Phospho-ATF4 (Ser245) Antibody

What is Phospho-ATF4 (Ser245) and its biological significance?

Phospho-ATF4 (Ser245) represents a post-translationally modified form of Activating Transcription Factor 4 (ATF4), specifically phosphorylated at the serine 245 residue. ATF4 is a member of the cAMP-responsive element-binding protein family of basic zipper-containing proteins, functioning as a transcription factor that regulates genes containing CRE elements .

The phosphorylation at serine 245 is particularly significant as it represents an active form of the protein. Research has demonstrated that RSK2-induced phosphorylation of ATF4 at serine 245 increases the expression of osteocalcin (BGLAP), suggesting a regulatory role in bone development . Additionally, increased expression of Phospho-ATF4 (Ser245) has been associated with cancer development, particularly in non-small cell lung cancer where it correlates with lymph node metastasis and advanced TNM stages .

How does Phospho-ATF4 (Ser245) differ from other phosphorylated forms of ATF4?

ATF4 can be phosphorylated at multiple sites, each with distinct functional outcomes:

Phospho-ATF4 (Ser245) specifically influences gene expression patterns distinct from other phosphorylated forms. While phosphorylation at serine 219 leads to protein degradation and threonine phosphorylation decreases pro-apoptotic gene transcription, serine 245 phosphorylation appears to enhance transcriptional activity for specific target genes . This site-specific phosphorylation contributes to the complex regulation of ATF4's diverse cellular functions.

What are the primary cellular locations of Phospho-ATF4 (Ser245)?

Phospho-ATF4 (Ser245) exhibits both nuclear and cytoplasmic localization patterns. Immunohistochemical and immunofluorescent analyses have confirmed expression in:

• Nucleus of cancer cells and normal lung epithelial cells

• Cytoplasm of cancer cells and normal lung epithelial cells

• Non-small cell lung cancer cell lines including NCI-H157 and LTE cells

The dual localization pattern suggests that Phospho-ATF4 (Ser245) may have functions beyond direct transcriptional regulation. ATF4 generally shuttles between the cytoplasm and nucleus, with heterodimerization with JUN being essential for nuclear localization . Under cellular stress conditions, ATF4 may relocalize to the cytoplasm, and in response to genotoxic stress, it can localize to the mitochondrial outer membrane . This dynamic localization contributes to its multifaceted roles in stress response and cellular homeostasis.

Experimental Applications and Methodologies

When designing experiments to detect Phospho-ATF4 (Ser245) in cancer tissues, researchers should consider several important methodological approaches:

• Tissue preparation and fixation:

  • For immunohistochemistry, use formalin-fixed, paraffin-embedded tissues

  • Maintain consistent fixation times to ensure phospho-epitope preservation

  • Consider antigen retrieval methods optimized for phospho-epitopes

• Antibody selection and validation:

  • Use antibodies specifically recognizing the phosphorylated serine 245 epitope

  • Confirm specificity with appropriate controls, including:

    • Dephosphorylation controls using phosphatases

    • Peptide competition assays with phosphorylated and non-phosphorylated peptides

    • Positive controls from tissues known to express Phospho-ATF4 (Ser245)

• Analytical approaches:

  • Implement tissue microarray techniques for high-throughput analysis

  • Use dual immunofluorescent staining to assess co-localization with other markers

  • Compare expression between tumor tissues and adjacent normal tissues

• Quantitative assessment:

  • Establish clear scoring criteria for positive staining

  • Consider both nuclear and cytoplasmic staining patterns

  • Correlate expression patterns with clinicopathological parameters

This experimental design has proven effective in studies of non-small cell lung cancer, where increased expression of Phospho-ATF4 (Ser245) was associated with lymph node metastasis and advanced TNM stages .

What are the optimal conditions for Western blot detection of Phospho-ATF4 (Ser245)?

For optimal Western blot detection of Phospho-ATF4 (Ser245), researchers should follow these methodological recommendations:

• Sample preparation:

  • Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in lysis buffers

  • Process samples quickly at cold temperatures to preserve phosphorylation status

  • Consider phospho-protein enrichment techniques for low-abundance samples

• Gel electrophoresis parameters:

  • Use 10-12% polyacrylamide gels for optimal resolution

  • Load appropriate positive controls (tissues/cells with known Phospho-ATF4 expression)

  • Target detection of a protein band at approximately 42 kDa

• Antibody incubation:

  • Utilize 1:1000 dilution of primary antibody (may vary by manufacturer)

  • Incubate overnight at 4°C for optimal binding

  • Include 5% BSA rather than milk in blocking/antibody solutions (milk contains phosphatases)

• Signal detection considerations:

  • Use enhanced chemiluminescence with appropriate exposure times

  • Consider phospho-specific positive controls for normalization

  • Include total ATF4 antibody detection on parallel blots for relative quantification

Remember that Phospho-ATF4 (Ser245) antibodies specifically detect endogenous ATF4 only when phosphorylated at serine 245 , so appropriate controls are essential for confirming specificity. The phospho-specific signal can be validated by pre-treating samples with lambda phosphatase, which should eliminate the signal.

