Phospho-PTK2 (Y397) Recombinant Monoclonal Antibody

What is Phospho-PTK2 (Y397) and why is it significant in cellular signaling?

Phospho-PTK2 (Y397) refers to the phosphorylated form of Protein Tyrosine Kinase 2 (PTK2), also known as Focal Adhesion Kinase (FAK), at tyrosine residue 397. This phosphorylation site represents the major autophosphorylation site of FAK and serves as a critical regulatory mechanism in multiple cellular processes.

Tyr-397 phosphorylation occurs upon activation of integrin signaling and promotes interaction with SRC and SRC family members, leading to subsequent phosphorylation at other tyrosine residues (Tyr-576, Tyr-577) and additional sites. This phosphorylation is particularly important for interactions with various signaling proteins including BMX, PIK3R1, and SHC1 . The phosphorylation status at Y397 is dynamically regulated depending on cellular adherence status, with phosphorylation typically occurring when cells are adherent, indicating its role in cell-matrix interactions .

Research has demonstrated connections between the FAK-pFAK-Y397 axis and the mTOR-S6K1 pathway, which plays a major role in carcinogenesis, highlighting its significance in cancer research .

How does Phospho-PTK2 (Y397) antibody specificity differ from total PTK2/FAK antibodies?

Phospho-PTK2 (Y397) antibodies specifically recognize FAK only when phosphorylated at the Y397 site, while total PTK2/FAK antibodies detect the protein regardless of its phosphorylation status. This critical distinction allows researchers to investigate the activation state of FAK within their experimental systems.

The specificity of pFAK-Y397 antibodies can be verified through peptide competition experiments. As described in published research, antibody specificity can be confirmed by blocking the primary antibody with a phosphorylated peptide (mouse FAK phospho Y397) in a molar excess ratio of 200-fold overnight . This approach demonstrates that the antibody is specifically recognizing the phosphorylated epitope rather than non-specifically binding to total FAK or other proteins.

When used in combination, total FAK and phospho-specific antibodies provide complementary information about both expression levels and activation status of the protein, enabling more comprehensive analysis of FAK signaling dynamics in experimental systems.

What are the validated applications for Phospho-PTK2 (Y397) antibodies in research?

Based on manufacturer validations and research publications, Phospho-PTK2 (Y397) antibodies have been validated for multiple experimental applications:

For immunohistochemical applications, these antibodies have been successfully employed in cancer research, particularly in studies examining FAK phosphorylation in epithelial ovarian cancer tissues using tissue microarrays . The subcellular localization pattern typically appears as granular cytoplasmic staining, characteristic of focal adhesion proteins .

When designing experiments, researchers should consider that the optimal working concentration may vary depending on the specific antibody clone, sample type, and experimental conditions, necessitating optimization for each research application.

How should researchers evaluate the specificity of Phospho-PTK2 (Y397) antibodies?

Researchers should employ multiple approaches to confirm the specificity of Phospho-PTK2 (Y397) antibodies:

• Peptide competition assays: Block the primary antibody with a phosphorylated peptide corresponding to the Y397 site (in molar excess of 200-fold) as a negative control. Loss of signal confirms specificity for the phospho-epitope .

• Phosphatase treatment: Treat half of your sample with lambda phosphatase before antibody incubation. The phospho-specific signal should disappear in the treated sample.

• Stimulation/inhibition experiments: Compare samples with known activation status (e.g., cells treated with integrin activators versus cells treated with FAK inhibitors) to verify responsive changes in phosphorylation.

• Colocalization studies: Perform immunofluorescence co-staining with total FAK and pFAK antibodies to verify proper subcellular localization at focal adhesions. As demonstrated in MCF7 and CaOV3 cell lines, both antibodies should clearly stain focal adhesions, though interestingly, pFAK may show additional nuclear localization in some cell types .

• Knockout/knockdown validation: Include FAK-null or FAK-knockdown samples as negative controls to confirm antibody specificity.

These validation steps are critical for ensuring reliable and reproducible results, particularly when studying subtle changes in phosphorylation status under different experimental conditions.

What are the proper storage and handling conditions for Phospho-PTK2 (Y397) antibodies?

