Enzymatic quantification of sphingosine in the picomole range in cultured cells

An enzymatic method to quantify the mass levels of free sphingosine in cellular lipid extracts was developed. The assay is based upon the observation that ceramide is phosphorylated by Escherichia coli diacylglycerol kinase. Although sphingosine is not recognized by the enzyme, it can be converted to a substrate by acylation with hexanoic anhydride. Using a mixed micellar assay, previously reported for the mass quantification of diacylglycerol, the short-chain ceramide (N-C6-sphingosine), generated by acylation, is quantitatively phosphorylated to N-C6-[32P]sphingosine phosphate. This assay allows quantification of sphingosine over a broad range from 25 to 5000 pmol. When this assay was applied to standard compounds, reverse-phase thin-layer chromatography of the reaction products was adequate to separate the phosphorylated derivatives of long-chain ceramide and N-C6-sphingosine. However, the presence of other lipids in extracts from biological samples (mainly monoalkylglycerols which are also a substrate for the diacylglycerol kinase) interfered and necessitated an additional purification step. The most efficient purification step devised was a combination of anion- and cation-exchange chromatography. The mass levels of free sphingoid bases in different cultured cells were quantified using this assay. Levels varied between 8 to 20 pmol/10(6) cells. When normalized to phospholipids, sphingosine levels varied between 0.01 and 0.04 mol%. The lowest levels were found in L929 cells, while Schwann cells derived from Twitcher mice contained the highest levels. These levels were significantly higher than those of Schwann cells derived from normal mice.     

An assay system for measuring the acute production of sphingosine 1-phosphate in intact monolayers

Sphingosine kinase (SK) is a signaling enzyme that phosphorylates sphingosine to produce sphingosine 1-phosphate. Sphingosine and sphingosine 1-phosphate (S1P) belong to a class of bioactive sphingolipid metabolites that are critical in a number of cellular processes, yet often have opposing biological functions. The intracellular localization of sphingosine kinase has been demonstrated in multiple studies to be a critical aspect of its signaling function. To date, assays of sphingosine kinase activity have been developed for measuring activity in lysates, where the effects of localization are lost. Here we outline a system in which the rate of production of S1P can be measured in intact cells using exogenously added radiolabeled ATP instead of tritiated sphingosine. The surprising ability of ATP to enter unpermeabilized monolayers is one aspect that makes this assay simple, efficient, and inexpensive, yet sensitive enough to measure endogenous enzyme activity. The assay is well behaved in terms of kinetics and substrate dependence. Overall, this assay is ideal for future studies to identify changes in S1P production in intact cells such as those that result from the differential intracellular targeting of sphingosine kinase.     

鞘氨醇-1-磷酸的定量测定——一种新的竞争性结合方法

鞘氨醇-1-磷酸（SPP）是重要的细胞第二信使,影响细胞的生长和死亡.通过培养和收集转染SPP受体-EDG-1的HEK293细胞,与标记及非标记SPP共孵育,利用它们与HEK293细胞的竞争性结合,测定细胞、血清和组织中SPP含量.该法无需特殊仪器,可以测到皮摩尔水平的低含量,批间差异小于15%（6次）.     

An assay for sphingosine kinase activity using biotinylated sphingosine and streptavidin-coated membranes

Sphingosine kinase catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, a lipid signaling molecule implicated in roles in a diverse range of mammalian cell processes through its action as both a ligand for G-protein-coupled cell-surface receptors and an apparent intracellular second messenger. This paper describes a rapid, sensitive, and reproducible assay for sphingosine kinase activity using biotinylated sphingosine (biotinyl-Sph) as a substrate and capturing the phosphorylated product with streptavidin-coated membranes. We have shown that both human sphingosine kinase 1 and 2 (hSK1 and hSK2) can efficiently phosphorylate biotinyl-Sph, with K(m) values similar to those of sphingosine. The assay utilizing this substrate has high sensitivity for hSK1 and hSK2, with detection limits in the low-femtomole range for both purified recombinant enzymes. Importantly, we have also demonstrated the capacity of this assay to measure endogenous sphingosine kinase activity in crude cell extracts and to follow changes in this activity following sphingosine kinase activation. Together, these results demonstrate the potential utility of this assay in both cell-based analysis of sphingosine kinase signaling pathways and high-throughput screens for agents affecting sphingosine kinase activity in vitro.     

