Phospho-EIF4EBP1 (Ser64) Antibody

What is EIF4EBP1 and what role does phosphorylation at Ser64/65 play in its function?

EIF4EBP1 (4E-BP1) is a translation repressor protein that acts as a suppressor of cap-dependent RNA translation by competitively associating with cap-bound eIF4E. Phosphorylation of 4E-BP1 causes its release from eIF4E, allowing cap-dependent translation to proceed .

The phosphorylation of 4E-BP1 occurs at multiple sites, with Ser64/65 being particularly significant. While phosphorylation by mTOR at Thr37 and Thr46 does not prevent the binding of 4E-BP1 to eIF4E, it is thought to prime 4E-BP1 for subsequent phosphorylation at Ser65 and Thr70 . The Ser64/65 phosphorylation is crucial for the complete dissociation of 4E-BP1 from eIF4E, enabling the formation of the translation initiation complex.

How do researchers differentiate between the various phosphorylation states of 4E-BP1?

Researchers employ several techniques to differentiate between 4E-BP1 phosphorylation states:

These methods allow researchers to track the complex phosphorylation patterns of 4E-BP1, including the hyperphosphorylated forms that do not bind to eIF4E and the less phosphorylated forms that maintain eIF4E binding capacity .

What are the established phosphorylation patterns of 4E-BP1 during the cell cycle?

The phosphorylation profile of 4E-BP1 changes dynamically throughout the cell cycle:

• Interphase: Lower levels of phosphorylation compared to mitosis, with multiple phospho-isoforms present .

• Mitosis: Greater fraction of 4E-BP1 becomes hyperphosphorylated (E and F isoforms) compared to asynchronous cells .

• Mitotic Arrest: Similar or modestly decreased levels of 4E-BP1 binding to eIF4E compared to interphase, with no eIF4E interaction detected with the most highly phosphorylated δ 4E-BP1 isoform .

Importantly, three phosphorylated, lower-molecular-weight 4E-BP1 bands (designated EB-α, -β, and -γ) coimmunoprecipitate with eIF4E, with the less abundant but slowest migrating 4E-BP1 band (EB-γ) being enriched in mitosis-arrested cell extracts .

How does mitotic phosphorylation of 4E-BP1 differ from interphase phosphorylation?

Mitotic phosphorylation of 4E-BP1 exhibits distinctive characteristics compared to interphase phosphorylation:

• Phosphorylation Sites: The eIF4E-unbound δ band of 4E-BP1 in mitotic cells is positive for Ser-83, Thr-37/Thr-46, Ser-65/Ser-101, and Thr-70 phosphorylations .

• Unique Mitotic Bands: The mitotic EB-γ band is positive for Ser-83 and Thr-70 phosphorylations but notably lacks the priming phosphorylations at Thr-37/Thr-46, suggesting an alternative phosphorylation mechanism during mitosis .

• Translation Regulation: Mitotic 5′-terminal oligopyrimidine RNA translation remains active and, unlike interphase translation, is resistant to mTOR inhibition, indicating different regulatory mechanisms .

• eIF4E:eIF4G Interaction: The eIF4E:eIF4G interaction is not inhibited but rather increased in mitotic cells, consistent with active translation initiation during mitosis .

These differences suggest specialized regulation of translation during mitosis that may involve alternative pathways beyond the canonical mTOR signaling.

What are the mTOR-independent mechanisms of 4E-BP1 phosphorylation?

Multiple kinases beyond mTOR have been implicated in 4E-BP1 phosphorylation:

• ATM Kinase: Phosphorylates 4E-BP1 at Ser111 in vitro .

• Insulin Signaling: Induces phosphorylation of 4E-BP1 at Ser112 in vivo .

• GSK3β: Suggested as a substitute for mTOR in phosphorylating 4E-BP1 in cancer cells that have developed resistance to mTOR inhibitors .

