Phospho-MET (Y1003) Antibody

What is the significance of MET Y1003 phosphorylation in receptor regulation?

Phosphorylation at tyrosine 1003 (Y1003) within the juxtamembrane domain of MET receptor plays a crucial role in receptor downregulation. When phosphorylated, Y1003 enables recruitment of the E3 ubiquitin ligase casitas B-lineage lymphoma (CBL), which promotes MET monoubiquitylation, receptor internalization, and subsequent lysosomal degradation . This mechanism serves as a negative regulatory pathway for the MET/SF (Scatter Factor) axis. Experimental evidence has demonstrated that mutation of this site (Y1003F) stabilizes the receptor by preventing this degradation pathway . Understanding Y1003 phosphorylation status is therefore essential for researchers investigating MET receptor dynamics, stability, and signaling duration.

How do researchers detect phospho-MET (Y1003) in experimental samples?

Detection of phospho-MET (Y1003) typically employs antibodies specifically designed to recognize this phosphorylation site. Common detection methods include:

• Western blotting: Cell lysates are analyzed using phospho-MET (Y1003) specific antibodies. For example, studies have shown successful detection in A431 human epithelial carcinoma cell lines treated with pervanadate to induce phosphorylation .

• Immunofluorescence: Fixed cells can be stained with phospho-MET (Y1003) antibodies to visualize the subcellular localization of phosphorylated receptors. This has been demonstrated in A431 cells using antibodies at concentrations of approximately 10 μg/mL .

• Immunohistochemistry: Tissue sections can be probed with these antibodies using standard IHC protocols with recommended dilutions of 1:100-1:300 .

• ELISA: For quantitative measurements, ELISA protocols using anti-phospho-MET (Y1003) can be employed with typical dilutions around 1:40000 .

What experimental controls should be included when working with phospho-MET (Y1003) antibodies?

When designing experiments with phospho-MET (Y1003) antibodies, several controls are critical:

• Positive control: Cells treated with pervanadate (100 μM for 10 minutes) to induce robust tyrosine phosphorylation, as demonstrated in A431 cell lines .

• Negative control: Untreated cells that exhibit minimal phosphorylation at Y1003 .

• Specificity control: Samples treated with lambda phosphatase to remove phosphorylation and confirm antibody specificity.

• Loading control: Total MET antibody should be used on parallel samples to normalize phosphorylation levels to total protein expression.

• Kinase inhibitor control: Pretreatment with specific c-MET tyrosine kinase inhibitors, such as PHA-665752 at concentrations of 1-100 nM, which has been shown to attenuate Y1003 phosphorylation in a dose-dependent manner .

How does LPS-induced phosphorylation of MET Y1003 differ from HGF-induced phosphorylation?

LPS (lipopolysaccharide) and HGF (Hepatocyte Growth Factor) induce distinct patterns of MET phosphorylation:

LPS treatment induces significant Y1003 phosphorylation in a time-dependent manner after 3 hours, without inducing phosphorylation at Y1230/1234/1235 and without altering MET expression levels . This phosphorylation is mediated by PKCα, as demonstrated by experiments where PKCα inhibition with Go-6976 or PKCα shRNA attenuated LPS-induced Y1003 phosphorylation . In contrast, HGF binding leads to auto-phosphorylation of multiple tyrosine residues, activating various signaling pathways before receptor downregulation.

What is the relationship between phospho-MET (Y1003) and epithelial barrier integrity?

Phosphorylation of MET at Y1003 has been linked to regulation of epithelial barrier integrity through several mechanisms:

• LPS-induced Y1003 phosphorylation leads to MET internalization, which correlates with reduced transepithelial electrical resistance (TER), indicating compromised epithelial barrier function .

• Inhibition of LPS-mediated Y1003 phosphorylation through:

  • Pretreatment with the MET inhibitor PHA-665752

  • Inhibition of PKCα

  • Overexpression of Y1003A mutant MET (phosphorylation-resistant)

These interventions attenuate LPS-induced reduction of TER, suggesting that Y1003 phosphorylation and subsequent MET internalization contribute to barrier dysfunction .

• Lysophosphatidic acid (LPA) treatment reverses LPS-induced Y1003 phosphorylation and promotes MET accumulation at cell-cell contacts, which enhances epithelial barrier integrity .