Proper storage and handling of Phospho-PTK2 (Y397) antibodies are essential for maintaining their activity and specificity. Based on manufacturer recommendations:

For long-term storage:

• Store at -20°C for up to one year

• Avoid repeated freeze-thaw cycles which can degrade antibody performance

For short-term storage and frequent use:

• Store at 4°C for up to one month

• Many antibodies are supplied in stabilizing buffers containing glycerol (typically 50%) and preservatives like sodium azide

When working with the antibody:

• Prepare working dilutions fresh on the day of the experiment

• Keep antibodies on ice when in use

• Centrifuge briefly before opening vials to collect all liquid at the bottom of the tube

Following these storage and handling guidelines helps maintain antibody performance and extends shelf life. Always refer to the specific manufacturer's instructions for your particular antibody product, as formulations and recommended handling procedures may vary between suppliers.

What factors can affect Phospho-PTK2 (Y397) detection in Western blot applications?

Several critical factors can influence the successful detection of Phospho-PTK2 (Y397) in Western blot applications:

• Sample preparation: Phosphorylation status can rapidly change during cell lysis. Use phosphatase inhibitors in lysis buffers, keep samples cold, and process quickly to preserve phosphorylation.

• Cell adherence status: As demonstrated in research findings, phosphorylation at Y397 typically occurs when cells are adherent. Experimental conditions that disrupt cell adhesion may lead to reduced phosphorylation signal .

• Transfer conditions: Inefficient protein transfer, particularly of high molecular weight proteins like FAK (~125 kDa), can significantly impact detection. Use appropriate transfer conditions (time, buffer, membrane type) optimized for large proteins.

• Antibody specificity and dilution: The recommended dilution for Western blot applications is typically around 1:1,000 , but this may require optimization for your specific system.

• Signal detection methods: Enhanced chemiluminescence (ECL) may not provide sufficient sensitivity for detecting low levels of phosphorylation. Consider using more sensitive detection methods such as enhanced ECL substrates or fluorescent secondary antibodies.

• Fresh vs. frozen samples: Phosphorylation can be lost during storage of samples. Whenever possible, analyze samples immediately after preparation.

• Post-translational modifications: Other modifications like sumoylation can enhance autophosphorylation , potentially affecting detection in experimental systems where these pathways are altered.

When troubleshooting, include appropriate positive controls (cells treated with stimuli known to induce FAK phosphorylation) and negative controls (phosphatase-treated samples) to validate assay performance.

How does Phospho-PTK2 (Y397) localization differ across cell types and experimental conditions?

Research has demonstrated interesting variations in Phospho-PTK2 (Y397) localization patterns that researchers should consider when designing and interpreting experiments:

These localization differences are functionally significant and may reflect distinct roles of phosphorylated FAK in different cellular compartments and processes. When designing immunofluorescence or immunohistochemistry experiments, researchers should consider these variations and include appropriate controls to properly interpret localization patterns.

What is the relationship between PTK2/FAK phosphorylation and cancer progression?

The relationship between PTK2/FAK phosphorylation and cancer progression is complex and sometimes paradoxical, as evidenced by research findings:

This complex relationship underscores the importance of studying FAK phosphorylation in specific cancer contexts rather than generalizing across all cancer types. The ambivalent role of pFAK-Y397 suggests that its function may be highly dependent on cancer type, stage, and molecular subtype, warranting careful interpretation of experimental findings in cancer research.

How can researchers distinguish between different phosphorylation sites on PTK2/FAK in their experiments?

Distinguishing between multiple phosphorylation sites on PTK2/FAK requires careful experimental design and specific tools:

• Site-specific phospho-antibodies: Use antibodies that specifically recognize distinct phosphorylation sites. Beyond Y397, other important sites include Tyr-576/577 (activated by SRC following Y397 phosphorylation), Tyr-861, and Tyr-925 (important for GRB2 interaction) .

• Phosphorylation site mutants: Employ FAK constructs with point mutations at specific tyrosine residues (e.g., Y397F, Y576F, Y577F) to validate antibody specificity and study site-specific functions.

• Mass spectrometry: For comprehensive phosphorylation profiling, use phospho-enrichment followed by mass spectrometry to identify and quantify all phosphorylation sites simultaneously.

• Sequential immunoprecipitation: Perform sequential immunoprecipitation with different phospho-specific antibodies to isolate populations of FAK with distinct phosphorylation patterns.

