Phospho-FLT3 (Y969) Antibody

What is the significance of the Y969 phosphorylation site in FLT3 receptor biology?

The Y969 residue is located at the extreme C-terminal region of the FLT3 receptor tyrosine kinase and serves as a critical docking site for SH2 domain-containing cytosolic signaling molecules . Unlike other tyrosine residues in FLT3 (such as Y768, Y842, and Y955), Y969 displays distinct phosphorylation patterns and regulatory mechanisms. While SYK (spleen tyrosine kinase) directly phosphorylates several tyrosine residues on FLT3, including Y768, Y842, and Y955, it notably does not increase the phosphorylation level of Y969 . This selective phosphorylation profile suggests Y969 may be regulated through alternative kinase pathways, establishing its unique role in FLT3 signaling.

How does Y969 phosphorylation contribute to normal versus oncogenic FLT3 signaling?

Y969 phosphorylation contributes to the complex signaling network of FLT3 in both normal and leukemic contexts. In structure-function analyses of FLT3, the C-terminal domain containing Y969 has been identified as functionally significant for signal transduction. While juxtamembrane domain tyrosines (particularly Y589 and Y591) are most critical for the transforming potential of FLT3 mutants and ligand-dependent activation of wild-type FLT3 , the C-terminal domain phosphorylation sites, including Y969, play complementary roles in signal propagation. The phosphorylation status of Y969 may affect how cells respond to FLT3 inhibitors, potentially contributing to treatment resistance mechanisms in acute myeloid leukemia (AML) patients.

What techniques are currently available for specific detection of phosphorylated FLT3 Y969?

Several complementary approaches can be employed to detect phosphorylated FLT3 Y969:

• Cell-Based ELISA: Colorimetric assays using antibodies specific to phospho-Y969 enable relative quantification of phosphorylation levels across different cell types or treatment conditions .

• Immunoaffinity-Mass Spectrometry (IAP-MS): This approach involves immunoprecipitation with phosphotyrosine antibodies followed by LC-MS/MS analysis, which has successfully detected Y969 phosphorylation in leukemia cell lines such as SEM cells .

• Phospho-mapping analysis: This technique can identify specific phosphorylation sites regulated by upstream kinases and has been used to characterize FLT3 phosphorylation patterns, including Y969 .

• Western blotting: Using phospho-specific antibodies against Y969 can provide semi-quantitative assessment of phosphorylation status in experimental models.

What are the critical considerations for optimizing phospho-FLT3 (Y969) detection in experimental systems?

For reliable detection of phospho-FLT3 (Y969), researchers should consider:

• Sample preparation: Cell lysis conditions significantly impact phosphoprotein preservation. Use phosphatase inhibitors immediately upon cell harvest to prevent dephosphorylation.

• Antibody validation: Confirm antibody specificity using appropriate controls, including FLT3-negative cells and Y969F mutants, to ensure signals accurately represent the phosphorylation state.

• Cell fixation protocols: When using cell-based assays, the fixation method is critical. For adherent cells, 4% formaldehyde is recommended, while suspension cells (common in leukemia research) require 8% formaldehyde to adequately preserve phosphoepitopes .

• Quantification normalization: For accurate interpretation, normalize phospho-Y969 signals to total FLT3 protein levels to distinguish between changes in phosphorylation versus altered protein expression.

• Cross-reactivity assessment: Ensure the phospho-Y969 antibody does not cross-react with other phosphorylated sites or species, as specificity is paramount for result interpretation .

How does Y969 phosphorylation interact with other FLT3 phosphorylation sites in signal transduction?

FLT3 contains multiple phosphorylation sites that form an interconnected regulatory network. From existing research:

• Differential regulation: While SYK directly phosphorylates Y768, Y842, and Y955 residues, Y969 phosphorylation appears to occur through independent mechanisms . This suggests distinct regulatory pathways controlling different regions of the receptor.

• Functional complementarity: The juxtamembrane domain tyrosines (Y566, Y572, Y589, Y591, Y597, Y599) are critical for transforming capacity, while interkinase domain (Y726, Y768) and C-terminal domain (Y955, Y969) sites like Y969 may modulate signal intensity or specificity .

