Related Experiment Video
Updated: Feb 2, 2026
Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis
Published on: June 25, 2020
Dynamic Phosphoproteomics Uncovers Signaling Pathways Modulated by Anti-oncogenic Sphingolipid Analogs
Peter Kubiniok1,2, Brendan T Finicle3, Fanny Piffaretti1
1From the ‡Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, H3C 3J7, Canada.
Abstract:
The anti-neoplastic sphingolipid analog SH-BC-893 starves cancer cells to death by down-regulating cell surface nutrient transporters and blocking lysosomal trafficking events. These effects are mediated by the activation of protein phosphatase 2A (PP2A). To identify putative PP2A substrates, we used quantitative phosphoproteomics to profile the temporal changes in protein phosphorylation in FL5.12 cells following incubation with SH-BC-893 or the specific PP2A inhibitor LB-100. These analyses enabled the profiling of more than 15,000 phosphorylation sites, of which 958 sites on 644 proteins were dynamically regulated. We identified 114 putative PP2A substrates including several nutrient transporter proteins, GTPase regulators (e.g. Agap2, Git1), and proteins associated with actin cytoskeletal remodeling (e.g. Vim, Pxn). To identify SH-BC-893-induced cell signaling events that disrupt lysosomal trafficking, we compared phosphorylation profiles in cells treated with SH-BC-893 or C2-ceramide, a non-vacuolating sphingolipid that does not impair lysosomal fusion. These analyses combined with functional assays uncovered the differential regulation of Akt and Gsk3b by SH-BC-893 (vacuolating) and C2-ceramide (non-vacuolating). Dynamic phosphoproteomics of cells treated with compounds affecting PP2A activity thus enabled the correlation of cell signaling with phenotypes to rationalize their mode of action.
Insights
The sphingolipid analog SH-BC-893 kills cancer cells by activating protein phosphatase 2A (PP2A), down-regulating nutrient transporters, and blocking lysosomal trafficking. This study identifies key PP2A substrates and signaling pathways involved in SH-BC-893
Area of Science:
- Oncology
- Cell Biology
- Biochemistry
Background:
- Sphingolipid analogs are investigated for anti-neoplastic properties.
- SH-BC-893, a sphingolipid analog, exhibits anti-cancer effects by targeting nutrient transporters and lysosomal trafficking.
- Protein phosphatase 2A (PP2A) activation is implicated in SH-BC-893's mechanism of action.
Purpose of the Study:
- To identify protein phosphatase 2A (PP2A) substrates regulated by SH-BC-893.
- To elucidate the cell signaling events underlying SH-BC-893-induced disruption of lysosomal trafficking.
- To correlate observed signaling changes with cellular phenotypes to understand SH-BC-893's mode of action.
Main Methods:
- Quantitative phosphoproteomics was employed to profile dynamic phosphorylation changes in cells treated with SH-BC-893 or a PP2A inhibitor (LB-100).
- Over 15,000 phosphorylation sites were analyzed, identifying 958 dynamically regulated sites on 644 proteins.
- Comparative phosphoproteomic analysis with C2-ceramide and functional assays were used to investigate lysosomal trafficking disruptions.
Main Results:
- 114 putative PP2A substrates were identified, including nutrient transporters, GTPase regulators (Agap2, Git1), and actin remodeling proteins (Vim, Pxn).
- SH-BC-893 and C2-ceramide differentially regulated Akt and Gsk3b, impacting lysosomal trafficking.
- Dynamic phosphoproteomics successfully correlated cell signaling events with specific cellular phenotypes.
Conclusions:
- SH-BC-893 exerts anti-neoplastic effects through PP2A activation, impacting nutrient transport and lysosomal function.
- The study identified novel PP2A substrates and signaling pathways critical to SH-BC-893's mechanism.
- Phosphoproteomics provides a powerful approach to link molecular signaling to cellular responses in drug discovery.
Related Concept Videos
03:14Notch Signaling Pathway
The Notch gene came into the limelight in 1914 after the discovery that its mutation in Drosophila melanogaster leads to a serrated (or "notched") wing margin phenotype. It was not...
02:33Hedgehog Signaling Pathway
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...
01:41Non-Canonical Wnt Signaling Pathways
02:54Canonical Wnt Signaling Pathway
02:26NF-κB-dependent Signaling Pathway
NF-κB-dependent Signaling Mechanism
The...