Phospho-KDR (Tyr951) Antibody

What is the significance of KDR phosphorylation at Tyrosine 951?

Phosphorylation of KDR (Kinase insert domain receptor, also known as VEGFR-2) at Tyrosine 951 plays a crucial role in VEGF-dependent signaling pathways. This specific phosphorylation is essential for:

• Interaction with SH2D2A/TSAD (T-cell-specific adapter protein)

• VEGFA-mediated reorganization of the actin cytoskeleton

• Regulation of endothelial cell migration

• Serving as a binding site for downstream effector molecules that activate the PI3K/Akt signaling pathway

Site-directed mutation studies have demonstrated that Tyr951 on VEGFR-2 can inhibit VEGF-mediated cytoskeletal reorganization and migration, confirming its functional importance in angiogenic responses .

How does Phospho-KDR (Tyr951) antibody specificity differ from other phospho-VEGFR-2 antibodies?

Phospho-KDR (Tyr951) antibodies are designed to detect endogenous levels of KDR/VEGFR-2 only when phosphorylated at Tyrosine 951, distinguishing it from antibodies targeting other phosphorylation sites:

When selecting an antibody, researchers should consider which specific phosphorylation event they aim to study within the VEGF signaling cascade .

What are the recommended applications for Phospho-KDR (Tyr951) antibodies?

Phospho-KDR (Tyr951) antibodies have been validated for various research applications:

For optimal results, researchers should titrate the antibody for their specific experimental conditions and sample types. Validation controls should include VEGF-stimulated endothelial cells (positive control) and non-stimulated cells (negative control) .

How does VEGF stimulation affect the association between KDR and other proteins in relation to Tyr951 phosphorylation?

VEGF stimulation significantly enhances the biochemical association between KDR and various partner proteins through Tyr951 phosphorylation. Research has revealed:

• Endogenous ECSCR/KDR co-immunoprecipitation is weak in starved cells but strongly enhanced following 10 minutes of VEGF stimulation

• Association levels return to basal levels after 30 minutes, suggesting dynamic regulation

• The time course of co-immunoprecipitation appears to lag behind receptor activation as measured by KDR phosphorylation on Tyr951

• VEGF-stimulated co-IP is completely blocked by the inhibitor SU5416

• VEGF165 stimulation shows more robust enhancement of KDR/protein interactions compared to VEGF121

Interestingly, the perinuclear immunoreactivity for ECSCR/KDR in VEGF-stimulated cells partially colocalizes with HRS, a component of the ESCRT complex implicated in trafficking of ubiquitinated activated receptors . This suggests Tyr951 phosphorylation may also influence receptor trafficking and degradation pathways.

What experimental approaches can resolve conflicting data regarding Phospho-KDR (Tyr951) signaling mechanisms?

Researchers occasionally encounter contradictory results when investigating Phospho-KDR (Tyr951) signaling. To resolve such conflicts, consider these methodological approaches:

• Temporal resolution studies: Implement time-course experiments with fine temporal resolution to capture the dynamic phosphorylation patterns

  • Example: ECSCR/KDR co-immunoprecipitation peaked at 10 minutes after VEGF stimulation before returning to baseline at 30 minutes

• Ligand-specific responses: Compare responses to different VEGF isoforms

  • VEGF165 induced stronger KDR/ECSCR co-IP than VEGF121 despite comparable receptor activation

• Domain-specific chimeric constructs: Generate chimeric proteins to identify specific domains mediating interactions

  • ECSCR transmembrane domain alone (TMswap) was sufficient for KDR co-IP, demonstrating the importance of this domain in protein-protein interactions

• Multiple detection methods: Employ complementary approaches

  • Co-immunoprecipitation plus immunofluorescence colocalization studies provide stronger evidence than either method alone

• Subcellular localization: Determine where phosphorylated KDR localizes using compartment markers

  • Phospho-KDR (Tyr951) and interacting proteins may be found in specific cellular compartments like HRS-positive endosomes but not RAB7-positive degradative compartments or RAB11-positive recycling vesicles

How does Phospho-KDR (Tyr951) signaling impact clinical outcomes in cancer patients?

Clinical studies have examined the relationship between phosphorylated VEGFR-2 and patient outcomes:

This multivariate analysis indicates that positive pVEGFR-2 expression is significantly associated with poorer prognosis in cancer patients (risk ratio: 1.507, p=0.045) . For researchers investigating clinical correlations, it's essential to:

• Employ standardized IHC protocols with validated antibodies

• Use consistent scoring systems for phospho-protein expression

• Correlate with multiple parameters including total receptor expression

• Account for treatment history, particularly anti-angiogenic therapies

• Consider the heterogeneity of tumor vasculature in sampling strategies

What are the optimal protocols for preserving phosphorylation status of Tyr951 during sample preparation?

