Phospho-EIF4B (Ser422) Antibody
Description
Introduction to Phospho-eIF4B (Ser422) Antibody
Phospho-eIF4B (Ser422) Antibody is a specialized immunological reagent designed to detect eukaryotic translation initiation factor 4B (eIF4B) protein exclusively when phosphorylated at the serine 422 residue. This high-specificity antibody enables researchers to monitor the phosphorylation state of eIF4B at this particular residue, which has been identified as a crucial regulatory site for translation initiation activity .
The antibody functions by recognizing the unique structural conformation created when a phosphate group is attached to the serine 422 amino acid of eIF4B. This specificity makes the antibody particularly valuable for studying signaling pathways involved in translation regulation, especially those connected to the mammalian target of rapamycin (mTOR) and S6 kinase (S6K) activity . The exquisite specificity of this antibody allows researchers to discriminate between phosphorylated and non-phosphorylated forms of eIF4B, providing a powerful tool for investigating the post-translational modifications that regulate protein synthesis.
Physiological Role in Translation Initiation
eIF4B plays a critical role in the translation initiation process by facilitating the binding of mRNA to ribosomes . It functions in close association with other translation factors, particularly eIF4F and eIF4A . eIF4B binds near the 5'-terminal cap of mRNA in the presence of eIF4F and ATP, and promotes the ATPase activity and ATP-dependent RNA unwinding activity of both eIF4A and eIF4F . This unwinding activity is crucial for resolving inhibitory secondary structures in the 5' untranslated region (5'UTR) of eukaryotic mRNAs, allowing for efficient ribosomal scanning and translation initiation .
eIF4B is particularly important for the translation of mRNAs with highly structured 5'UTRs, which often encode proteins involved in proliferation or anti-apoptotic functions. Consequently, eIF4B activity significantly affects cell growth and survival mechanisms .
Regulatory Mechanism and Signaling Pathways
Ser422 represents a major phosphorylation site on eIF4B that responds to serum stimulation and nutrient availability . Research has demonstrated that Ser422 phosphorylation is regulated by the S6 kinases (S6K1 and S6K2), which are downstream effectors of the phosphoinositide-3 kinase (PI3K) and mTOR signaling pathways . This phosphorylation event integrates signals from growth factors, hormones, and nutritional status to modulate translation efficiency.
The phosphorylation status of Ser422 is sensitive to pharmacological inhibitors of PI3K and mTOR, indicating that this modification serves as a convergence point for multiple upstream signaling cascades . The rapamycin sensitivity of Ser422 phosphorylation further confirms the involvement of the mTOR pathway in regulating this site .
Functional Impact on Translation
Phosphorylation of eIF4B at Ser422 enhances its activity in translation initiation. This post-translational modification is essential for the full translational activity of eIF4B, particularly its ability to stimulate the helicase activity of eIF4A . Mutation studies have shown that replacing Ser422 with alanine (preventing phosphorylation) diminishes eIF4B activity in in vitro translation assays .
The strategic location of Ser422 within an RNA-binding region of eIF4B suggests that phosphorylation at this site may directly influence the protein's interaction with mRNA or other components of the translation machinery . This modification therefore represents a critical regulatory switch that can rapidly modulate translational efficiency in response to changing cellular conditions.
Experimental Applications
| Application | Typical Dilution | Key Advantages |
|---|---|---|
| Western Blotting (WB) | 1:500 - 1:1000 | Detects phosphorylation status in cell/tissue lysates |
| Immunoprecipitation (IP) | 1:50 | Isolates phosphorylated eIF4B from complex mixtures |
| Immunofluorescence (IF) | 1:50 | Visualizes subcellular localization of phospho-eIF4B |
| Immunohistochemistry (IHC-P) | 1:50 | Assesses phosphorylation in tissue sections |
| ELISA | As recommended | Quantitative measurement in various sample types |
Phospho-eIF4B (Ser422) antibodies are versatile tools for studying eIF4B phosphorylation across multiple experimental contexts . In Western blotting, they allow researchers to monitor changes in Ser422 phosphorylation in response to various stimuli, inhibitors, or genetic manipulations. Immunocytochemistry and immunohistochemistry applications enable visualization of the spatial distribution of phosphorylated eIF4B within cells and tissues. These antibodies are also compatible with cell-based ELISAs for high-throughput screening of compounds that affect eIF4B phosphorylation .
