SphK1 and SphK2, Sphingosine Kinase Isoenzymes with Opposing Functions in Sphingolipid Metabolism

Abstract

The potent sphingolipid metabolite sphingosine 1-phosphate is produced by phosphorylation of sphingosine catalyzed by sphingosine kinase (SphK) types 1 and 2. In contrast to pro-survival SphK1, the putative BH3-only protein SphK2 inhibits cell growth and enhances apoptosis. Here we show that SphK2 catalytic activity also contributes to its ability to induce apoptosis. Overexpressed SphK2 also increased cytosolic free calcium induced by serum starvation. Transfer of calcium to mitochondria was required for SphK2-induced apoptosis, as cell death and cytochrome c release was abrogated by inhibition of the mitochondrial Ca 2؉ transporter. Serum starvation increased the proportion of SphK2 in the endoplasmic reticulum and targeting SphK1 to the endoplasmic reticulum converted it from anti-apoptotic to pro-apoptotic. Overexpression of SphK2 increased incorporation of [ 3 H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide, whereas SphK1 decreased it. Electrospray ionizationmass spectrometry/mass spectrometry also revealed an opposite effect on ceramide mass levels. Importantly, specific down-regulation of SphK2 reduced conversion of sphingosine to ceramide in the recycling pathway and conversely, down-regulation of SphK1 increased it. Our results demonstrate that SphK1 and SphK2 have opposing roles in the regulation of ceramide biosynthesis and suggest that the location of sphingosine 1-phosphate production dictates its functions.The sphingolipid metabolite, sphingosine 1-phosphate (S1P), 3 a ligand for a family of five specific G protein-coupled receptors, and regulates many important cellular processes including growth, survival, differentiation, cytoskeleton rearrangements, motility, angiogenesis, and immunity (reviewed in Refs. 1-3). Although there is no doubt that S1P acts extracellularly, several studies suggest that this potent lipid, like its precursors sphingosine (4) and ceramide (N-acylsphingosine) (5-7), may also have intracellular functions important for calcium homeostasis (8), cell growth (9, 10), and suppression of apoptosis (11)(12)(13)(14).Like other lipid mediators, S1P levels are tightly regulated by the balance between synthesis, catalyzed by sphingosine kinase (SphK), irreversible cleavage by S1P lyase, and reversible dephosphorylation to sphingosine by specific S1P phosphatases. Two distinct SphK isoforms, SphK1 and SphK2, have been cloned and characterized (15,16). Diverse external stimuli, particularly growth and survival factors, stimulate SphK1 and intracellularly generated S1P has been implicated in their mitogenic and anti-apoptotic effects (10,13,(17)(18)(19)(20)(21)(22)(23)(24). Expression of SphK1 enhanced proliferation and growth in soft agar, promoted the G 1 -S transition, protected cells from apoptosis (10,14,17), and induced tumor formation in mice (17, 18). However, although SphK1 and intracellularly generated S1P can signal "inside-out" to regulate cytoskeletal rearrangements and cell movement, remarkably, cell growth stimulation...

Hypoxia Enhances Sphingosine Kinase 2 Activity and Provokes Sphingosine-1-Phosphate-Mediated Chemoresistance in A549 Lung Cancer Cells

“…Activation of signaling pathways such as PI3K, extracellular signal-regulated kinase 1/2, or p38 MAPK, which are active under hypoxia (9,40), might therefore add to SphK2 regulation. Activation of SphK2 as seen in our study under hypoxia was cytoprotective rather than cell destructive as was observed before based on forced SphK2 overexpression (14). Differences might not only be the amount of protein being expressed by forced overexpression versus a moderated hypoxic increase but also different localization of the enzyme or the release of S1P to the extracellular space under hypoxia, which in turn can protect cells.…”

“…In mammals, two SphK isoforms have been identified, SphK1 and SphK2. Although generating the same product, both enzymes show opposed biological functions (14). Whereas SphK1 promotes survival, vascularization, and angiogenesis (15,16), SphK2 activity, mainly based on forced overexpression studies, is linked to a proapoptotic outcome (17,18).…”

Sphingosine kinase 1 and 2 regulate the capacity of mesangial cells to resist apoptotic stimuli in an opposing manner

“…In a previous study using cultured fibroblasts and HEK 293 cells, Maceyka et al (2005) proposed opposite regulatory effects of SK-1 and SK-2 on ceramide biosynthesis. By downregulating SK-2 by siRNA, they showed a decrease in ceramide formation, whereas downregulation of SK-1 by siRNA resulted in increased ceramide formation.…”

Targeting sphingosine-1-phosphate for cancer therapy

“…Thus, SPHK1 is thought to produce the extracellular S1P that protects cancer cells from proapoptotic cytotoxics (Figure 2). On the other hand, SPHK2 induces apoptosis and inhibits growth (Maceyka et al, 2005b). The SPHK2 is likely to produce S1P exclusively for intracellular signalling.…”