Phospho-LCK (Y393) Antibody

What is the significance of LCK phosphorylation at Y393 in T-cell signaling?

LCK (Lymphocyte-specific protein tyrosine kinase) is a non-receptor tyrosine kinase that plays an essential role in T-cell receptor (TCR) signaling. Phosphorylation at tyrosine 393 (Y393, sometimes referred to as Y394 in some literature) represents a critical activation mark that significantly enhances LCK's enzymatic activity. This phosphorylation event creates a stabilized open structure with increased kinase activity and substrate binding capacity .

In the TCR signaling cascade, activated LCK phosphorylates tyrosine residues within the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR-gamma chains and CD3 subunits, initiating the TCR/CD3 signaling pathway . Once activated, the TCR recruits the tyrosine kinase ZAP70, which becomes phosphorylated and activated by LCK, triggering downstream signaling events that ultimately lead to lymphokine production and T-cell activation .

Autophosphorylation of Y393 is balanced by several phosphatases, including CD45, SHP-1, PEP, and protein tyrosine phosphatase-PEST, creating a dynamic regulatory system . The phosphorylation state of Y393 is therefore a key indicator of TCR signaling integrity and T-cell activation status.

How should samples be prepared to maintain accurate LCK phosphorylation status?

Proper sample preparation is critical for maintaining authentic LCK phosphorylation status, as LCK can transautophosphorylate its Y393 site even after cell lysis, potentially resulting in artificially elevated activity when detected by Western blotting or immunoprecipitation . To preserve the native phosphorylation state, implement the following protocol:

• Supplement lysis buffer with SFK inhibitor PP2 (20 μM) combined with a protease-phosphatase inhibitor mixture during cell lysis and immunoprecipitation

• Process samples quickly at cold temperatures (4°C) to minimize enzymatic activity

• Add phosphatase inhibitors immediately upon lysis to prevent dephosphorylation

• Consider using pervanadate (phosphatase inhibitor) treatment as a positive control and PP2 (SFK inhibitor) as a negative control in experimental validation

When preparing cell lysates for Western blot analysis, avoid repeated freeze-thaw cycles that may affect phosphorylation status. For immunofluorescence applications, rapid fixation (e.g., with paraformaldehyde) is essential to preserve phosphorylation states .

Differentiating between total LCK protein expression and its activated (phosphorylated) form requires strategic experimental approaches:

• Dual antibody detection: Use both anti-total LCK and anti-phospho-LCK (Y393) antibodies on parallel samples or in sequential blotting of the same membrane after stripping

• Ratio calculation: Quantify the activation state by determining the ratio of pY394-LCK (active) to Y394-nonphospho-LCK (inactive) detected by Western blot

• Pharmacological manipulation: Treat samples with pervanadate (increases phosphorylation) or PP2 (decreases phosphorylation) to demonstrate antibody specificity and phosphorylation dynamics

• Mutant controls: Use Y394F LCK mutants that cannot be phosphorylated at this site as negative controls for phospho-specific antibody reactivity

• Fluorescence microscopy: Dual staining with total LCK (different fluorophore) and phospho-LCK antibodies can reveal spatial distribution of activated versus total LCK pools

This comprehensive approach allows researchers to determine whether observed changes reflect alterations in LCK expression or specific changes in its activation state.

How can phospho-LCK (Y393) antibodies distinguish between free and coreceptor-bound LCK activity?

Research has revealed significant functional differences between free LCK and coreceptor-bound LCK in T cells. Free LCK exhibits higher mobility and activity compared to coreceptor-bound LCK, with increased phosphorylation at the activating Y393 site regardless of TCR activation strength . Investigating these distinct LCK pools requires sophisticated experimental approaches:

• Sequential immunoprecipitation: First immunoprecipitate CD4/CD8 to isolate coreceptor-bound LCK, then immunoprecipitate remaining LCK from the supernatant to obtain the free LCK fraction

• Phosphorylation ratio analysis: For each fraction, determine the ratio of pY394/Y394-nonphospho LCK and pY505/total LCK to assess activation states

• Fluorescence microscopy: Perform co-localization studies with phospho-LCK (Y393) antibodies and CD4/CD8 antibodies to visualize spatial distribution

• Flow cytometry: Use multiparameter flow cytometry with surface staining for coreceptors and intracellular staining for phospho-LCK

This methodological approach revealed that "the pY394/Y394-nonphospho LCK ratio was higher, and the pY505/total LCK ratio was lower, in the free LCK compared to CD8-bound LCK," demonstrating differential activation states between these pools .

