Phospho-PTK2B (Tyr402) Antibody
Description
Overview of PTK2B and Phospho-Tyr402
PTK2B (PYK2/FAK2) is a non-receptor tyrosine kinase involved in cell adhesion, migration, and immune responses. Phosphorylation at Tyr402 is essential for its enzymatic activation, serving as a docking site for SH2 domain-containing proteins like Fyn and enabling downstream signaling cascades . This modification occurs in response to stimuli such as T-cell receptor activation, chemokines, or integrin engagement .
Antibody Characteristics
The Phospho-PTK2B (Tyr402) Antibody (Clone M269) is a mouse monoclonal IgG1 antibody generated against a phosphopeptide containing residues surrounding Tyr402. Key features include:
Signaling Pathway Analysis
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T-cell Activation: Detects PTK2B phosphorylation during T-cell receptor (TCR) engagement, critical for Fyn recruitment and downstream MAP kinase signaling .
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Immune Cell Migration: Used to study PTK2B’s role in macrophage and dendritic cell motility, which impacts antiviral responses .
Disease Relevance
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Cancer: PTK2B phosphorylation correlates with metastasis in hematopoietic cancers. The antibody identifies activated PTK2B in Burkitt’s lymphoma (Raji) and T-cell leukemia (Jurkat) models .
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Viral Infection: PTK2B regulates TBK1/STING oligomerization in antiviral innate immunity. Tyr402 phosphorylation may influence this process .
Western Blot Performance
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Induction: Strong signal in pervanadate-treated Raji/Jurkat cells (1 mM, 30 min) .
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Specificity: No cross-reactivity with unphosphorylated PTK2B or FAK (PTK2) .
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Reproducibility: Validated using Simple Western™, showing a single band at ~113 kDa .
Functional Insights from Studies
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PTK2B-deficient macrophages show reduced TBK1/IRF3 phosphorylation during HSV-1 infection, underscoring its role in antiviral signaling .
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Tyr402 phosphorylation is required for PTK2B’s interaction with ASC specks in inflammasome activation .
Research Implications
This antibody enables precise tracking of PTK2B activation in:
Product Specs
Target Background
In T-cells, PYK2 regulates cytoskeleton rearrangement and cell spreading, contributing to the regulation of T-cell responses. PYK2 promotes osteoclastic bone resorption, a process that requires both PYK2 and Src kinase. It may also inhibit the differentiation and activity of osteoprogenitor cells.
PYK2 functions in signaling downstream of multiple receptors, including integrin and collagen receptors, immune receptors, G-protein coupled receptors (GPCRs), cytokine, chemokine, and growth factor receptors. It mediates responses to cellular stress and forms multisubunit signaling complexes with Src and Src family members upon activation, leading to the phosphorylation of additional tyrosine residues, creating binding sites for scaffold proteins, effectors, and substrates. This intricate interaction allows PYK2 to regulate numerous signaling pathways.
PYK2 promotes the activation of phosphatidylinositol 3-kinase (PI3K) and the AKT1 signaling cascade. It also promotes the activation of nitric oxide synthase 3 (NOS3), regulates the production of the cellular messenger cyclic GMP (cGMP), and promotes the activation of the mitogen-activated protein kinase (MAPK) signaling cascade, including the activation of MAPK1/ERK2, MAPK3/ERK1, and MAPK8/JNK1. Furthermore, PYK2 promotes the activation of Rho family GTPases, such as RhoA and Rac1.
PYK2 acts as a scaffold, binding to both PDPK1 and Src, enabling Src to phosphorylate PDPK1 at tyrosine residues 9, 373, and 376. It also promotes the phosphorylation of NMDA receptors by Src family members, contributing to the regulation of NMDA receptor ion channel activity and intracellular calcium levels.
PYK2 may also regulate potassium ion transport by phosphorylating potassium channel subunits. It phosphorylates Src, enhancing Src kinase activity, and also phosphorylates other proteins, including ASAP1, NPHP1, KCNA2, SHC1, ASAP2, RHOU, and PXN. This phosphorylation of PXN requires both Src and PYK2.
