Phospho-ZAP70 (Tyr319) Antibody
Description
Biological Role of ZAP-70 and Tyr319 Phosphorylation
ZAP-70 (ζ-chain-associated protein kinase 70) is a cytoplasmic tyrosine kinase essential for TCR-mediated signal transduction. Upon TCR engagement:
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Phosphorylation of Tyr319: Occurs after immune receptor tyrosine-based activation motif (ITAM) phosphorylation by Lck kinase .
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Functional Impact: Phosphorylated Tyr319 facilitates Lck binding, enabling downstream phosphorylation of Tyr493 and activation of nuclear factor of activated T cells (NFAT) and interleukin-2 (IL-2) induction .
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Regulatory Roles:
Mechanism of Phospho-ZAP70 (Tyr319) Antibody Detection
The antibody operates via a homogeneous, no-wash FRET (Förster Resonance Energy Transfer) methodology :
| Detection Principle | Components | Signal Output |
|---|---|---|
| Phospho-specific antibody | Donor fluorophore-labeled anti-pTyr319 | FRET signal proportional to phosphorylation |
| Total ZAP-70 antibody | Acceptor fluorophore-labeled anti-ZAP-70 | Normalization control |
Key advantages over Western blot:
T-Cell Activation Studies
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PD-L1 Inhibition Assay: Pre-incubation of Jurkat cells with 40 µg/mL PD-L1 recombinant protein reduced phospho-Tyr319 levels by 27%, demonstrating its role in immunosuppression .
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Y319F Mutant Model: Mice expressing non-phosphorylatable Y319F ZAP-70 exhibited:
Signaling Pathway Analysis
| Phosphorylation Site | Functional Role | Downstream Effects |
|---|---|---|
| Tyr319 | Binds Lck kinase | NFAT activation, IL-2 production |
| Tyr493 | Kinase domain activation | PLC-γ1/Ras pathway initiation |
| Tyr292 | Suppresses ZAP-70 activity | Attenuates TCR signaling |
Comparative Advantages in Research
Product Specs
Target Background
- ZAP-70 emerged as a robust prognostic biomarker for patients with CLL. PMID: 29680229
- This research demonstrated that natural killer cells can reduce their functional role through downregulation of Syk and Zap70 kinases. PMID: 29263215
- The study identified a tight negative feedback loop where ZAP-70-activated p38 reciprocally phosphorylates ZAP-70 and destabilizes the signaling complex. PMID: 29440413
- A novel redox-active motif crucial for regulating Zap70 stability/activity was identified. This motif holds potential as a target for developing therapeutic tools to modulate the expression/activity of kinases. PMID: 28415650
- The expression of COBLL1, LPL, and ZAP70 correlated with patient prognosis and IGHV mutational status, although not definitively. When all three markers were combined and subjected to ROC analysis, the AUC increased compared to the AUC of individual gene expression. PMID: 27185377
- The study found that ZAP-70 selects its substrates using an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites surrounded by negatively-charged residues. PMID: 27700984
- The research describes the crucial early step of directional cell movement toward SDF-1, where ZAP-70 is recruited to the CXCR4 at the leading edge of the membrane and subsequently modulates lamellipodia/filopodia formation and integrin activation. PMID: 28846922
- The study established criteria for designing binders that specifically target either the Syk or the Zap-70 Tandem Src Homology 2 Domains, tSH2. While Syk tSH2 exhibits a broader substrate scope, ZAP-70 tSH2 required a proximal arrangement of the phosphotyrosine ligands in a defined strand orientation. PMID: 28767218
- ZAP-70 signaling was impaired by cholesterol depletion, further emphasizing the importance of membrane organization in TCR signaling. PMID: 27384937
- The findings suggest that ZAP70 plays a role in ALL cell homing to and/or survival in the CNS and that ZAP70 could be a therapeutic target. Furthermore, targeting CCR7/CXCR4 may be particularly promising in treating T-ALL. PMID: 27686375
- This study evaluated the expression of ZAP70 changes during disease progression, the intracellular interferon gamma (IFN-gamma) and IL-4 content of T and B lymphocytes and the CLL cell subset (CD5+CD19+) in CLL patients and healthy subjects, and the correlation of ZAP70 with cytokine production. PMID: 26376785
- This research uncovered a cycle of recruitment, activation, and release for Zap70 kinases at phosphorylated T-cell antigen receptors, transforming them into a 'catalytic unit' that amplified antigenic stimuli. PMID: 27869819
- The histological observations suggested that the patients represented diverse cases of NHL like mature B-cell type, mature T-cell type, and high-grade diffuse B-cell type NHL. The findings indicate that patients with NHL may also be analyzed for the status of PAX5, CD19, and ZAP70, and their transcriptional and post-translational variants for the differential diagnosis of NHL and therapy. PMID: 27748274
- Compound heterozygous mutations in the ZAP70 gene are associated with leaky severe combined immunodeficiency disorder. PMID: 28124082
