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At least 19 recordsLinked to original sources

PubMed2002

Diacylglyceride kinases, sphingosine kinases and NAD kinases: distant relatives of 6-phosphofructokinases.

Diacylglyceride kinases, sphingosine kinases, NAD kinases and 6-phosphofructokinases are thought to be related despite large evolution of their sequences. Discovery of a common signature has led to the suggestion that they possess a similar phosphate-donor-binding site and a similar phosphorylation mechanism. The substrate- and allosteric-binding sites are much more divergent and their delineation remains to be determined experimentally.

Allosteric Site↗

PubMed2006

Sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases.

Sphingolipids are ubiquitous components of cell membranes and their metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. Cer and Sph are associated with growth arrest and apoptosis. Many stress stimuli increase levels of Cer and Sph, whereas suppression of apoptosis is associated with increased intracellular levels of S1P. In addition, extracellular/secreted S1P regulates cellular processes by binding to five specific G protein coupled-receptors (GPCRs). S1P is generated by phosphorylation of Sph catalyzed by two isoforms of sphingosine kinases (SphK), type 1 and type 2, which are critical regulators of the "sphingolipid rheostat", producing pro-survival S1P and decreasing levels of pro-apoptotic Sph. Since sphingolipid metabolism is often dysregulated in many diseases, targeting SphKs is potentially clinically relevant. Here we review the growing recent literature on the regulation and the roles of SphKs and S1P in apoptosis and diseases.

Animals↗

PubMed2005

Sphingosine kinases, sphingosine-1-phosphate and sphingolipidomics.

It has become abundantly clear over the past decade that sphingolipids and their metabolites are key signaling molecules. Ceramide, the backbone of all sphingolipids, predominantly inhibits cell growth and induces apoptosis, while its metabolite, sphingosine-1-phosphate promotes growth and survival. Given the interconvertibility of these two opposing signaling molecules, it is essential that any study that examines the effects of one also look at the other. The newly available technology of liquid chromatography-tandem mass spectroscopy (LC-MS/MS) is increasingly being applied for this purpose, as it can quickly identify and measure many different sphingolipids simultaneously. An added benefit of LC-MS/MS is that it is several orders of magnitude more sensitive than enzymatic methods or more traditional chromatographic techniques, allowing smaller sample sizes and increased throughput. Here, we briefly discuss the importance of LC-MS/MS for measuring sphingolipid metabolites and some future directions researchers may take given the increasingly accessibility to this technology.

Animals↗

PubMed2002

Sphingosine kinase, sphingosine-1-phosphate, and apoptosis.

The sphingolipid metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) play an important role in the regulation of cell proliferation, survival, and cell death. Cer and Sph usually inhibit proliferation and promote apoptosis, while the further metabolite S1P stimulates growth and suppresses apoptosis. Because these metabolites are interconvertible, it has been proposed that it is not the absolute amounts of these metabolites but rather their relative levels that determines cell fate. The relevance of this "sphingolipid rheostat" and its role in regulating cell fate has been borne out by work in many labs using many different cell types and experimental manipulations. A central finding of these studies is that Sph kinase (SphK), the enzyme that phosphorylates Sph to form S1P, is a critical regulator of the sphingolipid rheostat, as it not only produces the pro-growth, anti-apoptotic messenger S1P, but also decreases levels of pro-apoptotic Cer and Sph. Given the role of the sphingolipid rheostat in regulating growth and apoptosis, it is not surprising that sphingolipid metabolism is often found to be disregulated in cancer, a disease characterized by enhanced cell growth, diminished cell death, or both. Anticancer therapeutics targeting SphK are potentially clinically relevant. Indeed, inhibition of SphK has been shown to suppress gastric tumor growth [Cancer Res. 51 (1991) 1613] and conversely, overexpression of SphK increases tumorigenicity [Curr. Biol. 10 (2000) 1527]. Moreover, S1P has also been shown to regulate angiogenesis, or new blood vessel formation [Cell 99 (1999) 301], which is critical for tumor progression. Furthermore, there is intriguing new evidence that S1P can act in an autocrine and/or paracrine fashion [Science 291 (2001) 1800] to regulate blood vessel formation [J. Clin. Invest. 106 (2000) 951]. Thus, SphK may not only protect tumors from apoptosis, it may also increase their vascularization, further enhancing growth. The cytoprotective effects of SphK/S1P may also be important for clinical benefit, as S1P has been shown to protect oocytes from radiation-induced cell death in vivo [Nat. Med. 6 (2000) 1109]. Here we review the growing literature on the regulation of SphK and the role of SphK and its product, S1P, in apoptosis.

