Phospho-LCK (Y505) Antibody

What is the biological significance of LCK Y505 phosphorylation in T-cell signaling?

Phosphorylation at tyrosine 505 (Y505) serves as a negative-regulatory mechanism for LCK activity. When Y505 is phosphorylated by C-terminal Src Kinase (CSK), LCK adopts a closed, inactive conformation through an intramolecular interaction between phosphorylated Y505 and its SH2 domain. This regulatory mechanism prevents inappropriate T-cell responses and maintains signaling homeostasis .

How can researchers distinguish between different LCK conformational states?

Researchers can distinguish between different LCK conformational states by using antibodies specific to different phosphorylation sites:

• Closed/inactive conformation: Use Phospho-LCK (Y505) antibodies to detect LCK with phosphorylated Y505, representing the inhibitory state.

• Open/active conformation: Use Phospho-LCK (Y394) antibodies to detect LCK with phosphorylated Y394, representing the active state.

• "Primed" state: This refers to LCK where both Y394 and Y505 are not phosphorylated .

For comprehensive analysis, researchers should calculate relative ratios:

• The pY394/Y394-nonphospho ratio indicates active LCK proportion

• The pY505/total LCK ratio indicates the proportion of LCK in closed conformation

Importantly, Y394 and Y505 double-phosphorylated LCK can still maintain an open conformation with kinase activity, despite Y505 phosphorylation .

What are the recommended applications for Phospho-LCK (Y505) antibodies?

Based on commercially available antibodies and published research protocols, recommended applications include:

For all applications, researchers should include appropriate controls, such as pervanadate-treated cells (increases phosphorylation) and PP2-treated cells (decreases phosphorylation) to validate specificity .

What techniques preserve phosphorylation status during cell lysis for accurate Phospho-LCK (Y505) detection?

Preserving phosphorylation states during cell lysis is critical for accurate assessment of LCK phosphorylation. Research indicates that LCK can undergo trans-autophosphorylation even after cell lysis, potentially confounding results . To prevent this artifact:

• Optimized lysis buffer: Supplement standard lysis buffer with:

  • SFK inhibitor PP2 (20μM)

  • Protease-phosphatase inhibitor mixture

  • Immediate processing on ice

• Hot lysis method: For maximum preservation of phosphorylation status:

  • Lyse cells directly in hot 2× sample buffer

  • Boil immediately

  • Sonicate lysates to shear DNA and reduce viscosity

• Rapid freezing: Flash-freeze samples in liquid nitrogen before processing if immediate analysis is not possible.

• Validation approach: Compare results with phosphoflow cytometry, which preserves phosphorylation states through rapid fixation .

These methodological considerations are essential as the ratio of pY394/Y394-nonphospho can be artificially altered during improper sample handling .

How does the relationship between free and coreceptor-bound LCK pools affect experimental design?

Research demonstrates that free LCK and coreceptor-bound LCK represent distinct pools with different phosphorylation patterns and biological activities. This has significant implications for experimental design:

• Differential phosphorylation status: Free LCK has higher pY394/Y394-nonphospho ratios and lower pY505/total LCK ratios compared to coreceptor-bound LCK, indicating greater activity in the free pool .

• Experimental separation techniques:

  • Sequential immunoprecipitation with anti-CD4/CD8 antibodies (3 rounds) to deplete coreceptor-bound LCK

  • Flow cytometry immunoprecipitation (FC-IP) to monitor depletion efficiency

  • Western blot analysis of supernatant (free LCK) versus immunoprecipitates (coreceptor-bound LCK)

• Mobility differences: Free LCK demonstrates higher mobility compared to coreceptor-bound LCK .

• Stimulus response variations: The phosphorylation status of free and CD8-bound LCK remains relatively constant regardless of TCR activation strength, suggesting intrinsic regulatory mechanisms beyond TCR signaling .

For comprehensive LCK analysis, researchers should separately evaluate these pools rather than analyzing total LCK in cell lysates, which may mask important biological differences.

What role does FAK play in regulating LCK Y505 phosphorylation and how should this be considered experimentally?

Focal adhesion kinase (FAK) functions as a negative regulator of TCR-mediated signaling by influencing LCK Y505 phosphorylation. This regulatory mechanism should be considered when designing experiments:

• Mechanism of regulation: FAK recruits C-terminal Src kinase (Csk) to the membrane and/or receptor complex following TCR activation, leading to inhibitory phosphorylation of LCK at Y505 .

