Phospho-PPP2CA (Y307) Recombinant Monoclonal Antibody

What is Phospho-PPP2CA (Y307) Recombinant Monoclonal Antibody and what are its basic characteristics?

Phospho-PPP2CA (Y307) Recombinant Monoclonal Antibody [3F11] is a primary antibody designed to recognize the phosphorylated tyrosine 307 residue of the catalytic subunit alpha isoform of protein phosphatase 2A (PPP2CA). The antibody is a recombinant monoclonal antibody produced in HEK293F cells with rabbit IgG isotype. It is unconjugated (not labeled with any tags or fluorophores) and reacts specifically with human samples .

The key specifications of this antibody are summarized in the following table:

What is the biological function of PP2A and its catalytic subunit PPP2CA?

Protein Phosphatase 2A (PP2A) is a major serine/threonine phosphatase involved in regulating numerous enzymes, signal transduction pathways, and cellular processes. PPP2CA is the catalytic subunit alpha isoform of PP2A.

PP2A's functions include:

• Serving as the major phosphatase for microtubule-associated proteins (MAPs)

• Modulating the activity of multiple kinases including phosphorylase B kinase, casein kinase 2, mitogen-stimulated S6 kinase, and MAP-2 kinase

• Cooperating with SGO2 to protect centromeric cohesin during meiosis I

• Dephosphorylating SV40 large T antigen and p53/TP53

• Activating RAF1 by dephosphorylating it at Ser-259

• Mediating dephosphorylation of WEE1, preventing its ubiquitin-mediated proteolysis and promoting the G2/M checkpoint

• Mediating dephosphorylation of MYC and FOXO3, promoting their ubiquitin-mediated proteolysis or stabilization

• Catalyzing dephosphorylation of NLRP3's pyrin domain, promoting inflammasome assembly

• Participating in striatin-interacting phosphatase and kinase (STRIPAK) complexes, which regulate multiple signaling pathways including Hippo, MAPK, nuclear receptor and cytoskeleton remodeling

How should researchers optimize Western blot protocols when using Phospho-PPP2CA (Y307) antibody?

To optimize Western blot protocols with Phospho-PPP2CA (Y307) antibody:

• Sample preparation: Include phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride) in lysis buffers to preserve phosphorylation status.

• Blocking: Use 5% BSA in TBST rather than milk, as milk contains phosphoproteins that may interfere with phospho-specific antibody binding.

• Antibody dilution: Start with a 1:1000 dilution within the recommended range (1:500-1:5000) . Optimize based on signal-to-noise ratio.

• Incubation time: Incubate with primary antibody overnight at 4°C to maximize specific binding.

• Controls:

  • Include a positive control (e.g., cells treated with pervanadate to inhibit phosphatases)

  • Include a negative control (e.g., sample treated with phosphatase)

  • Consider using PP2A Y307F mutant samples as specificity controls

• Signal detection: Use enhanced chemiluminescence (ECL) or fluorescence-based detection systems.

• Validation: Confirm specificity by using peptide competition assays or comparing with alternative detection methods.

What experimental controls are essential when studying PP2A phosphorylation at Y307?

When studying PP2A phosphorylation at Y307, the following controls are essential:

• Phosphatase treatment control: Treat a portion of your sample with lambda phosphatase to confirm the antibody is detecting phosphorylated protein.

• Stimulus controls: Include samples with known modulators of PP2A phosphorylation:

  • Treatment with pervanadate (phosphatase inhibitor) should increase Y307 phosphorylation

  • Treatment with SFK inhibitors (e.g., PP2) should decrease Y307 phosphorylation

• Mutant controls: Use PP2A constructs with site-directed mutations:

  • Y307F mutant as negative control

  • Comparative analysis with Y127F and Y284F mutants to assess cross-reactivity

• Immunoprecipitation controls: Validate phosphorylation using:

  • Anti-phosphotyrosine antibodies after PP2A immunoprecipitation

  • Reciprocal immunoprecipitation with anti-phosphotyrosine followed by PP2A detection

• Alternative methods: Confirm findings using:

  • Mass spectrometry to directly identify phosphorylation sites

  • Phos-tag gels to separate phosphorylated from non-phosphorylated forms

These controls are particularly important given the contradictory findings about Y307 phosphorylation discussed in section 3.

