChIP (Re-ChIP) Analysis:

• First immunoprecipitate with histone modification antibodies (e.g., γ-H2AX)

• Then perform second IP with phospho-BRCA1 (Ser1524) antibody

• Identifies genomic regions where phosphorylated BRCA1 colocalizes with specific chromatin marks

• Can reveal how chromatin context influences BRCA1 phosphorylation patterns

Live-Cell Imaging of Chromatin Dynamics:

• Utilize fluorescently tagged BRCA1 with phospho-sensors

• Track real-time changes in chromatin compaction at damage sites

• Correlate with recruitment of chromatin remodelers and modifiers

• Observe temporal relationship between BRCA1 phosphorylation and chromatin changes

Nascent Chromatin Capture:

• Isolate newly synthesized chromatin following DNA damage

• Assess phospho-BRCA1 recruitment during chromatin restoration

• Analyze histone modification patterns on nascent DNA

• Connect phosphorylation status to transcriptional restart following repair

Mass Spectrometry-Based Approaches:

• Chromatin-enriched proteomics to identify factors co-recruited with phospho-BRCA1

• Cross-linking mass spectrometry to map physical interactions in their native context

• Correlation of BRCA1 phosphorylation state with specific chromatin environments

CRISPR-Based Epigenome Editing:

• Target chromatin modifiers to specific genomic loci

• Assess how altered chromatin states affect BRCA1 phosphorylation

• Test whether heterochromatin versus euchromatin differentially influences phosphorylation at Ser1524

• Determine if pre-existing histone modifications predict BRCA1 phosphorylation efficiency