Phospho-CTNNB1 (Tyr333) Antibody

What is CTNNB1 and why is the Tyr333 phosphorylation site significant?

CTNNB1, also known as β-catenin, functions as a key downstream component of the canonical Wnt signaling pathway and plays crucial roles in cell adhesion as part of the E-cadherin:catenin complex . The Tyr333 phosphorylation site is particularly significant because phosphorylation at this position by SRC promotes interaction with the M2 isoform of PKM (PKM2), which enhances transcriptional activation . This represents a distinct regulatory mechanism from the well-characterized N-terminal serine/threonine phosphorylations that typically lead to protein degradation.

The phosphorylation status at Tyr333 influences CTNNB1's ability to regulate gene expression, making it a critical site for understanding how post-translational modifications modulate Wnt signaling outcomes. Unlike the destabilizing phosphorylation events mediated by GSK3B and CSNK1A1, tyrosine phosphorylation events often enhance CTNNB1's signaling capabilities through altered protein-protein interactions and subcellular localization.

What experimental applications are Phospho-CTNNB1 (Tyr333) antibodies used for?

Phospho-CTNNB1 (Tyr333) antibodies are primarily utilized in Western blot (WB) applications to detect endogenous levels of β-catenin specifically phosphorylated at tyrosine 333 . These antibodies provide researchers with a powerful tool to monitor this specific post-translational modification, which cannot be detected with total CTNNB1 antibodies.

Some Phospho-CTNNB1 (Tyr333) antibodies are also validated for immunohistochemistry (IHC) applications , enabling researchers to visualize the spatial distribution of phosphorylated CTNNB1 within tissues and cellular compartments. This can be particularly valuable for studying the localization of activated β-catenin in developmental processes, tissue homeostasis, and disease states such as cancer, where aberrant Wnt signaling is frequently observed.

How specific are Phospho-CTNNB1 (Tyr333) antibodies?

Phospho-CTNNB1 (Tyr333) antibodies are engineered to detect β-catenin only when phosphorylated at tyrosine 333, providing high specificity for this post-translational modification . These antibodies are typically produced through immunization of rabbits with synthetic phosphopeptides corresponding to the region surrounding Tyr333 of human β-catenin .

To ensure specificity, manufacturers employ rigorous purification methods including affinity chromatography with epitope-specific phosphopeptides . Crucially, non-phospho specific antibodies are removed through chromatography using non-phosphopeptides , which substantially reduces cross-reactivity with unphosphorylated CTNNB1. This specificity allows researchers to confidently assess the phosphorylation status at this particular residue without interference from unmodified protein or other phosphorylation sites.

Which species can be studied using commercially available Phospho-CTNNB1 (Tyr333) antibodies?

The species reactivity of Phospho-CTNNB1 (Tyr333) antibodies varies between products. Based on available information, some antibodies are specifically reactive with human samples only , while others demonstrate broader reactivity across human, mouse, and rat samples . This variability in cross-species reactivity stems from differences in the conservation of the sequence surrounding the Tyr333 residue.

When planning experiments, researchers should carefully review the manufacturer's specifications regarding species reactivity and consider validating the antibody in their specific experimental system. For studies involving less common model organisms, preliminary validation experiments comparing the antibody's performance in established models versus the experimental system are advisable to confirm cross-reactivity and specificity.

How does Tyr333 phosphorylation compare functionally with other CTNNB1 phosphorylation events?

CTNNB1 undergoes multiple phosphorylation events that differentially regulate its function, stability, and interactions. The table below compares key phosphorylation sites:

While N-terminal serine/threonine phosphorylation (particularly at Ser33, Ser37, Thr41, and Ser45) generally promotes CTNNB1 degradation and pathway inactivation, tyrosine phosphorylation at sites like Tyr333 often enhances signaling activity through altered protein interactions and increased nuclear function . This distinction highlights the complex, multi-layered regulation of CTNNB1 through site-specific phosphorylation events.

How is CTNNB1 Tyr333 phosphorylation integrated into Wnt signaling dynamics?

Tyr333 phosphorylation represents an additional regulatory layer within the canonical Wnt pathway. In the absence of Wnt, CTNNB1 is captured by a destruction complex containing AXIN1, AXIN2, APC, CSNK1A1, and GSK3B, which promotes its N-terminal phosphorylation and subsequent degradation . When Wnt is present, this destruction complex is inhibited, allowing CTNNB1 to accumulate and translocate to the nucleus.