Functional characterization of human sphingosine kinase-1

Sphingosine kinase catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (SPP), a novel lipid mediator with both intra- and extracellular functions. Based on sequence identity to murine sphingosine kinase (mSPHK1a), we cloned and characterized the first human sphingosine kinase (hSPHK1). The open reading frame of hSPHK1 encodes a 384 amino acid protein with 85% identity and 92% similarity to mSPHK1a at the amino acid level. Similar to mSPHK1a, when HEK293 cells were transfected with hSPHK1, there were marked increases in sphingosine kinase activity resulting in elevated SPP levels. hSPHK1 also specifically phosphorylated D-erythro-sphingosine and to a lesser extent sphinganine, but not other lipids, such as D,L-threo-dihydrosphingosine, N, N-dimethylsphingosine, diacylglycerol, ceramide, or phosphatidylinositol. Northern analysis revealed that hSPHK1 was widely expressed with highest levels in adult liver, kidney, heart and skeletal muscle. Thus, hSPHK1 belongs to a highly conserved unique lipid kinase family that regulates diverse biological functions.     

The role of sphingomyelin cycle metabolites in transduction of signals of cell proliferation, differentiation and death

Sphingomyelin cycle metabolites ceramide, sphingosine and sphingosine 1-phosphate play an important role in cell proliferation, differentiation, reception, oncogenesis and apoptosis. Ceramide is an intracellular second messenger for apoptosis activating proteases and specific phosphatases. Sphingosine is an endogenous inhibitor of protein kinase C and has an inhibitory effect on many cell functions depending on the activity of this enzyme. On the other hand, sphingosine can activate other kinases depending on the concentration, cell type and nature of a stimulus and release Ca2+ from intracellular stores thereby regulating cell proliferation. Sphingosine induces apoptosis and its level is increased in cells as a result of action of apoptotic inducers. A phosphorylated product of sphingosine, sphingosine 1-phosphate, mediates the mitogenic signal, induces Ca2+ mobilization and protects cells from apoptosis resulting from elevation of ceramide. The quantitative levels of sphingomyelin metabolites in the cell determine the dynamic balance between the apoptotic and mitogenic signals.     

Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival.

Sphingosine-1-phosphate (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein-coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. J. Cell Biol. 142:229-240). The recent cloning of sphingosine kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of sphingosine kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased sphingosine kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G(1)/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of sphingosine kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethylsphingosine, a competitive inhibitor of sphingosine kinase, blocked the effects of sphingosine kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of sphingosine kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate sphingosine kinase, our results indicate that SPP functions as a second messenger important for growth and survival of cells. Hence, SPP belongs to a novel class of lipid mediators that can function inside and outside cells.     

Sphingosine kinase expression regulates apoptosis and caspase activation in PC12 cells

Sphingosine-1-phosphate (SPP), a bioactive sphingolipid metabolite, suppresses apoptosis of many types of cells, including rat pheochromocytoma PC12 cells. Elucidating the molecular mechanism of action of SPP is complicated by many factors, including uptake and metabolism, as well as activation of specific G-protein-coupled SPP receptors, known as the endothelial differentiation gene-1 (EDG-1) family. In this study, we overexpressed type 1 sphingosine kinase (SPHK1), the enzyme that converts sphingosine to SPP, in order to examine more directly the role of intracellularly generated SPP in neuronal survival. Enforced expression of SPHK1 in PC12 cells resulted in significant increases in kinase activity, with corresponding increases in intracellular SPP levels and concomitant decreases in both sphingosine and ceramide, and marked suppression of apoptosis induced by trophic factor withdrawal or by C(2)-ceramide. NGF, which protects PC12 cells from serum withdrawal-induced apoptosis, also stimulated SPHK1 activity. Surprisingly, overexpression of SPHK1 had no effect on activation of two known NGF-stimulated survival pathways, extracellular signal regulated kinase ERK 1/2 and Akt. However, trophic withdrawal-induced activation of the stress activated protein kinase, c-Jun amino terminal kinase (SAPK/JNK), and activation of the executionary caspases 2, 3 and 7, were markedly suppressed. Moreover, this abrogation of caspase activation, which was prevented by the SPHK inhibitor N,N-dimethylsphingosine, was not affected by pertussis toxin treatment, indicating that the cytoprotective effect was likely not mediated by binding of SPP to cell surface G(i)-coupled SPP receptors. In agreement, there was no detectable release of SPP into the culture medium, even after substantially increasing cellular SPP levels by NGF or sphingosine treatment. In contrast to PC12 cells, C6 astroglioma cells secreted SPP, suggesting that SPP might be one of a multitude of known neurotrophic factors produced and secreted by glial cells. Collectively, our results indicate that SPHK/SPP may play an important role in neuronal survival by regulating activation of SAPKs and caspases.     