How do recent findings challenge the established view of Y307 as the primary phosphorylation site of PP2A?

Recent research significantly challenges the long-held dogma that Y307 is the primary phosphorylation site of PP2A catalytic subunit (PP2Ac), raising important considerations for researchers:

Mass spectrometry (MS) studies of Src-transformed mouse embryo fibroblasts (MEFs) have identified endogenous PP2Ac phosphorylation at Y127 and Y284, but not at Y307 . Multiple discovery-mode MS investigations across various oncogenic/transformed cells and human cancers have similarly identified Y284 phosphorylation, but not Y307 .

Even in targeted MS studies using HEK293T cells overexpressing both constitutively active Src and PP2Ac, Y307 phosphorylation was detected only at very low levels . Experiments using Y307F point mutants of PP2Ac revealed very similar levels of tyrosine phosphorylation compared to wild-type PP2Ac in response to Src activation or pervanadate treatment .

These findings suggest that:

• Y307 is not the predominant tyrosine phosphorylation site of PP2Ac

• Y127 and Y284 appear to be the major sites targeted by Src family kinases (SFKs)

• Most studies assessing PP2A Y307 phosphorylation have relied on antibodies that may lack specificity

This represents a paradigm shift in our understanding of PP2A regulation and raises questions about the utility and specificity of many commercially available "anti-pY307 PP2Ac" antibodies.

What is the comparative significance of Y127, Y284, and Y307 phosphorylation sites in PP2A regulation?

The comparative significance of these phosphorylation sites reveals a complex regulatory mechanism:

Y127 and Y284 phosphorylation:

• Primarily mediated by Src family kinases (SFKs)

• Src primarily phosphorylates both Y127 and Y284

• Fyn preferentially phosphorylates Y284

• These phosphorylation events enhance interaction between PP2Ac and SFKs

• Y284 phosphorylation particularly promotes dissociation of the regulatory Bα subunit, altering PP2A substrate specificity

• Mutation of these sites (Y127/284F and Y284F) prevents SFK-mediated phosphorylation of Tau at pSer202 (CP13 epitope), a pathological hallmark of Alzheimer's disease

• These mutations also prevent SFK-dependent activation of ERK, a major growth regulatory kinase upregulated in many cancers

Y307 phosphorylation:

• Previously believed to be the sole site of phosphorylation on PP2Ac

• Traditionally thought to be essential for catalytic inactivation of PP2A and v-Src-mediated cell transformation

• Recent evidence suggests this site is phosphorylated at very low levels, if at all, under physiological conditions

• Many commercially available "anti-pY307" antibodies have been shown to recognize both wild-type and Y307F mutant PP2Ac equally, raising questions about their specificity

What is the "Goldilocks phenomenon" observed with PPP2CA expression in cancer cells, and what are its implications for research?

The "Goldilocks phenomenon" refers to the observation that PPP2CA exhibits concentration-dependent opposing functions in cancer cells - specifically in neuroblastoma (NB). This phenomenon has significant implications for research:

Concentration-dependent functions of PPP2CA:

• At low expression levels, PPP2CA functions as an essential survival gene for cancer cells

• At high expression levels, PPP2CA acts as a tumor suppressor

Research findings demonstrating this phenomenon:

• Reduction of PPP2CA by knock-down decreased growth of neuroblastoma cells

• Complete ablation of PPP2CA by knock-out was not tolerated by these cells

• Neuroblastoma cells show addiction to PPP2CA, which is augmented by MYCN activation

• SET, an endogenous inhibitor of PP2A, was overexpressed in poor-prognosis neuroblastoma

• The SET inhibitor OP449 effectively decreased viability of neuroblastoma cells, consistent with a tumor suppressor function of PPP2CA when its activity is enhanced

Implications for research:

• Researchers must consider the baseline expression level of PPP2CA when interpreting experimental results

• Therapeutic strategies targeting PPP2CA must account for this dual functionality

• Simple activation or inhibition of PPP2CA may produce contradictory effects depending on cellular context

• Combined approaches that maintain PPP2CA within an optimal functional range may be necessary

This phenomenon explains why both PP2A inhibitors and activators have shown anti-cancer effects in different contexts and suggests that precise modulation, rather than simple activation or inhibition, may be required for therapeutic applications.

