Phospho-PTK2B (Y402) Antibody

What is PTK2B/PYK2 and what is the significance of Y402 phosphorylation?

PTK2B (Protein Tyrosine Kinase 2 Beta), also known as PYK2, FAK2, or RAFTK, is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases. It plays crucial roles in diverse cellular events downstream of integrin receptors, including cell migration, proliferation, and survival .

Phosphorylation at tyrosine 402 (Y402) is a critical regulatory event that creates a binding site for SH2 domain-containing proteins, particularly Src family kinases. This phosphorylation is associated with enzymatic activation, intercellular localization, cell growth, cell motility, and regulation of molecular associations . Importantly, Y402 phosphorylation is a prerequisite for subsequent phosphorylation at other sites like Y579, making it an essential step in the full activation of PYK2 .

What are the expression patterns of PYK2 in different tissues?

PYK2 shows tissue-specific expression patterns that are important to consider when designing experiments:

• Brain: Most abundant expression, with highest levels in amygdala and hippocampus

• Immune cells: Expressed in spleen and lymphocytes, particularly in B and T cells

• Vascular system: Present in endothelial cells and vascular smooth muscle cells

• Hematopoietic cells: Expressed in megakaryocytes

• Kidney: Low expression levels

This tissue distribution pattern suggests important roles in neuronal function, immune responses, and vascular biology, guiding research focus areas.

What are the validated applications for Phospho-PYK2 (Y402) antibodies?

Phospho-PYK2 (Y402) antibodies have been validated for multiple research applications:

For optimal results, these applications typically require specific sample preparation, including treatment with phosphatase inhibitors during lysis to preserve the phosphorylation state.

How do I optimize detection of phospho-PYK2 (Y402) in Western blotting applications?

Western blotting for phospho-PYK2 (Y402) requires specific methodological considerations:

• Sample preparation:

  • Include phosphatase inhibitors (e.g., pervanadate) in lysis buffers

  • For positive controls, treat cells with pervanadate (0.2-1 mM for 30 minutes)

  • For T cells, consider stimulation with anti-CD3 antibodies (10 μg/mL for 15 minutes)

• Electrophoresis conditions:

  • Use 8-10% gels for optimal separation of the 112-116 kDa PYK2 protein

  • Load appropriate protein amounts (typically 20-50 μg of total protein)

  • Include molecular weight markers covering 100-120 kDa range

• Transfer and detection:

  • Use PVDF membranes for optimal binding of high molecular weight proteins

  • Block with 5% BSA rather than milk to preserve phospho-epitopes

  • Primary antibody concentration: 0.5-5 μg/mL depending on specific antibody

  • Include both treated and untreated samples to demonstrate specificity

• Controls validation:

  • Include phosphatase-treated lysates as negative controls

  • Use phospho-blocking peptides to confirm band specificity

  • Consider using PYK2-deficient cell lines as additional negative controls

What is the unique relationship between Src kinase and PYK2 Y402 phosphorylation?

Research has revealed a novel role for Src kinase in PYK2 activation that differs from FAK regulation:

• Priming mechanism:

  • Src activity is indispensable for the initial PYK2 phosphorylation at Y402 site upon cell attachment to fibronectin

  • This contrasts with FAK, where the initial fibronectin-induced autophosphorylation at Y397 occurs in a Src-independent manner

• Molecular requirements:

  • The SH2-domain of Src is required for Src binding to PYK2

  • This binding is essential for PYK2 phosphorylation at sites Y402 and Y579

  • Y402 phosphorylation must occur before Y579 phosphorylation in a sequential process

• Two-phase activation model:

  • Initial phase: Src-dependent priming phosphorylation at Y402

  • Second phase: PYK2 autophosphorylation in trans for full activation

This unique mechanism suggests that experimental approaches to study PYK2 should account for Src activity, particularly when investigating integrin-mediated processes.

How can I distinguish between phospho-PYK2 (Y402) and other phosphorylated proteins in complex samples?

