Phospho-EIF4EBP1 (T37) Antibody

What is the biological significance of 4E-BP1 phosphorylation at threonine 37?

Phosphorylation of 4E-BP1 at threonine 37 serves as one of the critical priming sites in the hierarchical phosphorylation model of 4E-BP1 regulation. When 4E-BP1 is phosphorylated at the Thr-37/Thr-46 sites, it substantially weakens but does not completely block the protein's interaction with eIF4E . This initial phosphorylation event is necessary to facilitate subsequent phosphorylation at other sites, ultimately leading to the hyperphosphorylated state that fully prevents 4E-BP1 from sequestering eIF4E, thereby allowing cap-dependent translation initiation to proceed .

Research has shown that despite phosphorylation at Thr-37/Thr-46, some 4E-BP1 isoforms (such as EB-α and EB-β) can still bind to eIF4E, indicating that these priming phosphorylations alone are insufficient to completely disrupt the 4E-BP1:eIF4E interaction . This highlights the complex regulatory mechanism controlling translation initiation through the 4E-BP1/eIF4E axis.

How does the Phospho-EIF4EBP1 (T37) antibody differ from Phospho-EIF4EBP1 (T37/46) antibody?

The Phospho-EIF4EBP1 (T37) antibody specifically recognizes 4E-BP1 phosphorylated only at threonine 37, whereas the Phospho-EIF4EBP1 (T37/46) antibody detects 4E-BP1 molecules phosphorylated at both threonine 37 and threonine 46 sites . This distinction is crucial for researchers investigating the sequential phosphorylation events of 4E-BP1.

The Phospho-EIF4EBP1 (T37/46) antibody is commonly used in studies examining the priming phosphorylation events that initiate the cascade of 4E-BP1 modification. This dual-site antibody helps identify the population of 4E-BP1 molecules that have undergone the initial step in the phosphorylation process . In contrast, a single-site T37 antibody would enable more precise mapping of the phosphorylation sequence and potentially distinguish between partially phosphorylated states.

What are the recommended experimental applications for Phospho-EIF4EBP1 (T37/46) antibody?

The Phospho-EIF4EBP1 (T37/46) antibody has been validated for multiple experimental applications including:

The antibody shows reactivity with human, mouse, and rat samples, making it versatile for comparative studies across these species . For optimal results, researchers should validate the antibody in their specific experimental systems using positive control samples such as lysates from HeLa, NIH/3T3, or C6 cells .

What is the role of 4E-BP1 in regulating translation initiation?

4E-BP1 functions as a repressor of cap-dependent translation by competing with eIF4G for binding to eIF4E. In its hypophosphorylated state, 4E-BP1 strongly binds to eIF4E, preventing the formation of the eIF4F complex necessary for translation initiation . This inhibitory effect is relieved when 4E-BP1 undergoes hyperphosphorylation, which causes it to dissociate from eIF4E.

The regulation of 4E-BP1 phosphorylation is mediated through multiple signaling pathways, including the PI3-kinase/Akt pathway and the kinase FRAP/mTOR . Additionally, cell cycle-specific phosphorylation events occur, such as the CDK1/cyclin B-mediated phosphorylation of 4E-BP1 at Ser-83 during mitosis . This complex regulatory network allows cells to modulate protein synthesis rates in response to various stimuli and cellular conditions.

How can one effectively distinguish between different phosphorylated isoforms of 4E-BP1 in experimental settings?

Distinguishing between the various phosphorylated isoforms of 4E-BP1 (typically designated as α, β, γ, and δ bands on electrophoretic gels) requires a combination of techniques:

• Two-dimensional gel electrophoresis: This technique separates 4E-BP1 isoforms first by isoelectric point and then by molecular weight, providing better resolution of closely migrating phosphorylated forms compared to standard one-dimensional electrophoresis .

• Phospho-specific antibodies: Using a panel of antibodies specific to different phosphorylation sites (such as Thr-37/46, Thr-70, Ser-65, Ser-83, and Ser-101) in parallel Western blots or multiplexed assays allows identification of phosphorylation patterns unique to each isoform .