How can researchers validate the specificity of Phospho-PPP2CA (Y307) antibodies given recent concerns?

Given recent findings questioning the specificity of commercial pY307 antibodies , researchers should implement rigorous validation protocols:

• Genetic validation:

  • Express wild-type PP2A alongside Y307F mutant in a cellular system

  • If the antibody is specific for pY307, it should not recognize the Y307F mutant by Western blot or immunofluorescence

  • Note: Some commercial "anti-pY307" antibodies have been shown to recognize both wild-type and Y307F mutants equally

• Phosphatase treatment controls:

  • Treat cell lysates with lambda phosphatase and confirm loss of antibody recognition

  • Include both positive controls (untreated) and negative controls (phosphatase-treated)

• Peptide competition assays:

  • Pre-incubate antibody with phospho-Y307 peptide and non-phosphorylated control peptide

  • Specific binding should be blocked by phospho-peptide but not by non-phospho-peptide

• Mass spectrometry validation:

  • Perform targeted mass spectrometry to directly assess Y307 phosphorylation status

  • Compare MS results with antibody-based detection methods

• Multiple antibody approach:

  • Use antibodies from different vendors or clones

  • Compare detection patterns to identify potential non-specific binding

• Kinase manipulation:

  • Modulate SFK activity using inhibitors (PP2) or activators

  • Assess correlation between kinase activity and antibody signal

• Cross-reactivity assessment:

  • Test the antibody against Y127F, Y284F, and Y127/284F mutants

  • Evaluate whether the antibody cross-reacts with other phosphotyrosine sites

What methodological approaches can address the contradictions between commercial antibody specifications and recent research findings?

To address contradictions between commercial antibody specifications and recent research findings , researchers should employ these methodological approaches:

• Integrated multi-method detection strategy:

  • Combine antibody-based methods with mass spectrometry

  • Use Phos-tag SDS-PAGE to separate and identify phosphorylated species

  • Apply proximity ligation assays to detect and localize phosphorylated PP2A

• Site-directed mutagenesis comparative analysis:

  • Create a panel of mutants: Y307F, Y127F, Y284F, and Y127/284F

  • Compare phosphorylation patterns and functional outcomes

  • This approach helped uncover that Y127 and Y284 are the primary SFK-dependent phosphorylation sites

• Phospho-specific antibody generation and validation:

  • Develop new antibodies specifically validated against mutant controls

  • Characterize antibodies using multiple cell types and treatments

  • Confirm specificity using peptide competition and phosphatase treatment

• Functional correlation analysis:

  • Compare PP2A activity measurements with phosphorylation status detected by various methods

  • Assess correlation between detected phosphorylation and functional outcomes like regulatory subunit binding

• Quantitative phosphoproteomics:

  • Use SILAC or TMT labeling with phosphotyrosine enrichment

  • Quantify relative abundance of each phosphorylation site

  • This approach can reveal that Y127 and Y284 are more abundant than Y307

• Time-course and stimulus-response analysis:

  • Monitor phosphorylation kinetics after various stimuli

  • Compare detection by different methods at each timepoint

  • This revealed that Y127 and Y284 phosphorylation occurs rapidly after EGF stimulation

How can Phospho-PPP2CA antibodies be used to investigate the role of PP2A in neurodegenerative diseases?

