Phospho-BCR (Tyr177) Antibody

What is Phospho-BCR (Tyr177) Antibody and what cellular events does it detect?

Phospho-BCR (Tyr177) Antibody is a research tool that specifically detects endogenous levels of BCR protein only when phosphorylated at tyrosine 177. The antibody recognizes this specific post-translational modification, which plays a crucial role in cellular signaling pathways.

The phosphorylation of BCR at Tyr177 is a key regulatory event in several cellular processes:

• It creates a high-affinity docking site for the SH2 domain of GRB2

• This docking enables recruitment of SOS (son of sevenless), leading to activation of RAS signaling pathways

• It facilitates formation of the GRB2/GAB2 complex, which causes constitutive activation of the PI3K/AKT and ERK pathways in primary CML cells

Methodologically, researchers can use this antibody to track the activation status of BCR and BCR-ABL signaling in experimental systems.

What are the primary applications of Phospho-BCR (Tyr177) Antibody in research protocols?

Phospho-BCR (Tyr177) Antibody is versatile and can be employed in multiple experimental techniques:

For optimal results, researchers should:

• Include positive controls (e.g., K562 cells treated with H2O2)

• Use appropriate blocking peptides to confirm specificity

• Include normalization controls (e.g., total BCR or GAPDH)

How can I differentiate between normal BCR and BCR-ABL fusion protein when using Phospho-BCR (Tyr177) Antibody?

Differentiation between phosphorylated native BCR (160 kDa) and the BCR-ABL fusion protein (210 kDa) requires careful experimental design:

Methodological approach:

• Use SDS-PAGE with appropriate molecular weight markers that can resolve the 160 kDa and 210 kDa bands

• Run parallel Western blots with antibodies against:

  • Phospho-BCR (Tyr177) to detect phosphorylation status

  • Total BCR to detect both phosphorylated and non-phosphorylated forms

  • C-ABL to confirm the presence of BCR-ABL fusion protein

• Cell line controls:

  • Use K562 cells as a positive control for BCR-ABL fusion protein

  • Compare with cell lines expressing only native BCR

The molecular weight differences provide clear distinction: native BCR appears at 160 kDa while BCR-ABL fusion protein appears at 210 kDa when visualized on Western blots .

What signaling pathways are activated downstream of BCR Tyr177 phosphorylation?

Phosphorylation of BCR at Tyr177 initiates multiple signaling cascades essential for cellular transformation and leukemogenesis:

Primary signaling pathways:

• RAS pathway activation:

  • Phospho-Tyr177 serves as a docking site for GRB2

  • GRB2 recruits SOS, activating RAS signaling

  • This leads to activation of the RAF-MEK-ERK pathway

• PI3K/AKT pathway:

  • The GRB2/GAB2 complex formed at phospho-Tyr177 activates PI3K

  • This leads to constitutive activation of AKT

  • In chronic phase CML, MEK-ERK activation is cytokine-dependent but becomes constitutively activated in blast phase CML

• Cell cycle regulation:

  • BCR-ABL Y177F mutation (preventing phosphorylation) results in:

    • Reduced G1 phase and increased S-phase cell populations

    • Reduced phosphorylation of pRB on S780 and S807/S811 (CDK4 and CDK2 sites)

    • Restored p27 nuclear localization

These pathways collectively contribute to increased cell proliferation, survival, and leukemic transformation .

How does the Y177F mutation affect BCR-ABL function in experimental systems?