• Mass spectrometry analysis: This technique provides precise identification of specific phosphorylation sites and can detect combinatorial phosphorylation patterns that are difficult to resolve with antibody-based methods alone .

• Cap-binding assays: Using m7GTP cap pulldown assays or eIF4E immunoprecipitation followed by Western blotting with phospho-specific antibodies helps distinguish between eIF4E-bound and unbound 4E-BP1 isoforms and their phosphorylation status .

Research has shown that the eIF4E-unbound δ band is positive for Ser-83, Thr-37/Thr-46, Ser-65/Ser-101, and Thr-70 phosphorylations, while the eIF4E-bound EB-γ isoform present in mitotic cells is positive for Ser-83 and Thr-70 but negative for Thr-37/Thr-46 phosphorylations . These complex phosphorylation patterns highlight the need for comprehensive analytical approaches.

What are the optimal sample preparation methods for phospho-specific 4E-BP1 Western blot analysis?

For optimal detection of phosphorylated 4E-BP1 isoforms in Western blot analysis, the following sample preparation protocol is recommended:

• Rapid sample collection: Harvest cells directly into ice-cold lysis buffer to prevent dephosphorylation by cellular phosphatases. For tissues, snap freezing in liquid nitrogen followed by homogenization in lysis buffer is recommended.

• Phosphatase inhibitor cocktail: Include multiple phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate, and proprietary phosphatase inhibitor cocktails) in the lysis buffer to preserve phosphorylation status .

• Protein extraction buffer: Use a buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, supplemented with protease inhibitors and the aforementioned phosphatase inhibitors.

• Sample handling: Maintain samples at 4°C throughout processing and avoid multiple freeze-thaw cycles which can lead to dephosphorylation.

• Gel system selection: Use Phos-tag™ acrylamide gels or high-percentage (15-18%) SDS-PAGE gels for better separation of the closely migrating phosphorylated isoforms .

• Normalization controls: Include both phospho-specific loading controls and total 4E-BP1 antibody detection to accurately assess relative phosphorylation levels.

When analyzing specific cell cycle phases, synchronization methods (such as thymidine block for S phase or nocodazole treatment for mitosis) should be optimized to enrich for the particular 4E-BP1 phospho-isoforms of interest .

How can phosphorylation of 4E-BP1 at T37/46 be differentiated from other phosphorylation events in the context of cell cycle regulation?

Differentiating 4E-BP1 phosphorylation at T37/46 from other sites within the context of cell cycle regulation requires a multi-faceted experimental approach:

• Cell synchronization coupled with time-course analysis: Synchronize cells at different cell cycle phases (G1, S, G2, and M) using standard methods, and collect samples at defined intervals for phosphorylation analysis .

• Combinatorial phospho-specific antibody analysis: Use multiple phospho-specific antibodies (including T37/46, S65, T70, and S83) in parallel to create a comprehensive phosphorylation profile at each cell cycle stage .

• Kinase inhibitor studies: Employ specific inhibitors of mTOR (rapamycin, Torin), CDK1 (RO-3306), or other relevant kinases to distinguish between different phosphorylation pathways .

• Flow cytometry with phospho-specific antibodies: Combine DNA content analysis with phospho-specific antibody staining to directly correlate 4E-BP1 phosphorylation status with cell cycle position at the single-cell level.

• Mutational analysis: Use site-specific phospho-defective (T→A) or phospho-mimetic (T→E) 4E-BP1 mutants to assess the functional importance of T37/46 phosphorylation relative to other sites .

Research has demonstrated that while mTOR-dependent phosphorylation of 4E-BP1 at sites including T37/46 occurs throughout the cell cycle, mitosis-specific phosphorylation at S83 is mediated by CDK1/cyclin B . This creates distinct phospho-isoforms with different eIF4E-binding properties that can be distinguished using the techniques described above.

What are the technical considerations for using Phospho-EIF4EBP1 antibodies in immunohistochemistry of clinical samples?

