Phospho-EZR (Tyr353) Antibody

What is the biological significance of Ezrin Tyr-353 phosphorylation?

Phosphorylation at tyrosine 353 represents a key regulatory mechanism in the transition of ezrin from its dormant to active state. Ezrin, a cytoskeletal organizer and member of the ezrin-radixin-moesin (ERM) family, plays crucial roles in cell motility, adhesion, and signaling pathways. Unlike unphosphorylated ezrin which remains in a folded conformation through head-to-tail interaction (masking binding sites for other molecules), phosphorylation at Tyr-353 helps unlock ezrin's functional capacity.

Research demonstrates that Tyr-353 phosphorylation is linked to p85 interaction and Akt overexpression in the PI3-kinase/Akt pathway . Together with Thr-567, Tyr-353 phosphorylation regulates ezrin's transition to its active form, enabling it to serve as an intermediate between the plasma membrane and actin cytoskeleton .

In cancer research, Tyr-353 phosphorylation has been implicated in metastasis and tumor progression, making it a valuable biomarker for studying these processes .

How does Phospho-EZR (Tyr353) differ from Phospho-EZR (Thr567) in cellular localization and function?

These two phosphorylation sites show distinct differences in localization, regulation, and biological functions:

Research indicates that Thr-567 phosphorylation is essential for ezrin's full activation, allowing actin filament binding domains to interact with other proteins . Meanwhile, Tyr-353 phosphorylation appears to play complementary but distinct roles in activating ezrin's functions .

What are the recommended detection methods for Phospho-EZR (Tyr353)?

Several validated methods are available for detecting Phospho-EZR (Tyr353), each with specific applications and protocols:

• Western Blotting: The most common method, typically using dilutions of 1:500-1:1000. Expected molecular weight is ~80-81 kDa .

• Immunohistochemistry (IHC): Recommended dilutions range from 1:50-1:100 for formalin-fixed, paraffin-embedded tissues .

• Cell-Based ELISA: Provides a convenient, lysate-free approach for high-throughput screening of treatments affecting ezrin phosphorylation in cultured cells .

• Colorimetric Cell-Based ELISA: Offers high sensitivity and specificity for measuring EZRIN phosphorylation at Tyr-353 in cell-based assays with a dynamic range of >5000 cells .

When selecting an antibody, consider species reactivity (human, mouse, rat) and validation data. For optimal results, researchers should perform titration experiments to determine the optimal antibody concentration for their specific experimental conditions and sample types .

What is the relationship between estrogen signaling and Ezrin Tyr-353 phosphorylation?

17β-estradiol (E2) has been shown to enhance ezrin phosphorylation in breast cancer cells, promoting cell motility and invasion. The signaling cascade involves:

• Estrogen receptor (ER) interaction with the non-receptor tyrosine kinase c-Src

• Activation of the PI3K/Akt pathway

• Activation of the RhoA/ROCK-2 complex

• Subsequent phosphorylation of ezrin at key residues

Research indicates that E2 rapidly enhances ezrin phosphorylation in a time- and concentration-dependent manner . While the study primarily focused on Thr-567 phosphorylation, it also noted that E2 could potentially directly phosphorylate Tyr-353 via c-Src interaction with ezrin .

This estrogen-induced ezrin activation contributes to breast cancer cell movement and invasion, providing insights into estrogen's effects on breast cancer progression and highlighting potential therapeutic targets for endocrine-sensitive breast cancers .

How does tissue-specific context affect Ezrin Tyr-353 phosphorylation?

Ezrin phosphorylation patterns show remarkable tissue-specific differences:

Different tissues maintain distinct steady-state levels of ezrin phosphorylation. For example, renal proximal tubule cells and small intestinal enterocytes (both with rich brush border microvilli) maintain higher steady-state levels of ezrin phosphorylation compared to gastric parietal cells .

These tissue-specific differences in phosphorylation patterns may reflect the varying demands for membrane-cytoskeleton organization across different cell types. The turnover of phosphorylation appears to empower ezrin to relax and reposition membrane to the underlying cytoskeleton under varying conditions of filament growth or rapid membrane expansion/depletion .

