Phospho-VASP (Ser239) Antibody

What is the biological significance of VASP phosphorylation at Serine 239?

VASP phosphorylation at Serine 239 represents a crucial regulatory mechanism primarily mediated by cGMP-dependent protein kinase (PKG) and to a lesser extent by cAMP-dependent protein kinase (PKA). This specific phosphorylation site plays significant roles in:

• Regulation of cell motility, migration, and adhesion

• Modulation of the actin cytoskeleton and membrane protrusions

• Cardiovascular function, particularly in vascular smooth muscle cell relaxation

• Tumor suppression mechanisms through inhibition of invasive cell morphology

Unlike phosphorylation at Ser157, which is the preferred site for PKA, Ser239 is the preferred phosphorylation site for PKG/PRKG1. Importantly, while Ser157 phosphorylation causes a notable increase in apparent molecular mass (detected as a band shift in Western blots), Ser239 phosphorylation induces no change in apparent molecular mass .

How does phosphorylation at Ser239 differ functionally from other VASP phosphorylation sites?

VASP contains multiple phosphorylation sites with distinct functional consequences:

Phosphorylation at Ser239 specifically suppresses the invasive actin cytoskeleton of tumor cells and represents a potential tumor suppression mechanism . It achieves this by inducing VASP redistribution away from membrane protrusions, with studies showing a calculated half-life of 8.11 minutes for VASP removal from membrane projections following phosphorylation at this site .

What cellular processes are regulated by VASP Ser239 phosphorylation?

VASP Ser239 phosphorylation regulates several key cellular processes:

• Actin cytoskeleton dynamics: Phosphorylation at Ser239 interferes with EVH2-dependent processes at membrane leading edges, destabilizing VASP-actin interactions and inhibiting organelle protrusive dynamics .

• Cell morphology and migration: Studies demonstrate that cells expressing phosphomimetic VASP-S239D are smaller, rounder, and have fewer protrusions compared to wild-type VASP cells .

• Invasive behavior in cancer cells: Ser239 phosphorylation suppresses the formation of filopodia and invadopodia, which are critical for cancer cell invasion .

• Vascular smooth muscle relaxation: In vascular biology, Ser239 phosphorylation mediates NO-driven regulation of vascular smooth muscle cell invasion, adhesion, and matrix contraction .

What are the optimal detection methods for VASP phosphorylated at Ser239?

Several detection methods can be employed to measure VASP phosphorylation at Ser239, each with distinct advantages:

Western Blotting:

• Uses phospho-specific antibodies that recognize only VASP phosphorylated at Ser239

• Unlike Ser157 phosphorylation, Ser239 phosphorylation does not cause a band shift, so detection relies entirely on phospho-specific antibodies

• Recommended dilution ranges: 1:1000-2000 for most commercial antibodies

HTRF (Homogeneous Time Resolved Fluorescence) Assay:

• Plate-based quantitative method that does not require gels, electrophoresis, or transfer

• Employs two labeled antibodies: one with a donor fluorophore specific to phosphorylated Ser239, and another with an acceptor that recognizes VASP regardless of phosphorylation state

• FRET signal intensity is directly proportional to the concentration of phosphorylated protein

• Can be performed in a no-wash assay format

Immunofluorescence Microscopy:

• Allows visualization of subcellular localization of phosphorylated VASP

• Can be combined with F-actin staining to assess colocalization patterns

The choice between these methods depends on your specific research question, with Western blotting being suitable for basic detection, HTRF for quantitative cell-based assays, and immunofluorescence for localization studies.

How should samples be prepared for optimal detection of VASP Ser239 phosphorylation?

Sample preparation is critical for accurate detection of VASP Ser239 phosphorylation:

For Western Blotting:

• Rapid sample collection and immediate lysis are essential as phosphorylation states can change quickly

• Use phosphatase inhibitors in lysis buffers to prevent dephosphorylation during processing

• Thawed antibodies are stable for 48 hours at 2-8°C and can be refrozen (at ≤-16°C) and thawed at least one more time

• Control lysates should be included to validate the detection system

For HTRF Assays:
The assay can be performed using two different protocols:

• Two-plate protocol: Culture cells in a 96-well plate, lyse, then transfer lysates to a 384-well low volume detection plate before adding detection reagents. This allows monitoring of cell viability and confluence .

• Single-plate protocol: Perform culturing, stimulation, and detection in a single plate with no washing steps required .

