Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6

Abstract

Endogenous sphingolipids (ceramide) and related synthetic molecules (FTY720, SH-BC-893) reduce nutrient access by decreasing cell surface expression of a subset of nutrient transporter proteins. Here, we report that these sphingolipids disrupt endocytic recycling by inactivating the small GTPase ARF6. Consistent with reported roles for ARF6 in maintaining the tubular recycling endosome, MICAL-L1-positive tubules were lost from sphingolipid-treated cells. We propose that ARF6 inactivation may occur downstream of PP2A activation since: (1) sphingolipids that fail to activate PP2A did not reduce ARF6-GTP levels; (2) a structurally unrelated PP2A activator disrupted tubular recycling endosome morphology and transporter localization; and (3) overexpression of a phosphomimetic mutant of the ARF6 GEF GRP1 prevented nutrient transporter loss. ARF6 inhibition alone was not toxic; however, the ARF6 inhibitors SecinH3 and NAV2729 dramatically enhanced the killing of cancer cells by SH-BC-893 without increasing toxicity to peripheral blood mononuclear cells, suggesting that ARF6 inactivation contributes to the anti-neoplastic actions of sphingolipids. Taken together, these studies provide mechanistic insight into how ceramide and sphingolipid-like molecules limit nutrient access and suppress tumor cell growth and survival.

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

“…1B). These observations are consistent with published studies in multiple cell types (5,7,14,20,34). In addition to limiting cell surface CD98 expression, SH-BC-893, but not ceramide, prevented the fusion of endocytic vesicles and autophagosomes with lysosomes resulting in enlarged multivesicular bodies, MVB (5); LB-100 reversed this effect (Fig.…”

“…Interestingly, although Fingolimod (FTY720, Gilenya), pyrrolidine analogs such as SH-BC-893, and ceramide all induce nutrient transporter down-regulation downstream of PP2A activation, only FTY720 and SH-BC-893 produce PP2A-dependent cytoplasmic vacuolation (5). Ceramide, on the other hand, produces distinct effects from FTY720 and SH-BC-893 on the tubular recycling endosome, although whether these effects are PP2A-dependent is less certain (5,14). These observations suggest that these structurally-related molecules differentially activate PP2A, resulting in distinct patterns of dephosphorylation and different endolysosomal trafficking phenotypes.…”

“…Both SH-BC-893 and C2-ceramide (N-Acetyl-sphingosine, Fig. 1A) suppress cell growth and survival in part by downregulating nutrient transporter proteins through the activation of the cytosolic serine/threonine protein phosphatase PP2A (5,7,14,34). The cell surface antigen 4F2 heavy chain (solute carrier family 3 member 2, Slc3a2 or CD98) is a chaperone required for the surface expression of several associated light chains that transport amino-acids (36).…”

FTY720 induces non-canonical phosphatidylserine externalization and cell death in acute myeloid leukemia

“…Note that NSA displays high auto-fluorescence in the 488 nm laser and was excluded from analysis with this reagent. The staining of surface CD98 was adapted from Finicle et al 46 . Briefly, cells were harvested and washed twice with ice-cold FACS blocking buffer (10% FBS, 0.05% sodium azide in PBS).…”

An ionic control of metabolic-epigenetic reprogramming links inflammation to membrane potential-mediated nutrient acquisition

“…Last, nutrient transporters including GLUT1, CD98, and LAT1 are ARF6/GRP1 cargo proteins that can be recycled back to the plasma membrane via the tubular recycling endosome (Eyster et al, 2009; Finicle et al, 2018; Maldonado-Baez et al, 2013; McCracken and Edinger, 2013). Strikingly, a constitutively active mutant of GRP1 (S155D/T280D, GRP1 DD mutant), which forces the recycling of these transporters back to the plasma membrane and prevents their loss (Finicle et al, 2018; Li et al, 2012), largely restored the ML133-induced suppression of IL-1β ( Fig. 7K), further strengthening the contribution of surface nutrient transporters loss to the anti-in ammatory outcome of Kir2.1 blockade.…”