Phospho-LAT (Y191) Recombinant Monoclonal Antibody

What is LAT and why is phosphorylation at Y191 significant?

LAT (Linker for Activation of T-cells family member 1) is a critical palmitoylated and membrane-associated adaptor protein that becomes phosphorylated at multiple tyrosine residues upon T cell receptor (TCR) stimulation. Phosphorylation at Y191 is particularly significant as it serves as one of the key docking sites for signaling molecules that form the LAT signalosome. Upon TCR stimulation, the kinase ZAP-70 phosphorylates LAT at multiple tyrosine residues, including Y132, Y171, Y191, and Y226, allowing phosphorylated LAT to function as an essential scaffold for the assembly of TCR-coupled signaling complexes that mediate productive T cell activation . The Y191 phosphorylation site is part of the critical network that enables downstream signal propagation in T cells, making antibodies against this specific phosphorylation site valuable tools for investigating T cell signaling mechanisms.

How does a recombinant monoclonal antibody differ from conventional antibodies for detecting phospho-LAT?

Recombinant monoclonal antibodies against phospho-LAT (Y191) are produced through molecular biology techniques using plasmids containing the coding sequence of the LAT monoclonal antibody expressed in host cell lines like HEK293F . Unlike conventional antibodies produced from hybridomas, these recombinant antibodies offer several advantages for phospho-LAT research, including:

• Higher reproducibility between batches due to the defined genetic sequence

• Greater specificity for the phosphorylated Y191 residue due to targeted design

• Reduced background binding and cross-reactivity

• Consistent affinity and performance characteristics

• More sustainable production without animal immunization for each batch

The recombinant nature ensures that researchers receive a consistent research tool with predictable binding properties across experiments and over time, which is especially important when studying dynamic phosphorylation events in signaling cascades .

What are the optimal applications for Phospho-LAT (Y191) antibodies in T cell signaling research?

Phospho-LAT (Y191) antibodies are versatile tools for investigating T cell activation and signaling pathways with several recommended applications:

The antibody is particularly valuable in studies examining the formation of the immunological synapse, temporal dynamics of LAT phosphorylation following TCR engagement, and assembly of signaling complexes at the membrane . When designing experiments, researchers should consider the kinetics of phosphorylation at Y191, which can be transient and stimulus-dependent, requiring careful timing of sample collection after T cell activation.

How should experiments be designed to accurately capture LAT phosphorylation dynamics?

Accurately capturing LAT phosphorylation dynamics requires careful experimental design due to the rapid and transient nature of phosphorylation events. Based on current research methodologies:

• Time course considerations: LAT phosphorylation at Y191 occurs within minutes of TCR stimulation. Design experiments with multiple early time points (30 seconds, 1, 2, 5, 10 minutes) to capture rapid phosphorylation kinetics .

• Stimulation protocols: Use physiologically relevant stimulation methods:

  • Anti-CD3/CD28 antibodies for polyclonal activation

  • Peptide-loaded APCs for antigen-specific responses

  • Surface-bound stimuli for studying immunological synapse formation

• Sample preparation: Quick sample processing with immediate lysis in phosphatase inhibitor-containing buffers is essential to prevent dephosphorylation artifacts.

• Complementary techniques: TIRF microscopy can be used alongside antibody-based detection to visualize LAT condensate formation in real-time, as studies have shown Grb2 recruitment (a marker of LAT phosphorylation) occurs simultaneously with LAT condensation approximately 22.1 ± 2.0 seconds after TCR engagement .

• Controls: Include both positive controls (strongly activated T cells) and negative controls (phosphatase-treated samples or Y191F LAT mutants) to establish detection specificity and dynamic range .

To accurately assess signaling dynamics, researchers should consider that phosphorylation at Y191 may be dependent on previous phosphorylation events at other residues, emphasizing the importance of comprehensive phosphorylation analysis across multiple sites .

How does LAT Y191 phosphorylation relate to phase separation and condensate formation in T cell signaling?

Recent research has revealed that LAT phosphorylation, including at Y191, plays a critical role in facilitating phase separation and condensate formation during T cell activation. These findings represent a paradigm shift in understanding T cell signaling:

• LAT condensate formation: Phosphorylated LAT (pLAT) forms discrete, mobile condensates following TCR stimulation. These condensates represent high-density protein assemblies that facilitate signal transduction. Studies using TIRF microscopy have shown that LAT condensates emerge with a mean delay time of approximately 22 seconds after TCR engagement .

• Molecular basis: The phosphorylation of multiple tyrosine residues, including Y191, creates multivalent binding sites for SH2 domain-containing proteins like Grb2. When Grb2 and SOS are present in solution with phosphorylated LAT, macroscopic network assemblies form on membranes, with estimated densities of ~4,000 molecules per square micron .

• Dynamic properties: These LAT condensates demonstrate liquid-like properties with internal protein mobility. FRAP (Fluorescence Recovery After Photobleaching) measurements reveal recovery of intensity in photobleached spots with a diffusion coefficient (Deffective) of approximately 0.20 μm²/s .