How does Phospho-ATF4 (Ser245) status influence the integrated stress response outcomes?

The integrated stress response (ISR) can lead to contradicting cellular outcomes: pro-survival or apoptosis. Phospho-ATF4 (Ser245) may play a key role in determining these divergent pathways:

• Pro-survival functions:

  • RSK2-induced phosphorylation of ATF4 at serine 245 increases expression of osteocalcin (BGLAP) , suggesting a role in promoting specialized cellular functions rather than cell death

  • Phosphorylation states may influence ATF4's ability to upregulate genes involved in amino acid metabolism, redox homeostasis, and protein folding – all supportive of cell survival

• Apoptotic pathway regulation:

  • Phosphorylation status at different sites affects ATF4's transcriptional activity toward pro-apoptotic genes

  • While RET kinase phosphorylation of ATF4 at threonine residues decreases transcription of pro-apoptotic genes NOXA and PUMA , the specific effect of serine 245 phosphorylation on these genes remains to be elucidated

• Integration with other signaling pathways:

  • Serine 245 phosphorylation may influence ATF4's interaction with dimerization partners

  • Different phosphorylation patterns could create distinct binding preferences among potential heterodimerization partners, affecting target gene selection

The interplay between different phosphorylation sites creates a complex regulatory network. The integrated stress response outcome appears to depend on the specific combination of post-translational modifications, dimerization partners, and chromatin architecture surrounding ATF4 target genes . Understanding Phospho-ATF4 (Ser245)'s precise role in this decision-making process requires further research examining its target gene specificity under different stress conditions.

What are the challenges in distinguishing between different phosphorylated forms of ATF4 in complex biological samples?

Researchers face several technical and biological challenges when attempting to distinguish between different phosphorylated forms of ATF4:

• Antibody cross-reactivity:

  • Ensuring absolute specificity for phospho-serine 245 versus other phosphorylated residues

  • Potential cross-reactivity with similar phosphorylated motifs in other proteins

  • Need for rigorous validation through peptide competition assays and phosphatase treatments

• Temporal dynamics of phosphorylation:

  • Phosphorylation states may change rapidly during sample processing

  • Different phosphorylation sites may have distinct temporal dynamics following stress induction

  • Need for rapid sample processing and preservation techniques

• Context-dependent phosphorylation patterns:

  • Different cell types may exhibit distinct ATF4 phosphorylation patterns

  • Stress-specific phosphorylation profiles may complicate interpretation

  • Multiple kinases may target the same residues under different conditions

• Technical detection limitations:

  • Low abundance of specific phosphorylated forms

  • Co-migration of differently modified forms on standard gels

  • Need for phospho-enrichment strategies or high-resolution separation techniques

To overcome these challenges, researchers should consider employing complementary approaches such as phospho-specific antibodies, mass spectrometry-based phosphopeptide mapping, and kinase inhibitor studies to comprehensively characterize ATF4 phosphorylation status in their experimental systems.

How can researchers investigate the kinase-substrate relationship between RSK2 and ATF4 at serine 245?

To investigate the RSK2-ATF4 kinase-substrate relationship at serine 245, researchers should implement a multi-faceted experimental approach:

• In vitro kinase assays:

  • Perform in vitro kinase reactions using purified RSK2 and ATF4 proteins

  • Include [γ-32P]ATP to monitor phosphate incorporation

  • Use point mutants (S245A) as negative controls

  • Analyze phosphorylation by autoradiography and phospho-specific Western blotting

• Cellular manipulation approaches:

  • Utilize RSK2 inhibitors (e.g., BI-D1870) to block phosphorylation

  • Implement RSK2 knockdown/knockout strategies using siRNA or CRISPR-Cas9

  • Overexpress constitutively active RSK2 mutants to enhance phosphorylation

  • Analyze effects on ATF4 serine 245 phosphorylation status

• Phosphorylation site mapping:

  • Employ mass spectrometry to confirm phosphorylation at serine 245

  • Conduct phosphopeptide mapping following in vitro and in vivo phosphorylation

  • Use phospho-mimetic (S245D/E) and phospho-null (S245A) ATF4 mutants to assess functional consequences

• Functional readouts:

  • Measure transcriptional activity of ATF4 using reporter gene assays

  • Assess expression of known ATF4 target genes (e.g., osteocalcin/BGLAP)

  • Evaluate cell proliferation, survival, and differentiation phenotypes

• Contextual analysis:

  • Investigate cell type-specific effects of RSK2-mediated phosphorylation

  • Examine stress-dependent modulation of the RSK2-ATF4 relationship

  • Assess competition or cooperation with other ATF4-targeting kinases

This comprehensive approach would provide mechanistic insights into how RSK2 phosphorylates ATF4 at serine 245 and the functional consequences of this specific modification in various cellular contexts.

