Phospho-DOK1 (Y398) Antibody

What is DOK1 and what cellular functions does it regulate?

DOK1 (Docking protein 1, also known as p62dok or pp62) is an enzymatically inert adaptor or scaffolding protein that provides a docking platform for the assembly of multimolecular signaling complexes . DOK1 is an abundant Ras-GTPase-activating protein-associated tyrosine kinase substrate that plays several important regulatory roles in cellular signaling :

• It negatively regulates cell growth and proliferation

• It promotes cell migration and motility

• It appears to function as a negative regulator of the insulin signaling pathway

• It modulates integrin activation by competing with talin for the same binding site on ITGB3

• It regulates platelet-derived growth factor (PDGF)-BB-stimulated glioma cell motility

DOK1's activity is primarily regulated through phosphorylation at multiple tyrosine and serine residues, with Y398 being one of the critical regulatory sites.

What is the significance of Y398 phosphorylation in DOK1?

Y398 is a key tyrosine phosphorylation site in DOK1 that becomes phosphorylated in response to various stimuli. Research indicates that phosphorylation at this site is crucial for DOK1's downstream signaling functions. Specifically:

• PDGF-BB stimulates DOK1 phosphorylation on both Tyr362 and Tyr398

• This phosphorylation is critical for PDGF-BB-stimulated tyrosine phosphorylation of p130Cas, a key component in cell migration signaling pathways

• Phosphorylation at Y398 plays an essential role in DOK1's ability to regulate cell motility and invasion

• It may influence DOK1's interaction with other signaling molecules in various cellular contexts

This site-specific phosphorylation represents a critical regulatory mechanism that determines DOK1's functional activity in various signaling pathways.

How should Phospho-DOK1 (Y398) antibody be stored and handled for optimal results?

For optimal antibody performance and longevity, follow these research-validated storage and handling recommendations:

When working with this antibody, it's advisable to make small aliquots upon first thaw to minimize freeze-thaw cycles, which can degrade antibody performance over time.

What validation methods confirm the specificity of Phospho-DOK1 (Y398) antibody?

The specificity of Phospho-DOK1 (Y398) antibody has been validated through multiple complementary approaches:

• Western blot analysis: The antibody detects a specific band at approximately 62 kDa corresponding to phosphorylated DOK1 in extracts from K562 cells

• Immunohistochemistry: Specific staining has been demonstrated in paraffin-embedded human breast carcinoma tissues

• Peptide competition assays: Similar to validation approaches used for other phospho-specific DOK1 antibodies, specificity can be confirmed by demonstrating loss of signal when the antibody is pre-incubated with the phosphorylated immunizing peptide

• Phosphatase treatment controls: As demonstrated with other phospho-DOK1 antibodies, lambda phosphatase treatment eliminates the specific band, confirming phospho-specificity

These validation approaches collectively establish that the antibody specifically recognizes the phosphorylated Y398 epitope of DOK1 without significant cross-reactivity to unphosphorylated DOK1 or other phosphorylated proteins.

What are the recommended protocols for using Phospho-DOK1 (Y398) antibody in Western blotting?

For optimal Western blot results with Phospho-DOK1 (Y398) antibody, the following protocol elements are recommended:

• Sample preparation:

  • Cells should be lysed in buffer containing phosphatase inhibitors to preserve phosphorylation status

  • Include protease inhibitors to prevent protein degradation

  • Quantify protein concentration and load 25-50 μg per lane

• Electrophoresis and transfer:

  • Use 8-10% SDS-PAGE gels to achieve optimal separation around the 62 kDa region

  • Transfer to PVDF or nitrocellulose membrane using standard protocols

• Antibody incubation:

  • Blocking: 3% BSA in TBST (preferred over milk, which contains phosphatases)

  • Primary antibody dilution: 1:500-1:2000 in blocking buffer

  • Incubation: Overnight at 4°C with gentle agitation

  • Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:10,000 dilution

• Detection:

  • Use enhanced chemiluminescence (ECL) detection systems

  • Expected molecular weight: 62 kDa

• Controls:

  • Positive control: Extracts from cells known to express phosphorylated DOK1 (e.g., K562 cells)

  • Negative control: Lambda phosphatase-treated samples

  • Peptide competition control: Antibody pre-incubated with immunizing phosphopeptide

What are the recommended protocols for Immunohistochemistry using Phospho-DOK1 (Y398) antibody?