When performing immunohistochemistry (IHC) with phospho-specific 4E-BP1 antibodies on clinical samples, several critical factors must be addressed:

• Fixation and processing: Phospho-epitopes are sensitive to fixation conditions. For FFPE (formalin-fixed, paraffin-embedded) tissues, limit fixation time to 24 hours in 10% neutral buffered formalin to preserve phospho-epitopes. For phospho-T37/46 detection, a recommended dilution range of 1:50 - 1:200 should be tested .

• Antigen retrieval: Optimize antigen retrieval conditions; for phospho-4E-BP1, heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes typically yields the best results.

• Signal amplification: Consider using polymer-based detection systems or tyramide signal amplification to enhance sensitivity for detecting phosphorylated epitopes.

• Controls: Include both positive controls (tissues known to express phospho-4E-BP1) and negative controls (phosphatase-treated sections and/or primary antibody omission). Tumors with activated mTOR signaling typically show strong phospho-4E-BP1 staining .

• Scoring system standardization: Develop a consistent scoring method for phospho-4E-BP1 positivity. Studies in clear cell renal cell carcinoma defined positivity as moderate to strong staining in >10% of tumor cells .

Research on clear cell renal cell carcinoma has demonstrated the prognostic value of phospho-4E-BP1 and eIF4E co-expression, with patients whose tumors expressed both markers having significantly worse disease-free survival (2.9 years) compared to those expressing neither or only one marker (5.4-5.6 years) . This highlights the potential clinical utility of these markers when properly assessed.

What are common causes of inconsistent results when using Phospho-EIF4EBP1 (T37/46) antibody in Western blot applications?

Several factors can contribute to inconsistent results when using phospho-specific 4E-BP1 antibodies:

• Phosphatase activity: Insufficient phosphatase inhibition during sample preparation is a primary cause of inconsistent phospho-signal detection. Use a robust combination of phosphatase inhibitors including sodium fluoride (50 mM), sodium orthovanadate (1 mM), and β-glycerophosphate (10 mM) .

• Sample handling time: Extended processing time between sample collection and lysis can result in dephosphorylation. Samples should be collected and lysed as quickly as possible or snap-frozen immediately.

• Antibody specificity issues: Some phospho-specific antibodies may cross-react with similar phospho-epitopes. Validate specificity using phosphatase-treated controls and phospho-defective mutants .

• Variable phosphorylation levels due to cell cycle distribution: Asynchronous cell populations will have variable 4E-BP1 phosphorylation patterns. Consider synchronizing cells if studying specific cell cycle-dependent phosphorylation events .

• Protein loading: Overloading protein can cause band distortion and poor resolution of the closely migrating phospho-isoforms. Use 20-40 μg of total protein and a 15-18% acrylamide gel for optimal separation.

• Antibody dilution optimization: The recommended working dilution for Western blot (1:500-1:1000) should be empirically tested and optimized for each experimental system .

• Membrane blocking conditions: Excessive blocking can mask phospho-epitopes. Use 3-5% BSA in TBST rather than milk (which contains phosphatases) for blocking and antibody dilution.

How can researchers verify the specificity of Phospho-EIF4EBP1 antibody detection in their experimental system?

To verify the specificity of phospho-4E-BP1 antibody detection, researchers should implement the following validation steps:

• Phosphatase treatment control: Treat duplicate samples with lambda phosphatase before immunoblotting. The phospho-specific signal should be eliminated or substantially reduced in treated samples.

• Competing peptide assay: Pre-incubate the antibody with a synthetic phosphopeptide containing the target phosphorylation site (e.g., the immunogen sequence STTP GGTL FSTT PG for the T37/46 antibody) . This should ablate specific antibody binding.

• Kinase inhibition experiments: Treat cells with specific inhibitors of relevant kinases (e.g., rapamycin or Torin for mTOR) to reduce phosphorylation at the target site .

• siRNA knockdown or CRISPR knockout: Reduce or eliminate 4E-BP1 expression to confirm the identity of detected bands.

• Phospho-defective mutants: If using overexpression systems, include phospho-defective mutants (T37A/T46A) as negative controls for phospho-specific detection.

