Phospho-PPP1R2 (Ser120/Ser121) Antibody

Basic Research Questions

• What is PPP1R2 and what role does the Ser120/Ser121 phosphorylation site play?

PPP1R2 (Protein Phosphatase 1 Regulatory Inhibitor Subunit 2), also known as Inhibitor-2 (I2) or IPP-2, is a regulatory subunit that binds to the catalytic subunit of Protein Phosphatase 1 (PP1), strongly inhibiting its activity . PPP1R2 is a 23 kDa protein (calculated molecular weight) that forms part of the protein phosphatase inhibitor family .

The Ser120/Ser121 phosphorylation sites are located in the C-terminal region of PPP1R2. While the exact function of phosphorylation at these specific sites is not fully characterized in the literature, phosphorylation states of PPP1R2 are critical for regulating PPP1R2-PP1 interactions . Research indicates that PPP1R2 phosphorylation directs PP1 recruitment to specific subcellular locations, particularly during cell division .

• How should I validate the specificity of a Phospho-PPP1R2 (Ser120/Ser121) Antibody?

Validating the specificity of Phospho-PPP1R2 (Ser120/Ser121) Antibody requires multiple complementary approaches:

• Peptide competition assay: Pre-incubate the antibody with the phosphopeptide used as immunogen (sequence around phosphorylation site of Ser120/Ser121) . Loss of signal confirms specificity for the phospho-epitope.

• Phosphatase treatment: Treat half of your sample with lambda phosphatase before immunoblotting. The phospho-specific signal should disappear in the treated sample .

• Phosphorylation mutants: Compare antibody reactivity between wild-type PPP1R2 and Ser120/121Ala (phospho-null) or Ser120/121Glu (phospho-mimetic) mutants. The antibody should not recognize the phospho-null mutant .

• Kinase activation/inhibition: Stimulate or inhibit kinases known to target these sites and verify corresponding changes in antibody reactivity.

• Cross-reactivity testing: Test against related phosphoproteins, especially other PPP1R family members, to ensure specificity.

• What are the recommended applications and protocols for Phospho-PPP1R2 (Ser120/Ser121) Antibody?

Based on the validated applications from multiple sources, the recommended applications include:

Most suppliers provide the antibody in liquid form, in PBS with 50% glycerol, 0.5% BSA and 0.02% sodium azide, with a recommended storage temperature of -20°C .

Intermediate Research Questions

• How does PPP1R2 phosphorylation state influence its regulatory effect on PP1 activity?

PPP1R2 has traditionally been characterized as an inhibitor of PP1, but recent research reveals a more complex relationship where phosphorylation status plays a crucial regulatory role:

• Unphosphorylated state: Forms a stable complex with PP1 catalytic subunit (PPP1C), blocking the active site and potently inhibiting its activity .

• Phosphorylated state: Depending on the specific phosphorylation site, can either enhance inhibition or trigger reactivation of PP1. Notably, phosphorylation at some sites directs PP1 recruitment to specific subcellular locations .

• Paradoxical effects: Recent research shows that PPP1R2 overexpression increases PP1 activity rather than inhibiting it, an effect dependent on PPP1R2's C-terminus .

The functional consequences of phosphorylation at Ser120/Ser121 specifically are still being elucidated, but research suggests PPP1R2 phosphorylation helps target PP1 to the midbody during cell division, with the phosphomimetic mutant increasing PP1 localization at this structure .

• What are the methodological approaches for studying PPP1R2-PP1 interactions in living cells?

Several methodologies can be employed to study PPP1R2-PP1 interactions in living cells:

• Fluorescence microscopy techniques:

  • FRET (Förster Resonance Energy Transfer) to monitor real-time interactions

  • Fluorescence recovery after photobleaching (FRAP) to assess binding dynamics

  • Bimolecular Fluorescence Complementation (BiFC) for visualizing protein interactions

• Proximity-based labeling:

  • BioID or TurboID to identify proteins in close proximity to PPP1R2

  • APEX2-based proximity labeling for temporal control

• Pharmacological manipulation:

  • Calyculin A treatment for PP1 inhibition, followed by staurosporin (kinase inhibitor) for kinase shutoff assays

  • Specific kinase inhibitors to determine which kinases regulate PPP1R2 phosphorylation

• Genetic approaches:

  • Expression of phospho-mutants (phospho-null, phospho-mimetic)

  • CRISPR/Cas9 gene editing to create endogenous mutations at Ser120/Ser121

  • PPP1R2 depletion studies showing hyperphosphorylation of PP1 substrates

In one study applying these approaches, researchers performed a kinase shutoff assay where they first hyperphosphorylated PP1 with calyculin A, then monitored dephosphorylation after adding staurosporin. In PPP1R2-depleted cells, PP1 dephosphorylation occurred much more slowly, indicating decreased PP1 activity .

• How do phosphorylation patterns of PPP1R2 change during cell cycle progression?

PPP1R2 phosphorylation undergoes dynamic changes during cell cycle progression, with important functional consequences:

• G2/M transition:

  • Increased phosphorylation of PP1α at Thr320 was observed in G2/M-arrested (RO3306-treated) PPP1R2-depleted cells

  • This hyperphosphorylation was maintained for 2 hours after RO3306-washout

• Mitosis:

  • PPP1R2 facilitates PP1:RepoMan complex formation, which is crucial for chromosome segregation

  • PPP1R2 depletion results in increased phosphorylation of histone H3 at Thr3 (H3T3), a substrate of PP1:RepoMan

• Cytokinesis:

  • Both PPP1R2 and PP1 localize to the midbody

  • PP1's localization there depends on both N- and C-terminal domains of PPP1R2

  • Full-length PPP1R2 enhances PP1 localization at the midbody

  • The phosphomimetic mutant of PPP1R2 increases PP1 localization, supporting a role for PPP1R2 phosphorylation in targeting PP1 during cytokinesis

This suggests that proper regulation of PPP1R2 phosphorylation is essential for accurate progression through mitosis and cell division.

