Phospho-PTK2 (Tyr861) Antibody

What is Phospho-PTK2 (Tyr861) Antibody and what signaling pathway does it detect?

Phospho-PTK2 (Tyr861) antibody specifically recognizes focal adhesion kinase 1 (FAK1, encoded by PTK2 gene) only when phosphorylated at tyrosine 861. FAK is a cytoplasmic protein tyrosine kinase that localizes to cell adhesion sites and becomes activated by integrin receptor binding to extracellular matrix proteins . The antibody detects a protein of approximately 125 kDa on Western blots .

FAK Y861 phosphorylation occurs downstream in the FAK signaling cascade. While FAK Y397 is the primary autophosphorylation site that initiates signaling, Y861 phosphorylation is predominantly mediated by Src family kinases following initial FAK activation . This creates a signaling complex that regulates cell migration, adhesion, and survival pathways.

What applications can Phospho-PTK2 (Tyr861) antibody be used for?

Most commercially available Phospho-PTK2 (Tyr861) antibodies have been validated for the following applications:

The specificity is typically validated through phospho-peptide competition assays, where the antibody signal is blocked with phosphorylated peptide but not with the non-phosphorylated version .

What are the recommended storage conditions for optimal antibody performance?

For long-term preservation of antibody activity:

• Store at -20°C for up to one year from date of receipt

• For frequent use, store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles as this may compromise antibody activity

• Most preparations contain 50% glycerol, 0.5% BSA, and 0.02% sodium azide in PBS

Proper storage is critical for maintaining phospho-specific recognition, as degradation can lead to increased non-specific binding and decreased phospho-specificity.

How does FAK Y861 phosphorylation differ functionally from other FAK phosphorylation sites?

FAK contains multiple phosphorylation sites with distinct functions in the signaling cascade:

Y861 phosphorylation has been specifically implicated in prostate cancer metastasis, where increased phosphorylation correlates with enhanced migration, invasion, and metastatic potential . Research has shown that while Y397 phosphorylation is essential for initial FAK activation, Y861 phosphorylation may represent a distinct signaling node that specifically regulates migratory and invasive behaviors.

What is the relationship between Src family kinases and FAK Y861 phosphorylation?

FAK Y861 is predominantly phosphorylated by Src family kinases, but recent research has revealed specificity among family members:

Yes kinase has been specifically implicated in FAK Y861 phosphorylation in prostate cancer cells, where it promotes migration and lymph node metastasis . In a study using highly migratory prostate cancer cell variants (PC3 Mig-3 and DU145 Mig-3), researchers observed:

• Specifically increased phosphorylation of FAK Y861 (but not other FAK phosphorylation sites)

• Increased Yes expression (but not other Src family kinases)

• Overexpression of Yes selectively increased FAK Y861 phosphorylation and cell migration

• Knockdown of Yes decreased migration and lymph node metastasis in orthotopic mouse models

This suggests that while multiple Src family kinases can potentially phosphorylate FAK Y861, there may be context-specific preferences that impact biological outcomes.

How can I validate the specificity of Phospho-PTK2 (Tyr861) antibody in my experimental system?

Proper validation ensures reliable experimental results:

• Phosphatase treatment control: Treat lysates with lambda phosphatase to remove phosphorylation and confirm signal loss.

• Phosphopeptide competition: Pre-incubate antibody with phosphorylated and non-phosphorylated peptides:

  • Signal should be blocked with phosphopeptide but not with non-phosphopeptide

  • Example: "Western blot analysis of lysates from HeLa cells, Jurkat cells and COS cells, using FAK (Phospho-Tyr861) antibody. The lane on the right is blocked with the phospho peptide."

• FAK knockdown/knockout: Validate using control and FAK-deficient samples to confirm signal specificity.

• Src inhibition: Treatment with Src family kinase inhibitors should reduce Y861 phosphorylation signal if the antibody is specific.

• Multiple detection methods: Cross-validate findings using different techniques (WB, IF, ELISA) to ensure consistent results.

What methodological considerations should be addressed when examining FAK Y861 phosphorylation in tumor tissues versus cell lines?