• Cell Cycle-Specific Kinases: Evidence suggests specialized kinases may regulate 4E-BP1 during mitosis, as the mitotic EB-γ band shows a distinct phosphorylation pattern lacking the typical priming sites .

This multifactorial regulation suggests that the control of 4E-BP1 is highly dependent on cellular context, with cancer cells potentially adapting by deregulating additional kinases that substitute for mTOR .

How does 4E-BP1 phosphorylation status relate to protein stability and expression levels?

The relationship between 4E-BP1 phosphorylation and protein stability is complex:

• Stabilization Effect: Hyperphosphorylation of 4E-BP1 at multiple sites may play an important role in its stabilization and overexpression in cancer cells .

• Multi-site Phosphorylation Mechanism: Because more than one kinase is involved in the phosphorylation of multiple sites, dephosphorylation of a single site is not sufficient for ubiquitination and degradation. Multi-site phosphorylation could set a higher threshold for susceptibility to degradation .

• Transcriptional Regulation: High 4E-BP1 mRNA levels, independent of phosphorylation status, have been associated with adverse outcomes in breast cancer. Different transcription factors regulate 4E-BP1 accumulation:

  • cMyc binds to the 4E-BP1 gene promoter in prostate cancer

  • ATF4 mediates induction in pancreatic beta-cells under endoplasmic reticulum stress

  • HIF-1α and SMAD4 trigger 4E-BP1 induction in pancreatic cancer cell lines under hypoxia

This interconnection between phosphorylation and expression points to a sophisticated regulatory network that cancer cells may exploit.

What are the optimal protocols for detecting Phospho-EIF4EBP1 (Ser64/65) in different experimental systems?

Researchers should consider the following protocol optimizations:

Western Blotting:

• Recommended dilution: 1:1000 for Cell Signaling antibody #9451

• Alternative dilution range: 1:500-1:3000 for other commercially available antibodies

• Molecular weight detection range: 15-20 kDa

• Buffer conditions: Phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol

Immunoprecipitation:

• Recommended dilution: 1:50

• Sample preparation: Permeabilize samples in PBS containing 0.1% Triton X-100 for 30 min and incubate in blocking buffer for 1 hr at room temperature before applying primary antibody

Immunohistochemistry:

• Recommended dilution: 1:50-1:100

• Controls: Include phospho-defective substitution controls (such as at Ser-83) to validate specificity

How can researchers validate the specificity of Phospho-EIF4EBP1 (Ser64/65) antibodies?

To ensure antibody specificity, researchers should implement these validation strategies:

• Phosphatase Treatment: Treat duplicate samples with lambda phosphatase to confirm that signal loss occurs when phosphorylation is removed.

• Phospho-defective Mutants: Compare antibody reactivity between wild-type and phospho-defective substitution mutants of 4E-BP1 .

• Multiple Antibody Comparison: Use antibodies from different sources or clones that recognize the same phosphorylation site to confirm consistent patterns.

• Correlation with Functional Assays: Combine antibody detection with functional assays such as m⁷GTP cap pulldown to correlate phosphorylation status with eIF4E binding capacity .

• Isoform Analysis: Examine the pattern of multiple phospho-isoforms (such as the α, β, γ, and δ bands) to ensure the expected phosphorylation profile is observed .

What experimental conditions might affect the detection of 4E-BP1 phosphorylation?

Several experimental factors can significantly impact phospho-4E-BP1 detection:

• Cell Synchronization Methods: Different synchronization techniques (such as STLC treatment) may affect phosphorylation patterns .

• Kinase Inhibitors: mTOR inhibitors will affect phosphorylation differently during interphase versus mitosis, with mitotic 5′-terminal oligopyrimidine RNA translation being resistant to mTOR inhibition .

• Sample Preparation Timing: Phosphorylation states can change rapidly; samples should be processed immediately or stored at 4°C to preserve phosphorylation status .

• Permeabilization Conditions: Optimal detection requires proper permeabilization (0.1% Triton X-100 for 30 min) .

