Fpk1/2 kinases regulate cellular sphingoid long‐chain base abundance and alter cellular resistance toLCBelevation or depletion

Abstract

Sphingolipids are a family of eukaryotic lipids biosynthesized from sphingoid long-chain bases (LCBs). Sphingolipids are an essential class of lipids, as their depletion results in cell death. However, acute LCB supplementation is also toxic; thus, proper cellular LCB levels should be maintained. To characterize the “sphingolipid-signaling intercross,” we performed a kinome screening assay in which budding yeast protein kinase-knockout strains were screened for resistance to ISP-1, a potent inhibitor of LCB biosynthesis. Here, one pair of such DIR (deletion-mediated ISP-1 resistance) genes, FPK1 and FPK2, was further characterized. Cellular LCB levels increased in the fpk1/2Δ strain, which was hypersensitive to phytosphingosine (PHS), a major LCB species of yeast cells. Concomitantly, this strain acquired resistance to ISP-1. Fpk1 and Fpk2 were involved in two downstream events; that is, ISP-1 uptake due to aminophospholipid flippase and LCB degradation due to LCB4 expression. RSK3, which belongs to the p90-S6K subfamily, was identified as a functional counterpart of Fpk1/2 in mammalian cells as the RSK3 gene functionally complemented the ISP-1-resistant phenotype of fpk1/2Δ cells.

Protein kinase Gin4 negatively regulates flippase function and controls plasma membrane asymmetry

“…3 C). Indeed, in agreement with the observation that flippases regulate the permeability of the yeast PM to xenobiotics (Yamane-Sando et al, 2014), we found that 400 ng/ml of Myr drastically reduced formation of complex sphingolipids in wild-type cells, whereas even 600 ng/ml had no effect in dnf1 dnf2 cells (Fig. S3 A).…”

Control of Plasma Membrane Permeability by ABC Transporters

“…Introduction of the lem3⌬ allele into either wild-type or pdr5⌬ yor1 strains led to a large increase in tolerance to Myr while causing a dramatic sensitization to AbA exposure. The elevated resistance to Myr caused by loss of Lem3 has been noted by two other groups (36,54). The epistatic relationship between the LEM3 and PDR5/YOR1 genes is most consistent with Lem3 acting downstream from the ABC transporters.…”

“…Based on work from two other labs, Myr appears to enter the cell via the action of flippase (54,69). The role of Lem3-dependent flippase as the importer of Myr would explain virtually all the genetic effects we have seen for this inhibitor.…”

“…Lem3-dependent flippase activity is required for wildtype PHS resistance (11), likely through its role in the normal delivery of Tat2 to the plasma membrane (68). Mutants lacking Lem3 are highly sensitive to AbA but also extremely resistant to Myr (54,69). These same phenotypes are seen when a lem3⌬ mutation is introduced into a pdr5⌬ yor1 strain.…”

“…These two related genes, FPK1 and FPK2, encode protein kinases that stimulate flip activity (55). Loss of Fpk1 is sufficient to cause Myr resistance and a marked decrease in flip (54,55). Loss of both Fpk1 and Fpk2 caused elevated Myr resistance and AbA hypersensitivity.…”