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Updated: Feb 22, 2026
A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors
Published on: April 29, 2022
Phosphoproteomics Profiling of Nonsmall Cell Lung Cancer Cells Treated with a Novel Phosphatase Activator
Danica D Wiredja1, Marzieh Ayati2, Sahar Mazhar3
1Center for Proteomics and Bioinformatics, Department of Nutrition, Case Western Reserve University, Cleveland, OH, USA.
Abstract:
Activation of protein phosphatase 2A (PP2A) is a promising anticancer therapeutic strategy, as this tumor suppressor has the ability to coordinately downregulate multiple pathways involved in the regulation of cellular growth and proliferation. In order to understand the systems-level perturbations mediated by PP2A activation, we carried out mass spectrometry-based phosphoproteomic analysis of two KRAS mutated non-small cell lung cancer (NSCLC) cell lines (A549 and H358) treated with a novel small molecule activator of PP2A (SMAP). Overall, this permitted quantification of differential signaling across over 1600 phosphoproteins and 3000 phosphosites. Kinase activity assessment and pathway enrichment implicate collective downregulation of RAS and cell cycle kinases in the case of both cell lines upon PP2A activation. However, the effects on RAS-related signaling are attenuated for A549 compared to H358, while the effects on cell cycle-related kinases are noticeably more prominent in A549. Network-based analyses and validation experiments confirm these detailed differences in signaling. These studies reveal the power of phosphoproteomics studies, coupled to computational systems biology, to elucidate global patterns of phosphatase activation and understand the variations in response to PP2A activation across genetically similar NSCLC cell lines.
Insights
Activating protein phosphatase 2A (PP2A) shows anticancer potential by downregulating growth pathways. This study used phosphoproteomics to reveal distinct signaling changes in KRAS-mutated lung cancer cells, highlighting differences in response to PP2A activation.
Area of Science:
- Oncology
- Biochemistry
- Systems Biology
Background:
- Protein phosphatase 2A (PP2A) acts as a tumor suppressor, regulating cellular growth and proliferation.
- PP2A activation is a potential anticancer strategy targeting multiple signaling pathways.
- KRAS-mutated non-small cell lung cancer (NSCLC) presents a significant therapeutic challenge.
Purpose of the Study:
- To investigate the systems-level effects of PP2A activation in KRAS-mutated NSCLC.
- To compare the differential signaling perturbations induced by a novel PP2A activator (SMAP) in two distinct NSCLC cell lines (A549 and H358).
- To elucidate the variations in cellular response to PP2A activation based on genetic similarities.
Main Methods:
- Mass spectrometry-based phosphoproteomic analysis was employed to quantify differential signaling.
- Over 1600 phosphoproteins and 3000 phosphosites were analyzed.
- Kinase activity assessment, pathway enrichment, network-based analyses, and validation experiments were performed.
Main Results:
- PP2A activation led to the collective downregulation of RAS and cell cycle kinases in both A549 and H358 cell lines.
- RAS-related signaling downregulation was attenuated in A549 cells compared to H358 cells.
- Cell cycle-related kinase downregulation was more prominent in A549 cells than in H358 cells.
Conclusions:
- Phosphoproteomics coupled with computational systems biology effectively elucidates global phosphatase activation patterns.
- Significant, yet distinct, variations in signaling responses to PP2A activation exist even in genetically similar NSCLC cell lines.
- Understanding these nuanced differences is crucial for developing targeted PP2A-based anticancer therapies.