For immunohistochemical detection of phosphorylated DOK1 (Y398) in tissue samples:

• Sample preparation:

  • Fix tissues in formalin and embed in paraffin

  • Section tissues at 4-6 μm thickness

  • Mount on positively charged slides

• Deparaffinization and antigen retrieval:

  • Deparaffinize in xylene and rehydrate through graded alcohols

  • Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Cool slides to room temperature

• Staining procedure:

  • Block endogenous peroxidase activity with 3% H₂O₂

  • Block non-specific binding with 5% normal goat serum

  • Primary antibody dilution: 1:50-1:100

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Detect using an appropriate detection system (e.g., HRP-polymer and DAB)

  • Counterstain with hematoxylin, dehydrate, and mount

• Controls:

  • Positive control: Human breast carcinoma tissue

  • Negative control: Primary antibody omission or non-immune IgG

  • Peptide competition: Pre-incubation with immunizing phosphopeptide

How does DOK1 Y398 phosphorylation integrate with PDGF-BB signaling pathways?

DOK1 Y398 phosphorylation plays a crucial role in PDGF-BB signaling cascades, particularly in the context of cell motility and migration:

• Activation mechanism:

  • PDGF-BB stimulation leads to rapid phosphorylation of DOK1 at tyrosine residues 362 and 398

  • This phosphorylation is partially dependent on PI3K activity, as treatment with the PI3K inhibitor LY294002 reduces PDGF-stimulated DOK1 phosphorylation at these sites

• Downstream effectors:

  • Phosphorylated DOK1 mediates PDGF-BB-stimulated tyrosine phosphorylation of p130Cas, a key adaptor protein in cell migration

  • DOK1 Y398 phosphorylation is crucial for the activation of Rap1, a small GTPase involved in cytoskeletal reorganization

• Functional consequences:

  • When Y362 and Y398 are mutated to phenylalanine (DOK1FF), PDGF-BB-stimulated p130Cas tyrosine phosphorylation is significantly decreased

  • DOK1FF expression also results in decreased Rap1 activation in response to PDGF-BB

  • These molecular changes translate to inhibition of cell motility and invasiveness

This signaling axis represents a critical mechanism by which PDGF-BB promotes cell migration in various cell types, including glioma cells, with potential implications for understanding tumor cell invasion.

What is the relationship between tyrosine and serine phosphorylation of DOK1?

DOK1 undergoes complex regulation through both tyrosine and serine phosphorylation, with cross-talk between these modifications:

• Tyrosine phosphorylation sites:

  • Key sites include Y362 and Y398, which are phosphorylated in response to receptor tyrosine kinase activation

  • These modifications are critical for DOK1's scaffolding functions and interactions with downstream effectors

• Serine phosphorylation sites:

  • IKKβ phosphorylates DOK1 at serines 439, 443, 446, and 450

  • These modifications occur in response to TNF-α, IL-1, or γ-radiation

  • Tyrosine phosphorylation may enhance the association of DOK1 with IKKβ, potentially promoting subsequent serine phosphorylation

• Functional interplay:

  • Serine phosphorylation is required for DOK1's ability to inhibit PDGF-induced ERK1/2 activation and cell proliferation

  • DOK1 with mutations in these serine residues (S439A, S443A, S446A, S450A) fails to inhibit cell proliferation or promote cell motility

  • Phosphorylation of Y450 may mimic the acidic residues that facilitate IKKβ recognition and enhance the association of tyrosine-phosphorylated DOK1 with IKKβ

This dual phosphorylation system represents a sophisticated regulatory mechanism allowing DOK1 to integrate signals from multiple upstream pathways and modulate diverse cellular responses.

How do mutations in DOK1 phosphorylation sites affect its biological functions?