• Kinase inhibitor studies: Different kinases preferentially phosphorylate specific sites; for example, FER promotes phosphorylation at Tyr-577, Tyr-861, and Tyr-925, while FGR promotes phosphorylation at Tyr-397 and Tyr-576 . Using specific kinase inhibitors can help distinguish site-specific phosphorylation events.

Understanding the temporal sequence of phosphorylation is also important - Tyr-397 autophosphorylation generally precedes and enables subsequent phosphorylation at other sites by recruited kinases like SRC family members. This sequential activation creates distinct signaling outputs through different phosphorylation combinations.

What experimental conditions influence Phospho-PTK2 (Y397) levels?

Several experimental conditions significantly impact Phospho-PTK2 (Y397) levels, which researchers should carefully control in their experiments:

• Cell adhesion status: Tyr-397 phosphorylation typically occurs when cells are adherent. Research has shown that Tyr-397, Tyr-576, and Ser-722 are phosphorylated only when cells are adherent .

• ECM composition: The specific extracellular matrix proteins cells are grown on (fibronectin, collagen, laminin, etc.) differentially affect FAK phosphorylation levels.

• Cell density: Confluency affects cell-cell contacts and can influence FAK phosphorylation independently of cell-matrix interactions.

• Growth factor stimulation: Various growth factors can induce FAK phosphorylation through receptor crosstalk mechanisms, even in the absence of integrin engagement.

• Microtubule dynamics: Microtubule-induced dephosphorylation at Tyr-397 is crucial for focal adhesion disassembly, potentially mediated by PTPN11 and regulated by ZFYVE21 .

• Mechanical forces: Mechanical stimulation, including substrate stiffness and applied forces, significantly affects FAK phosphorylation levels.

• Sumoylation status: Post-translational modification by SUMO enhances FAK autophosphorylation , representing another level of regulation.

To maintain experimental consistency, researchers should standardize cell density, substrate coating, serum levels, and time points for analysis. For studying FAK activation dynamics, consider using live-cell imaging with phospho-specific biosensors rather than fixed timepoint analyses.

What is the significance of focal adhesion localization versus nuclear localization of Phospho-PTK2 (Y397)?

The differential localization of Phospho-PTK2 (Y397) to focal adhesions versus the nucleus has important functional implications that researchers should consider:

• Focal adhesion localization:

  • Represents the classical role of pFAK in integrin-mediated signaling and cell-matrix adhesion

  • Facilitates recruitment of signaling proteins to adhesion sites

  • Typically appears as a granular cytoplasmic staining pattern in immunohistochemistry

  • Critical for regulating cell migration, invasion, and mechanosensing

• Nuclear localization:

  • Observed in specific cell types like MCF7 breast cancer cells but not in others like CaOV3 ovarian cancer cells

  • Previously reported in colon and breast cancer tissues by Murata et al.

  • Suggests non-canonical functions of pFAK in transcriptional regulation

  • May represent kinase-independent scaffold functions in the nucleus

  • Could influence cell survival and gene expression programs

The translocation of pFAK between these compartments likely represents a regulated process that allows the protein to coordinate cytoplasmic signaling with nuclear events. The dual localization may explain some of the context-dependent functions of FAK in cancer, where it can simultaneously influence both adhesion-dependent processes and transcriptional programs.

When analyzing experimental results, researchers should carefully document the subcellular distribution patterns of pFAK-Y397 as these may provide insights into the specific cellular processes being regulated in their experimental system.

How can researchers accurately quantify Phospho-PTK2 (Y397) levels in tissue samples?

Accurate quantification of Phospho-PTK2 (Y397) in tissue samples requires rigorous methodological approaches:

• Standardized scoring systems: For immunohistochemistry, implement a scoring system that considers both staining intensity (0-3) and percentage of positive cells, as used in published research . Since FAK and pFAK staining is often homogeneous within epithelial ovarian cancer tissue, intensity scoring may be the primary determinant.

• Tissue microarrays (TMAs): Consider using TMAs with duplicate cores (e.g., two 1 mm-diameter cores from each tumor sample) to account for tumor heterogeneity while maintaining standardized staining conditions .

• Proper controls:

  • Positive controls: Include tissues known to express pFAK (e.g., breast cancer or kidney tissues)

  • Negative controls: Process serial sections without primary antibody

  • Peptide competition: Block antibody with phosphorylated peptide to confirm specificity

• Blinded assessment: Have samples examined by two independent observers blinded to clinical data to reduce bias, including a pathologist specialized in the relevant field .