Recent studies using quantitative live-cell imaging and computational modeling reveal that Wnt pathway activation regulates the dynamic distribution of CTNNB1 across different functional pools through three regulatory nodes: cytoplasmic destruction, nucleocytoplasmic shuttling, and nuclear retention . Tyr333 phosphorylation by SRC potentially influences these dynamics by enhancing interaction with nuclear PKM2, thereby affecting nuclear retention and transcriptional activity .

The phosphorylation status of CTNNB1 also affects its subcellular distribution and mobility. FCS (Fluorescence Correlation Spectroscopy) and N&B (Number and Brightness) analyses demonstrate that WNT stimulation alters the size and mobility of CTNNB1-containing complexes , suggesting that post-translational modifications like Tyr333 phosphorylation may modulate these biophysical properties to influence signaling outcomes.

What methodological challenges exist in detecting phospho-CTNNB1 (Tyr333) in experimental systems?

Detecting phospho-CTNNB1 (Tyr333) presents several technical challenges that researchers should address:

• Transience of phosphorylation: Tyrosine phosphorylation events are often rapid and transient, requiring careful timing of experiments and inclusion of phosphatase inhibitors in lysis buffers to preserve the modification.

• Low abundance: Only a fraction of the total CTNNB1 pool may be phosphorylated at Tyr333 at any given time, necessitating sensitive detection methods and potentially enrichment strategies like immunoprecipitation prior to Western blotting.

• Antibody cross-reactivity: Ensuring antibody specificity is crucial, as CTNNB1 contains multiple tyrosine phosphorylation sites (Tyr64, Tyr142, Tyr331) that could potentially cross-react with Tyr333-specific antibodies .

• Dynamic subcellular distribution: CTNNB1 exhibits complex shuttling between cytoplasmic and nuclear pools , which can affect detection depending on the cellular fractionation method used.

• Context-dependent regulation: The phosphorylation of Tyr333 may be regulated differently across cell types and tissues, requiring optimization of experimental conditions for each biological system.

These challenges necessitate rigorous validation of antibody specificity and careful optimization of experimental protocols to ensure reliable detection of phospho-CTNNB1 (Tyr333).

What is the relationship between CTNNB1 Tyr333 phosphorylation and cancer progression?

The relationship between CTNNB1 Tyr333 phosphorylation and cancer progression involves several interconnected mechanisms:

• Enhanced transcriptional activity: Phosphorylation at Tyr333 by SRC promotes interaction with PKM2, enhancing transcriptional activation . This increased transcriptional activity could upregulate oncogenic target genes involved in proliferation, invasion, and metastasis.

• Resistance to anoikis: CTNNB1 blocks anoikis (programmed cell death triggered by detachment from extracellular matrix) in malignant kidney and intestinal epithelial cells by down-regulating DAPK2 . This promotes anchorage-independent growth, a hallmark of metastatic capability.

• Intersection with oncogenic pathways: SRC kinase, responsible for Tyr333 phosphorylation, is frequently hyperactivated in various cancers. Enhanced Tyr333 phosphorylation could represent a mechanism by which oncogenic SRC signaling promotes cancer progression through CTNNB1-mediated transcription.

• Altered complex formation: Research using SGFP2-CTNNB1 S45F mutant cells (mimicking oncogenic activation) revealed altered dynamics of CTNNB1-containing complexes compared to physiological WNT stimulation . Tyr333 phosphorylation may further modify the composition and function of these complexes in cancer cells.

Understanding how Tyr333 phosphorylation contributes to these oncogenic processes could potentially identify new therapeutic targets for cancers driven by aberrant Wnt/β-catenin signaling.

What are the optimal protocols for using Phospho-CTNNB1 (Tyr333) antibodies in Western blot?

For optimal detection of phospho-CTNNB1 (Tyr333) by Western blot, researchers should consider the following protocol recommendations:

• Sample preparation:

  • Rapidly harvest cells and immediately lyse in buffer containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride, and phosphatase inhibitor cocktails)

  • Maintain samples at 4°C throughout processing to minimize dephosphorylation

  • Consider using positive controls such as cells treated with EGF, which stimulates tyrosine phosphorylation

• Protein separation and transfer:

  • Use freshly prepared SDS-PAGE gels (8-10%) for optimal resolution of CTNNB1 (~92 kDa)

  • Employ wet transfer methods for more consistent results with phospho-proteins

  • Use PVDF membranes, which typically provide better retention of phospho-proteins than nitrocellulose

• Antibody incubation:

  • Block membranes with BSA (3-5%) rather than milk, as milk contains phosphoproteins that may interfere with detection

  • Use antibody dilutions between 1:500 and 1:2000 as recommended by manufacturers