- The findings suggest that genetic polymorphism in the 3' UTR of ZAP-70 is associated with rheumatoid arthritis susceptibility in southern Taiwanese. PMID: 26245723
- Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. PMID: 27052731
- Whole-exome sequencing performed on five family members revealed two affected siblings to be compound heterozygous for two unique missense mutations in the 70-kD T cell receptor zeta-chain associated protein (ZAP-70). PMID: 26783323
- The data suggest that ZO-1, along with CD38 and Zap-70, plays a role in cell cycle regulation in chronic B cell leukemia, and may be used as a prognostic marker in disease monitoring. PMID: 26306999
- A distinct set of protein interaction partners required for chemokine-directed T cell migration is attracted by phosphotyrosine 571 of ADAP, including ZAP70. PMID: 26246585
- Activation of innate immune receptors induces an antiapoptotic signal and proliferation in ZAP-70-positive chronic lymphocytic leukemia dependent on Syk activation. PMID: 26508782
- In all, this study demonstrates that miR-631 decreases PCa cell migration and invasion by dampening ZAP70 expression. PMID: 26620225
- The study examined the differential requirements of ZAP70 and SYK during thymic development. PMID: 26187144
- Blockade of CXCR7 suppressed MIF-mediated ERK- and zeta-chain-associated protein kinase (ZAP)-70 activation. PMID: 26139098
- The kinase activity of ZAP-70 stimulates negative feedback pathways that target Lck and thereby modulate the phosphorylation patterns of the immunoreceptor tyrosine-based activation motifs of T cell receptors. PMID: 25990959
- Low ZAP-70 expression is associated with B-cell chronic lymphoid leukemia. PMID: 25743836
- The study observed decreased CD3 surface expression, reduced ZAP-70 abundance, and increased histone H3-acetylation in activated T lymphocytes after 5 minutes of clinorotation, as well as a transient downregulation of CD3 and stable downregulation of IL-2R. PMID: 25661802
- These findings confirm the role of PTPN22 and CD28 involved in the T cell activation pathway in the development of T1D in Tunisian families. Interestingly, ZAP70 and TCRbeta/CD3z appear to contribute to the susceptibility to the disease in this population. PMID: 25448703
- In ZAP-70(+) patients with B-cell chronic lymphocytic leukaemia, the CD4/CD8 ratio was significantly below the norm, indicating an active disease process. PMID: 25804237
- ZAP-70 and CD 38 positivity were detected in 25% and 36%, respectively, in Chronic lymphocytic leukemia patients with a concordance rate of 56%, which is higher than Western literature. PMID: 24369212
- The results provide the first evidence for differential expression of CD27 among CLL prognostic groups, suggest a role for ZAP-70 dependent signaling in CD27 induction, and implicate CD27 in cell-cell interactions with the lymphoid tissue microenvironment. PMID: 26002513
- The novel biosensor ROZA-XL exhibits a 3-4 times greater dynamic range than its predecessor and possesses a robust baseline FRET value for ZAP-70. PMID: 25735979
- Data indicate a substantial decline in ZAP-70 and biomarker protein levels in Vacutainer cell processing tubes (CPTs)-isolated chronic lymphocytic leukemia (CLL) cells. PMID: 25124785
- Data indicate that the decreasing trend in the expression level of TCRzeta chain, ZAP-70 kinase, and epsilon Fc Receptors FcvarepsilonRIgamma was significantly associated with disease progression. PMID: 25513989
- A specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys39 in ZAP-70, Cys206 in Syk) that is necessary for inhibition by thiol-reactive compounds was identified. PMID: 25287889
- The corrected mean fluorescence intensity (CorrMFI) represents the most promising method currently available in a routine diagnostic setting for the assessment of ZAP-70 expression in CLL patients. PMID: 24127306
- In early-stage B-CLL patients, ZAP-70 upregulation is associated with distinct patterns of activation/differentiation stage subset distribution and of cytokine expression in CD4 T lymphocytes. PMID: 24166938
- ZAP-70 CpG+223 methylation represents a superior biomarker in risk-stratification of chronic lymphocytic leukemia. PMID: 24868078
- ZAP-70 enhances the migration of malignant B-cells into the supportive microenvironment found in the bone marrow mainly by enhancing signaling and migration after CXCR4 stimulation. PMID: 24312539
- LAT is a modulator of CD3zeta and ZAP-70 tyrosine phosphorylation. PMID: 24204825
- Zap70 mutation reducing protein stability demonstrates a rate-limiting threshold for Zap70 protein levels exists at which signaling capacity switches from nearly intact to effectively null. PMID: 24164480
- Both drugs significantly decreased the expressions of CD5 and ZAP-70. PMID: 23686733
- A deficient lipid rafts recruitment of CD3zeta/ZAP-70/Grb2, and these proteins do not merge with GM1 within the lipid rafts. PMID: 23916875
- Data indicate that inflammatory cytokine-induced increases in chronic lymphocytic leukemia (CLL) cell adhesion to stromal cells are correlated with ZAP-70 expression and blocked by PI3K inhibitor. PMID: 23981382