Analyzing FAK phosphorylation in different experimental contexts requires specific methodological adaptations:

Cell Line Analysis:

• Standardize cell culture conditions (confluence, time after plating, matrix protein) as these affect FAK phosphorylation status

• Consider analyzing cells in both adherent and suspension conditions as Y861 may be differentially phosphorylated

• FER kinase can promote Y861 phosphorylation even when cells are not adherent

Tumor Tissue Analysis:

• Rapid tissue preservation is critical as phosphorylation is rapidly lost post-excision

• Consider snap-freezing versus phosphatase inhibitor-supplemented fixatives

• Optimize antigen retrieval protocols specifically for phospho-epitopes

• Use adjacent sections for total FAK detection to normalize phospho-signal

• Include region-matched normal tissue controls

A recent study demonstrated that "elevated FAK tyrosine phosphorylation is common in tumors, including pancreatic and ovarian cancers, where it is associated with decreased survival" . In prostate cancer, "increased pFAK Y861 expression in lymph node metastases correlated with poor prognosis" , highlighting the potential prognostic value of this marker.

How can Phospho-PTK2 (Tyr861) antibody be used to investigate the relationship between FAK signaling and cancer progression?

FAK signaling impacts multiple aspects of cancer progression that can be investigated using phospho-specific antibodies:

Metastasis Research Applications:

• Track Y861 phosphorylation in paired primary tumors and metastases

• Correlate with migration/invasion assays in matched cell lines

• Use in orthotopic mouse models to predict metastatic potential

Research has shown that "Yes expression was increased in lymph node metastases relative to paired primary tumors from the same patient, and increased pFAK Y861 expression in lymph node metastases correlated with poor prognosis" . This suggests that monitoring FAK Y861 phosphorylation may provide valuable insights into metastatic progression.

Drug Resistance Studies:

• Monitor changes in Y861 phosphorylation during development of resistance

• Combine with analysis of other FAK phosphorylation sites

• Correlate with activation of downstream survival pathways

Recent clinical trials are targeting FAK in ovarian cancer, where it acts as a "master regulator of drug resistance" . Phospho-specific antibodies can help identify responsive patient populations and monitor treatment efficacy.

What are the technical challenges in distinguishing roles of different Src family kinases in FAK Y861 phosphorylation?

Dissecting the specific contributions of Src family kinases to FAK Y861 phosphorylation presents several technical challenges:

• Kinase redundancy: Multiple Src family kinases can potentially phosphorylate the same site, requiring selective inhibition or depletion strategies:

  • Use of SFK-null cells (src-/-fyn-/-yes-/- fibroblasts) with selective re-expression

  • RNA interference targeting specific family members

  • CRISPR/Cas9-mediated knockout of individual kinases

• Context-dependent activation: The dominant kinase may vary by cell type or condition:

  • In prostate cancer, Yes kinase specifically mediates FAK Y861 phosphorylation and migration

  • Expression levels of different SFKs vary across tissue types

• Antibody cross-reactivity: Phospho-specific antibodies must be validated for specificity:

  • Confirm specificity using phosphopeptide competition assays

  • Validate in knockout/knockdown systems

Research strategies have included "overexpression of Yes in PC3 parental cells and src-/-fyn-/-yes-/- fibroblasts" which "selectively increased FAK Y861 phosphorylation, and increased migration" , demonstrating a methodological approach to address these challenges.

What considerations are important when using Phospho-PTK2 (Tyr861) antibodies in drug development research?

FAK inhibitors are currently in clinical trials, and phospho-specific antibodies are critical for assessing target engagement:

Pharmacodynamic Biomarker Applications:

• Monitor FAK Y397 (autophosphorylation) as direct measure of FAK activity

• Assess Y861 phosphorylation as marker of downstream signaling inhibition

• Compare with total FAK levels to detect compensatory expression changes

Clinical Trial Considerations:

• "Several different small-molecule ATP-competitive inhibitors to FAK have been developed that exhibit high specificity, can be delivered orally (and in pill form to patients) and exhibit on-target FAK Y397 phosphorylation reduction in tumor and stromal cells"

• Some inhibitors (ifebemtinib, narmafotinib) are selective for FAK, while others (defactinib) equally inhibit both FAK and Pyk2 kinase activities

• Phospho-antibodies can distinguish between target engagement (reduced Y397) and downstream efficacy (reduced Y861)

Emerging Therapeutic Approaches:

• New FAK-targeting approaches include proteolysis-targeting chimeras (PROTACs): "FAK PROTAC addition to tumor cells in vitro at sub-micromolar concentrations recapitulates many of the FAK inhibitor-associated cell phenotypes"

• Phospho-specific antibodies can validate target degradation versus inhibition outcomes

Understanding the dynamics of various FAK phosphorylation sites provides critical insight into the efficacy of different therapeutic approaches.