Animals↗

PubMed1996

Effect of acidic phospholipids on sphingosine kinase.

Sphingosine-1-phosphate (SPP) is a unique sphingolipid metabolite involved in cell growth regulation and signal transduction. SPP is formed from sphingosine in cells by the action of sphingosine kinase, an enzyme whose activity can be stimulated by growth factors. Little is known of the mechanisms by which sphingosine kinase is regulated. We found that acidic phospholipids, particularly phosphatidylserine, induced a dose-dependent increase in sphingosine kinase activity due to an increase in the apparent Vmax of the enzyme. Other acidic phospholipids, such as phosphatidylinositol, phosphatidic acid, phosphatidylinositol bisphosphate, and cardiolipin stimulated sphingosine kinase activity to a lesser extent than phosphatidylserine, whereas neutral phospholipids had no effect. Diacylglycerol, a structurally similar molecule which differs from phosphatidic acid in the absence of the phosphate group, failed to induce any changes in sphingosine kinase activity. Our results suggest that the presence of negative charges on the lipid molecules is important for the potentiation of sphingosine kinase activity, but the effect does not directly correlate with the number of negative charges. These results also support the notion that the polar group confers specificity in the stimulation of sphingosine kinase by acidic glycerophospholipids. The presence of a fatty acid chain in position 2 of the glycerol backbone was not critical since lysophosphatidylserine also stimulated sphingosine kinase, although it was somewhat less potent. Dioleoylphosphatidylserine was the most potent species, including a fourfold stimulation, whereas distearoyl phosphatidylserine was completely inactive. Thus, the degree of saturation of the fatty acid chain of the phospholipids may also play a role in the activation of sphingosine kinase.

3T3 Cells↗

PubMed1998

Molecular cloning and functional characterization of murine sphingosine kinase.

Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosine or N, N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.

3T3 Cells↗

PubMed1998

Purification and characterization of rat kidney sphingosine kinase.

Sphingosine kinase catalyzes the formation of the bioactive sphingolipid metabolite sphingosine 1-phosphate, which plays important roles in numerous physiological processes, including growth, survival, and motility. We have purified rat kidney sphingosine kinase 6 x 10(5)-fold to apparent homogeneity. The purification procedure involved ammonium sulfate precipitation followed by chromatography on an anion exchange column. Partially purified sphingosine kinase was found to be stabilized by the presence of high salt, and thus, a scheme was developed to purify sphingosine kinase using sequential dye-ligand chromatography steps (since the enzyme bound to these matrices even in the presence of salt) followed by EAH-Sepharose chromatography. This 385-fold purified sphingosine kinase bound tightly to calmodulin-Sepharose and could be eluted in high yield with EGTA in the presence of 1 M NaCl. After concentration, the calmodulin eluate was further purified by successive high pressure liquid chromatography separations on hydroxylapatite, Mono Q, and Superdex 75 gel filtration columns. Purified sphingosine kinase has an apparent molecular mass of approximately 49 kDa under denaturing conditions on SDS-polyacrylamide gel, which is similar to the molecular mass determined by gel filtration, suggesting that the active form is a monomer. Sphingosine kinase shows substrate specificity for D-erythro-sphingosine and does not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, DL-threo-dihydrosphingosine, or N,N-dimethylsphingosine. However, the latter two sphingolipids were potent competitive inhibitors. With sphingosine as substrate, the enzyme had a broad pH optimum of 6.6-7.5 and showed Michaelis-Menten kinetics, with Km values of 5 and 93 microM for sphingosine and ATP, respectively. This study provides the basis for molecular characterization of a key enzyme in sphingolipid signaling.

