Phospho-JUN (S63) Recombinant Monoclonal Antibody

What is the biological significance of c-JUN phosphorylation at Ser63?

Phosphorylation of c-JUN at Ser63 represents a critical post-translational modification that directly regulates its transcriptional activity. This phosphorylation occurs in the NH2-terminal transactivation domain and is essential for c-JUN's function as a major component of the Activator Protein 1 (AP-1) complex. When phosphorylated at Ser63, c-JUN can effectively dimerize and bind to target DNA sequences, thereby influencing gene transcription. This phosphorylation event plays a crucial regulatory role in multiple cellular processes including proliferation, differentiation, malignant transformation, and apoptosis . The phosphorylation state of c-JUN at Ser63 serves as an important biomarker for JNK pathway activation and cellular stress response mechanisms.

How does c-JUN Ser63 phosphorylation differ kinetically from other c-JUN phosphorylation sites?

c-JUN phosphorylation by JNK exhibits a defined temporal order with distinctive kinetics. Research demonstrates that Ser63 phosphorylation occurs most rapidly, followed by Ser73, with threonine sites (T91 and T93) being phosphorylated significantly slower. In vitro and in vivo studies reveal the following phosphorylation order: S63 > S73 > T91 ≈ T93 . This temporal pattern has been confirmed through time-resolved NMR studies and immunoblotting with phosphorylation-specific antibodies.

Under physiological conditions with anisomycin stimulation, S63 and S73 phosphorylation occurs with half-times of approximately 12 minutes, while T91 and T93 phosphorylation proceeds more slowly with half-times of 15-17 minutes . These distinct kinetics have important implications for the temporal regulation of c-JUN activity and downstream signaling events.

What is the molecular structure of c-JUN and how does phosphorylation affect it?

c-JUN (36 kDa, 331 amino acids) belongs to the bZIP family and Jun subfamily of transcription factors. The transactivation domain (TAD) containing the phosphorylation sites is intrinsically disordered, as evidenced by 2D 1H, 15N correlation spectroscopy showing narrow chemical shift dispersion .

The protein contains four major phosphorylation sites (S63, S73, T91, T93) and a MAPK binding motif (D-motif, residues 32-50) that controls phosphorylation of these sites. Upon phosphorylation, particularly at Ser63 and Ser73, c-JUN undergoes conformational changes that facilitate dimerization and DNA binding, activating its transcriptional functions. Secondary chemical shift analyses show no significant populations of secondary structure in the unphosphorylated state, consistent with the protein's intrinsically disordered nature .

What are the optimal methods for detecting phosphorylated c-JUN at Ser63 in cell-based assays?

Several methodologies can be employed for detecting phosphorylated c-JUN at Ser63, each with distinct advantages:

• HTRF (Homogeneous Time-Resolved Fluorescence) Assay:

  • Offers a streamlined mix-and-read no-wash protocol

  • Demonstrated 8-fold higher sensitivity compared to Western blot

  • Can be performed in single-plate or two-plate formats

  • Requires only 16 μL sample volume

  • Enables quantitative detection directly in cell lysates without electrophoresis or transfer

• Western Blot:

  • Recommended antibody dilutions: 1:500-1:5000

  • Allows visualization of protein size (observed at 40-48 kDa)

  • Useful for comparing relative phosphorylation levels

• Immunohistochemistry (IHC):

  • Recommended antibody dilutions: 1:50-1:200

  • Enables in situ detection in tissue sections

  • Valuable for spatial distribution analysis

• Immunofluorescence (IF):

  • Recommended antibody dilutions: 1:20-1:200

  • Allows subcellular localization studies

  • Can be combined with other markers for co-localization analysis

For high-throughput screening applications, the HTRF assay provides significant advantages in terms of sensitivity, throughput, and reduced sample requirements. The choice of method should be determined by the specific research question, required sensitivity, and available instrumentation.