Phosphorylation Mechanism and Regulation
Research using Phospho-eIF4B (Ser422) antibodies has yielded significant insights into the mechanisms controlling eIF4B phosphorylation. Phosphopeptide mapping and immunoblotting with these antibodies have demonstrated that Ser422 is a major serum-responsive phosphorylation site on eIF4B .
Studies have established that S6K1 and S6K2 are the primary kinases responsible for phosphorylating Ser422 both in vitro and in vivo . Using mutation analysis, researchers found that replacing Ser422 with alanine or aspartic acid eliminated phosphorylation at this site . Interestingly, even conservative substitution of serine with threonine abrogated phosphorylation, indicating highly specific substrate recognition by S6 kinases .
Experiments with rapamycin, an mTOR inhibitor, have shown that Ser422 phosphorylation is significantly diminished when mTOR signaling is inhibited . This finding establishes a direct link between the mTOR pathway and eIF4B activity, positioning eIF4B as an important effector in mTOR-regulated translation control.
Role in Cancer and Disease
False expression and abnormal phosphorylation levels of eIF4B have been implicated in several types of cancer, including breast cancer, cell lymphoblastic leukemia, and diffuse large B-cell lymphoma . Phospho-eIF4B (Ser422) antibodies have been instrumental in establishing these connections by enabling researchers to assess phosphorylation status in various tumor tissues.
The involvement of eIF4B in the translation of mRNAs with highly structured 5'UTRs, which often encode proliferative or anti-apoptotic proteins, suggests that its phosphorylation may contribute to cancer progression by enhancing the synthesis of oncogenic proteins . This makes eIF4B phosphorylation a potential therapeutic target and biomarker for certain types of cancer.
Emerging Applications and Technologies
Phospho-eIF4B (Ser422) antibodies continue to evolve, with newer formats such as recombinant antibodies offering improved consistency and reproducibility compared to traditional polyclonal antibodies . Cell-based ELISA kits incorporating these antibodies provide high-throughput methods for screening compounds that affect eIF4B phosphorylation, potentially accelerating drug discovery efforts targeting the translation machinery .
The development of multiplex assays that simultaneously detect multiple phosphorylation sites on eIF4B and related proteins will enable more comprehensive analysis of translation regulation networks. Integration of phospho-eIF4B detection with advanced imaging techniques, such as super-resolution microscopy, may reveal new insights into the spatial organization of translation initiation complexes within cells.
Product Specs
Target Background
- FASN-induced S6 kinase facilitates USP11-eIF4B complex formation for sustained oncogenic translation in diffuse large B-cell lymphoma. PMID: 29483509
- The synthesis of MCL1, an antiapoptotic protein known to be involved in cancer cell survival during cell division, relies on the function of MELK-elF4B signaling. PMID: 27528663
- Our research shows that muscarinic acetylcholine receptors (mAChRs) modulate eIF4B phosphorylation through the ERK1/2 and PKC signaling pathways in SNU-407 colon cancer cells. PMID: 27773818
- Our findings indicate that eIF4B integrates signals from the Pim and PI3K/Akt/mTOR pathways in Abl-expressing leukemic cells. PMID: 26848623
- MicroRNA-216a inhibits the growth and metastasis of oral squamous cell carcinoma by targeting eIF4B. PMID: 25955794
- S6K1 enhances c-Myc translation efficiency by modulating the phosphorylation of eukaryotic initiation factor eIF4B, which is crucial for unwinding its structured 5' untranslated region. PMID: 25220053
- eIF4B is the RSK downstream effector responsible for elevated Lamgamma2 and MYC protein levels in neoplastic epithelial cells. PMID: 24205356
- The protein level of eukaryotic translation initiation factor 4B (eIF4B) was significantly reduced in A549 cells, as well as in the lung, spleen, and thymus of mice infected with influenza A virus. PMID: 24829357
- eIF4B-driven expression of these key survival proteins is directly correlated with patient outcome, and eIF4B, DAXX, and ERCC5 are identified as novel prognostic markers for poor survival in DLBCL. PMID: 24135829
- This research reports EIF4B binding within the IRES domain V of the coxsackie virus B3 mutant strain. PMID: 24063684
- Overexpression of pim-2 may inhibit the apoptosis of prostate cancer cells through phosphorylation of eIF4B, thus promoting tumorigenesis. PMID: 23813671
- Our results identify eIF4B as a critical substrate of Pim kinases in mediating the activity of Abl oncogenes. PMID: 23749639
- The ORF45/RSK axis-induced eIF4B phosphorylation is involved in translational regulation and is required for optimal KSHV lytic replication. PMID: 21994950
- The solution structure of the eIF4B RNA recognition motif (RRM) domain is reported; the core RRM domain provides only a relatively weak interaction with RNA targets and appears to require extensions at the N- and C-terminus for high-affinity binding. PMID: 12885229
- eIF4B may mediate some of the effects of the ribosomal protein S6 kinases on translation. PMID: 15071500
- Data indicate that eIF4B and 4H stimulate the nuclease activity of herpes simplex virus vhs, and they provide evidence that additional mammalian factors are required for targeting to the encephalomyocarditis virus IRES. PMID: 15078951
- Data show that reduced levels of eIF4B, eIF4H, or polyA-binding protein also trigger stress granule formation. PMID: 19369421
- Phosphorylation of eIF4B on Ser422 by p90 ribosomal protein S6 kinase (RSK) and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3. PMID: 16763566
Q&A
What is the biological significance of eIF4B Ser422 phosphorylation?
