Phospho-KIT (Y936) Antibody

What is KIT and what role does the Y936 phosphorylation site play in signaling pathways?

KIT (CD117) is a receptor tyrosine kinase belonging to the subfamily that includes PDGF, CSF-1, and FLT3/flk-2 receptors. It plays critical roles in cell survival, proliferation, hematopoiesis, stem cell maintenance, gametogenesis, melanocyte development, and mast cell function and migration .

The Y936 phosphorylation site is located in the C-terminal tail of the KIT receptor and serves as a key docking site for signaling molecules. When phosphorylated, this site:

• Binds to the SH2 domain of adaptor protein growth factor receptor-bound protein 2 (Grb2)

• Activates the MAPK/ERK pathway, which regulates gene transcription and cell proliferation

• May interact with PLC-γ1, contributing to downstream signaling effects

Full activation of KIT occurs through sequential phosphorylation events across multiple domains, with Y936 being one of the later sites phosphorylated after initial activation at the juxtamembrane domain (Y568/Y570) and activation loop (Y823) .

What are the typical applications of Phospho-KIT (Y936) antibodies in research?

Phospho-KIT (Y936) antibodies are versatile tools in research with several established applications:

These applications enable researchers to:

• Monitor KIT activation status in various cell types and tissues

• Evaluate effects of inhibitors or activators on KIT signaling

• Study KIT phosphorylation in disease contexts, particularly cancer models

• Investigate downstream pathway activation in response to KIT ligand (SCF) binding

What cell lines or tissue samples are most suitable for studying KIT Y936 phosphorylation?

Based on research data, the following models have demonstrated detectable levels of phosphorylated KIT at Y936:

When selecting experimental models, researchers should consider:

• Basal KIT expression levels (often high in mast cells, melanocytes, hematopoietic stem cells)

• Stimulation conditions (SCF treatment may be necessary to induce phosphorylation)

• Potential mutations that might affect signaling dynamics

How should samples be prepared to preserve KIT Y936 phosphorylation for accurate detection?

Phosphorylation is a dynamic post-translational modification that can be rapidly lost through phosphatase activity. To ensure accurate detection of KIT Y936 phosphorylation:

Immediate processing is critical:

• Process samples immediately after collection to prevent dephosphorylation

• If immediate processing is impossible, use phosphatase inhibitors in collection buffers

• For cell culture experiments, lyse cells directly in buffer containing phosphatase inhibitors

Recommended lysis procedure:

• Use ice-cold lysis buffer containing:

  • 1% NP-40 or Triton X-100

  • Protease inhibitor cocktail

  • Phosphatase inhibitor cocktail (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • EDTA to chelate metal ions required for phosphatase activity

• Maintain samples at 4°C throughout processing

• Clarify lysates by centrifugation (14,000g for 10 minutes at 4°C)

• Quantify protein concentration

• Immediately add denaturing SDS-PAGE sample buffer and heat to 95°C for 5 minutes

• Store at -80°C until analysis or proceed directly to gel electrophoresis

For tissue samples:

• Flash freeze tissues in liquid nitrogen immediately after collection

• Store at -80°C until processing

• During processing, maintain phosphatase inhibition throughout

How can I validate the specificity of a Phospho-KIT (Y936) antibody?

Validating antibody specificity is crucial for accurate interpretation of results. For Phospho-KIT (Y936) antibodies, use these approaches:

Peptide competition assay:

• Pre-incubate the antibody with excess phospho-peptide (the immunogen)

• In parallel, run the antibody without peptide pre-incubation

• A significant reduction in signal with the phospho-peptide confirms specificity

• Also test with non-phosphorylated peptide to confirm phospho-specificity

Phosphatase treatment:

• Split your sample and treat half with lambda phosphatase

• The signal should disappear in the phosphatase-treated sample

• This confirms the antibody detects only phosphorylated epitopes

Stimulation/inhibition experiments:

• Stimulate cells with SCF (KIT ligand) to increase phosphorylation

• Treat cells with tyrosine kinase inhibitors to decrease phosphorylation

• The antibody signal should increase with stimulation and decrease with inhibition

Genetic approaches:

• Use KIT knockout or knockdown cells as negative controls

• Express wild-type KIT versus Y936F mutant (cannot be phosphorylated)

• The antibody should not detect the Y936F mutant

What are the most effective blocking conditions for Western blot applications with Phospho-KIT (Y936) antibodies?

