Phospho-EIF4E (Ser209) Antibody

What is eIF4E and why is its phosphorylation at Ser209 significant?

eIF4E is the mRNA 5' cap-binding protein crucial for translation initiation. Phosphorylation at Ser209 is mediated primarily by MAPK-interacting protein kinases (MNKs) downstream of the MAPK/ERK and p38 MAPK pathways . This post-translational modification alters eIF4E's functionality in regulating mRNA translation, particularly for specific subsets of mRNAs involved in inflammation, extracellular matrix remodeling, and serotonin pathway regulation . Rather than globally affecting protein synthesis, phosphorylation at Ser209 selectively influences the translation of certain mRNAs, making it a critical regulatory mechanism for specific cellular responses.

Which experimental methods are most reliable for detecting phospho-eIF4E (Ser209)?

Western blotting remains the gold standard for detecting phospho-eIF4E (Ser209) with commercially available antibodies demonstrating high specificity . For optimal results:

• Use phospho-specific antibodies validated with appropriate controls (phosphopeptide competition, phosphatase treatment)

• Include total eIF4E detection to normalize phosphorylation levels

• Consider using recombinant monoclonal antibodies for superior lot-to-lot consistency

For cellular assays, HTRF (Homogeneous Time Resolved Fluorescence) technology offers a plate-based alternative that doesn't require electrophoresis or protein transfer . This method uses two labeled antibodies: one specific to the phosphorylated motif and another recognizing the protein regardless of phosphorylation state. The proximity of these antibodies generates a FRET signal proportional to phospho-eIF4E concentration, enabling quantitative measurement in a no-wash assay format .

How can I validate the specificity of a phospho-eIF4E (Ser209) antibody?

Proper validation should include:

• Peptide competition assays – signal should be blocked only by the phosphopeptide corresponding to eIF4E (Ser209) but not by non-phosphorylated peptides or unrelated phosphopeptides

• Phosphatase treatment – signal should be eliminated following phosphatase treatment

• Genetic controls – using tissues or cells from eIF4E S209A knock-in models as negative controls

• Stimulation experiments – serum starvation followed by serum stimulation should modulate phosphorylation levels

For example, antibody validation data shows that treatment with the phosphopeptide immunogen blocks antibody signal while non-phosphopeptides or generic phosphoserine-containing peptides fail to compete, confirming specificity for the phospho-Ser209 epitope .

Which kinases are responsible for eIF4E phosphorylation at Ser209?

eIF4E is primarily phosphorylated by MAPK-interacting kinases (MNK1 and MNK2), which are activated downstream of ERK1/2 and p38 MAPK pathways . Specifically:

• MNK1 is mainly activated by ERK and p38 MAPK in response to growth factors, mitogens, and stress

• MNK2a appears to be the dominant kinase phosphorylating eIF4E in certain cancers, such as renal cell carcinoma

• Two ERK and p38 MAPK phosphorylation sites in mouse Mnk1 (Thr197 and Thr202) are essential for Mnk1 kinase activity

• Protein kinase C can also phosphorylate eIF4E at Ser209, though this is inhibited by 4E-binding proteins

Interestingly, in mouse models, complete ablation of eIF4E phosphorylation (Ser209A mutation) does not impair viability, indicating this phosphorylation is not essential for basic cellular functions but rather involved in more specialized regulatory processes .

How does phospho-eIF4E (Ser209) affect the mRNA cap binding and translation?

Phosphorylation of eIF4E at Ser209 has complex effects on translation:

• Surprisingly, phosphorylation markedly reduces eIF4E's affinity for capped mRNA , yet it enhances translation of specific mRNAs

• It selectively promotes translation of a subset of mRNAs rather than affecting global protein synthesis

• In neuronal cells, phosphorylation triggers remodeling of the mRNA cap-binding complex, causing release of translational repressors and recruitment of β-catenin to the cap complex

This mechanism creates "translational selectivity" where certain mRNAs (particularly those with complex 5'UTRs) are preferentially translated. For example, research has shown that eIF4E phosphorylation selectively enhances translation of mRNAs encoding:

• SNAIL and MMP-3 (involved in invasion and EMT)

• Wnt pathway components (involved in neuronal plasticity)

• Inflammatory mediators and modulators of the serotonin pathway

What is the relationship between eIF4E phosphorylation and the mTOR pathway?

