Phospho-RET (Y905) Antibody

What is the functional significance of RET Y905 phosphorylation in cellular signaling?

RET Y905 phosphorylation represents a critical regulatory event in RET signaling pathways. Unlike traditional activation loop phosphorylation sites in other receptor tyrosine kinases, Y905 phosphorylation in RET exhibits unique characteristics:

• Y905 undergoes delayed autophosphorylation compared to other sites like Y687

• When phosphorylated, Y905 tethers several basic side chains including R770 from the αC helix and residues R897 and K907 from the activation loop

• Y905 phosphorylation is associated with activation of downstream signaling pathways that regulate cell proliferation and survival

• Mutation studies (Y905F) demonstrated that Y905 phosphorylation is critical for RET's catalytic activity, as its absence greatly decreases autophosphorylation and kinase activity

Interestingly, while Y905 is important, it should not be considered purely "activating" in the traditional sense, as it undergoes delayed autophosphorylation and is not absolutely catalytically required . This distinguishes RET from many other receptor tyrosine kinases.

How can researchers reliably detect phosphorylated RET Y905 in experimental systems?

Detection of phosphorylated RET Y905 requires strategic approaches depending on your experimental system:

Western Blot Analysis:

• Use validated phospho-specific antibodies at recommended dilutions (typically 1:500 - 1:2000)

• Include appropriate controls: positive control (pervanadate-treated cells), negative control (inhibitor-treated cells or Y905F mutants)

• Use PVDF membranes for optimal protein retention and signal

• Block with 5% non-fat dry milk (NFDM) in TBST

• For enhanced detection, consider immunoprecipitation followed by Western blot

Cell Line Selection:

• TT (human thyroid carcinoma epithelial cells) treated with pervanadate show robust Y905 phosphorylation

• MDA-MB-468 (human breast cancer cells) respond well to pervanadate treatment for Y905 phosphorylation detection

• LC-2/ad cells (human lung epithelial cells) express constitutively active CCDC6-RET fusion with detectable Y905 phosphorylation

• K-562 cells have been validated as positive samples for several commercial antibodies

Treatment Conditions:

• Pervanadate treatment (1mM for 30 minutes or 100μM for 10 minutes) strongly induces Y905 phosphorylation

• GDNF treatment upregulates Y905 phosphorylation

• Ponatinib treatment (300nM for 2 hours) downregulates Y905 phosphorylation in cells expressing RET fusions

What are the relationships between Y905 and other phosphorylation sites in RET?

The interplay between RET phosphorylation sites creates a complex regulatory network:

• Y900 and Y905 Cooperation: Mutation studies revealed that while Y905F mutation alone greatly decreases RET activity, the double mutant Y900F/Y905F completely abolished both kinase activity and autophosphorylation

• Y687 and Y905 Kinetics: The JM segment Y687 undergoes faster autophosphorylation than activation-loop residues Y900 and Y905, suggesting a sequential activation mechanism

• S909 and Y905 Displacement: In a fascinating regulatory mechanism, phospho-S909 can displace phospho-Y905 from its binding pocket. When this occurs, phospho-S909 engages activation segment residues R897 and R912, as well as R873 from the HRD motif, while Y905 projects away from the kinase body becoming more solvent-accessible

• Y928 and Y905 Interaction: Phospho-Y928 forms hydrogen bonds with side chains of R873 and R897, positioned beneath phospho-S909, further disrupting interactions of phospho-Y905 with the activation loop

This complex network of phosphorylation events underscores the sophisticated regulation of RET signaling.

How does RET's unique dual-specificity kinase activity impact experimental design and data interpretation?