Phospho-PPP2CA antibodies can provide valuable insights into neurodegenerative disease mechanisms through carefully designed experimental approaches:

• Tau phosphorylation studies in Alzheimer's disease (AD):

  • Recent research has shown that SFK-mediated phosphorylation of PP2Ac at Y284 alters substrate specificity and influences Tau phosphorylation at the CP13 (pSer202) epitope, a pathological hallmark of AD

  • Researchers can use phospho-specific antibodies alongside PP2A mutants (Y127F, Y284F) to investigate how PP2A tyrosine phosphorylation impacts Tau hyperphosphorylation

  • Comparative immunohistochemistry of phospho-PP2A and phospho-Tau in AD brain sections can reveal spatial relationships

• PP2A-SFK interaction analysis:

  • Phospho-PP2A antibodies can be used in co-immunoprecipitation studies to assess how PP2A phosphorylation affects its interaction with Src family kinases in neuronal models

  • Proximity ligation assays can visualize these interactions in situ in brain tissue

• Regulatory subunit displacement mechanism:

  • As Y284 phosphorylation promotes dissociation of the regulatory Bα subunit , researchers can use Y284-phospho-specific antibodies to track this process in neurodegeneration models

  • This can be combined with FRET-based approaches to monitor PP2A holoenzyme assembly/disassembly in living neurons

• In vivo phosphorylation monitoring:

  • Tracking PP2A phosphorylation status in animal models of neurodegeneration at different disease stages

  • Correlating changes in PP2A phosphorylation with cognitive deficits and neuropathology

• Therapeutic intervention assessment:

  • Evaluating how potential therapeutic compounds affect PP2A phosphorylation status and downstream substrate phosphorylation

  • Using phospho-PP2A antibodies as pharmacodynamic markers in preclinical studies

What are the implications of the newly discovered SFK-mediated PP2A regulatory mechanism for cancer research?

The newly discovered SFK-mediated PP2A regulatory mechanism has profound implications for cancer research, challenging existing paradigms and opening new therapeutic avenues:

• Revised understanding of PP2A regulation in cancer:

  • The finding that SFKs primarily phosphorylate PP2A at Y127 and Y284 rather than Y307 fundamentally changes our understanding of how oncogenic kinases regulate this crucial phosphatase

  • Rather than simply inhibiting catalytic activity, SFK-mediated phosphorylation appears to alter PP2A substrate specificity by modulating regulatory subunit binding

• Impact on SFK-ERK signaling axis:

  • PP2A Y127/284F and Y284F mutants prevent SFK-dependent activation of ERK, a major growth regulatory kinase upregulated in many cancers

  • This suggests that SFK-mediated PP2A phosphorylation is required for full ERK activation, revealing a previously unknown regulatory mechanism

• Dual role of PP2A in cancer (Goldilocks phenomenon):

  • PP2A exhibits concentration-dependent opposing functions in cancer cells - at low levels it's essential for survival, while at high levels it acts as a tumor suppressor

  • This explains why both PP2A inhibitors and activators have shown anti-cancer effects in different contexts

• PP2A addiction in cancer cells:

  • Neuroblastoma cells show addiction to PPP2CA, particularly in MYCN-amplified cases

  • Complete PPP2CA knockout is not tolerated, while partial knockdown decreases growth

  • This suggests a therapeutic window for PP2A-targeted interventions

• SET inhibition as a therapeutic strategy:

  • SET, an endogenous PP2A inhibitor, is overexpressed in poor-prognosis neuroblastoma

  • SET inhibitors like OP449 effectively decrease cancer cell viability by enhancing PP2A activity

  • Understanding phosphorylation-dependent changes in PP2A-SET interaction could lead to more targeted interventions

• Biomarker potential:

  • The phosphorylation status of PP2A at Y127 and Y284 could serve as biomarkers for SFK activity and potential response to SFK inhibitors in cancer

  • Monitoring these sites might better predict therapeutic outcomes than the previously used Y307 phosphorylation

What sample preparation methods best preserve PP2A phosphorylation status for antibody-based detection?