Mutation studies have provided critical insights into the functional significance of specific DOK1 phosphorylation sites:

• Tyrosine site mutations:

  • Mutation of Y362 and Y398 to phenylalanine (DOK1FF) prevents PDGF-BB-stimulated phosphorylation

  • DOK1FF acts in a dominant-negative manner, competing with endogenous DOK1 binding partners to form non-functional complexes

  • This mutant significantly decreases PDGF-BB-stimulated p130Cas tyrosine phosphorylation and Rap1 activation

  • Functionally, DOK1FF inhibits spheroid outgrowth, cell motility, and invasion

• Serine site mutations:

  • Mutation of S439, S443, S446, and S450 to alanine (DOK1-AAAA) prevents IKKβ-mediated phosphorylation

  • DOK1-AAAA fails to inhibit PDGF-induced ERK1/2 phosphorylation or cell proliferation

  • This mutant is unable to increase cell motility, unlike wild-type DOK1

• Phosphomimetic mutations:

  • Conversion of S439, S443, S446, and S450 to glutamic acid (DOK1-EEEE) to mimic constitutive phosphorylation enhances DOK1's ability to promote cell motility beyond wild-type levels

These mutation studies collectively demonstrate that both tyrosine and serine phosphorylation events are essential for DOK1's biological functions, particularly in regulating cell proliferation, motility, and migration.

How can phospho-DOK1 (Y398) antibody be used to study tumor cell invasion and migration?

The phospho-DOK1 (Y398) antibody serves as a valuable tool for investigating tumor cell invasion and migration through several experimental approaches:

• Correlation with invasive phenotypes:

  • Immunohistochemical analysis of tumor tissues can reveal whether enhanced DOK1 Y398 phosphorylation correlates with invasive phenotypes or metastatic potential

  • Comparative analyses between primary tumors and metastatic lesions can determine if phospho-DOK1 levels change during disease progression

• Pathway analysis in 3D models:

  • In spheroid invasion assays, phospho-DOK1 (Y398) detection can track activation of migration pathways as cells invade surrounding matrices

  • Time-course experiments can determine the temporal relationship between DOK1 phosphorylation and the initiation of invasive behavior

• Response to microenvironmental cues:

  • Western blotting with phospho-DOK1 (Y398) antibody can measure how various extracellular stimuli (growth factors, ECM components, inflammatory mediators) trigger DOK1 activation

  • Co-immunoprecipitation coupled with phospho-DOK1 detection can identify context-specific protein complexes formed during invasion

• Therapeutic intervention assessment:

  • Monitoring changes in DOK1 Y398 phosphorylation following treatment with kinase inhibitors or other therapeutic agents can provide mechanistic insights into drug effects on cell migration

  • This approach has been demonstrated in studies using PI3K inhibitors like LY294002, which reduce PDGF-stimulated DOK1 phosphorylation

These applications make phospho-DOK1 (Y398) antibody an important tool for understanding the molecular mechanisms underlying tumor cell invasion and for developing therapeutic strategies targeting metastasis.

What techniques can be employed to study the structural consequences of DOK1 Y398 phosphorylation?

Understanding the structural changes induced by Y398 phosphorylation requires sophisticated biophysical and biochemical approaches:

• Protein crystallography and structural biology:

  • While the crystal structure of the DOK1 PTB domain has been determined , similar approaches could be applied to study larger DOK1 fragments containing the Y398 site

  • Comparative crystallography of phosphorylated versus non-phosphorylated DOK1 could reveal conformational changes induced by Y398 phosphorylation

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • This technique can identify regions of DOK1 that undergo changes in solvent accessibility upon Y398 phosphorylation

  • HDX-MS can reveal allosteric effects of phosphorylation on distant protein domains

• Nuclear magnetic resonance (NMR) spectroscopy:

  • NMR can provide dynamic information about conformational changes induced by phosphorylation

  • Chemical shift perturbation experiments can map the effects of Y398 phosphorylation on the entire protein structure

• Molecular dynamics simulations:

  • Computational approaches can model the structural consequences of Y398 phosphorylation

  • Simulations can predict changes in protein flexibility, surface properties, and binding interfaces

• Protein-protein interaction studies:

  • Phospho-specific pull-down assays using synthetic peptides containing phosphorylated Y398 can identify phosphorylation-dependent binding partners

  • Comparison with similar assays using non-phosphorylated peptides can define phosphorylation-specific interactions

These approaches provide complementary information about how Y398 phosphorylation affects DOK1 structure and function, potentially revealing mechanisms by which this modification regulates DOK1's scaffolding activities.