• Normalization strategies: When quantifying by Western blot, normalize phospho-signal to total FAK rather than to housekeeping proteins, to account for variations in total FAK expression.

• Digital pathology tools: Consider using digital image analysis software for objective quantification of staining intensity and distribution, which can reduce inter-observer variability.

For categorical analysis, grouping samples into "low" (scores 0-1; not stained at all and low expression) and "high" (scores 2-3; moderate to high expression) categories facilitates statistical comparisons with clinical outcomes .

How does the prognostic value of Phospho-PTK2 (Y397) vary across different cancer types?

The prognostic significance of Phospho-PTK2 (Y397) shows remarkable variation across cancer types, reflecting its context-dependent roles:

This heterogeneity underscores the importance of:

• Analyzing pFAK prognostic value in the context of specific cancer types and subtypes

• Considering multivariate models that account for treatment regimens

• Integrating pFAK status with other molecular markers for more accurate prognostication

Researchers studying pFAK as a biomarker should be cautious about generalizing findings across cancer types and should validate prognostic associations in independent cohorts.

What are the molecular mechanisms connecting Phospho-PTK2 (Y397) to the mTOR-S6K1 pathway?

The molecular linkage between Phospho-PTK2 (Y397) and the mTOR-S6K1 pathway represents an important signaling axis in cell growth regulation and cancer:

• PI3K activation: Phosphorylation at Tyr-397 is important for interaction with the p85 regulatory subunit of PI3K (PIK3R1) , leading to PI3K activation and subsequent PIP3 production at the membrane.

• AKT recruitment and activation: PIP3 recruits AKT to the membrane, where it becomes activated by phosphorylation. Activated AKT can then regulate mTORC1 through inhibition of the TSC1/2 complex.

• mTORC1 regulation: The FAK-PI3K-AKT axis promotes mTORC1 activation, which directly phosphorylates S6K1, leading to increased protein synthesis and cell growth.

• Feedback mechanisms: S6K1 can provide feedback regulation to multiple components of this pathway, potentially influencing FAK activity through indirect mechanisms.

• Microenvironmental sensing: Both FAK and mTOR function as sensors of cellular microenvironment - FAK responds to adhesion and mechanical cues while mTOR responds to nutrient availability, suggesting coordinated regulation of cell growth based on multiple environmental inputs.

Gene expression analysis in epithelial ovarian cancer patients has revealed a connection between the FAK-pFAK-Y397 axis and the mTOR-S6K1 pathway , supporting the functional relevance of this signaling network in cancer pathogenesis.

Understanding this molecular interplay has important implications for combination therapy approaches targeting both pathways simultaneously, which might overcome resistance mechanisms observed with single-pathway inhibition.

What methodological considerations are important when using Phospho-PTK2 (Y397) antibodies for immunofluorescence studies?

When conducting immunofluorescence studies with Phospho-PTK2 (Y397) antibodies, researchers should consider these critical methodological factors:

• Fixation method: Paraformaldehyde fixation (typically 4%) preserves phospho-epitopes while maintaining cellular architecture, as successfully used in published studies .

• Antibody validation: Confirm specificity using controls such as:

  • Competition with phosphorylated peptide

  • FAK-null cells

  • Phosphatase-treated samples

• Optimal dilution ranges: For immunofluorescence applications, the recommended dilution range is typically 1:50-1:200 , though optimization may be required for specific experimental systems.

• Co-staining considerations: When performing co-localization studies with total FAK:

  • Use compatible host species for primary antibodies (e.g., rabbit anti-pFAK with mouse anti-total FAK)

  • Select appropriate fluorescent secondary antibodies with minimal spectral overlap (e.g., AlexaFluor® 488 for anti-rabbit and AlexaFluor® 568 for anti-mouse)

  • Include DAPI for nuclear counterstaining to assess nuclear localization

• Interpretation of patterns: Be aware that different cell types may show distinct localization patterns. For example, MCF7 cells show both focal adhesion and nuclear staining of pFAK, while CaOV3 cells primarily show focal adhesion staining .