• Storage Conditions: Antibodies should be stored at -20°C or -80°C, and repeated freeze/thaw cycles should be avoided .

What is the significance of 4E-BP1 phosphorylation in cancer research?

4E-BP1 phosphorylation has emerged as a critical factor in cancer biology:

• Hyperphosphorylation Patterns: Hyperphosphorylated 4E-BP1 and its overexpression occur simultaneously in various human cancers, suggesting a correlation between phosphorylation status and protein levels .

• Therapeutic Resistance: Cancer cells may adapt by deregulating additional kinases (such as GSK3β) that substitute for mTOR in phosphorylating 4E-BP1, contributing to resistance against mTOR inhibitors .

• Prognostic Value: High 4E-BP1 mRNA levels, independent of phosphorylation status, have been associated with adverse outcomes in breast cancer, particularly in ER-positive subgroups .

• Potential Therapeutic Target: Finding alternative kinases responsible for 4E-BP1 phosphorylation represents an important opportunity for identifying new cancer therapy targets .

How can Phospho-EIF4EBP1 (Ser64/65) antibodies be utilized in studying treatment responses?

These antibodies provide valuable tools for monitoring treatment efficacy:

• mTOR Inhibitor Efficacy: Monitoring changes in Ser64/65 phosphorylation can help assess the effectiveness of mTOR inhibitors, especially identifying cases of resistance where phosphorylation persists despite treatment .

• Cell Cycle-Specific Drug Effects: Since 4E-BP1 phosphorylation changes throughout the cell cycle, these antibodies can help determine if treatments are affecting specific cell cycle phases differently .

• Combination Therapy Assessment: For therapies targeting multiple kinases, these antibodies can help determine which phosphorylation sites remain active, guiding the selection of additional targeted agents .

• Patient Stratification: Phosphorylation patterns could potentially help stratify patients for clinical trials based on their likelihood of responding to translation-targeting therapies.

What are the emerging areas of research regarding 4E-BP1 phosphorylation beyond translation control?

Research is expanding beyond the canonical role of 4E-BP1 in translation regulation:

• Cell Cycle Regulation: Evidence suggests a complex interplay between 4E-BP1 phosphorylation and cell cycle control. Depression of 4E-BP1 by shRNA strategy has been shown to result in incomplete G2 arrest .

• DNA Damage Response: 4E-BP1 has been implicated in the DNA damage response pathway, with potential connections to CHK2 phosphorylation .

• Spatial Regulation: Antibodies against Ser64-phosphorylated EIF4EBP1 have shown localization to the germinal vesicle and cytoplasm, with intense spots also visible within the cytoplasm, suggesting compartment-specific functions .

• Spindle-Associated Translation: cDNA microarray analysis has shown enrichment for maternal mRNAs encoding spindle proteins and other proteins on the mouse oocyte MII spindle, with EIF4EBP1 phosphorylation potentially playing a regulatory role in localized translation .

These emerging areas suggest that 4E-BP1 phosphorylation may have multifunctional roles beyond its established position in the translation initiation pathway.

What technological advances might improve the study of 4E-BP1 phosphorylation states?

Several technological developments show promise for advancing 4E-BP1 research:

• Site-Specific Phospho-Sensors: Development of fluorescent biosensors that can report on specific phosphorylation events in live cells.

• Mass Spectrometry Approaches: Advanced mass spectrometry techniques can provide comprehensive mapping of all phosphorylation sites simultaneously, revealing the interplay between different modifications.

• Single-Cell Analysis: Methods to detect phosphorylation states at the single-cell level would reveal heterogeneity within populations and potentially identify rare cellular states.

• In Situ Proximity Ligation Assays: These could reveal the spatial relationships between phosphorylated 4E-BP1 and its binding partners in intact cells or tissues.

• CRISPR-Based Phosphorylation Site Editing: Precise genome editing to create endogenous phospho-mimetic or phospho-deficient mutations would allow study of specific sites without overexpression artifacts.