Animals↗

PubMed2004

Early activation of sphingosine kinase in mast cells and recruitment to FcepsilonRI are mediated by its interaction with Lyn kinase.

Sphingosine kinase has been recognized as an essential signaling molecule that mediates the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase and generation of sphingosine-1-phosphate have been linked to the initial rise in Ca(2+), released from internal stores, and to degranulation. These events either precede or are concomitant with the activation of phospholipase C-gamma and the generation of inositol trisphosphate. Here we show that sphingosine kinase type 1 (SPHK1) interacts directly with the tyrosine kinase Lyn and that this interaction leads to the recruitment of this lipid kinase to the high-affinity receptor for immunoglobulin E (FcepsilonRI). The interaction of SPHK1 with Lyn caused enhanced lipid and tyrosine kinase activity. After FcepsilonRI triggering, enhanced sphingosine kinase activity was associated with FcepsilonRI in sphingolipid-enriched rafts of mast cells. Bone marrow-derived mast cells from Lyn(-/)(-) mice, compared to syngeneic wild-type cells, were defective in the initial induction of SPHK1 activity, and the defect was overcome by retroviral Lyn expression. These findings position the activation of SPHK1 as an FcepsilonRI proximal event.

Animals↗

PubMed1999

Formyl peptide receptor signaling in HL-60 cells through sphingosine kinase.

Sphingosine-1-phosphate (SPP) produced from sphingosine by sphingosine kinase has recently been reported to act as intracellular second messenger for a number of plasma membrane receptors. In the present study, we investigated whether the sphingosine kinase/SPP pathway is involved in cellular signaling of the Gi protein-coupled formyl peptide receptor in myeloid differentiated human leukemia (HL-60) cells. Receptor activation resulted in rapid and transient production of SPP by sphingosine kinase, which was abolished after pertussis toxin treatment. Direct activation of heterotrimeric G proteins by AlF4- also rapidly increased SPP formation in intact HL-60 cells. In cytosolic preparations of HL-60 cells, sphingosine kinase activity was stimulated by the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate). Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine did not affect phospholipase C stimulation and superoxide production but markedly inhibited receptor-stimulated Ca2+ mobilization and enzyme release. We conclude that the formyl peptide receptor stimulates through Gi-type G proteins SPP production by sphingosine kinase, that the enzyme is also stimulated by direct G protein activation, and that the sphingosine kinase/SPP pathway apparently plays an important role in chemoattractant signaling in myeloid differentiated HL-60 cells.

Calcium↗

PubMed2004

Fluorescence-based assay of sphingosine kinases.

Sphingosine kinase enzymatic activity is commonly measured using radiolabeled substrates, with thin-layer chromatography and/or solvent extraction needed to detect the reaction product sphingosine-1-phosphate. We developed a fluorescence-based assay, using a sphingosine derivative labeled with a 7-nitrobenz-2-oxa-1,3-diazole moiety (15-NBD-Sph). Separation of substrate (15-NBD-Sph) from product (the corresponding phosphate) is achieved by extraction with chloroform/methanol at pH 8.5. The phosphate derivative is recovered by >98% in the aqueous phase and is directly detected and quantified by its fluorescence. 15-NBD-Sph is readily phosphorylated by human and murine sphingosine kinases 1 and 2. The suitability of the assay for measuring the activity of the kinases, both in the purified state and when contained in lysates of mammalian cells, was demonstrated. The present method is a convenient alternative to the radiometric assays and is particularly suited to the search for inhibitors of sphingosine kinases.

Animals↗

PubMed2003

PDGF upregulates delayed rectifier via Src family kinases and sphingosine kinase in oligodendroglial progenitors.

