Phospho-FOXO4 (Thr451) Antibody

What is FOXO4 and what is the significance of its Thr451 phosphorylation?

FOXO4 (Forkhead box O4) is a transcription factor encoded by the FOXO4 gene located on the X chromosome. It belongs to the O class of forkhead/winged helix family of transcription factors, along with FOXO1, FOXO3, and FOXO6. FOXO4 is abundantly expressed in skeletal muscle and adipose tissue, where it plays crucial roles in insulin signaling, oxidative stress response, and cell cycle regulation. The protein is also known as AFX1 or MLLT7 (Myeloid/Lymphoid or Mixed-Lineage Leukemia Translocated To 7) .

Phosphorylation at Threonine 451 (Thr451) represents a critical post-translational modification that regulates FOXO4's transcriptional activity. Unlike some phosphorylation events that inhibit FOXO4, phosphorylation at Thr451 has been shown to enhance FOXO4 transcriptional activity. This site becomes phosphorylated in response to oxidative stress (e.g., H₂O₂ treatment) through a JNK-dependent mechanism, making it an important marker for stress-activated FOXO4 function .

How does Thr451 phosphorylation differ from other FOXO4 phosphorylation events?

Thr451 phosphorylation fundamentally differs from other FOXO4 phosphorylation events in both its regulatory mechanism and functional outcome. While Akt-mediated phosphorylation of FOXO4 (on sites like Thr28, Ser193, and Ser258) typically promotes cytoplasmic retention through 14-3-3 binding and reduces transcriptional activity, Thr451 phosphorylation enhances FOXO4's transcriptional function .

The Thr451 site, along with the nearby Thr447 site, becomes phosphorylated independently of PKB/Akt activation. These phosphorylation events occur through a distinct pathway involving the small GTPase Ral and JNK, particularly in response to oxidative stress conditions. Mutational analysis has revealed that both sites are critical, as mutation of either Thr447 or Thr451 to alanine almost completely blocks transcriptional activity of FOXO4, while phospho-mimicking mutations (T447E or T451E) enhance its activity .

What is the molecular pathway leading to FOXO4 Thr451 phosphorylation?

The molecular pathway leading to FOXO4 Thr451 phosphorylation involves a stress-responsive signaling cascade. When cells are exposed to oxidative stress (even at concentrations as low as 20 μM H₂O₂), the small GTPase Ral is rapidly activated. Activated Ral is both necessary and sufficient to induce phosphorylation of Thr451 on FOXO4, as demonstrated by experiments with dominant-negative Ral (RalN28) that completely blocks this phosphorylation .

Downstream of Ral activation, c-Jun N-terminal kinase (JNK) serves as the direct kinase responsible for phosphorylating Thr451. This has been conclusively demonstrated through multiple lines of evidence: JNK1,2-/- MEFs fail to induce Thr451 phosphorylation in response to H₂O₂ treatment; reintroduction of either JNK1 or JNK3 restores this phosphorylation; JNK physically binds to FOXO4 upon oxidative stress; and active JNK1 can directly phosphorylate Thr451 in vitro .

What are the optimal conditions for detecting FOXO4 Thr451 phosphorylation in cell culture experiments?

For optimal detection of FOXO4 Thr451 phosphorylation in cell culture experiments, researchers should consider several critical parameters. Based on published research, H₂O₂ treatment at concentrations as low as 20 μM is sufficient to induce phosphorylation of FOXO4 at Thr451 . This concentration is particularly significant because it induces FOXO4 phosphorylation without activating the PKB/Akt pathway (which occurs at higher concentrations, around 200 μM).

The recommended experimental protocol includes:

• Culture cells to 70-80% confluence

• Serum-starve cells for 4-6 hours to reduce baseline phosphorylation

• Treat cells with 20-50 μM H₂O₂ for 15-30 minutes

• Immediately lyse cells in appropriate buffer containing phosphatase inhibitors

• Proceed with Western blot analysis using the Phospho-FOXO4 (Thr451) antibody at a dilution of 1:500-1:1000

For endogenous FOXO4 detection, mouse C2C12 cells provide a suitable model as they express detectable levels of FOXO4, though researchers should note that mouse FOXO4 differs from human FOXO4 at the Thr451 position .

How can I validate the specificity of Phospho-FOXO4 (Thr451) Antibody in my experimental system?