What is the recommended protocol for stimulating c-JUN phosphorylation at Ser63 in cell culture experiments?

Based on established research protocols, the following methodology is recommended for stimulating c-JUN phosphorylation at Ser63 in cell culture:

• Cell Selection and Culture:

  • HEK293, NIH-3T3, or other relevant cell lines can be used

  • Culture cells to 70-80% confluence (approximately 50,000 cells/well in a 96-well format)

• Stimulation Protocol:

  • Anisomycin treatment (500nM) is highly effective for inducing c-JUN phosphorylation

  • Optimal stimulation time is 30 minutes at 37°C

  • JNK activation typically begins after a 5-minute lag period and reaches maximum activity within 12 minutes

• Cell Lysis and Detection:

  • After stimulation, remove culture medium and add appropriate lysis buffer

  • Incubate for 30 minutes to ensure complete lysis

  • Collect supernatants following centrifugation

  • Proceed with detection using HTRF, Western blot, or other methods

This protocol has been validated to produce robust and reproducible c-JUN phosphorylation that can be readily detected using phospho-specific antibodies. The stimulation conditions can be adjusted based on the specific cell type and experimental requirements.

How can I validate the specificity of phospho-JUN (S63) antibody in my experimental system?

Validation of phospho-JUN (S63) antibody specificity is crucial for ensuring reliable research outcomes. A comprehensive validation approach includes:

• Alanine Substitution Mutants:

  • Generate S63A mutants as negative controls

  • Compare antibody reactivity between wild-type and mutant samples

  • This approach has been successfully used to verify the site specificity of phospho-antibodies

• Peptide Competition Assay:

  • Pre-incubate antibody with synthetic phosphopeptides containing the S63 phosphorylation site

  • Test whether this competition blocks antibody binding in your assay

  • A cell-permeable c-Jun peptide has been used successfully as a competitive inhibitor

• Phosphatase Treatment Control:

  • Treat positive samples with lambda phosphatase

  • Confirm loss of antibody reactivity after phosphate group removal

• Stimulation/Inhibition Controls:

  • Compare samples from unstimulated cells, anisomycin-stimulated cells, and cells pre-treated with JNK inhibitors

  • A dose-dependent response should be observed with anisomycin stimulation

  • NIH-3T3 cells stimulated with increasing concentrations of anisomycin show proportional increases in S63 phosphorylation

• Antibody Dilution Series:

  • Perform serial dilutions to establish the linear range of detection

  • Ensure your experimental conditions fall within this range

These validation steps collectively provide strong evidence for antibody specificity and help identify potential cross-reactivity issues or artifacts in your experimental system.

How does the spatial proximity of the D-motif affect c-JUN phosphorylation kinetics at different sites?

Research using engineered c-JUN TAD constructs has revealed complex relationships between D-motif positioning and phosphorylation kinetics. The D-motif (residues 32-50) serves as the MAPK binding site that controls phosphorylation of S63, S73, T91, and T93 sites.

In wild-type c-JUN, the D-motif is positioned N-terminal to the phosphorylation sites, with S63 and S73 being closer to the D-motif than T91 and T93. Experiments with transposed D-motif variants (mDock and cDock) revealed that:

• Moving the D-motif closer to T91/T93 (mDock variant) did not accelerate their phosphorylation rates

• Sites positioned N-terminal to the transposed D-motif showed strongly suppressed phosphorylation

• The normally fast S63 site exhibited very slow modification in both mDock and cDock variants

These findings indicate that while spatial proximity to the D-motif contributes to phosphorylation efficiency, it is not the sole determinant. The three-dimensional structural interaction between JNK1 and c-JUN, as well as the primary amino acid sequence surrounding the phosphorylation sites, play crucial roles in determining site-specific phosphorylation kinetics.

What is the relationship between c-JUN Ser63 phosphorylation and its role in cellular pathways beyond transcriptional activation?