- LFA-1 cross-linking recruits and activates FAK1 and PYK2 to phosphorylate LAT selectively on a single Y-171 site that binds to the GRB2-SKAP1 complex and limits dwell times of T-cells with dendritic cells. PMID: 28699640
- Pyk2 plays a role in spine structure and synaptic function; its deficit contributes to Huntington's disease cognitive impairments. PMID: 28555636
- Results provide evidence that Pyk2 phosphorylates STIM1 at its Y361 residue, thereby activating store-operated Ca(2+) entry. PMID: 28218251
- The authors' findings identify Pyk2 as a unique mediator of invadopodium formation and function, providing a novel insight into the mechanisms by which Pyk2 mediates tumor cell invasion. PMID: 29133485
- Results show that VEGFA induces Pyk2 activation in mediating human retinal microvascular endothelial cell migration, sprouting, and tube formation. Additionally, Pyk2-mediated STAT3 activation is required for hypoxia-induced retinal neovascularization. PMID: 27210483
- Interestingly, rs2279590 locus has a widespread enhancer effect on two nearby genes, protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolase-2 (EPHX2), both of which have been previously associated with Alzheimer's disease (AD) as risk factors. PMID: 28973302
- Multiple myeloma that is driven by deregulated iron homeostasis and/or Pyk2/beta-catenin signaling is susceptible to deferasirox-induced apoptosis. PMID: 27602957
- In summary, our data suggest that PYK2 via S6K1 activation modulated AR function and growth properties in prostate cancer cells. Thus, PYK2 and S6K1 may potentially serve as therapeutic targets for prostate cancer treatment. PMID: 27492635
- Our findings suggest that Pyk2 plays an important role in the coordination of beta-catenin stabilization in the crosstalk between Wnt/beta-catenin and Wnt/Ca(2+) signaling pathways upon Wnt3a stimulation in differentiating human neural progenitor cells (hNPCs). PMID: 28694190
- STIM1-induced Ca(2+) signaling activates Pyk2 to inhibit the interaction of VE-PTP and VE-cadherin, thereby increasing endothelial permeability. PMID: 28385807
- Ascites and CCL18 stimulate the phosphorylation and expression of Pyk2, which positively regulates ascites-induced ovarian cancer cell migration. PMID: 27613122
- We demonstrated trophoblast cytoprotection by intervention with supraphysiological concentrations of relaxin, a process in part mediated through the PI3-kinase-Akt/PKB cell survival pathway. These results provide further rationale for clinical investigation of relaxin as a potential therapeutic in preeclampsia. PMID: 28122716
- PTK2B polymorphism (rs28834970) could modify the risk of late-onset Alzheimer's disease (LOAD), and PTK2B polymorphism (rs28834970) and APOE may interact to increase LOAD risk in a Han Chinese population. PMID: 27080426
- Studies suggest that PYK2 is a common downstream effector of ErbB and IL8 receptors, and that PYK2 integrates their signaling pathways through a positive feedback loop to potentiate breast cancer invasion. PMID: 26084289
- Pyk2 is a key downstream signaling molecules of CCR7 in squamous cell carcinoma of the head and neck (SCCHN), which promotes SCCHN tumorigenesis and progression. PMID: 26352169
- Phosphoproteomic analysis identifies FAK2 as a potential therapeutic target for tamoxifen resistance in breast cancer. PMID: 26330541
- Pyk2-focal adhesion targeting domain interacts with and binds to leupaxin. PMID: 26866573
- Src has a role in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins. PMID: 26866924
- Data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and alpha4beta1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation. PMID: 26202465
- FAK and PYK2 functioned redundantly to promote the Wnt/beta-catenin pathway by phosphorylating GSK3beta(Y216) to reinforce pathway output-beta-catenin accumulation and intestinal tumorigenesis. PMID: 26274564
- This study identified Pyk2 as a cellular component required for the intracellular trafficking of HPV16 during infection. PMID: 26109718
- These results suggest that melatonin exerts anti-migratory and anti-invasive effects on glioma cells in response to hypoxia via reactive oxygen species-alphavbeta3 integrin-FAK/Pyk2 signaling pathways. PMID: 25889845
- Data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway. PMID: 25415317
- Orai1/Pyk2 pathway is essential for glioma migration and invasion. PMID: 25433371
- Pyk2-NDRG1 axis is possibly involved in conveying the anti-proliferative effect of beta-ionone in prostate cancer cells. PMID: 25219547
- Pyk2 has a role in integrin regulation of size and dynamics of signaling microclusters. PMID: 25778396
- PYK2 is an intermediary component of Ca(2+) signaling between PKA-mediated and Tyr phosphorylations that is required for achieving functional human sperm capacitation. PMID: 25180269
- Data indicate that chicken paxillin leucine-aspartate LD2 and LD4 motifs preferentially dock at the helix 2/3 binding site of human Proline-rich tyrosine kinase 2 (Pyk2)-focal adhesion kinase (FAK). PMID: 25174335
- These data demonstrate that Pyk2 is a critical regulator of PI3K function downstream of the TCR. PMID: 25387834
- Data indicate the tumor-promoting role of proline-rich tyrosine kinase 2 (Pyk2) in multiple myeloma (MM), suggesting tyrosine kinase inhibitor as a therapeutic option in MM. PMID: 25217697
