Phospho-PTK2 (Tyr925) Antibody

What is the molecular significance of PTK2/FAK phosphorylation at Tyr925?

Phosphorylation of PTK2 (also known as Focal Adhesion Kinase or FAK) at tyrosine 925 represents a critical signal transduction event that creates a specific binding site for the Grb2/SH2 domain. This phosphorylation event triggers a Ras-dependent activation of the MAP kinase pathway . The molecular significance includes:

• Creation of a protein-protein interaction interface for Grb2 adaptor protein

• Initiation of downstream MAPK/ERK signaling cascade

• Regulation of cell migration, adhesion, and proliferation mechanisms

• Indication of active Src-FAK signaling complex in cells

This site-specific phosphorylation acts as a molecular switch that connects integrin and growth factor receptor signaling to intracellular responses mediating cell behavior and fate.

How does Src kinase activity relate to PTK2/FAK Tyr925 phosphorylation?

Phosphorylation of FAK Tyr925 is predominantly Src-dependent, making it a valuable marker for Src-FAK pathway activation . The relationship involves:

• Src directly phosphorylates FAK at Tyr925 following FAK activation

• This phosphorylation event occurs downstream of initial FAK autophosphorylation at Tyr397

• Tyr925 phosphorylation specifically requires Src family kinase activity, unlike some other FAK phosphorylation sites

• In v-Src-transformed NIH3T3 cells, the association of v-Src, Grb2, and Sos with FAK occurs independently of cell adhesion to fibronectin

Monitoring Tyr925 phosphorylation provides a useful indicator of increased signaling through the Src-FAK signaling complex in experimental systems and tumors .

What distinguishes the biological function of Tyr925 phosphorylation from other FAK phosphorylation sites?

FAK contains multiple phosphorylation sites with distinct functional outcomes:

Uniquely, Tyr925 phosphorylation:

• Creates a specific binding site for Grb2's SH2 domain

• Has been identified as the binding site through site-directed mutagenesis studies

• Promotes MAPK pathway activation distinct from other phosphorylation events

• May serve as a biomarker for increased Src-FAK signaling in tumors

• Is subject to dephosphorylation by PTPN11, which is recruited to PTK2 via phosphorylated EPHA2

What are the optimal conditions for detecting Phospho-PTK2 (Tyr925) in Western blotting experiments?

Based on manufacturer protocols and research practices, optimal Western blotting conditions include:

Sample Preparation:

• Use cellular lysates from adherent cells or tissues where FAK is activated

• Pervanadate (1 mM for 5 minutes) treatment can enhance phosphorylation signal

• Include phosphatase inhibitors in lysis buffer to preserve phosphorylation status

Technical Parameters:

• Antibody dilutions: Generally 1:500-1:2000 for most polyclonal antibodies

• Specific recommendations vary by manufacturer: 1:1000 for Cell Signaling antibodies

• Reducing conditions using appropriate buffer systems (e.g., Immunoblot Buffer Group 1)

• PVDF membrane typically yields better results than nitrocellulose for phospho-epitopes

• Expected molecular weight: 125 kDa band is characteristic of phosphorylated full-length FAK

Controls:

• Untreated versus phosphatase inhibitor-treated samples

• Peptide competition assays to confirm specificity

• Phospho-blocking peptide preincubation controls

Optimal detection typically reveals a distinct band at approximately 125 kDa that increases in intensity upon cellular stimulation with integrin engagement or growth factors.

How can I validate the specificity of a Phospho-PTK2 (Tyr925) antibody in my experimental system?

Rigorous validation of phospho-specific antibodies requires multiple complementary approaches:

• Peptide competition assays:

  • Preincubate the antibody with synthetic phosphorylated peptide

  • Compare signal between blocked and unblocked antibody samples

  • A specific antibody will show diminished signal when preincubated with phosphopeptide

• Phosphatase treatment controls:

  • Treat half of your sample with lambda phosphatase

  • Compare signal between treated and untreated samples

  • Loss of signal in phosphatase-treated samples confirms phospho-specificity

• Site-directed mutagenesis:

  • Express wild-type FAK and Y925F mutant constructs

  • Stimulate cells to induce phosphorylation

  • Absence of signal in Y925F mutant confirms site-specificity

• Stimulation experiments:

  • Compare unstimulated cells versus cells treated with known FAK activators

  • HUVEC cells treated with pervanadate (1 mM for 5 minutes) show increased phosphorylation

  • Integrin engagement via fibronectin adhesion should increase signal

• Antibody source comparison:

  • Use antibodies from different vendors or different clones

  • Consistent results across antibody sources strengthen validity of findings

Validation results should be documented with appropriate controls and included in publications to demonstrate antibody specificity.