• Image acquisition parameters: Use consistent exposure settings across samples, and consider acquiring Z-stacks to fully capture focal adhesion structures, which may not all be in the same focal plane.

Adherence to these methodological considerations will help ensure reliable and reproducible results when studying pFAK localization and abundance via immunofluorescence.

How can researchers differentiate between auto-phosphorylation and trans-phosphorylation of PTK2/FAK at Y397?

Distinguishing between auto-phosphorylation and trans-phosphorylation at the Y397 site requires specialized experimental approaches:

• Kinase-dead mutants: Express a kinase-dead mutant of FAK (K454R) in cells. Any Y397 phosphorylation observed in this mutant must be due to trans-phosphorylation by other kinases, as the mutant lacks catalytic activity for auto-phosphorylation.

• In vitro kinase assays: Perform in vitro kinase assays with:

  • Purified FAK protein alone (to assess auto-phosphorylation)

  • Purified FAK with candidate kinases (to assess trans-phosphorylation potential)

  • Kinase-dead FAK with candidate kinases (to confirm trans-phosphorylation)

• Kinase inhibitor studies: While Tyr-397 is primarily an autophosphorylation site, other kinases like FGR can promote phosphorylation at this residue . Use specific kinase inhibitors to determine contribution of different kinases to Y397 phosphorylation in your system.

• Proximity ligation assays: This technique can detect close association between proteins, potentially identifying kinases in proximity to FAK during phosphorylation events.

• Phosphorylation kinetics: Auto-phosphorylation and trans-phosphorylation may have different kinetics. Time-course experiments after stimulation can help distinguish these mechanisms.

Understanding the source of Y397 phosphorylation is important because:

• Auto-phosphorylation typically occurs through FAK clustering and trans-activation between FAK molecules

• Trans-phosphorylation may represent cross-talk from other signaling pathways

• Different phosphorylation mechanisms may have distinct functional outcomes

This differentiation is particularly relevant when studying FAK in cancer contexts, where altered expression or activity of various kinases may shift the balance between auto- and trans-phosphorylation mechanisms.

What are the best experimental models for studying Phospho-PTK2 (Y397) dynamics in cancer research?

Several experimental models offer distinct advantages for investigating Phospho-PTK2 (Y397) dynamics in cancer research:

• Cell line models:

  • 2D cultures: Cell lines like MCF7 (breast cancer) and CaOV3 (ovarian cancer) have been successfully used to study pFAK localization

  • 3D cultures: Spheroids or organoids better recapitulate tissue architecture and cell-matrix interactions, providing more physiologically relevant models for studying FAK phosphorylation

• Patient-derived materials:

  • Tissue microarrays (TMAs): Enable high-throughput analysis of pFAK in large patient cohorts

  • Patient-derived xenografts (PDX): Maintain tumor heterogeneity and microenvironment, allowing for more translational studies of pFAK dynamics

• In vivo models:

  • Genetically engineered mouse models with FAK mutations (e.g., Y397F knock-in)

  • Orthotopic tumor models that preserve tissue-specific microenvironments

• Live cell imaging models:

  • FRET-based biosensors for real-time visualization of FAK phosphorylation events

  • Photoactivatable or optogenetic FAK constructs to study spatiotemporal dynamics

• Chemical genetic approaches:

  • Analog-sensitive FAK mutants that allow specific inhibition or labeling

When selecting models, consider these factors:

• The specific cancer type being studied (e.g., epithelial ovarian cancer shows high pFAK abundance in 36.9% of cases )

• The research question (mechanistic studies vs. biomarker validation)

• The need to recapitulate tumor microenvironment components that influence FAK phosphorylation

For translational relevance, combining multiple model systems (e.g., in vitro mechanistic studies validated in patient samples) provides the most comprehensive understanding of pFAK dynamics in cancer.

How do PTK2/FAK phosphorylation patterns differ between normal and cancer tissues?