eIF4B Ser422 phosphorylation represents a critical regulatory mechanism in the process of translation initiation. This specific modification occurs within an RNA-binding region required for eIF4A helicase-promoting activity . Functionally, phosphorylation at this site enhances eIF4B's ability to stimulate the helicase activity of eIF4A, which is essential for unwinding inhibitory secondary structures in the 5' untranslated region (UTR) of eukaryotic mRNAs .
When eIF4B Ser422 is phosphorylated, it shows increased recruitment to the pre-initiation complex , thereby facilitating the binding of mRNA to ribosomes and enhancing translation efficiency, particularly for mRNAs with structured 5' UTRs that often encode proteins involved in critical cellular processes .
How does eIF4B Ser422 phosphorylation differ from other phosphorylation sites on eIF4B?
eIF4B contains multiple phosphorylation sites, but Ser422 and Ser406 are the most extensively characterized. These sites differ in their regulatory kinases and response patterns:
| Phosphorylation Site | Primary Regulatory Kinases | Response Pattern | Location | Functional Impact |
|---|---|---|---|---|
| Ser422 | S6K1/S6K2, PKB/Akt | Serum-responsive, rapamycin-sensitive | RNA-binding region | Critical for translation activity |
| Ser406 | RSK, p70S6K | Primarily mitogen-regulated | N-terminal region | Less impact on translation activity |
| Ser504 | Casein kinases | Sensitive to metabotropic glutamate receptor activation | - | Influences synaptic localization |
Unlike Ser406, Ser422 phosphorylation shows high sensitivity to rapamycin treatment and amino acid deprivation . Mutation studies have demonstrated that substitution of Ser422 with alanine (creating a non-phosphorylatable mutant) results in significant loss of eIF4B activity in translation assays, while similar mutations of Ser406 have less dramatic effects .
What are the validated applications for Phospho-EIF4B (Ser422) antibodies in research?
Phospho-EIF4B (Ser422) antibodies have been validated for multiple experimental applications:
| Application | Validation Status | Common Protocols | Key Considerations |
|---|---|---|---|
| Western Blot (WB) | Extensively validated | 1:500 dilution typically used | 69-80 kDa band observed |
| Immunohistochemistry (IHC) | Validated | 1:50 dilution recommended | Works on paraffin-embedded tissues |
| Immunofluorescence (IF) | Validated | Standard IF protocols applicable | Can detect endogenous levels |
| ELISA | Validated | Various formats available | Suitable for quantitative analysis |
| Flow Cytometry (FCM) | Partially validated | Requires optimization | Less commonly used |
When using these antibodies for Western blot applications, researchers should be aware that the observed molecular weight is typically 69-80 kDa . For immunohistochemistry applications, optimization of antigen retrieval methods may be necessary depending on the tissue fixation protocol .
How can researchers verify the specificity of Phospho-EIF4B (Ser422) antibodies?
Verifying antibody specificity is crucial for reliable experimental results. Several approaches are recommended:
-
Peptide competition assay: Pre-incubation of the antibody with the phosphorylated peptide should eliminate the signal, while pre-incubation with the corresponding unphosphorylated peptide should not affect signal detection . This was demonstrated in Figure 5A of the Raught et al. study, where the signal detected by Ser422 phosphospecific antiserum was specifically depleted by preincubation with the eIF4B 416–427 phosphopeptide but not by the unphosphorylated peptide .