Preserving PP2A phosphorylation status requires specific sample preparation considerations:

• Lysis buffer optimization:

  • Use buffers containing phosphatase inhibitors: sodium orthovanadate (1-2 mM), sodium fluoride (10 mM), β-glycerophosphate (10 mM), and sodium pyrophosphate (5 mM)

  • Include protease inhibitors to prevent degradation

  • Maintain cold temperature throughout processing (4°C)

  • Consider using commercial phospho-protein preservation buffers

• Cell harvesting techniques:

  • Direct lysis in the culture dish is preferred over trypsinization

  • If cell scraping is necessary, perform quickly and transfer immediately to cold lysis buffer

  • For tissues, snap-freeze in liquid nitrogen immediately after collection

• Denaturing conditions:

  • Include sufficient detergent (1% NP-40 or 0.5% Triton X-100) to solubilize membrane-associated PP2A

  • Add SDS (0.1%) to denature phosphatases that might dephosphorylate during processing

  • Heat samples at 95°C for 5 minutes in Laemmli buffer containing 5% β-mercaptoethanol

• Phosphatase treatment controls:

  • Process parallel samples with and without lambda phosphatase treatment

  • This provides a negative control for phospho-specific antibody detection

• Stabilization strategies:

  • Consider pre-treating cells with pervanadate (100 μM for 15-30 minutes) to enhance detection of low-abundance phosphorylation sites

  • For tissue samples, perfuse with phosphatase inhibitors before collection when possible

• Fractionation considerations:

  • If performing subcellular fractionation, maintain phosphatase inhibitors in all buffers

  • Validate phosphorylation status in each fraction with appropriate controls

• Storage conditions:

  • Store lysates at -80°C with phosphatase inhibitors

  • Avoid multiple freeze-thaw cycles

  • Add glycerol (10%) for cryoprotection if multiple uses are anticipated

How can researchers determine the optimal experimental conditions for studying the different PP2A phosphorylation sites?

Determining optimal experimental conditions for studying PP2A phosphorylation requires systematic optimization:

• Cell model selection:

  • For SFK-mediated phosphorylation at Y127/Y284, use:

    • Src-transformed fibroblasts

    • HEK293 or Cos-7 cells transfected with constitutively active Src or Fyn

    • Neuroblastoma cells (e.g., N2a) for Fyn-mediated Y284 phosphorylation

  • For potential Y307 phosphorylation, use:

    • HEK293T cells with overexpression of both Src and PP2A

• Stimulus optimization:

  • SFK activation: Transfection with constitutively active Src (Src CA) or Fyn (Fyn CA)

  • Growth factor stimulation: EGF treatment (50-100 ng/ml) combined with pervanadate to trap transient phosphorylation

  • Phosphatase inhibition: Pervanadate treatment (100 μM for 15-30 minutes)

  • Kinase inhibition: PP2 (SFK inhibitor) at 10 μM

• Time-course analysis:

  • Monitor phosphorylation at different time points (5, 15, 30, 60 minutes) after stimulation

  • For EGF stimulation, focus on early time points (5-15 minutes)

  • For constitutively active kinase expression, 24-48 hours post-transfection is typically optimal

• Detection method selection:

  • Western blotting: Optimize antibody dilution (start with 1:1000) and incubation conditions

  • Immunoprecipitation: Use either PP2A antibodies followed by phosphotyrosine detection or vice versa

  • Phos-tag SDS-PAGE: Optimize acrylamide percentage (6-8%) and Phos-tag concentration (25-100 μM)

  • Mass spectrometry: Consider SILAC or TMT labeling with phosphotyrosine enrichment

• Mutant analysis system:

  • Express wild-type PP2A alongside Y127F, Y284F, Y307F, and Y127/284F mutants

  • Compare phosphorylation patterns under various stimulation conditions

  • Assess functional outcomes (e.g., regulatory subunit binding, substrate phosphorylation)

• Comparative kinase analysis:

  • Directly compare Src CA and Fyn CA effects to identify kinase-specific phosphorylation patterns

  • Include other SFK family members (Yes, Lck) to establish broader patterns

This systematic approach will help researchers determine the optimal conditions for studying each phosphorylation site while avoiding potential pitfalls based on outdated models of PP2A regulation.

What emerging technologies could advance our understanding of PP2A phosphorylation dynamics?