What stimuli effectively induce PTK2/FAK phosphorylation at Tyr925 in experimental settings?

Several stimuli and conditions reliably induce FAK Tyr925 phosphorylation:

Integrin-Mediated Activation:

• Cell adhesion to fibronectin or other extracellular matrix proteins

• Integrin clustering induced by antibody cross-linking

• Cell spreading on appropriate substrates

Growth Factor Receptor Activation:

• Epidermal growth factor (EGF) treatment

• Platelet-derived growth factor (PDGF) stimulation

• Vascular endothelial growth factor (VEGF) in endothelial cells

G-Protein Coupled Receptor (GPCR) Signaling:

• Bombesin treatment

• Lysophosphatidic acid (LPA) stimulation

Experimental Inducers:

• Pervanadate treatment (1 mM for 5 minutes) in HUVEC cells

• Src overexpression or constitutively active Src mutants

• v-Src transformation of NIH3T3 cells

Other Activators:

• LDL receptor occupancy

• Mechanical stress or cell stretching

• Hyperosmotic shock

The choice of stimulus should align with your experimental system and research question. For example, integrin-mediated activation is most relevant for studies on cell adhesion and migration, while growth factor stimulation may be more appropriate for proliferation studies.

Why might I observe different results when detecting Phospho-PTK2 (Tyr925) using Western blotting versus immunofluorescence?

Discrepancies between Western blotting and immunofluorescence detection of phospho-FAK (Tyr925) can result from several factors:

Methodological Differences:

• Western blotting detects denatured proteins, while immunofluorescence detects native conformations

• Epitope accessibility may differ between methods due to protein folding or complex formation

• Different fixation methods in immunofluorescence can affect phospho-epitope preservation

• Antibody concentration requirements differ: typically 1:500-1:1000 for Western blotting versus 1:100-1:200 for immunofluorescence

Biological Considerations:

• Subcellular localization of phospho-FAK is spatially restricted in cells (focal adhesions)

• Rapid turnover of phosphorylation status may affect detection in fixed versus lysed samples

• Western blotting provides population-averaged data, while immunofluorescence reveals cell-to-cell heterogeneity and spatial information

Technical Issues:

• Methanol fixation is often recommended for phospho-epitope preservation in immunofluorescence

• Different blocking reagents may affect antibody performance differently between methods

• Western blotting may detect cross-reactive proteins at similar molecular weights

When encountering discrepancies, consider validating with alternative techniques (e.g., ELISA, proximity ligation assay) and optimizing each method independently with appropriate controls.

What controls should be included when analyzing Phospho-PTK2 (Tyr925) levels in experimental samples?

Robust experimental design requires several controls when analyzing phospho-FAK (Tyr925):

Essential Controls:

• Positive Control:

  • Cells treated with pervanadate (1 mM, 5 minutes)

  • Cells adhered to fibronectin

  • Cells expressing constitutively active Src

• Negative Controls:

  • Unstimulated/serum-starved cells

  • Cells treated with FAK or Src inhibitors

  • Cells expressing FAK Y925F mutant

• Antibody Specificity Controls:

  • Phosphopeptide competition assay

  • Phosphatase-treated samples

  • Secondary antibody-only control

• Loading/Normalization Controls:

  • Total FAK protein detection on stripped and reprobed membrane

  • Housekeeping protein detection (β-actin, GAPDH)

  • Total protein stain (Ponceau S, REVERT)

• Technical Validation:

  • Replicate biological and technical samples

  • Independent experimental repeats

  • Multiple antibody sources when possible

Data Presentation Requirements:

• Show both phospho-FAK and total FAK levels

• Present quantitative analysis as phospho-FAK/total FAK ratio

• Include statistical analysis of replicate experiments

Proper controls allow confident interpretation of results and facilitate troubleshooting when unexpected patterns emerge.