Optimal blocking conditions can significantly improve signal-to-noise ratio when using Phospho-KIT (Y936) antibodies:

Important considerations:

• Avoid milk for blocking when detecting phosphorylated proteins, as it contains phospho-proteins and phosphatases that can interfere with detection

• BSA is the preferred blocking agent for phospho-epitope detection

• For challenging samples, consider specialized blocking buffers formulated for phospho-protein detection

• Optimize antibody concentration (typically 1:500-1:2000 dilution range for Western blot)

How does KIT Y936 phosphorylation differ from other KIT phosphorylation sites in terms of downstream signaling?

KIT activation involves a sequential and site-specific phosphorylation cascade that activates distinct downstream pathways. Y936 has unique signaling characteristics compared to other phosphorylation sites:

Y936 signaling specificity:

• Unlike Y721 (which primarily activates PI3K/AKT), Y936 predominantly activates the MAPK/ERK pathway

• While both Y703 and Y936 bind Grb2, they may do so with different affinities or in different cellular contexts

• Y936 phosphorylation typically occurs after the initial activation events (Y568/Y570 and Y823)

• Designer receptor studies demonstrate that replacing Y936 with other tyrosine motifs alters downstream signaling specificity

Understanding these site-specific differences allows researchers to target specific KIT-mediated pathways in experimental or therapeutic contexts.

What methodologies can be used to study the dynamics of KIT Y936 phosphorylation in response to stimuli?

Several advanced methodologies can reveal the temporal and spatial dynamics of KIT Y936 phosphorylation:

Real-time phosphorylation monitoring:

• Phospho-flow cytometry to quantify phosphorylation in intact cells

• FRET-based biosensors incorporating the KIT Y936 region and SH2 domains

• Live-cell imaging with fluorescently tagged SH2 domains that bind specifically to phospho-Y936

Quantitative phosphoproteomics:

• Mass spectrometry-based approaches to quantify site-specific phosphorylation

• SILAC or TMT labeling for comparing phosphorylation levels across conditions

• Enrichment strategies using phosphotyrosine antibodies before MS analysis

Time-course experimental design:

• Stimulate cells with SCF at physiologically relevant concentrations (10-100 ng/mL)

• Collect samples at multiple timepoints (0, 1, 5, 15, 30, 60, 120 min)

• Analyze phosphorylation using Western blotting or MS

• Plot phosphorylation kinetics to determine rate constants

• Compare Y936 phosphorylation kinetics with other sites and downstream events

Inhibitor studies:

• Use selective inhibitors to block specific pathways and assess feedback on Y936 phosphorylation

• Apply kinase inhibitors in time-course experiments to determine the persistence of phosphorylation

• Combine with genetic approaches (e.g., site-directed mutagenesis) to validate results

How can designer receptor approaches utilizing KIT Y936 phosphorylation be implemented for signaling pathway interrogation?

Designer receptor approaches offer powerful tools for dissecting KIT signaling with high specificity. Based on research findings, the following methodology can be implemented:

Construction of designer KIT receptors:

• Start with a backbone containing:

  • The kinase domain (for tyrosine phosphorylation)

  • FKBP(F36V) (for ligand-dependent dimerization)

  • Specific tyrosine motifs for signal transducer binding

• Engineer the receptor by:

  • Replacing native tyrosine motifs with specific signaling motifs

  • Selecting the optimal position for motif insertion (N-terminus, Y730 position, Y747 position, or C-terminus work best)

  • Maintaining the juxtamembrane domain (JMD) structure to preserve kinase activity

Implementation strategy:

• Express the designer receptor in appropriate cell lines (preferably with low endogenous KIT)

• Induce dimerization using AP20187 (for FKBP(F36V) systems) or light stimulation (for optogenetic systems)

• Measure pathway activation using phospho-specific antibodies

• Verify specificity by assessing activation of target versus non-target pathways

Applications of this approach:

• Dissect the contribution of individual signaling pathways to biological outcomes

• Create receptors that activate only specific pathways (e.g., STAT3 but not PI3K)

• Design receptors that activate multiple specific pathways simultaneously

• Develop optogenetic systems for spatiotemporally controlled pathway activation

The research by Harada et al. demonstrates that designer receptors can achieve pathway-specific signaling, with the positioning of tyrosine motifs being critical for functionality .

What is the relationship between KIT Y936 phosphorylation and cancer progression or therapeutic response?