Advanced Research Questions

• How does PPP1R2 modify PP1 holoenzyme assembly and substrate selectivity?

Recent research has transformed our understanding of how PPP1R2 affects PP1 function beyond simple inhibition. PPP1R2 modulates PP1 through several sophisticated mechanisms:

• Holoenzyme stabilization: PPP1R2 stabilizes a subgroup of PP1 holoenzymes, exemplified by PP1:RepoMan, thereby promoting the dephosphorylation of their substrates .

• Structural modifications: Mechanistically, PPP1R2 recruitment disrupts an inhibitory, fuzzy interaction between the C-terminal tail and catalytic domain of PP1, generating an additional C-terminal RepoMan-interaction site .

• Cooperative interactions: The resulting holoenzyme is further stabilized by a direct PPP1R2:RepoMan interaction, which renders it refractory to competitive disruption by RIPPOs (Regulatory-Interactors-of-Protein-Phosphatase-One) that do not interact with PPP1R2 .

• Substrate selectivity: Global phosphoproteome analysis of PPP1R2-depleted HCT116 cells identified 2,450 hyperphosphorylated and 925 hypophosphorylated tryptic peptides, indicating that PPP1R2 depletion mainly results in protein hyperphosphorylation .

These findings indicate that rather than being a simple inhibitor, PPP1R2 functions as a modulator that alters the balance between different PP1 holoenzymes by stabilizing specific subunit interactions.

• What are the experimental challenges in studying Phospho-PPP1R2 (Ser120/Ser121) and how can they be addressed?

Researchers face several challenges when studying Phospho-PPP1R2 (Ser120/Ser121):

• Pseudogene interference: Ten related PPP1R2 pseudogenes exist throughout the human genome (e.g., PPP1R2P1, PPP1R2P3) . This creates specificity challenges for both antibody-based detection and genetic manipulation.

  Solution: Use highly specific primers designed to distinguish between PPP1R2 and its pseudogenes; validate antibody specificity against recombinant pseudogene products.

• Dynamic phosphorylation: PPP1R2 has multiple phosphorylation sites (at least 14 documented sites including S4, S7, T19, S20, S23, S24, S28, S44, T55, Y56, Y63, S72, T73, Y75, S77, S87) with rapid turnover.

  Solution: Use phosphatase inhibitors in all buffers; employ phospho-specific antibodies for different sites; utilize mass spectrometry-based phosphoproteomics for comprehensive analysis.

• Context-dependent function: PPP1R2 functions differently depending on cell type, cell cycle stage, and interaction partners.

  Solution: Study PPP1R2 in synchronized cell populations; use cell-type specific conditional knockouts; perform interaction studies in relevant physiological contexts.

• Antibody cross-reactivity: Given the high sequence conservation and multiple phosphorylation sites, ensuring specific detection of Ser120/Ser121 phosphorylation is challenging.

  Solution: Validate antibodies using phospho-null mutants; perform peptide competition assays; use orthogonal detection methods like mass spectrometry.

• How does PPP1R2 contribute to the regulation of cytokinesis and what role does Ser120/Ser121 phosphorylation play?

PPP1R2 has emerged as a critical regulator of cytokinesis through several mechanisms:

• PP1 targeting to the midbody:

  • PPP1R2 and PP1 both localize to the midbody during cytokinesis

  • PP1's localization there depends on both N- and C-terminal domains of PPP1R2

  • Full-length PPP1R2 enhances PP1 localization at the midbody

• Phosphorylation-dependent regulation:

  • The phosphomimetic mutant of PPP1R2 increased PP1 localization at the midbody

  • This supports a role for PPP1R2 phosphorylation in targeting PP1 during cytokinesis

• Midbody structure regulation:

  • Overexpression of PPP1R2 phosphomimetic and truncation mutants resulted in misshaped and elongated midbody structures

  • This suggests PPP1R2 has a role in regulating kinase and phosphatase activity to control central spindle architecture

• Consequences of dysregulation:

  • Increased centrosome numbers observed after PPP1R2 overexpression result from incomplete cytokinesis

  • This appears to be caused by increased phosphatase activity at the midbody

• What is the relationship between PPP1R2, PP1, and Aurora kinases in cell cycle regulation?

Research has revealed an intricate regulatory network involving PPP1R2, PP1, and Aurora kinases during cell cycle regulation:

• PPP1R2-AURKA-PP1 interaction:

  • PPP1R2 interacts with both Aurora Kinase A (AURKA) and PP1

  • This interaction regulates phosphorylation and activity of both AURKA and PP1 at the centrosome

• Paradoxical activation of PP1:

  • Despite its traditional characterization as an inhibitor, PPP1R2 overexpression stimulates PP1 activity

  • Simultaneously, PPP1R2 overexpression reduces AURKA activity to undetectable levels

  • These effects were independent of protein amount or phosphorylation state

• Domain-specific regulation:

  • The C-terminus of PPP1R2 (which contains Ser120/Ser121) is critical for this regulatory function

  • Overexpression of PPP1R2 truncated at its C-terminal domain did not change the activity of either PP1 or AURKA

  • Only PPP1R2 containing the C-terminus caused a decrease in phosphorylated PP1 and phosphorylated AURKA levels at the centrosome

• Phosphorylation changes:

  • Both AURKA and PP1 were less phosphorylated at the centrosome when PPP1R2 was overexpressed

  • This corresponds to increased PP1 activity and reduced AURKA activity