How can Phospho-PTK2 (Tyr925) antibodies be utilized to study cancer cell migration and invasion?

Phospho-FAK (Tyr925) antibodies offer powerful tools for investigating cancer cell migration and invasion:

Experimental Approaches:

• Quantitative Analysis of Signaling Dynamics:

  • Time-course analysis of Tyr925 phosphorylation during migration/invasion

  • Correlation with other phosphorylation sites (Tyr397, Tyr576/577)

  • Comparative analysis across cancer cell lines with different metastatic potential

• Visualization of Spatial Signaling:

  • Immunofluorescence imaging of phospho-FAK localization at invasive protrusions

  • Co-localization with other focal adhesion components (paxillin, vinculin)

  • Live-cell imaging using phospho-sensor technologies

• Functional Intervention Studies:

  • Assess effects of FAK inhibitors on Tyr925 phosphorylation and migration

  • Correlate migration/invasion capacity with phosphorylation levels

  • Rescue experiments with phospho-mimetic or phospho-deficient FAK mutants

• Clinical Correlation:

  • Analysis of Tyr925 phosphorylation in patient tumor samples

  • Correlation with tumor grade, stage, and metastatic potential

  • Potential biomarker development for tumor aggressiveness

Research Significance:
FAK is implicated in cancer cell behavior, with increased levels of FAK and Src proteins found in tumors . Phosphorylation of Tyr925 may provide a useful indicator of increased signaling through the Src-FAK complex in tumors, potentially serving as a biomarker for aggressive disease .

What is the relationship between PTK2/FAK Tyr925 phosphorylation and the RET-FAK signaling axis?

The relationship between FAK Tyr925 phosphorylation and RET kinase reveals an interesting signaling specificity:

Key Findings:

• A RET-FAK transactivation mechanism exists consisting of direct phosphorylation of FAK Tyr-576/577 by RET, but notably NOT Tyr925

• This creates a reciprocal phosphorylation relationship where FAK can phosphorylate RET

• The selective phosphorylation pattern distinguishes RET-mediated FAK activation from Src-mediated FAK activation, which includes Tyr925

Signaling Implications:

• RET activates FAK through a mechanism distinct from the classical Src-FAK pathway

• Tyr925 phosphorylation status can distinguish between RET-mediated versus Src-mediated FAK activation

• This provides a potential biomarker for determining the dominant upstream kinase in a given cellular context

Methodological Application:
Researchers can use phospho-specific antibodies against different FAK tyrosine residues (particularly comparing Tyr576/577 versus Tyr925) to determine the relative contributions of RET versus Src in FAK activation under different experimental conditions.

What methodologies can be combined with Phospho-PTK2 (Tyr925) antibodies to study protein-protein interactions at focal adhesions?

Advanced research on focal adhesion signaling complexes can leverage Phospho-FAK (Tyr925) antibodies in combination with several sophisticated methodologies:

Proximity-Based Interaction Methods:

• Proximity Ligation Assay (PLA):

  • Detects interactions between phospho-FAK(Tyr925) and binding partners (e.g., Grb2)

  • Provides spatial information within cells

  • Allows quantification of interaction events

• FRET/BRET Approaches:

  • Measures direct protein interactions in living cells

  • Can track temporal dynamics of FAK-Grb2 interactions

  • Requires fluorescent or bioluminescent protein tagging

Pull-Down and Co-Immunoprecipitation:

• SH2 Domain Pull-Downs:

  • Use recombinant Grb2 SH2 domains to isolate phospho-Tyr925 FAK

  • Compare binding efficiency across experimental conditions

• Immunoprecipitation Strategies:

  • Anti-phospho-FAK(Tyr925) antibodies can be used for IP at 1:50 dilution

  • Co-IP followed by mass spectrometry to identify novel interaction partners

  • Sequential IP to isolate multi-protein complexes

Advanced Imaging Approaches:

• Super-Resolution Microscopy:

  • Nanoscale visualization of phospho-FAK localization within focal adhesions

  • Multi-color imaging with other focal adhesion components

• Correlative Light-Electron Microscopy:

  • Combines immunofluorescence of phospho-FAK with ultrastructural analysis

  • Provides context for protein localization at the nanoscale

Proteomic Approaches:

• Phospho-Proteomic Analysis:

  • Global analysis of phosphorylation events downstream of FAK Tyr925

  • Identification of signaling networks activated by this specific phosphorylation event

• BioID or APEX Proximity Labeling:

  • FAK fusion proteins to identify proteins in proximity to FAK

  • Can be combined with phospho-specific antibodies to compare interaction partners of phosphorylated versus non-phosphorylated FAK

These methodologies, when combined with phospho-specific antibodies, provide comprehensive insights into the spatial, temporal, and functional aspects of FAK signaling at focal adhesions.

How does PTK2/FAK Tyr925 phosphorylation contribute to cell cycle progression and proliferation?

FAK Tyr925 phosphorylation plays multifaceted roles in cell cycle regulation and proliferation through several interconnected mechanisms:

Signaling Pathway Integration:

• Phosphorylation of Tyr925 creates a binding site for the Grb2/SH2 domain

• This triggers Ras-dependent activation of the MAP kinase pathway , which promotes cell cycle progression

• Activation of MAPK1/ERK2 and MAPK3/ERK1 signaling cascades drives proliferative gene expression

Cell Cycle Phase Regulation:

• FAK phosphorylation status changes during different cell cycle phases

• Contributes to acceleration of G1 to S phase transition

• Regulates cell cycle checkpoints through nuclear and cytoplasmic signaling

Nuclear Functions:

• FAK can shuttle between focal adhesions and the nucleus

• Phosphorylated FAK recruits the ubiquitin ligase MDM2 to p53 in the nucleus

• This regulates p53 activity, ubiquitination, and proteasomal degradation

• Suppression of p53-mediated growth inhibition and apoptosis promotes proliferation

Growth Factor and Integrin Signaling Coordination:

• Integration of adhesion-dependent and growth factor-dependent signals

• Cross-talk between integrin and receptor tyrosine kinase pathways

• Coordination of cell adhesion status with proliferative decisions

Experimental Approaches to Study This Function:

• Cell cycle synchronization followed by phospho-FAK analysis at different cell cycle phases

• FAK inhibitor studies with cell cycle markers and proliferation assays

• Y925F mutant expression compared to wild-type FAK in proliferation assays

• Combined inhibition of FAK and MEK/ERK pathway components

Understanding FAK Tyr925 phosphorylation in proliferation contexts is particularly relevant for cancer research, as dysregulated FAK signaling contributes to uncontrolled proliferation in multiple tumor types.

What factors affect the reproducibility of Phospho-PTK2 (Tyr925) detection across experiments?

Achieving consistent detection of phospho-FAK (Tyr925) requires attention to several critical factors:

Biological Variables:

• Cell Density and Confluence:

  • Overcrowded cells show altered FAK phosphorylation patterns

  • Standardize seeding density and experiment at consistent confluence

• Cell Passage Number:

  • FAK signaling can change with cellular aging

  • Use cells within a defined passage range

• Growth Factor Exposure:

  • Serum components activate FAK signaling pathways

  • Standardize serum starvation protocols before stimulation

Sample Processing:

• Lysis Conditions:

  • Rapid lysis is critical to preserve phosphorylation status

  • Temperature matters: keep samples cold throughout processing

• Phosphatase Inhibitors:

  • Must be fresh and at correct concentrations

  • Include multiple inhibitor types (e.g., serine/threonine and tyrosine phosphatase inhibitors)

• Protein Degradation:

  • Include protease inhibitors

  • Process samples quickly to prevent degradation

Technical Parameters:

• Antibody Storage and Handling:

  • Avoid repeated freeze-thaw cycles of antibodies

  • Aliquot antibodies for long-term storage at -20°C

  • Store working dilutions at 4°C for no more than one week

• Detection Methods:

  • Consistent exposure times for chemiluminescence

  • Regular calibration of imaging equipment

  • Use of fluorescent secondary antibodies may provide better quantitative linearity

• Signal Normalization:

  • Always normalize to total FAK levels

  • Consider normalizing to a loading control and total FAK

Documentation for Reproducibility:
Maintain detailed records of all variables including cell culture conditions, lysis buffer composition, antibody lot numbers, and imaging parameters to ensure experimental reproducibility.