The Y177F mutation (tyrosine to phenylalanine substitution) at position 177 of BCR-ABL has profound effects on its leukemogenic potential:

Experimental findings:

• Biochemical effects:

  • Largely abolishes GRB2 binding to BCR-ABL

  • Diminishes BCR-ABL-induced RAS activation

  • Impairs the transformation of primary bone marrow cultures despite preservation of ABL kinase activity

• Cellular effects in CD34+ cells:

  • Markedly reduced proliferation of BCR-ABL expressing CD34+ cells

  • Restoration of normal p27 nuclear localization (versus cytoplasmic in wild-type BCR-ABL)

  • Reduced p27 expression compared to wild-type BCR-ABL expressing cells

• In vivo effects:

  • Limits the induction of myeloproliferative disorders in murine stem cell transplantation models of CML

  • Reverses abnormal cytoplasmic localization of p27

Methodological considerations:

• When studying Y177F mutants, researchers should examine both kinase activity and protein-protein interactions

• Complementary approaches including co-immunoprecipitation, subcellular fractionation, and immunofluorescence provide comprehensive understanding of the mutation's effects

What are the optimal sample preparation methods for detecting Phospho-BCR (Tyr177) by Western blot?

Detecting phosphorylated proteins requires careful sample preparation to preserve phosphorylation status:

Recommended protocol:

• Cell lysis:

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

  • Include protease inhibitors to prevent protein degradation

  • Maintain cold conditions throughout processing

• Sample preparation:

  • Determine optimal protein loading (typically 20-50 μg total protein)

  • Heat samples at 95°C for 5 minutes in Laemmli buffer containing 2-mercaptoethanol

  • Use freshly prepared samples when possible

• Gel electrophoresis and transfer:

  • Use 7.5% SDS-PAGE gels to effectively resolve high molecular weight proteins (160-210 kDa)

  • Transfer to PVDF membrane at 100V for 2 hours or 30V overnight at 4°C

• Antibody incubation:

  • Block with 5% BSA in TBST (not milk, which contains phosphatases)

  • Incubate with Phospho-BCR (Tyr177) antibody at recommended dilution (1:1000)

  • Include positive controls (K562 cells) and treatment controls (e.g., H2O2 treated vs. untreated)

• Detection optimization:

  • Use enhanced chemiluminescence with sensitive detection systems

  • Consider longer exposure times if signal is weak

These methods maximize detection sensitivity while preserving phosphorylation-specific signals .

What is the relationship between JAK2 and BCR-Tyr177 phosphorylation in CML pathogenesis?

JAK2 plays a crucial role in the phosphorylation of BCR-ABL at Tyr177, representing a key regulatory mechanism in CML pathogenesis:

Research findings:

• JAK2 as the primary kinase:

  • JAK2, not BCR-ABL itself, phosphorylates Tyr177 of BCR-ABL

  • Tyr177 of BCR-ABL (YVNV) has the JAK2 consensus target sequence (YxxV/L/I)

  • JAK2 inhibition by TG101209 or WP1193 rapidly reduces phosphorylation of Tyr177 in BCR-ABL-positive cell lines

• JAK2 inhibition effects:

  • Reduces Grb2 binding to BCR-ABL

  • Diminishes activation of RAS and PI-3 kinase pathways within two hours

  • Effective in both imatinib-sensitive and -resistant cell lines

• Mechanistic significance:

  • JAK2 knockdown with specific siRNA reduces levels of phospho-Tyr177 BCR-ABL and total BCR-ABL protein

  • Rescue experiments that reverse JAK2 knockdown stimulate phospho-Tyr177 levels

  • JAK2 inhibitors, but not imatinib, inhibit phosphorylation of synthetic BCR peptides containing Tyr177

This relationship suggests JAK2 inhibition as a potential therapeutic strategy to overcome imatinib resistance in CML by targeting the critical Tyr177 phosphorylation event .

How can researchers quantitatively assess phosphorylation dynamics of BCR Tyr177?