These findings indicate that phospho-MET (Y1003) status influences epithelial barrier function, with phosphorylation and internalization associated with barrier disruption, while MET localization at cell-cell contacts promotes barrier integrity.

How can researchers differentiate between different phosphorylation sites on the MET receptor?

Distinguishing between various MET phosphorylation sites requires specific methodological approaches:

• Site-specific antibodies: Use antibodies that specifically recognize distinct phosphorylation sites:

  • Phospho-Y1003 antibodies detect the CBL-binding site in the juxtamembrane domain

  • Phospho-Y1234/1235 antibodies detect activation loop phosphorylation

  • Phospho-Y1349/1356 antibodies detect docking site phosphorylation

• Western blot pattern analysis: Different phosphorylation sites exhibit distinct phosphorylation kinetics and patterns in response to stimuli. For example, LPS induces Y1003 phosphorylation without Y1230/1234/1235 phosphorylation , while HGF induces phosphorylation at multiple sites.

• Mutational analysis: Expressing MET mutants where specific tyrosine residues are replaced with phenylalanine (e.g., Y1003F) to prevent phosphorylation at individual sites can help determine site-specific functions .

• Mass spectrometry: For comprehensive phosphorylation site mapping, immunoprecipitated MET can be analyzed by LC-MS/MS to identify and quantify phosphorylation at multiple sites simultaneously.

• Functional readouts: Different phosphorylation sites activate distinct downstream pathways:

  • Y1349 phosphorylation → PI3K/AKT pathway (migration/survival)

  • Y1356 phosphorylation → RAS/MAPK pathway (proliferation/cell cycle)

How does the phosphorylation status of MET Y1003 influence response to MET-targeted therapies in cancer?

The phosphorylation status of MET Y1003 can significantly impact therapeutic responses through several mechanisms:

• Receptor internalization and degradation: Y1003 phosphorylation normally promotes receptor downregulation through CBL-mediated ubiquitination . Tumors with defective Y1003 phosphorylation or mutations at this site may exhibit prolonged MET signaling and potentially reduced efficacy of kinase inhibitors.

• Predictive biomarker potential: Assessment of phospho-Y1003 status in tumor samples may predict responsiveness to different classes of MET inhibitors:

  • Tumors with high Y1003 phosphorylation may indicate active receptor cycling and potentially better response to kinase inhibitors

  • Tumors with low Y1003 phosphorylation despite high MET expression might suggest defective receptor downregulation and potential resistance

• Combined therapeutic approaches: Understanding that Y1003 phosphorylation regulates receptor trafficking suggests potential synergistic approaches:

  • Combining MET kinase inhibitors with agents that promote Y1003 phosphorylation could enhance receptor downregulation

  • For tumors with defective Y1003 phosphorylation, therapies targeting degradation through alternative pathways might be beneficial

• Resistance mechanisms: Acquired resistance to MET inhibitors has been observed in clinical settings , and alterations in Y1003 phosphorylation status could contribute to these resistance mechanisms through stabilization of the receptor.

Researchers should consider incorporating phospho-Y1003 analysis in clinical samples when evaluating MET-targeted therapies, as this may provide valuable insights into treatment response and resistance mechanisms.

What cellular pathways regulate PKCα-mediated phosphorylation of MET Y1003, and how might these be exploited therapeutically?

The regulation of PKCα-mediated MET Y1003 phosphorylation involves complex cellular pathways:

• LPS/TLR4 pathway: LPS activates PKCα, leading to Y1003 phosphorylation . This suggests inflammatory signaling through Toll-like receptors can cross-talk with MET regulation.

• Calcium signaling: Classical PKCs like PKCα are calcium-dependent, indicating that calcium flux may influence Y1003 phosphorylation.

• Diacylglycerol (DAG) signaling: PKCα activation requires DAG, linking phospholipid metabolism to MET regulation.

• LPA receptor signaling: Lysophosphatidic acid (LPA) reverses LPS-induced Y1003 phosphorylation , suggesting antagonistic signaling pathways that modulate PKCα activity toward MET.