When designing experiments involving ezrin phosphorylation, researchers should consider these tissue-specific contexts, as they may significantly impact experimental outcomes and interpretation of results .

What are the optimal experimental conditions for detecting dynamic changes in Ezrin Tyr-353 phosphorylation?

To effectively detect dynamic changes in Ezrin Tyr-353 phosphorylation:

Experimental Design Considerations:

• Time Course Experiments: Monitor phosphorylation at multiple timepoints (2-30 minutes) following stimulation. Research shows that E2 treatment leads to increased phosphorylation between 2-10 minutes, declining after 20 minutes .

• Concentration Gradients: Test multiple concentrations of stimulants. For example, E2 has been shown to increase phosphorylation at concentrations ranging from 10^-10 to 10^-6 M .

• Positive and Negative Controls:

  • Positive control: Treatment with protein phosphatase inhibitors like calyculin A (CLA) to maximize phosphorylation levels

  • Negative control: Treatment with metabolic inhibitors like sodium azide to minimize phosphorylation by depleting ATP

• Pathway Inhibitors: Include c-Src inhibitors (e.g., PP2) or PI3K inhibitors (e.g., dominant negative forms of p85α) to confirm the signaling pathway's involvement .

Detection Protocol Optimization:

• For Western blotting, ensure rapid sample preparation in cold conditions with phosphatase inhibitors to prevent dephosphorylation during processing

• For immunofluorescence, fix cells quickly after stimulation to capture transient phosphorylation events

• Consider using phospho-specific antibodies from multiple sources to validate findings

Remember that ezrin phosphorylation is highly dynamic and context-dependent, so experimental conditions should be carefully controlled to obtain reproducible results .

How can researchers effectively design silencing experiments to study Ezrin Tyr-353 phosphorylation functions?

When designing RNA interference or CRISPR-based approaches to study Ezrin Tyr-353 phosphorylation:

Strategic Approach:

• Target Selection Options:

  • Silence total ezrin protein expression using siRNAs targeting the EZR gene

  • Use site-specific mutagenesis to create phospho-null (Y353F) or phospho-mimetic (Y353D/E) ezrin mutants

  • Target upstream kinases in the PI3K/Akt pathway that regulate Tyr-353 phosphorylation

• Validation Methods:

  • Confirm knockdown efficiency via Western blot for total ezrin

  • Verify phosphorylation status using phospho-specific antibodies

  • Assess functional outcomes through migration/invasion assays

Experimental Design Considerations:

• Include appropriate controls:

  • Non-targeting siRNA controls

  • Wild-type ezrin expression constructs

  • Rescue experiments with RNAi-resistant ezrin constructs

• Timing considerations:

  • For transient knockdown, assess 48-72h post-transfection

  • For stable knockdown, select clones with confirmed ezrin reduction

• Functional readouts to consider:

  • Cell adhesion assays

  • Migration/invasion assays (as demonstrated in T47-D breast cancer cells)

  • 3D matrix invasion assays to better mimic in vivo conditions

Research has shown that E2-enhanced horizontal cell migration and invasion of breast cancer cells in three-dimensional matrices is reversed by transfection with specific ezrin siRNAs, demonstrating the effectiveness of this approach .

What are the clinical implications of aberrant Ezrin Tyr-353 phosphorylation in cancer progression?

Ezrin Tyr-353 phosphorylation has significant implications for cancer progression and potential therapeutic approaches:

Clinical Correlations:

• Increased ezrin Tyr-353 levels correlate with late stage and poor differentiation in NSCLC .

• Ezrin phosphorylation is significantly upregulated in cancer tissues compared to normal counterparts .

