Phosphoproteome and drug-response effects mediated by the three protein phosphatase 2A inhibitor proteins CIP2A, SET, and PME-1
Related Experiment Video
Updated: Dec 28, 2025
A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors
Published on: April 29, 2022
Otto Kauko1, Susumu Y Imanishi2, Evgeny Kulesskiy3
1Turku Bioscience Centre, University of Turku and Åbo Akademi University, 20500 Turku, Finland; Institute of Biomedicine, University of Turku, 20500 Turku, Finland; TuBS and TuDMM Doctoral Programmes, University of Turku, 20500 Turku, Finland.
Abstract:
Protein phosphatase 2A (PP2A) critically regulates cell signaling and is a human tumor suppressor. PP2A complexes are modulated by proteins such as cancerous inhibitor of protein phosphatase 2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and SET nuclear proto-oncogene (SET) that often are deregulated in cancers. However, how they impact cellular phosphorylation and how redundant they are in cellular regulation is poorly understood. Here, we conducted a systematic phosphoproteomics screen for phosphotargets modulated by siRNA-mediated depletion of CIP2A, PME-1, and SET (to reactivate PP2A) or the scaffolding A-subunit of PP2A (PPP2R1A) (to inhibit PP2A) in HeLa cells. We identified PP2A-modulated targets in diverse cellular pathways, including kinase signaling, cytoskeleton, RNA splicing, DNA repair, and nuclear lamina. The results indicate nonredundancy among CIP2A, PME-1, and SET in phosphotarget regulation. Notably, PP2A inhibition or reactivation affected largely distinct phosphopeptides, introducing a concept of nonoverlapping phosphatase inhibition- and activation-responsive sites (PIRS and PARS, respectively). This phenomenon is explained by the PPP2R1A inhibition impacting primarily dephosphorylated threonines, whereas PP2A reactivation results in dephosphorylation of clustered and acidophilic sites. Using comprehensive drug-sensitivity screening in PP2A-modulated cells to evaluate the functional impact of PP2A across diverse cellular pathways targeted by these drugs, we found that consistent with global phosphoproteome effects, PP2A modulations broadly affect responses to more than 200 drugs inhibiting a broad spectrum of cancer-relevant targets. These findings advance our understanding of the phosphoproteins, pharmacological responses, and cellular processes regulated by PP2A modulation and may enable the development of combination therapies.
Insights
Protein phosphatase 2A (PP2A) regulates cell signaling and suppresses tumors. This study reveals PP2A regulators CIP2A, PME-1, and SET are nonredundant, impacting distinct phosphosites and drug responses, offering new therapeutic avenues.
Area of Science:
- Cellular Biology
- Molecular Oncology
- Biochemistry
Background:
- Protein phosphatase 2A (PP2A) is a crucial regulator of cell signaling and a known human tumor suppressor.
- Proteins like cancerous inhibitor of protein phosphatase 2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and SET nuclear proto-oncogene (SET) modulate PP2A activity and are frequently deregulated in cancers.
- The precise impact of these modulators on cellular phosphorylation and their degree of functional redundancy remain poorly understood.
Purpose of the Study:
- To systematically identify phosphotargets modulated by PP2A regulators (CIP2A, PME-1, SET) and the PP2A scaffolding subunit (PPP2R1A).
- To investigate the nonredundancy of CIP2A, PME-1, and SET in regulating cellular phosphorylation.
- To explore the functional consequences of PP2A modulation on cellular pathways and drug sensitivity.
Main Methods:
- A systematic phosphoproteomics screen was performed in HeLa cells using siRNA-mediated depletion of CIP2A, PME-1, and SET to reactivate PP2A, or by inhibiting PP2A via PPP2R1A.
- Identification of PP2A-modulated phosphotargets across diverse cellular pathways.
- Comprehensive drug-sensitivity screening in PP2A-modulated cells.
Main Results:
- Identified PP2A-modulated targets involved in kinase signaling, cytoskeleton dynamics, RNA splicing, DNA repair, and nuclear lamina organization.
- Demonstrated nonredundancy among CIP2A, PME-1, and SET in phosphotarget regulation.
- Revealed distinct phosphopeptide modulation upon PP2A inhibition versus reactivation, introducing concepts of phosphatase inhibition-responsive sites (PIRS) and activation-responsive sites (PARS).
- Showed that PP2A modulation broadly affects cellular responses to over 200 drugs targeting cancer-relevant pathways.
Conclusions:
- CIP2A, PME-1, and SET exhibit nonredundant roles in regulating cellular phosphoproteome.
- PP2A inhibition and reactivation target distinct phosphorylation sites, suggesting specific regulatory mechanisms.
- PP2A modulation significantly impacts drug sensitivity, highlighting its potential as a therapeutic target and informing combination therapy strategies.
Related Concept Videos
01:42Phosphoinositides and PIPs
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane...
02:54Protein Kinases and Phosphatases
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
01:27Transducer Mechanism: Enzyme-Linked Receptors
Major types that are helpful drug targets include:
01:19Interactions Between Signaling Pathways
Convergence and divergence, and cross-talk between signaling pathways
Two distinct signaling pathways can converge on a single functional unit, which may either be a single protein or a complex of proteins. The response is either functionally distinct or synergistic between the two pathways but different from the response...
01:22Amplifying Signals via Enzymatic Cascade
01:22PI3K/mTOR/AKT Signaling Pathway