Phospho-DDX5 (Y593) Antibody

What is the biological significance of DDX5 phosphorylation at Y593?

Phosphorylation of DDX5 at the Y593 residue is a critical post-translational modification that significantly alters its functionality. When phosphorylated at Y593, DDX5 exhibits enhanced coactivation of androgen receptor transcription . More significantly, this phosphorylation promotes the dissociation of histone deacetylase 1 (HDAC1) from the Snail1 promoter, thereby activating Snail1 transcription, inhibiting E-cadherin expression, and ultimately promoting epithelial-mesenchymal transition (EMT) .

Additionally, Y593-phosphorylated DDX5 facilitates the nuclear translocation of β-catenin by blocking GSK-3β-induced β-catenin phosphorylation and replacing Axin in the β-catenin complex . This leads to activation of β-catenin target genes including cyclin D1 and c-Myc, stimulating cell proliferation . In glioblastoma, dual phosphorylation at Y593/Y595 inhibits cell apoptosis by suppressing XAF1 expression .

How does phosphorylated DDX5 (Y593) differ functionally from unphosphorylated DDX5?

Unphosphorylated DDX5 functions primarily as an RNA helicase involved in RNA metabolism, including pre-mRNA splicing, where its RNA helicase activity increases tau exon 10 inclusion . It also serves as a transcriptional regulator independent of its RNA helicase activity .

In contrast, Y593-phosphorylated DDX5 acquires additional oncogenic functions that significantly alter cellular processes:

• Enhanced transcriptional coactivation: Phospho-DDX5 (Y593) exhibits increased coactivation of androgen receptor transcription

• Altered protein interactions: Forms complexes with β-catenin, blocking its degradation and enabling nuclear translocation

• Epigenetic regulation: Promotes dissociation of HDAC1 from target promoters, altering transcriptional landscapes

• Cell survival promotion: Inhibits apoptotic pathways, particularly when dual-phosphorylated at Y593/Y595

• Metastatic potential: Activates EMT-related genes through Snail1 upregulation

These phosphorylation-dependent functions make phospho-DDX5 (Y593) a critical player in cancer progression and potential therapeutic target .

What are the optimal storage conditions for phospho-DDX5 (Y593) antibodies?

To maintain optimal antibody activity, phospho-DDX5 (Y593) antibodies should be stored according to the following evidence-based recommendations:

• Short-term storage (up to 2 weeks): Maintain at 4°C in refrigerated conditions

• Long-term storage: Store at -20°C or -80°C

• Aliquoting: Divide into small aliquots to avoid repeated freeze-thaw cycles, which can degrade antibody quality

• Buffer composition: Most commercial antibodies come in optimized buffers that typically include:

  • 30-50% glycerol (to prevent freezing damage)

  • Phosphate buffered saline (PBS) or Tris-buffered saline (TBS)

  • 0.01-0.02% sodium azide (as preservative)

  • Sometimes includes 0.5% BSA (as stabilizer)

• Temperature fluctuations: Avoid repeated freeze-thaw cycles which demonstrably reduce antibody efficacy

Testing has shown that antibodies stored under these conditions maintain their specificity and reactivity for the duration of their expected shelf life (typically 12 months from receipt) .

What are the recommended protocols for detecting phospho-DDX5 (Y593) in different experimental systems?

Based on validated research protocols, the following are recommended approaches for detecting phospho-DDX5 (Y593) in various experimental systems:

Western Blot Analysis:

• Dilution range: 1:500-1:2000

• Sample preparation: Total cell lysates (40μg protein typically sufficient)

• Recommended positive controls: MCF-7, A375, U-87MG, CT-26, C6 cell lines

• Molecular weight: Expected band at approximately 70 kDa

• Blocking: 5% BSA in TBST recommended over milk (phospho-epitopes can be masked by milk proteins)

Immunohistochemistry:

• Dilution range: 1:100-1:200

• Sample preparation: Formalin-fixed paraffin-embedded tissue sections

• Antigen retrieval: Heat-induced epitope retrieval with citrate buffer (pH 6.0)

• Detection: ABC method or polymer-based detection systems

• Validated tissues: Human prostate cancer has shown clear phospho-DDX5 (Y593) staining

Immunofluorescence:

• Dilution range: 1:100-1:500

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: 0.1% Triton X-100 for 5 minutes

• Expected subcellular localization: Predominantly nuclear staining

ELISA:

• Validated for detecting phospho-DDX5 (Y593)

• Capture antibody concentration: 1-2 μg/ml

• Detection antibody dilution: 1:1000-1:5000

How can I reliably quantify changes in DDX5 phosphorylation status following experimental treatments?

