Phospho-CASP9 (Y153) Antibody

What is the biological significance of Caspase-9 Y153 phosphorylation in apoptotic pathways?

Caspase-9 serves as a critical initiator caspase in the intrinsic apoptotic pathway. It forms part of the apoptosome complex that activates executioner caspases such as caspase-3 and caspase-7, thereby initiating the cell death cascade. Phosphorylation at Y153 has been implicated in the response of cells to DNA damage .

When investigating phosphorylation at this site, researchers should consider that:

• Y153 forms a hydrogen bond with Asp-350 in the L2′ loop in the dimeric, substrate-bound structure of caspase-9

• This interaction supports the position of L2′ as it interacts with L2 and L4 to form the substrate binding groove and catalytic site

• Modification at this position may therefore disrupt the protein's catalytic function

What applications are optimal for Phospho-CASP9 (Y153) antibodies and what experimental conditions yield the best results?

Phospho-CASP9 (Y153) antibodies are validated for several research applications with specific dilution recommendations:

For optimal results in Western blotting:

• Ensure complete protein transfer to the membrane

• Block thoroughly to reduce background

• Use freshly prepared phosphatase inhibitors in all buffers during sample preparation to prevent dephosphorylation

• Consider using phospho-enrichment strategies for low abundance targets

The antibodies are typically generated against synthetic peptides derived from human Caspase-9 around the phosphorylation site of Y153, within the amino acid range 119-168 . Most commercially available antibodies are rabbit polyclonal antibodies that detect endogenous levels of Caspase-9 protein only when phosphorylated at Y153 .

How should researchers validate the specificity of Phospho-CASP9 (Y153) antibodies?

Thorough validation is essential due to the controversy surrounding Y153 phosphorylation. Recommended approaches include:

• Phosphatase treatment controls: Treating half of your sample with lambda phosphatase should eliminate the signal if the antibody is truly phospho-specific

• Phosphomimetic mutants: Y153E or Y153D mutants can serve as positive controls, though note that these substitutions inhibit catalytic activity

• Unphosphorylatable mutants: Y153F substitution can serve as a negative control for phosphorylation

• Phospho-peptide competition: Pre-incubating the antibody with the phospho-peptide immunogen should block specific binding

• Kinase activation/inhibition: Treatment with c-Abl activators (like DPH/vanadate) or inhibitors (like imatinib) should modulate the signal if Y153 is indeed a c-Abl target

• Mass spectrometry validation: For definitive confirmation of the phosphorylation site, LC-MS/MS analysis following enrichment of phosphopeptides should be considered

It's important to note that researchers have raised doubts about Tyr-153 as a phosphorylation target of c-Abl, with compelling evidence suggesting Tyr-397 may be the primary site instead .

What sample preparation methods are critical for detecting authentic Phospho-CASP9 (Y153) signals?

Successful detection of phosphorylated caspase-9 requires meticulous attention to sample preparation:

• Cell stimulation protocol:

  • For DNA damage-induced phosphorylation studies, consider etoposide or radiation treatment

  • For c-Abl activation, dipyridyl hydroxymethylpyrimidine (DPH) combined with vanadate has been validated

• Lysis buffer composition:

  • Use a buffer containing both phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) and protease inhibitors

  • Include 0.1% SDS or other strong detergents to ensure complete solubilization

• Phosphatase prevention strategies:

  • Keep samples on ice throughout preparation

  • Use freshly prepared inhibitors at appropriate concentrations

  • Avoid repeated freeze-thaw cycles

• Enrichment approaches:

  • For low abundance phosphoproteins, consider immunoprecipitation before Western blotting

  • Phosphotyrosine-specific antibodies can be used for initial enrichment

• Control samples:

  • Include both positive controls (cells treated with phosphatase inhibitors) and negative controls (phosphatase-treated samples)

  • Consider using catalytically inactive C287A caspase-9 to prevent auto-processing during experiments

What is the controversy surrounding c-Abl phosphorylation of Caspase-9 at Y153 versus Y397?

A significant controversy exists regarding the true site of c-Abl-mediated phosphorylation of caspase-9. This represents an important consideration for researchers using Y153 phospho-specific antibodies:

• No evidence for Y153 phosphorylation: Using in vitro kinase assays with purified c-Abl and caspase-9, researchers observed no phosphorylation in the CARD+Large region where Y153 is located, but instead found clear phosphorylation of the small subunit .

• Y397 identified as the dominant site: LC-MS/MS analysis identified Tyr-397 as the site of c-Abl phosphorylation with high confidence. The Y397F substitution almost completely eliminated c-Abl-mediated phosphorylation of caspase-9 .

• Sequence recognition preference: Tyr-397 is contained within a much more ideal c-Abl recognition site than Tyr-153, and is more surface-exposed .

• Cellular validation: In cells treated with c-Abl activators (DPH/vanadate), the Y397F caspase-9 variant showed significantly reduced phosphorylation compared to wild-type caspase-9 .

• Functional consequences: Contrary to the activation reported for Y153 phosphorylation, phosphorylation at Y397 (or phosphomimetic Y397E) was found to inhibit caspase-9 activity .

This controversy highlights the importance of rigorous validation when using phospho-specific antibodies in research.