KIT Y936 phosphorylation has significant implications for cancer biology and therapeutic approaches:

Cancer-relevant findings:

• KIT mutations can lead to constitutive activation and are associated with various cancers including gastrointestinal stromal tumors (GISTs), acute myeloid leukemia, and mastocytosis

• Y936 phosphorylation activates the MAPK/ERK pathway, which drives cell proliferation and can contribute to oncogenic signaling

• Phosphorylation at Y936 creates binding sites for adaptor proteins that can influence cancer cell growth, survival, and migration

Therapeutic implications:

• Tyrosine kinase inhibitors targeting KIT (e.g., imatinib) may have different efficacies depending on phosphorylation patterns

• Phosphorylation status at Y936 could potentially serve as a biomarker for KIT activation in cancer samples

• In FLT3-ITD acute myeloid leukemia, kinase phosphorylation profiles (including sites analogous to KIT Y936) may predict response to tyrosine kinase inhibitors

Research directions:

• Investigate whether Y936 phosphorylation status correlates with response to KIT inhibitors

• Determine if resistance mechanisms involve altered phosphorylation at Y936 or its downstream targets

• Explore combination therapies targeting both KIT and Y936-activated pathways

• Develop phosphorylation-specific inhibitors that could target cancers with high Y936 phosphorylation

The research by Cucchi (2021) suggests that phosphorylation profiling may provide valuable information for predicting therapeutic responses in diseases where receptor tyrosine kinase signaling is dysregulated .

What are common issues encountered when using Phospho-KIT (Y936) antibodies and how can they be resolved?

Researchers using Phospho-KIT (Y936) antibodies may encounter several challenges:

Additional optimization strategies:

• For Western blotting, consider using gradient gels to better resolve KIT (145 kDa)

• For immunoprecipitation, use a total KIT antibody for IP followed by Phospho-KIT (Y936) for detection

• Validate results with complementary techniques (e.g., mass spectrometry)

How can I distinguish between specific and non-specific signals when studying KIT Y936 phosphorylation?

Distinguishing specific from non-specific signals is crucial for accurate interpretation. Implement these validation strategies:

Essential controls:

• Positive control: Include a sample known to contain phospho-KIT (Y936), such as SCF-stimulated cells expressing KIT

• Negative controls:

  • Unstimulated samples (minimal phosphorylation)

  • Samples treated with tyrosine kinase inhibitors

  • KIT-negative cell lines

  • Phosphatase-treated samples

Specificity validation:

• Peptide competition: Pre-incubate antibody with:

  • Phospho-Y936 peptide (specific signal should decrease)

  • Non-phospho Y936 peptide (specific signal should remain)

  • Unrelated phospho-tyrosine peptide (specific signal should remain)

• Multiple antibody approach: Use different antibodies targeting:

  • The same phospho-epitope from different vendors

  • Different phospho-sites on KIT

  • Total KIT protein

Molecular validation:

• Express wild-type KIT vs. Y936F mutant (cannot be phosphorylated)

• Use siRNA/shRNA to knock down KIT and confirm signal reduction

• If possible, perform mass spectrometry validation of the phosphorylation site

These strategies, when used in combination, provide robust validation of Phospho-KIT (Y936) antibody specificity.

What are the best approaches for quantifying KIT Y936 phosphorylation in research samples?

Accurate quantification of KIT Y936 phosphorylation requires careful methodological consideration:

Western Blot Quantification:

• Use a standard curve of phosphorylated recombinant protein (if available)

• Always normalize phospho-signal to total KIT protein

• Use infrared fluorescence-based detection systems (e.g., LI-COR) for wider linear range

• Include multiple exposure times to ensure linearity of signal

• Use image analysis software with background subtraction capabilities

ELISA-Based Quantification:

• Commercial sandwich ELISA kits specific for phospho-KIT (Y936)

• Custom ELISA using capture antibody (total KIT) and detection antibody (phospho-Y936)

• Phospho-specific multiplex immunoassays (e.g., Luminex platform)

Mass Spectrometry Approaches:

• Absolute quantification using synthetic phosphopeptide standards

• Relative quantification comparing samples across conditions

• SILAC or TMT labeling for multiplexed quantification

• Selected reaction monitoring (SRM) for targeted quantification

Flow Cytometry:

• Single-cell quantification using permeabilization and phospho-KIT (Y936) antibody

• Particularly useful for heterogeneous samples (e.g., bone marrow aspirates)

• Can be combined with other markers for multiparametric analysis

Critical considerations for quantification:

• Always run technical and biological replicates

• Include internal controls for normalization across experiments

• Consider the dynamic range of your detection method

• Account for potential variations in antibody affinity across different sample types

How can phospho-KIT (Y936) analysis be integrated into broader phosphoproteomic studies?