• Reversibility: Introduction of tyrosine phosphatases (such as YopH) rapidly reverses condensate formation within minutes, demonstrating the dynamic and phosphorylation-dependent nature of these structures .

• Signal regulation: The formation of these condensates appears to encode antigen information, with single-molecule binding events triggering distinct LAT condensation patterns that may contribute to the ability of T cells to discriminate between antigens .

Understanding these phase separation dynamics has profound implications for how we conceptualize and investigate signaling pathways in immune cells, suggesting that spatial organization through condensation may represent a fundamental regulatory mechanism in cell signaling .

What is the interplay between tyrosine and serine phosphorylation in regulating LAT function?

Recent research has uncovered a complex interplay between tyrosine and serine phosphorylation in LAT, revealing a previously underappreciated regulatory mechanism in T cell signaling:

• DNA-PKcs as a LAT kinase: Recent phospho-mass spectrometry studies have identified DNA-PKcs as a kinase that phosphorylates LAT at serine residues, particularly S224 and S241. These serine residues are located within conserved S/Q motifs, known DNA-PKcs consensus sites .

• Hierarchical regulation: Phosphorylation at S224 and S241 appears to be critical for the subsequent phosphorylation of tyrosine residues, including Y191. Studies using LAT mutants (S224A, S241A, and S2A double mutant) revealed that phosphorylation at these serine residues is essential for phosphorylation at Y132, Y171, and Y191 following TCR stimulation .

• Functional impact: LAT mutants lacking these serine phosphorylation sites showed normal proliferation but failed to rescue IL-2 production in LAT-deficient cells, indicating these sites are crucial for certain signaling outcomes .

• Signalosome composition effects: Mass spectrometry analysis revealed that serine phosphorylation, especially at S241, regulates not only which proteins bind to LAT but also the release of proteins during T cell activation. This suggests a dual role in both recruitment and dissociation of signaling components .

• Cell type differences: DNA-PKcs deficiency shows differential impacts on CD4+ versus CD8+ T cells, with a more pronounced effect on the cytotoxic functions of CD8+ T cells, suggesting context-dependent roles for this phosphorylation pathway .

This emerging understanding of the hierarchical relationship between serine and tyrosine phosphorylation reveals new layers of complexity in LAT signaling and may provide novel therapeutic opportunities for modulating specific T cell responses .

What are common issues when working with phospho-specific antibodies and how can they be addressed?

Working with phospho-specific antibodies like anti-phospho-LAT (Y191) presents several technical challenges that researchers should anticipate:

• Loss of phosphorylation signal:

  • Problem: Rapid dephosphorylation during sample preparation

  • Solution: Include comprehensive phosphatase inhibitor cocktails in lysis buffers; maintain samples at 4°C during processing; consider shorter lysis times and immediate denaturation in SDS sample buffer for Western blotting applications

• Non-specific binding:

  • Problem: Cross-reactivity with similar phospho-epitopes

  • Solution: Validate specificity using LAT-deficient cells (such as J.LAT) or Y191F mutants as negative controls; optimize antibody dilution as recommended (1:500-1:5000 for WB, 1:50-1:200 for IHC)

• Variable signal strength:

  • Problem: Inconsistent stimulation of cells leading to variable phosphorylation levels

  • Solution: Standardize stimulation protocols; include positive controls with strong stimulation; consider time course experiments to identify optimal detection windows

• Epitope masking:

  • Problem: Protein-protein interactions at the signalosome may obscure the Y191 epitope

  • Solution: Test different lysis conditions and detergents; consider gentle fixation methods for IF/IHC that preserve phospho-epitopes while maintaining cellular architecture

• Sensitivity limitations:

  • Problem: Low detection sensitivity in samples with minimal phosphorylation

  • Solution: Consider signal amplification methods; use complementary approaches like Phos-tag gels or mass spectrometry for confirmation; implement proximity ligation assays for detection of low-abundance phosphorylated proteins

For optimal results, researchers should perform preliminary experiments to establish the kinetics of LAT phosphorylation in their specific experimental system, as timing is critical for capturing this transient modification .

How can phospho-LAT (Y191) antibodies be integrated with other methodologies to provide comprehensive signaling analysis?