Beyond its well-established role in transcriptional activation, phosphorylation of c-JUN at Ser63 mediates several additional cellular functions:

• Apoptosis Regulation:

  • c-JUN phosphorylation has been linked to apoptosis mediation when survival signals are interrupted

  • The balance between pro-survival and pro-apoptotic functions appears to be context-dependent

• Protein Stability and Turnover:

  • Phosphorylation at Ser63 can affect c-JUN protein stability and half-life

  • This post-translational modification may protect c-JUN from ubiquitin-mediated degradation

• Protein-Protein Interactions:

  • Phosphorylated c-JUN can recruit specific co-factors that modulate its activity

  • Phosphorylation may alter interaction patterns with other transcription factors and regulatory proteins

• Cellular Stress Response:

  • Ser63 phosphorylation serves as a marker for cellular stress response

  • Different stressors (UV radiation, inflammatory cytokines, oxidative stress) can trigger this phosphorylation through JNK activation

• Tissue Morphogenesis:

  • c-JUN phosphorylation contributes to developmental processes and tissue organization

  • This function appears to be regulated by both JNK-dependent and JNK-independent mechanisms

These diverse functions highlight the central role of c-JUN Ser63 phosphorylation as a regulatory node connecting multiple cellular signaling networks, extending well beyond simple transcriptional control.

How do the kinetics of c-JUN phosphorylation at Ser63 compare between different JNK isoforms?

The kinetics of c-JUN phosphorylation at Ser63 show subtle but important differences between JNK isoforms. Comparative analyses using recombinant active JNK1 and JNK2 with c-JUN TAD have revealed:

These kinetic differences may have significant implications for understanding the specific roles of different JNK isoforms in various physiological and pathological contexts, providing potential targets for isoform-selective therapeutic interventions.

What are the common technical challenges when detecting phospho-JUN (S63) and how can they be addressed?

Researchers frequently encounter several technical challenges when working with phospho-JUN (S63) detection. Here are the most common issues and recommended solutions:

• Low Signal Intensity:

  • Cause: Insufficient stimulation, rapid dephosphorylation, or low antibody sensitivity

  • Solution: Optimize stimulation conditions (500nM anisomycin for 30 minutes works well); include phosphatase inhibitors in lysis buffer; consider using HTRF assay which offers 8-fold higher sensitivity than Western blot

• High Background:

  • Cause: Non-specific antibody binding or insufficient blocking

  • Solution: Optimize antibody dilution (1:500-1:5000 for WB); increase blocking time/concentration; include validated negative controls (S63A mutants)

• Inconsistent Phosphorylation:

  • Cause: Cell density variations, heterogeneous stimulation, or unstable stimulants

  • Solution: Standardize cell seeding density (50,000 cells/well recommended); ensure uniform reagent distribution; prepare fresh stimulants before each experiment

• Rapid Dephosphorylation:

  • Cause: Phosphatase activity during sample preparation

  • Solution: Work quickly on ice; use comprehensive phosphatase inhibitor cocktails; validate inhibitor effectiveness in your system

• Cross-Reactivity with Other Phosphorylation Sites:

  • Cause: Antibody recognizing similar epitopes around other phosphorylation sites

  • Solution: Validate antibody specificity using alanine mutants for each phosphorylation site; consider using specific blocking peptides

• Antibody Detection Outside Linear Range:

  • Cause: Over-saturated signal or insufficient protein loading

  • Solution: Perform back-dilution series of phosphorylated samples to ensure antibody signals remain within the linear detection range

Implementing these solutions can significantly improve the reliability and reproducibility of phospho-JUN (S63) detection across different experimental platforms.

How can I quantitatively compare phospho-JUN (S63) levels across different experimental conditions?

