Phospho-EIF4EBP1 (Thr45) Antibody

What is EIF4EBP1 and what role does it play in protein translation?

EIF4EBP1 (also known as 4E-BP1) is a translation repressor protein that regulates cap-dependent translation by binding to eukaryotic translation initiation factor 4E (eIF4E). In its hypophosphorylated state, 4E-BP1 competes with eIF4G1/eIF4G3 and binds strongly to eIF4E, repressing translation. When hyperphosphorylated, 4E-BP1 dissociates from eIF4E, allowing interaction between eIF4G1/eIF4G3 and eIF4E, thus enabling translation initiation. This protein mediates the regulation of protein translation by hormones, growth factors, and other stimuli that signal through the MAP kinase and mTORC1 pathways .

Why is the Thr45 phosphorylation site significant for research?

Thr45 phosphorylation (numbered as Thr46 in human 4E-BP1) represents a critical regulatory site that works in concert with other phosphorylation events to control 4E-BP1 function. Phosphorylation at Thr45 is primarily mediated by mTORC1 and serves as one of the priming events required for subsequent phosphorylation at other sites. Research has shown that Thr45 phosphorylation occurs early in the sequential phosphorylation cascade and is necessary for the hyperphosphorylation that ultimately leads to 4E-BP1 dissociation from eIF4E . Studies using phospho-specific antibodies targeting this site allow researchers to monitor mTORC1 signaling activity and early events in translational activation .

What is the hierarchical phosphorylation pattern of 4E-BP1?

Research has established a clear hierarchical pattern of 4E-BP1 phosphorylation:

• Thr37 and Thr46 (human numbering) are phosphorylated first, primarily by mTORC1

• This priming phosphorylation is followed by Thr70 phosphorylation

• Ser65 is phosphorylated last

This sequential phosphorylation has been confirmed through various techniques including mass spectrometry and two-dimensional isoelectric focusing/SDS-PAGE combined with phospho-specific antibody detection . Importantly, phosphorylation of Thr37/Thr46 alone is not sufficient to dissociate 4E-BP1 from eIF4E but serves as a required priming step for the subsequent phosphorylation events that ultimately lead to eIF4E release .

What research applications are recommended for Phospho-EIF4EBP1 (Thr45) antibodies?

Phospho-EIF4EBP1 (Thr45) antibodies are suitable for multiple experimental applications:

When selecting an application, researchers should consider that phosphorylation-specific signals may vary depending on cell type and treatment conditions. The molecular weight of 4E-BP1 typically appears between 15-20 kDa on Western blots, but multiple bands representing different phosphorylation states are often observed .

How should researchers design controls for experiments using Phospho-EIF4EBP1 (Thr45) antibodies?

Proper experimental controls are essential when working with phospho-specific antibodies:

• Positive controls: Use cell lines or tissues with known mTORC1 activation (e.g., serum-stimulated cells) to confirm antibody reactivity .

• Negative controls: Include samples treated with mTORC1 inhibitors (e.g., rapamycin) or PI3K inhibitors (LY294002, wortmannin) that reduce Thr45 phosphorylation .

• Dephosphorylation controls: Treat lysates with phosphatases to confirm phospho-specificity.

• Loading controls: Use total 4E-BP1 antibodies in parallel to normalize phospho-signals and account for expression differences .

• Mutation controls: When possible, include 4E-BP1 constructs with T45A mutations to verify antibody specificity .

What experimental approaches can distinguish between different phosphorylated forms of 4E-BP1?

Several techniques can help distinguish between different phosphorylated forms:

• Two-dimensional analysis: Combining isoelectric focusing with SDS-PAGE provides excellent resolution of different phosphorylated species, which can then be detected with phospho-specific antibodies .

• Phosphopeptide mapping: This approach allows identification of specific phosphorylated residues and can be combined with mass spectrometry for precise site mapping .

• Mobility shift assays: Different phosphorylation states of 4E-BP1 demonstrate characteristic mobility patterns on SDS-PAGE, with hyperphosphorylated forms migrating more slowly (appearing as α, β, and γ bands) .

• Cap-binding assays: These functional assays can distinguish 4E-BP1 forms based on their ability to bind to eIF4E, which correlates with phosphorylation status .