What controls should be included when using phospho-LCK (Y393) antibodies?

Rigorous experimental design for phospho-LCK (Y393) studies requires comprehensive controls:

When using phospho-specific antibodies in Western blot applications, it's particularly informative to show paired lanes of untreated (-) and pervanadate-treated (+) samples to demonstrate specificity, as seen in multiple validation studies .

How do Y393 and Y505 phosphorylation states interact to regulate LCK activity?

LCK activity is regulated through a complex interplay between phosphorylation at the activating Y393 site and the inhibitory Y505 site, creating a sophisticated molecular switch system:

• When Y505 is phosphorylated by C-terminal Src kinase (CSK), it associates with LCK's own SH2 domain, creating a "closed" conformation with inhibited kinase activity

• Dephosphorylation of Y505 by CD45 results in conformational opening, making Y393 accessible for autophosphorylation

• Autophosphorylation of Y393 stabilizes the open structure, enhancing kinase activity and substrate binding

• Dephosphorylation of Y393 by phosphatases (CD45, SHP-1, PTPN2, DUSP22) negatively regulates TCR signaling

This regulation creates four possible LCK states based on phosphorylation combinations:

Notably, "the Y394 and Y505 double phosphorylated LCK has similar activity to the Y394 single-phosphorylated LCK," indicating that Y393 phosphorylation can override the inhibitory effect of Y505 phosphorylation . This equilibrium is maintained by coordinated actions of kinases and phosphatases in resting T cells and can shift during activation.

What are potential pitfalls in interpreting phospho-LCK (Y393) data?

Several technical and biological factors can complicate the interpretation of phospho-LCK (Y393) experimental data:

• Post-lysis autophosphorylation: LCK can transautophosphorylate at Y393 even after cell lysis, potentially yielding artificially elevated activity in Western blotting or immunoprecipitation assays . Solution: Include SFK inhibitors in lysis buffers.

• Antibody cross-reactivity: Due to high sequence homology, some phospho-LCK (Y393) antibodies may cross-react with other phosphorylated Src family members . Solution: Validate with LCK-deficient cells or LCK knockdown.

• Heterogeneous LCK pools: Total LCK comprises approximately equal fractions of differently phosphorylated species (Y393-/Y505-, Y393-/Y505+, Y393+/Y505-, Y393+/Y505+) . Solution: Use antibodies specific for each phosphorylation state.

• Cell line versus primary cell differences: Jurkat cells (commonly used in research) have different baseline LCK phosphorylation patterns than primary T cells . Solution: Validate findings in primary cells.

• Rapid phosphorylation dynamics: LCK phosphorylation states change quickly after stimulation, making timing critical . Solution: Include detailed time-course experiments.

• Protein expression variations: LCK protein levels may vary significantly between samples, confounding phosphorylation analysis . Solution: Always normalize phospho-LCK to total LCK.

How can phospho-LCK (Y393) antibodies investigate T-cell receptor signaling defects?

Phospho-LCK (Y393) antibodies are valuable tools for investigating TCR signaling defects in both research and clinical contexts:

• Comparing patient samples: In cases of suspected T cell immunodeficiencies, phospho-LCK (Y393) antibodies can assess LCK activation in patient T cells versus healthy controls following TCR stimulation

• Structure-function analysis: Using site-directed mutagenesis to create LCK variants (like the clinically relevant P440S variant) followed by phospho-Y393 detection can reveal how structural changes affect kinase activation

• Signaling pathway mapping: Phospho-LCK (Y393) analysis in combination with inhibitors of upstream or downstream components can elucidate pathway dependencies and potential compensatory mechanisms

• Therapeutic target validation: Monitoring phospho-LCK (Y393) levels following treatment with experimental compounds can confirm on-target activity of drugs designed to modulate TCR signaling

• Zap70 regulation studies: Phospho-LCK (Y393) antibodies can help investigate the relationship between LCK activity and Zap70 expression/phosphorylation, revealing regulatory mechanisms in T cell signaling

A recent study demonstrated that "a partial human LCK defect causes a T cell immunodeficiency," using phospho-LCK detection to characterize signaling defects . In silico 3D protein modeling suggested that the P440S LCK variant caused "distortion of the activating loops, likely resulting in significant instability of the region surrounding the P440S variant and altered interaction of Y394 with neighboring amino acids" .

