Phospho-CBL (Y774) Antibody

What is c-CBL and why is phosphorylation at Y774 significant?

c-CBL is a proto-oncogene that encodes a RING finger E3 ubiquitin ligase essential for targeting substrates for proteasomal degradation. It functions as both an E3 ligase and scaffolding protein with three major phosphorylation sites: Y700, Y731, and Y774 . Phosphorylation at Y774 is particularly significant as it regulates interactions with SH2-domain containing proteins, specifically Crk/CrkL family proteins . This phosphorylation event serves as a critical regulatory mechanism in multiple signaling cascades including receptor tyrosine kinase signaling, cellular growth, differentiation, and immune responses .

Which kinases are responsible for c-CBL Y774 phosphorylation?

c-CBL Y774 phosphorylation is regulated by multiple kinases in a context-dependent manner:

• Src Family Kinases (SFKs): In platelets, inhibitor studies using PP2 (SFK inhibitor) demonstrated that SFKs are upstream regulators of Y774 phosphorylation .

• Syk-dependent phosphorylation: When treated with OXSI-2 (a Syk inhibitor), Y774 phosphorylation is significantly reduced, indicating Syk plays a major role in phosphorylating this residue .

• Other kinases: Research has shown that ALK, EGFR, FYN, ZAP70, as well as FLT1, KIT, INSR, FGR, FGFR3, CSF1R, PDGFRA, PDGFRB, and HCK can all contribute to tyrosine phosphorylation of c-CBL, potentially including the Y774 site .

The hierarchy appears to be that SFKs act upstream, activating Syk, which then primarily phosphorylates Y774 and Y700, while Y731 phosphorylation follows a separate regulatory pathway .

What are the optimal conditions for detecting phospho-CBL (Y774) in Western blot experiments?

For optimal detection of phospho-CBL (Y774) in Western blot experiments:

• Sample preparation: Use fresh cell lysates from appropriate positive control samples (e.g., Jurkat cells) or cells treated with appropriate stimuli that induce CBL phosphorylation.

• Recommended dilutions: Most commercial phospho-CBL (Y774) antibodies work optimally at dilutions between 1:500-1:2000 for Western blotting .

• Expected molecular weight: Look for bands at approximately 110kDa, which corresponds to the observed molecular weight of phosphorylated c-CBL, although the calculated molecular weight is around 100kDa .

• Blocking and washing: Use a standard TBST buffer with 5% BSA (rather than milk) for blocking and antibody dilution to preserve phospho-epitope recognition.

• Positive controls: Include samples from cells treated with pervanadate or receptor tyrosine kinase activators known to induce c-CBL phosphorylation .

• Negative controls: Consider including samples treated with phosphatase inhibitors or specific kinase inhibitors (PP2 for SFKs or OXSI-2 for Syk) to demonstrate specificity .

How can I validate the specificity of phospho-CBL (Y774) antibody signals?

Multiple approaches can be used to validate specificity:

• Phosphatase treatment: Treating duplicate samples with lambda phosphatase should eliminate the specific phospho-Y774 signal.

• Kinase inhibitors: Pre-treatment of cells with specific inhibitors like PP2 (SFK inhibitor) or OXSI-2 (Syk inhibitor) should reduce Y774 phosphorylation signals in contexts where these kinases are responsible for the phosphorylation .

• Mutant expression: Express Y774F mutant (tyrosine-to-phenylalanine) in cells and compare with wild-type c-CBL. The mutant should not be recognized by the phospho-specific antibody .

• Peptide competition: Pre-incubation of the antibody with the immunizing phosphopeptide (typically DGYDV sequence surrounding Y774) should block specific signals .

• Knockdown/knockout controls: Use c-CBL knockdown or knockout samples as negative controls to confirm signal specificity .

• Cross-reactivity testing: Test the antibody against related proteins that might contain similar phosphorylation motifs to ensure it doesn't cross-react .

What signaling pathways involve phospho-CBL (Y774) in platelets?