- The study investigated, using the Sequenom MassARRAY platform, polymorphisms of sixteen single-nucleotide polymorphisms (SNPs) belonging to PTPN22, CD28, CTLA-4, and ZAP-70 genes in 76 T1D patients and 162 unrelated healthy controls. PMID: 24103478
- Expression of ZAP70 in CLL cells increases expression of the NF-kappaB target genes interleukin-1beta, IL6 & IL8 upon BCR triggering. ZAP70 directly amplifies NF-kappaB signaling in CLL cells, which could be an underlying mechanism for its poor prognosis. PMID: 24219331
- ZAP70 expression in regulatory T cells in allergic rhinitis: effect of immunotherapy. PMID: 23786282
- CBAP can indeed function as a novel signaling component within the ZAP70/Vav1/talin complex and plays an important role in regulating chemokine-promoted T-cell trafficking. PMID: 23620790
- The study evaluated the expression of CD74 in chronic lymphocytic leukemia patients. CD74 expression was significantly higher in the CLL group than in controls. There was a positive correlation between CD74 and ZAP70 expression. PMID: 23572149
- These results suggest the involvement of the ZAP70 and PTPN6 genes in the genetic component conferring a general susceptibility to Crohn's disease and ulcerative colitis, respectively. PMID: 23406209
- Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker. Autoinhibition of ZAP-70 is fully released upon phosphorylation by Src family kinases. PMID: 23530057
Q&A
Basic Research Questions
What are optimal experimental conditions for detecting phospho-ZAP70 (Tyr319) in primary T cells?
Successful detection of phospho-ZAP70 (Tyr319) in primary T cells requires careful optimization of several experimental parameters:
For HTRF and LANCE Ultra assays:
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Cell density: Typically 50,000-100,000 cells per well in 96-well format before transfer to detection plates
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Stimulation: Anti-CD3 antibodies at appropriate concentrations (typically 1-10 μg/mL)
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Timing: Rapid phosphorylation occurs within minutes of stimulation
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Lysis conditions: Must preserve phosphorylation status with appropriate phosphatase inhibitors
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Controls: Include unstimulated cells and positive controls such as PP2 (Lck inhibitor)
The 2-plate protocol commonly used for HTRF assays involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before adding phospho-ZAP-70 detection reagents. This approach enables monitoring of cell viability and confluence prior to analysis . For Western blotting applications, optimization of antibody dilutions (typically 1:1000) and proper selection of positive controls are essential for reliable results .
How can researchers troubleshoot weak or inconsistent phospho-ZAP70 (Tyr319) signals?
Weak or inconsistent phospho-ZAP70 (Tyr319) signals present a common challenge in research. Several approaches can help troubleshoot these issues:
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Method comparison: Phosphoflow techniques may show weaker responses compared to HTRF methods. In one study, researchers observed a low phospho-Tyr-319-ZAP70 response in anti-CD3–stimulated cells detected by phosphoflow, while HTRF methods showed "a rapid, severalfold signal increase following anti-CD3 stimulation" .
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Signal validation: Use multiple detection methods to confirm results, especially when signals appear weak. Include relevant positive controls (e.g., the Lck inhibitor PP2) to validate the detection system .
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Timing optimization: ZAP70 phosphorylation occurs rapidly after stimulation but may be transient. Establish a detailed time course to identify optimal detection windows.
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Preservation of phosphorylation: Ensure rapid sample processing with adequate phosphatase inhibitors to prevent loss of phosphorylation during preparation.
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Antibody selection: Different antibody clones may have varying sensitivity for detecting phospho-ZAP70 (Tyr319). Compare results with multiple validated antibodies .
When unexpected results occur, as in the case described where a compound (AX-024) appeared to lack effect in phosphoflow assays but showed clear dose-dependent inhibition in HTRF assays, methodological differences may explain the discrepancy .
What are the key considerations for ensuring specificity in phospho-ZAP70 (Tyr319) detection?
Ensuring specificity in phospho-ZAP70 (Tyr319) detection requires careful attention to several factors:
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Cross-reactivity: The sequence homology between ZAP70 and related kinases like Syk must be considered. Some antibodies may cross-react with the orthologous Tyr352 residue in Syk . This is particularly important when studying mixed cell populations that may express both proteins.