Therapeutic exploitation of these pathways could include:

• PKCα modulators: Compounds that inhibit PKCα could prevent excessive Y1003 phosphorylation in inflammatory conditions where MET internalization contributes to tissue damage.

• LPA receptor agonists: In conditions where epithelial barrier integrity is compromised due to MET internalization, LPA receptor agonists might promote MET stabilization at cell-cell contacts.

• Tailored combination therapies: For tumors dependent on MET signaling, combining PKCα activators with MET kinase inhibitors might enhance receptor downregulation and improve therapeutic efficacy.

• Inflammatory pathway modulation: In diseases where inflammation-induced MET internalization contributes to pathology, targeting upstream inflammatory mediators could preserve MET localization and function.

How can researchers effectively distinguish between MET activation and MET degradation signals when analyzing phospho-MET (Y1003) data?

Distinguishing between activation and degradation signals requires comprehensive analytical approaches:

• Temporal analysis: Monitor phosphorylation kinetics at multiple sites:

  • Early phosphorylation events (minutes): Y1234/1235 (activation loop) indicates activation

  • Delayed phosphorylation (hours): Isolated Y1003 phosphorylation may indicate degradation signaling

• Multi-site phosphorylation profiling: Analyze multiple phosphorylation sites simultaneously:

  Phosphorylation Pattern	Interpretation
  Y1003+ / Y1234/1235+ / Y1349/1356+	Full activation with feedback regulation
  Y1003+ / Y1234/1235- / Y1349/1356-	Degradation signal without activation
  Y1003- / Y1234/1235+ / Y1349/1356+	Activation without degradation (potential oncogenic)

• Receptor localization studies: Combine phosphorylation analysis with subcellular localization:

  • Membrane-localized phospho-Y1003: Early stage of receptor regulation

  • Endosomal phospho-Y1003: Internalization and potential degradation

  • Cell-cell junction phospho-Y1003: Associated with barrier function

• Downstream signaling analysis: Measure activation of:

  • PI3K/AKT pathway: Indicates functional signaling

  • MAPK pathway: Indicates functional signaling

  • Ubiquitination status: Indicates degradation pathway engagement

• Inhibitor studies: Use selective inhibitors to dissect pathways:

  • PKCα inhibitors (e.g., Go-6976): Block degradation-associated Y1003 phosphorylation

  • MET kinase inhibitors (e.g., PHA-665752): Block activation-associated phosphorylation

This multi-parameter approach allows researchers to determine whether observed Y1003 phosphorylation represents activation with feedback regulation or a primary degradation signal.

What are the optimal fixation and staining protocols for phospho-MET (Y1003) immunofluorescence studies?

For optimal phospho-MET (Y1003) immunofluorescence, the following protocol is recommended based on published research:

• Fixation:

  • Use 4% paraformaldehyde in PBS for 15 minutes at room temperature

  • Alternative: methanol fixation for 10 minutes at -20°C may better preserve phospho-epitopes

• Permeabilization:

  • 0.1-0.5% Triton X-100 in PBS for 5-10 minutes at room temperature

  • Avoid over-permeabilization which can extract membrane proteins

• Blocking:

  • 5% normal serum (matching secondary antibody species) with 1% BSA in PBS for 1 hour

• Primary antibody:

  • Dilute phospho-MET (Y1003) antibody to 10 μg/mL in blocking buffer

  • Incubate for 3 hours at room temperature or overnight at 4°C

• Secondary antibody:

  • Fluorophore-conjugated secondary antibody (e.g., NorthernLights 557-conjugated Anti-Rabbit IgG)

  • Dilute according to manufacturer's recommendations

  • Incubate for 1 hour at room temperature in the dark

• Counterstaining:

  • DAPI (1 μg/mL) for nuclear visualization

  • Optional: phalloidin staining for F-actin to visualize cell boundaries

• Mounting:

  • Use anti-fade mounting medium to preserve fluorescence

  • Seal coverslips with nail polish for long-term storage

• Controls:

  • Include pervanadate-treated cells (100 μM, 10 minutes) as positive control

  • Include untreated cells as negative control

  • Include phosphatase-treated samples to confirm antibody specificity

How should researchers quantify changes in phospho-MET (Y1003) levels in complex experimental designs?

