PP2A4 Antibody

What is PP2A and what specific protein does a PP2A4 antibody detect?

Anti-PP2A antibodies detect the protein encoded by the gene PTPA (Protein Phosphatase 2 Phosphatase Activator) in humans. This is a 358-amino acid protein belonging to the PTPA-type PPIase family with both cytoplasmic and nuclear localization . PPP2R4 (another name for PTPA) acts as a regulatory subunit for serine/threonine-protein phosphatase 2A (PP2A) and modulates its activity or substrate specificity by inducing conformational changes in the catalytic subunit .

What are the common applications for PP2A4 antibodies in research?

PP2A4 antibodies are commonly used in several experimental applications:

• Western Blot (WB): For detecting and quantifying PP2A4 expression levels

• Immunohistochemistry (IHC): For visualizing PP2A4 in tissue sections

• Flow Cytometry: For analyzing PP2A4 expression in cell populations

• Immunoprecipitation (IP): For isolating PP2A4-containing complexes

Different antibodies show varying reactivity across species, with many being validated for human, mouse, and rat samples .

How does PP2A4 regulate phosphatase activity?

PP2A4 (PTPA) serves as a regulatory subunit that modulates PP2A activity through several mechanisms:

• It can reactivate inactive phosphatase PP2A-phosphatase methylesterase complexes (PP2A(i)) in the presence of ATP and Mg²⁺

• It reversibly stimulates the variable phosphotyrosyl phosphatase activity of PP2A core heterodimer (PP2A(D)) in the presence of ATP and Mg²⁺

• The phosphotyrosyl phosphatase activity is dependent on an ATPase activity of the PP2A(D):PPP2R4 complex

This regulatory function is essential for ensuring proper phosphorylation balance within the cell, thereby playing a significant role in cellular homeostasis .

What cellular processes are influenced by PP2A4 activity?

PP2A4 is involved in multiple critical cellular processes:

• DNA damage response and repair: Cells with functional α4 (which stabilizes PP2A) can more efficiently reverse the phosphorylation of H2AX and p53 following DNA damage

• Apoptosis regulation: PPP2R4 is involved in apoptotic processes, and this function appears to be independent from PP2A in some contexts

• Cell viability during stress: Enhanced α4 expression protects cells from genotoxic reagents and nutrient deprivation

How do experimental conditions affect PP2A complex stability during immunoprecipitation assays?

Recommended Protocol:

• For studying A/C core enzyme: Perform immunoprecipitation at 4°C

• For studying α4/C interactions: Consider comparative analysis at different temperatures (4°C, room temperature)

• Always include temperature controls when comparing different experimental conditions

What mechanisms regulate PP2A4 stability and activity during cellular stress responses?

During cellular stress responses, particularly following DNA damage, complex regulatory mechanisms control PP2A4 stability and activity:

• α4 plays a crucial role in stabilizing newly-synthesized PP2A-C subunit by preventing its ubiquitination

• Following DNA damage (e.g., doxorubicin treatment), control cells display increased PP2A activity as phosphorylation of DNA repair proteins declines

• In α4-depleted cells, there is a >70% decline in total PP2A activity prior to treatment, and activity decreases further following genotoxic treatment

• Overexpression of α4 significantly improves cell viability following exposure to DNA damaging agents like doxorubicin and camptothecin

This indicates that α4-mediated stabilization of PP2A is a critical regulatory mechanism during cellular stress responses.

What technical challenges exist when overexpressing PP2A catalytic subunits in experimental systems?

Enhanced expression of PP2A-C in cells has been historically difficult to achieve because cells tightly regulate PP2A protein levels. Key technical challenges include:

• Transfection of C subunit expression plasmid alone leads to only minimal increases in PP2A-C expression

• Overexpressed C subunit undergoes rapid ubiquitination when expressed alone

• Co-expression with A subunit fails to enhance PP2A-C expression or prevent its ubiquitination

Experimental Solution:
Co-expression of α4 significantly increases protein levels of the C subunit, and this increase correlates with the amount of α4 co-expressed. Additionally, α4 co-expression results in substantial reduction in ubiquitinated C subunit . For optimal PP2A-C expression:

• Co-transfect with α4 expression plasmid

• Consider triple transfection (A, C, and α4) for maximizing stable holoenzyme formation

• Monitor ubiquitination status as a quality control measure

How can researchers distinguish between different PP2A phosphatase activities in experimental assays?

PP2A complexes can possess different substrate specificities, making activity assessment complex. Researchers should consider multiple approaches:

• Use diverse substrates in phosphatase activity assays:

  • Phospho-peptides with different sequences

  • p-Nitrophenyl Phosphate (pNPP)

  • Specific phosphorylated protein substrates relevant to the pathway under investigation

• Normalize activity for the level of precipitated catalytic C subunit when comparing different PP2A complexes

• Evaluate temperature-dependence of complex formation and activity

• Consider ATP and Mg²⁺ requirements, as some PP2A activities (particularly those involving PPP2R4) are dependent on ATP and Mg²⁺

What considerations are important when selecting PP2A4 antibodies for specific research applications?

