1. Academic Validation
  2. Dynamic Phosphoproteomics Uncovers Signaling Pathways Modulated by Anti-oncogenic Sphingolipid Analogs

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

  • Mol Cell Proteomics. 2019 Mar;18(3):408-422. doi: 10.1074/mcp.RA118.001053.
Peter Kubiniok 1 2 Brendan T Finicle 3 Fanny Piffaretti 1 Alison N McCracken 3 Michael Perryman 2 Stephen Hanessian 2 Aimee L Edinger 4 Pierre Thibault 5 2 6
Affiliations

Affiliations

  • 1 From the ‡Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, H3C 3J7, Canada.
  • 2 §Department of Chemistry, Université de Montréal, Quebec, H3C 3J7, Canada.
  • 3 ¶Department of Developmental and Cell Biology, University of California Irvine, Irvine CA 92697.
  • 4 ¶Department of Developmental and Cell Biology, University of California Irvine, Irvine CA 92697; pierre.thibault@umontreal.ca.
  • 5 From the ‡Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, H3C 3J7, Canada; aedinger@uci.edu.
  • 6 ‖Department of Biochemistry, 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.

Keywords

Cancer Biology*; Cell biology*; Phosphoproteome; Phosphorylation; Quantification.

Figures
Products