• Phospho-specific antibody panels: Using antibodies against multiple phosphorylation sites (Thr37/46, Thr70, Ser65) in parallel allows researchers to track the complete phosphorylation state .

How do researchers interpret contradictory phosphorylation data across different experimental systems?

Contradictory phosphorylation data may arise from several factors:

• Cell-type specific regulation: Different cell types may exhibit varying basal levels and responses of 4E-BP1 phosphorylation. For example, some studies indicate all phosphorylation sites are sensitive to serum and rapamycin, while others observe differential sensitivity across sites .

• Temporal dynamics: The timing of phosphorylation events is critical. Studies show that Thr37/46 phosphorylation increases only 1.3-1.8 fold after serum stimulation, while other sites show more dramatic changes. Time-course experiments are essential to capture these dynamics .

• Methodological differences: Detection methods vary in sensitivity and specificity. Mass spectrometry provides definitive site identification but may miss low-abundance forms, while phospho-specific antibodies might have cross-reactivity issues .

• Inhibitor specificity: Rapamycin and other inhibitors may have incomplete effects on mTORC1 depending on concentration, duration, and cell type .

To address these contradictions, researchers should: (a) validate findings across multiple cell types, (b) perform detailed time-course analyses, (c) use complementary techniques to confirm results, and (d) clearly report experimental conditions to facilitate comparison across studies.

What is the relationship between 4E-BP1 phosphorylation states and its binding to eIF4E?

The relationship between phosphorylation and eIF4E binding follows a complex pattern:

Studies using mutational analysis have shown that 4E-BP1 can be phosphorylated by FRAP/mTOR even when bound to eIF4E, confirming that the early phosphorylation events occur while 4E-BP1 is still in complex with eIF4E .

How do researchers distinguish between mTORC1-dependent and independent phosphorylation of 4E-BP1?

Distinguishing between mTORC1-dependent and independent phosphorylation requires careful experimental design:

• Pharmacological approaches: Use rapamycin (an mTORC1 inhibitor) alongside more comprehensive mTOR inhibitors (like Torin) to distinguish mTORC1-specific effects from those of other kinases. Phosphorylation of Thr37/46 is inhibited by PI3K inhibitors like LY294002 and wortmannin, confirming the upstream regulatory pathway .

• Genetic approaches: Employ cells with RAPTOR or mTOR knockdown/knockout to validate mTORC1-dependent phosphorylation. The TOS motif in 4E-BP1 mediates interaction with RAPTOR, promoting phosphorylation by the mTORC1 complex .

• Site-specific analysis: Research has identified that multiple kinases can phosphorylate 4E-BP1 at different sites. For instance, FRAP/mTOR phosphorylates Thr37 and Thr46, while ATM phosphorylates Ser111 . Other kinases implicated include:

  • MAPK1/ERK2 for Thr37, Thr45/46, Ser65, Thr70

  • CDK1 for Thr37, Thr46, Ser65, Thr70

  • MAPK14/p38 for Thr37, Thr46, Ser65, Thr70

  • AKT1 for Thr37, Thr46, Ser65

• Kinase activity assays: In vitro kinase assays using recombinant or immunoprecipitated mTORC1 can directly test which sites are phosphorylated by mTORC1. Studies using recombinant FRAP/mTOR protein and FRAP/mTOR immunoprecipitate in in vitro kinase assays have confirmed that these specifically phosphorylate Thr37 and Thr46 .

What factors affect the specificity of Phospho-EIF4EBP1 (Thr45) antibodies?

Several factors can influence antibody specificity:

• Antibody source and type: Different antibodies (monoclonal vs. polyclonal) may have varying specificity. For example, the V3NTY24 monoclonal antibody recognizes both human and mouse 4E-BP1 when phosphorylated at threonine 37 and/or threonine 46 .

• Cross-reactivity with related phosphorylation sites: Due to sequence similarity around phosphorylation sites, some antibodies may detect multiple phosphorylated threonines. Some antibodies detect both Thr37 and Thr46 phosphorylation together .

• Cross-reactivity with related proteins: 4E-BP family includes 4E-BP1, 4E-BP2, and 4E-BP3, which share sequence homology that may lead to cross-reactivity .

• Sample preparation: Inadequate fixation or denaturing conditions can expose epitopes differentially, leading to inconsistent results.