• Cross-validation with mass spectrometry: For critical experiments, confirm phosphorylation status using phospho-proteomic mass spectrometry analysis .

• Positive control samples: Include samples known to contain high levels of phosphorylated 4E-BP1, such as serum-stimulated HeLa cells or insulin-treated cells .

How should researchers interpret conflicting data between phosphorylation status and functional outcomes in 4E-BP1 studies?

When facing conflicting data between 4E-BP1 phosphorylation status and expected functional outcomes, consider these analytical approaches:

• Comprehensive phosphorylation profiling: Instead of focusing on a single phosphorylation site, analyze all major phosphorylation sites (T37/46, S65, T70, S83, S101) to create a complete phosphorylation profile. Research has shown that certain combinations of phosphorylation, rather than individual sites, determine functional outcomes .

• Direct eIF4E binding assessment: Since the functional consequence of 4E-BP1 phosphorylation is altered eIF4E binding, directly measure this interaction using cap-binding assays (m7GTP pulldown) or co-immunoprecipitation. This provides functional data that may clarify seemingly conflicting phosphorylation results .

• Translational activity measurements: Measure actual translational output using polysome profiling, ribosome profiling, or reporter assays that monitor cap-dependent translation. This establishes whether observed phosphorylation changes correlate with translation regulation.

• Cell cycle context consideration: The functional significance of 4E-BP1 phosphorylation differs across cell cycle phases. For instance, a mitotic EB-γ isoform positive for Ser-83 and Thr-70 phosphorylation but negative for Thr-37/46 can still bind eIF4E . Analysis should account for cell cycle distribution.

• Isoform-specific analysis: 4E-BP1 exists in multiple isoforms (α, β, γ, δ) with different phosphorylation patterns and eIF4E-binding properties. Two-dimensional gel electrophoresis or isoform-specific analysis may resolve apparent contradictions in the data .

• Stoichiometry assessment: Consider the relative abundance of phosphorylated versus non-phosphorylated forms, and of 4E-BP1 versus eIF4E. Functional outcomes depend on the stoichiometric balance between these players .

How does the phosphorylation of 4E-BP1 at T37/46 relate to cancer progression and therapeutic response?

The relationship between 4E-BP1 phosphorylation at T37/46 and cancer progression is multifaceted:

• Prognostic significance: Studies in clear cell renal cell carcinoma have demonstrated that co-expression of phosphorylated 4E-BP1 and eIF4E is associated with significantly worse disease-free survival (2.9 years vs. 5.7 years for patients with expression of only one or neither marker) . This phosphorylation pattern serves as an independent prognostic indicator with a hazard ratio of 4.2 (CI = 2.1-8.6; P < 0.001) .

• Therapeutic resistance mechanisms: Phosphorylation of 4E-BP1 at T37/46 represents a key step in the activation of cap-dependent translation, which can drive the synthesis of proteins involved in cell survival, proliferation, and metastasis. Enhanced phosphorylation at these sites has been linked to resistance to mTOR inhibitors in various cancer types .

• Biomarker potential: The phosphorylation status of 4E-BP1 at T37/46 sites serves as a reliable biomarker of mTOR pathway activation and potentially as a predictive marker for response to targeted therapies. Clinical studies have shown phosphorylated 4E-BP1 to be an accurate single biomarker of mTOR pathway activity for predicting disease progression in carcinomas of the ovary, brain, and prostate .

• Cell cycle-specific roles: While T37/46 phosphorylation occurs throughout the cell cycle, research has revealed that mitotic-specific phosphorylation patterns play distinct roles in regulating translation during cell division, potentially contributing to the proliferative capacity of cancer cells .

• Therapeutic targeting strategies: Dual targeting of both the phosphorylation mechanism (through mTOR inhibitors) and the downstream effects (through eIF4E inhibitors) may provide more effective therapeutic approaches than single-agent strategies, particularly given the synergistic effect observed in tumors expressing both phosphorylated 4E-BP1 and elevated eIF4E .