Validating antibody specificity is crucial for reliable research outcomes. For Phospho-FOXO4 (Thr451) Antibody, several validation approaches are recommended:

• Peptide competition assay: Pre-incubate the antibody with the immunizing phosphopeptide (sequence around L-G-T(p)-P-V). This should abolish specific signal in Western blots, as demonstrated in validation experiments .

• Phosphatase treatment control: Treat half of your protein lysate with lambda phosphatase before immunoblotting. The phospho-specific signal should disappear in the phosphatase-treated samples.

• Mutant expression: Express wild-type FOXO4 alongside a T451A mutant. The antibody should detect the wild-type protein after appropriate stimulation but not the T451A mutant .

• Positive control: Include lysates from cells treated with EGF, which has been documented to induce Thr451 phosphorylation, as shown in validation Western blots .

• Kinase inhibitor control: Pre-treat cells with a JNK inhibitor before stimulation, which should prevent Thr451 phosphorylation, confirming the specificity of the signal and the pathway involved .

What are the recommended applications and experimental parameters for Phospho-FOXO4 (Thr451) Antibody?

The Phospho-FOXO4 (Thr451) Antibody has been validated for several applications with specific recommended parameters:

Western Blotting (WB):

• Recommended dilution: 1:500-1:1000

• Expected molecular weight: 55 kDa and 70 kDa (endogenous protein)

• Sample preparation: Use RIPA buffer supplemented with protease and phosphatase inhibitors

• Loading control: Total FOXO4 on a separate blot or after stripping

Enzyme-Linked Immunosorbent Assay (ELISA):

• Recommended dilution: 1:2000-1:10000

• Coating antigen: Phospho-peptide around the Thr451 site (L-G-T(p)-P-V)

The antibody shows reactivity to both human and mouse samples, though it's important to note that the Thr451 site is not conserved between human and mouse FOXO4, so experimental design must account for species-specific differences .

How should I interpret changes in FOXO4 Thr451 phosphorylation in the context of cellular stress responses?

Interpreting changes in FOXO4 Thr451 phosphorylation requires careful consideration of the cellular context and stress conditions. Increased phosphorylation at Thr451 generally indicates activation of the stress-responsive JNK pathway and suggests enhanced transcriptional activity of FOXO4 .

When analyzing oxidative stress responses, consider these interpretation guidelines:

• Rapid, transient phosphorylation: Typically occurs within 15-30 minutes of H₂O₂ exposure, reflecting immediate stress response

• Dose-dependent effects: Low doses (20 μM H₂O₂) induce Thr451 phosphorylation without PKB/Akt activation, while higher doses (200 μM) activate multiple pathways

• Correlation with transcriptional activity: Increased Thr451 phosphorylation should correlate with enhanced FOXO4-mediated transcription of target genes

• Relationship to cellular protection: Enhanced Thr451 phosphorylation typically associates with increased cellular protection against stressors like glucose deprivation

For comprehensive interpretation, always compare Thr451 phosphorylation with total FOXO4 levels and examine the activation status of upstream regulators (Ral, JNK) and downstream targets.

What are the known downstream effects of FOXO4 Thr451 phosphorylation?

The phosphorylation of FOXO4 at Thr451 has several documented downstream effects that impact cellular physiology:

• Enhanced transcriptional activity: Phosphorylation at Thr451 significantly increases FOXO4's ability to activate transcription, as demonstrated by reporter gene assays. This enhanced activity is completely blocked by dominant-negative Ral expression, but phospho-mimicking mutants (T451E) remain active even in the presence of RalN28 .

• Target gene expression: Activated FOXO4 regulates multiple target genes, including:

  • IGFBP1 (Insulin-like growth factor-binding protein 1)

  • HIF1A (Hypoxia-inducible factor 1-alpha) - FOXO4 downregulates HIF1A expression and suppresses hypoxia-induced transcriptional activation

  • PSMD11 - Activated in embryonic stem cells, enhancing 26S proteasome assembly and activity

• Cellular protection: FOXO4 with phosphorylated Thr451 provides enhanced protection against glucose deprivation-induced mitochondrial membrane instability. The T447/451A mutant shows reduced protection, while the phospho-mimicking T447/451D mutant displays slightly enhanced protection .

• Cell cycle regulation: Activated FOXO4 contributes to negative regulation of the cell cycle, potentially mediating stress-induced cell cycle arrest .

How does FOXO4 Thr451 phosphorylation differ across cell types and experimental conditions?

