Phospho-CSK (S364) Antibody

What is CSK and why is the phosphorylation at Serine 364 significant?

C-terminal Src kinase (CSK) is a non-receptor tyrosine-protein kinase that plays a crucial role in regulating cell growth, differentiation, migration, and immune response. CSK functions primarily as an inhibitory kinase for Src family kinases (SFKs) by phosphorylating tyrosine residues in their C-terminal tails, which leads to their inactivation .

The phosphorylation of CSK at Serine 364 (S364) by Protein Kinase A (PKA) is particularly significant because it increases CSK's kinase activity . Phosphoamino acid analysis of CSK phosphorylated by PKA has demonstrated strong labeling on phosphoserine, with tryptic peptide mapping revealing two major radioactive spots containing phosphoserine . When researchers created a CSK-S364A mutant, it was only weakly phosphorylated by PKA, confirming this site's importance in PKA-mediated regulation .

This phosphorylation event represents a critical regulatory mechanism where PKA can enhance CSK activity, which subsequently increases inhibition of SFKs, creating an important cross-talk between different signaling pathways.

How does Phospho-CSK (S364) contribute to immune cell regulation?

In immune cells, particularly T cells, the phosphorylation of CSK at S364 by PKA provides a mechanism for cAMP-dependent regulation of T cell activity. Research has demonstrated that this phosphorylation event is necessary for PKA regulation of CSK in intact T cells .

The regulatory importance is evident in functional studies where PKA activation leads to enhanced CSK activity. When Lck (a Src family kinase crucial in T cell signaling) was used as a substrate, CSK-mediated tyrosine phosphorylation of Lck was 4.8-fold stronger in the presence of PKA compared to its absence . This increased phosphorylation of Lck leads to its inactivation, subsequently dampening T cell receptor signaling.

This mechanism provides a molecular explanation for how elevated cAMP levels, which activate PKA, can suppress T cell activation – a finding with significant implications for understanding immune regulation and potential therapeutic targets in autoimmune disorders.

What structural elements of the antibody ensure specificity for the phosphorylated form?

The Phospho-CSK (S364) antibody's specificity derives from several key elements:

• Immunogen design: The antibody is generated using a synthesized phosphopeptide derived specifically from human CSK around the phosphorylation site of Serine 364 . This targeted approach ensures recognition of the phosphorylated epitope.

• Affinity purification: The antibody undergoes affinity-chromatography purification using epitope-specific immunogen, which enriches for antibodies that specifically recognize the phospho-S364 epitope .

• Validation protocols: Manufacturers test the antibody's ability to detect endogenous levels of CSK protein only when phosphorylated at S364, confirming its phospho-specificity .

The antibody's ability to discriminate between phosphorylated and non-phosphorylated forms is critical for studying the dynamic regulation of CSK in various signaling contexts and cellular responses.

What are the optimal conditions for using Phospho-CSK (S364) antibody in Western blot experiments?

For optimal Western blot detection of phosphorylated CSK at S364, researchers should consider the following protocol elements:

When conducting Western blot analysis, various cell lines have been successfully used to detect Phospho-CSK (S364), including CT-26, C6, MCF-7, and HeLa cell lysates . The antibody has demonstrated consistent detection at approximately 51 kDa, which corresponds to the calculated molecular weight of CSK .

To ensure specificity, always include appropriate controls such as phosphatase-treated samples and, when possible, samples where PKA activation has been stimulated or inhibited to modulate S364 phosphorylation.

How can I effectively use Phospho-CSK (S364) antibody in immunohistochemistry applications?

For successful immunohistochemistry (IHC) applications with Phospho-CSK (S364) antibody, consider this methodological approach:

• Sample preparation: Fix tissues in 4% paraformaldehyde and embed in paraffin. Alternatively, frozen sections may preserve phospho-epitopes better in some contexts.

• Antigen retrieval: Use citrate buffer (pH 6.0) with heat-induced epitope retrieval to maximize exposure of the phospho-epitope.

• Antibody dilution: Begin with a 1:100-1:300 dilution range as recommended by manufacturers . Optimize for your specific tissue type.

• Blocking strategy: Use a blocking solution containing normal serum from the same species as the secondary antibody, with added BSA (3-5%) to reduce background.

• Primary antibody incubation: Incubate overnight at 4°C to improve sensitivity and specificity.

• Detection system: Use a polymer-based detection system rather than avidin-biotin, as this often provides cleaner results for phospho-specific antibodies.

• Controls: Include negative controls (primary antibody omitted) and, when possible, tissues from experimental models where PKA has been activated or inhibited to alter S364 phosphorylation levels.

For quantitative assessment, digitize stained sections and use image analysis software with appropriate thresholding to measure staining intensity relative to total CSK staining in serial sections.

What methodological considerations are important for cell-based ELISA using Phospho-CSK (S364) antibody?