Quantification of phospho-MET (Y1003) requires rigorous approaches for reliable results:

• Western blot quantification:

  • Always normalize phospho-MET (Y1003) signal to total MET protein

  • Use housekeeping proteins (β-actin, GAPDH) as loading controls

  • Employ at least three biological replicates for statistical analysis

  • Use digital image analysis software with dynamic range verification

• Immunofluorescence quantification:

  • Measure mean fluorescence intensity within defined cellular regions

  • Analyze at least 50-100 cells per condition

  • Use automated image analysis to reduce bias

  • Consider ratio imaging with total MET antibody in different channel

• Statistical considerations:

  • Perform appropriate statistical tests (t-test, ANOVA) with correction for multiple comparisons

  • Report both statistical significance and effect size

  • Consider variability between experimental batches

• Normalization strategies for complex designs:

  Experimental Design	Recommended Normalization
  Time-course	Express as fold-change relative to t=0
  Dose-response	Express as percent of maximum response
  Multiple treatments	Normalize to common positive control
  Patient samples	Normalize to pooled reference sample

• Advanced quantification approaches:

  • Consider phosphorylation site stoichiometry calculations

  • Use phospho-flow cytometry for single-cell analysis in heterogeneous populations

  • Employ targeted mass spectrometry for absolute quantification

What are the best approaches for studying the dynamics between phospho-MET (Y1003) and CBL-mediated receptor internalization?

Studying the dynamics between Y1003 phosphorylation and CBL-mediated internalization requires specialized methodologies:

• Temporal analysis of protein interactions:

  • Co-immunoprecipitation of MET and CBL at different time points after stimulation

  • Proximity ligation assay to visualize MET-CBL interactions in situ

  • FRET/BRET biosensors to monitor interaction dynamics in living cells

• Receptor internalization assays:

  • Surface biotinylation with Sulfo-NHS-SS-Biotin followed by internalization period and surface biotin stripping

  • Cell surface ELISA using antibodies against extracellular MET epitopes

  • Flow cytometry with non-permeabilized cells to quantify surface MET

• Genetic approaches:

  • Expression of MET Y1003A mutant to prevent CBL binding

  • CBL knockdown or knockout to assess dependency of internalization on CBL

  • Structure-function analysis with chimeric receptors

• Advanced imaging techniques:

  • Live-cell imaging with fluorescently tagged MET and CBL

  • Super-resolution microscopy to visualize endocytic structures

  • Correlative light and electron microscopy for ultrastructural analysis

• Pharmacological intervention:

  • Clathrin-dependent endocytosis inhibitors (e.g., chlorpromazine)

  • Dynamin inhibitors (e.g., dynasore)

  • Lysosomal inhibitors (e.g., chloroquine) to assess degradation

• Quantitative modeling:

  • Kinetic modeling of phosphorylation, CBL recruitment, and internalization

  • Computational approaches to predict the impact of mutations or interventions

What steps should researchers take when phospho-MET (Y1003) antibody yields inconsistent detection in Western blot applications?

Inconsistent phospho-MET (Y1003) detection can stem from several factors:

• Sample preparation issues:

  • Ensure rapid sample processing to prevent dephosphorylation

  • Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Maintain samples at 4°C and avoid repeated freeze-thaw cycles

  • Verify protein extraction efficiency for membrane proteins

• Technical optimization:

  • Test different blocking agents (5% BSA often superior to milk for phospho-epitopes)

  • Optimize antibody concentration (1:500-1:2000 dilution range recommended)

  • Extend primary antibody incubation (overnight at 4°C)

  • Test different transfer methods (wet transfer often superior for large proteins like MET)

• Positive control strategies:

  • Include pervanadate-treated cells (100 μM for 10 minutes) as positive control

  • Use recombinant phosphorylated peptide standards when available

  • Consider including known positive cell lines (e.g., A431 cells)

• Validation approaches:

  • Verify with alternative phospho-MET (Y1003) antibodies from different vendors

  • Confirm with phosphatase treatment to demonstrate specificity

  • Perform immunoprecipitation before Western blotting for enrichment

• Signal enhancement techniques:

  • Consider signal amplification systems for low abundance detection

  • Use high-sensitivity chemiluminescent substrates

  • Try fluorescent secondary antibodies with digital imaging systems

How can researchers determine whether observed changes in phospho-MET (Y1003) are biologically significant versus technical artifacts?