FOXO4 Thr451 phosphorylation exhibits notable variations across different cell types and experimental conditions, which researchers must consider when designing experiments and interpreting results:

Cell Type Variations:

• FOXO4 is abundantly expressed in skeletal muscle and adipose tissue, making these tissues particularly suitable for studying endogenous Thr451 phosphorylation

• Mouse C2C12 myoblast cells express detectable levels of endogenous FOXO4, though the Thr451 site is not conserved between human and mouse FOXO4

• HUVEC (Human Umbilical Vein Endothelial Cells) have been used successfully to study EGF-induced FOXO4 phosphorylation

Experimental Condition Variations:

• Oxidative stress: H₂O₂ treatment (20-200 μM) induces dose-dependent phosphorylation

• Growth factor stimulation: EGF treatment induces Thr451 phosphorylation through a pathway that may differ from oxidative stress

• Ral activation: Expression of active Ras (RasV12) or active Ral guanine nucleotide exchange factors (RlfCAAX and RalGEF2) increases Thr451 phosphorylation independent of stress stimulation

Phosphorylation kinetics also vary, with oxidative stress typically inducing more rapid phosphorylation compared to growth factor stimulation. These variations highlight the importance of establishing cell type-specific baseline and positive controls.

How can I use Phospho-FOXO4 (Thr451) Antibody to investigate cross-talk between oxidative stress and insulin signaling pathways?

Investigating the cross-talk between oxidative stress and insulin signaling pathways using Phospho-FOXO4 (Thr451) Antibody requires a strategic experimental approach:

• Dual stimulation experiments: Treat cells with insulin/IGF-1 followed by H₂O₂ (or vice versa) and monitor:

  • Thr451 phosphorylation (JNK pathway)

  • Canonical FOXO4 phosphorylation sites (Thr28, Ser193, Ser258) (Akt pathway)

  • Subcellular localization of FOXO4

  • Transcriptional activity using reporter assays

• Inhibitor studies: Use specific inhibitors to dissect pathway interactions:

  • JNK inhibitors to block stress-induced phosphorylation

  • PI3K/Akt inhibitors (e.g., LY294002) to block insulin signaling

  • Analyze how inhibiting one pathway affects the other's response

• Ral activation analysis: Since Ral is involved in both insulin signaling and oxidative stress response, measure Ral-GTP levels under various conditions:

  • Insulin alone

  • H₂O₂ alone

  • Combined stimulation

• Transcriptional output profiling: Compare transcriptional targets regulated by:

  • Akt-mediated FOXO4 inhibition

  • JNK-mediated FOXO4 activation through Thr451 phosphorylation

  • Dual pathway activation

This approach can reveal how cells integrate conflicting signals (insulin-mediated inhibition vs. stress-mediated activation) to fine-tune FOXO4 activity and downstream physiological responses.

What approaches can I use to study the temporal dynamics of FOXO4 Thr451 phosphorylation in live cells?

Studying the temporal dynamics of FOXO4 Thr451 phosphorylation in live cells presents technical challenges but can be approached through several advanced methods:

• Phospho-specific FRET sensors:

  • Design FRET-based biosensors incorporating the FOXO4 region around Thr451

  • The sensor would change conformation upon phosphorylation, altering FRET efficiency

  • This allows real-time visualization of phosphorylation events in living cells

• Phospho-specific nanobodies:

  • Develop fluorescently labeled nanobodies that specifically recognize phosphorylated Thr451

  • Express these in cells alongside fluorescently tagged FOXO4

  • Monitor binding events in real-time using advanced microscopy

• Time-course immunofluorescence:

  • While not truly "live," fix cells at short time intervals after stimulation

  • Perform immunofluorescence with Phospho-FOXO4 (Thr451) Antibody

  • Use high-content imaging to quantify phosphorylation dynamics across many cells

• Split-luciferase complementation assays:

  • Engineer a system where luciferase fragments reconstitute when phospho-binding domains recognize phosphorylated Thr451

  • This allows semi-quantitative monitoring of phosphorylation events

When designing these experiments, researchers should consider that H₂O₂ treatment induces rapid Ral activation within minutes, followed by JNK-mediated phosphorylation of FOXO4 at Thr451 . The temporal resolution of the chosen method should be sufficient to capture these rapid signaling events.

How can I investigate the relationship between FOXO4 Thr451 phosphorylation and transcriptional target specificity?

