Phospho-FLT3 (Y599) Antibody

What is the biological significance of Y599 phosphorylation in FLT3 signaling?

Y599 is one of several critical autophosphorylation sites identified in the juxtamembrane domain of FLT3. Research has confirmed that Y599, along with Y572, Y589, and Y591, serves as an in vivo autophosphorylation site in human FLT3 . These phosphorylation sites play crucial roles in regulating FLT3 activity and downstream signaling.

Methodologically, researchers can detect Y599 phosphorylation using phospho-specific antibodies in techniques such as Western blot, ELISA, and immunofluorescence. When designing experiments, it's important to include appropriate controls, such as FLT3 tyrosine kinase inhibitors (TKIs) like quizartinib or midostaurin, which affect phosphorylation status.

How does Y599 phosphorylation relate to other key phosphorylation sites in FLT3?

Y599 functions within a network of phosphorylation sites that collectively regulate FLT3 signaling. Research has demonstrated that Y599 is part of a tetrad of tyrosine residues (Y589, Y591, Y597, and Y599) in the juxtamembrane region that is essential for physical interactions with other proteins . Among these sites, Y589 and Y591 have been identified as particularly critical for both ligand-dependent activation of wild-type FLT3 and the transforming potential of oncogenic FLT3 mutants .

The following table summarizes key FLT3 phosphorylation sites and their known functions:

What are the recommended experimental conditions for Phospho-FLT3 (Y599) antibody in Western blotting?

When using Phospho-FLT3 (Y599) antibody for Western blotting, researchers should follow these methodological guidelines:

For optimal results, a working dilution range of 1:500-1:2000 is recommended . Sample preparation should include phosphatase inhibitors to preserve phosphorylation status. Controls should include both positive samples (e.g., cells treated with growth factors such as EGF at 200ng/ml for 30 minutes) and negative controls (untreated cells or phospho-peptide blocked antibody) .

To validate specificity, researchers should perform peptide competition assays by pre-incubating the antibody with the phospho-peptide used as immunogen. As demonstrated in available validation data, the phospho-peptide blocking should eliminate or significantly reduce the specific signal in Western blot .

How does the subcellular localization of FLT3 affect Y599 phosphorylation status?

Research has revealed important differences in subcellular localization between wild-type FLT3 (FLT3-wt) and FLT3 with internal tandem duplication (FLT3-ITD), a common mutation in acute myeloid leukemia (AML). While FLT3-wt primarily localizes to the plasma membrane, FLT3-ITD accumulates in the perinuclear region, specifically in the Golgi apparatus .

This aberrant localization affects signaling dynamics, as FLT3-ITD activates different downstream pathways depending on its location: STAT5 activation occurs in the endoplasmic reticulum (ER), while AKT and ERK activation occurs in the Golgi apparatus . The phosphorylation status of Y599 in these different compartments represents an important research question.

Methodologically, researchers investigating this relationship should employ subcellular fractionation combined with phospho-specific antibodies, or immunofluorescence co-localization studies. Importantly, tyrosine kinase inhibitors (TKIs) can alter this localization pattern, decreasing Golgi retention and increasing plasma membrane levels of FLT3-ITD , which may affect the phosphorylation profile.

What is the relationship between SYK kinase and FLT3 Y599 phosphorylation?

SYK (spleen tyrosine kinase) has been identified as a critical regulator of FLT3 phosphorylation. Research has demonstrated that SYK directly phosphorylates several tyrosine residues in FLT3, including Y768, Y842, and Y955, but not Y969 .

While the direct effect of SYK on Y599 phosphorylation is not explicitly stated in available research, Y599 is part of a tetrad of tyrosine residues (Y589, Y591, Y597, and Y599) in the juxtamembrane domain that is essential for physical interaction with SYK . Importantly, SYK shows greater affinity for FLT3-ITD than for FLT3-wt , suggesting a potential mechanism for enhanced signaling in FLT3-ITD-positive AML.

For researchers investigating this relationship, methodological approaches should include co-immunoprecipitation experiments, in vitro kinase assays, and the use of SYK inhibitors to assess the impact on Y599 phosphorylation. Mutation studies replacing Y599 with phenylalanine (Y599F) can help determine the specific contribution of this residue to SYK-FLT3 interactions.

How do FLT3 inhibitors affect the phosphorylation status of Y599 compared to other phosphorylation sites?

FLT3 inhibitors such as quizartinib (AC220) and midostaurin (PKC412) are important therapeutic agents for FLT3-mutated AML. These tyrosine kinase inhibitors (TKIs) affect not only the kinase activity but also the subcellular localization of FLT3-ITD, markedly decreasing its retention in the Golgi apparatus and increasing plasma membrane levels .

The differential effects of these inhibitors on specific phosphorylation sites, including Y599, represent an important area of research. Understanding which phosphorylation sites are most sensitive to inhibition could provide insights into mechanisms of drug action and resistance.