How can researchers integrate phospho-DOK1 (Y398) detection with other signaling markers for comprehensive pathway analysis?

Integrating phospho-DOK1 (Y398) detection with other signaling markers enables comprehensive mapping of interconnected pathways:

• Multiplex immunofluorescence or immunohistochemistry:

  • Co-staining tissues for phospho-DOK1 (Y398) and other phosphorylated signaling proteins (e.g., phospho-p130Cas, phospho-AKT, phospho-ERK) can reveal spatial relationships between different activated pathways

  • This approach can identify cell populations with coordinated signaling activation within heterogeneous tissues

• Phosphoproteomic analysis:

  • Mass spectrometry-based phosphoproteomics can quantify changes in DOK1 Y398 phosphorylation alongside hundreds of other phosphorylation events

  • This global approach can identify novel signaling relationships between DOK1 and other pathways

  • Phosphoproteomic time-course experiments can establish the temporal sequence of phosphorylation events following stimulus

• Multi-parameter flow cytometry:

  • For single-cell analysis, phospho-flow cytometry can simultaneously measure DOK1 Y398 phosphorylation and other phosphoproteins

  • This technique is particularly valuable for analyzing heterogeneous cell populations like tumor samples or mixed immune cells

• Reverse-phase protein arrays (RPPA):

  • RPPA allows high-throughput, quantitative analysis of many phosphorylated proteins, including phospho-DOK1 (Y398), across large sample sets

  • This approach is ideal for screening the effects of multiple treatments or genetic perturbations on DOK1 signaling in the context of broader pathway activation

• Computational pathway analysis:

  • Integration of phospho-DOK1 data with other phosphoprotein measurements allows computational reconstruction of signaling networks

  • Machine learning approaches can identify patterns of coordinated phosphorylation events associated with specific cellular phenotypes

These integrative approaches provide a systems-level understanding of how DOK1 Y398 phosphorylation fits into broader signaling networks regulating cell behavior.

What are the common challenges when detecting phospho-DOK1 (Y398) and how can they be addressed?

Researchers may encounter several challenges when working with phospho-DOK1 (Y398) antibodies:

• Low signal intensity:

  • Cause: Rapid dephosphorylation during sample preparation or low abundance of phosphorylated protein

  • Solution: Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) in lysis buffers; enrich phosphoproteins using immunoprecipitation prior to Western blotting

• High background or non-specific bands:

  • Cause: Insufficient blocking, cross-reactivity with similar phosphoepitopes, or incomplete antibody optimization

  • Solution: Use alternative blocking agents (5% BSA is preferred over milk for phospho-antibodies); increase washing time and volume; optimize antibody dilution through titration experiments

• Inconsistent phosphorylation levels:

  • Cause: Variation in cell culture conditions or serum starvation protocols affecting basal phosphorylation

  • Solution: Standardize culture conditions; implement consistent serum starvation periods (typically 16-24 hours) before stimulation

• Phospho-epitope masking in IHC applications:

  • Cause: Formalin fixation can mask phospho-epitopes through protein cross-linking

  • Solution: Optimize antigen retrieval methods; test both citrate (pH 6.0) and EDTA (pH 9.0) buffers; extend retrieval time if necessary

• Variable phosphorylation kinetics:

  • Cause: Phosphorylation may be transient or have cell type-specific temporal patterns

  • Solution: Perform detailed time-course experiments to determine optimal time points for phosphorylation detection after stimulation

Implementing these technical solutions can significantly improve the reliability and sensitivity of phospho-DOK1 (Y398) detection in experimental applications.

How can researchers validate the specificity of phospho-DOK1 (Y398) detection in their experimental systems?