-
Mutant controls: Testing the antibody against cells expressing Ser422 mutants (Ser422Ala, Ser422Asp, or Ser422Thr) should show no signal, as demonstrated in multiple studies .
-
Phosphatase treatment: Sample treatment with lambda phosphatase should abolish the signal if the antibody is truly phospho-specific.
-
Stimulation experiments: Serum stimulation typically increases Ser422 phosphorylation dramatically, providing a positive control for antibody specificity .
Which signaling pathways regulate eIF4B Ser422 phosphorylation?
eIF4B Ser422 phosphorylation is regulated by multiple interconnected signaling pathways:
| Signaling Pathway | Key Kinases | Activating Stimuli | Inhibitors |
|---|---|---|---|
| PI3K/Akt/mTOR | PKB/Akt, S6K1/S6K2 | Serum, insulin, IL-3 | LY294002, rapamycin |
| MAPK/ERK | p90RSK | Mitogens | U0126 |
| Amino acid sensing | S6K1/S6K2 | Amino acid availability | Leucine deprivation |
| Pim kinase pathway | Pim-1, Pim-2 | Abl transformation | Pim inhibitors |
The PI3K/Akt/mTOR pathway appears to be the dominant regulator of Ser422 phosphorylation. Research shows that treatment with the PI3K inhibitor LY294002 or the mTOR inhibitor rapamycin significantly reduces Ser422 phosphorylation . Additionally, amino acid deprivation, particularly leucine deprivation, leads to a gradual decrease in Ser422 phosphorylation, consistent with the role of amino acid-responsive S6Ks in mediating this modification .
In Abl-transformed cells, Pim kinases and Akt have been identified as convergent regulators of eIF4B Ser422 phosphorylation, highlighting the complexity of this regulatory network .
How does rapamycin treatment affect eIF4B Ser422 phosphorylation?
Rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin), significantly diminishes serum-stimulated phosphorylation of eIF4B Ser422 . This effect occurs through inhibition of S6K1/S6K2 activity, which directly phosphorylates Ser422.
Experimental evidence demonstrates that:
-
Pretreatment with rapamycin significantly reduces serum-stimulated Ser422 phosphorylation, without affecting eIF4B protein expression levels .
-
This reduction in Ser422 phosphorylation correlates with decreased phosphorylation of S6K1 Thr389, which is required for S6K activity .
-
Expression of rapamycin-resistant S6K1 mutants (D3E-E389 or ΔCT104) confers rapamycin resistance upon eIF4B Ser422 phosphorylation, providing direct evidence for S6K-mediated regulation .
These findings collectively indicate that the mTOR-S6K signaling axis is a primary regulator of eIF4B Ser422 phosphorylation in response to serum stimulation.
How does eIF4B Ser422 phosphorylation affect protein translation?
eIF4B Ser422 phosphorylation significantly impacts translation efficiency through several mechanisms:
-
Enhanced RNA binding and helicase activity: Ser422 is located within an RNA-binding region required for promoting eIF4A helicase activity . Phosphorylation at this site enhances the ability of eIF4B to stimulate eIF4A-mediated unwinding of structured mRNA 5' UTRs.
-
Increased recruitment to pre-initiation complex: Studies have shown that Ser422 phosphorylation increases eIF4B recruitment to the translation pre-initiation complex, facilitating mRNA binding to ribosomes .
-
Differential effects on mRNA translation: The stimulation provided by eIF4B is different for different mRNAs, with structured 5' UTRs showing higher dependency on eIF4B activity .
-
Impact on specific protein synthesis: Ser422 phosphorylation modulates the translation of specific proteins such as protein kinase Mζ, which has implications for synaptic plasticity .
Experimental evidence from in vivo translation assays demonstrates that substitution of Ser422 with alanine (creating a non-phosphorylatable mutant) results in significant loss of eIF4B activity in translation inhibition assays, while phosphomimetic substitutions (Ser422Glu) maintain activity similar to the wild-type protein .
Do in vitro and in vivo studies show discrepancies regarding eIF4B Ser422 phosphorylation effects?