RET's dual-specificity kinase activity (ability to phosphorylate both tyrosine and serine residues) has significant implications for researchers:

Experimental Considerations:

• Standard tyrosine kinase inhibitor screens may miss critical aspects of RET regulation

• Phospho-proteomics experiments should account for both tyrosine and serine phosphorylation events

• When evaluating RET activation, researchers should monitor both Y905 and S909 phosphorylation states

Structural Implications:

• Phospho-S909 displaces phospho-Y905 and adopts an approximately equivalent position by engaging different residues in the activation segment

• This displacement mechanism creates a dynamic conformational switch that affects RET signaling

Evolutionary Context:

• This dual-specificity appears conserved, as Drosophila RET also requires an equivalent serine for signaling

• Understanding this dual-specificity offers new perspectives on RTK evolution and regulation

When designing experiments to study RET signaling or developing RET-targeted therapeutics, researchers must account for this unusual dual phosphorylation mechanism, which differs significantly from the canonical RTK activation paradigm.

What structural and conformational changes accompany Y905 phosphorylation and how do they regulate RET activity?

Y905 phosphorylation triggers distinct structural rearrangements that modulate RET catalytic activity:

Conformational Changes:

• When phosphorylated, Y905 tethers several basic side chains including R770 from the αC helix and residues R897 and K907 from the activation loop

• These interactions stabilize a catalytically active conformation of the kinase domain

• The activation loop detaches from the body of the catalytic core upon phosphorylation

Regulatory Mechanisms:

• In the presence of phospho-S909, phospho-Y905 is displaced and projects away from the RET kinase body, becoming more solvent-accessible

• Y905 does not engage with αC helix R770 when displaced by phospho-S909, creating an alternative active conformation

• Phospho-Y928 positions beneath tethered phospho-S909, further disrupting phospho-Y905 interactions with the activation loop

Allosteric Regulation:

• The juxtamembrane (JM) segment influences RET catalytic activity, with full-length JM starting at residue 661 (JM661) required for maximal activity

• There appears to be cross-talk between the JM hinge, αC helix, and serine-phosphorylated activation loop

These structural insights are critical for understanding RET function and for structure-based drug design efforts targeting specific RET conformations.

How do Y905F mutations impact RET signaling pathways in different experimental models?

Mutation studies involving Y905F have revealed critical insights into RET signaling:

Effects on Kinase Activity:

• Y905F mutation greatly decreases RET autophosphorylation and kinase activity

• While Y900F mutation alone has minimal impact, the double Y900F/Y905F mutation completely abolishes activity, suggesting functional cooperation

Differential Effects in RET Variants:

• In constitutively active RET-MEN2A, Y905F mutation greatly decreases both autophosphorylation and kinase activity

• This indicates that even in oncogenic RET variants, Y905 phosphorylation remains critical for signaling

Downstream Signaling Consequences:

Understanding these mutation effects is particularly important when:

• Designing kinase-dead controls for experiments

• Interpreting resistance mechanisms to RET-targeted therapies

• Developing phosphorylation site-specific biosensors for RET activity

What are the optimal conditions for detecting Y905 phosphorylation by Western blot?

To achieve sensitive and specific detection of phosphorylated RET Y905, researchers should follow these optimized protocols:

Sample Preparation:

• Treatment conditions: 1mM pervanadate for 30 minutes or 100μM for 10 minutes strongly induces Y905 phosphorylation

• GDNF treatment can be used to induce physiological RET activation

• For inhibition studies, ponatinib treatment (300nM for 2 hours) reliably downregulates Y905 phosphorylation

Western Blot Protocol:

• Membrane: PVDF is recommended for optimal protein retention and phospho-epitope preservation

• Blocking: 5% non-fat dry milk (NFDM) in TBST for 1 hour at room temperature

• Primary antibody dilutions:

  • Rabbit monoclonal: 1:1000 (0.521 μg/ml)

  • Rabbit polyclonal: 1:500 - 1:2000 or 0.5 μg/mL

• Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:10,000 - 1:100,000 dilution

• Exposure time: Longer exposures (approximately 180 seconds) may be necessary for optimal signal detection

Validation Controls:

• Positive control: Pervanadate-treated cells (TT, MDA-MB-468, K-562)

• Negative control: Untreated cells, Y905F mutant cells, or inhibitor-treated cells

• Peptide competition: Using phospho-Y905 peptide to verify antibody specificity

• Loading control: GAPDH antibody at 1:200,000 dilution is suitable

How can researchers validate the specificity of phospho-RET (Y905) antibodies?