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Antibody validation: Confirm antibody specificity using appropriate controls such as:
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Unstimulated cells (negative for phosphorylation)
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Stimulated cells treated with kinase inhibitors
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Cells expressing mutant ZAP70 (Tyr319F)
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Total protein normalization: Always measure total ZAP70 alongside phosphorylated forms to distinguish between changes in phosphorylation status versus changes in protein expression .
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Cell type considerations: Human, mouse, and rat ZAP70 have high sequence homology, but validation across species is necessary. Available antibodies show reactivity with human samples, with some also cross-reacting with mouse and rat samples .
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Assay format selection: HTRF and LANCE Ultra assays use two labeled antibodies—one specific to the phosphorylated motif and another recognizing the protein independent of phosphorylation state—providing an internal control for specificity .
How can phospho-ZAP70 (Tyr319) assays be applied to study T-cell receptor signaling modulation?
Phospho-ZAP70 (Tyr319) assays provide powerful tools for studying modulation of TCR signaling pathways:
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Compound screening: These assays enable high-throughput screening of compounds that modulate T-cell activation. For example, the compound AX-024 was studied using phospho-ZAP70 (Tyr319) as a readout, demonstrating "a reproducible dose-dependent inhibition by AX-024, with no impact on total ZAP70" .
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Quantitative analysis: HTRF and LANCE Ultra assays generate quantitative data where "intensity is directly proportional to the concentration of phosphorylated protein present in the sample" . This enables precise dose-response studies and kinetic analyses.
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Mechanistic studies: Because Tyr319 phosphorylation is required for the assembly of signaling complexes leading to activation of PLC-gamma1-dependent and Ras-dependent pathways, monitoring this site provides insight into early signaling events .
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Integration with other readouts: Combining phospho-ZAP70 (Tyr319) measurements with analysis of downstream effectors can establish signaling relationships and identify points of intervention in TCR pathways.
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Cell-based assays: The HTRF phospho-ZAP70 (Tyr319) cellular assay is "ideal for monitoring T-cell activation" and allows researchers to observe the effects of various stimuli or inhibitors on this critical signaling event .
What are the relationships between ZAP70 phosphorylation at Tyr319 and clinical implications in immunological disorders?
ZAP70 phosphorylation status has important clinical implications:
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T-cell functional assessment: Since phosphorylation at Tyr319 is essential for ZAP70's positive function in T-lymphocyte activation, measuring this modification provides insight into T-cell functional capacity in various disease states .
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Biomarker potential: ZAP70 expression and phosphorylation patterns in B-cells can serve as indicators for various forms of leukemia, making phospho-specific assays valuable diagnostic tools .
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Immunotherapeutic development: As a key component of TCR signaling, ZAP70 phosphorylation represents a potential target for therapeutic intervention in autoimmune disorders and transplant rejection.
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Monitoring T-cell receptor engagement: The LANCE Ultra and HTRF assays for phospho-ZAP70 (Tyr319) enable researchers to monitor T-cell activation in response to various stimuli, facilitating the development of immunomodulatory strategies .
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Correlation with disease activity: Changes in the phosphorylation pattern of ZAP70 may correlate with disease activity in T-cell mediated autoimmune conditions, offering potential prognostic value.
What methodological advances are improving the study of ZAP70 phosphorylation dynamics?
Recent methodological advances have significantly enhanced our ability to study ZAP70 phosphorylation dynamics:
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Homogeneous assay formats: Both HTRF and LANCE Ultra technologies provide no-wash, homogeneous assay formats that preserve the native state of protein complexes and enable higher throughput analysis .
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TR-FRET technology: Time-resolved fluorescence resonance energy transfer in LANCE Ultra assays utilizes "one antibody labeled with a donor fluorophore (a LANCE Europium chelate) and the second antibody labeled with an acceptor fluorophore [ULight™ dye]" . This approach reduces background and increases sensitivity.
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Dual-parameter detection: The ability to simultaneously measure both phospho-ZAP70 (Tyr319) and total ZAP70 enables more reliable interpretation of phosphorylation status independent of expression level variations.
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Multi-site phosphorylation analysis: Antibody duos that detect multiple phosphorylation sites, such as the "Phospho ZAP70 Antibody Duo (pY319, pY493)," allow researchers to monitor different regulatory phosphorylation events simultaneously .
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Integrated protocols: The development of standardized protocols that integrate cell culture, stimulation, lysis, and detection steps has simplified the workflow while improving reproducibility. For example, the 2-plate protocol for HTRF assays enables monitoring of "cells' viability and confluence" alongside phosphorylation analysis .
These advances collectively facilitate more comprehensive and reliable analysis of the complex signaling events involved in T-cell activation, contributing to our understanding of basic immunology and potential therapeutic interventions.