Quantitative assessment of BCR Tyr177 phosphorylation dynamics requires sophisticated experimental approaches:

Methodological strategies:

• Cell-Based ELISA methods:

  • Commercially available kits allow determination of relative phosphorylation levels

  • Multiple normalization methods can be employed:

    • Anti-GAPDH antibody as internal positive control

    • Crystal Violet whole-cell staining to adjust for cell density differences

    • Normalization to total BCR levels

• Sandwich ELISA approach:

  • Use c-Abl mouse monoclonal antibody as capture antibody

  • Detect with Phospho-BCR (Tyr177) rabbit detection antibody

  • Visualize with anti-rabbit IgG, HRP-linked antibody and TMB substrate

  • Quantify by measuring absorbance proportional to phosphorylated protein levels

• Time-course experiments:

  • Treat cells with inhibitors (JAK2 inhibitors or imatinib) and harvest at multiple time points

  • Analyze phosphorylation kinetics to understand temporal dynamics

  • Compare inhibitor efficacy based on half-life of phosphorylation

• Phosphorylation site-specific quantification:

  • Use synthetic peptide standards containing phosphorylated Tyr177

  • Develop calibration curves for absolute quantification

  • Apply mass spectrometry for multiplexed analysis of phosphorylation sites

These approaches provide complementary information about the dynamics, stoichiometry, and regulation of BCR Tyr177 phosphorylation in various experimental contexts .

How does BCR Tyr177 phosphorylation affect cell cycle regulation mechanisms?

BCR Tyr177 phosphorylation has significant impacts on cell cycle regulation through multiple mechanisms:

Experimental evidence:

• Effects on p27 localization and function:

  • BCR-ABL expression results in abnormal cytoplasmic localization of p27 (CDKN1B)

  • The Y177F mutation restores nuclear localization of p27

  • Nuclear p27 functions to inhibit CDK2 and CDK4 activity

• Impact on RB phosphorylation:

  • BCR-ABL transformed cells show increased phosphorylation of pRB on:

    • S780 (CDK4 site)

    • S807/S811 (CDK2 sites)

  • This suggests reduced nuclear p27 is associated with increased CDK activity

  • BCR-ABL Y177F expressing cells demonstrate reduced pRB phosphorylation

• Cell cycle distribution changes:

  • BCR-ABL expression leads to reduced G1 and increased S-phase populations compared to controls

  • These changes are reversed in cells expressing BCR-ABL Y177F

• p27 phosphorylation status:

  • p27 mRNA levels are similar in cells expressing Y177F and wild-type BCR-ABL

  • Suggesting post-transcriptional regulation mechanisms

These findings demonstrate that BCR Tyr177 phosphorylation mediates cell cycle dysregulation through p27 mislocalization and altered CDK activity, contributing to the leukemogenic potential of BCR-ABL .

What experimental approaches can be used to study the interaction between phosphorylated BCR Tyr177 and its binding partners?

Investigating interactions between phosphorylated BCR Tyr177 and its binding partners requires sophisticated techniques:

Recommended experimental approaches:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate with Phospho-BCR (Tyr177) antibody

  • Blot for interaction partners (e.g., GRB2, GAB2)

  • Reverse Co-IP: immunoprecipitate GRB2 and blot for phospho-BCR

  • JAK2 inhibition studies show reduced GRB2 binding to BCR-ABL within two hours of treatment

• Proximity ligation assay (PLA):

  • Allows visualization of protein-protein interactions in situ

  • Use antibodies against phospho-BCR (Tyr177) and potential binding partners

  • Quantify interaction signals at single-molecule resolution

• Peptide pull-down assays:

  • Synthesize BCR peptides containing phosphorylated Tyr177 and surrounding sequences

  • Use as bait to pull down binding partners from cell lysates

  • Compare binding efficiency with non-phosphorylated peptides

  • JAK2 inhibitors strongly inhibit phosphorylation of synthetic BCR peptides containing Tyr177

• Fluorescence resonance energy transfer (FRET):

  • Tag BCR and interaction partners with compatible fluorophores

  • Measure FRET efficiency as indicator of protein proximity

  • Monitor interactions in living cells in real time

• Surface plasmon resonance (SPR):

  • Immobilize phosphorylated BCR peptides or proteins

  • Measure binding kinetics and affinity constants with purified GRB2

  • Compare binding parameters between wild-type and mutant proteins

These complementary approaches provide comprehensive characterization of phospho-Tyr177-dependent interactions that drive downstream signaling events in normal and pathological contexts .