For reliable quantification of DDX5 phosphorylation at Y593 following experimental treatments, researchers should implement multiple complementary approaches:

Western Blot Quantification:

• Always run parallel blots with both phospho-specific and total DDX5 antibodies

• Calculate the phospho-DDX5/total DDX5 ratio to normalize for variations in total protein expression

• Include appropriate loading controls (β-actin, GAPDH)

• Use digital image analysis software (ImageJ, Li-Cor, etc.) for densitometric analysis

• Perform at least three independent experiments for statistical validity

Phosphorylation-Specific Flow Cytometry:

• Fix cells with 4% paraformaldehyde

• Permeabilize with cold methanol

• Stain with fluorophore-conjugated phospho-DDX5 (Y593) antibodies (like Cy5.5 conjugated antibodies)

• This allows quantification at single-cell resolution and detection of heterogeneous responses

Mass Spectrometry-Based Approaches:

• Immunoprecipitate DDX5 using total DDX5 antibodies

• Perform tryptic digestion

• Use targeted mass spectrometry to quantify the absolute stoichiometry of Y593 phosphorylation

• This provides the most accurate measurement of phosphorylation levels

Proximity Ligation Assay (PLA):

• Use antibodies against total DDX5 and phosphotyrosine

• PLA signals will only appear when DDX5 is phosphorylated

• This approach allows in situ visualization of phosphorylation events in intact cells

Research has demonstrated that integrating multiple quantification methods provides the most reliable assessment of phosphorylation changes, particularly when studying signaling dynamics or drug responses .

What are the most effective strategies for validating phospho-DDX5 (Y593) antibody specificity?

Rigorous validation of phospho-DDX5 (Y593) antibody specificity is critical for reliable research outcomes. Based on established research practices, implementation of the following comprehensive strategy is recommended:

1. Peptide Competition Assays:

• Pre-incubate antibody with phosphorylated and non-phosphorylated Y593 peptides

• Specific binding should be blocked only by the phospho-peptide and not by non-phosphorylated peptide

• This confirms phospho-specificity of the antibody

2. Phosphatase Treatment Controls:

• Treat one sample set with lambda phosphatase before immunoblotting

• Signal should be eliminated in phosphatase-treated samples while total DDX5 remains detectable

• This verifies that the antibody specifically recognizes the phosphorylated form

3. Genetic Validation Approaches:

• Use CRISPR/Cas9 to generate Y593F mutant cell lines (phenylalanine cannot be phosphorylated)

• Compare antibody reactivity between wild-type and Y593F mutant samples

• Absence of signal in Y593F mutants confirms specificity

4. Kinase Modulation:

• Treat cells with kinase inhibitors known to affect DDX5 phosphorylation

• Alternatively, overexpress kinases that target DDX5

• Observe corresponding changes in phospho-Y593 signal

5. Cross-Reactivity Assessment:

• Test antibody against recombinant phosphorylated and non-phosphorylated DDX5

• Include the highly homologous DDX17 to ensure no cross-reactivity

• Western blot analysis should show a single band at the expected molecular weight (~70 kDa)

6. Application-Specific Validation:

• Validate separately for each application (WB, IHC, IF, etc.)

• Use positive controls with known DDX5 Y593 phosphorylation (e.g., cells treated with growth factors)

• Include negative controls (untransfected cells, secondary antibody only)

Implementation of these validation strategies ensures that experimental findings truly reflect specific phospho-DDX5 (Y593) detection rather than non-specific or artifactual signals.

How does the phosphorylation status of DDX5 at Y593 impact its role in β-catenin signaling in cancer?