Several emerging technologies hold promise for advancing our understanding of PP2A phosphorylation dynamics:

• Genetically encoded phosphorylation sensors:

  • FRET-based sensors specifically designed for PP2A phosphorylation sites

  • These would allow real-time monitoring of phosphorylation/dephosphorylation events in living cells

  • Could reveal compartment-specific dynamics and temporal regulation

• CRISPR-based phosphorylation site editing:

  • Precise genome editing to mutate endogenous PP2A phosphorylation sites (Y127, Y284, Y307)

  • Avoids overexpression artifacts associated with transfection-based approaches

  • Can be combined with conditional systems to study tissue-specific effects

• Proximity-dependent labeling technologies:

  • BioID or TurboID fused to PP2A to identify proximity interactors dependent on phosphorylation status

  • APEX2 labeling to map the spatiotemporal organization of phosphorylated PP2A pools

  • Would reveal how phosphorylation affects PP2A interaction networks

• Single-molecule tracking:

  • Tracking individual PP2A molecules in living cells using quantum dots or photoactivatable fluorophores

  • Could reveal how phosphorylation affects PP2A diffusion, localization, and complex formation

  • May uncover heterogeneity in PP2A populations that bulk analyses miss

• Advanced mass spectrometry techniques:

  • Targeted parallel reaction monitoring (PRM) for absolute quantification of site-specific phosphorylation

  • Crosslinking mass spectrometry to capture phosphorylation-dependent structural changes

  • Top-down proteomics to analyze intact PP2A complexes with multiple modifications

• Optogenetic control of kinase activity:

  • Light-controlled activation of SFKs to achieve temporal precision in PP2A phosphorylation

  • Would enable study of rapid phosphorylation dynamics without chemical perturbations

  • Could be combined with live-cell imaging to correlate phosphorylation with functional outcomes

• Cryo-EM structural analysis:

  • High-resolution structures of phosphorylated versus non-phosphorylated PP2A complexes

  • Would reveal how phosphorylation at different sites affects holoenzyme assembly

  • Could guide structure-based drug design for phosphorylation-specific modulators

How might the revised understanding of PP2A phosphorylation inform development of new therapeutic strategies?

The revised understanding of PP2A phosphorylation offers several promising avenues for therapeutic development:

• Site-specific PP2A modulators:

  • Rather than general PP2A activators or inhibitors, develop compounds that selectively interfere with Y127 or Y284 phosphorylation

  • These could modulate PP2A substrate specificity without completely inhibiting catalytic activity

  • Potential to disrupt specific pathological PP2A functions while preserving essential ones

• Targeted degradation approaches:

  • Develop PROTACs (proteolysis targeting chimeras) that selectively target phosphorylated PP2A pools

  • This could allow for elimination of specific PP2A subpopulations while preserving others

  • Would address the "Goldilocks phenomenon" by fine-tuning PP2A levels rather than complete inhibition

• SFK-PP2A interface disruptors:

  • Design peptides or small molecules that specifically disrupt the interaction between SFKs and PP2A

  • This would prevent phosphorylation while potentially preserving PP2A activity

  • Could selectively block pathological SFK-mediated PP2A regulation

• Regulatory subunit-focused strategies:

  • Since Y284 phosphorylation promotes dissociation of the Bα regulatory subunit , develop compounds that prevent this dissociation

  • This would maintain normal PP2A holoenzyme assembly even in the presence of activated SFKs

  • Could restore proper substrate targeting in disease states

• Dual-targeting approaches:

  • Combine SFK inhibitors with SET inhibitors to simultaneously reduce aberrant PP2A phosphorylation and enhance PP2A activity

  • This two-pronged approach could be particularly effective in cancers with high SFK activity and SET expression

  • Would address both catalytic inhibition and substrate redirection mechanisms

• Biomarker-guided therapies:

  • Use PP2A phosphorylation status (Y127/Y284 vs. Y307) as biomarkers to guide therapeutic decisions

  • Select patients most likely to benefit from SFK inhibitors or PP2A modulators based on phosphorylation profile

  • Monitor treatment efficacy through changes in phosphorylation status

• Stabilization of regulatory interactions:

  • Develop molecules that stabilize specific PP2A holoenzyme complexes even in the presence of Y284 phosphorylation

  • This approach could preserve critical tumor suppressor functions of PP2A

  • May help address the concentration-dependent opposing functions of PP2A in cancer cells