Phospho-FANCD2 (S222) Antibody

What is the functional significance of FANCD2 S222 phosphorylation in DNA damage response pathways?

FANCD2 S222 phosphorylation plays a critical role in the cellular response to DNA damage. Research indicates that S222 phosphorylation occurs in response to DNA damage, particularly during the later phases of the damage response (4-8 hours post-irradiation), unlike other phosphorylation sites that respond more rapidly . This phosphorylation site is part of a regulatory network that mediates FANCD2's functions in DNA repair.

Methodologically, researchers can investigate S222 phosphorylation dynamics by:

• Conducting time-course experiments following DNA damage induction

• Using phospho-specific antibodies to monitor S222 phosphorylation status

• Comparing S222 phosphorylation with other DNA damage response markers

The temporal pattern of S222 phosphorylation (later than other sites) suggests it may function in the resolution phase of DNA damage response rather than the initial activation phase .

How does FANCD2 S222 phosphorylation relate to other phosphorylation sites on FANCD2?

FANCD2 undergoes phosphorylation at multiple sites, each with distinct regulatory functions and kinetics:

While S592 phosphorylation is primarily cell cycle-dependent and mediated by CDK2-Cyclin A during S-phase, S222 phosphorylation appears to be more strongly associated with the DNA damage response . Notably, phosphorylation at T691 and S717 occurs rapidly after DNA damage (within 1 hour), whereas S222 phosphorylation is detected more prominently at later timepoints (4-8 hours) .

For comprehensive analysis, researchers should monitor multiple phosphorylation sites simultaneously to understand their interdependence and sequential regulation.

What experimental methods should be used to study FANCD2 S222 phosphorylation?

Multiple complementary approaches are recommended for rigorous FANCD2 S222 phosphorylation research:

Antibody-Based Detection:

• Western blotting with phospho-specific antibodies (anti-pS222)

• Immunohistochemistry (IHC) for tissue samples

• Immunofluorescence (IF/ICC) for cellular localization

Phosphorylation Site Validation:

• Lambda phosphatase treatment to confirm phosphorylation-dependent band shifts

• Phospho-null (S222A) and phospho-mimetic (S222D) mutants for functional studies

Mass Spectrometry Analysis:

• LC-MS/MS of immunoprecipitated FANCD2 for comprehensive phosphorylation profiling

• SILAC (Stable Isotope Labeling with Amino acids in Cell culture) for quantitative phosphoproteomic analysis

For synchronization experiments to study cell cycle-dependent phosphorylation, researchers can employ:

• Double-thymidine block for S-phase synchronization

• Nocodazole treatment for M-phase synchronization

Which kinases are responsible for FANCD2 S222 phosphorylation and how can their activity be verified?

FANCD2 S222 phosphorylation appears to be mediated primarily by the ATM and ATR kinases . Unlike S592, which is primarily phosphorylated by CDK2, S222 phosphorylation occurs in response to DNA damage rather than as part of normal cell cycle progression .

To verify kinase involvement, researchers can employ:

Kinase Inhibition Approaches:

• ATR inhibitors (e.g., VE-821, AZD6738)

• ATM inhibitors (e.g., KU-55933)

• CDK inhibitors (e.g., RO3306) as controls

Genetic Approaches:

• siRNA/shRNA knockdown of candidate kinases

• CRISPR-Cas9 knockout cell lines

• Kinase-dead dominant negative constructs

In Vitro Kinase Assays:

• Recombinant kinases with FANCD2 substrate

• Phosphorylation site mapping by mass spectrometry

Importantly, researchers should establish the specificity of kinase-phosphorylation site relationships by monitoring multiple phosphorylation sites simultaneously and conducting careful time-course experiments.

What is the relationship between FANCD2 S222 phosphorylation and its monoubiquitination?

The relationship between FANCD2 phosphorylation and monoubiquitination is complex and site-specific. For S222, the relationship differs from other phosphorylation sites:

• Phosphorylation-Ubiquitination Sequence:

  • S222 phosphorylation occurs even in the absence of FANCD2 monoubiquitination, as demonstrated in experiments with the FANCD2(K561R) mutant, which cannot be monoubiquitinated

  • This suggests S222 phosphorylation may precede or occur independently of monoubiquitination

• Functional Interdependence:

  • While S592 phosphorylation promotes S-phase monoubiquitination of FANCD2 , the relationship between S222 phosphorylation and monoubiquitination is less clear

  • Research suggests phosphorylation at T691 and S717 may promote the accumulation of monoubiquitinated FANCD2

To investigate this relationship, researchers should:

• Use site-specific phospho-null mutants (S222A) and phospho-mimetic mutants (S222D)

• Perform time-course experiments with synchronized cells

• Employ dual detection of phosphorylation and ubiquitination states using specific antibodies

• Compare wild-type FANCD2 with the K561R mutant to dissect dependency relationships

How can researchers troubleshoot phospho-specific antibody validation for FANCD2 S222 in different experimental contexts?