Rigorous validation of phospho-specificity is essential for reliable interpretation of experimental results:

• Peptide competition assays:

  • Pre-incubate the antibody with the phosphorylated immunizing peptide before application

  • A specific signal should be abolished or substantially reduced

  • Pre-incubation with the corresponding non-phosphorylated peptide should not affect signal intensity

• Phosphatase treatment controls:

  • Treat one sample aliquot with lambda phosphatase to remove phosphate groups

  • Compare antibody reactivity between treated and untreated samples

  • Specific phospho-signals should be eliminated by phosphatase treatment

• siRNA or CRISPR knockdown validation:

  • Reduce DOK1 expression using RNA interference or gene editing

  • A specific signal should decrease proportionally to the reduction in total DOK1

  • This approach confirms that the detected signal derives from DOK1 rather than cross-reactive proteins

• Stimulation experiments:

  • Compare phospho-DOK1 (Y398) levels in cells before and after treatments known to induce phosphorylation (e.g., PDGF-BB stimulation)

  • Treatments should increase phospho-signal without affecting total DOK1 levels

  • Include appropriate time points based on expected phosphorylation kinetics

• Mutation studies:

  • Express wild-type DOK1 or Y398F mutant in cells with endogenous DOK1 knockdown

  • Only wild-type DOK1 should show reactivity with the phospho-specific antibody

  • This approach definitively confirms epitope specificity

These validation strategies provide complementary evidence for phospho-specificity and should be selected based on the specific experimental context and available resources.

What experimental considerations are important when studying both tyrosine and serine phosphorylation of DOK1?

Studying the interplay between tyrosine and serine phosphorylation of DOK1 requires careful experimental design:

• Buffer optimization for comprehensive phosphorylation detection:

  • Include inhibitors targeting both tyrosine and serine/threonine phosphatases (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Protect from degradation using protease inhibitors

  • Maintain sample temperature at 4°C during processing to minimize dephosphorylation

• Sequential immunoprecipitation strategies:

  • Initial immunoprecipitation with phospho-tyrosine antibodies followed by immunoblotting with phospho-serine antibodies (or vice versa)

  • This approach can reveal populations of DOK1 with both modifications

  • Alternatively, immunoprecipitate with total DOK1 antibodies and probe with phospho-specific antibodies

• Time-course experiments:

  • Design experiments to capture the temporal relationship between tyrosine and serine phosphorylation

  • Data from the literature suggests tyrosine phosphorylation (Y362, Y398) may precede and potentially facilitate serine phosphorylation (S439, S443, S446, S450)

• Pathway inhibitor studies:

  • Use specific inhibitors to dissect the regulation of each phosphorylation type

  • For example, PI3K inhibitors reduce PDGF-stimulated DOK1 tyrosine phosphorylation

  • IKK inhibitors would be expected to reduce serine phosphorylation

• Mass spectrometry-based approaches:

  • Phosphopeptide mapping can simultaneously identify multiple phosphorylation sites

  • This approach can determine whether specific tyrosine and serine phosphorylations co-occur on the same DOK1 molecule

  • Quantitative MS can measure the stoichiometry of different phosphorylation events

These considerations help researchers design experiments that capture the complex interplay between different phosphorylation events on DOK1 and their functional consequences.

What are the emerging areas of DOK1 phosphorylation research relevant to cancer biology?

Several promising research directions are emerging at the intersection of DOK1 phosphorylation and cancer biology:

• DOK1 as a biomarker for therapeutic response:

  • Investigating whether phospho-DOK1 (Y398) levels predict response to receptor tyrosine kinase inhibitors or other targeted therapies

  • Determining if DOK1 phosphorylation status correlates with response to immunotherapy or conventional chemotherapy

  • This could lead to the development of companion diagnostic assays using phospho-DOK1 antibodies

• DOK1 in the tumor microenvironment:

  • Exploring how DOK1 phosphorylation in stromal cells (fibroblasts, immune cells) influences tumor-stroma interactions

  • Investigating whether cancer cells and stromal cells exhibit different patterns of DOK1 phosphorylation

  • This could reveal new mechanisms of tumor-stroma communication

• DOK1 in therapy resistance mechanisms:

  • Determining if altered DOK1 phosphorylation contributes to acquired resistance to targeted therapies

  • Investigating whether combined inhibition of pathways that regulate DOK1 phosphorylation can overcome resistance

  • This may lead to rational combination therapy approaches

• DOK1 in cancer stem cell biology:

  • Examining whether cancer stem cells exhibit distinct patterns of DOK1 phosphorylation

  • Investigating if DOK1 signaling contributes to stem cell maintenance or differentiation in tumors

  • This could identify new therapeutic vulnerabilities in treatment-resistant cancer stem cells

• Integration with immune checkpoint regulation:

  • Exploring potential roles of DOK1 phosphorylation in immune cell function within the tumor microenvironment

  • Investigating whether DOK1 signaling interacts with immune checkpoint pathways

  • This may reveal unexpected connections between DOK1 and immunotherapy response

These emerging research areas highlight the potential significance of DOK1 phosphorylation in various aspects of cancer biology and therapeutic response.

How might novel technologies advance our understanding of DOK1 phosphorylation dynamics?

Cutting-edge technologies are poised to revolutionize our understanding of DOK1 phosphorylation:

• Live-cell phosphorylation sensors:

  • Genetically encoded FRET-based biosensors can monitor DOK1 phosphorylation in real-time in living cells

  • This approach would reveal spatiotemporal dynamics of DOK1 activation with subcellular resolution

  • Could identify previously unknown compartmentalization of DOK1 signaling

• Single-cell phosphoproteomics:

  • Emerging mass spectrometry techniques allow phosphoproteomic analysis at the single-cell level

  • This could reveal heterogeneity in DOK1 phosphorylation within cell populations

  • May identify rare cell subpopulations with distinct DOK1 activation patterns

• CRISPR-based phosphorylation screens:

  • CRISPR activation/interference libraries targeting kinases and phosphatases

  • Readout based on phospho-DOK1 (Y398) levels

  • This approach could identify novel regulators of DOK1 phosphorylation

• Intravital microscopy with phospho-specific probes:

  • In vivo imaging of DOK1 phosphorylation in tumor models

  • Could reveal dynamic changes in DOK1 activation during processes like metastatic invasion

  • May identify spatiotemporal relationships between DOK1 activation and tissue microenvironments

• Proximity labeling proteomics:

  • BioID or APEX2 fused to DOK1 to identify proteins that interact specifically with phosphorylated DOK1

  • Comparing interactomes of wild-type versus phospho-deficient DOK1 mutants

  • This approach could map phosphorylation-dependent protein interaction networks

These technological advances will provide unprecedented insights into the dynamics, regulation, and functional consequences of DOK1 phosphorylation in normal and disease states.

What interdisciplinary approaches might yield new insights into the role of DOK1 Y398 phosphorylation in disease progression?

Interdisciplinary research strategies hold particular promise for advancing our understanding of DOK1 Y398 phosphorylation:

• Systems biology and computational modeling:

  • Integration of phosphoproteomic data with transcriptomic and metabolomic datasets

  • Mathematical modeling of DOK1 signaling networks to predict system responses to perturbations

  • This approach could identify non-intuitive relationships between DOK1 phosphorylation and cellular phenotypes

• Patient-derived models and precision medicine:

  • Analysis of DOK1 phosphorylation in patient-derived xenografts and organoids

  • Correlation with treatment response and clinical outcomes

  • Could lead to personalized therapeutic strategies based on DOK1 phosphorylation status

• Structural biology and drug discovery:

  • Structure-based design of compounds that specifically target phosphorylated DOK1 or its interaction interfaces

  • Development of proteolysis-targeting chimeras (PROTACs) that selectively degrade phosphorylated DOK1

  • This could yield first-in-class therapeutics targeting DOK1-dependent pathways

• Immunology and cancer biology integration:

  • Investigation of DOK1 phosphorylation in tumor-infiltrating immune cells

  • Examination of how tumor-derived factors affect DOK1 phosphorylation in immune cells

  • May reveal immunomodulatory roles of DOK1 signaling in the tumor microenvironment

• Developmental biology perspectives:

  • Comparative analysis of DOK1 phosphorylation in embryonic development and cancer progression

  • Identification of developmental programs that are aberrantly reactivated in cancer

  • Could reveal fundamental mechanisms underlying DOK1's role in cell migration and invasion

These interdisciplinary approaches combine diverse methodologies and perspectives to address complex questions about DOK1 phosphorylation that cannot be answered through any single discipline alone.