Integration of phospho-LAT (Y191) antibody detection with complementary methodologies can provide a more comprehensive understanding of T cell signaling dynamics:

• Multi-parametric flow cytometry:

  • Combine phospho-LAT (Y191) detection with other phospho-specific antibodies (phospho-ZAP70, phospho-PLCγ1, phospho-ERK) to assess signaling pathway activation at the single-cell level

  • Allows correlation of LAT phosphorylation with cellular phenotypes and functional outcomes

• Advanced microscopy approaches:

  • TIRF microscopy enables visualization of membrane-proximal LAT phosphorylation events and condensate formation in real-time

  • Super-resolution microscopy (STORM, PALM) can resolve nanoscale organization of LAT signalosomes

  • Live-cell imaging with fluorescent reporters can track temporal dynamics of LAT recruitment and phosphorylation

• Proximity-based assays:

  • Proximity ligation assays (PLA) can detect specific protein-protein interactions dependent on Y191 phosphorylation

  • FRET-based sensors can monitor conformational changes and protein associations resulting from LAT phosphorylation

• Proteomic approaches:

  • Immunoprecipitation with phospho-LAT (Y191) antibodies followed by mass spectrometry can identify novel interaction partners

  • Phospho-proteomics can map the signaling networks downstream of LAT Y191 phosphorylation

• Genetic approaches:

  • CRISPR-Cas9 editing to create Y191F mutants for functional studies

  • Reconstitution experiments in LAT-deficient cells to assess the specific contribution of Y191 phosphorylation to various T cell functions

By integrating these methodologies, researchers can move beyond simple detection of LAT phosphorylation to understand its spatial organization, temporal dynamics, and functional consequences in various physiological and pathological contexts .

How might differential phosphorylation patterns of LAT relate to T cell fate decisions?

Recent research suggests that differential phosphorylation patterns of LAT, including at Y191, may play a crucial role in determining T cell fate decisions and functional outcomes:

• Kinetic proofreading and antigen discrimination:

  • The timing and magnitude of LAT phosphorylation appears to encode information about antigen quality. Studies have shown that LAT condensation occurs with a characteristic delay (~22 seconds) following TCR engagement .

  • This timing mechanism may contribute to the ability of T cells to discriminate between self and foreign antigens, with strong agonists inducing more rapid and robust LAT phosphorylation.

• CD4+ vs. CD8+ T cell responses:

  • Differential requirements for LAT phosphorylation have been observed between CD4+ and CD8+ T cells. DNA-PKcs-dependent LAT phosphorylation appears more critical for CD8+ T cell cytotoxic functions compared to CD4+ T cells .

  • This suggests that distinct phosphorylation patterns might contribute to lineage-specific functions.

• Effector vs. memory T cell differentiation:

  • Emerging evidence suggests that the amplitude and duration of LAT signaling may influence T cell differentiation decisions between effector and memory fates.

  • Weaker or more transient LAT phosphorylation might favor memory cell development, while stronger, sustained phosphorylation may drive terminal effector differentiation.

• Cytokine production vs. proliferation:

  • LAT mutants lacking serine phosphorylation sites (S224A, S241A) show normal proliferation but impaired IL-2 production, indicating specific phosphorylation patterns may selectively regulate certain functional outputs .

  • The composition of the LAT signalosome appears to be differentially affected by specific phosphorylation patterns, potentially explaining these selective functional effects.

Future research should explore how combinatorial phosphorylation patterns across multiple LAT residues collectively influence T cell fate decisions in various immunological contexts, potentially leading to new therapeutic strategies for modulating specific immune responses .

What are the methodological considerations for studying LAT condensates in relation to phosphorylation status?

Studying the relationship between LAT phosphorylation and condensate formation requires specialized methodological approaches that preserve the dynamic and often transient nature of these structures:

• Live-cell imaging systems:

  • TIRF microscopy is essential for visualizing membrane-proximal LAT condensates with minimal background

  • Spinning disk confocal microscopy allows for rapid 3D acquisition to capture condensate dynamics

  • Implement temperature-controlled stages (37°C) to maintain physiological conditions that support condensate formation

• Fluorescent protein tagging strategies:

  • Use monomeric fluorescent proteins (mNeonGreen, mScarlet) that minimize artifacts in phase separation studies

  • Consider fluorescent protein location (N- vs C-terminal) to avoid interfering with LAT membrane localization

  • Implement bicistronic P2A vectors containing LAT-mScarleti and mNeonGreen-GRB2 to simultaneously monitor LAT and binding partners

• Quantitative analysis approaches:

  • Develop automated image analysis pipelines to identify and track condensates over time

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to measure protein mobility within condensates

  • Calculate diffusion coefficients to characterize the material properties of LAT condensates (reported Deffective of 0.20 μm²/s)

• Reconstitution systems:

  • Consider in vitro reconstitution of LAT condensates using purified components on supported lipid bilayers

  • Systematically vary the phosphorylation state of LAT to determine the minimal requirements for condensate formation

  • Control the concentration of binding partners (e.g., 5.8 μM Grb2 and 1.45 μM SOS) to study concentration-dependent effects

• Verification controls:

  • Use phosphatase treatments (e.g., 10 μM YopH) to confirm phosphorylation-dependence of observed condensates

  • Implement LAT mutants with altered phosphorylation sites to determine the contribution of specific residues

  • Consider LAT(4F) mutants with Y136, Y175, Y195, and Y235 mutated to phenylalanine as negative controls

These methodological considerations ensure that researchers can accurately capture and characterize the complex relationship between LAT phosphorylation status and its organization into functional condensates that mediate T cell activation .