Methodologically, researchers should employ time-course experiments with various inhibitor concentrations, monitoring multiple phosphorylation sites simultaneously when possible. Phospho-specific antibodies against Y599 and other sites, combined with total FLT3 detection, allow for quantitative assessment of inhibitor effects. Correlating changes in phosphorylation patterns with functional outcomes (proliferation, survival, differentiation) can help establish the biological significance of these molecular changes.

How can researchers distinguish between phosphorylation at Y599 and other nearby tyrosine residues?

Distinguishing between phosphorylation at closely spaced tyrosine residues in the juxtamembrane domain (Y589, Y591, Y597, and Y599) presents technical challenges. Researchers should consider these methodological approaches:

• Antibody validation: Use highly specific antibodies validated against synthetic phospho-peptides containing only the site of interest. Perform peptide competition assays with different phospho-peptides to confirm specificity .

• Mutational analysis: Generate Y→F mutants of each site (Y589F, Y591F, Y597F, Y599F) and test with site-specific antibodies. If an antibody shows reduced signal only with the Y599F mutant but not with other mutants, it is specific for pY599.

• Mass spectrometry: Use immunoprecipitation followed by tryptic digestion and targeted mass spectrometry approaches such as parallel reaction monitoring (PRM) to specifically identify and quantify peptides containing each phosphorylation site.

• Inhibitor profiles: Different kinases may preferentially phosphorylate specific sites. Establish profiles of phosphorylation changes in response to different kinase inhibitors to help distinguish between sites.

What controls are essential when studying FLT3 Y599 phosphorylation?

Robust experimental design for studying FLT3 Y599 phosphorylation requires several critical controls:

• Positive controls:

  • Cells treated with FLT3 ligand (FL) to stimulate receptor activation

  • Cell lines with constitutively active FLT3, such as FLT3-ITD-expressing cells

  • Growth factor stimulation (e.g., EGF treatment has been shown to induce phosphorylation)

• Negative controls:

  • FLT3 tyrosine kinase inhibitors (quizartinib, midostaurin) to reduce phosphorylation

  • Y599F mutant FLT3 (non-phosphorylatable at this site)

  • Phosphatase treatment of lysates to remove phosphorylation

• Specificity controls:

  • Peptide competition with the phospho-peptide used as immunogen

  • Comparison with a total FLT3 antibody to normalize for expression levels

  • Isotype control antibodies to assess non-specific binding

• Technical controls:

  • Loading controls (β-actin, GAPDH) for Western blotting

  • Multiple antibody dilutions to ensure detection is in the linear range

  • Inclusion of standards with known concentrations for quantitative assays

How can phospho-FLT3 (Y599) detection be integrated into multiparameter analyses of FLT3 signaling?

Modern research approaches often require integration of multiple parameters to fully understand signaling networks. For FLT3 signaling, researchers can implement these methodological strategies:

• Multiplexed Western blotting: Use systems that allow detection of multiple proteins on the same membrane through sequential probing or spectrally distinct fluorescent secondary antibodies. This enables simultaneous detection of pY599 alongside other phosphorylation sites and downstream effectors.

• Phospho-flow cytometry: Optimize protocols for detecting phospho-FLT3 (Y599) in conjunction with other phospho-proteins (pSTAT5, pERK, pAKT) using multicolor flow cytometry. This approach allows for single-cell analysis and can reveal heterogeneity in signaling responses.

• Mass cytometry (CyTOF): Develop panels that include phospho-FLT3 (Y599) and multiple other markers for high-dimensional analysis of signaling networks at the single-cell level.

• Proximity ligation assays: Detect interactions between phospho-FLT3 (Y599) and potential binding partners in situ, providing spatial information about signaling complexes.

• Computational integration: Apply statistical and machine learning approaches to integrate phosphorylation data with other parameters (gene expression, mutation status, clinical outcomes) to develop comprehensive models of FLT3 signaling.

What are common challenges in detecting phospho-FLT3 (Y599) and how can they be addressed?

Researchers may encounter several challenges when detecting phospho-FLT3 (Y599). Here are methodological solutions to common problems:

• Low signal intensity:

  • Increase protein loading (50-100 μg per lane)

  • Optimize antibody concentration (try a range from 1:500 to 1:2000)

  • Employ signal enhancement systems

  • Consider immunoprecipitation of FLT3 before Western blotting to concentrate the target

• High background:

  • Optimize blocking conditions (try BSA instead of milk for phospho-epitopes)

  • Increase washing stringency without compromising specific signal

  • Test different antibody dilutions

  • Use phospho-peptide competition to distinguish specific from non-specific signals

• Rapid dephosphorylation:

  • Ensure samples are processed rapidly and kept cold

  • Include phosphatase inhibitors in all buffers

  • Consider stabilizing phosphorylation with crosslinking agents

  • Optimize lysis conditions to inactivate endogenous phosphatases quickly

• Antibody cross-reactivity:

  • Validate with Y599F mutants

  • Perform peptide competition with phospho-peptides representing nearby sites

  • Test the antibody in FLT3-negative cell lines to assess non-specific binding

How can researchers validate that their phospho-FLT3 (Y599) antibody is detecting the intended epitope?