How does the Y153 phosphorylation state affect Caspase-9's catalytic activity and what are the kinetic parameters?

The catalytic consequences of Y153 phosphorylation or modification have been thoroughly characterized through kinetic analyses:

These kinetic parameters reveal several important insights:

• Y153E and Y153D phosphomimetic substitutions completely abolish caspase-9 catalytic activity, with substantial increases in Km (>3,000 μM) and dramatic reductions in kcat (<0.01 s-1) .

• Even the conservative Y153F substitution (removing only the hydroxyl group) severely impairs catalytic efficiency, reducing it by approximately 150-fold compared to wild-type .

• The inhibitory effect persists regardless of whether caspase-9 is in its zymogen form or its cleaved, mature form, indicating that the inhibition is an inherent consequence of modification at this position .

This data contradicts earlier reports suggesting that Y153 phosphorylation activates caspase-9, and instead demonstrates that modification at this position is strongly inhibitory to catalytic function .

What methodological approaches can resolve conflicting data regarding Caspase-9 phosphorylation?

When faced with contradictory findings regarding caspase-9 phosphorylation, researchers should consider implementing the following methodological strategies:

• Orthogonal detection methods:

  • Combine antibody-based detection with mass spectrometry

  • Use phospho-enrichment approaches prior to MS analysis to increase sensitivity

  • Consider targeted MS approaches for known phosphorylation sites

• Comprehensive mutagenesis:

  • Create a complete panel of phosphomimetic (Y to E/D) and phospho-null (Y to F) mutants

  • Assess both structural and functional consequences of each substitution

  • Test mutants in multiple cellular contexts

• Activity-based assays:

  • Measure enzymatic activity with defined substrates

  • Determine kinetic parameters (Km, kcat) for wild-type and mutant proteins

  • Consider both zymogen and cleaved forms of caspase-9

• Structural analysis:

  • Utilize structural modeling to predict effects of phosphorylation

  • Consider how phosphorylation might affect interactions with binding partners like Apaf-1

  • Examine potential allosteric effects on the catalytic site

• Context-dependent analysis:

  • Test under different apoptotic stimuli (intrinsic vs. extrinsic)

  • Assess in various cell types with different c-Abl expression levels

  • Consider the subcellular localization of both kinase and substrate

How can researchers accurately model the effects of Y153 phosphorylation on Caspase-9 structure and function?

To accurately model the structural and functional impacts of Y153 phosphorylation, researchers should employ a multi-faceted approach:

• Structural analysis and modeling:

  • In the dimeric, substrate-bound structure of caspase-9, Y153 forms a hydrogen bond with Asp-350 in the L2′ loop

  • This interaction supports the position of L2′ as it interacts with L2 and L4 to form the substrate binding groove

  • Computational modeling with phosphotyrosine at position 153 can predict steric and electrostatic effects on substrate binding

• Biochemical characterization:

  • Express and purify recombinant phosphomimetic variants (Y153E/D)

  • Assess oligomerization state using analytical ultracentrifugation or size exclusion chromatography

  • Examine thermal stability using differential scanning fluorimetry

• Interaction studies:

  • Evaluate binding to Apaf-1 using co-immunoprecipitation or surface plasmon resonance

  • Assess incorporation into the apoptosome complex

  • Examine interactions with other caspases, particularly executioner caspases 3 and 7

• Functional assays in cellular systems:

  • Develop cell lines expressing Y153 variants using CRISPR-Cas9 knock-in

  • Measure apoptotic responses to various stimuli

  • Assess caspase activation cascades using activity-based probes

• Temporal dynamics:

  • Develop phospho-specific biosensors to monitor phosphorylation in real-time

  • Track the kinetics of phosphorylation and dephosphorylation events

  • Correlate phosphorylation status with apoptotic progression

This comprehensive approach can help resolve contradictions in the literature regarding the role of Y153 phosphorylation in caspase-9 regulation .

What are the most effective phospho-enrichment strategies for studying low-abundance Caspase-9 phosphorylation events?

For studying low-abundance phosphorylation events in caspase-9, researchers should consider these specialized enrichment strategies:

• Immunoprecipitation-based approaches:

  • Use phosphotyrosine-specific antibodies for initial enrichment

  • Follow with caspase-9-specific antibodies for sequential IP

  • Consider crosslinking antibodies to beads to reduce background

• Phosphopeptide enrichment for mass spectrometry:

  • Immobilized metal affinity chromatography (IMAC) using Fe3+ or Ga3+

  • Titanium dioxide (TiO2) enrichment for phosphopeptides

  • Phosphotyrosine-specific antibody enrichment prior to MS analysis

• Cellular strategies:

  • Treatment with phosphatase inhibitors to preserve phosphorylation status

  • Use of catalytically inactive C287A caspase-9 to prevent auto-processing

  • c-Abl activation using DPH combined with vanadate

• Genetic approaches:

  • Overexpression systems with epitope-tagged constructs

  • CRISPR-Cas9 knock-in of tagged endogenous caspase-9

  • Inducible expression systems for temporal control

• Advanced MS techniques:

  • Parallel reaction monitoring (PRM) for targeted phosphopeptide detection

  • Data-independent acquisition (DIA) for comprehensive phosphoproteome analysis

  • Heavy-labeled synthetic phosphopeptide standards for absolute quantification