Sample Volume Requirements:

• Typical HTRF assays require 16 μL sample volume

• Western blotting typically requires 40 μg protein in SDS loading buffer

What controls should be included when working with Phospho-VASP (Ser239) antibodies?

Proper controls are essential for reliable interpretation of results with Phospho-VASP (Ser239) antibodies:

Positive Controls:

• Samples treated with cGMP analogs (e.g., 8-br-cGMP) or NO donors, which activate PKG and induce VASP Ser239 phosphorylation

• Commercial positive control lysates designed specifically for phospho-VASP detection

Negative Controls:

• Samples treated with PKG inhibitors

• Samples from VASP knockout cells (when available)

• Samples with VASP-S239A mutation (cannot be phosphorylated at Ser239)

Specificity Controls:

• Peptide competition assays to confirm antibody specificity

• Dual detection with a separate antibody that recognizes total VASP regardless of phosphorylation state

• Comparison with other VASP phosphorylation sites (e.g., Ser157) to confirm site specificity

Experimental Validation:
Researchers have validated phospho-VASP (Ser239) antibody specificity using cells expressing VASP phosphomutants including:

• Wild-type VASP (control)

• VASP-S239A (cannot be phosphorylated at Ser239)

• VASP-S157A (cannot be phosphorylated at Ser157)

• VASP-AA (double mutant)

How can Phospho-VASP (Ser239) antibodies be used to study NO/cGMP signaling pathways?

Phospho-VASP (Ser239) antibodies serve as valuable tools for investigating NO/cGMP signaling pathways:

Mechanism of Action:

• Nitric oxide (NO) activates soluble guanylyl cyclase, increasing cGMP levels

• cGMP activates PKG, which phosphorylates VASP at Ser239

• This phosphorylation can be used as a biomarker for PKG activation

Experimental Applications:

• Monitoring PKG activation: The phospho-VASP (Ser239) assay is ideal for monitoring PKG activation in response to various stimuli .

• Cardiovascular research: Used to study vascular relaxation mechanisms, as VASP phosphorylation correlates with vascular relaxation effects .

• Cancer research: NO-driven regulation of VASP phosphorylation can be studied to understand its effects on tumor cell invasion and migration .

• Platelet activation studies: Both cGMP and cAMP pathways converge on VASP phosphorylation, with distinct functional outcomes that can be monitored using phospho-specific antibodies .

Research has demonstrated that cGMP-dependent VASP Ser239 phosphorylation suppresses the invasive actin cytoskeleton of tumor cells, making it a potential biomarker for cancer progression and metastasis .

What are the experimental challenges in distinguishing between different VASP phosphorylation sites?

Distinguishing between different VASP phosphorylation sites presents several experimental challenges:

Detection Challenges:

• Differential migration patterns: While Ser157 phosphorylation causes a band shift in Western blots, Ser239 phosphorylation does not change apparent molecular mass, requiring phospho-specific antibodies for detection .

• Temporal dynamics: Different sites can be phosphorylated with different kinetics in response to the same stimulus, requiring careful time-course experiments.

• Sequential phosphorylation: Some sites (e.g., Thr278) require prior phosphorylation at other sites (Ser157 and Ser239), creating complex interdependencies .

Analytical Solutions:

• Phospho-mutant expression: Generate cells expressing VASP with mutations at specific phosphorylation sites (e.g., S239A, S157A) to confirm antibody specificity and study site-specific functions .

• Phospho-mimetic mutants: Express VASP with mutations that mimic constitutive phosphorylation (e.g., S239D) to study the effects of phosphorylation at specific sites without upstream signaling complications .

• Site-specific kinase activators: Use selective activators of PKA (mainly targets Ser157) versus PKG (primarily targets Ser239) to differentially phosphorylate VASP sites .

• Phosphoproteomics approaches: Mass spectrometry-based methods can provide unambiguous identification and quantification of phosphorylation at multiple sites simultaneously.

How can Phospho-VASP (Ser239) antibodies be used to investigate cancer cell invasion and metastasis?

Phospho-VASP (Ser239) antibodies provide valuable insights into cancer cell invasion and metastasis mechanisms:

Research Applications:

• Monitoring invasive structures: VASP phosphorylation at Ser239 suppresses the formation of invasive membrane protrusions like filopodia and invadopodia, which can be visualized and quantified .

• Real-time imaging: Live cell imaging with GFP-VASP constructs and phospho-specific antibody staining can reveal how VASP phosphorylation dynamically regulates the actin cytoskeleton during invasion .

