Phospho-BRAF (T401) Recombinant Monoclonal Antibody

What is the biological significance of BRAF T401 phosphorylation?

BRAF T401 phosphorylation represents a critical regulatory modification that influences BRAF's role in the transduction of mitogenic signals from the cell membrane to the nucleus. BRAF functions as a serine/threonine-protein kinase that phosphorylates downstream targets including MAP2K1, thereby activating the MAP kinase signal transduction pathway . Recent research has revealed that T401 phosphorylation is regulated by the mTOR pathway, rather than exclusively by ERK as previously believed, providing new insights into its biological significance .

The phosphorylation at T401 appears to be constitutively present at substantial levels in various cell types even without acute stimulation, suggesting it plays a role in maintaining baseline BRAF function rather than solely responding to acute pathway activation . This contrasts with the traditional view of T401 as primarily an ERK substrate following growth factor stimulation.

How does the regulation of T401 phosphorylation differ from other BRAF phosphorylation sites?

Unlike some other BRAF phosphorylation sites that are primarily regulated by ERK-dependent feedback mechanisms, T401 phosphorylation shows distinct regulatory patterns. Recent research demonstrates that prominent T401 phosphorylation of endogenous BRAF occurs in the absence of acute stimulation in multiple cell lines of both murine and human origin .

Importantly, while BRAF/RAF1 inhibitors (e.g., naporafenib), MEK inhibitors (e.g., trametinib), and ERK inhibitors (e.g., ulixertinib) fail to reduce T401 phosphorylation levels, mTOR inhibitors like torin1 and dual-specific PI3K/mTOR inhibitors like dactolisib significantly suppress T401 phosphorylation in all investigated cell types in both time- and concentration-dependent manners . This contrasts with other regulatory phosphorylation sites such as S446, S729, and S750, which are not affected by mTOR inhibition.

For comparison, other phosphorylation sites such as S732 in BRAF (and the homologous S624 in CRAF) show different regulatory mechanisms, with phosphorylation at these sites appearing to disrupt 14-3-3 binding and dimerization, ultimately affecting MEK phosphorylation .

What experimental approaches can reveal T401 phosphorylation mechanisms?

To investigate T401 phosphorylation mechanisms, researchers employ several complementary approaches:

• Phosphoproteomic analysis: Using TiO2 chromatography and LC-MS/MS to identify and quantify phosphopeptides, researchers can detect changes in T401 phosphorylation status under different conditions . This technique allowed researchers to detect that T401 phosphorylation is maintained independently of ERK activity .

• Genetic manipulation: Creating cell lines with specific pathway activations, such as expressing oncogenic RHEB (Q64L) and mTOR (S2215Y and R2505P) mutants, can confirm the role of mTOR in T401 phosphorylation .

• Pharmacological inhibition: Treating cells with various kinase inhibitors (mTOR, PI3K, MEK, ERK, RAF inhibitors) at different concentrations and timepoints allows researchers to identify which pathways regulate T401 phosphorylation .

• shRNA-mediated knockdown: Depletion of specific complex components (e.g., Raptor for mTORC1, Rictor for mTORC2) can further delineate which complex primarily regulates T401 phosphorylation .

What are the validated applications for Phospho-BRAF (T401) antibodies?

Based on the provided information, Phospho-BRAF (T401) recombinant monoclonal antibodies have been validated primarily for Western blotting applications . The antibody has been tested and confirmed to work with rat samples, with evidence suggesting it may be applicable to other species with strong homology .

When using this antibody for Western blotting, optimal dilution ratios of approximately 1/5000 have been demonstrated to be effective, as shown in experiments with PC-12 cell lysates (both untreated and TPA-treated) .

Beyond Western blotting, phospho-specific antibodies like this can potentially be used in:

• Immunoprecipitation followed by mass spectrometry analysis to identify interaction partners

• Fluorescence microscopy to examine subcellular localization of phosphorylated BRAF

• Flow cytometry to quantify phosphorylation levels at the single-cell level

How should mass spectrometry be optimized for phospho-BRAF T401 detection?

For optimal detection of BRAF T401 phosphorylation by mass spectrometry, researchers should consider the following methodological approaches:

• Sample preparation: Immunoprecipitation of BRAF using either tagged constructs (e.g., FLAG-tagged BRAF) or specific antibodies followed by digestion with trypsin or chymotrypsin is effective for generating phosphopeptides suitable for LC-MS/MS analysis .

• Phosphopeptide enrichment: TiO2 chromatography has been successfully employed for phosphopeptide enrichment prior to LC-MS/MS analysis. Micro-column-based approaches are particularly effective for enhancing detection sensitivity .