While eIF4E phosphorylation by MNKs and the mTOR pathway both regulate translation, they represent distinct regulatory mechanisms:

• The mTOR pathway controls eIF4E availability by regulating 4E-BP1 phosphorylation, which determines whether eIF4E can participate in cap-dependent translation

• MNK-mediated eIF4E phosphorylation affects its function within the translation initiation complex

• The HTRF phospho-eIF4E (Ser209) kit can be used as a readout for mTOR pathway activation

What role does phospho-eIF4E (Ser209) play in synaptic plasticity and memory?

Research using eIF4E S209A knock-in mice (where eIF4E cannot be phosphorylated) has revealed:

• These mice are profoundly impaired in dentate gyrus long-term potentiation (LTP) maintenance in vivo, while basal transmission and LTP induction remain intact

• Phosphorylation is required for synaptic activity-induced remodeling of translation initiation complexes, specifically:

  • Removal of translational repressors from eIF4E

  • Recruitment of β-catenin to the eIF4E cap complex

• Ribosome profiling identified selective, phospho-eIF4E-dependent translation of Wnt signaling pathway components during in vivo LTP

Interestingly, while phospho-eIF4E is critical for dentate gyrus LTP maintenance, classical forms of hippocampal LTP and spatial or contextual fear memory appear unaffected in these mice , suggesting region-specific roles in neuroplasticity.

How does phospho-eIF4E (Ser209) contribute to depression and antidepressant responses?

Mice lacking eIF4E phosphorylation (4Eki mice) display:

• Depression and anxiety-like behaviors

• Exaggerated inflammatory responses

• Reduced serotonin levels

• Resistance to the chronic antidepressant effects of fluoxetine (SSRI)

Mechanistically, phospho-eIF4E differentially regulates translation of mRNAs linked to:

• Inflammation

• Extracellular matrix components

• Pituitary hormones

• The serotonin pathway

This suggests a novel translational control mechanism involving the GAIT complex (gamma IFN activated inhibitor of translation) that connects inflammation regulation and depression, which could be exploited for developing new antidepressant approaches .

What evidence links phospho-eIF4E (Ser209) to tumor development and metastasis?

Extensive research demonstrates connections between eIF4E phosphorylation and oncogenesis:

• Mouse embryonic fibroblasts (MEFs) from eIF4E S209A knock-in mice show marked resistance to oncogene-induced transformation

• eIF4E S209A knock-in mice are resistant to PTEN loss-induced prostate cancer development

• eIF4E phosphorylation promotes epithelial-mesenchymal transition (EMT) and metastasis by enhancing translation of specific mRNAs including SNAIL and MMP-3

• Restoration of SNAIL and MMP-3 levels in eIF4E S209A MEFs rescues their invasion capability

How can phospho-eIF4E (Ser209) levels be modulated in experimental models of cancer?

Researchers can modulate phospho-eIF4E levels through several approaches:

• Pharmacological inhibition:

  • MNK inhibitors such as CGP57380 or ETP45835

  • These decrease phospho-eIF4E levels without affecting global protein synthesis

• Genetic approaches:

  • siRNA targeting MNK1/2 to reduce phosphorylation

  • Expression of eIF4E S209A mutant to create phosphorylation-deficient models

  • Overexpression of wild-type eIF4E to restore phosphorylation levels

• Pathway modulation:

  • TGFβ treatment induces EMT and increases phospho-eIF4E levels

  • Serum starvation followed by stimulation modulates phosphorylation

For example, in 786-O and A-498 renal cell carcinoma lines, MNK inhibition decreased phospho-eIF4E and increased vimentin and N-cadherin expression, while MNK2a inhibition via siRNA reduced phospho-eIF4E and enhanced vimentin translation, cell migration, and invasion .

How should phospho-eIF4E (Ser209) levels be quantified in tumor samples for clinical correlation studies?