The phosphorylation of DDX5 at Y593 creates a critical molecular switch that fundamentally alters β-catenin signaling with profound implications for cancer progression. Mechanistically, phospho-DDX5 (Y593) promotes β-catenin nuclear translocation through multiple coordinated pathways:

Direct Mechanism of Action:

• Phospho-DDX5 (Y593) physically blocks GSK-3β-mediated phosphorylation of β-catenin

• This prevents β-catenin targeting for proteasomal degradation

• Phospho-DDX5 (Y593) replaces Axin in the β-catenin destruction complex, further stabilizing β-catenin

• The stabilized β-catenin accumulates and translocates to the nucleus

• In the nucleus, phospho-DDX5 (Y593) enhances β-catenin/TCF transcriptional activity

Oncogenic Consequences:

Therapeutic Targeting:
Compounds like RX-5902 (a quinoxalinyl-piperazine compound) have been developed to specifically:

• Attenuate phosphorylation of DDX5 at Y593

• Lead to cytoplasmic accumulation of β-catenin (preventing nuclear activity)

• Downregulate c-MYC expression

• Show promise in AML treatment by targeting this specific mechanism

This phosphorylation-dependent regulation represents a targetable vulnerability in cancers dependent on aberrant β-catenin signaling, including AML, colorectal cancer, and several other malignancies .

What is the relationship between DDX5 Y593 phosphorylation and tumor immune microenvironment modulation?

Recent single-cell and multi-omic analyses have uncovered a previously unrecognized relationship between DDX5 Y593 phosphorylation status and tumor immune microenvironment (TIME) modulation, with potentially significant implications for cancer immunotherapy.

Impact on Immune Cell Infiltration:

• High DDX5 expression tumors demonstrate:

  • Significantly reduced B cell infiltration

  • Reduced macrophage infiltration, particularly M2-type

  • Increased T cell infiltration

• DDX5 expression shows a strong negative correlation specifically with M2 macrophage (Macro_spp1) infiltration in the tumor microenvironment, as verified by multi-immunofluorescence staining

Mechanistic Insights:
Analysis of macrophage subtype distribution in relation to DDX5 expression reveals:

• Elevation of Macro_apoec3a macrophages in DDX5-high tumors

• Significant reduction in Macro_spp1, Macro_thbs1, and Macro_ccnl1 macrophages in DDX5-high tumors

• These alterations in macrophage polarization may explain the decreased pro-tumor immune environment in DDX5-high contexts

Clinical Correlations:
Multi-immunofluorescence staining of clinical tongue cancer samples confirmed a negative association between DDX5 expression and M2-type macrophage infiltration in the tumor microenvironment

This evidence suggests that phospho-DDX5 status may serve as a biomarker for immunotherapy responsiveness and raises the possibility that targeting DDX5 phosphorylation could potentially reprogram the tumor immune microenvironment. The specific molecular mechanisms connecting Y593 phosphorylation to these immune modulatory effects requires further investigation, but appears to involve alteration of cancer cell-derived signals that influence immune cell recruitment and polarization .

How do different phosphorylation sites on DDX5 interact with Y593 phosphorylation to regulate its function?

DDX5 function is regulated by a complex interplay of multiple phosphorylation sites that can work synergistically, antagonistically, or independently of Y593 phosphorylation. This combinatorial code creates a sophisticated regulatory network with context-dependent outcomes.

Multiple Phosphorylation Sites and Their Interactions:

Regulatory Complexity:

• Phosphorylation at different residues can exert opposite effects even within the same protein

  • Y593/Y595 phosphorylation promotes cell survival

  • T564/T446 phosphorylation promotes apoptosis during chemotherapy

• The temporal sequence of phosphorylation events matters:

  • Y593 phosphorylation appears to be a primary event that enables subsequent protein interactions

  • Secondary phosphorylation events may fine-tune these interactions

• Context-dependent outcomes:

  • In some cancer types, certain phosphorylation combinations predominate

  • The kinase/phosphatase balance in different cellular contexts determines which sites are phosphorylated

This complex phosphorylation interplay highlights the need for comprehensive phosphoproteomic analysis when studying DDX5 function, as focusing solely on Y593 may miss critical regulatory mechanisms. Additionally, therapeutic strategies targeting DDX5 should consider this multisite phosphorylation network to achieve desired specificity .

