Phospho-KIT (Y703) Antibody

What is the significance of KIT Y703 phosphorylation in cellular signaling?

KIT (CD117) is a receptor tyrosine kinase that plays essential roles in cell survival, proliferation, hematopoiesis, stem cell maintenance, and melanogenesis. Tyr703 is located in the kinase insert domain of c-kit. Its phosphorylation provides a crucial docking site for Grb2 binding, which mediates downstream signaling through the RAS/RAF/MAPK pathway .

When activated by its ligand stem cell factor (SCF), KIT undergoes dimerization and trans-phosphorylation at multiple tyrosine residues, including Y703. This phosphorylation event is a critical step in normal KIT signaling that enables the recruitment of signaling adaptor proteins and activation of downstream pathways, including the AKT1 signaling pathway, RAS/RAF/MAPK cascade, and STAT activation .

How can I validate the specificity of a Phospho-KIT (Y703) antibody in my experiments?

Validating specificity requires multiple approaches:

• Inhibitor treatment: Treat cells expressing KIT with specific KIT inhibitors (e.g., regorafenib, imatinib) and confirm that Y703 phosphorylation is reduced while total KIT remains unchanged .

• siRNA knockdown: Transfect cells with siRNA targeting KIT and verify that both total KIT and phospho-KIT are reduced .

• Stimulation experiments: Treat KIT-expressing cells with SCF and confirm increased Y703 phosphorylation using western blot. For example, in MO7e human megakaryocytic leukemic cell line, treating with 100 ng/mL of recombinant human SCF for 10 minutes should induce detectable phosphorylation at Y703 .

• Molecular weight verification: The detected band should be at approximately 145 kDa, which corresponds to the mature glycosylated form of KIT .

• Negative controls: Include samples from cell lines that do not express KIT to confirm antibody specificity.

What are the recommended applications and protocols for Phospho-KIT (Y703) antibodies?

Based on validated commercial antibodies, the following applications and protocols are recommended:

For Western blotting:

• Use PVDF membrane for optimal results

• For detection, HRP-conjugated secondary antibodies are recommended

• The expected molecular weight is approximately 145 kDa

For immunofluorescence:

• Fixation methods should preserve phosphorylation status

• Co-staining with total KIT antibodies can provide valuable comparative data

How does Phospho-KIT (Y703) signaling differ between wild-type KIT and oncogenic KIT mutants?

The phosphorylation pattern of Y703 differs significantly between wild-type and mutant KIT:

In wild-type KIT, Y703 phosphorylation is:

• Ligand (SCF) dependent and transient

• Effectively inhibited by imatinib in sensitive cells

• Primarily localized to the cytoplasm

In oncogenic KIT mutants:

• Y703 is often constitutively phosphorylated, especially in GIST tumor cells

• Shows differential sensitivity to tyrosine kinase inhibitors (TKIs) based on mutation location

• May exhibit altered subcellular distribution, including nuclear localization

Research has demonstrated that in GIST48 and GIST430 cells with KIT mutations (exon 17 D820A and exon 13 V654A), both cytoplasmic and nuclear KIT Y703 were only partially inhibited by imatinib, while in imatinib-sensitive GIST-T1 cells (KIT exon 13 K642E mutation), both cytoplasmic and nuclear KIT Y703 were strongly inhibited .

What is the significance of nuclear Phospho-KIT (Y703) in cancer research?

Nuclear localization of phosphorylated KIT represents an important area of cancer research with significant implications:

• Association with disease risk: Immunofluorescence studies on 96 GIST patient samples revealed that nuclear KIT Y703 expression levels were significantly higher in high-risk GISTs compared to moderate- and low-/very low-risk GISTs .

• Correlation with NFKBIB expression: The expression levels of nuclear KIT Y703 significantly correlate with NFKBIB expression in GISTs, suggesting a potential autoregulatory loop .

• Therapeutic implications: Nuclear KIT Y703 may contribute to drug resistance mechanisms, as evidenced by its continued phosphorylation in imatinib-resistant GIST cells. Targeting the NFKB pathway with valproic acid (VPA) has shown promise in reducing phospho-KIT levels and inhibiting tumor growth in GIST430 xenograft models, comparable to high-dose VPA alone .

The H-score analysis of patient samples demonstrated a statistically significant correlation between nuclear KIT Y703 expression and NFKBIB levels, providing strong clinical evidence for this signaling axis in GIST pathogenesis .

How can SILAC be used to analyze changes in Phospho-KIT (Y703) and related signaling networks?

Stable Isotope Labeling by Amino acids in Cell culture (SILAC) provides a powerful approach for analyzing phosphorylation dynamics:

• Experimental design:

  • Culture cells expressing different KIT mutants in media containing light, medium, or heavy isotope-labeled amino acids

  • Stimulate with SCF or leave unstimulated

  • Lyse cells and combine lysates for phosphoproteomic analysis

• Data analysis:

  • Identify phospho-peptides mapped to KIT, including pY-KIT 703

  • Quantify relative phosphorylation levels across different conditions

  • Use bioinformatics tools like DAVID for GO term analysis of differentially phosphorylated proteins

In a study comparing PHM KIT-V559D and PHM KIT-D816H cells, SILAC analysis revealed that Y-KIT 703 site was significantly more phosphorylated in PHM KIT-D816H compared to PHM KIT-V559D, suggesting mutation-specific effects on phosphorylation patterns .

What controls should be included when studying Phospho-KIT (Y703) in experimental systems?