- Inhibition of FAK, PYK2, and BCL-XL synergistically enhances apoptosis in ovarian clear cell carcinoma cell lines. PMID: 24523919
- Pyk2 and Src are important in CCL18-induced breast cancer metastasis. PMID: 24142406
- High expression of proline-rich tyrosine kinase 2 is associated with hepatocellular carcinoma via regulating phosphatidylinositol 3-kinase/AKT pathway. PMID: 22618716
- Development of a coordinated allo T cell and auto B cell response against autosomal PTK2B after allogeneic hematopoietic stem cell transplantation. PMID: 24097630
- Pyk2 is essential for skin wound reepithelialization in vivo and in vitro and that it regulates epidermal keratinocyte migration via a pathway that requires PKCdelta and MMP functions. PMID: 24598361
- Pyk2 is a shared key mediator of targeted-therapy induced adhesion and migration; Targeting Pyk2 may serve as an effective therapeutic strategy to reduce extramedullar relapse in acute promyelocytic leukemia and chronic myeloid leukemia. PMID: 24176282
- We hypothesize a potential direct or indirect role for SRC, RAF1, PTK2B genes in neurotransmission and in central nervous system signaling processes. PMID: 24108181
- Determine a crucial role of LRP1-mediated Pyk2 phosphorylation on hypoxia-induced MMP-9 activation and human vascular smooth muscle cell (hVSMC) migration and therefore in hypoxia-induced vascular remodeling. PMID: 24072693
- Both Pyk2 and phosphorylated Pyk2[pY881] are potential prognostic factors and therapeutic targets for non-small-cell lung cancer. PMID: 23922106
- Our results confirm that receptor tyrosine kinases (RTKs) are frequently altered in chordomas. PMID: 23618355
- Results indicate that in vitro Pyk2 might function to regulate cell adhesion and motility following all-trans-retinoic acid (ATRA). PMID: 23587524
- We conclude that the effects of selenoprotein H on mitochondrial biogenesis and mitochondrial function are probably mediated through protein kinase A-CREB-PGC-1alpha and Akt/protein kinase B-CREB-PGC-1alpha pathways. PMID: 23220172
- Data indicate protein secretion pathways activated by monosodium urate (MSU) in macrophages, and reveal a novel role for cathepsin B and Src, Pyk2, PI3 kinases in the activation of unconventional protein secretion. PMID: 23292187
- These results identify a novel pathway of integrin alphaIIbbeta3 outside-in signaling and recognize the tyrosine kinase Pyk2 as a major regulator of platelet adhesion and spreading on fibrinogen. PMID: 23216754
- The non-catalytic functions of the kinases Fyn and Pyk2 were required for late stage human T cell adhesion. PMID: 23300847
- SOCS3 definitely plays roles in regulating Pyk2 signaling and cell motility in A549 cells. PMID: 23302305
- PAC1 regulates PYK-2 tyrosine phosphorylation in a calcium-dependent manner in lung cancer cell lines. PMID: 22581436
- Findings showed that Pyk2 is overexpressed in squamous cell carcinoma of the head and neck; data suggest that CCR7 via Pyk2 and cofilin regulates the chemotaxis and migration ability of metastatic squamous cell carcinoma of the head and neck cells. PMID: 22923218
- Down-regulation of both miR-517a and miR-517c contribute to hepatocellular carcinoma cells development through regulating Pyk2. PMID: 23142219
- Pyk2, which was strongly activated by IGF-I, was critical for IGF-IR-dependent motility. PMID: 22859931
Q&A
What is the significance of Tyr402 phosphorylation in PTK2B?
Phosphorylation at Tyr402 in PTK2B plays a pivotal role in its activation and function within cellular signaling pathways. This residue acts as a major autophosphorylation site, facilitating interactions with SRC family kinases and other signaling molecules. The phosphorylation of Tyr402 is essential for downstream signaling events that regulate cytoskeletal reorganization, cell migration, adhesion, and immune responses . In particular, this modification promotes the recruitment of SH2 domain-containing proteins such as Fyn and GRB2, which are integral to T-cell activation and other immune functions .
How can Phospho-PTK2B (Tyr402) antibodies be used to study signal transduction pathways?
Phospho-PTK2B (Tyr402) antibodies are highly specific tools for detecting the phosphorylated form of PTK2B at Tyr402. They are commonly employed in techniques such as Western blotting, immunoprecipitation, immunohistochemistry (IHC), and immunofluorescence (IF). These applications allow researchers to monitor the activation status of PTK2B under various experimental conditions . For example:
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Western blotting can quantify phosphorylation levels across different treatments or time points.
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Immunoprecipitation enables the isolation of PTK2B complexes for further analysis.
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IHC and IF provide spatial information about PTK2B activation within tissues or cells.
These methods are particularly useful for studying signaling pathways involved in immune responses, cancer progression, and cellular stress responses .
What experimental controls are necessary when using Phospho-PTK2B (Tyr402) antibodies?