Understanding the differences in PTK2/FAK phosphorylation between normal and cancer tissues provides important insights into cancer biology:

• Expression and phosphorylation levels:

  • In epithelial ovarian cancer (EOC), high FAK expression was detected in 92.2% of samples, while high pFAK-Y397 abundance was found in 36.9% of cases

  • Normal adult tissues typically show lower baseline levels of both FAK expression and phosphorylation compared to cancer tissues

• Subcellular localization differences:

  • Nuclear localization of pFAK has been reported in certain cancer types (colon and breast cancer) but may be absent in corresponding normal tissues

  • Focal adhesion localization patterns may be altered in cancer cells due to disrupted adhesion structures

• Correlation with invasive properties:

  • High pFAK abundance in cancer tissues correlates with nodal positivity and/or distant metastasis (p = 0.030) , suggesting enhanced activation during cancer progression

• Relationship to tissue architecture:

  • Normal tissues maintain organized architecture with regulated FAK phosphorylation

  • In cancer, disrupted tissue architecture can lead to dysregulated FAK phosphorylation

  • This is reflected in the homogeneous staining observed within EOC tissue sections

• Microenvironmental influences:

  • Cancer-associated changes in extracellular matrix composition and stiffness directly affect FAK phosphorylation

  • Inflammatory components in the tumor microenvironment can influence FAK activation patterns

These differences highlight why pFAK-Y397 is being investigated as both a biomarker and therapeutic target in cancer, with potential value in distinguishing between normal and malignant tissues.

What technical considerations are important when designing experiments to study the interplay between PTK2/FAK and SRC kinases?

The PTK2/FAK-SRC signaling axis represents a critical node in cellular signaling that requires careful experimental design:

• Sequential phosphorylation events:

  • Tyr-397 autophosphorylation promotes interaction with SRC and SRC family members, leading to subsequent phosphorylation at Tyr-576 and Tyr-577

  • Time-course experiments are essential to capture this sequential activation

• Antibody selection:

  • Use antibodies specific for different phosphorylation sites (pY397-FAK, pY576/577-FAK, pY416-SRC)

  • Consider multi-color immunofluorescence to simultaneously visualize different phosphorylation events

• Inhibitor strategies:

  • Use FAK inhibitors (targeting the kinase domain) versus SRC inhibitors to distinguish respective contributions

  • Consider dual FAK/SRC inhibitors versus single inhibitors to study pathway interdependence

• Expression systems:

  • FAK mutants (Y397F) to prevent SRC recruitment and activation

  • SRC mutants (kinase-dead) to prevent FAK phosphorylation at Y576/577

  • Expression of FRNK (FAK-related non-kinase) which acts as a dominant-negative

• Proximity analysis:

  • Proximity ligation assays to detect FAK-SRC interactions

  • FRET-based approaches to measure direct interactions in living cells

• Stimulus selection:

  • Different stimuli activate this pathway through distinct mechanisms

  • Integrin engagement, growth factor stimulation, and mechanical forces may differentially regulate FAK-SRC interactions

• Cell type considerations:

  • Different cell types express distinct profiles of SRC family kinases

  • Some cell types may rely more heavily on FGR for Tyr-397 and Tyr-576 phosphorylation

Understanding this interplay is particularly important for cancer research, as FAK-SRC co-targeting strategies are being explored therapeutically. Careful experimental design allows researchers to determine whether observed effects are FAK-dependent, SRC-dependent, or require both kinases.

How can gene expression data be used to interpret Phospho-PTK2 (Y397) abundance in experimental systems?

Gene expression analysis provides valuable context for interpreting Phospho-PTK2 (Y397) data in research:

• Correlation with pathway components:

  • Gene expression data from samples with high versus low pFAK abundance can reveal co-regulated pathway components

  • Research has demonstrated connections between the FAK-pFAK-Y397 axis and the mTOR-S6K1 pathway through this approach

• Methodological approach:

  • First, categorize samples based on pFAK immunohistochemistry status (high vs. low)

  • Apply statistical methods such as shrinkage with non-parametric prior calculation to identify differentially expressed genes

  • Use Bayesian False Discovery Rate (BFDR) values below 10% as significance threshold

• Functional analysis tools:

  • Databases like DAVID (Database for Annotation, Visualization and Integrated Discovery) can be used for functional analysis of differentially expressed genes

  • Gene Set Enrichment Analysis (GSEA) using databases like MSigDB helps identify pathway enrichment

• Computational approaches:

  • Use functions like "romer" from R-package limma for gene set analysis

  • These approaches can reveal biological processes associated with FAK phosphorylation

• Integration with phosphorylation data:

  • Combined analysis of phosphoproteomic and transcriptomic data provides insights into both immediate (phosphorylation) and longer-term (transcriptional) consequences of FAK activation