EIF4EBP1 Human

What is the fundamental role of EIF4EBP1 in protein translation regulation?

EIF4EBP1 encodes 4E-BP1, a translation repressor protein that acts as a negative regulator of cap-dependent mRNA translation. The protein functions by directly binding to eukaryotic translation initiation factor 4E (eIF4E), which is a limiting component of the multisubunit complex that recruits 40S ribosomal subunits to the 5' end of mRNAs. When 4E-BP1 binds to eIF4E, it prevents the assembly of the translation initiation complex and inhibits cap-dependent translation. Phosphorylation of 4E-BP1 results in its dissociation from eIF4E, allowing cap-dependent translation to proceed, thereby increasing the rate of protein synthesis .

Methodologically, researchers investigating this function typically employ techniques such as m7GTP cap pull-down assays to examine cap-binding complex formation, co-immunoprecipitation to detect protein-protein interactions, and translation reporter assays to measure the effect of 4E-BP1 on protein synthesis rates.

What are the critical phosphorylation sites in 4E-BP1 and their functional significance?

4E-BP1 has seven phosphorylation sites, with three being particularly crucial for its function:

These phosphorylation events occur in a hierarchical fashion. Research has shown that phosphorylation of Ser65 and Thr70 alone is not sufficient to block the inhibitory function of 4E-BP1, indicating that multiple phosphorylation events must occur in combination to increase protein synthesis rates .

For experimental approaches, phospho-specific antibodies targeting these sites are commonly used in Western blotting, while phospho-proteomic mass spectrometry offers a comprehensive analysis of all phosphorylation sites simultaneously.

How does 4E-BP1 interact with the mTOR signaling pathway?

4E-BP1 is a direct substrate of the mechanistic target of rapamycin complex 1 (mTORC1), a central regulator of cell growth and metabolism. The phosphorylation status of 4E-BP1 is considered a reliable marker of mTORC1 activity. When mTORC1 is activated in response to nutrients, growth factors, or energy signals, it phosphorylates 4E-BP1 at multiple sites, causing 4E-BP1 to dissociate from eIF4E and allowing cap-dependent translation to proceed .

In research contexts, the level of phosphorylated 4E-BP1 is often used as a readout for mTORC1 activity, making it a valuable biomarker in studies investigating cellular growth control, nutrient sensing, and cancer pathways. Different mTOR inhibitors (ATP-competitive vs. rapamycin) can provide insights into the regulation of different 4E-BP1 phosphorylation sites.

What protein interaction partners have been identified for EIF4EBP1?

The principal known interaction partners of EIF4EBP1 include:

• EIF4E (Eukaryotic translation initiation factor 4E): The primary binding partner, with this interaction directly inhibiting cap-dependent translation

• mTOR (Mammalian target of rapamycin): The kinase responsible for phosphorylating 4E-BP1

• KIAA1303 (also known as RAPTOR): A component of the mTORC1 complex that may facilitate the interaction between mTOR and 4E-BP1

For researchers studying these protein-protein interactions, techniques such as co-immunoprecipitation, proximity ligation assays, and FRET-based approaches offer valuable insights. The phosphorylation state of 4E-BP1 dramatically affects its binding properties, particularly with eIF4E.

How is EIF4EBP1 expression regulated at the transcriptional level?

Research on transcriptional regulation of EIF4EBP1, particularly in cancer contexts, has identified several transcription factors that can regulate its expression:

• In gliomas, MYBL2 and ETS1 have been identified as transcriptional drivers of enhanced EIF4EBP1 expression

• Other transcription factors shown to bind to the EIF4EBP1 promoter include HIF-1A and E2F6, though their functional significance may vary by cell type

For investigating transcriptional regulation of EIF4EBP1, chromatin immunoprecipitation (ChIP) assays, luciferase reporter assays, and transcription factor knockdown/overexpression studies have proven useful. The research suggests that EIF4EBP1 overexpression in malignant gliomas results from aberrant transcriptional activation rather than gene amplification or altered DNA methylation .

What evidence exists for altered EIF4EBP1 expression during cellular senescence?

Studies have demonstrated that 4E-BP1 expression changes during cellular aging and senescence:

• 4E-BP1 protein levels decrease in replicatively senescent human mesenchymal stem cells (hMSCs)

• Primary hMSCs isolated from aged individuals show lower 4E-BP1 expression compared to those from young individuals

• Experimental depletion of 4E-BP1 accelerates senescence in hMSCs

RNA-sequencing analysis reveals that 4E-BP1 depletion affects the expression of genes associated with mitotic cell cycle, DNA repair, and response to reactive oxygen species, while upregulating genes related to cell adhesion, negative regulation of catalytic activity, and aging .

What are the optimal approaches for studying different phosphorylation states of 4E-BP1?

Several complementary approaches are recommended for comprehensive analysis of 4E-BP1 phosphorylation:

• Western blotting with phospho-specific antibodies: For site-specific phosphorylation detection at Thr37/46, Thr70, and Ser65

• Phospho-proteomic mass spectrometry: For unbiased, comprehensive identification of all phosphorylation sites

• Phos-tag SDS-PAGE: For separation of different phosphorylated forms of 4E-BP1

• CRISPR/Cas9-mediated generation of phospho-site mutants: For functional studies of specific phosphorylation events

When designing experiments, researchers should consider that 4E-BP1 phosphorylation is dynamic and sensitive to culture conditions, including serum levels, cell density, and nutrient availability. Time course experiments are often necessary to capture the sequential nature of phosphorylation events.

What gene-editing strategies are most effective for EIF4EBP1 functional studies?

