Phospho-LCK (Tyr394) Antibody

What is the biological significance of LCK Tyr394 phosphorylation in T cell signaling?

LCK (lymphocyte-specific protein tyrosine kinase) is a critical Src family tyrosine kinase that regulates T cell activation by associating with CD4 and CD8 surface receptors. Phosphorylation at Tyr394 is essential for enhancing kinase activity. Studies have demonstrated that the phosphorylation of Tyr394 plays a role beyond merely inducing an open conformation of LCK - it is required for initiating TCR signaling events . The enzymatic activity of LCK is tightly controlled by differential phosphorylation of two key tyrosine residues: Tyr394 (activating) and Tyr505 (inhibitory). While phosphorylation of Tyr505 generates a closed, inactive conformation, phosphorylation of Tyr394 stabilizes the activation loop in an active conformation .

When investigating T cell activation mechanisms, it's important to understand that approximately 20% of LCK in T lymphocytes exists in a double-phosphorylated form (both Tyr394 and Tyr505), which remains catalytically active despite the theoretically inhibitory phosphorylation at Tyr505 .

How does the phosphorylation state of LCK regulate TCR signaling initiation?

The regulation of TCR signaling by LCK involves a complex interplay between its phosphorylation state and conformational changes:

• Activation mechanism: Upon TCR triggering, LCK phosphorylates the ITAM motifs in the TCR's zeta subunits, establishing binding sites for ZAP70's SH2 domains

• Regulatory phosphorylation sites: The inhibitory Tyr505 is phosphorylated by C-terminal Src kinase (Csk) and dephosphorylated by CD45, while the activating Tyr394 undergoes transphosphorylation by LCK itself

• Conformational requirements: Research has shown that both conformational opening of LCK and phosphorylation of Tyr394 are required for T cell activation - neither is sufficient alone

Fluorescence lifetime imaging microscopy (FLIM) studies with LCK biosensors have demonstrated that TCR-stimulated phosphorylation of LCK at Tyr394 occurs preferentially at the plasma membrane of both Jurkat cells and primary human T cells .

What are the most effective methods for detecting phospho-LCK (Tyr394) in different experimental contexts?

Several validated methods exist for detecting phospho-LCK (Tyr394), each with specific advantages depending on your experimental goals:

For optimal phospho-LCK (Tyr394) detection, consider these methodological guidelines:

• When using Western blot, Jurkat (JK) cells serve as an effective positive control

• For MILLIPLEX MAP assays, Ramos cells treated with pervanadate demonstrate strong phospho-LCK (Tyr394) signal as shown in experimental data

• When designing immunofluorescence experiments, remember that TCR-stimulated phosphorylation of LCK at Tyr394 occurs preferentially at the plasma membrane

How can I optimize cell lysis conditions to preserve phospho-LCK (Tyr394) for downstream analysis?

Maintaining phosphorylation status during cell lysis is critical for accurate analysis of phospho-LCK (Tyr394). Follow these methodological steps:

• Rapid sample processing: Minimize time between cell stimulation and lysis to prevent dephosphorylation

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

• Temperature control: Perform lysis on ice and maintain cold temperatures throughout processing

• Lysis buffer composition: Use buffers containing 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris (pH 7.4), and 1 mM EDTA supplemented with protease inhibitors

• Positive controls: Consider pervanadate treatment of cells as a positive control, as it markedly enhances phosphorylation of LCK at Tyr394

Research has shown that suppression of phosphatase activity by pervanadate treatment significantly increases phosphorylation of Tyr394 and promotes T cell activation, making this an effective positive control for phospho-LCK studies .

How do conformational changes and phosphorylation states of LCK interact to regulate its function?

The relationship between LCK conformation and phosphorylation is more complex than initially understood:

• Conformational states: LCK exists in closed (inactive) and open (active) conformations, regulated by intramolecular interactions involving SH2 and SH3 domains

• Regulatory phosphorylation: Phosphorylation of Tyr505 stabilizes the closed conformation through interaction with the SH2 domain, while Tyr394 phosphorylation stabilizes the activation loop in an active conformation

• Unexpected findings: Contrary to previous assumptions, recent research using FLIM with sensitive biosensors has demonstrated that conformational opening of LCK alone is insufficient to initiate T cell signaling events; phosphorylation of Tyr394 is additionally required

What are the most effective strategies for studying LCK activation in primary T cells versus cell lines?

Research approaches differ when studying LCK in primary T cells compared to established cell lines:

When studying primary T cells:

• Isolation considerations: Use negative selection to avoid pre-activation of cells

• Stimulation protocol: For physiological activation, use plate-bound anti-CD3/CD28 or APCs

• Imaging approach: Optimized immunofluorescence microscopy protocols reveal that TCR-stimulated phosphorylation of LCK at Tyr394 occurs preferentially at the plasma membrane of primary human T cells

• Temporal considerations: Primary cells may exhibit different kinetics of LCK phosphorylation compared to cell lines

How can I distinguish between LCK Tyr394 phosphorylation and other Src family kinases with similar phosphorylation sites?