Robust experimental design requires comprehensive controls:

• Positive controls:

  • SCF-stimulated cells expressing wild-type KIT (e.g., MO7e cells treated with 100 ng/mL SCF for 10 minutes)

  • GIST cell lines with constitutive KIT activation (e.g., GIST48, GIST430)

• Negative controls:

  • Unstimulated cells expressing wild-type KIT

  • KIT-null cell lines

  • Samples treated with phosphatase to remove phosphorylation

• Inhibitor controls:

  • Cells treated with KIT inhibitors (imatinib, regorafenib)

  • Concentration-dependent inhibition curves to demonstrate specificity

• Specificity controls:

  • siRNA knockdown of KIT

  • Competing peptide controls

  • Pre-absorption with phosphorylated and non-phosphorylated peptides

When validating antibody specificity, it's essential to demonstrate that in cells treated with KIT inhibitors, only phospho-KIT (Y703) is downregulated while total KIT remains unchanged, whereas in siRNA-treated cells, both total and phospho-KIT should be reduced .

How should samples be prepared to preserve Phospho-KIT (Y703) for detection?

Preserving phosphorylation status is critical for accurate analysis:

• Cell lysis:

  • Use lysis buffers containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Perform lysis at 4°C to minimize enzymatic activity

  • Process samples quickly to minimize dephosphorylation

• Subcellular fractionation:

  • For studying nuclear vs. cytoplasmic Phospho-KIT, use validated fractionation protocols

  • Verify fractionation purity using compartment-specific markers (e.g., LMNB1 for nuclear envelope)

• Sample storage:

  • Store lysates at -80°C with phosphatase inhibitors

  • Avoid repeated freeze-thaw cycles

  • For long-term storage, consider adding protease inhibitors as well

• Fixation for immunofluorescence:

  • Use paraformaldehyde fixation followed by permeabilization

  • For z-stack imaging to confirm nuclear localization, careful confocal microscopy settings are essential

• Tissue samples:

  • Flash-freeze tissues immediately after collection

  • Consider using phospho-epitope preservation methods during fixation for IHC applications

What are the key factors affecting detection sensitivity when working with Phospho-KIT (Y703) antibodies?

Several factors influence detection sensitivity:

• Antibody selection:

  • Monoclonal antibodies often provide higher specificity than polyclonal antibodies

  • Consider validated recombinant antibodies for superior lot-to-lot consistency

  • Verify the exact epitope recognized by the antibody

• Detection methods:

  • Enhanced chemiluminescence (ECL) provides good sensitivity for western blot

  • For quantitative analysis, consider fluorescence-based detection systems

  • For low abundance samples, consider using Simple Western™ systems which can provide higher sensitivity

• Sample enrichment:

  • Immunoprecipitation of total KIT followed by phospho-Y703 detection

  • Phospho-tyrosine enrichment followed by KIT detection

  • Cell stimulation with SCF to increase phosphorylation signal

• Signal amplification:

  • Consider tyramide signal amplification for immunohistochemistry

  • Use highly sensitive ECL substrates for western blot

  • For multiplexed analysis, consider Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) technologies

• Background reduction:

  • Optimize blocking conditions (5% BSA often works better than milk for phospho-epitopes)

  • Include appropriate washing steps

  • Use antibody dilutions optimized for specific applications (1:1000 for WB, 1:50 for IP)

How can Phospho-KIT (Y703) be used as a biomarker in cancer research?

Phospho-KIT (Y703) has several applications as a cancer biomarker:

• GIST risk stratification:

  • High nuclear phospho-KIT (Y703) expression correlates with high-risk GIST classification according to NCCN guidelines

  • H-score estimation can be used for quantitative analysis of expression levels

• Treatment response monitoring:

  • In xenograft models, KIT Y703 immunoexpression levels were significantly reduced after treatment with high-dose VPA or imatinib combined with low-dose VPA

  • This reduction correlated with inhibitory effects on tumor growth

• Resistance mechanisms:

  • Persistent Y703 phosphorylation despite TKI treatment may indicate resistance

  • Different patterns of Y703 phosphorylation between cytoplasmic and nuclear compartments may provide insights into resistance mechanisms

• Mutation-specific responses:

  • Y703 phosphorylation patterns differ between various KIT mutants (e.g., exon 11, 13, and 17 mutations)

  • These differences may help predict treatment responses to various TKIs

What are common pitfalls in Phospho-KIT (Y703) detection and how can they be addressed?

Several technical challenges may arise when working with phospho-epitopes:

• Loss of phosphorylation during sample handling:

  • Solution: Add phosphatase inhibitors immediately during sample collection and preparation

  • Maintain samples at 4°C throughout processing

  • Process samples quickly to minimize dephosphorylation

• Antibody cross-reactivity:

  • Solution: Validate antibody specificity using multiple approaches (inhibitors, siRNA)

  • Include appropriate positive and negative controls

  • Consider using recombinant antibodies with defined epitope specificity

• Variable results between experiments:

  • Solution: Standardize protocols, including cell stimulation times, lysis conditions

  • Use recombinant antibodies for superior lot-to-lot consistency

  • Include internal loading controls and normalization methods

• Difficulty detecting low-abundance phosphorylation:

  • Solution: Consider enrichment steps (IP before WB)

  • Use more sensitive detection methods (Simple Western™)

  • Optimize cell stimulation conditions to maximize phosphorylation

• Interference from nearby phosphorylation sites:

  • Solution: Use antibodies validated for specificity even in the presence of other phosphorylation events

  • Confirm key findings with multiple antibodies or other techniques (mass spectrometry)