How does phospho-DDX5 (Y593) contribute differently to solid tumors versus hematological malignancies?

Phospho-DDX5 (Y593) exhibits distinct mechanisms of action and clinical implications in solid tumors compared to hematological malignancies, highlighting the context-dependent nature of its oncogenic functions.

Solid Tumors:

• Epithelial-Mesenchymal Transition (EMT) Regulation:

  • In solid tumors, phospho-DDX5 (Y593) promotes EMT by activating Snail1 transcription through dissociation of HDAC1 from the Snail1 promoter

  • This leads to repression of E-cadherin and increased invasiveness

  • This mechanism is particularly important in carcinomas where EMT drives metastasis

• Hormone Receptor Coactivation:

  • Enhances transcriptional coactivation of androgen receptor in prostate cancer

  • Modulates estrogen receptor signaling in breast cancer

  • This hormone-dependent regulation is specific to certain solid tumors

• Tissue-Specific Effects:

  • Acts as a tumor suppressor in tongue cancer, with high expression associated with better outcomes

  • Functions as an oncogene in glioblastoma through dual Y593/Y595 phosphorylation

  • Required for proliferation in colon cancer cell lines like HCT116

Hematological Malignancies (AML):

These distinctions highlight the importance of cancer-specific approaches when targeting phospho-DDX5 (Y593), as strategies effective in hematological malignancies may not translate directly to solid tumors due to these fundamental differences in molecular mechanism and cellular context .

What are the most effective experimental models for studying phospho-DDX5 (Y593) function in different cancer types?

Selecting appropriate experimental models is crucial for investigating phospho-DDX5 (Y593) functions in cancer research. Based on validated research approaches, the following models offer distinct advantages for different research questions:

Cell Line Models:

Genetic Modification Approaches:

• CRISPR/Cas9 Y593F mutant lines: Replace tyrosine with phenylalanine to prevent phosphorylation

• Phosphomimetic Y593E mutants: Replace tyrosine with glutamic acid to mimic constitutive phosphorylation

• Inducible DDX5 knockdown/knockout systems: Temporal control over DDX5 depletion

• Site-specific phosphorylation sensors: FRET-based reporters to monitor Y593 phosphorylation dynamics

Animal Models:

• Xenograft models using phospho-DDX5 (Y593) modified cell lines

• Patient-derived xenografts (PDXs) from tumors with high phospho-DDX5 (Y593)

• Genetically engineered mouse models with DDX5 Y593F knock-in mutations

• Orthotopic models for tissue-specific microenvironment interactions

3D/Organoid Models:

• Patient-derived organoids maintaining original tumor phospho-DDX5 status

• Spheroid co-culture systems with immune cells to study microenvironment effects

• Scaffold-based 3D cultures to assess invasion properties dependent on phospho-DDX5 (Y593)

Clinical Sample Analysis:

• Multi-immunofluorescence tissue staining for phospho-DDX5 (Y593) and interacting partners

• Single-cell RNA sequencing combined with phosphoproteomic analysis

• Tissue microarrays with paired primary and metastatic samples

Researchers should select models based on specific research questions, considering the distinct phospho-DDX5 (Y593) functions observed in different cancer contexts .

How can phospho-DDX5 (Y593) status be utilized as a biomarker for cancer prognosis or treatment response?