Modern gene-editing approaches for manipulating EIF4EBP1 include:

• CRISPR/Cas9 gene editing: For complete knockout or introduction of specific mutations, as demonstrated in the generation of EIF4EBP1-/- human embryonic stem cells

• Inducible expression systems: For temporal control of 4E-BP1 expression

• Structure-guided mutagenesis: For studying specific functional domains

• Phospho-mimetic and phospho-deficient mutants: To study the effects of specific phosphorylation events

When using these techniques, researchers should verify genomic integrity through karyotyping and genome-wide copy number variation (CNV) analyses and check for potential off-target effects at predicted genomic loci .

How is EIF4EBP1 involved in cancer progression and prognosis?

4E-BP1 plays significant roles in cancer:

• High levels of phosphorylated 4E-BP1 have been widely reported in human cancers and are associated with worse outcomes in several malignancies

• In breast cancer, EIF4EBP1 is significantly upregulated in tumor tissues compared to normal tissues

• Elevated EIF4EBP1 expression is associated with tamoxifen resistance in breast cancer patients

For cancer researchers, analyzing both total and phosphorylated 4E-BP1 levels is crucial, as they may have different prognostic implications. Patient-derived samples and models can capture the heterogeneity of 4E-BP1's role across different cancer types and genetic backgrounds.

What challenges exist in targeting the 4E-BP1 pathway for cancer therapeutics?

Despite promising associations between 4E-BP1 phosphorylation and cancer outcomes, several challenges exist:

• Specificity concerns: 4E-BP1 is a downstream effector of mTOR, which has numerous substrates

• Feedback mechanisms: Inhibition of mTOR can lead to compensatory activation of upstream pathways

• Context-dependent roles: 4E-BP1 may function differently across cancer types

Current research approaches include:

• Developing selective modulators of 4E-BP1 phosphorylation

• Identifying synthetic lethal interactions with 4E-BP1-related pathways

• Investigating combination therapies targeting both 4E-BP1 and complementary pathways

How does 4E-BP1 contribute to mitochondrial function?

Recent research has uncovered important links between 4E-BP1 and mitochondrial function:

These findings indicate that 4E-BP1 plays an important role in maintaining mitochondrial fitness. Loss of 4E-BP1 leads to impaired mitochondrial respiration and increased reactive oxygen species, which may contribute to cellular senescence .

How can researchers effectively measure translational changes mediated by 4E-BP1?

To quantify translational changes regulated by 4E-BP1, researchers can employ these methodologies:

• Polysome profiling combined with RNA-seq: To identify differentially translated mRNAs

• Ribosome profiling (Ribo-seq): For genome-wide assessment of ribosome occupancy

• SUnSET assay: For measuring global protein synthesis rates

• Bicistronic reporter assays: To distinguish cap-dependent and cap-independent translation

• Translational efficiency calculation: The ratio of ribosome-associated mRNA to total mRNA

What recombinant EIF4EBP1 protein resources are available for in vitro studies?

For researchers conducting in vitro studies with recombinant 4E-BP1, commercially available options include:

• Human 4E-BP1/EIF4EBP1 recombinant proteins with various tags (e.g., His-tag)

• Proteins covering the full sequence (Met 1-Ile118) or specific regions

• Expression systems typically include E. coli for producing recombinant human 4E-BP1

Specifications for typical recombinant 4E-BP1:

• Molecular mass: 14.7 kDa (theoretical); 17 kDa (apparent on SDS-PAGE)

• Purity: >90% as determined by reducing SDS-PAGE

• Storage: Lyophilized proteins stable for up to 12 months at -20 to -80°C; reconstituted solutions at 4-8°C for 2-7 days; aliquots at <-20°C for 3 months

These resources are valuable for binding studies, enzyme assays, and structural analyses.

How can contradictory findings about 4E-BP1's role in different biological contexts be reconciled?

To address contradictory findings regarding 4E-BP1's function across different studies, consider:

• Phosphorylation state: Total 4E-BP1 vs. phosphorylated 4E-BP1 may have different implications

• Tissue specificity: The role of 4E-BP1 varies across tissue types and cellular contexts

• Genetic background: The function of 4E-BP1 may depend on the mutational landscape

• Temporal dynamics: Short-term vs. long-term effects of 4E-BP1 modulation

Research strategies to address these contradictions include:

• Multi-omics approaches integrating transcriptomics, proteomics, and phospho-proteomics

• Longitudinal studies examining 4E-BP1's role at different disease or developmental stages

• Considering 4E-BP1 in the context of broader signaling networks

• Clearly specifying experimental conditions and models when reporting results

What are the emerging areas of EIF4EBP1 research?

Emerging areas in EIF4EBP1 research include:

• Role in cellular senescence and aging biology

• Contributions to mitochondrial function and metabolic regulation

• Non-canonical functions beyond translation regulation

• Tissue-specific and context-dependent roles

• Potential as a therapeutic target in various diseases

• Role in drug resistance mechanisms, particularly in cancer

These areas represent promising avenues for researchers investigating the multifaceted functions of this important translational regulator.

What methodological advances are needed to further EIF4EBP1 research?

Advancements that would benefit future EIF4EBP1 research include:

• Development of more selective inhibitors or activators of the 4E-BP1 pathway

• Improved tools for visualizing 4E-BP1-eIF4E interactions in living cells

• Better methods to distinguish between the roles of different 4E-BP family members

• Advanced computational models to predict the impact of 4E-BP1 on translatome composition

• Single-cell approaches to capture cellular heterogeneity in 4E-BP1 function

• Physiologically relevant 3D model systems for studying 4E-BP1 in tissue contexts