There are notable discrepancies between in vitro and in vivo studies regarding the effects of eIF4B Ser422 phosphorylation:
-
Differences in recombinant vs. native protein activity: Native eIF4B purified from HeLa cells shows significantly higher activity in both reconstituted translation initiation assays and complete cell-free translation systems compared to recombinant wild-type or phosphomimetic mutants (S422D, S422E) .
-
Cell-free system limitations: In cell-free translation systems, phosphomimetic mutations (S422D, S422E) do not show enhanced activity compared to wild-type eIF4B, despite their demonstrated effects in living cells .
-
Potential requirement for additional modifications: The effects of S422D and S422E substitutions observed in living cells may require additional protein modifications or may only be manifested in intact cellular environments .
This suggests that while Ser422 phosphorylation is necessary for optimal eIF4B function, it may not be sufficient alone to fully activate the protein, pointing to the complexity of eIF4B regulation in vivo.
How is eIF4B Ser422 phosphorylation implicated in disease states?
eIF4B Ser422 phosphorylation has been implicated in several pathological conditions:
| Disease | Alteration in eIF4B Ser422 Phosphorylation | Proposed Mechanism | Research Implications |
|---|---|---|---|
| Cancer | Increased phosphorylation | Hyperactivation of PI3K/Akt/mTOR pathway | Potential biomarker and therapeutic target |
| Epilepsy | Increased phosphorylation during epileptogenesis | Altered translation driving maladaptive synaptic changes | Link between neuronal activity and translation |
| Leukemia | Elevated levels in Abl transformants | Convergent regulation by Pim and Akt kinases | Critical effector of oncogenic signaling |
In cancer cells, eIF4B is frequently overexpressed and hyperphosphorylated at Ser422, contributing to dysregulated translation of oncogenic proteins . In a rat model of epilepsy, increased Ser422 phosphorylation was observed during epileptogenesis, suggesting a role in the maladaptive synaptic changes that occur during this process .
Understanding the precise role of eIF4B Ser422 phosphorylation in these disease contexts may reveal new therapeutic opportunities targeting translation regulation.
How do intrinsically disordered regions of eIF4B influence its phosphorylation and function?
Recent research has revealed that eIF4B contains a high degree of intrinsic disorder, which significantly impacts its function:
-
Structural flexibility: Due to its high degree of intrinsic disorder, eIF4B is rarely observed in cryo-EM structures of translation complexes, with only its single structured RNA recognition motif (RRM) domain typically visible .
-
Dynamic self-association: The intrinsically disordered region (IDR) of eIF4B orchestrates a transition from monomers to a condensed phase, forming dynamic oligomers before mesoscopic phase separation .
-
Phosphorylation within disordered regions: Both Ser422 and Ser406 are located within intrinsically disordered regions, suggesting that phosphorylation may influence the conformational dynamics of these regions.
-
Environmental sensitivity: The self-association landscape of eIF4B shows sensitivity to ionic strength and molecular crowding, suggesting potential regulation through protein modifications, binding partners, or changes in the cellular environment .
These findings suggest that the intrinsically disordered nature of eIF4B may facilitate its ability to integrate multiple regulatory inputs, including phosphorylation at sites like Ser422, to fine-tune its role in translation initiation.
What methodological advances are improving the study of eIF4B phosphorylation dynamics?
Several methodological advances have enhanced our ability to study eIF4B phosphorylation:
-
Phosphospecific antibodies: Development of highly specific antibodies against phosphorylated Ser422 has enabled precise monitoring of phosphorylation status in response to various stimuli .
-
Colorimetric Cell-Based ELISA: Techniques like the eIF4B (Phospho-Ser422) Colorimetric Cell-Based ELISA provide a convenient, lysate-free, high-throughput method for detecting eIF4B phosphorylation and expression profiles in cells .
-
Single-molecule spectroscopy: Integration of single-molecule spectroscopy with molecular simulations has enabled characterization of conformational ensembles and underlying dynamics of eIF4B across oligomerization transitions .
-
Phosphomimetic and phospho-deficient mutants: Generation of Ser422Ala (phospho-deficient) and Ser422Glu/Asp (phosphomimetic) mutants has facilitated functional studies of this phosphorylation site .
-
Reconstituted translation initiation assays: In vitro systems using purified components allow for mechanistic studies of how eIF4B phosphorylation affects 48S complex formation and translation initiation .
These methodological advances provide researchers with a comprehensive toolkit for investigating the complex regulation and function of eIF4B phosphorylation in translation control.