Emerging evidence supports the potential of phospho-DDX5 (Y593) as a clinically relevant biomarker for both prognostication and prediction of treatment response across several cancer types. Implementation of phospho-DDX5 (Y593) as a biomarker requires consideration of multiple factors:

Prognostic Value:

Predictive Biomarker Applications:

• β-catenin Pathway Inhibitors:

  • Tumors with high phospho-DDX5 (Y593) show enhanced β-catenin nuclear localization

  • These tumors may be particularly responsive to β-catenin pathway inhibitors

  • RX-5902, which attenuates DDX5 Y593 phosphorylation, shows promise in AML with high phospho-DDX5 levels

• Rational Combination Therapy Selection:

  • Phospho-DDX5 (Y593) status may help identify tumors that would benefit from combining standard therapies with targeted phospho-DDX5 inhibitors

  • The antagonistic relationship between Y593/Y595 and T564/T446 phosphorylation suggests the potential for phosphorylation site-specific targeting

• Immunotherapy Response Prediction:

  • DDX5 expression levels correlate with immune cell infiltration patterns

  • Low phospho-DDX5 (Y593) tumors show increased M2 macrophage infiltration, suggesting potential resistance to certain immunotherapies

  • This could inform patient selection for immunotherapy approaches

Methodological Considerations for Clinical Implementation:

• Tissue-based Detection:

  • Immunohistochemistry with validated phospho-specific antibodies

  • Quantitative scoring systems needed (H-score or digital image analysis)

  • Standardized cutoff values for "high" vs "low" expression

• Liquid Biopsy Potential:

  • Investigation of circulating tumor cells (CTCs) for phospho-DDX5 (Y593) status

  • Exosomal phospho-DDX5 as a non-invasive biomarker

• Combinatorial Biomarker Approaches:

  • Integrating phospho-DDX5 (Y593) with other phosphorylation sites (Y595, T564, etc.)

  • Combining with markers of related pathways (β-catenin, AR signaling)

  • Multiparametric assessment for improved predictive power

These biomarker applications require further validation in prospective clinical studies to establish standardized protocols and clinically meaningful cutoffs before routine implementation .

How can potential cross-reactivity with other phosphorylated proteins be minimized when using phospho-DDX5 (Y593) antibodies?

Minimizing cross-reactivity is essential for accurate phospho-DDX5 (Y593) detection. Researchers should implement the following comprehensive strategies based on established immunological principles and phospho-specific antibody validation approaches:

Antibody Selection and Validation:

• Choose antibodies raised against longer phosphopeptides (>10 amino acids) surrounding Y593

• Select antibodies validated against multiple species to confirm recognition of the conserved epitope

• Verify manufacturer's validation data that explicitly tests for cross-reactivity with similar proteins (especially DDX17)

• Perform in-house validation with phosphatase treatment controls and Y593F mutants

Experimental Design Optimizations:

• Implement a sequential immunoprecipitation approach:

  • First immunoprecipitate with total DDX5 antibody

  • Then probe with phospho-specific antibody

  • This ensures the detected phosphoprotein is specifically DDX5

• Use DDX5 knockdown or knockout controls:

  • Include DDX5 siRNA/shRNA treated samples

  • Any remaining signal in knockdown samples indicates potential cross-reactivity

• Peptide competition assays with graduated specificity:

  • Include phospho-Y593 DDX5 peptide

  • Include non-phosphorylated Y593 DDX5 peptide

  • Include phosphopeptides from proteins with similar sequences

  • Signal should be blocked only by the specific phospho-Y593 DDX5 peptide

Technical Adjustments:

• Increase stringency of wash buffers:

  • Use higher salt concentrations (250-500 mM NaCl)

  • Add non-ionic detergents (0.1-0.5% Triton X-100)

  • These reduce non-specific binding

• Optimize antibody dilutions:

  • Perform titration experiments to find minimum effective concentration

  • Working at lower antibody concentrations reduces non-specific binding

• For blotting applications:

  • Block with 5% BSA rather than milk (phospho-epitopes can be masked by milk)

  • Add phosphatase inhibitors to all buffers

  • Consider using PVDF membranes for higher protein retention and signal-to-noise ratio

• For immunofluorescence:

  • Include double staining with total DDX5 antibody from different species

  • True phospho-DDX5 (Y593) signal should colocalize with total DDX5

Implementing these strategies ensures that signals detected truly represent phospho-DDX5 (Y593) and not cross-reactive phosphoproteins, which is particularly important given the conserved nature of many phosphorylation motifs .

What are the advantages and limitations of different detection methods for phospho-DDX5 (Y593) in complex biological samples?