Ensuring specificity when detecting phospho-PYK2 (Y402) requires multiple validation approaches:

• Antibody validation techniques:

  • Use phospho-blocking peptides in parallel experiments

  • Compare results with multiple antibodies targeting different epitopes

  • Perform side-by-side analysis with phospho-FAK antibodies to distinguish between these related proteins

• Genetic approaches:

  • Use siRNA knockdown or CRISPR knockout of PYK2 to confirm band identity

  • Employ Y402F mutant PYK2 expression as a negative control

  • Consider rescue experiments with wild-type PYK2 in knockout cells

• Biochemical verification:

  • Immunoprecipitate with total PYK2 antibody followed by phospho-specific detection

  • Perform phosphatase treatment to demonstrate phospho-specificity

  • Use pervanadate treatment as a positive control

• Analytical considerations:

  • For Western blot, compare band migrations with predicted molecular weights (112-116 kDa)

  • In Simple Western assays, verify the peak at approximately 113 kDa

  • For ELISA, validate with known positive and negative controls

What experimental approaches can measure dynamic changes in PYK2 Y402 phosphorylation?

To effectively capture the temporal dynamics of PYK2 Y402 phosphorylation:

• Time-course experimental design:

  • Short intervals (30 seconds to 60 minutes) to capture rapid phosphorylation changes

  • Synchronized stimulation using:

    • Pervanadate treatment (0.2-1 mM)

    • Anti-CD3 antibody stimulation for T cells

    • Calcium mobilization via ionophores

    • Integrin engagement through cell attachment protocols

• Quantitative detection methods:

  • Sandwich ELISA for high sensitivity and throughput

    • Using anti-pan PYK2 antibody for capture and mouse anti-PYK2 (Y402) antibody for detection

    • Allowing simultaneous measurement of phospho-PYK2 and total PYK2

  • Simple Western for automated, reproducible quantification

  • Western blot with densitometry analysis normalized to total PYK2

• Single-cell analysis techniques:

  • Immunofluorescence microscopy with phospho-specific antibodies

  • Flow cytometry with cell permeabilization protocols

  • Analysis of subcellular localization changes upon phosphorylation

Why might I observe inconsistent phospho-PYK2 (Y402) signal in different cell types?

Variability in phospho-PYK2 (Y402) detection across cell types can stem from several factors:

• Cell type-specific activation mechanisms:

  • Lymphoid cells (e.g., Raji, Jurkat) show strong response to pervanadate and receptor stimulation

  • Adherent cells may require different stimuli related to integrin engagement

  • Neuronal cells might respond primarily to calcium signaling given high PYK2 expression in brain tissue

• Pathway differences:

  • Varying levels of Src family kinases impact the efficiency of Y402 phosphorylation

  • Different phosphatase activities can affect steady-state phosphorylation levels

  • Cell-specific adaptor protein expression influences PYK2 activation

• Technical considerations:

  • Optimize lysis conditions for specific cell types (adherent vs. suspension)

  • Adjust stimulation protocols to reflect physiologically relevant activation mechanisms

  • Consider the timing of phosphorylation events, which may differ between cell types

• Sample handling:

  • Rapid processing is essential as phosphorylation states can change quickly

  • Ensure consistent phosphatase inhibitor use across experiments

  • Account for differences in protein extraction efficiency between cell types

How do I interpret results when phospho-PYK2 (Y402) and total PYK2 data appear discordant?

When facing discrepancies between phospho-PYK2 (Y402) and total PYK2 measurements:

• Biological explanations:

  • Actual changes in the proportion of phosphorylated protein

  • Subcellular relocalization of phosphorylated form affecting extraction

  • Phosphorylation-induced changes in protein stability or turnover

• Technical considerations:

  • Epitope masking in the total protein due to protein-protein interactions

  • Different antibody affinities between phospho-specific and total antibodies

  • Phosphorylation-dependent changes in protein solubility

• Analytical approaches:

  • Always normalize phospho-signal to total protein within the same experiment

  • Use multiple detection methods (e.g., compare ELISA with Western blot results)

  • Consider immunoprecipitation with total antibody followed by phospho-detection

  • Verify with phosphatase treatment to confirm specificity of phospho-signal

• Controls to include:

  • Pervanadate-treated positive controls to establish maximum phosphorylation

  • Phosphatase-treated negative controls to demonstrate specificity

  • Dilution series to ensure linear range of detection for both antibodies

What are the critical controls needed when studying Src-dependent PYK2 phosphorylation?