• Quantification strategies: Labeling strategies such as reductive dimethylation using light and heavy isotopes enable comparative quantification between different treatment conditions . For label-free approaches, analysis of total ion intensities over elution peaks can provide semi-quantitative measurements of phosphorylation changes .

• Confidence assessment: For proper site localization, confidence thresholds (e.g., >75% localization probability) should be applied. In studies examining BRAF phosphorylation, approximately 76% of phosphorylation sites could be localized with high confidence using these criteria .

• Validation: Following identification by MS, validation using phospho-specific antibodies or site-directed mutagenesis (e.g., S→A phospho-deficient or S→E/D phosphomimetic mutations) is crucial to confirm the functional significance of the identified phosphorylation events .

What controls should be included when working with phospho-BRAF (T401) antibodies?

When working with phospho-BRAF (T401) antibodies, the following controls are essential to ensure experimental validity:

• Phosphatase treatment controls: Treating a portion of your sample with lambda phosphatase will dephosphorylate the target residue, providing a negative control to confirm antibody specificity.

• Pathway activation/inhibition controls: Including samples treated with mTOR pathway inhibitors (e.g., torin1, dactolisib) which are known to reduce T401 phosphorylation, or activators of the mTOR pathway which increase phosphorylation .

• Cell treatment controls: Using treatments known to affect the phosphorylation status, such as TPA treatment for PC-12 cells, which has been demonstrated in Western blot validations .

• Loading and transfer controls: Probing for total BRAF expression in parallel with phospho-specific detection to normalize phosphorylation signals to total protein levels.

• Blocking peptide competition: Using a phosphorylated peptide containing the T401 site to compete with epitope recognition can demonstrate specificity.

• Genetic controls: Where possible, using cells expressing BRAF T401A (phospho-deficient) or BRAF T401E/D (phosphomimetic) mutants provides definitive controls for antibody specificity.

How does the mTOR pathway regulate BRAF T401 phosphorylation?

Recent research has established a novel connection between the mTOR pathway and BRAF T401 phosphorylation. The mTOR pathway appears to maintain T401 phosphorylation independently of the traditional ERK-mediated feedback mechanisms .

Key findings include:

• mTOR inhibition reduces T401 phosphorylation: The mTOR inhibitor torin1 and dual PI3K/mTOR inhibitor dactolisib significantly suppress T401 phosphorylation in multiple cell types in both a time- and concentration-dependent manner .

• Genetic mTOR activation increases T401 phosphorylation: Expression of oncogenic RHEB (Q64L) and mTOR (S2215Y and R2505P) mutants substantially increases T401 phosphorylation, an effect reversed by mTOR inhibitors but not by MEK inhibitors .

• mTORC1 is primarily responsible: While both mTOR complexes may contribute, knockdown of Raptor (mTORC1 component) more significantly enhances the suppression of T401 phosphorylation by low-dose torin1 compared to Rictor (mTORC2 component) knockdown .

• Selective phosphorylation pattern: Mass spectrometry evidence shows that mTOR inhibition suppresses phosphorylation of T401, S405, and S409, but not other regulatory phosphorylation sites such as S446, S729, and S750 .

This mTOR-dependent regulation of T401 phosphorylation represents a previously unrecognized crosstalk between the PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways, with potential implications for cancer therapies targeting these pathways.

How does T401 phosphorylation compare with other regulatory phosphorylation sites on BRAF?

BRAF contains multiple phosphorylation sites that regulate its activity and interactions through different mechanisms:

• T401 phosphorylation (mTOR-regulated): Maintains baseline phosphorylation even without acute stimulation. Not significantly affected by BRAF/RAF1, MEK, or ERK inhibitors, but reduced by mTOR inhibitors .

• S729 phosphorylation (AMPK-regulated): Promotes association with 14-3-3 proteins and disrupts BRAF interaction with KSR1 scaffolding protein, attenuating MEK-ERK signaling. AMPK activation increases S729 phosphorylation from approximately 30% to 70% .

• S732 phosphorylation (negative regulatory): Phosphorylation at S732 appears to disrupt 14-3-3 binding and decrease BRAF homodimerization and BRAF:CRAF heterodimerization. Phosphomimetic S732E mutants show decreased activation loop phosphorylation and reduced MEK phosphorylation .

• T599 phosphorylation (activation loop): Phospho-deficient BRAF S732A shows higher activation loop phosphorylation (T599) compared to S732E phosphomimetic mutants, suggesting interconnections between different phosphorylation sites .

