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Molecular & Cellular Proteomics

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Research

Dynamic Phosphoproteomics Uncovers Signaling Pathways Modulated by Anti-oncogenic Sphingolipid Analogs

Peter Kubiniok, Brendan T. Finicle, Fanny Piffaretti, Alison N. McCracken, Michael Perryman, Stephen Hanessian, Aimee L. Edinger  Correspondence email and Pierre Thibault  Correspondence email
Molecular & Cellular Proteomics March 1, 2019, First published on November 27, 2018, 18 (3) 408-422; https://doi.org/10.1074/mcp.RA118.001053
Peter Kubiniok
From the ‡Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, H3C 3J7, Canada; §Department of Chemistry, Université de Montréal, Quebec, H3C 3J7, Canada;
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Brendan T. Finicle
¶Department of Developmental and Cell Biology, University of California Irvine, Irvine CA 92697;
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Fanny Piffaretti
From the ‡Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, H3C 3J7, Canada;
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Alison N. McCracken
¶Department of Developmental and Cell Biology, University of California Irvine, Irvine CA 92697;
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Michael Perryman
§Department of Chemistry, Université de Montréal, Quebec, H3C 3J7, Canada;
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Stephen Hanessian
§Department of Chemistry, Université de Montréal, Quebec, H3C 3J7, Canada;
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Aimee L. Edinger
¶Department of Developmental and Cell Biology, University of California Irvine, Irvine CA 92697;
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  • For correspondence: pierre.thibault@umontreal.ca
Pierre Thibault
From the ‡Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, H3C 3J7, Canada; §Department of Chemistry, Université de Montréal, Quebec, H3C 3J7, Canada; ‖Department of Biochemistry, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, H3C 3J7, Canada
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  • For correspondence: aedinger@uci.edu
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Graphical Abstract

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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.

  • Phosphoproteome
  • Phosphorylation
  • Quantification
  • Cancer Biology*
  • Cell biology*

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.

  • ↵Embedded Image 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.

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Dynamic Phosphoproteomics Uncovers Signaling Pathways Modulated by Anti-oncogenic Sphingolipid Analogs
Peter Kubiniok, Brendan T. Finicle, Fanny Piffaretti, Alison N. McCracken, Michael Perryman, Stephen Hanessian, Aimee L. Edinger, Pierre Thibault
Molecular & Cellular Proteomics March 1, 2019, First published on November 27, 2018, 18 (3) 408-422; DOI: 10.1074/mcp.RA118.001053

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Dynamic Phosphoproteomics Uncovers Signaling Pathways Modulated by Anti-oncogenic Sphingolipid Analogs
Peter Kubiniok, Brendan T. Finicle, Fanny Piffaretti, Alison N. McCracken, Michael Perryman, Stephen Hanessian, Aimee L. Edinger, Pierre Thibault
Molecular & Cellular Proteomics March 1, 2019, First published on November 27, 2018, 18 (3) 408-422; DOI: 10.1074/mcp.RA118.001053
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Molecular & Cellular Proteomics: 18 (3)
Molecular & Cellular Proteomics
Vol. 18, Issue 3
1 Mar 2019
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