What is the correlation between Phospho-ATF4 (Ser245) expression and cancer progression?

Research on Phospho-ATF4 (Ser245) expression in cancer has revealed significant correlations with disease progression, particularly in non-small cell lung cancer (NSCLC):

These findings suggest that Phospho-ATF4 (Ser245) may play a role in cancer progression through several potential mechanisms:

• Influence on cell survival pathways:

  • Phosphorylation may alter ATF4's ability to regulate genes involved in cellular stress responses

  • May contribute to cancer cell survival under stressful tumor microenvironment conditions

• Metastatic potential:

  • Association with lymph node metastasis suggests involvement in invasion and migration

  • May regulate genes involved in epithelial-mesenchymal transition or extracellular matrix remodeling

• Stress adaptation:

  • Could enable adaptation to hypoxia, nutrient deprivation, or other stresses in the tumor microenvironment

  • May influence the integrated stress response toward pro-survival outcomes in cancer cells

These correlations position Phospho-ATF4 (Ser245) as a potential biomarker for cancer progression and suggest it may be a therapeutic target worth exploring further .

How does Phospho-ATF4 (Ser245) regulate gene expression in normal versus disease states?

Phospho-ATF4 (Ser245) appears to differentially regulate gene expression between normal and disease states through several mechanisms:

• Normal physiological functions:

  • In normal cells, RSK2-induced phosphorylation of ATF4 at serine 245 increases expression of osteocalcin (BGLAP)

  • This suggests a role in bone development and homeostasis

  • Low baseline expression in normal lung epithelium and submucosal glands (14.6% positive rate)

• Disease state alterations:

  • Significantly increased expression in non-small cell lung cancer cells (42.7% positive rate)

  • May contribute to altered gene expression profiles supporting cancer development

  • Could shift the balance of ATF4 target genes toward those supporting cell survival and proliferation

• Mechanistic differences:

  • Phosphorylation may alter ATF4's dimerization partner preferences

  • Changes in nuclear localization patterns between normal and cancer cells

  • Potential differences in chromatin accessibility at target genes in disease states

• Target gene selectivity:

  • Different sets of genes may be regulated in normal versus cancer cells

  • In cancer, may preferentially upregulate genes involved in stress adaptation

  • Competitive binding with other transcription factors may be altered in disease contexts

The transition from normal regulation to disease-associated patterns may involve quantitative changes (increased expression levels) as well as qualitative shifts in target gene preferences and transcriptional outcomes. These differences make Phospho-ATF4 (Ser245) an interesting subject for comparative studies between normal and pathological states.

Can Phospho-ATF4 (Ser245) serve as a therapeutic target in cancer and other diseases?

The potential of Phospho-ATF4 (Ser245) as a therapeutic target warrants careful consideration based on current evidence:

• Rationale for targeting:

  • Increased expression correlates with advanced cancer stages and metastasis

  • Specific phosphorylation state may be more prevalent in disease than normal tissues

  • Targeting a specific post-translational modification offers potential for precision

• Potential therapeutic strategies:

  • Inhibitors of RSK2 or other kinases responsible for serine 245 phosphorylation

  • Phosphorylation-specific degraders utilizing proteolysis-targeting chimera (PROTAC) technology

  • Disruption of phosphorylation-dependent protein-protein interactions

  • Blocking nuclear localization of phosphorylated ATF4

• Challenges and considerations:

  • Need to maintain normal ATF4 functions while targeting disease-specific activities

  • Potential for compensatory mechanisms through other phosphorylation sites

  • Complex integration with the broader integrated stress response network

  • Requirement for context-specific targeting to avoid disrupting beneficial stress responses

• Biomarker applications:

  • Phospho-ATF4 (Ser245) could serve as a prognostic biomarker for cancer progression

  • May help identify patients likely to respond to therapies targeting this pathway

  • Could be monitored as a pharmacodynamic marker during treatment

Translating these findings into therapeutic applications will require additional research to understand the specific contexts where targeting Phospho-ATF4 (Ser245) would provide benefit while minimizing potential adverse effects from disrupting normal ATF4 functions.

What high-throughput methods could advance our understanding of Phospho-ATF4 (Ser245) function?