In platelets, phospho-CBL (Y774) plays a critical role in outside-in signaling:

• Integrin αIIbβ3 pathway: Upon platelet adhesion to immobilized fibrinogen, c-CBL undergoes phosphorylation at Y774 downstream of integrin αIIbβ3 engagement .

• Kinase cascade: The signaling follows a hierarchical pattern where SFKs are activated first, leading to Syk activation, which then phosphorylates c-CBL at Y774 .

• CrkL/C3G recruitment: Phosphorylated Y774 serves as a docking site for the SH2 domain of Crk/CrkL proteins, which then recruit C3G (a Rap1 GEF), potentially activating Rap1 signaling .

• Functional outcomes: Studies using c-CBL knockout mice demonstrate that these phosphorylation events regulate platelet spreading and clot retraction, critical processes in hemostasis .

• GPCR independence: Interestingly, direct GPCR activation doesn't significantly contribute to c-CBL phosphorylation; rather, it occurs primarily through integrin-mediated outside-in signaling .

How does phospho-CBL (Y774) interact with the Crk/CrkII signaling axis in immune cells?

The phospho-CBL (Y774)-Crk signaling axis in immune cells functions as follows:

• Binding mechanism: Upon phosphorylation of Y774, this site serves as a specific docking point for the SH2 domain of CrkII adapter proteins .

• Complex formation: The phospho-Y774-CrkII interaction facilitates formation of a signaling complex that can include C3G (a Rap1 GEF) and other signaling molecules .

• Regulation by inhibitory receptors: In natural killer (NK) cells, inhibitory Killer Immunoglobulin-like Receptors (KIRs) can disrupt this interaction through two potential mechanisms:

  • Direct dephosphorylation of Y774 via SHP-1 phosphatase recruitment

  • Phosphorylation of CrkII at Y221 by c-Abl, causing CrkII autoinhibition through intramolecular binding of its own SH2 domain

• Functional consequences: Disruption of the phospho-CBL (Y774)-CrkII-C3G complex contributes to inhibition of NK cell cytotoxicity, demonstrating its importance in immune cell function .

How does the differential regulation of Y774 versus Y731 phosphorylation impact c-CBL function?

The differential regulation of Y774 and Y731 phosphorylation represents a sophisticated control mechanism for c-CBL function:

• Kinase specificity: Y774 (along with Y700) is primarily phosphorylated in a Syk-dependent manner, while Y731 phosphorylation is regulated directly by SFKs and is Syk-independent .

• Protein interactions: Each phosphorylation site recruits distinct binding partners:

  • Y774 binds Crk/CrkL proteins

  • Y731 interacts with PI3K

  • Y700 engages Vav proteins

• Wnt signaling implications: Y731 phosphorylation (but not Y774 or Y700) is crucial for c-CBL's role in Wnt signaling, enhancing c-CBL dimerization and binding to β-catenin. Y731F mutants show substantially reduced interaction with β-catenin during Wnt activation .

• Functional consequences: In platelet studies, Y731 phosphorylation correlates with both cell spreading and clot retraction, while Y774 phosphorylation appears less critical for clot retraction .

• Cellular localization: Y731 phosphorylation, but not Y774, is required for c-CBL's Wnt-mediated nuclear translocation, highlighting its unique role in subcellular trafficking .

This differential regulation allows c-CBL to participate in multiple signaling pathways simultaneously with distinct functional outcomes.

What experimental approaches can resolve contradictory findings regarding phospho-CBL (Y774) in different cell types?

To resolve contradictions in phospho-CBL (Y774) research across different cell types:

• Standardized activation protocols:

  • For platelets: Use consistent fibrinogen concentrations for integrin activation

  • For T cells: Standardize TCR/CD3 stimulation protocols and pervanadate treatments

  • For other cell types: Establish clear activation parameters using relevant receptor ligands

• Kinetics analysis:

  • Perform detailed time-course experiments to capture transient phosphorylation events

  • Compare early vs. late phosphorylation patterns across cell types

  • Note that in some contexts, phosphorylation occurs only after 60 seconds of activation

• Cell-type specific kinase inhibition:

  • Systematically test the effects of inhibiting SFKs, Syk, and other relevant kinases