• Biomarker development: VASP Ser239 phosphorylation status may serve as a biomarker for tumor invasiveness and metastatic potential .

Experimental Findings:
Studies have demonstrated that cGMP signaling through phosphorylation of VASP at Ser239 significantly:

• Reduces the number and length of migratory (filopodia) and invasive (invadopodia) actin-based protrusions

• Promotes rapid removal of VASP from membrane projections with a calculated half-life of 8.11 minutes

• Causes VASP redistribution to inner cell compartments away from membrane tips

These findings suggest that VASP Ser239 phosphorylation represents a potential "invasion suppressor" that could be exploited for cancer diagnostics and therapeutics. Notably, cancer cells expressing the VASP-S239A mutant (resistant to Ser239 phosphorylation) maintained their invasive phenotype even when exposed to cGMP pathway activators, highlighting the critical role of this specific phosphorylation site .

What are common issues when detecting VASP Ser239 phosphorylation and how can they be resolved?

Researchers may encounter several challenges when detecting VASP Ser239 phosphorylation:

Issue 1: Low or No Signal

• Potential Causes: Rapid dephosphorylation during sample preparation; inadequate stimulation of PKG pathway; improper antibody storage

• Solutions:

  • Use phosphatase inhibitors in all buffers

  • Ensure proper storage of antibodies (store at -20°C for up to 1 year; avoid repeated freeze-thaw cycles)

  • Increase stimulation time or concentration of PKG activators

  • Verify antibody activity with positive control samples

Issue 2: Non-specific Binding

• Potential Causes: Insufficient blocking; cross-reactivity with other phosphorylated proteins; suboptimal antibody dilution

• Solutions:

  • Optimize blocking conditions (5% milk in TBS-Tween is commonly used)

  • Validate antibody specificity using phospho-mutant controls (S239A)

  • Adjust antibody dilution (typically 1:1000-2000 for Western blotting)

Issue 3: Inconsistent Results Between Experiments

• Potential Causes: Variations in cell stimulation; inconsistent sample handling; changes in phosphorylation during processing

• Solutions:

  • Standardize stimulation protocols

  • Process all samples simultaneously

  • Include internal controls for normalization

  • Consider using HTRF assays which provide more consistent quantification

How should experimental conditions be optimized for studying VASP Ser239 phosphorylation in different cell types?

Optimizing experimental conditions for VASP Ser239 phosphorylation studies requires cell type-specific considerations:

General Optimization Strategy:

• Baseline phosphorylation assessment: Determine basal Ser239 phosphorylation levels in your cell type before stimulation

• Time-course experiments: Establish optimal stimulation times, as phosphorylation kinetics vary between cell types

• Dose-response curves: Determine effective concentrations of PKG activators (e.g., 8-br-cGMP) for your specific cell type

Cell Type-Specific Considerations:

Validated Experimental Approaches:
Research has successfully employed GFP-VASP constructs (wild-type and phospho-mutants) to study phosphorylation dynamics in live cells, allowing real-time visualization of VASP redistribution following phosphorylation at Ser239 .

How can phosphorylation of VASP at multiple sites be analyzed simultaneously?

Analyzing multiple VASP phosphorylation sites simultaneously presents both challenges and opportunities:

Methodological Approaches:

• Sequential immunoblotting:

  • Strip and reprobe membranes with different phospho-specific antibodies

  • Start with phospho-Ser239 detection (which shows no band shift) before detecting phospho-Ser157 (which does show a band shift)

  • Include total VASP detection to normalize phosphorylation levels

• Multiplex fluorescent Western blotting:

  • Use phospho-specific primary antibodies from different species

  • Detect with spectrally distinct fluorescent secondary antibodies

  • Analyze signals in different channels on fluorescent imaging systems

• Phospho-mutant expression studies:

  • Generate cells expressing combinations of phospho-mutants:

    • VASP-S239A (cannot be phosphorylated at Ser239)

    • VASP-S157A (cannot be phosphorylated at Ser157)

    • VASP-AA (double mutant)

  • Compare functional outcomes to dissect site-specific roles

• Mass spectrometry-based phosphoproteomics:

  • Provides unbiased detection of all phosphorylation sites

  • Enables quantitative comparison of phosphorylation stoichiometry

  • Can reveal novel phosphorylation sites and their dynamics

Interpretational Considerations:

• Consider the hierarchical nature of phosphorylation (some sites require prior phosphorylation at other sites)

• Account for different kinetics of phosphorylation/dephosphorylation at each site

• Evaluate cross-talk between different phosphorylation events and signaling pathways

Research has demonstrated that VASP Ser157 and Ser239 phosphorylation can have distinct and sometimes opposing effects on cell function, highlighting the importance of analyzing multiple phosphorylation sites to fully understand VASP regulation .