Distinguishing biological significance from artifacts requires multiple controls and validation steps:

• Dose-response relationships:

  • Biological effects typically show dose-dependent responses

  • Technical artifacts often show random or all-or-nothing patterns

• Time-course analysis:

  • Biological phosphorylation events follow predictable kinetics

  • Examine both phosphorylation and dephosphorylation phases

• Functional validation:

  • Correlate phosphorylation changes with functional outcomes (e.g., receptor internalization)

  • Use MET Y1003A mutants to determine if preventing phosphorylation blocks the biological effect

• Multiple detection methods:

  • Confirm findings using different techniques (Western blot, immunofluorescence, ELISA)

  • Artifacts are less likely to appear consistently across different methods

• Genetic approaches:

  • siRNA/shRNA knockdown of upstream regulators should reduce phosphorylation

  • Expression of constitutively active upstream kinases should enhance phosphorylation

• Biological replicates and statistics:

  • Perform experiments with multiple biological replicates (n≥3)

  • Calculate effect sizes and confidence intervals, not just p-values

  • Consider thresholds for biological significance (e.g., >2-fold change)

How might single-cell analysis of phospho-MET (Y1003) advance our understanding of heterogeneous receptor dynamics in tumor microenvironments?

Single-cell analysis of phospho-MET (Y1003) offers promising insights into tumor heterogeneity:

• Technological approaches:

  • Mass cytometry (CyTOF) with phospho-MET (Y1003) antibodies for multi-parameter analysis

  • Single-cell Western blotting for protein-level quantification

  • Imaging mass cytometry for spatial distribution in tissue context

  • Single-cell RNA-seq combined with phosphoproteomic analysis

• Potential research questions:

  • How does phospho-MET (Y1003) status vary between cancer cells within a tumor?

  • Do specific tumor microenvironmental niches show distinct patterns of Y1003 phosphorylation?

  • Can rare cell populations with altered Y1003 phosphorylation predict treatment resistance?

  • How does Y1003 phosphorylation correlate with other phosphorylation sites at single-cell resolution?

• Implications for precision medicine:

  • Identification of cellular subpopulations that might respond differently to MET inhibitors

  • Development of combinatorial treatment strategies targeting cells with different phosphorylation profiles

  • Biomarker discovery based on cellular heterogeneity patterns

• Technical challenges to address:

  • Preservation of phosphorylation status during single-cell isolation

  • Antibody specificity at single-cell sensitivity levels

  • Computational methods for analyzing high-dimensional phosphorylation data

  • Integration with spatial information in tissue context

What is the potential significance of studying phospho-MET (Y1003) in non-cancer pathologies where epithelial barrier function is compromised?

The role of phospho-MET (Y1003) extends beyond cancer to conditions involving epithelial barrier dysfunction:

• Inflammatory bowel diseases:

  • LPS from gut microbiota could trigger Y1003 phosphorylation and barrier disruption

  • MET signaling has been implicated in intestinal epithelial regeneration

  • Research could explore how inflammatory mediators affect MET phosphorylation and localization

• Acute lung injury and ARDS:

  • LPS-induced Y1003 phosphorylation has been shown to reduce lung epithelial barrier function

  • Understanding this pathway could identify intervention points to preserve barrier integrity

  • HGF is known to have protective effects in lung injury models

• Chronic kidney disease:

  • Tubular epithelial barrier function depends on proper cell-cell adhesion

  • MET is expressed in renal tubular epithelium and may regulate barrier function

  • Phospho-MET (Y1003) status might influence progression of renal fibrosis

• Neurodegenerative diseases:

  • Blood-brain barrier integrity could be influenced by MET phosphorylation status

  • Neuroinflammation might trigger pathological MET internalization through Y1003 phosphorylation

  • Understanding these mechanisms could identify novel therapeutic targets

• Methodological approaches:

  • Animal models of epithelial injury with phospho-MET (Y1003) monitoring

  • Organoid systems to study barrier function in controlled environments

  • Translational studies examining phospho-MET status in patient biopsies