Technical Considerations

Thorough validation ensures reliable experimental results with phospho-LCK (Y393) antibodies:

• Pervanadate treatment: Treating cells with pervanadate (phosphatase inhibitor) should increase phospho-LCK (Y393) signal, confirming phospho-specificity

• SFK inhibitor treatment: PP2 treatment should reduce phospho-LCK (Y393) signal by inhibiting autophosphorylation

• Peptide competition: Pre-incubating the antibody with phospho-Y393 peptide should block specific binding and eliminate signal

• Y393F mutant expression: Cells expressing LCK with Y393F mutation should show no reactivity with phospho-specific antibodies regardless of treatment

• siRNA/CRISPR knockdown: LCK-depleted cells should show minimal or absent signal

• Western blot molecular weight confirmation: Phospho-LCK (Y393) should appear at approximately 56 kDa

• Cell type specificity: Strong signals in T cells (Jurkat) with minimal background in non-T cells

In published validations, Western blots typically show paired lanes of untreated (-) and pervanadate-treated (+) samples, with a specific band detected for phospho-LCK (Y393) at approximately 56 kDa, confirming both specificity and molecular weight .

How can phospho-LCK (Y393) analysis contribute to understanding human immunodeficiencies?

Phospho-LCK (Y393) antibodies have proven valuable in characterizing novel immunodeficiencies and T cell disorders:

Recent research identified a partial human LCK deficiency causing T cell immunodeficiency with defective TCR signaling. In this study, phospho-LCK (Y393) analysis revealed how a specific LCK variant (P440S) affected kinase function . In silico protein modeling suggested that this variant caused "distortion of the activating loops" and "altered interaction of Y394 with neighboring amino acids in the kinase pseudo substrate domain" .

Methodologically, researchers can implement:

• Comparative phospho-flow cytometry between patient and healthy control T cells following TCR stimulation

• Time-course analysis of phospho-LCK kinetics in response to activation signals

• Correlation of phospho-LCK levels with clinical phenotypes and downstream signaling events

• Structure-function studies using patient-derived mutations expressed in model systems

• Therapeutic targeting strategies based on phosphorylation profiles

These approaches can identify novel signaling defects, characterize disease mechanisms, and potentially suggest therapeutic interventions for T cell disorders.

What is the relationship between LCK phosphorylation at Y393 and ZAP70 regulation?

The interplay between LCK and ZAP70 represents a critical regulatory node in T cell signaling, with recent findings revealing unexpected complexity:

Research has discovered that "Zap70 expression is negatively regulated by Lck activity: augmented Lck activity resulting in severe diminution in total Zap70" . This negative feedback mechanism may help maintain signaling homeostasis in T cells.

Experimental approaches to investigate this relationship include:

• Co-transfection studies with plasmids encoding ZAP70 and LCK variants, followed by flow cytometry analysis

• Mutation of key tyrosine residues (Y315F/Y319F) in ZAP70 to determine phosphorylation dependencies

• Pharmacological manipulation of LCK activity using inhibitors or activators, followed by ZAP70 protein level assessment

• Time-course analysis of phospho-LCK (Y393) and total/phospho-ZAP70 following TCR stimulation

• Analysis of protein turnover rates in cells with varying levels of LCK activity

This relationship forms "the mechanistic basis" for intricate T-cell signaling regulation and highlights the importance of studying phosphorylation networks rather than isolated proteins .

What are emerging technologies for phospho-LCK (Y393) detection in research and clinical applications?

Recent technological advances offer new opportunities for phospho-LCK (Y393) analysis:

• Single-cell phospho-proteomics: Enables analysis of phospho-LCK (Y393) in individual cells within heterogeneous populations, revealing signaling diversity not detectable in bulk analyses

• LANCE Ultra homogeneous assays: No-wash proximity assay technology that combines time resolution with fluorescence, streamlining phospho-LCK detection in high-throughput formats

• Multiplex phosphorylation analysis: Simultaneous detection of phospho-LCK (Y393) alongside other phosphorylation sites (Y505) and downstream signaling molecules

• Automated cell-based ELISAs: High-throughput screening systems that enable rapid analysis of phospho-LCK responses to various stimuli or drug candidates

• In vivo imaging: Development of techniques to visualize phospho-LCK dynamics in living cells or tissues using phosphorylation-sensitive fluorescent reporters