What emerging technologies might enhance the study of VASP Ser239 phosphorylation?

Several emerging technologies show promise for advancing VASP Ser239 phosphorylation research:

CRISPR-based Phosphorylation Reporters:

• Gene editing to tag endogenous VASP with fluorescent sensors

• Creating knock-in cell lines with phospho-mutant VASP at endogenous levels

• Developing CRISPR activation/inhibition systems to modulate kinases controlling VASP phosphorylation

Biosensors for Live Cell Imaging:

• Phosphorylation-sensitive FRET-based biosensors for real-time monitoring

• Optogenetic tools to spatiotemporally control VASP phosphorylation

• Genetically encoded fluorescent biosensors that change conformation upon VASP phosphorylation

High-throughput Screening Platforms:

• Microfluidic systems for rapid assessment of VASP phosphorylation in response to drug libraries

• Automated image analysis for quantifying changes in cell morphology associated with VASP phosphorylation

Single-cell Analysis Technologies:

• Single-cell phosphoproteomics to reveal cell-to-cell variability in VASP phosphorylation

• Spatial transcriptomics combined with phospho-protein analysis to map VASP activity in complex tissues

What are the therapeutic implications of targeting VASP Ser239 phosphorylation in disease?

The therapeutic implications of targeting VASP Ser239 phosphorylation span several disease areas:

Cardiovascular Disease:

• VASP Ser239 phosphorylation correlates with vascular relaxation, suggesting therapeutic potential in hypertension and other vascular disorders

• NO-cGMP-PKG pathway activators could be optimized to enhance VASP phosphorylation in vascular smooth muscle cells

• Monitoring VASP Ser239 phosphorylation may serve as a biomarker for drug efficacy in cardiovascular therapies

Cancer:

• VASP Ser239 phosphorylation suppresses invasive membrane protrusions and could represent an "invasion suppressor" mechanism

• Strategies to enhance VASP Ser239 phosphorylation might reduce cancer cell invasion and metastasis

• Research suggests that "ligand-dependent GCC signaling may prevent colon cancer metastasis by neutralizing oncogenic VASP functions at the invasive cancer front"

Inflammatory Conditions:

• VASP's role in cell motility and migration extends to immune cell function

• Modulating VASP Ser239 phosphorylation could potentially regulate inflammatory cell recruitment

Platelet Dysfunction:

• VASP phosphorylation inhibits platelet aggregation

• Targeted enhancement of VASP Ser239 phosphorylation represents a potential antithrombotic strategy

Research has shown that "VASP Ser239 phosphorylation, a simple intracellular biochemical reaction, could represent a unique invasion suppressor with sophisticated regulatory dynamics, an inducible mechanism embedded into signal transduction networks shaping tumor cell metastasis" , highlighting its potential as a therapeutic target.

How might the interplay between different VASP phosphorylation sites be better understood?

Understanding the complex interplay between different VASP phosphorylation sites requires sophisticated approaches:

Systems Biology Approaches:

• Mathematical modeling of phosphorylation dynamics at multiple sites

• Network analysis to identify key regulatory nodes controlling site-specific phosphorylation

• Computational prediction of structural changes induced by combinations of phosphorylation events

Advanced Structural Biology:

• Cryo-EM or X-ray crystallography of VASP in different phosphorylation states

• Molecular dynamics simulations to predict how phosphorylation alters protein conformation and function

• NMR studies to analyze dynamic structural changes upon phosphorylation

Combinatorial Mutagenesis:

• Generate comprehensive libraries of VASP phospho-mutants with combinations of sites mutated

• Use high-throughput functional assays to map the phenotypic consequences of each combination

• Identify synergistic, antagonistic, or hierarchical relationships between phosphorylation sites

Temporal Analysis:

• Single-molecule tracking to monitor VASP dynamics in real-time following phosphorylation

• High-temporal resolution phosphoproteomics to track the sequence of phosphorylation events

• Optogenetic tools to precisely control the timing of phosphorylation at specific sites