Investigating how Thr451 phosphorylation influences FOXO4's transcriptional target specificity requires approaches that connect phosphorylation status with genome-wide binding and transcriptional activation:

• Phosphorylation-specific ChIP-seq:

  • Perform chromatin immunoprecipitation using Phospho-FOXO4 (Thr451) Antibody

  • Compare binding sites with those identified using total FOXO4 antibodies

  • Identify genomic regions specifically bound by phosphorylated FOXO4

• Phospho-mimetic vs. phospho-deficient mutant comparisons:

  • Express wild-type FOXO4, T451A (phospho-deficient), and T451E (phospho-mimetic) variants

  • Perform RNA-seq to compare transcriptional profiles

  • Use ChIP-seq to compare genomic binding patterns

  • This approach can identify genes specifically regulated by Thr451 phosphorylation status

• Integrative analysis with co-factor binding:

  • Since phosphorylation may alter co-factor recruitment, perform sequential ChIP (ChIP-reChIP)

  • First immunoprecipitate with Phospho-FOXO4 (Thr451) Antibody

  • Follow with antibodies against potential co-factors

  • This identifies co-factor associations specific to phosphorylated FOXO4

• Promoter-specific reporter assays:

  • Create luciferase reporters with various FOXO4 target promoters

  • Test activation by wild-type vs. phospho-mutant FOXO4 variants

  • This can reveal target genes particularly sensitive to Thr451 phosphorylation

Previous research has shown that FOXO4 phosphorylated at Thr451 regulates genes involved in oxidative stress response and can enhance cellular protection against glucose deprivation . The approaches outlined above would expand our understanding of the complete transcriptional program regulated by this specific phosphorylation event.

What are common technical issues when detecting FOXO4 Thr451 phosphorylation by Western blot?

When detecting FOXO4 Thr451 phosphorylation by Western blot, researchers commonly encounter several technical challenges:

• High background signal:

  • Possible causes: Insufficient blocking, too high antibody concentration, cross-reactivity

  • Solution: Optimize blocking conditions (5% BSA often works better than milk for phospho-specific antibodies), titrate antibody concentration (start with 1:1000 dilution), and include proper controls

• Weak or absent phospho-signal:

  • Possible causes: Phosphatase activity during sample preparation, insufficient stimulation, low endogenous FOXO4 expression

  • Solution: Use fresh phosphatase inhibitors in lysis buffer, optimize stimulation conditions (20-50 μM H₂O₂ for 15-30 minutes), consider transfecting cells with FOXO4 if endogenous levels are low

• Multiple bands or unexpected molecular weight:

  • Possible causes: Post-translational modifications, proteolytic cleavage, isoforms

  • Solution: The antibody should detect endogenous protein at molecular weights of 55 and 70 kDa . Confirm specificity with peptide competition or T451A mutant expression

• Poor reproducibility:

  • Possible causes: Variability in stimulation conditions, cell density, passage number

  • Solution: Standardize experimental protocols, use low-passage cells, include positive controls (HUVEC cells treated with EGF)

• Cross-reactivity with other FOXO family members:

  • Possible causes: Sequence similarity between FOXO proteins

  • Solution: Validate specificity with FOXO4 knockdown/knockout controls, as the T451 region is not highly conserved among FOXO family members

How can I optimize immunofluorescence protocols for detecting phosphorylated FOXO4 in tissue sections?

Optimizing immunofluorescence protocols for detecting phosphorylated FOXO4 in tissue sections requires careful attention to several critical parameters:

• Tissue fixation and antigen retrieval:

  • Use fresh tissue samples and fix immediately in 4% paraformaldehyde (10-12 hours)

  • Test multiple antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0 is often effective for phospho-epitopes)

  • For phospho-epitopes, sodium orthovanadate (1mM) in the retrieval buffer helps preserve phosphorylation

• Blocking and antibody incubation:

  • Block with 5-10% normal serum from the secondary antibody host species plus 0.1-0.3% Triton X-100

  • For phospho-specific staining, add 1mM sodium orthovanadate to blocking solution

  • Use Phospho-FOXO4 (Thr451) Antibody at 1:100-1:200 dilution

  • Extend primary antibody incubation to overnight at 4°C

• Signal amplification and detection:

  • Consider using tyramide signal amplification for low-abundance phospho-epitopes

  • Test fluorophore-conjugated secondary antibodies with different brightness profiles

  • Include DAPI counterstain to visualize nuclei (important for evaluating FOXO4 localization)

• Validation controls:

  • Adjacent sections stained with antibody pre-absorbed with phospho-peptide

  • Sections from tissues known to express FOXO4 (skeletal muscle, adipose tissue)

  • If possible, tissue from models with JNK pathway activation/inhibition

• Co-staining recommendations:

  • Co-stain with total FOXO4 antibody (using antibodies raised in different species)

  • Consider co-staining with phospho-JNK to correlate with upstream pathway activation

  • Tissue-specific markers to identify FOXO4-expressing cell types

What controls should I include when studying the functional significance of FOXO4 Thr451 phosphorylation?

