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

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.

Graphical abstract

Fig. 1.

SH-BC-893 and C2-ceramide cause PP2A-dependent disruptions in endolysosomal trafficking. A, Structures of the PP2A activators FTY720 (Fingolimod), C2-ceramide, SH-BC-893 and the PP2A inhibitor LB-100. B, Surface CD98 levels in FL5.12 cells pre-treated with LB-100 (100 μm) for 1.5 h before addition of DMSO, SH-BC-893 (5 μm), or C2-ceramide (50 μm) for 1 h. Using Student's t test (n.s.) not significant; *, p ≤ 0.05; **, p ≤ 0.01; and ***, p ≤ 0.001 C, DIC microscopy of FL5.12 cells pre-treated with LB-100 (100 μm) for 1.5 h before addition of DMSO, vehicle, SH-BC-893 (5 μm), or C2-ceramide (50 μm) for 2 h.

Fig. 2.

Triple SILAC phosphoproteomics workflow. Light cells are treated with vehicle, medium cells were treated with C2-ceramide (50 μm) or LB-100 (10 μm), and heavy cells were treated with SH-BC-893 (5 μm). Cells were collected every 5 min from 0–60 min of stimulation by snap freezing in pre-cooled ethanol. Cells were lysed in urea, digested with trypsin and phosphopeptides were enriched using titanium dioxide. Strong cation fractionation (SCX) was performed before LC-MS/MS analysis. Phosphopeptide identification and quantitation was performed using MaxQuant, and only kinetic profiles of sites with localization confidence > 0.75 and measured in at least 6 time points were selected for further analysis. Polynomial fitting was used to define regulated phosphosites, and only phosphopeptide profiles with FDR < 1% were selected and used for fuzzy c-means clustering and subsequent analyses.

Fig. 3.

Phosphoproteomics results from the comparison of treatments with LB-100 (10 μm) and SH-BC-893 (5 μm). A, Inter quartile range (IQR) shows the global change in phosphorylation on treatment. Unmodified peptides (control) do not change over time whereas LB-100 and SH-BC-893 treatments show a progressive increase in IQR with time. B, Heatmap of all 390 phosphosites regulated by SH-BC-893. Different kinetic behaviors are indicated. C, Kinetic trends extracted from LB-100 and SH-BC-893 treatments using fuzzy means clustering; numbers correspond to profiles observed with a particular kinetic trend.

Fig. 4.

Comparison of dynamic phosphorylation sites following SH-BC-893 and LB-100 treatments. A, Venn diagram showing that 292 phosphosites are common to both treatments whereas 253 and 98 phosphosites are uniquely regulated by LB-100 and SH-BC-893, respectively. Of the 292 common sites, 116 sites showed opposite (Bidirectional) tends and 176 sites are regulated in the same direction (Monodirectional). B, Phosphorylation motifs extracted from motif-X. Monodirectional phosphosites display PxSP and SP motifs. Bidirectional sites that show rapid changes in phosphorylation are present on Serine-rich peptides. C, Gene Ontology enrichment of proteins corresponding to the 214 bidirectional and unidirectional (for SH-BC-893) phosphosites.

Fig. 5.

Identification of putative PP2A targets. A, Comparative analysis of the list of candidate PP2A substrates identified from various proteomic studies. The comparison was done at the protein level because not all data sets reported the position of the phosphorylated sites. B, A network of phosphorylation sites identified as direct PP2A targets based on comparative literature review. Color coding indicates if the proteins are involved in actin reorganization. C, Dynamic profiles of sites from putative PP2A targets involved in actin reorganization.

Fig. 6.

Dynamic phosphoproteomics identifies signaling events differently regulated by C2-ceramide and SH-BC-893 treatments. A, Heatmap corresponding to 380 phosphorylation sites that were dynamically phosphorylated on C2-ceramide treatment. Kinetic trends are indicated. B, Dynamic profiles extracted from C2-ceramide and SH-BC-893 treatments using fuzzy means clustering. Numbers correspond to profiles observed for a given cluster. C, Venn diagram of dynamic phosphosites showing that 254 phosphosites are common, 126 and 48 phosphosites are uniquely regulated by C2-ceramide and SH-BC-893, respectively. Of the 254 commonly regulated sites, 51 show opposite change in phosphorylation (bidirectional) and 203 are regulated in the same direction (monodirectional). D, Radar plot showing the enrichment of GO terms associated with specific groups highlighted in different colors. E, Motif-X analyses of bidirectional sites showing an over-representation of basophilic motifs. F, Network of putative Akt substrates and known interacting members.

Fig. 7.

C2-ceramide and SH-BC-893 promote actin polymerization. A, FL5.12 cells treated with SH-BC-893 (10 μm) or C2-ceramide (50 μm) for 1 h and then stained for F-actin with Alexa Fluor 488-conjugated phalloidin. B, Same as in A, except cells pre-treated with vehicle or PP2A inhibitors calyculinA (calyA, 5 nm) or LB-100 (100 μm) for 1.5 h before SH-BC-893 addition. C, Same as in A, except cells pre-treated with vehicle or actin polymerization inhibitor latrunculinA (LatA, 1 μm) for 30 min before SH-BC-893 (2.5 μm) or C2-ceramide (30 μm) addition. D, FL5.12 cells pre-treated (30 min) with LatA (1 μm) were treated with SH-BC-893 (2.5 or 5 μm) or C2-ceramide (30 or 50 μm) for 1 h and surface CD98 quantified by flow cytometry. E, FL5.12 cells pre-treated with LatA (30 min, 1 μm) were treated with SH-BC-893 (5 μm) for 2 h and imaged by DIC microscopy to visualize vacuoles. n = 3 in D and n ≥ 30 cells per condition in E. A two-tailed t test was used to compare SH-BC-893 -treated cells to combination of LatA and SH-BC-893, *** = p ≤ 0.001.

Fig. 8.

Ceramide inhibits vacuolation by reducing Akt activity. A, FL5.12 cells were treated for 3 h with SH-BC-893 (2.5 μm) and/or C2-ceramide (50 μm) as indicated and imaged by DIC microscopy. B, Western blotting measuring the phosphorylation of the AKT substrate PRAS40 (total and phospho-Thr246) in FL5.12 cells treated for 30 min with SH-BC-893 (5 μm), C2-ceramide (50 μm), and/or MK2206 (1 μm) as indicated. C, FL5.12 cells treated for 3 h with SH-BC-893 (2.5 μm) and/or AKT inhibitor MK2206 (1 μm) as indicated and visualized as in (A). D, Rictor WT and KO murine embryonic fibroblasts (MEFs) treated for 6 h with vehicle or SH-BC-893 (5 μm) then visualized by phase contrast microscopy. E, FL5.12 cells treated for 3 h with SH-BC-893 (2.5 μm) or C2-ceramide (50 μm) and the Gsk3b inhibitor CHIR99021 (10 μm) as indicated; visualized as in (A). In (A, C, E), ≥ 400 cells per condition were analyzed from a total of three independent experiments; using a two-tailed t test to compare either SH-BC-893 or C2-ceramide to the vehicle control (in E) or SH-BC-893 alone to the combination of SH-BC-893 with C2-ceramide (A), MK2206 (C), or CHIR99021 (E), n.s. = not significant, ** = p ≤ 0.01, or *** = p ≤ 0.001.