What novel methodologies are being developed to study dynamic 4E-BP1 phosphorylation in living cells?

Emerging methodologies for studying dynamic 4E-BP1 phosphorylation in living cells include:

• Phosphorylation-specific FRET biosensors: Genetically encoded fluorescence resonance energy transfer (FRET) biosensors designed to detect specific phosphorylation events in real-time. These constructs typically contain the 4E-BP1 sequence flanked by fluorescent proteins that undergo FRET changes upon phosphorylation-induced conformational alterations.

• Optogenetic control of kinase activity: Light-controlled activation of kinases involved in 4E-BP1 phosphorylation (such as mTOR) allows precise temporal control of phosphorylation events and real-time monitoring of downstream effects.

• Fluorescence lifetime imaging microscopy (FLIM): FLIM-based approaches combined with phospho-specific antibody fragment-based sensors can detect phosphorylation events with high spatial and temporal resolution in living cells.

• Live-cell compatible phospho-specific nanobodies: Development of cell-permeable phospho-specific nanobodies conjugated to fluorophores enables tracking of phosphorylation events without cell fixation.

• Phosphorylation-dependent protein translocation assays: Systems that link 4E-BP1 phosphorylation status to subcellular localization changes of reporter proteins, allowing visual assessment of phosphorylation dynamics.

• CRISPR-based endogenous tagging strategies: Knock-in of fluorescent tags at the endogenous 4E-BP1 locus coupled with phospho-specific detection methods provides physiologically relevant monitoring of phosphorylation events.

These innovative approaches are enabling researchers to move beyond static measurements of 4E-BP1 phosphorylation toward understanding the spatiotemporal dynamics of this process in the context of different cellular compartments, cell cycle phases, and in response to various stimuli.

How do the combinatorial phosphorylation patterns of 4E-BP1 create a "phosphorylation code" that regulates translation?

The concept of a "phosphorylation code" in 4E-BP1 regulation refers to how specific combinations of phosphorylation events, rather than individual site modifications, determine functional outcomes:

• Hierarchical phosphorylation model: Research has established that 4E-BP1 phosphorylation follows a specific sequence, with phosphorylation at T37/46 serving as priming events that enable subsequent phosphorylation at other sites (S65, T70, S83, S101) . This creates a stepwise activation model where multiple phosphorylation events must occur in the correct order to fully inhibit 4E-BP1's repressive function.

• Distinct phospho-isoforms with different functionality: Studies have identified multiple phospho-isoforms of 4E-BP1 (α, β, γ, δ) with distinct phosphorylation patterns and eIF4E-binding properties. For example, the mitotic EB-γ band is positive for Ser-83 and Thr-70 phosphorylations but negative for T37/46, yet can still bind eIF4E. In contrast, the δ isoform with phosphorylation at T37/46, S65/101, T70 and S83 does not bind eIF4E .

• Cell cycle-specific phosphorylation patterns: Different kinases target 4E-BP1 during different cell cycle phases, creating phase-specific phosphorylation codes. During mitosis, CDK1/cyclin B phosphorylates S83, creating a phosphorylation pattern distinct from the mTOR-mediated phosphorylation predominant in other phases .

• Integration of multiple signaling pathways: The phosphorylation state of 4E-BP1 integrates inputs from multiple signaling pathways, including PI3K/Akt/mTOR, MAPK, and cell cycle-regulatory pathways . This allows translation regulation to respond to diverse cellular conditions.

• Resistance to single-pathway inhibition: The combinatorial nature of 4E-BP1 phosphorylation explains why inhibition of a single pathway (e.g., using rapamycin to inhibit mTORC1) may not fully block 4E-BP1 phosphorylation and translation initiation, particularly during mitosis when alternative phosphorylation mechanisms are active .

This complex phosphorylation code provides cells with a sophisticated mechanism to fine-tune translation initiation in response to diverse physiological contexts, energy states, stress conditions, and cell cycle positions.

How does 4E-BP1 phosphorylation interact with other translation regulatory mechanisms?