These different phosphorylation events create a complex regulatory network that fine-tunes BRAF activity in response to various cellular signals and energy states.

What are the implications of BRAF T401 phosphorylation for cancer research and therapeutic development?

The discovery that BRAF T401 phosphorylation is regulated by the mTOR pathway has significant implications for cancer research and therapeutic strategies:

• Dual pathway targeting: Since T401 phosphorylation involves crosstalk between the mTOR and MAPK pathways, combination therapies targeting both pathways might be more effective than single-pathway inhibition in certain contexts .

• Resistance mechanisms: In melanomas with acquired resistance to BRAF inhibitors, reactivation of MAPK signaling is a central paradigm . Understanding how T401 phosphorylation contributes to pathway reactivation could provide insights into resistance mechanisms.

• Biomarker potential: The phosphorylation status of T401 might serve as a biomarker for mTOR pathway activation in tumors and potentially predict response to targeted therapies.

• Novel therapeutic targets: Identifying the specific kinases and phosphatases that directly modify T401 could reveal new therapeutic targets in the treatment of BRAF-driven cancers.

• Pharmacodynamic markers: Changes in T401 phosphorylation could serve as pharmacodynamic markers to monitor the effectiveness of mTOR pathway inhibitors in clinical settings.

This research highlights the importance of understanding phosphorylation-based signaling events beyond the canonical MAPK pathway activation in the context of targeted cancer therapies.

How can researchers address variability in phospho-BRAF (T401) detection across different cell types?

Variability in phospho-BRAF (T401) detection across different cell types can arise from multiple factors. Researchers can address these challenges through the following approaches:

• Optimization of lysis conditions: Different cell types may require adjusted lysis buffers with appropriate phosphatase inhibitors to preserve phosphorylation status. Testing different lysis conditions (RIPA, NP-40, Triton X-100 based) with various phosphatase inhibitor cocktails can help optimize detection.

• Cell-type specific pathway activation: Baseline activation of the mTOR pathway varies across cell types, affecting T401 phosphorylation levels. Establishing baseline phosphorylation in each cell type through systematic treatment with pathway activators and inhibitors can provide context for interpreting results .

• Antibody validation: Validating the phospho-BRAF (T401) antibody specificity in each cell type using phosphatase treatment, blocking peptides, or genetic approaches (T401A mutants) ensures reliable detection.

• Quantification methods: Using both phospho-specific and total BRAF antibodies to calculate phosphorylation ratios rather than absolute phospho-signal can normalize for expression differences across cell types.

• Multiple detection methodologies: Complementing Western blot analysis with mass spectrometry-based approaches provides orthogonal validation of phosphorylation status .

What techniques can resolve contradictory findings about T401 phosphorylation regulation?

When faced with contradictory findings regarding T401 phosphorylation regulation, researchers can employ several strategies:

• Temporal dynamics analysis: Assessing phosphorylation at multiple timepoints after treatment can resolve apparent contradictions that result from different kinetics of phosphorylation/dephosphorylation .

• Dose-response relationships: Testing a range of inhibitor concentrations can reveal threshold effects, as demonstrated by the concentration-dependent effects of mTOR inhibitors on T401 phosphorylation .

• Genetic approaches: Using RNA interference or CRISPR-based methods to knock down or knock out specific components of signaling pathways provides more definitive evidence than pharmacological inhibitors, which may have off-target effects .

• Cell-state considerations: Cell confluency, serum starvation conditions, and passage number can affect signaling pathway status. Standardizing these conditions or systematically testing their impact can help resolve contradictions.

• Context-specific regulation: T401 phosphorylation regulation may differ in normal versus oncogenic contexts. Parallel studies in both settings can clarify context-dependent regulation mechanisms .

How can researchers distinguish between direct and indirect effects on BRAF T401 phosphorylation?

Distinguishing direct from indirect effects on BRAF T401 phosphorylation requires rigorous experimental approaches:

• In vitro kinase assays: Using purified recombinant kinases with immunoprecipitated BRAF (preferably kinase-dead mutants to avoid auto-phosphorylation) can demonstrate direct phosphorylation, as shown for AMPK-mediated phosphorylation of BRAF at S729 .

• Phosphorylation site mutants: Creating phospho-deficient (T401A) or phosphomimetic (T401D/E) BRAF mutants allows assessment of downstream effects and pathway dependencies .

• Rapid kinetics: Examining the temporal order of phosphorylation events following pathway activation or inhibition can help establish causality.