Cell-based ELISA represents a valuable high-throughput approach for quantifying changes in CSK phosphorylation. Based on the Cell-Based Colorimetric ELISA Kit methodology , researchers should consider:

• Cell seeding density: Optimize to achieve 70-90% confluency at the time of fixation. This ensures adequate signal while maintaining individual cell morphology.

• Stimulation protocols: Design time-course experiments to capture the dynamic nature of S364 phosphorylation, particularly in response to PKA activators.

• Normalization strategy: Use crystal violet staining for cell number normalization, which allows adjustment for plating differences . This is critical for accurate comparison between conditions.

• Controls and specificity: Include both phospho-specific (S364) and total CSK antibodies to calculate the phosphorylation ratio, which provides more reliable data than absolute phospho-signal alone .

• Signal development: Use One-Step TMB substrate for colorimetric detection, with careful monitoring of the reaction to avoid saturation .

• Data analysis: Calculate the ratio of phospho-CSK to total CSK after normalizing both to cell number, and compare this ratio across experimental conditions rather than raw values.

This methodology allows for rapid, quantitative assessment of CSK phosphorylation status across multiple experimental conditions, making it ideal for signaling studies and inhibitor screening.

How can FRET biosensors be designed to monitor CSK phosphorylation in live cells?

Fluorescence resonance energy transfer (FRET) biosensors offer powerful tools for visualizing CSK activity dynamics in living cells. Based on recent developments , researchers can design and implement FRET biosensors for CSK using these principles:

• Biosensor design strategy: Create a construct containing:

  • A donor fluorophore (ECFP)

  • An SH2 domain that recognizes phosphorylated tyrosine

  • A specific substrate peptide containing the target tyrosine

  • An acceptor fluorophore (YPet)

• Mechanism of action: In the unphosphorylated state, the FRET efficiency is high due to proximity of fluorophores. Upon phosphorylation by active CSK, the phospho-tyrosine binds to the SH2 domain, causing conformational change that separates the fluorophores and decreases FRET efficiency .

• Cellular targeting: Add specific localization sequences to direct the biosensor to relevant subcellular compartments where CSK activity is of interest, particularly membrane regions where CSK interacts with SFKs.

• Measurement approach: Use ratiometric imaging (donor/acceptor emission ratio) to monitor FRET changes that reflect CSK activity independently of biosensor concentration.

• Controls and validation: Include a non-phosphorylatable mutant version as a negative control, and validate biosensor response using specific activators and inhibitors of the PKA-CSK pathway.

This approach enables researchers to visualize CSK activity with high spatiotemporal resolution, revealing dynamic regulation that would be missed by fixed-cell techniques. The biosensor can be particularly valuable for studying how S364 phosphorylation affects CSK's subcellular localization and activity in real-time.

What experimental approaches can elucidate the relationship between PKA-mediated phosphorylation of CSK at S364 and T cell activation?

To investigate the functional relationship between CSK S364 phosphorylation and T cell activation, researchers can employ these methodological approaches:

• Reconstitution experiments with phospho-mutants: Generate CSK knockout T cells and reconstitute with either wild-type CSK, phospho-mimetic (S364D/E), or phospho-deficient (S364A) mutants. Measure functional outcomes such as IL-2 production following T cell receptor stimulation .

• Pharmacological modulation: Use specific PKA activators (e.g., forskolin) or inhibitors (e.g., H-89) to modulate S364 phosphorylation and correlate with functional T cell responses including:

  • Calcium flux

  • IL-2 secretion measured by ELISA

  • CD69 upregulation by flow cytometry

  • Proliferation assays

• Phosphorylation dynamics: Establish the temporal relationship between PKA activation, CSK S364 phosphorylation, and SFK tail phosphorylation using time-course experiments with Phospho-CSK (S364) antibody detection.

• Subcellular localization studies: Use cell fractionation or imaging approaches to determine if S364 phosphorylation affects CSK recruitment to the plasma membrane or lipid rafts where it can access SFK substrates.

• Quantitative kinase assays: Compare the kinase activity of immunoprecipitated wild-type versus S364A mutant CSK toward purified SFK substrates, with and without prior PKA treatment .

When combined, these approaches can establish whether S364 phosphorylation is necessary and/or sufficient for PKA-mediated suppression of T cell activation, providing mechanistic insight into this important regulatory pathway.

What are common issues when detecting Phospho-CSK (S364) and how can they be resolved?

For optimal results, researchers should store the Phospho-CSK (S364) antibody at -20°C and avoid repeated freeze-thaw cycles . The storage buffer (PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide) helps maintain antibody integrity , but creating single-use aliquots is still recommended for long-term studies.

How can researchers validate the specificity of Phospho-CSK (S364) antibody in their experimental system?

Establishing antibody specificity is crucial for reliable research findings. Implement these validation strategies:

• Phosphatase treatment: Divide your sample and treat half with lambda phosphatase before immunoblotting. The phospho-specific signal should disappear in the treated sample.