The phosphorylation of 4E-BP1 functions within a broader network of translation regulatory mechanisms:

• Coordination with eIF4E phosphorylation: While 4E-BP1 phosphorylation regulates eIF4E availability, eIF4E itself is regulated through phosphorylation at S209 by MNK kinases. Studies suggest these parallel modifications can work synergistically to regulate translation, particularly in cancer contexts where both phosphorylated 4E-BP1 and elevated eIF4E levels correlate with poor prognosis .

• Integration with S6K signaling: Both 4E-BP1 and S6K are major downstream effectors of mTORC1, but they can be differentially regulated. In some contexts, rapamycin treatment inhibits S6K phosphorylation more effectively than 4E-BP1 phosphorylation, creating distinct translational outputs .

• Interplay with eIF2α phosphorylation: While 4E-BP1 regulates cap-dependent translation initiation through eIF4E availability, eIF2α phosphorylation controls the ternary complex formation. These two mechanisms can function independently or in concert to modulate global and mRNA-specific translation rates.

• Complementary regulation with PDCD4: Programmed cell death protein 4 (PDCD4) inhibits eIF4A helicase activity within the eIF4F complex. The coordinated regulation of both 4E-BP1 and PDCD4 phosphorylation (both of which are degraded upon phosphorylation) provides multilevel control of eIF4F complex formation.

• Selective mRNA translation regulation: The 4E-BP1/eIF4E axis preferentially regulates the translation of mRNAs with complex 5' UTR structures, including many oncogenes and growth factors. This selective regulation works in parallel with other mechanisms controlling different subsets of mRNAs.

• Alternative translation initiation mechanisms: Under conditions where cap-dependent translation is inhibited by hypophosphorylated 4E-BP1, cells can utilize cap-independent translation mechanisms including internal ribosome entry sites (IRES), creating a complex regulatory landscape.

Understanding these integrated regulatory mechanisms is essential for developing effective therapeutic strategies targeting translation in diseases like cancer, where multiple pathways may be dysregulated simultaneously.

What are the methodological approaches for studying the relationship between mTOR signaling and 4E-BP1 phosphorylation?

Investigating the relationship between mTOR signaling and 4E-BP1 phosphorylation requires multifaceted experimental approaches:

• Pharmacological inhibition studies: Use mTOR inhibitors with different mechanisms of action:

  • Rapamycin and rapalogs (allosteric mTORC1 inhibitors)

  • Torin1 and other ATP-competitive mTOR inhibitors (targeting both mTORC1 and mTORC2)

  • Dual PI3K/mTOR inhibitors

  Combined with time-course analysis of 4E-BP1 phosphorylation at multiple sites, these studies can distinguish between rapamycin-sensitive and rapamycin-resistant phosphorylation events .

• Genetic manipulation of mTOR pathway components:

  • siRNA/shRNA knockdown of mTOR, Raptor, Rictor

  • CRISPR/Cas9-mediated knockout

  • Expression of constitutively active or dominant-negative mutants

  These approaches help establish direct causality between specific mTOR complex components and 4E-BP1 phosphorylation.

• In vitro kinase assays: Reconstituted systems using purified components:

  • Immunoprecipitated mTOR complexes

  • Recombinant 4E-BP1 (wild-type and mutants)

  • Phospho-specific antibody detection or mass spectrometry analysis

  These assays can determine direct phosphorylation events and identify phosphorylation site preferences .

• Nutrient and growth factor signaling manipulation:

  • Amino acid starvation/repletion protocols

  • Glucose deprivation

  • Serum starvation/stimulation

  • Insulin or growth factor treatment

  These conditions modulate mTOR activity through physiological mechanisms and allow assessment of pathway dynamics.

• Translation status correlation:

  • Parallel analysis of 4E-BP1 phosphorylation and cap-binding activity

  • Polysome profiling to assess translation efficiency

  • Metabolic labeling to measure protein synthesis rates

  These functional readouts connect phosphorylation events to their physiological outcomes.

These methodological approaches, used in combination, provide comprehensive insights into the complex relationship between mTOR signaling and 4E-BP1 phosphorylation in various physiological and pathological contexts.