• Experimental approaches to study this relationship:

  • Correlation analysis: Measure relationship between FAK expression and LCK Y505 phosphorylation levels

  • FAK suppression experiments: Monitor changes in LCK phosphorylation ratios

  • Normalized quantification: Express data as ratio of pY505/total LCK

• Technical considerations:

  • Use anti-CD3/CD28 coated beads with IL-2 for T cell activation

  • Normalize expression data to actin for valid comparisons

  • Apply Pearson Correlation Coefficient and linear regression analysis to evaluate relationships

Understanding this regulatory axis is important when studying T cell malignancies and autoimmune diseases where abnormal T cell responses may involve dysregulation of this pathway .

How can researchers effectively measure dynamic changes in LCK Y505 phosphorylation during T cell activation?

Measuring dynamic phosphorylation changes requires specialized approaches:

• Time-course experiments with rapid sampling:

  • Stimulate T cells with anti-CD3/CD28 or peptide-MHC complexes

  • Collect samples at closely spaced time points (seconds to minutes)

  • Immediately fix to preserve phosphorylation state

• Phosphoflow cytometry optimization:

  • Superior temporal resolution compared to Western blotting

  • Enables simultaneous measurement of multiple parameters

  • Can distinguish phosphorylation in specific cell subsets

  • Requires careful validation with positive controls (pervanadate treatment)

• Specialized techniques for membrane dynamics:

  • FRET-based approaches to monitor LCK conformational changes

  • Live-cell imaging with fluorescently tagged proteins

  • Single-molecule tracking to follow individual LCK molecules

• Quantification strategies:

  • Calculate pY505/total LCK ratio changes over time

  • Compare with pY394/Y394-nonphospho ratios to assess net LCK activity

  • Correlate with downstream markers of T cell activation

These approaches allow researchers to correlate changes in LCK Y505 phosphorylation with functional outcomes in T cell biology.

How do Phospho-LCK (Y505) levels correlate with TCR condensation and signal propagation?

Recent research reveals a dynamic relationship between LCK phosphorylation states, T cell receptor condensation, and signal propagation:

• Conformational influence on condensation:

  • LCK conformation affects CD3ε/LCK condensation efficiency

  • Open-conformation LCK (Y505F mutation) forms larger condensates than wild-type LCK

  • These condensates enhance TCR signaling through spatial organization

• Spatiotemporal dynamics during T cell activation:

  • LCK with pY394 (active) localizes with TCR in peripheral clusters

  • LCK with pY505 (inactive) predominantly clusters at the central supramolecular activation complex (cSMAC)

  • This spatial segregation creates distinct signaling zones

• Csk-mediated regulation:

  • After CD3ε phosphorylation, Csk is recruited to phosphorylated CD3ε

  • This recruitment dissolves CD3ε/LCK condensates

  • The process provides a negative feedback mechanism to limit TCR signaling duration

  • Mutation of CD3ε ITAM tyrosines (CD3ε-YYFF) prevents Csk-mediated condensate dissolution

• Functional outcomes:

  • TCRs carrying CD3ε-YYFF mutations show increased microcluster formation

  • Greater colocalization with active LCK (pY394)

  • Reduced colocalization with inactive LCK (pY505)

  • Enhanced TCR signaling and increased IL-2 production

These findings highlight the importance of considering LCK Y505 phosphorylation within the broader context of membrane dynamics and protein condensation during T cell activation.

What are the methodological considerations for studying pathological alterations in LCK Y505 phosphorylation?

Studying pathological alterations in LCK Y505 phosphorylation, particularly in disease contexts, requires specialized approaches:

• Patient sample handling for phosphorylation preservation:

  • Process samples immediately after collection

  • Cryopreserve cells in media containing phosphatase inhibitors

  • Validate phospho-protein stability in frozen samples

• Disease-specific considerations:

  • In PAX5 translocated leukemia: Enhanced LCK activity correlates with reduced pY505, driving aberrant STAT5 signaling

  • Evaluate potential therapeutic targeting with LCK inhibitors like BIBF1120

  • Monitor both basal and cytokine-stimulated (IL-7) STAT5 activation patterns

• Experimental controls for pathological samples:

  • Compare with matched healthy donor samples

  • Include disease controls (e.g., PAX5 wildtype and PAX5 deleted for leukemia studies)

  • Standardize activation conditions (co-culture on OP9 stroma for ALL samples)

• Analysis parameters:

  • Measure percentage of cells positive for STAT5 Y694 phosphorylation

  • Evaluate response to pathway inhibitors

  • Consider dual-phosphorylation status (pY394/pY505 ratio)

These methodological approaches enable researchers to investigate LCK dysregulation in pathological conditions and evaluate potential therapeutic interventions targeting the LCK pathway.