  • Incubate with primary antibody overnight at 4°C for optimal signal-to-noise ratio

• Detection and visualization:

  • Employ sensitive detection methods like enhanced chemiluminescence (ECL) or fluorescent secondary antibodies

  • Consider stripping and reprobing with total CTNNB1 antibody to calculate the phosphorylated/total ratio

• Controls and validation:

  • Include phosphatase-treated samples as negative controls

  • Use SRC inhibitor-treated samples to confirm specificity

  • Consider parallel detection with multiple phospho-specific antibodies

Following these optimized protocols will maximize the likelihood of successfully detecting phospho-CTNNB1 (Tyr333) in experimental samples.

How can researchers validate the specificity of Phospho-CTNNB1 (Tyr333) antibody detection?

Validating antibody specificity is crucial for ensuring reliable results. Researchers should implement multiple validation strategies:

• Peptide competition assays:

  • Pre-incubate the antibody with excess phospho-peptide (containing the Tyr333 phosphorylation site)

  • Perform parallel Western blots with competed and non-competed antibody

  • Specific signal should be eliminated or substantially reduced in the competed condition

• Phosphatase treatment controls:

  • Divide lysate samples and treat one set with lambda phosphatase

  • Compare detection between phosphatase-treated and untreated samples

  • Phospho-specific signal should be eliminated in phosphatase-treated samples

• Genetic modification approaches:

  • Generate cell lines expressing CTNNB1 with Tyr333 mutated to phenylalanine (Y333F)

  • Compare antibody reactivity between wild-type and Y333F mutant cells

  • No signal should be detected in the Y333F mutant despite equivalent total CTNNB1 expression

• Kinase manipulation:

  • Treat cells with SRC kinase inhibitors to prevent Tyr333 phosphorylation

  • Alternatively, activate SRC using EGF stimulation or expression of constitutively active SRC

  • Observe corresponding changes in phospho-signal intensity

• Mass spectrometry validation:

  • Perform immunoprecipitation with the phospho-specific antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm the presence of the phosphorylated Tyr333 peptide

These comprehensive validation approaches ensure that the observed signal genuinely represents Tyr333-phosphorylated CTNNB1 rather than cross-reactivity or non-specific binding.

What experimental systems are ideal for studying CTNNB1 Tyr333 phosphorylation dynamics?

Several experimental systems are particularly well-suited for investigating the dynamics of CTNNB1 Tyr333 phosphorylation:

• Cell culture models:

  • Cell lines with active Wnt signaling (e.g., colorectal cancer cell lines)

  • HAP1 cells with CRISPR/Cas9-edited fluorescently tagged CTNNB1 for live imaging

  • C2C12 and NIH/3T3 cells, which have been reported as positive samples for phospho-CTNNB1 detection

• Advanced imaging techniques:

  • Fluorescence Correlation Spectroscopy (FCS) for measuring mobility and concentration of CTNNB1 in living cells

  • Number and Brightness (N&B) analysis for characterizing CTNNB1-containing complexes

  • Fluorescence Recovery After Photobleaching (FRAP) for studying nuclear-cytoplasmic shuttling dynamics

• Computational modeling integration:

  • Combining experimental data with computational models to predict CTNNB1 distribution and dynamics

  • Utilizing the interactive model available at https://wntlab.shinyapps.io/WNT_minimal_model/ to explore parameter variations

• Targeted perturbations:

  • SRC activation through EGF stimulation or expression of constitutively active SRC

  • GSK3 inhibition using CHIR99021 to block N-terminal phosphorylation and mimic Wnt activation

  • Introduction of oncogenic mutations like S45F to study pathway dysregulation

These complementary approaches allow researchers to examine multiple aspects of CTNNB1 Tyr333 phosphorylation, from molecular interactions to system-level dynamics, providing a comprehensive understanding of this regulatory mechanism.

What techniques can be combined with Phospho-CTNNB1 (Tyr333) antibody detection for comprehensive pathway analysis?