When studying the functional significance of FOXO4 Thr451 phosphorylation, a comprehensive set of controls is essential for robust and interpretable results:

• Phosphorylation site mutants:

  • T451A (phospho-deficient): Should show reduced transcriptional activity and cellular protection

  • T451E (phospho-mimetic): Should show enhanced transcriptional activity even without stimulation

  • T447/451A double mutant: Should show more complete loss of function

  • T447/451E double mutant: Should show enhanced activity resistant to RalN28 inhibition

• Pathway manipulation controls:

  • Dominant-negative Ral (RalN28): Should block H₂O₂-induced Thr451 phosphorylation

  • Constitutively active Ral or RalGEF: Should enhance Thr451 phosphorylation even without stimulation

  • JNK inhibition (chemical inhibitors or JNK1,2-/- cells): Should prevent Thr451 phosphorylation

  • JNK rescue experiments: Reintroduction of JNK1 or JNK3 should restore phosphorylation capacity

• Functional readout controls:

  • Transcriptional activity: Compare luciferase reporter assays with endogenous target gene expression

  • Cellular protection assays: Include positive controls (e.g., antioxidants) and negative controls

  • For glucose deprivation experiments: Include both wild-type and phospho-mutant FOXO4 variants

• Specificity controls:

  • FOXO4 knockdown/knockout: Confirms signal specificity

  • Other FOXO family members: Test whether effects are FOXO4-specific or general to the FOXO family

  • Phospho-specific vs. total FOXO4: Always compare phosphorylation with total protein levels

These controls collectively ensure that any observed functional effects can be specifically attributed to Thr451 phosphorylation rather than other variables in the experimental system.

How does FOXO4 Thr451 phosphorylation compare to regulatory mechanisms of other FOXO family members?

FOXO4 Thr451 phosphorylation represents a distinct regulatory mechanism within the broader context of FOXO family regulation:

While the Thr451 site is not conserved across all FOXO members, functional equivalency exists. As noted in search result , "We are currently investigating whether JNK can induce a phosphorylation-dependent activation of FOXO1 and FOXO3a upon treatment with H₂O₂, and which of the phosphorylation sites are involved, and thus are functionally equivalent to T447/451."

This comparative analysis reveals a common theme: FOXO transcription factors integrate multiple phosphorylation events to determine their subcellular localization, stability, and transcriptional activity. Stress-activated kinases like JNK generally promote FOXO activity, while growth factor-activated kinases like Akt typically inhibit FOXO function .

What is the evolutionary conservation of the FOXO4 Thr451 phosphorylation site across species?

The evolutionary conservation of the FOXO4 Thr451 phosphorylation site shows interesting patterns across species:

This pattern of conservation suggests that while the specific Thr451 site may not be conserved across all species, the functional mechanism of stress-activated, JNK-mediated phosphorylation of FOXO transcription factors appears to be evolutionarily conserved. As noted in the search results, the regulation of FOXO4 via Thr451 phosphorylation "bears striking similarities to the proposed role and regulation of DAF-16," the C. elegans homolog of mammalian FOXO transcription factors .

The lack of conservation of the specific Thr451 site between human and mouse FOXO4 has important implications for research, as it suggests that mouse models may not perfectly recapitulate human FOXO4 regulation through this specific phosphorylation event. Researchers should be cautious when extrapolating findings between species and consider using humanized mouse models or human cell lines when specifically studying Thr451 phosphorylation.

How does the JNK-FOXO4 pathway integrate with other stress response mechanisms?