Maintaining phosphorylation integrity is critical for accurate detection of Phospho-KDR (Tyr951):

• Immediate sample processing:

  • Process tissue samples immediately after collection

  • Flash-freeze in liquid nitrogen for later analysis

  • For cell cultures, stimulate with VEGF (5 minutes; 1 nM) immediately before lysis

• Phosphatase inhibitor cocktail components:

  • Include sodium orthovanadate (1 mM) to inhibit tyrosine phosphatases

  • Add sodium fluoride (10 mM) to inhibit serine/threonine phosphatases

  • Include β-glycerophosphate (20 mM) for broad phosphatase inhibition

• Lysis buffer optimization:

  • Use complete lysis buffer freshly prepared just prior to sample dilution

  • Avoid high concentrations of reducing agents like DTT

  • Minimize use of ionic detergents like SDS that may interfere with antibody binding

• Storage conditions:

  • Store antibody solutions at -20°C for up to 1 year

  • For frequent use, store at 4°C for up to one month

  • Avoid repeated freeze-thaw cycles

• Validation controls:

  • Include paired positive controls (VEGF-stimulated cells) and negative controls (unstimulated cells)

  • Logarithmically growing HEK-KDR cells treated with VEGF (5 minutes; 1 nM) provide reliable positive controls

How does the biochemical association between ECSCR and KDR influence Tyr951 phosphorylation and downstream signaling?

Research on ECSCR (Endothelial Cell-Specific Chemotaxis Receptor) and KDR interaction provides insights into Tyr951 phosphorylation regulation:

• Association characteristics:

  • Both full-length ECSCR (ECSCR-FL) and truncation mutants lacking the conserved cytoplasmic domain (ECSCR-ΔC) associate with KDR

  • This association is selective for KDR and not detected with FLT1 or chimeric receptors

  • Multiple regions of ECSCR contribute to KDR association, with both transmembrane and cytoplasmic sequences playing roles

• Functional consequences:

  • ECSCR overexpression reduces KDR/PAE cell migration toward VEGF

  • ECSCR influences KDR protein stability, with a ~40% increase in KDR protein observed in HUVECs overexpressing ECSCR-FL

  • ECSCR overexpression increases surface availability of KDR by approximately 30%

• Localization:

  • After VEGF stimulation, ECSCR and KDR co-localize in perinuclear regions

  • This co-localization partially overlaps with HRS-positive endosomal compartments but not with RAB7 or RAB11-positive vesicles

These findings suggest ECSCR functions as a regulatory partner of KDR, potentially modulating Tyr951 phosphorylation and subsequent signaling events. Researchers investigating Phospho-KDR (Tyr951) should consider the influence of endogenous ECSCR expression in their experimental systems.

What techniques can effectively quantify changes in Phospho-KDR (Tyr951) levels in response to experimental treatments?

Several quantitative approaches can accurately measure changes in Phospho-KDR (Tyr951) levels:

• Sandwich immunoassay/ELISA:

  • Provides precise quantification with higher sensitivity than Western blotting

  • Can detect changes across a wider dynamic range

  • Sample data from a phospho-VEGFR-2 assay shows clear differentiation:

  Lysate (µg)	Positive Signal	Negative Signal	P/N Ratio
  0.16	848 ± 76	295 ± 22	2.9
  0.31	1148 ± 84	349 ± 18	3.3
  0.63	1555 ± 70	393 ± 22	4.0
  1.3	2053 ± 149	467 ± 32	4.4
  2.5	2773 ± 26	508 ± 26	5.5
  5.0	3665 ± 101	526 ± 22	7.0
  10	6018 ± 239	522 ± 13	12
  20	12207 ± 1065	647 ± 30	19

  Data shows increasing signal-to-background ratio with increasing lysate concentration

• Quantitative immunofluorescence:

  • Allows assessment of subcellular localization

  • Can be combined with markers for specific cellular compartments

  • Enables correlation of phosphorylation with protein-protein interactions

• Phospho-flow cytometry:

  • Provides single-cell resolution of phosphorylation events

  • Allows simultaneous assessment of multiple parameters

  • Useful for heterogeneous cell populations

• Western blotting with densitometry:

  • Standard approach for semi-quantitative analysis

  • Use recommended dilutions (1:500-1:2000) for optimal results

  • Include loading controls and normalize to total KDR levels

• Proximity ligation assay:

  • Detects protein-protein interactions dependent on phosphorylation

  • Provides spatial information about signaling complexes

  • Highly sensitive for detecting low-abundance phosphoproteins

How does sMEK1 inhibit VEGF-induced phosphorylation of KDR at Tyr951?

Research has identified sMEK1 (secreted MEK1) as a novel regulatory protein that inhibits VEGF-induced KDR phosphorylation at Tyr951:

• Binding specificity:

  • sMEK1 specifically binds to VEGFR-2 but not VEGFR-1

  • This interaction was verified through yeast two-hybrid screening (β-galactosidase activity: 92.31±0.99 for sMEK1-VEGFR-2 vs. 2.08±0.84 for sMEK1-VEGFR-1)

  • Co-immunoprecipitation experiments confirmed direct interaction between sMEK1 and VEGFR-2

• Phosphorylation inhibition:

  • Ectopic expression of sMEK1 inhibits VEGF-induced VEGFR-2 phosphorylation at Tyr951 in a dose-dependent manner

  • Notably, VEGFR-2 phosphorylation at Tyr1175 was not affected by sMEK1

  • sMEK1-siRNA reverses this inhibitory effect

• Functional consequences:

  • sMEK1 reduces VEGF-stimulated endothelial cell proliferation to ~65% of control levels

  • It inhibits VEGF-induced endothelial cell migration in Transwell assays

  • sMEK1 suppresses phosphorylation of downstream components in the VEGFR-2/PI3K signaling pathway, including Akt and eNOS

• Pathway specificity:

  • sMEK1 inhibits VEGF-stimulated phosphorylation of Akt at both Ser473 and Thr308

  • This suggests sMEK1 specifically targets the Tyr951-dependent signaling axis while sparing other VEGFR-2 signaling pathways