  • Compare inhibition profiles across cell types to identify cell-specific regulatory mechanisms

  • Use genetic approaches (knockouts, kinase-dead mutants) alongside pharmacological inhibitors

• Cross-validation strategies:

  • Employ multiple phospho-specific antibodies from different vendors

  • Confirm with mass spectrometry analysis of phosphorylation sites

  • Use phosphomimetic (Y774E) and phospho-deficient (Y774F) mutants in rescue experiments

• Context consideration:

  • Analyze the activation state of upstream kinases (SFKs, Syk) in each cell type

  • Assess the expression levels of potential binding partners (Crk/CrkL)

  • Consider the influence of other simultaneously phosphorylated residues (Y700, Y731)

How can phospho-CBL (Y774) be used as a biomarker in disease states?

Phospho-CBL (Y774) has potential as a biomarker in several disease contexts:

• Hematological disorders:

  • Given its role in platelet function, aberrant Y774 phosphorylation could indicate platelet dysfunction in bleeding disorders or thrombotic conditions

  • Analysis of phospho-CBL (Y774) levels in patient platelets could reveal signaling defects

• Cancer research applications:

  • c-CBL functions as a proto-oncogene, and dysregulation of its E3 ligase activity through altered phosphorylation may contribute to oncogenesis

  • Phospho-CBL (Y774) levels could indicate aberrant receptor tyrosine kinase signaling in cancer cells

  • Monitoring phospho-CBL (Y774) might help predict response to tyrosine kinase inhibitor therapies

• Inflammatory conditions:

  • c-CBL regulates intestinal inflammation through various mechanisms

  • Phospho-CBL (Y774) levels could serve as indicators of inflammatory pathway activation

  • Therapeutic monitoring could include assessment of phospho-CBL levels in immune cells

• Methodological considerations:

  • Development of high-throughput assays (ELISA, flow cytometry) using phospho-specific antibodies

  • Correlation with other established biomarkers to validate clinical relevance

  • Standardization of sample collection and processing to ensure consistent results

What factors affect the detection sensitivity of phospho-CBL (Y774) in different experimental systems?

Several factors can influence detection sensitivity:

• Antibody selection and quality:

  • Different commercial antibodies (Assay Genie, StJohns Labs, Boster Bio) may have varying specificities and sensitivities

  • Lot-to-lot variations can affect performance; validation with positive controls is essential

  • Storage conditions (-20°C, avoid freeze-thaw cycles) affect antibody stability

• Sample preparation considerations:

  • Rapid sample processing is critical as phosphorylation can be transient

  • Use of phosphatase inhibitors (sodium orthovanadate, phosphatase inhibitor cocktails)

  • Proper lysis buffers that preserve phospho-epitopes (RIPA or NP-40 based buffers)

• Cell/tissue-specific factors:

  • Expression levels of total c-CBL vary across cell types

  • Background phosphorylation states differ between resting cells

  • Presence of phosphatases that might dephosphorylate Y774 during sample processing

• Stimulation protocols:

  • Optimization of stimulation time (phosphorylation of Y774 may be transient)

  • Concentration of stimuli affects phosphorylation efficiency

  • The nature of the stimulus (e.g., integrin engagement vs. receptor activation)

• Detection method optimization:

  • For Western blotting: Transfer efficiency, membrane type, blocking agents

  • For immunoprecipitation: Choice of lysis buffers, antibody binding conditions

  • For immunofluorescence: Fixation methods, permeabilization conditions

How can I design experiments to investigate the relationship between phospho-CBL (Y774) and specific receptor tyrosine kinases?