• Signaling node interactions: Y969 phosphorylation status may influence the activation of downstream pathways differently than other phosphorylation sites, potentially explaining differences in biological outcomes when specific sites are mutated.

What is known about the role of Y969 phosphorylation in FLT3-driven leukemias?

Y969 phosphorylation has been detected in leukemia cells, including SEM cells , suggesting its relevance in malignant contexts. As a docking site for SH2 domain-containing proteins , phosphorylated Y969 likely contributes to aberrant signaling in leukemia cells through:

• Mutant FLT3 signaling: While mutations like FLT3-ITD and D835Y dramatically alter FLT3 signaling primarily through enhanced phosphorylation at sites like Y591, Y768, and Y955 , Y969 phosphorylation may provide additional oncogenic signaling capacity.

• Therapeutic implications: The phosphorylation status of Y969 could potentially influence response to FLT3 inhibitors. Resistance mechanisms might involve altered phosphorylation patterns that maintain signaling despite inhibitor binding.

• Biomarker potential: Assessment of Y969 phosphorylation, alongside other phosphorylation sites, might provide prognostic or predictive information in AML patients.

How should researchers design experiments to investigate the specific functions of Y969 phosphorylation?

To elucidate the role of Y969 phosphorylation in FLT3 biology:

• Site-directed mutagenesis: Generate Y969F mutants (non-phosphorylatable) and compare functional outcomes with wild-type FLT3. This approach was effectively used for other tyrosine residues in FLT3 .

• Phosphomimetic mutations: Create Y969E or Y969D mutations to mimic constitutive phosphorylation and assess consequences on signaling and cellular transformation.

• Domain-specific analysis: Compare Y969 functions with other C-terminal phosphorylation sites like Y955 to understand domain-specific contributions to signaling.

• Interactome studies: Use proteomics approaches to identify proteins that specifically interact with phosphorylated Y969, as it serves as a docking site for SH2 domain-containing proteins .

• Combinatorial mutation analysis: Generate multiple mutations (e.g., Y969F combined with mutations at other sites) to understand the interplay between different phosphorylation sites.

What controls and validation steps are essential when using phospho-FLT3 (Y969) antibodies?

For rigorous experimental design using phospho-FLT3 (Y969) antibodies:

• Antibody validation controls:

  • Use Y969F mutant FLT3 as a negative control

  • Include FLT3-negative cell lines to confirm specificity

  • Validate with phosphatase treatment to demonstrate phospho-specificity

• Stimulus controls:

  • Compare FLT3 ligand-stimulated versus basal conditions

  • Include time-course analysis to capture dynamic phosphorylation events

• Inhibitor controls:

  • Use FLT3 kinase inhibitors to demonstrate signal reduction

  • Include inhibitors of upstream kinases to identify regulatory pathways

• Cell line selection:

  • Use cell lines with defined FLT3 status (wild-type, ITD, TKD mutations)

  • Include primary patient samples when possible for clinical relevance

• Quantification methodology:

  • Always normalize phospho-signal to total FLT3 protein levels

  • Use appropriate statistical analysis for comparative studies

What are common challenges in detecting FLT3 Y969 phosphorylation and their solutions?

How can phospho-FLT3 (Y969) analysis be integrated into broader FLT3 signaling studies?

For comprehensive understanding of FLT3 signaling:

• Multi-site phosphorylation profiling: Simultaneously analyze Y969 alongside other critical phosphorylation sites (Y591, Y768, Y955) to understand their coordinated regulation and interdependence.

• Pathway integration analysis: Correlate Y969 phosphorylation with activation of downstream pathways including STAT5, ERK1/2, and AKT to establish signaling connectivity .

• Structure-function correlation: Relate Y969 phosphorylation to receptor conformational changes using molecular modeling or hydrogen-deuterium exchange mass spectrometry.

• Pharmacological intervention studies: Examine how different classes of FLT3 inhibitors affect Y969 phosphorylation versus other sites to understand inhibitor mechanism of action.

• Resistance mechanism investigation: Compare Y969 phosphorylation in inhibitor-sensitive versus resistant cells to identify adaptive signaling changes.