• Pathway reconstruction: Reconstituting minimal pathway components in simplified systems (e.g., in vitro or in yeast) can eliminate confounding influences from complex mammalian signaling networks.

• Structural biology approaches: Understanding how T401 phosphorylation affects BRAF structure and interactions through techniques like hydrogen-deuterium exchange mass spectrometry or cryo-electron microscopy can provide mechanistic insights into direct effects .

How can phospho-BRAF (T401) analysis contribute to understanding drug resistance mechanisms?

Phospho-BRAF (T401) analysis can provide valuable insights into drug resistance mechanisms through several approaches:

• Temporal profiling during resistance development: Monitoring T401 phosphorylation status during the development of resistance to BRAF inhibitors can reveal adaptive pathway changes. This approach was used to generate resistant cell lines (e.g., LM-MEL-28R) that showed threefold reduced sensitivity to BRAF inhibitors compared to parental lines .

• Phosphoproteomic integration: Combining T401 phosphorylation data with broader phosphoproteomic analyses can identify compensatory pathway activation. Studies have shown that phosphoproteomics can reveal signaling events in proteins associated with established pathways of drug resistance (IGFR2, IRS1, PKC, and GEFs) .

• Correlation with paradoxical activation: Investigating the relationship between T401 phosphorylation and paradoxical MEK/ERK activation by BRAF inhibitors at different doses could provide mechanistic insights into resistance. Similar approaches with other phosphorylation sites (e.g., S732) have revealed phosphorylation-dependent differences in paradoxical activation .

• Patient-derived samples: Analyzing T401 phosphorylation in patient samples before treatment and at relapse can identify clinically relevant resistance mechanisms. Phosphoproteomic methods can be applied to tumors biopsied before, during, and after treatment to provide direct readouts for potential drug-able targets in relapsed patients .

What novel insights can be gained by examining BRAF T401 phosphorylation in different subcellular compartments?

Examining BRAF T401 phosphorylation in different subcellular compartments can provide novel insights into spatial regulation of RAF signaling:

• Membrane versus cytosolic fractionation: Since inactive RAF is believed to be monomeric, autoinhibited, and cytosolic, while activated RAF is recruited to the membrane, analyzing T401 phosphorylation in these different fractions can reveal compartment-specific regulation .

• Nuclear localization: Though primarily considered a cytoplasmic signaling molecule, BRAF may have nuclear functions. Assessing nuclear T401 phosphorylation could uncover previously unrecognized roles.

• Scaffold protein associations: T401 phosphorylation may affect BRAF association with scaffold proteins like KSR1, which organize signaling complexes in specific subcellular locations . Immunoprecipitation of these scaffolds followed by phospho-T401 detection can reveal spatial organization principles.

• Endosomal signaling: MAPK signaling can occur from endosomal compartments. Examining T401 phosphorylation in purified endosomal fractions could reveal distinct regulatory mechanisms in these compartments.

• Cell-cell junctions: In epithelial cells, analyzing T401 phosphorylation at cell-cell junctions versus cytoplasmic pools might reveal context-specific regulation relevant to epithelial-mesenchymal transition in cancer.

How does BRAF T401 phosphorylation interact with BRAF dimerization and 14-3-3 protein binding?

While direct evidence for T401 phosphorylation's effect on dimerization is limited in the provided sources, we can draw insights from studies of other phosphorylation sites:

• Potential regulatory similarities: Like S732 phosphorylation, T401 phosphorylation might influence BRAF interactions with 14-3-3 proteins and dimerization capabilities. S732 phosphorylation has been shown to decrease 14-3-3 binding and reduce BRAF homodimerization and BRAF:CRAF heterodimerization .

• Dimerization consequences: Changes in dimerization affect MEK phosphorylation, with phosphomimetic S732E showing reduced MEK1/2 phosphorylation compared to phospho-deficient S732A . Similar effects might exist for T401 phosphorylation.

• Drug sensitivity correlation: Phosphorylation status correlates with drug sensitivity, as seen with the S732 site. Phosphomimetic BRAF S732E requires higher concentrations of BRAF inhibitors than BRAF S732A to induce paradoxical activation . T401 phosphorylation may similarly influence drug responses.

• Structural considerations: T401 is located in a different region than the C-terminal 14-3-3 binding sites, so its effects on dimerization and 14-3-3 binding may involve distinct mechanisms, possibly affecting the activation segment or other regulatory interfaces.

• Combinatorial effects: T401 phosphorylation may work in concert with other phosphorylation events to create a phosphorylation code that collectively determines BRAF's dimerization capacity and interactions with regulatory proteins.