How should researchers interpret contradictory results between different phospho-specific PTK2/FAK antibodies?

Contradictory results between different phospho-specific FAK antibodies require systematic investigation:

Common Causes of Discrepancies:

• Epitope Accessibility Differences:

  • Different antibodies may recognize slightly different regions around Tyr925

  • Protein conformation or interacting proteins may affect epitope access

• Cross-Reactivity Profiles:

  • Some antibodies may cross-react with other phospho-tyrosine sites

  • Secondary recognition of similar phosphorylation motifs in other proteins

• Sensitivity Differences:

  • Varying detection thresholds between antibody clones

  • Different signal-to-noise ratios affecting interpretation

Resolution Strategies:

• Validation with Functional Assays:

  • Correlate antibody signals with known functional outcomes

  • Use FAK inhibitors or Y925F mutants to validate specificity

• Phosphopeptide Competition:

  • Test each antibody with phospho-Tyr925 peptide competition

  • Compare blocking efficiency to identify most specific antibody

• Multiple Detection Methods:

  • Compare Western blot, ELISA, and immunofluorescence results

  • Use mass spectrometry-based phosphorylation site analysis as a gold standard

• Antibody Characterization Table:
  Create a detailed comparison table:

  Antibody	Source	Clone/ID	Immunogen	Specificity Testing Method	Cross-Reactivity	Recommended Dilution
  Antibody 1	Vendor A	Rabbit pAb	KVY(p)EN	Peptide competition	Human, Mouse, Rat	WB: 1:1000
  Antibody 2	Vendor B	Mouse mAb	Longer peptide	Y925F mutant cells	Human only	WB: 1:500

• Literature Consensus:

  • Review multiple publications to identify most reliable antibodies

  • Contact authors of key papers for technical advice

When publishing results, transparently report antibody validation methods and acknowledge any discrepancies between different antibodies. This approach strengthens data interpretation and contributes to better reproducibility in the field.

What methodologies can quantitatively assess changes in PTK2/FAK Tyr925 phosphorylation in response to experimental manipulations?

Quantitative assessment of FAK Tyr925 phosphorylation changes requires rigorous methodologies:

Western Blot-Based Quantification:

• Densitometric Analysis:

  • Use phospho-FAK/total FAK ratio for normalization

  • Employ linear range detection methods (fluorescent secondaries preferred)

  • Analyze multiple exposures to ensure linearity of signal

• Multiplexed Western Blotting:

  • Simultaneous detection of phospho-FAK and total FAK using different fluorophores

  • Eliminates stripping and reprobing variability

  • Allows precise ratio calculation from single membrane

ELISA-Based Methods:

• Phospho-Specific ELISA:

  • Sandwich ELISA with capture/detection antibody pairs

  • Higher throughput than Western blotting

  • Suitable for screening multiple conditions

• Bead-Based Multiplex Assays:

  • Simultaneous measurement of multiple phosphorylation sites

  • Requires smaller sample volumes

  • Allows correlation analysis between different phosphorylation events

Cellular Imaging Quantification:

• High-Content Imaging:

  • Automated microscopy with phospho-FAK antibodies

  • Single-cell resolution across populations

  • Spatial information on phosphorylation patterns

• Phospho-Flow Cytometry:

  • Single-cell quantification of phospho-epitopes

  • High-throughput analysis of cell populations

  • Can be combined with other cellular markers

Mass Spectrometry-Based Approaches:

• Targeted MS Approaches:

  • Selected/multiple reaction monitoring (SRM/MRM)

  • Absolute quantification using isotope-labeled peptide standards

  • Highest specificity for phosphorylation site determination

• Global Phosphoproteomics:

  • Unbiased analysis of phosphorylation changes

  • Detection of compensatory phosphorylation events

  • Requires specialized equipment and expertise

Data Analysis Requirements:

• Statistical comparison across multiple biological replicates

• Time-course analyses to capture phosphorylation dynamics

• Dose-response relationships for pharmacological interventions

• Correlation with functional endpoints (migration, proliferation)