Rigorous validation of phospho-specific antibodies is critical for reliable research outcomes. For FANCD2 S222 phospho-antibodies, comprehensive validation strategies include:

Antibody Specificity Assessment:

• Compare signal between wild-type cells and FANCD2-deficient cells (FA-D2 patient cells or CRISPR knockout lines)

• Test antibody reactivity with phospho-null mutants (S222A) as negative controls

• Evaluate cross-reactivity with other phosphorylation sites using peptide competition assays

Phosphorylation Verification:

• Treat samples with lambda phosphatase to confirm phosphorylation-dependent signal

• Use kinase inhibitors to reduce phosphorylation and confirm specificity

• Induce DNA damage to enhance phosphorylation signal in a time-dependent manner

Technical Optimization:

• Optimize antibody concentration for each application (WB, IHC, IF/ICC)

• Adjust blocking conditions to minimize background

• Employ enhanced chemiluminescence detection systems for low-abundance phosphorylated species

Common Challenges and Solutions:

• For weak signals: Enrich FANCD2 by immunoprecipitation before detection

• For high background: Increase washing stringency and optimize blocking conditions

• For inconsistent results: Standardize cell synchronization and damage induction protocols

What experimental design considerations are necessary when studying temporal relationships between FANCD2 phosphorylation events?

The different FANCD2 phosphorylation sites exhibit distinct temporal patterns, necessitating careful experimental design:

Synchronization Approaches:

• Double-thymidine block for S-phase synchronization offers good temporal resolution for studying S-phase-specific events

• Nocodazole treatment for M-phase synchronization allows study of post-mitotic regulation

• Release from synchronization should be carefully timed with sample collection

Multi-parameter Analysis:

• Simultaneously monitor multiple phosphorylation sites (S222, S592, T691, S717)

• Include cell cycle markers (e.g., H3 pS10 for mitosis)

• Track FANCD2 monoubiquitination status in parallel

Kinetics Consideration:

• Include early timepoints (1-2h) to capture rapid phosphorylation events (T691, S717)

• Extend to later timepoints (4-8h) to fully capture delayed phosphorylation events (S222)

• Use both untreated and DNA damage-induced conditions

Data Analysis Framework:

• Quantify band intensities relative to total FANCD2 protein

• Plot phosphorylation kinetics with rigorous statistical analysis

• Consider mathematical modeling to infer sequential dependencies between modifications

How does FANCD2 S222 phosphorylation contribute to mitotic fidelity and chromosomal stability?

FANCD2 phosphorylation affects mitotic fidelity through multiple mechanisms:

Experimental Evidence:

• Mutation of phosphorylation sites (like S592) leads to increased levels of micronuclei, nucleoplasmic bridges, and bi/multi-nucleated cells

• FANCD2 phosphorylation status affects the persistence of mitotic markers like H3 pS10

• FA-D2 cells complemented with phospho-site mutants show altered cell cycle progression and genomic instability

Methodological Approaches for Assessment:

• Microscopy-Based Analysis:

  • Quantification of micronuclei formation

  • Assessment of nucleoplasmic bridges

  • Enumeration of bi- and multi-nucleated cells

• Cell Cycle Analysis:

  • Flow cytometry to detect cell cycle perturbations

  • Real-time cell proliferation assays (e.g., xCELLigence system)

  • Mitotic index determination using phospho-histone H3 (Ser10)

• DNA Damage Response Assays:

  • Measure sensitivity to replication stress inducers (aphidicolin, hydroxyurea)

  • Assess DNA damage checkpoint activation

  • Quantify chromosomal aberrations using metaphase spreads

While S592 phosphorylation has been directly linked to mitotic fidelity , the specific contribution of S222 phosphorylation to these processes requires further investigation using similar methodological approaches.

How can researchers integrate FANCD2 S222 phosphorylation analysis with other DNA repair pathway markers?

Comprehensive analysis of DNA repair pathways requires integration of multiple markers:

Multiplexed Detection Strategies:

• Dual immunofluorescence for co-localization studies

• Sequential reprobing of Western blots

• Multi-parameter flow cytometry for quantitative analysis

Key DNA Repair Pathway Markers to Include:

• FA Pathway Components:

  • FANCI phosphorylation and monoubiquitination

  • FANCD2 monoubiquitination (distinct from phosphorylation)

  • FA core complex activation markers

• Related DNA Repair Pathways:

  • Homologous recombination markers (RAD51 foci, BRCA1/2)

  • Non-homologous end joining factors (53BP1, Ku70/80)

  • Replication stress indicators (RPA32 phosphorylation)

• Cell Cycle Checkpoints:

  • Chk1 and Chk2 phosphorylation

  • p53 activation markers

  • Cyclin levels and CDK activity indicators

Experimental Design Considerations:

• Include appropriate positive controls (DNA damaging agents)

• Use synchronized cell populations when feasible

• Employ genetic models (knockouts, patient-derived cells) for pathway validation

This integrated approach allows researchers to place FANCD2 S222 phosphorylation within the broader context of DNA damage response signaling networks and determine its relative importance compared to other phosphorylation events.

What are the optimal experimental conditions for detecting FANCD2 S222 phosphorylation in different cellular contexts?

Protocol Optimization Notes:

• For Western blotting: Use freshly prepared lysates and include phosphatase inhibitors

• For immunoprecipitation: Pre-clear lysates thoroughly to reduce background

• For immunofluorescence: Optimize fixation conditions (formaldehyde vs. methanol)

• For all applications: Run phospho-null mutants (S222A) as negative controls