The JNK-FOXO4 pathway represents one component of an integrated cellular stress response network that coordinates adaptive responses to various environmental challenges:

• Cross-talk with other stress-responsive pathways:

  • p38 MAPK pathway: While p38α cannot phosphorylate FOXO4 at Thr451 in vitro , both JNK and p38 are activated by similar stressors and may coordinate complementary responses

  • NRF2 pathway: Both FOXO4 and NRF2 are activated by oxidative stress and regulate overlapping sets of antioxidant genes

  • Heat shock response: Heat shock factors (HSFs) and FOXOs can cooperatively regulate stress response genes

• Integration with metabolic sensing:

  • AMPK pathway: Energy stress activates AMPK, which can directly phosphorylate FOXOs

  • mTOR signaling: Nutrient availability signals through mTOR, which inhibits FOXO activity

  • Insulin/IGF signaling: Counteracts stress-activated FOXO function through Akt-mediated inhibitory phosphorylation

• Temporal coordination:

  • Immediate response: JNK activation and FOXO4 Thr451 phosphorylation occur rapidly (within minutes)

  • Sustained response: Transcriptional changes induced by activated FOXO4 persist longer

  • Adaptation phase: Eventually, feedback mechanisms reset the system to baseline

• Functional outcomes:

  • Antioxidant defense: Upregulation of enzymes that detoxify reactive oxygen species

  • Cell cycle regulation: Stress-induced cell cycle arrest to prevent propagation of damaged cells

  • Metabolic adaptation: Shift to catabolic metabolism during stress

  • Protection against specific stressors: For example, FOXO4 provides protection against glucose deprivation-induced mitochondrial damage

The integration of these pathways allows cells to mount appropriate stress responses tailored to specific environmental challenges, with FOXO4 Thr451 phosphorylation serving as a critical node in this complex network.

What are the key considerations for researchers planning to use Phospho-FOXO4 (Thr451) Antibody in their studies?

Researchers planning to use Phospho-FOXO4 (Thr451) Antibody should consider several key factors to ensure successful experimental outcomes:

• Antibody validation: Thoroughly validate the antibody in your specific experimental system using controls like phosphatase treatment, peptide competition, and phospho-deficient mutants (T451A) .

• Species considerations: Be aware that the Thr451 site is not conserved between human and mouse FOXO4, which may affect experimental design and interpretation when working with different species .

• Stimulation conditions: Optimize stimulation parameters for inducing Thr451 phosphorylation, with H₂O₂ at 20-50 μM being effective while avoiding activation of other pathways that occur at higher concentrations .

• Sample preparation: Include phosphatase inhibitors in all buffers to preserve phosphorylation status, and process samples quickly to prevent dephosphorylation.

• Appropriate controls: Always include positive controls (H₂O₂ or EGF-treated samples), negative controls, and compare phospho-FOXO4 levels to total FOXO4 expression .

• Context interpretation: Interpret Thr451 phosphorylation in the context of the full signaling network, including Ral activation, JNK activation, and functional outcomes like transcriptional activity and cellular protection .

• Application-specific optimization: Different applications (Western blot, ELISA, immunofluorescence) require specific optimization of antibody concentration and protocol parameters .

By carefully addressing these considerations, researchers can generate reliable and meaningful data on FOXO4 Thr451 phosphorylation in their specific research contexts.

What future research directions might further elucidate the role of FOXO4 Thr451 phosphorylation in cellular physiology and disease?

Several promising research directions could significantly advance our understanding of FOXO4 Thr451 phosphorylation's role in cellular physiology and disease:

• System-wide phospho-proteomics: Comprehensive mapping of the phosphorylation state of FOXO4 and its interacting partners under various stress conditions would provide a more complete picture of how Thr451 phosphorylation integrates with other signaling events.

• Single-cell analysis: Investigating cell-to-cell variability in FOXO4 Thr451 phosphorylation responses could reveal how individual cells within a population differentially respond to stress signals.

• In vivo models: Developing knock-in mouse models with humanized FOXO4 containing the Thr451 site would allow investigation of this phosphorylation event in whole-organism physiology and disease models.

• Disease relevance: Exploring the status of FOXO4 Thr451 phosphorylation in various disease contexts, particularly:

  • Diabetes and metabolic disorders (given FOXO4's role in insulin signaling)

  • Cancer (given FOXO4's role in cell cycle regulation)

  • Neurodegenerative diseases (where oxidative stress plays a significant role)

  • Aging-related conditions (as FOXO transcription factors are implicated in longevity)

• Therapeutic targeting: Developing compounds that specifically modulate the JNK-FOXO4 pathway could provide novel therapeutic approaches for diseases characterized by dysregulated stress responses or FOXO4 activity.

• Mechanistic studies: Further investigation into how Thr451 phosphorylation alters FOXO4's interaction with DNA, co-factors, and the transcriptional machinery would deepen our understanding of the molecular mechanisms involved.