How can researchers accurately quantify the ratio between different LCK phosphorylation states?

Accurate quantification of LCK phosphorylation states is essential for understanding its functional status in T cells:

• Western blot quantification approaches:

  • Use antibodies specific for pY394, pY505, Y394-nonphosphorylated, and total LCK

  • Calculate ratios: pY394/Y394-nonphospho (activation indicator) and pY505/total LCK (inhibition indicator)

  • Include pervanadate (phosphatase inhibitor) and PP2 (SFK inhibitor) treated controls to establish dynamic range

  • Normalize to whole cell lysate for consistent comparison

• Flow cytometry quantification:

  • Use fluorescently conjugated antibodies (PE-conjugated for optimal resolution)

  • Report data as percentage of positive cells or mean fluorescence intensity

  • Consider dual staining for both pY394 and pY505 to identify cell populations with different LCK activation states

  • Apply proper compensation when using multiple fluorophores

• Standardization approaches:

  • Use Jurkat cells with known LCK phosphorylation states as reference standards

  • Normalize to total LCK expression when comparing different samples

  • Account for cell-specific expression levels of regulatory proteins (CD45, CSK)

• Statistical analysis:

  • Apply Pearson Correlation Coefficient and linear regression for relationship analysis

  • Use appropriate statistical tests to determine significance of differences between experimental conditions

  • Consider biological variability when interpreting results

These quantification strategies provide robust assessment of LCK's activation state and regulatory mechanisms in experimental settings.

What are common pitfalls when working with Phospho-LCK (Y505) antibodies and how can they be overcome?

Researchers frequently encounter several challenges when working with Phospho-LCK (Y505) antibodies:

• Post-lysis phosphorylation artifacts:

  • Problem: LCK can undergo trans-autophosphorylation even after cell lysis

  • Solution: Add SFK inhibitor PP2 (20μM) to lysis buffer; use rapid denaturation protocols

• Cross-reactivity with other SFK family members:

  • Problem: Structural similarities between SFK family proteins can lead to antibody cross-reactivity

  • Solution: Validate antibody specificity using knockout controls or SFK-deficient cell lines; use peptide competition assays

• Variable phospho-epitope accessibility:

  • Problem: Conformational changes can mask epitopes in intact proteins

  • Solution: Optimize denaturation conditions; consider dot blot analysis with phosphopeptides

• Rapid phosphatase activity during sample preparation:

  • Problem: Loss of phosphorylation signal during processing

  • Solution: Use phosphatase inhibitor cocktails; process samples rapidly on ice; validate results with multiple techniques

• Background in immunofluorescence applications:

  • Problem: Non-specific staining in flow cytometry or microscopy

  • Solution: Optimize fixation and permeabilization conditions; include fluorescence-minus-one (FMO) controls; validate with phosphorylation-modifying treatments

Implementing these solutions will improve data quality and reliability when working with Phospho-LCK (Y505) antibodies.

What controls should be included when validating Phospho-LCK (Y505) antibody specificity?

Proper validation requires a comprehensive set of controls:

• Positive controls:

  • Pervanadate treatment: Inhibits phosphatases, increasing pY505 levels

  • H₂O₂ treatment: Activates signaling pathways leading to increased pY505

  • Jurkat cells: Well-characterized T cell line with established LCK expression and phosphorylation

• Negative controls:

  • PP2 treatment: SFK inhibitor that prevents Y505 phosphorylation

  • Y505F mutant LCK: Site-directed mutagenesis eliminates phosphorylation site

  • Phosphatase treatment of lysates: Enzymatically removes phosphorylation

• Specificity controls:

  • Peptide competition assays: Pre-incubation with phospho-Y505 peptide should block antibody binding

  • Immunodepletion: Sequential immunoprecipitation should reduce signal

  • Cross-reactivity testing: Evaluate antibody against other SFK family members

• Application-specific controls:

  • For Western blot: Include molecular weight markers and total LCK antibody

  • For flow cytometry: Include fluorescence-minus-one and isotype controls

  • For immunoprecipitation: Use IgG control and verify with Western blot