Cross-reactivity with other Src family kinases (SFKs) is a significant concern when studying phospho-LCK (Tyr394), as the activation loop is highly conserved across this family:

• Antibody selection: Some antibodies recognize multiple phosphorylated SFKs. For example, the E5L3D antibody detects phosphorylated forms of LYN (Tyr397), LCK (Tyr394), HCK (Tyr411), and BLK (Tyr389)

• Specificity validation: Verify antibody specificity using:

  • Knockout/knockdown controls

  • Phosphorylation-deficient mutants (e.g., Y394F)

  • Competition with phospho-peptides

• Complementary approaches: Combine immunological detection with:

  • Immunoprecipitation to isolate specific SFKs before phospho-detection

  • Mass spectrometry to distinguish specific phosphorylated residues

  • Kinase-specific inhibitors to validate functional effects

For maximum specificity when working with multiple SFKs, consider using phosphorylation-deficient mutants such as Y394F, Y505F, or the double mutant Y394F/Y505F as controls in your experimental system .

What are common causes of inconsistent phospho-LCK (Tyr394) detection, and how can they be addressed?

Inconsistent phospho-LCK (Tyr394) detection can result from several factors:

• Rapid dephosphorylation: LCK Tyr394 is subject to rapid dephosphorylation by phosphatases including SHP-1 and CD45

  • Solution: Ensure phosphatase inhibitors (e.g., sodium orthovanadate) are fresh and at effective concentrations

• Fixation artifacts: Inadequate fixation can lead to epitope masking or loss of phosphorylation

  • Solution: Optimize fixation protocols; for immunofluorescence, an optimized protocol revealed that TCR-stimulated phosphorylation of LCK at Tyr394 occurred preferentially at the plasma membrane

• Antibody specificity issues: Cross-reactivity with other SFKs can complicate interpretation

  • Solution: Use phosphorylation-deficient mutants (Y394F) as controls and validate with multiple detection methods

• Cell type variations: Different T cell subsets or cell lines may have varying levels of baseline LCK phosphorylation

  • Solution: Include appropriate cell type-specific positive and negative controls; for Western blot, JK cells are recommended as positive controls

• Stimulation conditions: Suboptimal TCR stimulation may yield inconsistent results

  • Solution: Titrate stimulation conditions and include pervanadate treatment as a positive control, which markedly enhances phosphorylation of Tyr394

How should I interpret changes in phospho-LCK (Tyr394) levels in the context of T cell activation experiments?

Interpretation of phospho-LCK (Tyr394) data requires consideration of several factors:

When designing experiments to study LCK activation, remember that both conformational opening of LCK and phosphorylation of Tyr394 are required for T cell activation - neither is sufficient alone .

How are phospho-LCK (Tyr394) antibodies being used to study T cell immunotherapies and immune checkpoint modulation?

Phospho-LCK (Tyr394) antibodies are increasingly valuable tools for studying immunotherapeutic approaches:

• CAR-T cell engineering: Monitoring LCK activation in CAR-T cells provides insights into:

  • Signaling efficiency of different CAR constructs

  • Mechanisms of CAR-T exhaustion or dysfunction

  • Optimization of CAR costimulatory domains

• Immune checkpoint inhibition: Phospho-LCK (Tyr394) detection helps elucidate:

  • How PD-1 and CTLA-4 signaling suppress TCR-induced LCK activation

  • Mechanisms of checkpoint inhibitor resistance

  • Biomarkers for checkpoint inhibitor response

• Combination immunotherapies: Analyses of LCK phosphorylation patterns can guide:

  • Rational design of drug combinations

  • Identification of optimal sequencing of therapies

  • Development of novel immune modulators targeting the LCK pathway

For these emerging applications, it's critical to develop standardized assays for phospho-LCK detection in clinical samples, which may involve optimization of fixation and permeabilization protocols for various sample types.

What novel techniques are being developed to study the spatial and temporal dynamics of LCK activation in intact T cells?

Advanced imaging and analytical techniques are transforming our understanding of LCK dynamics:

• Live-cell FRET/FLIM biosensors: These sensitive tools can:

  • Monitor real-time conformational changes in LCK

  • Distinguish conformational changes from phosphorylation states

  • Reveal that both conformational opening and Tyr394 phosphorylation are required for T cell activation

• Super-resolution microscopy: Techniques like STORM and PALM enable:

  • Visualization of LCK nanoclusters at the immunological synapse

  • Tracking of single-molecule LCK diffusion and activation

  • Correlation of spatial organization with TCR triggering events

• Mass cytometry (CyTOF): This approach allows:

  • Simultaneous detection of multiple phosphorylation sites on LCK and other signaling molecules

  • Analysis of signaling heterogeneity in complex T cell populations

  • Correlation of LCK activation with cellular phenotypes

• Optogenetic control of LCK: Emerging techniques permit:

  • Spatiotemporal control of LCK activation in specific subcellular regions

  • Dissection of feedback mechanisms in TCR signaling

  • Testing of mechanistic models of TCR triggering