Antibody validation is critical for ensuring reliable experimental results. For phospho-RET (Y905) antibodies, consider these validation approaches:

Dot Blot Analysis:

• Test antibody against:

  • Phospho-Y905 peptides (multiple variants if available)

  • Non-phosphorylated RET peptide (negative control)

  • Other phosphorylated RET peptides (e.g., phospho-Y900) to test cross-reactivity

Cellular Validation:

• Compare untreated cells versus cells treated with:

  • Pervanadate (phosphatase inhibitor that increases phosphorylation)

  • GDNF (physiological RET activator)

  • RET inhibitors like ponatinib

Genetic Validation:

• Use Y905F mutant cells as negative controls

• The double Y900F/Y905F mutant provides an even more stringent control

Immunoprecipitation:

• Perform immunoprecipitation from pervanadate-treated cells followed by western blot with the same or different phospho-RET antibody

• This approach confirms the antibody recognizes the native protein

What approaches can be used to study the interplay between Y905 phosphorylation and other RET phosphorylation events?

Investigating the complex relationships between RET phosphorylation sites requires sophisticated methodological approaches:

Label-Free Quantitative Mass Spectrometry (LFQMS):

• Enables temporal analysis of multiple phosphorylation events simultaneously

• Can track phosphorylation kinetics for Y687, Y826, Y900, Y905, and other sites

• Standardize phosphorylated peptides to their non-phosphorylated counterparts and plot relative to a zero time point

Time-Course Autophosphorylation Assays:

Enzymatic Assays:

• Measure kcat/KM constants toward substrates to assess catalytic efficiency

• Compare wild-type RET with constructs containing mutations at different phosphorylation sites

Structural Studies:

• X-ray crystallography can reveal how phospho-S909 displaces phospho-Y905

• Molecular dynamics simulations can provide insights into the dynamic interplay between phosphorylation sites

These complementary approaches provide a comprehensive view of how different phosphorylation events on RET influence each other and collectively regulate RET's function.

What are common technical challenges when working with phospho-RET (Y905) antibodies and how can they be addressed?

Researchers frequently encounter several challenges when detecting phospho-RET (Y905):

High Background Signal:

• Problem: Nonspecific binding leading to high background

• Solutions:

  • Increase blocking time/concentration (try 5% BSA instead of milk)

  • Optimize antibody concentration (test dilutions from 1:500 to 1:2000)

  • Include phosphatase inhibitors in all buffers to prevent dephosphorylation

  • Ensure thorough washing between steps (at least 3×10 minutes with TBST)

Weak or No Signal:

• Problem: Insufficient phosphorylation or sensitivity issues

• Solutions:

  • Verify RET expression in your cell line

  • Confirm phosphorylation status with pervanadate treatment (positive control)

  • Increase exposure time (up to 180 seconds)

  • Consider immunoprecipitation to concentrate the target protein

  • Test different antibody clones or formats

Multiple Bands or Unexpected Molecular Weight:

• Problem: Nonspecific binding or RET variants/degradation

• Solutions:

  • RET typically appears at 124-175 kDa depending on glycosylation state and fusion status

  • Use Y905F mutant cells as negative control to identify specific bands

  • Include molecular weight markers and positive control samples

Inconsistent Results:

• Problem: Variability between experiments

• Solutions:

  • Standardize cell culture conditions and treatment protocols

  • Prepare fresh lysates with phosphatase inhibitors

  • Document lot numbers of antibodies and control for batch variations

  • Consider quantitative approaches like LFQMS for more consistent measurements

How can researchers optimize experimental design to study the dual-specificity kinase activity of RET?