When selecting PP2A4 antibodies, researchers should consider:

• Antibody Specificity: Some antibodies target PP2A alpha + beta, while others are specific to particular subunits

• Species Reactivity: Verify cross-reactivity with your experimental model (human, mouse, rat, etc.)

• Validated Applications: Ensure the antibody has been validated for your specific application (WB, IHC, Flow Cytometry)

• Epitope Information: Consider whether the antibody recognizes specific domains or phosphorylation states

• Clone Type: Monoclonal antibodies (like clone 4D9) provide high specificity for particular epitopes, while polyclonal antibodies may offer higher sensitivity but potentially lower specificity

What are the optimal protocols for measuring PP2A activity in cellular extracts?

For accurate measurement of PP2A activity in cellular extracts:

• Sample Preparation:

  • Prepare extracts in phosphatase-friendly buffer (low or no phosphate)

  • Include protease inhibitors but avoid phosphatase inhibitors

  • Consider temperature effects on complex stability

• Activity Assay Options:

  • Use multiple substrates to assess different aspects of PP2A activity

  • For studying α4-associated PP2A-C activity, immunoprecipitate with α4 antibodies

  • For studying holoenzyme activity, immunoprecipitate with A subunit antibodies

• Controls and Normalization:

  • Always normalize for the amount of catalytic C subunit present

  • Include positive controls (purified PP2A) and negative controls (heat-inactivated samples)

  • Consider using specific PP2A inhibitors (e.g., okadaic acid) to confirm specificity

How can researchers effectively track changes in PP2A complex formation during stress responses?

To monitor PP2A complex dynamics during stress responses:

• Time-course Experiments:

  • Track PP2A activity at multiple timepoints following stress induction

  • Monitor parallel changes in A, C subunit levels and their association

  • Assess α4-C interactions simultaneously

• Subcellular Fractionation:

  • Separate nuclear and cytoplasmic fractions to assess compartment-specific changes

  • Monitor PP2A subunit redistribution following stress

• Proximity Ligation Assays:

  • Consider using in situ proximity ligation to visualize changes in protein-protein interactions within intact cells

• Correlation with Functional Outcomes:

  • Link observed changes in PP2A complex formation with functional outcomes such as dephosphorylation of target proteins (e.g., H2AX, p53)

How does PP2A4 function in DNA damage response pathways?

PP2A4 and its associated proteins play critical roles in DNA damage response:

• In cells with functional α4, induced levels of H2AX, ATM, and p53 phosphorylation are efficiently reversed following treatment with genotoxic agents

• α4-deficient cells show:

  • Persistent H2AX phosphorylation after camptothecin treatment

  • Greater extent and duration of ATM, H2AX, and p53 phosphorylation after doxorubicin treatment

  • Significant decrease in total PP2A activity (>70% decline)

• α4 overexpression enhances cell survival after DNA damage by:

  • More rapidly reversing induced phosphorylation of H2AX and p53

  • Displaying reduced induction of p21

  • Providing significantly greater increases in PP2A activity during recovery

These findings suggest PP2A4 could be a potential therapeutic target for enhancing DNA damage repair or sensitizing cancer cells to genotoxic therapies.

What emerging techniques are advancing our understanding of PP2A4 biology?

Several emerging approaches are enhancing our understanding of PP2A4 biology:

• Structural Biology:

  • Cryo-EM studies of PP2A complexes with different regulatory subunits

  • Structural analysis of the PP2A-PPP2R4 interaction interfaces

• Proximity-based Proteomics:

  • BioID or APEX2-based mapping of PP2A4 interaction networks

  • Defining dynamic changes in PP2A4 interactome during stress responses

• Single-cell Analysis:

  • Examining cell-to-cell variability in PP2A4 expression and activity

  • Correlating PP2A4 levels with cell fate decisions following stress

• CRISPR-based Approaches:

  • Creation of separation-of-function mutations to dissect specific aspects of PP2A4 function

  • Genome-wide screens for synthetic lethal interactions with PP2A4 manipulation

What quality control measures should researchers implement when working with PP2A4 antibodies?

To ensure robust and reproducible results with PP2A4 antibodies:

• Validation Across Applications:

  • Verify antibody performance in your specific experimental system

  • Include appropriate positive and negative controls

  • Consider using multiple antibodies targeting different epitopes

• Experimental Conditions:

  • Be mindful of temperature effects on PP2A complex stability

  • Consider buffer composition effects on antibody performance

  • Document lot-to-lot variations in antibody performance

• Data Interpretation:

  • Correlate antibody-based detection with functional assays of PP2A activity

  • Consider the dynamic nature of PP2A complexes when interpreting results

  • Account for potential post-translational modifications affecting epitope accessibility