For comprehensive analysis of CTNNB1 signaling and the functional impact of Tyr333 phosphorylation, researchers should integrate multiple complementary techniques:

• Phosphoproteomic analyses:

  • Mass spectrometry-based phosphoproteomics to identify and quantify multiple phosphorylation sites on CTNNB1

  • Targeted multiple reaction monitoring (MRM) for quantitative analysis of specific phospho-sites

  • Correlation of Tyr333 phosphorylation with other post-translational modifications

• Interaction studies:

  • Co-immunoprecipitation with phospho-CTNNB1 (Tyr333) antibodies to identify interacting partners

  • Proximity ligation assay (PLA) to visualize and quantify interactions between phospho-CTNNB1 and partners like PKM2

  • FRET-based approaches to monitor interactions in living cells

• Transcriptional activity assessment:

  • Chromatin immunoprecipitation (ChIP) using phospho-specific antibodies to identify target genes

  • TCF/LEF reporter assays to quantify transcriptional activation

  • RNA-seq analysis to determine global transcriptional changes associated with Tyr333 phosphorylation

• Cellular localization studies:

  • Subcellular fractionation combined with Western blotting for phospho-CTNNB1 (Tyr333)

  • Immunofluorescence microscopy to visualize the spatial distribution of phosphorylated protein

  • Live cell imaging with genetically encoded reporters to track dynamic changes

• Functional assays:

  • Cell proliferation, migration, and invasion assays to assess biological outcomes

  • Anoikis resistance tests to evaluate effects on anchorage-independent growth

  • 3D organoid cultures to examine effects in more physiologically relevant contexts

Integrating these multidisciplinary approaches provides a comprehensive understanding of how Tyr333 phosphorylation influences CTNNB1 function across different biological contexts and signaling states.

What are the emerging technologies for studying CTNNB1 phosphorylation dynamics?

Several cutting-edge technologies are poised to advance our understanding of CTNNB1 Tyr333 phosphorylation:

• Single-molecule imaging approaches:

  • Super-resolution microscopy techniques (STORM, PALM) for visualizing individual phosphorylated CTNNB1 molecules

  • Single-molecule tracking to follow the fate of Tyr333-phosphorylated CTNNB1 in living cells with nanometer precision

  • Single-molecule pull-down (SiMPull) assays to analyze composition of protein complexes containing phospho-CTNNB1

• Genetically encoded biosensors:

  • FRET-based sensors that report on conformational changes associated with Tyr333 phosphorylation

  • Split fluorescent protein complementation systems to visualize interaction between phospho-CTNNB1 and binding partners

  • Optogenetic tools to spatiotemporally control SRC activity and induce Tyr333 phosphorylation

• Proximity labeling approaches:

  • BioID or APEX2 fusion proteins to identify the proximal proteome of phosphorylated CTNNB1

  • Spatial-specific variants to distinguish interaction partners in different subcellular compartments

  • TurboID for rapid labeling to capture transient interactions

• CRISPR-based technologies:

  • Base editing to introduce precise phospho-mimetic (Y333D/E) or phospho-deficient (Y333F) mutations

  • CRISPR activation/interference systems to modulate expression of regulatory kinases and phosphatases

  • CRISPR knock-in of split fluorescent tags for endogenous visualization of CTNNB1 dynamics

These advanced technologies will provide unprecedented insights into the spatial and temporal dynamics of CTNNB1 Tyr333 phosphorylation and its functional consequences in both physiological and pathological contexts.

What are the key unanswered questions about CTNNB1 Tyr333 phosphorylation in cancer and development?

Despite advances in understanding CTNNB1 regulation, several critical questions about Tyr333 phosphorylation remain unresolved:

• Regulatory mechanisms:

  • What upstream signals specifically trigger SRC-mediated phosphorylation of CTNNB1 at Tyr333?

  • Which phosphatases dephosphorylate Tyr333, and how is this process regulated?

  • How does Tyr333 phosphorylation interact with other post-translational modifications of CTNNB1?

• Structural consequences:

  • How does Tyr333 phosphorylation alter CTNNB1's conformation to promote specific protein interactions?

  • Does Tyr333 phosphorylation affect the armadillo repeat structure that mediates most CTNNB1 interactions?

  • Can structural changes induced by Tyr333 phosphorylation affect N-terminal phosphorylation and subsequent degradation?

• Developmental significance:

  • What is the role of Tyr333 phosphorylation during embryonic development and tissue homeostasis?

  • How does it contribute to the balance between proliferation and differentiation in stem cell compartments?

  • Are there tissue-specific functions of Tyr333 phosphorylation in development?

• Cancer implications:

  • Is Tyr333 phosphorylation altered in cancers with aberrant Wnt signaling but without mutations in canonical pathway components?

  • Could targeting the SRC-CTNNB1-PKM2 axis represent a therapeutic strategy for Wnt-driven cancers?

  • Does Tyr333 phosphorylation contribute to therapy resistance mechanisms in cancer?

Addressing these questions will significantly advance our understanding of CTNNB1 regulation and potentially reveal new therapeutic approaches for diseases with dysregulated Wnt signaling.