Investigating RET's unique dual-specificity kinase activity requires careful experimental design:

Comprehensive Phosphorylation Analysis:

• Use antibodies against both phospho-tyrosine and phospho-serine residues

• Employ mass spectrometry to detect all phosphorylation events simultaneously

• Consider targeted phospho-proteomics focusing on known RET phosphorylation sites

Structural Studies:

• X-ray crystallography can reveal how phospho-S909 displaces phospho-Y905

• Analyze how these conformational changes affect binding of regulators or substrates

Mutation Analysis:

• Generate S909A and Y905F single and double mutants

• Compare kinase activity, substrate specificity, and cellular phenotypes

Molecular Dynamics Simulations:

• Model the dynamic interplay between tyrosine and serine phosphorylation

• Simulate transitions between different phosphorylation states

Biophysical Assays:

• Thermal shift assays to measure stabilization upon different phosphorylation events

• Surface plasmon resonance to analyze how different phosphorylation patterns affect protein-protein interactions

By combining these approaches, researchers can build a comprehensive understanding of RET's dual-specificity kinase activity and its biological significance.

How can phospho-RET (Y905) antibodies be used to evaluate RET inhibitor efficacy and specificity?

Phospho-RET (Y905) antibodies serve as valuable tools for assessing RET inhibitor properties:

Inhibitor Screening:

• Monitor Y905 phosphorylation to evaluate target engagement

• Compare inhibition patterns against wild-type versus mutant RET (including oncogenic variants)

• Determine IC₅₀ values for Y905 phosphorylation inhibition across inhibitor candidates

Mechanism of Action Studies:

• Analyze how different inhibitors affect Y905 versus other phosphorylation sites

• For example, ponatinib treatment (300nM for 2 hours) effectively downregulates Y905 phosphorylation in cells expressing RET fusions

• Compare type I (ATP-competitive) versus type II (inactive conformation-binding) inhibitors

Resistance Mechanism Investigation:

• Track changes in Y905 phosphorylation patterns in cells developing resistance

• Compare with phosphorylation of other RTKs to assess inhibitor specificity

• Evaluate bypass mechanisms through downstream signaling pathways

Structure-Activity Relationship Analysis:

• Correlate structural properties of RET inhibitors with their ability to prevent Y905 phosphorylation

• Use molecular dynamics simulations to understand how inhibitors affect the conformation of the activation loop and Y905 accessibility

These applications make phospho-RET (Y905) antibodies essential tools in developing next-generation RET-targeted therapeutics with improved efficacy and specificity.

What are emerging research directions involving RET Y905 phosphorylation and its role in disease mechanisms?

Current research is expanding our understanding of RET Y905 phosphorylation in several exciting directions:

Cancer Biology:

• Investigation of Y905 phosphorylation status in RET fusion-positive cancers (thyroid, lung, colon)

• Assessment of Y905 phosphorylation as a biomarker for response to RET inhibitors

• Understanding how Y905 phosphorylation affects oncogenic signaling in different cellular contexts

Structural Biology:

• Further characterization of the "phosphorylation code" involving Y905, S909, and other sites

• Investigation of allosteric networks connecting the juxtamembrane region to Y905 phosphorylation

• Development of conformation-specific antibodies that distinguish different Y905 states

Developmental Biology:

• Studies on how Y905 phosphorylation regulates RET's role in neural crest development

• Investigation of Y905 phosphorylation in GDNF-mediated kidney development

• Analysis of how disrupted Y905 phosphorylation contributes to Hirschsprung's disease

Drug Discovery:

• Design of inhibitors specifically targeting Y905-phosphorylated RET conformations

• Development of degraders that preferentially target phosphorylated RET species

• Creation of Y905 phosphorylation-based biosensors for high-throughput screening

These emerging research directions highlight the continuing importance of Y905 phosphorylation in understanding RET biology and developing targeted therapies for RET-driven diseases.