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Graphical Abstract
Highlights
Quantitative phosphoproteomics of cells treated with sphingolipid analogs or PP2A inhibitor identify novel protein targets of PP2A.
PP2A substrates include several nutrient transporter proteins, GTPase regulators and proteins associated with actin cytoskeletal remodeling.
Differential regulation of Akt and Gsk3b account for the difference in vacuolating phenotype observed between SH-BC-893 and C2-ceramide.
Dynamic phosphoproteomics enabled the correlation of cell signaling with phenotypes to rationalize their mode of action.
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.
Footnotes
Author contributions: P.T., A.L.E., and S.H. designed the research; P.K., B.F., F.P., A.N.M., and M.P. performed the research, conducted the experiments. P.K. and B.F. analyzed the data; and P.K., B.F., A.L.E., and P.T. wrote the paper. All authors approved the content and submission of the paper.
↵* This work was funded in part by the Natural Sciences and Engineering Research Council (NSERC) (P.T. 311598; S.H. 04726), grants to A.L.E. from the NIH (R01 GM089919, R21 CA178230), CDMRP (W81XWH-15-1-0010), the American Cancer Society (RSG-11-111-01-CDD), and the UCI CORCL. The Institute for Research in Immunology and Cancer (IRIC) receives infrastructure support from the Canadian Center of Excellence in Commercialization and Research, the Canadian Foundation for Innovation, and the Fonds de recherche du Québec - Santé (FRQS). Proteomics analyses were performed at the Center for Advanced Proteomic and Chemogenomics Analyses (CAPCA), a Node of the Genomic Technology Platform supported by the Canadian Government through Genome Canada. Imaging was performed in the Optical Biology Core at UCI which is supported in part by NIH P30 CA062203.
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This article contains supplemental material. We declare no competing financial interests.
- Received August 23, 2018.
- Revision received October 23, 2018.
- © 2019 Kubiniok et al.
Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.