To investigate relationships between phospho-CBL (Y774) and receptor tyrosine kinases (RTKs):

• Co-immunoprecipitation approach:

  • Immunoprecipitate specific RTKs (EGFR, PDGFR, etc.) and probe for phospho-CBL (Y774)

  • Reverse IP: Immunoprecipitate with phospho-CBL (Y774) antibody and probe for RTKs

  • Compare results across multiple stimulation timepoints to establish kinetics

• Pharmacological intervention:

  • Use specific RTK inhibitors to block kinase activity

  • Monitor effects on phospho-CBL (Y774) levels by Western blot

  • Create dose-response curves to determine IC50 values for inhibition of Y774 phosphorylation

• Genetic manipulation strategies:

  • Express kinase-dead RTK mutants and assess effects on Y774 phosphorylation

  • Use CRISPR/Cas9 to knockout specific RTKs and measure phospho-CBL (Y774)

  • Create Y774F CBL mutants to study functional consequences of blocking this phosphorylation

• Proximity ligation assays:

  • Use in situ techniques to visualize direct interactions between RTKs and phospho-CBL

  • Quantify cellular localization of these interactions

  • Determine how stimulation affects co-localization patterns

• Functional readouts:

  • Assess ubiquitination of RTKs in relation to Y774 phosphorylation status

  • Monitor receptor internalization and degradation rates

  • Measure downstream signaling events (MAPK, PI3K/Akt activation)

What are the emerging techniques for studying phospho-CBL (Y774) dynamics in live cells?

Emerging techniques for studying phospho-CBL (Y774) dynamics include:

• Phospho-specific biosensors:

  • FRET-based reporters that change conformation upon Y774 phosphorylation

  • Bioluminescence resonance energy transfer (BRET) sensors for real-time monitoring

  • Split fluorescent protein systems that assemble upon phospho-dependent protein interactions

• Optogenetic approaches:

  • Light-inducible kinase systems to trigger specific phosphorylation events

  • Photocaged phosphoamino acids for temporal control of phosphorylation status

  • Optogenetic control of upstream regulators (SFKs, Syk) to induce Y774 phosphorylation

• Advanced microscopy techniques:

  • Super-resolution microscopy to visualize nanoscale organization of signaling complexes

  • Light sheet microscopy for 3D visualization of phosphorylation dynamics

  • Fluorescence correlation spectroscopy to measure diffusion rates of phosphorylated vs. non-phosphorylated CBL

• Mass spectrometry innovations:

  • Targeted phosphoproteomics to quantify Y774 phosphorylation alongside other sites

  • Crosslinking mass spectrometry to identify interaction partners specific to phospho-Y774

  • SILAC or TMT labeling for quantitative comparison across conditions

• CRISPR-based approaches:

  • Base editing to introduce phosphomimetic mutations (Y to E/D) at endogenous loci

  • CRISPRa/i systems to modulate expression of kinases/phosphatases affecting Y774

  • CRISPR knock-in of fluorescent tags at endogenous c-CBL for physiological expression levels

How might understanding phospho-CBL (Y774) signaling lead to novel therapeutic approaches?

Therapeutic applications based on phospho-CBL (Y774) signaling could include:

• Targeting platelet dysfunction:

  • Modulating c-CBL Y774 phosphorylation could potentially regulate platelet spreading and clot retraction

  • This could lead to novel approaches for bleeding disorders or thrombotic conditions

  • Personalized medicine approaches based on patient phospho-CBL profiles

• Cancer therapeutics:

  • Development of small molecules that specifically disrupt phospho-Y774-CrkL interactions

  • Combination therapies targeting both RTKs and downstream c-CBL-dependent signaling

  • Biomarker-guided therapy selection based on phospho-CBL status

• Immunomodulatory approaches:

  • Targeting the phospho-CBL (Y774)-Crk axis in NK cells could enhance or inhibit cytotoxicity

  • Potential applications in autoimmunity, transplantation, and cancer immunotherapy

  • Development of peptide mimetics that compete for SH2 domain binding

• Inflammatory disorders:

  • Given c-CBL's role in intestinal inflammation , targeting Y774-dependent pathways could yield new anti-inflammatory approaches

  • Development of small molecules that modulate Y774 phosphorylation without affecting other c-CBL functions

  • Cell-specific delivery of inhibitors to affected tissues

• Drug development considerations:

  • Structure-based design of compounds targeting the Y774 pocket of c-CBL

  • Allosteric modulators affecting kinase access to Y774

  • Development of proteolysis-targeting chimeras (PROTACs) that leverage c-CBL's E3 ligase activity in a phosphorylation-dependent manner