• Genetic approaches:

  • Use CRISPR/Cas9 to generate CSK knockout cells as a negative control

  • Compare wild-type CSK with S364A mutant expression in knockout background

• Pharmacological manipulation:

  • Treat cells with PKA activators (e.g., forskolin, cAMP analogs) to increase S364 phosphorylation

  • Use PKA inhibitors (e.g., H-89, PKI) to reduce phosphorylation

• Peptide competition: Pre-incubate the antibody with excess phospho-S364 peptide before using in your application. This should block specific binding and eliminate the target signal.

• Cross-validation: Compare results using alternative detection methods or antibodies from different sources when available.

• Assessment across multiple applications: Confirm consistent patterns of detection across different techniques (e.g., Western blot, IHC, IF) to strengthen confidence in specificity.

Document these validation steps thoroughly as they significantly enhance the credibility of research findings involving phospho-specific antibodies.

What is the current understanding of how CSK S364 phosphorylation influences its spatial regulation and membrane recruitment?

CSK is a cytoplasmic protein that must be recruited to the plasma membrane to effectively regulate SFKs . Recent research has begun to elucidate how S364 phosphorylation affects this spatial regulation:

CSK distributes primarily in the cytosol as a ~50 kDa protein, but a fraction localizes to lipid rafts through interaction with PAG (CSK binding protein), also known as Csk-binding protein . This membrane recruitment is critical for CSK to access and phosphorylate SFKs at their C-terminal inhibitory tyrosine residues.

Phosphorylation at S364 by PKA increases CSK activity , but research also suggests it may influence binding to adaptor proteins that facilitate membrane recruitment. FRET biosensor development has enabled visualization of CSK activity in live cells , revealing dynamic regulation that may connect phosphorylation status with subcellular localization.

Future research directions should focus on:

• Determining whether S364 phosphorylation directly affects CSK's affinity for membrane adaptor proteins

• Investigating potential conformational changes induced by S364 phosphorylation that might alter protein-protein interactions

• Developing spatially-targeted biosensors to monitor CSK activity specifically at membrane microdomains versus cytosolic regions

These approaches will help clarify how phosphorylation and localization collectively regulate CSK function in different cellular contexts.

How might advances in phosphoproteomic approaches complement antibody-based detection of Phospho-CSK (S364)?

While antibody-based detection of Phospho-CSK (S364) remains valuable for targeted studies, emerging phosphoproteomic technologies offer complementary advantages:

• Mass spectrometry-based phosphoproteomics:

  • Enables unbiased detection of multiple phosphorylation sites on CSK simultaneously

  • Can reveal previously unknown phosphorylation events that may crosstalk with S364

  • Provides absolute quantification of phosphorylation stoichiometry when combined with stable isotope labeling

• Multiplexed approaches:

  • Techniques like mass cytometry (CyTOF) with phospho-specific antibodies enable single-cell analysis of CSK phosphorylation in heterogeneous populations

  • Allow correlation of S364 phosphorylation with numerous other signaling events

• Proximity labeling proteomics:

  • BioID or APEX2 fused to CSK can identify proteins that interact specifically with phosphorylated versus non-phosphorylated CSK

  • Helps map the phospho-regulated interactome

• Integrative data analysis:

  • Combining phosphoproteomics with transcriptomics and functional assays can place S364 phosphorylation in broader signaling networks

  • Network analysis may reveal unexpected signaling nodes influenced by CSK phosphorylation

These advanced approaches extend beyond the binary "phosphorylated/non-phosphorylated" information provided by antibodies alone, offering deeper mechanistic insights into CSK regulation and function in complex biological systems.

What methodological advances are enhancing the study of CSK phosphorylation dynamics in complex signaling networks?

Recent methodological innovations are transforming how researchers study CSK phosphorylation in signaling networks:

• Optogenetic control of PKA activity: Light-activated adenylate cyclase or directly photoactivatable PKA constructs enable precise spatiotemporal control of S364 phosphorylation, allowing researchers to dissect immediate downstream consequences.

• Kinase translocation reporters (KTRs): These genetically encoded reporters allow visualization of CSK substrate phosphorylation in real-time through nuclear-cytoplasmic shuttling, complementing direct measurements of S364 phosphorylation.

• Single-molecule tracking: Super-resolution microscopy combined with photo-convertible CSK fusion proteins can reveal how S364 phosphorylation affects the dwelling time and diffusion characteristics of CSK at membrane microdomains.

• Microfluidic approaches: Rapid stimulation and fixation devices enable capturing transient phosphorylation events with millisecond resolution, critical for understanding the kinetics of CSK regulation.

• CRISPR-based screening: Focused CRISPR libraries targeting components of PKA-CSK-SFK signaling networks, combined with phospho-flow cytometry, can identify novel regulators of CSK phosphorylation.

• Mathematical modeling: Quantitative models incorporating phosphorylation/dephosphorylation rates, diffusion, and enzyme kinetics help predict system-level behaviors of CSK regulation networks.

These methodological advances collectively enable more precise, quantitative, and systems-level understanding of how CSK phosphorylation integrates into broader cellular signaling networks.