• While both Tyr-353 and Thr-567 phosphorylation correlate with poor prognosis, they show distinct patterns of association with clinical parameters:

  • Only ezrin Thr-567 correlates with lymph node metastasis

  • Ezrin Tyr-353 shows distinct cytosolic staining patterns compared to the cytosolic and membranous patterns of Thr-567

Mechanistic Insights:

• Phosphorylation at Tyr-353 is linked to p85 interaction and Akt pathway activation, which drives:

  • Enhanced cell survival

  • Increased invasive capacity

  • Promotion of metastatic behavior

• In breast cancer, estrogen stimulation leads to ezrin phosphorylation via:

  • ER interaction with c-Src

  • Activation of PI3K/Akt pathway

  • Subsequent enhancement of cell motility and invasion

Therapeutic Implications:
Research on anti-metastatic small molecules targeting ezrin phosphorylation (such as NSC30587 and NSC668394) demonstrates the potential for developing targeted therapies that inhibit ezrin's actin-binding activity, which could offer new therapeutic directions for clinical cancer interventions .

How does the interplay between Thr-567 and Tyr-353 phosphorylation regulate complete Ezrin activation?

The coordinated phosphorylation of ezrin at multiple sites creates a sophisticated regulatory mechanism:

Sequential Activation Model:

• Initial Membrane Recruitment: PIP2 binding initially recruits ezrin to the plasma membrane

• Conformational Changes: Phosphorylation events then induce critical conformational changes:

  • Thr-567 phosphorylation (regulated by p38 MAP-kinase) breaks the head-to-tail association, exposing binding sites for other molecules

  • Tyr-353 phosphorylation (regulated through PI3K/Akt pathway) enhances interaction with signaling partners like p85

• Functional Specialization:

  • Thr-567 phosphorylation primarily affects ezrin's interaction with the actin cytoskeleton

  • Tyr-353 phosphorylation appears more involved in signaling pathway activation

Temporal Dynamics:
Research indicates that these phosphorylation events may occur with different temporal patterns. For example, in response to estrogen stimulation, phosphorylation of c-Src occurs within 2-10 minutes and then declines after 20 minutes, potentially affecting the timing of downstream ezrin phosphorylation events .

Tissue-Specific Regulation:
The relative importance of each phosphorylation site varies by tissue context. Studies comparing gastric glands, renal proximal tubules, and small intestine show distinct patterns of phosphorylation regulation across these tissues .

This multi-site phosphorylation system likely provides redundancy, specificity, and fine-tuning of ezrin's functions in different cellular contexts .

What methodological approaches can resolve contradictory findings about Ezrin Tyr-353 phosphorylation across different experimental systems?

Researchers encountering contradictory findings about Ezrin Tyr-353 phosphorylation should consider several methodological approaches:

Source of Variability Analysis:

• Tissue/Cell Type Differences: Different tissues maintain distinct steady-state phosphorylation levels and regulatory mechanisms. For example, renal proximal tubules and small intestinal enterocytes show different ezrin phosphorylation patterns compared to gastric glands .

• Experimental Conditions:

  • Culture conditions (serum components, confluence)

  • Sample preparation methods (lysis buffers, phosphatase inhibitors)

  • Detection methods (antibody specificity, protocol variations)

• Temporal Dynamics: Phosphorylation events are often transient. Sampling at different timepoints may yield contradictory results .

Resolution Strategies:

• Standardized Protocols:

  • Implement consistent cell culture conditions

  • Use standardized stimulation protocols with precise timing

  • Apply identical sample preparation methods with appropriate controls

• Multi-Method Validation:

  • Employ multiple antibodies from different vendors

  • Utilize complementary techniques (Western blot, immunofluorescence, mass spectrometry)

  • Confirm with genetic approaches (phospho-null or phospho-mimetic mutants)

• Context Documentation:

  • Thoroughly document experimental conditions

  • Consider the activation state of upstream pathways

  • Account for cross-talk between signaling cascades

• Positive and Negative Controls:

  • Include phosphatase inhibitors (like CLA) as positive controls

  • Use metabolic inhibitors (like sodium azide) as negative controls

  • Include genetic models with known phosphorylation patterns

By systematically addressing these variables, researchers can better understand the context-dependent nature of ezrin phosphorylation and reconcile apparently contradictory findings across different experimental systems.