Experimental controls are crucial to ensure specificity and reliability when using Phospho-PTK2B (Tyr402) antibodies. Recommended controls include:
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Negative controls: Samples treated with phosphatase to remove phosphorylation at Tyr402 can confirm antibody specificity for the phosphorylated form.
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Positive controls: Cells or tissues known to exhibit high levels of PTK2B phosphorylation at Tyr402 under specific conditions serve as benchmarks.
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Isotype controls: These verify that observed signals are not due to nonspecific binding of the antibody's isotype.
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Loading controls: Proteins such as actin or tubulin ensure equal sample loading in Western blot experiments .
Additionally, using blocking peptides corresponding to the phosphopeptide immunogen can validate antibody specificity .
What are the challenges in detecting endogenous levels of Phospho-PTK2B (Tyr402)?
Detecting endogenous levels of phosphorylated PTK2B can be challenging due to low abundance or transient phosphorylation events. Strategies to overcome these challenges include:
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Optimizing antibody dilution: Start with recommended dilutions (e.g., 1:1000 for Western blotting) and adjust based on signal intensity .
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Enhancing signal detection: Use sensitive detection systems such as chemiluminescence or fluorescence-based methods.
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Stimulating cells: Treat cells with agents like pervanadate or cytokines to enhance tyrosine phosphorylation before analysis .
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Improving sample preparation: Employ rapid lysis protocols with phosphatase inhibitors to preserve phosphorylation states during extraction .
How do you interpret contradictory data when studying PTK2B phosphorylation?
Contradictory data may arise due to differences in experimental conditions, antibody specificity, or biological variability. To address such discrepancies:
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Verify antibody specificity: Confirm that the antibody exclusively detects Tyr402-phosphorylated PTK2B using blocking peptides or phosphatase-treated samples.
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Standardize experimental conditions: Ensure consistency in cell lines, treatments, and incubation times across experiments.
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Analyze biological variability: Consider genetic or environmental factors that may influence PTK2B phosphorylation levels.
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Replicate experiments: Perform independent replicates to assess reproducibility .
What role does PTK2B play in immune cell activation?
PTK2B is highly expressed in immune cells and becomes activated upon stimulation by antigen receptors or chemokine receptors. Phosphorylation at Tyr402 is critical for recruiting signaling molecules such as Fyn SH2 domains, which mediate downstream events like T-cell activation and cytokine production . Additionally, PTK2B regulates inflammasome responses by phosphorylating ASC proteins, thereby modulating proinflammatory gene expression induced by TNF-alpha and IL-1β .
How can Phospho-PTK2B (Tyr402) antibodies be used in cancer research?
In cancer research, Phospho-PTK2B (Tyr402) antibodies are valuable for studying aberrant signaling pathways associated with tumor progression and metastasis. Elevated expression of phosphorylated PTK2B has been observed in gliomas, hepatocellular carcinoma, lung cancer, and breast cancer . Researchers can use these antibodies to:
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Assess PTK2B activation status in tumor samples via IHC or IF.
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Investigate its role in promoting cancer cell migration and invasion through Western blotting.
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Explore therapeutic targets by analyzing interactions between phosphorylated PTK2B and other oncogenic proteins .
What methodologies are available for quantifying Phospho-PTK2B (Tyr402)?
Quantification of Phospho-PTK2B (Tyr402) can be achieved through several methodologies:
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Western blotting: Provides relative quantification based on band intensity normalized against loading controls.
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ELISA: Offers high sensitivity for absolute quantification using specific capture and detection antibodies.
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Mass spectrometry: Enables precise measurement of phosphorylation levels at Tyr402 along with identification of other post-translational modifications .
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Flow cytometry: Allows quantification at a single-cell level when combined with fluorescently labeled antibodies.
Each method has its advantages depending on the research context.
How does PTK2B interact with other signaling molecules?
PTK2B interacts with various signaling molecules through its phosphorylated Tyr402 residue. This interaction facilitates complex formation with SRC family kinases, TBK1, STING, and other proteins involved in immune responses or cellular stress pathways . Mechanistically:
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Tyr402 phosphorylation promotes SRC-mediated phosphorylation at additional sites like Tyr579 and Tyr881.
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Binding to TBK1 enhances its oligomerization and activation during antiviral signaling .
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Association with STING regulates innate immunity independently of kinase activity .
Domain-mapping experiments have further elucidated these interactions.
What are the storage requirements for Phospho-PTK2B (Tyr402) antibodies?
Proper storage is essential to maintain antibody stability:
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Short-term storage: Keep undiluted antibodies at 4°C for up to one week.
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Long-term storage: Aliquot antibodies into smaller volumes and store at -20°C to prevent repeated freeze-thaw cycles .
Avoid frost-free freezers due to temperature fluctuations that may degrade antibody integrity.