An increase in the expression of the delayed rectifier current (I(K)) has been shown to correlate with mitogenesis in many cell types. However, pathways involved in the upregulation of I(K) by growth factors in oligodendroglial progenitors (OPs) have not been well-elucidated. In this study, we found that treatment with platelet-derived growth factor (PDGF) and basic fibroblast growth factor but not ciliary neurotrophic factor resulted in increased I(K) density and upregulation of Kv1.5 and Kv1.6 mRNA transcripts. The effect of PDGF on I(K) was blocked by mimosine, a cell cycle inhibitor, and by genistein, a tyrosine kinase inhibitor. Using inhibitors of PDGF-activated pathways, we found that PDGF-induced upregulation of Kv1.5 and I(K) density involves Src family tyrosine kinases, sphingosine kinase, and intracellular Ca(2+) but not ERK1/2 or phosphatidylinositol 3-kinase pathways. Furthermore, agents that were effective inhibitors of PDGF-induced I(K) upregulation also attenuated OP proliferation, supporting the concept that I(K) is an important link between PDGF-activated signaling cascades and cell cycle progression.

Animals↗

PubMed2000

An oncogenic role of sphingosine kinase.

Sphingosine kinase (SphK) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P/SphK has been implicated as a signalling pathway to regulate diverse cellular functions [1-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.

3T3 Cells↗

PubMed2006

Cellular signalling by sphingosine kinase and sphingosine 1-phosphate.

Sphingosine kinases, through the formation of the bioactive phospholipid sphingosine 1-phosphate, have been implicated in a diverse range of cellular processes, including cell proliferation, apoptosis, calcium homeostasis, angiogenesis and vascular maturation. The last few years have seen a number of significant advances in understanding of the mechanisms of action, activation, cellular localisation and biological roles of these enzymes. Here we review the current understanding of the regulation of and cellular signalling by sphingosine kinase and sphingosine 1-phosphate and discuss recent findings implicating sphingosine kinase as a potential therapeutic target for the control of cancer, inflammation and a number of other diseases. We suggest that, since the activation and subcellular localization of these enzymes appear to play critical roles in their biological functions, targeting these processes may provide more specific therapeutic options than direct catalytic inhibitors.

Animals↗

PubMed2002

Sphingosine kinases: a novel family of lipid kinases.

Sphingosine kinase (SPHK) catalyzes the formation of sphingosine-1-phosphate (S11). S1P plays an important role in regulation of a variety of biological processes through intracellular and extracellular actions. S1P has recently been shown to be the ligand for the EDG-1 family of G-protein-coupled receptors. To date, seven cloned SPHKs have been reported with confirmed SPHK activity, including human, mouse, yeast, and plant. A computer search of various databases suggests that a new SPHK family is emerging. The cloning and manipulation of SPHK genes will no doubt provide us with important information about the functions of S1P in a wide range of organisms.

Amino Acid Sequence↗

PubMed2005

Synthesis and evaluation of sphingoid analogs as inhibitors of sphingosine kinases.

Sphingosine 1-phosphate (S1P), a product of sphingosine kinases (SphK), mediates diverse biological processes such as cell differentiation, proliferation, motility, and apoptosis. In an effort to search and identify specific inhibitors of human SphK, the inhibitory effects of synthetic sphingoid analogs on kinase activity were examined. Among the analogs tested, we found two, SG12 and SG14, that have specific inhibitory effects on hSphK2. N,N-Dimethylsphingosine (DMS), a well-known SphK inhibitor, displayed inhibitory effects for both SphK1 and SphK2, as well as protein kinase C. In contrast, SG12 and SG14 exhibited selective inhibitory effects on hSphK2. Furthermore, SG14 did not affect PKC. In isolated platelets, SG14 blocked the conversion of sphingosine into sphingosine 1-phosphate significantly. This is the first report on the identification of a hSphK2-specific inhibitor, which may provide a useful tool for studying the biological functions of hSphK2.

Animals↗

PubMed2000

Expression of a catalytically inactive sphingosine kinase mutant blocks agonist-induced sphingosine kinase activation. A dominant-negative sphingosine kinase.

Sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a lipid messenger that plays an important role in a variety of mammalian cell processes, including inhibition of apoptosis and stimulation of cell proliferation. Basal levels of S1P in cells are generally low but can increase rapidly when cells are exposed to various agonists through rapid and transient activation of SK activity. To date, elucidation of the exact signaling pathways affected by these elevated S1P levels has relied on the use of SK inhibitors that are known to have direct effects on other enzymes in the cell. Furthermore, these inhibitors block basal SK activity, which is thought to have a housekeeping function in the cell. To produce a specific inhibitor of SK activation we sought to generate a catalytically inactive, dominant-negative SK. This was accomplished by site-directed mutagenesis of Gly(82) to Asp of the human SK, a residue identified through sequence similarity to the putative catalytic domain of diacylglycerol kinase. This mutant had no detectable SK activity when expressed at high levels in HEK293T cells. Activation of endogenous SK activity by tumor necrosis factor-alpha (TNFalpha), interleukin-1beta, and phorbol esters in HEK293T cells was blocked by expression of this inactive sphingosine kinase (hSK(G82D)). Basal SK activity was unaffected by expression of hSK(G82D). Expression of hSK(G82D) had no effect on TNFalpha-induced activation of protein kinase C and sphingomyelinase activities. Thus, hSK(G82D) acts as a specific dominant-negative SK to block SK activation. This discovery provides a powerful tool for the elucidation of the exact signaling pathways affected by elevated S1P levels following SK activation. To this end we have employed the dominant-negative SK to demonstrate that TNFalpha activation of extracellular signal-regulated kinases 1 and 2 (ERK1,2) is dependent on SK activation.

Amino Acid Sequence↗

PubMed2006

Sphingosine kinase and sphingosine-1-phosphate regulate migration, endocytosis and apoptosis of dendritic cells.

Dendritic cells (DC) are inducers of primary immune responses and represent an attractive vector for cancer immunotherapy. Sphingosine kinase (SphK) and its product sphingosine-1-phosphate (S1P) play an important role in the regulation of immune cells and cancer, affecting processes such as differentiation, growth or migration. We studied the role of SphK and S1P on migration of DC. RT-PCR showed mRNA expression of SphK in DC, declining from immature (iDC) to mature DC (mDC) to antigen-loaded mDC. Expression of S1P receptors was S1P(1) > S1P(2) = S1P(3), unrelated to maturation or antigen uptake. In transwell assays, iDC migrated towards SDF-1, MIP-1alpha, MCP and S1P, whereby S1P combined with a chemokine had a synergistic effect. mDC migrated towards 6Ckine and MIP-3beta, but not towards S1P. The SphK-inhibitor dihydro-sphingosine (DHS) reduced migration of iDC but not of mDC. In addition S1P(3)-inhibitor suramin inhibited DC migration in response to S1P. DHS had a reverse effect on endocytosis, enhancing the uptake of FITC dextran. We also observed an anti-apoptotic effect of S1P on mDC for the first time. This indicates that SphK/S1P may play a role in accumulation of peripheral iDC at the location of antigen and subsequent antigen-uptake. These findings may help to optimise DC-based cancer immunotherapy by modulation of SphK/S1P.

Apoptosis↗

PubMed1995

Partial purification of two forms of choline kinase and separation of choline kinase from sphingosine kinase of rat brain.

Choline kinase of rat brain was purified approximately 200,000 fold using acid precipitation, ammonium sulphate fractionation, Q-Sepharose, Octyl-Sepharose and AH-Sepharose chromatography. The ability of this enzyme to catalyze the phosphorylation of choline, ethanolamine (Etn), monomethylethanolamine (MeEtn), dimethylethanolamine (Me2Etn) and sphingosine was investigated. Choline kinase was separated from sphingosine kinase. The fraction with highly purified choline kinase had four major polypeptides with different molecular masses and possessed activities towards choline, Etn, MeEtn and Me2Etn. Two forms of choline kinase were obtained when the enzymatically active fractions eluted from the Q-Sepharose column were subjected to a horizontal isoelectrofocusing electrophoresis. One form focused around pH 4.7 and is able to phosphorylate choline, Etn, MeEtn and Me2Etn. The other form focused around pH 10 and possessed only choline kinase activity. The latter form of choline kinase did not display classical Michaelis-Menten's mechanism but revealed a positive co-operative pattern for two choline binding sites. This form was purified to apparent homogeneity with a approximate molecular mass of 14.4 kDa.

Animals↗