Validating antibody specificity for phospho-FLT3 (Y599) requires multiple complementary approaches:

• Peptide competition assays: Pre-incubate the antibody with the phospho-peptide used as immunogen before application to the Western blot or other assay. The specific signal should be eliminated or significantly reduced, as demonstrated in validation data for commercial antibodies .

• Genetic approaches: Compare detection in wild-type FLT3 versus a Y599F mutant. A truly specific antibody will show signal with wild-type FLT3 but not with the Y599F mutant. Additionally, testing in FLT3 knockout or knockdown models can confirm specificity.

• Pharmacological validation: Treat cells with FLT3 tyrosine kinase inhibitors (e.g., quizartinib, midostaurin) and confirm decreased signal. Time-course and dose-response experiments can provide additional confidence in specificity.

• Correlation with other detection methods: When possible, compare results with orthogonal methods such as mass spectrometry-based phospho-proteomics to confirm the detection of Y599 phosphorylation.

• Cross-reactivity testing: Test the antibody against related receptor tyrosine kinases (e.g., c-KIT, PDGFR) to ensure it does not detect similar phospho-epitopes in other proteins.

How can phospho-FLT3 (Y599) detection contribute to understanding mechanisms of resistance to FLT3 inhibitors?

FLT3 inhibitor resistance represents a significant clinical challenge in AML treatment. Phospho-FLT3 (Y599) detection can provide valuable insights into resistance mechanisms:

• On-target resistance: Persistent Y599 phosphorylation despite FLT3 inhibitor treatment may indicate mutations in the FLT3 kinase domain that prevent inhibitor binding while preserving kinase activity. Comparing Y599 phosphorylation patterns before treatment and at relapse can help identify such mechanisms.

• Bypass pathway activation: In some cases, alternative signaling pathways may maintain downstream signaling despite FLT3 inhibition. Monitoring Y599 phosphorylation alongside markers of alternative pathways (e.g., phospho-SYK) can reveal such bypass mechanisms.

• Pharmacodynamic assessment: Insufficient target inhibition due to pharmacokinetic issues can be detected through incomplete suppression of Y599 phosphorylation. Time-course analysis after drug administration can identify suboptimal inhibition.

• Clonal evolution: Single-cell approaches to monitor Y599 phosphorylation can detect resistant subpopulations before they become clinically apparent, allowing for early intervention strategies.

Methodologically, researchers should implement longitudinal monitoring with standardized assays to track changes in phosphorylation patterns over time and in response to treatment.

What is the potential for using phospho-FLT3 (Y599) as a biomarker in precision medicine approaches for AML?

The phosphorylation status of FLT3 at Y599 holds promise as a biomarker in several precision medicine applications for AML:

• Patient stratification: Different patterns of FLT3 phosphorylation might correlate with clinical outcomes or response to specific therapies. Baseline assessment of Y599 phosphorylation could potentially identify patients most likely to benefit from FLT3-targeted therapies.

• Real-time treatment monitoring: Periodic assessment of Y599 phosphorylation during treatment could provide early indications of response or resistance, allowing for timely therapeutic adjustments.

• Rational combination design: Understanding the effects of various drugs on Y599 phosphorylation can inform the development of synergistic combinations. For example, if a particular agent incompletely inhibits Y599 phosphorylation, adding a complementary drug targeting residual signaling might improve efficacy.

• Minimal residual disease (MRD) assessment: Detection of phospho-FLT3 (Y599) in rare leukemic cells could provide functional information about persistent disease beyond traditional genetic or immunophenotypic MRD approaches.

For clinical implementation, standardized protocols must be developed to ensure reproducible results across different laboratories and time points. Reference standards and quality control materials will be essential for assay validation and harmonization.

How might advances in single-cell phospho-protein detection impact research on FLT3 Y599 phosphorylation?

Emerging single-cell technologies are transforming our understanding of cellular heterogeneity in signaling responses. For FLT3 Y599 phosphorylation research, these advances offer several opportunities:

• Heterogeneity assessment: Single-cell approaches can reveal subpopulations with distinct phosphorylation patterns that would be masked in bulk analysis. This could identify therapy-resistant clones or cells primed for differentiation versus proliferation.

• Rare cell analysis: Techniques like mass cytometry or imaging mass cytometry can detect phospho-FLT3 (Y599) in rare leukemic stem cells or minimal residual disease, providing insights into the biology of these clinically significant populations.

• Spatial context integration: New methods combining phospho-protein detection with spatial transcriptomics or multiplexed imaging can reveal how microenvironmental factors influence FLT3 Y599 phosphorylation in different bone marrow niches.

• Dynamic signaling analysis: Live-cell reporters for kinase activity, though technically challenging for specific phosphosites, could eventually allow real-time monitoring of FLT3 signaling dynamics in response to therapeutic interventions.

Methodologically, researchers should consider:

• Optimizing fixation and permeabilization protocols to preserve phospho-epitopes while enabling single-cell resolution

• Developing standardized panels that include phospho-FLT3 (Y599) alongside other relevant markers

• Implementing computational approaches to integrate phospho-protein data with other single-cell omics datasets

• Validating findings from single-cell studies in appropriate in vivo models to confirm clinical relevance