To optimize specificity, researchers should:

• Validate antibodies using phosphatase treatments

• Include appropriate positive and negative controls

• Test antibodies on cells with mutated phosphorylation sites when possible

• Consider using antibodies that have been validated through multiple applications

How can researchers optimize detection of Thr45 phosphorylation in various experimental systems?

Optimizing detection requires tailored approaches for different techniques:

• For Western blotting:

  • Ensure complete protein denaturation

  • Use fresh phosphatase inhibitors in lysis buffers

  • Consider using Phos-tag gels for enhanced separation of phosphorylated species

  • Recommended dilution: 1:1000

• For immunoprecipitation:

  • Optimize antibody:lysate ratio (typically 1:50)

  • Use mild detergents to preserve protein complexes

  • Consider cross-linking antibodies to beads to prevent interference

• For flow cytometry:

  • Optimize fixation and permeabilization protocols

  • Use 5 μL (0.06 μg) antibody per test with 10^5-10^8 cells in 100 μL volume

  • Include appropriate isotype controls

• For cell-based assays:

  • Include normalization controls (GAPDH, total 4E-BP1, cell number)

  • Consider rapid sample processing to prevent phosphatase activity

  • Validate results across multiple cell types

• For all applications:

  • Confirm antibody reactivity across species of interest (Human, Mouse, Rat, etc.)

  • Test different stimulation conditions (serum, insulin, growth factors)

  • Consider both time and dose-dependent effects on phosphorylation

What are the best approaches to quantify changes in 4E-BP1 phosphorylation?

Quantifying phosphorylation changes requires careful consideration of normalization methods:

• Western blot quantification:

  • Use phospho-specific signal normalized to total 4E-BP1

  • Consider the ratio between different phosphorylated bands (α, β, γ)

  • Use fluorescent secondary antibodies for wider linear range of detection

  • Include standard curves with known concentrations when possible

• Flow cytometry quantification:

  • Report median fluorescence intensity (MFI)

  • Use fold-change relative to unstimulated controls

  • Consider single-cell analysis to capture population heterogeneity

• Cell-based ELISA quantification:

  • Multiple normalization options:
    a. Normalize to GAPDH as internal control
    b. Use Crystal Violet staining to normalize to cell number
    c. Normalize phospho-signal to total 4E-BP1

• Mass spectrometry approaches:

  • Use stable isotope labeling (SILAC, TMT) for precise quantification

  • Consider analyzing the ratio of phosphorylated to non-phosphorylated peptides

  • Include multiple technical and biological replicates

When reporting quantitative changes, researchers should indicate fold changes with statistical analysis. For example, studies have shown that Thr37/46 phosphorylation increases only 1.3-1.8 fold after serum stimulation, while other sites show more dramatic changes .

How do researchers study the relationship between different 4E-BP1 phosphorylation sites?

Advanced approaches to study interrelationships between phosphorylation sites include:

What recent technological advances have improved the study of 4E-BP1 phosphorylation?

Recent technological innovations have expanded research capabilities:

• Proximity Ligation Assay (PLA): This technique allows visualization of individual phosphorylated proteins within cells. Each red dot represents one single phosphorylated protein, enabling quantitative spatial analysis of phosphorylation events at the single-molecule level .

• Phospho-specific flow cytometry: Flow cytometry with phospho-specific antibodies enables single-cell analysis of phosphorylation across heterogeneous populations and can detect subtle changes in signaling pathways .

• Small molecule modulators: Compounds like DT-061, which activate B56-PP2A, have been developed to manipulate 4E-BP1 phosphorylation states in living cells. These tools help establish causality in phosphorylation cascades by promoting loss of phosphorylation at specific sites like Ser64 and Thr45 .

• CRISPR-based phosphorylation site editing: Precise genome editing to modify endogenous phosphorylation sites avoids artifacts associated with overexpression systems.

• Mass spectrometry advances: Improved sensitivity and throughput in mass spectrometry have enabled comprehensive mapping of phosphorylation sites and their dynamics across different conditions, helping resolve contradictions in earlier studies .

These technologies collectively provide unprecedented resolution for studying the complex phosphorylation patterns of 4E-BP1 and their functional consequences in translation regulation.