For clinical studies, standardized assessment of phospho-eIF4E levels is crucial:

• Immunohistochemical (IHC) evaluation:

  • Score both staining area (≤50% = score "-"; >50% = score "1+") and intensity (negative = "-"; moderate = "1+"; strong = "2+")

  • Final scores are obtained by adding area and intensity scores

  • High expression is typically defined as strong positive intensity with >50% staining area (final score = "3+")

• Consider phosphorylation levels in relation to total eIF4E:

  • High eIF4E with low phospho-eIF4E = hypophosphorylation

  • Low eIF4E with high phospho-eIF4E = hyperphosphorylation

  • Low eIF4E with low phospho-eIF4E or high eIF4E with high phospho-eIF4E = intermediate phosphorylation

• For Western blotting quantification:

  • Normalize phospho-eIF4E signal to total eIF4E (not β-actin) to account for variations in eIF4E expression levels

  • Include positive controls (serum-stimulated cells) and negative controls (phosphatase-treated samples)

These standardized approaches enable meaningful correlation with clinical outcomes such as recurrence-free interval in cancer patients .

Why might I observe multiple bands when using phospho-eIF4E (Ser209) antibodies in Western blotting?

Multiple bands in Western blots with phospho-eIF4E antibodies may occur for several reasons:

• Post-translational modifications – In addition to phosphorylation, eIF4E can undergo other modifications that alter migration

• Isoforms – Two bands around 25 and 28 kDa corresponding to eIF4E have been observed across cell lines

• Non-specific binding – Less likely with well-validated antibodies but can occur with suboptimal blocking/washing conditions

• Degradation products – Improper sample handling may result in proteolytic fragments

To address this:

• Include proper controls (phospho-peptide competition)

• Use phospho-specific and total eIF4E antibodies on parallel blots

• Ensure consistent sample preparation and optimal transfer conditions

• Consider using phosphatase treatment as a negative control

What are the optimal cell stimulation conditions to detect phospho-eIF4E (Ser209)?

To maximize phospho-eIF4E detection:

• Serum stimulation protocol:

  • Serum-starve cells overnight (12-16 hours)

  • Stimulate with 10-20% serum for 30 minutes to 2 hours

  • This approach shows clear phosphorylation differences in HeLa cells

• Growth factor/mitogen stimulation:

  • TPA (12-O-tetradecanoylphorbol-13-acetate) activates MNK and induces a twofold increase in eIF4E phosphorylation

  • TGFβ treatment (used for EMT induction) increases phospho-eIF4E levels

• Stress pathway activation:

  • p38 MAPK activators can increase phosphorylation via MNK activation

  • Remember that different cell types may respond differently to stimulation

For HTRF-based phospho-eIF4E detection, a 2-plate protocol is recommended: culture cells in a 96-well plate before lysis, then transfer lysates to a 384-well low volume detection plate before adding HTRF detection reagents (16 μL sample volume) .

How can I determine if eIF4E phosphorylation is regulating translation of my specific mRNA of interest?

To investigate whether phospho-eIF4E regulates translation of a specific mRNA:

• Polysome profiling analysis:

  • Compare mRNA distribution across sucrose density gradients in wild-type vs. eIF4E S209A mutant cells

  • Shift toward lighter polysomes in S209A mutants indicates translational control (as seen with SNAIL and MMP-3 mRNAs)

  • No difference in global polysome profiles indicates specific rather than global translation effects

• Functional rescue experiments:

  • Express your protein of interest from constructs lacking 5' and 3' UTRs in phospho-deficient cells

  • If this restores the phenotype, it suggests translational control

• Ribosome profiling:

  • Provides genome-wide translatome analysis to identify eIF4E phosphorylation-dependent translation

  • Has been used to identify selective, phospho-eIF4E-dependent translation of the Wnt signaling pathway

• Analysis of protein levels compared to mRNA levels:

  • Discrepancies between transcript abundance and protein levels may indicate translational control

  • Western blotting for protein levels combined with qRT-PCR for mRNA levels can reveal such differences