For rigorous quantitative comparison of phospho-JUN (S63) levels across experimental conditions, consider the following methodological approaches:

• HTRF Quantitative Assay:

  • Provides direct quantitative measurement of phosphorylated c-JUN

  • Signal intensity is directly proportional to phosphorylated protein concentration

  • Allows for high-throughput, plate-based analysis

  • Standard curves can be generated using recombinant phosphorylated standards

• Normalization Strategies:

  • Total c-JUN Normalization: Express phospho-JUN (S63) signal relative to total c-JUN levels

  • Housekeeping Protein Normalization: Normalize to stable reference proteins (β-actin, GAPDH)

  • Loading Control Normalization: Use total protein stains (Ponceau S, REVERT)

• Dose-Response Analysis:

  • Generate dose-response curves using varying concentrations of stimulus

  • NIH-3T3 cells stimulated with increasing anisomycin concentrations show proportional increases in S63 phosphorylation

  • Calculate EC50 values for different stimuli or inhibitors

• Time-Course Analysis:

  • Measure phosphorylation at multiple time points (0-30 minutes recommended)

  • Calculate rates and half-times of phosphorylation

  • Allows comparison of phosphorylation kinetics under different conditions

  • In vivo studies show half-times of approximately 12 minutes for S63 phosphorylation following anisomycin treatment

• Quantitative Image Analysis:

  • For IHC/IF applications, use software-based quantification

  • Include internal controls in each experiment

  • Consider automated high-content imaging for large-scale analysis

Each of these approaches offers distinct advantages depending on your experimental system and research questions. Combining multiple quantitative methods provides the most robust comparative analysis across experimental conditions.

What considerations are important when interpreting phospho-JUN (S63) data in the context of complex signaling networks?

Interpreting phospho-JUN (S63) data requires careful consideration of the broader signaling context. Key factors to consider include:

• Temporal Dynamics:

  • c-JUN phosphorylation follows specific temporal patterns

  • S63 is phosphorylated rapidly (half-time ~12 min) following JNK activation

  • Transient versus sustained phosphorylation may lead to different functional outcomes

  • Consider collecting data at multiple time points to capture the complete dynamics

• Multiple Phosphorylation Sites:

  • c-JUN contains multiple phosphorylation sites (S63, S73, T91, T93)

  • These sites are phosphorylated with different kinetics (S63 > S73 > T91 ≈ T93)

  • The combinatorial pattern of phosphorylation may determine specific functional outcomes

  • Consider analyzing multiple phosphorylation sites simultaneously

• Pathway Crosstalk:

  • JNK is not the only kinase that can influence c-JUN activity

  • Plk3 can phosphorylate c-JUN independently of JNK

  • UV irradiation-induced phosphorylation of c-JUN and apoptosis were not significantly affected by JNK knockdown in some systems

  • Consider analyzing multiple pathway components to determine the specific contribution of each

• Cell Type-Specific Regulation:

  • Different cell types may exhibit distinct patterns of c-JUN phosphorylation

  • Expression levels of JNK isoforms, phosphatases, and regulatory proteins vary across cell types

  • Validate findings across multiple relevant cell systems

• Functional Correlation:

  • Phosphorylation at S63 is necessary but not always sufficient for transcriptional activation

  • Consider complementing phosphorylation data with functional readouts (AP-1 reporter assays, target gene expression)

  • Establish causality through inhibitor studies or mutation of phosphorylation sites

By integrating these considerations into your data analysis, you can develop a more comprehensive understanding of how phospho-JUN (S63) signaling contributes to specific cellular responses within complex regulatory networks.

How does the sensitivity and specificity of HTRF technology compare with traditional Western blot for phospho-JUN (S63) detection?

Comparative analysis between HTRF and Western blot technologies for phospho-JUN (S63) detection reveals significant differences in performance characteristics:

Experimental validation with HEK293 cells stimulated with 500nM anisomycin for 30 min demonstrated that the HTRF assay could detect phospho-JUN (S63) at significantly lower concentrations than Western blot, making it particularly valuable for experiments with limited sample availability or when detecting subtle changes in phosphorylation levels .

What are the advanced experimental approaches for studying the relationship between c-JUN phosphorylation kinetics and transcriptional outcomes?