When investigating the Src-PYK2 relationship, include these essential controls:

• Pharmacological controls:

  • Src inhibitor dose-response (e.g., PP2, dasatinib) to establish dependency

  • Pervanadate treatment as positive control for maximum phosphorylation

  • Phosphatase treatment as negative control

• Genetic controls:

  • Src family kinase knockdown/knockout cells

  • PYK2 Y402F mutant expression to prevent phosphorylation at this site

  • SH2 domain mutants of Src that cannot bind to phospho-Y402

• Activation controls:

  • Fibronectin attachment time course to monitor integrin-dependent activation

  • Comparison with FAK Y397 phosphorylation, which occurs Src-independently

  • Sequential stimulation protocols to establish pathway order

• Sample preparation controls:

  • Consistent cell density and growth conditions

  • Standardized lysis procedures to maintain phosphorylation status

  • Fresh preparation of inhibitors and stimulants for each experiment

How can phospho-PYK2 (Y402) antibodies be utilized in cancer research?

Phospho-PYK2 (Y402) antibodies offer valuable insights into cancer biology:

• Diagnostic and prognostic applications:

  • Immunohistochemical detection in tumor samples, as demonstrated in breast carcinoma tissue

  • Correlation of phosphorylation levels with clinical outcomes

  • Potential biomarker development through ELISA-based detection in patient samples

• Mechanistic studies:

  • Investigation of PYK2's role in cancer cell migration and invasion

  • Analysis of PYK2-dependent survival signaling in cancer cells

  • Examination of cross-talk with oncogenic pathways

• Therapeutic target validation:

  • Monitoring PYK2 inhibition in response to targeted therapies

  • Assessing combination approaches targeting both PYK2 and Src family kinases

  • Studying resistance mechanisms involving PYK2 reactivation

• Methodological approaches:

  • Tissue microarray analysis of phospho-PYK2 across tumor types

  • Patient-derived xenograft models with phospho-PYK2 monitoring

  • Correlation of phospho-PYK2 with other activation markers

What are effective approaches for studying phospho-PYK2 (Y402) in neuronal systems?

Given PYK2's high expression in brain tissue, especially in amygdala and hippocampus , specialized approaches for neuronal research include:

• Tissue-specific techniques:

  • Optimize fixation methods for brain tissue to preserve phospho-epitopes

  • Utilize micro-dissection of specific brain regions before analysis

  • Employ slice cultures for pharmacological manipulation with preserved architecture

• Neuronal activation paradigms:

  • Glutamate receptor stimulation to induce calcium-dependent PYK2 activation

  • Neuronal activity models (e.g., LTP induction protocols)

  • Excitotoxicity models to study stress-induced PYK2 phosphorylation

• Visualization methods:

  • Co-localization with synaptic markers to determine subcellular distribution

  • High-resolution imaging of dendritic spines to examine local phosphorylation

  • In vivo phospho-imaging using cleared brain tissue techniques

• Functional correlations:

  • Relate PYK2 phosphorylation to electrophysiological measurements

  • Assess consequences of phosphorylation on neuronal morphology

  • Connect phosphorylation status to learning and memory paradigms

How can multiplexed detection methods improve phospho-PYK2 research?

Multiplexed approaches provide richer context for phospho-PYK2 (Y402) signaling:

• Multi-parameter phospho-profiling:

  • Simultaneous detection of multiple phosphorylation sites (Y402, Y579)

  • Correlation with upstream regulators (Src activity) and downstream effectors

  • Integration with broader pathway analysis (MAPK activation, calcium signaling)

• Technical implementations:

  • Multiplex Western blotting with differently labeled secondary antibodies

  • Sandwich ELISA formats that can measure both phospho and total PYK2 in the same sample

  • Mass spectrometry-based phospho-proteomics for unbiased analysis

• Single-cell approaches:

  • Multi-parameter flow cytometry with phospho-specific antibodies

  • Multiplexed immunofluorescence for tissue sections

  • Single-cell Western technologies for heterogeneous populations

• Data integration strategies:

  • Correlation analysis between phosphorylation sites

  • Pathway modeling incorporating multiple phosphorylation events

  • Machine learning approaches to identify patterns in complex phosphorylation data