Sphingosine 1-phosphate phosphatase 2 is induced during inflammatory responses

Sphingosine 1-phosphate (S1P) levels in cells and, consequently, its bioactivity as a signalling molecule are controlled by the action of enzymes responsible for its synthesis and degradation. In the present report, we examined alterations in expression patterns of enzymes involved in S1P-metabolism (sphingosine kinases including their splice variants, sphingosine 1-phosphate phosphatases, and sphingosine 1-phosphate lyase) under certain inflammatory conditions. We found that sphingosine kinase type 1 (SPHK1) mRNA could be triggered in a cell type-specific manner; individual SPHK1 splice variants were induced with similar kinetics. Remarkably, expression and activity of S1P phosphatase 2 (SPP2) was found to be highly upregulated by inflammatory stimuli in a variety of cells (e.g., neutrophils, endothelial cells). Bandshift analysis using oligonucleotides spanning predicted NFkappaB sites within the SPP2 promoter and silencing of NFkappaB/RelA via RelA-directed siRNA demonstrated that SPP2 is an NFkappaB-dependent gene. Silencing of SPP2 expression in endothelial cells, in turn, led to a marked reduction of TNF-alpha-induced IL-1beta mRNA and protein and to a partial reduction of induced IL-8, suggesting a pro-inflammatory role of SPP2. Notably, up-regulation of SPP2 was detected in samples of lesional skin of patients with psoriasis, an inflammatory skin disease. This study provides detailed insights into the regulation of SPP2 gene expression and suggests that SPP2 might be a novel player in pro-inflammatory signalling.     

Identification and characterization of RPK118, a novel sphingosine kinase-1-binding protein

Sphingosine kinase (SPHK) is a key enzyme catalyzing the formation of sphingosine 1 phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through intracellular as well as extracellular mechanisms. However, the molecular mechanism of the intracellular actions of SPP remains unclear. Here we have cloned a novel sphingosine kinase-1 (SPHK1)-binding protein, RPK118, by yeast two-hybrid screening. RPK118 contains several functional domains whose sequences are homologous to other known proteins including the phox homology domain and pseudokinase 1 and 2 domains and is shown to be a member of an evolutionarily highly conserved gene family. The pseudokinase 2 domain of RPK118 is responsible for SPHK1 binding as judged by yeast two-hybrid screening and immunoprecipitation studies. RPK118 is also shown to co-localize with SPHK1 on early endosomes in COS7 cells expressing both recombinant proteins. Furthermore, RPK118 specifically binds to phosphatidylinositol 3-phosphate. These results strongly suggest that RPK118 is a novel SPHK1-binding protein that may be involved in transmitting SPP-mediated signaling into the cell.     

Sphingosine 1-Phosphate Regulates Heat Shock Protein 27 Induction by a p38 MAP Kinase-Dependent Mechanism in Aortic Smooth Muscle Cells

In an aortic smooth muscle cell line, A10 cells, we investigated the effect of sphingosine 1-phosphate on the induction of heat shock protein 27 (HSP27), a low-molecular-weight heat shock protein. Sphingosine 1-phosphate significantly induced the accumulation of HSP27 in a pertussis toxin-sensitive manner. The effect was dose-dependent in the range between 0.1 and 30 microM. Sphingosine 1-phosphate stimulated an increase in the levels of mRNA for HSP27. Sphingosine 1-phosphate stimulated both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase activation. PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase, did not affect sphingosine 1-phosphate-stimulated HSP27 induction. In contrast, SB203580, an inhibitor of p38 MAP kinase, reduced sphingosine 1-phosphate-induced HSP27 induction. SB203580 reduced the levels of mRNA for HSP27 induced by sphingosine 1-phosphate. These results indicate that sphingosine 1-phosphate stimulates the induction of HSP27 via p38 MAP kinase activation in aortic smooth muscle cells.     