Different detection methodologies for phospho-DDX5 (Y593) offer distinct advantages and limitations when applied to complex biological samples. Understanding these tradeoffs enables researchers to select the most appropriate technique based on specific experimental objectives:

Method Selection Guidelines:

• For initial phosphorylation status assessment: Western blot provides robust detection and semi-quantification

• For spatial distribution in tissues: IHC or IF are preferred methods

• For absolute quantification: Mass spectrometry provides the most definitive measurement

• For heterogeneous populations: Phospho-flow cytometry enables single-cell analysis

• For protein interaction studies: Proximity ligation assay or co-immunoprecipitation followed by western blot

Each method requires proper controls and validation to ensure specificity for phospho-DDX5 (Y593) detection in complex biological samples .

What are the critical considerations for designing experiments to study the functional impact of DDX5 Y593 phosphorylation?

Designing rigorous experiments to delineate the functional impact of DDX5 Y593 phosphorylation requires careful consideration of multiple factors to ensure valid and reproducible results. Based on published research approaches, the following critical considerations should guide experimental design:

1. Phosphorylation Site Manipulation Strategies:

A. Genetic Approaches:

• Y593F mutant (cannot be phosphorylated): Essential for loss-of-function studies

• Y593E mutant (phosphomimetic): Simulates constitutive phosphorylation

• Y593A mutant: Alternative neutral substitution to control for structural changes

• Double mutants (Y593F/Y595F): Control for potential compensatory phosphorylation at adjacent sites

B. Expression System Selection:

• Inducible expression systems to control timing and expression level

• Rescue experiments in DDX5 knockout backgrounds to eliminate endogenous protein interference

• CRISPR knock-in mutations for studying at endogenous expression levels

2. Kinase and Phosphatase Modulation:

A. Kinase Identification and Inhibition:

• Identify kinases responsible for Y593 phosphorylation (c-Abl, Src family implicated)

• Use specific kinase inhibitors to modulate phosphorylation status

• Develop inducible kinase expression systems to trigger phosphorylation

B. Phosphatase Considerations:

• Identify phosphatases that dephosphorylate Y593

• Use phosphatase inhibitors to maintain phosphorylation

• Consider temporal dynamics of phosphorylation/dephosphorylation cycles

3. Functional Readout Selection:

A. Target Gene Expression:

• β-catenin target genes (c-Myc, cyclin D1)

• Snail1 and EMT markers (E-cadherin)

• Androgen receptor target genes

B. Cellular Phenotypes:

• Proliferation assays (optimized for cell type-specific growth characteristics)

• Apoptosis assessment (particularly relevant for dual Y593/Y595 phosphorylation)

• Cell migration and invasion assays (relevant for EMT functions)

• Nuclear/cytoplasmic fractionation (for β-catenin localization studies)

C. Molecular Interactions:

• Co-immunoprecipitation with interaction partners (β-catenin, HDAC1, Axin)

• Chromatin immunoprecipitation for transcriptional targets

• RNA immunoprecipitation for RNA binding targets (if relevant)

4. Context Dependency Considerations:

A. Cell Type Selection:

• Include multiple cell lines representing different cancer types

• Compare cells where DDX5 acts as oncogene vs. tumor suppressor

• Include normal cell counterparts as controls

B. Stress and Microenvironmental Factors:

• Test under different growth conditions (serum starvation, hypoxia)

• Include immune cell co-culture models when studying microenvironment effects

• Assess phosphorylation changes during drug treatments or radiation

5. Technical Controls and Validation:

A. Phosphorylation Status Verification:

• Always confirm phosphorylation status with validated phospho-specific antibodies

• Include phosphatase-treated controls in key experiments

• Monitor total DDX5 levels alongside phosphorylation status

B. Specificity Controls:

• Include DDX17 controls to ensure observed effects are DDX5-specific

• Use multiple independent siRNAs/shRNAs for knockdown studies

• Perform rescue experiments with phosphorylation site mutants

By addressing these critical considerations, researchers can design comprehensive experiments that provide meaningful insights into the specific functional consequences of DDX5 Y593 phosphorylation in different biological contexts .