Integrating phospho-KIT (Y936) analysis into comprehensive phosphoproteomic studies provides valuable insights into signaling networks:

Integration strategies:

• Multi-level phosphorylation analysis:

  • Examine Y936 phosphorylation in context with other KIT phosphorylation sites

  • Compare with phosphorylation patterns of related RTKs (e.g., PDGFR, FLT3)

  • Correlate with downstream effector phosphorylation (MAPK/ERK pathway components)

• Phosphoproteomics workflow integration:

  • Include phospho-specific enrichment strategies (phosphotyrosine antibodies, IMAC, TiO2)

  • Use targeted MS approaches to ensure detection of the Y936 phosphopeptide

  • Apply sequential enrichment to first capture KIT and then analyze phosphorylation patterns

• Systems biology approaches:

  • Build pathway models incorporating Y936 signaling nodes

  • Apply computational approaches to predict network responses to perturbations

  • Use mathematical modeling to understand phosphorylation dynamics

Methodological considerations:

• Consider using both antibody-based and MS-based approaches for complementary data

• Implement standardized sample handling to preserve phosphorylation status

• Apply appropriate normalization strategies when comparing across samples

• Consider temporal dynamics in experimental design

Research by Cucchi (2021) demonstrates how phosphoproteomic approaches can reveal meaningful patterns in tyrosine kinase signaling that correlate with biological outcomes in diseases like acute myeloid leukemia .

What insights can be gained from studying KIT Y936 phosphorylation in stem cell biology and development?

KIT signaling plays crucial roles in stem cell function and development, with Y936 phosphorylation contributing to specific aspects:

Stem cell functions influenced by KIT signaling:

• Hematopoietic stem cell self-renewal and differentiation

• Germ cell development and migration

• Melanocyte development and survival

• Interstitial cells of Cajal development (gut pacemaker cells)

Y936 phosphorylation in developmental contexts:

• Activation of the MAPK/ERK pathway through Y936 phosphorylation may regulate proliferation versus differentiation decisions in stem cells

• The Grb2 adapter protein, which binds to phospho-Y936, is involved in multiple developmental signaling pathways

• Temporal regulation of Y936 phosphorylation may help coordinate developmental timing

Research applications:

• Stem cell maintenance and expansion:

  • Monitor Y936 phosphorylation as a marker of KIT activation in culture

  • Correlate phosphorylation patterns with self-renewal versus differentiation

  • Use phospho-Y936 status to optimize culture conditions

• Developmental biology:

  • Track Y936 phosphorylation during critical developmental windows

  • Create phospho-mimetic or phospho-deficient Y936 mutations to study functional consequences

  • Use designer receptor approaches to selectively activate Y936-dependent pathways

• Disease modeling:

  • Study how KIT mutations affect Y936 phosphorylation in developmental disorders

  • Investigate the role of Y936-dependent signaling in developmental malignancies

  • Explore therapeutic approaches targeting Y936-dependent pathways

Understanding Y936 phosphorylation dynamics in stem cells may provide insights into both normal development and pathological conditions arising from dysregulated KIT signaling.

How can computational approaches enhance our understanding of KIT Y936 phosphorylation networks?

Computational approaches provide powerful tools for understanding the complex networks involving KIT Y936 phosphorylation:

Structural modeling applications:

• 3D structural modeling of the full-length KIT cytoplasmic domain can reveal insights into Y936 accessibility and interactions

• Molecular dynamics simulations can predict how Y936 phosphorylation affects protein conformation

• Docking studies can model interactions between phospho-Y936 and SH2 domain-containing proteins like Grb2

Network modeling approaches:

• Signaling pathway reconstruction:

  • Integrate phosphoproteomics data to build KIT-centered signaling networks

  • Map Y936-specific interactions and downstream effects

  • Identify potential feedback mechanisms and cross-talk with other pathways

• Dynamical systems modeling:

  • Create mathematical models of phosphorylation kinetics

  • Simulate temporal responses to different stimuli (e.g., varying SCF concentrations)

  • Predict system behavior under different perturbations (inhibitors, mutations)

• Machine learning applications:

  • Use phosphorylation signatures to predict cellular responses or disease outcomes

  • Identify patterns correlating Y936 phosphorylation with biological phenotypes

  • Develop predictive models for therapeutic responses based on phosphorylation profiles

Integration with experimental data:

• Use computational predictions to guide experimental design

• Refine models iteratively with experimental validation

• Develop hybrid approaches combining computational and experimental methods

The study by Cucchi (2021) demonstrates how phosphoproteomic data can be integrated with computational approaches to identify patterns that predict therapeutic responses in diseases involving tyrosine kinase signaling .