Advanced experimental strategies can elucidate the complex relationship between c-JUN phosphorylation kinetics and downstream transcriptional regulation:

• Engineered c-JUN Variants:

  • Create "swap" variants where sequences containing fast phosphorylation sites (S63/S73) are exchanged with sequences containing slower sites (T91/T93)

  • Generate phosphomimetic mutants (S63D, S73D) to simulate constitutive phosphorylation

  • Develop phospho-deficient mutants (S63A, S73A) as negative controls

  • These engineered variants help dissect the specific contribution of each phosphorylation site to transcriptional activity

• Time-Resolved Transcriptomics:

  • Combine time-course phosphorylation analysis with RNA-seq at multiple time points

  • Identify temporal relationships between specific phosphorylation events and transcriptional changes

  • Group target genes based on their activation kinetics relative to c-JUN phosphorylation

• Chromatin Immunoprecipitation (ChIP) with Phospho-Specific Antibodies:

  • Use phospho-JUN (S63) antibodies for ChIP experiments

  • Determine the genomic binding sites of specifically S63-phosphorylated c-JUN

  • Compare with binding profiles of total c-JUN or other phosphorylated forms

• Live-Cell Imaging with Phosphorylation Biosensors:

  • Develop FRET-based biosensors for real-time visualization of c-JUN phosphorylation

  • Correlate spatiotemporal patterns of phosphorylation with transcriptional reporter activity

  • Analyze single-cell dynamics to capture heterogeneity in response

• Targeted Inhibition of Specific Kinases:

  • Compare the effects of different JNK isoform-specific inhibitors on c-JUN phosphorylation kinetics

  • Use cell-permeable c-Jun peptide inhibitors that demonstrate dose-dependent inhibition of c-Jun phosphorylation following anisomycin stimulation

These advanced approaches collectively provide a comprehensive view of how the temporal dynamics of c-JUN phosphorylation translate into specific patterns of gene expression, offering insights into the encoding of signal specificity in the JNK pathway.

How can phospho-JUN (S63) detection be integrated into multi-parameter analyses of stress response pathways?

Integration of phospho-JUN (S63) detection into multi-parameter analyses provides a more comprehensive understanding of stress response signaling networks:

• Multiplexed Phospho-Protein Profiling:

  • Combine phospho-JUN (S63) detection with analysis of other JNK substrates

  • Include upstream activators (MKK4/7 phosphorylation) and parallel MAPK pathways (p38, ERK)

  • HTRF technology allows development of multiplexed assays using different fluorophore combinations

• Single-Cell Multi-Parameter Analysis:

  • Utilize phospho-flow cytometry to simultaneously detect phospho-JUN (S63) and other pathway components

  • Combine with cell cycle markers or apoptosis indicators

  • Reveals cell-to-cell heterogeneity and identifies distinct cellular subpopulations

• Pathway Activity Correlation:

  • Correlate phospho-JUN (S63) levels with functional readouts:

    • AP-1 transcriptional reporter activity

    • Expression of c-JUN target genes

    • Apoptosis markers

    • Cell proliferation indices

• Dynamic Pathway Modeling:

  • Develop mathematical models incorporating the temporal kinetics of c-JUN phosphorylation

  • S63 is phosphorylated most rapidly, followed by S73, with T91/T93 phosphorylated more slowly

  • Model how these differential kinetics contribute to signal encoding and specificity

• Integration with -Omics Approaches:

  • Correlate phospho-JUN (S63) levels with:

    • Transcriptomic profiles (RNA-seq)

    • Chromatin accessibility changes (ATAC-seq)

    • Global phosphoproteomic alterations (phospho-MS)

  • Identify broader network changes associated with specific phosphorylation patterns

This integrated approach provides a systems-level understanding of how phospho-JUN (S63) signaling contributes to complex cellular responses to various stressors, potentially revealing novel therapeutic targets or biomarkers for stress-related pathologies.