Several cutting-edge high-throughput approaches could significantly advance our understanding of Phospho-ATF4 (Ser245) function:

• Phosphoproteomics:

  • Mass spectrometry-based global phosphoproteome analysis following ATF4 modulation

  • Identification of co-regulated phosphorylation events in the ATF4 signaling network

  • Temporal mapping of phosphorylation dynamics under various stress conditions

• ChIP-seq and Cut&Run:

  • Chromatin immunoprecipitation using Phospho-ATF4 (Ser245) specific antibodies

  • Genome-wide mapping of phosphorylation-dependent binding sites

  • Comparative analysis of binding patterns between phosphorylated and non-phosphorylated ATF4

• Proximity labeling approaches:

  • BioID or TurboID fusion proteins to identify phosphorylation-specific interactors

  • Spatial mapping of Phospho-ATF4 (Ser245) in different cellular compartments

  • Temporal changes in the interactome following stress induction

• Single-cell technologies:

  • Single-cell phospho-flow cytometry to assess heterogeneity in phosphorylation status

  • Single-cell RNA-seq following manipulation of ATF4 phosphorylation

  • Spatial transcriptomics to correlate Phospho-ATF4 (Ser245) with gene expression in tissue context

• CRISPR screens:

  • Genome-wide CRISPR screens using Phospho-ATF4 (Ser245) levels as readout

  • Identification of regulators and downstream effectors

  • Synthetic lethality screens in the context of ATF4 phosphorylation states

These approaches would provide a systems-level understanding of how Phospho-ATF4 (Ser245) functions within complex cellular networks and how its regulation contributes to normal physiology and disease states.

How might contradictory findings regarding Phospho-ATF4 (Ser245) be reconciled across different experimental systems?

Researchers investigating Phospho-ATF4 (Ser245) may encounter contradictory findings across different experimental systems. These discrepancies can be addressed through several methodological approaches:

• Standardization of detection methods:

  • Use of consistent antibody clones and validation protocols

  • Adoption of standardized phospho-protein preservation techniques

  • Implementation of quantitative methods with appropriate normalization

• Context-specific analysis:

  • Explicit characterization of cell type-specific effects

  • Consideration of species differences in ATF4 sequence and regulation

  • Evaluation of the influence of different stress stimuli on phosphorylation outcomes

• Temporal resolution:

  • Analysis of phosphorylation dynamics rather than single time points

  • Consideration of sequential phosphorylation events and their interdependence

  • Investigation of feedback mechanisms affecting phosphorylation status

• Integration with other modifications:

  • Examination of the interplay between serine 245 phosphorylation and other post-translational modifications

  • Analysis of combinatorial effects of multiple phosphorylation sites

  • Investigation of modification crosstalk (phosphorylation influencing ubiquitination, etc.)

• Mechanistic dissection:

  • Generation of phospho-mimetic and phospho-null mutants for functional studies

  • Investigation of upstream signaling diversity leading to serine 245 phosphorylation

  • Analysis of downstream effects with pathway-specific readouts

By implementing these approaches, researchers can develop a more nuanced understanding of Phospho-ATF4 (Ser245) function that accounts for context-dependent variation while identifying conserved core mechanisms.

What are the emerging techniques for studying phosphorylation dynamics of ATF4 in living cells?

Several cutting-edge techniques are emerging for real-time study of ATF4 phosphorylation dynamics in living cells:

• Genetically encoded phosphorylation sensors:

  • FRET-based reporters designed specifically for serine 245 phosphorylation

  • Phosphorylation-dependent conformational changes detected through fluorescence changes

  • Enables real-time visualization of phosphorylation status in living cells

• Protein complementation assays:

  • Split fluorescent protein systems that reassemble upon phosphorylation

  • Phospho-binding domains that recognize phosphorylated serine 245

  • Provides spatial information about phosphorylation events

• Advanced microscopy approaches:

  • Live-cell super-resolution microscopy for subcellular localization

  • Fluorescence correlation spectroscopy to assess phosphorylation-dependent mobility

  • Light-sheet microscopy for 3D visualization of phosphorylation patterns

• Optogenetic manipulation:

  • Photoinducible kinases or phosphatases targeting ATF4

  • Spatiotemporal control of phosphorylation status

  • Assessment of acute functional consequences of phosphorylation changes

• Mass spectrometry of living cells:

  • Nanoscale secondary ion mass spectrometry (NanoSIMS) for elemental analysis

  • Matrix-assisted laser desorption/ionization (MALDI) imaging for spatial mapping

  • Development of cell-permeable mass tags for dynamic phosphoproteomics

These emerging techniques promise to transform our understanding of ATF4 phosphorylation from static snapshots to dynamic processes within living cellular systems, providing unprecedented insights into the temporal and spatial regulation of this critical transcription factor.