Identification and characterization of a novel human sphingosine-1-phosphate phosphohydrolase,hSPP2

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts as both an extracellular signaling mediator and an intracellular second messenger. S1P is synthesized from sphingosine by sphingosine kinase and is degraded either by S1P lyase or by S1P phosphohydrolase. Recently, mammalian S1P phosphohydrolase (SPP1) was identified and shown to constitute a novel lipid phosphohydrolase family, the SPP family. In this study we have identified a second human S1P phosphohydrolase, SPP2, based on sequence homology to human SPP1. SPP2 exhibited high phosphohydrolase activity against S1P and dihydrosphingosine 1-phosphate. The dihydrosphingosine-1-phosphate phosphohydrolase activity was efficiently inhibited by excess S1P but not by lysophosphatidic acid, phosphatidic acid, or glycerol 3-phosphate, indicating that SPP2 is highly specific to sphingoid base 1-phosphates. Immunofluorescence microscopic analysis demonstrated that SPP2 is localized to the endoplasmic reticulum. Although the enzymatic properties and localization of SPP2 were similar to those of SPP1, the tissue-specific expression pattern of SPP2 was different from that of SPP1. Thus, SPP2 is another member of the SPP family that may play a role in attenuating intracellular S1P signaling.     

Sphingosine induces apoptosis in hippocampal neurons and astrocytes by activating caspase-3/-9 via a mitochondrial pathway linked to SDK/14-3-3 protein/Bax/cytochrome c

The present study examined sphingosine-induced apoptosis in cultured rat hippocampal neurons and astrocytes. Sphingosine induced apoptosis in a concentration (1-100 μM)-dependent manner, that is inhibited by the PKC-δ inhibitor rottlerin, and a similar effect was obtained with the sphingosine kinase inhibitors, to raise intracellular sphingosine concentrations. Sphingosine increased presence of sphingosine-dependent protein kinase (SDK), and the effect was suppressed by rottlerin. Sphingosine increased phosphorylated 14-3-3 protein, thereby transforming the protein from a dimeric structure into a monomeric structure. Sphingosine accumulated Bax in the mitochondria and stimulated cytochrome c release into the cytosol, and those effects were inhibited by rottlerin. Sphingosine disrupted mitochondrial membrane potentials, that was abolished by silencing the PKC-δ-targeted gene. Moreover, sphingosine activated caspase-9 and the effector caspase-3 in a PKC-δ-dependent manner. Taken together, the results of the present study indicate that sphingosine activates SDK, produced through proteolytic processing of an active form of PKC-δ, to phosphorylate 14-3-3 protein and transform into a monomeric structure, causing Bax dissociation from 14-3-3 protein and accumulation in the mitochondria, which perturbs mitochondrial membrane potentials allowing cytochrome c release into the cytosol, to activate caspase-9 and the effector caspase-3, responsible for apoptosis in hippocampal neurons and astrocytes.     

Involvement of sphingosine kinase in TNF-alpha-stimulated tetrahydrobiopterin biosynthesis in C6 glioma cells

In C6 glioma cells, the sphingolipid second messenger ceramide potentiates expression of inducible nitric-oxide synthase (iNOS) induced by tumor necrosis factor alpha (TNF-alpha) without affecting GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the biosynthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH(4)), a cofactor required for iNOS activity. TNF-alpha also stimulates sphingosine kinase, the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (SPP), a further metabolite of ceramide. Several clones of C6 cells, expressing widely varying levels of sphingosine kinase, were used to examine the role of SPP in regulation of GTPCH and BH(4) biosynthesis. Overexpression of sphingosine kinase, with concomitant increased endogenous SPP levels, potentiated the effect of TNF-alpha on GTPCH expression and activity and BH(4) biosynthesis. In contrast, enforced expression of sphingosine kinase had no effect on iNOS expression or NO formation. Furthermore, N,N-dimethylsphingosine, a potent sphingosine kinase inhibitor, completely eliminated the increased GTPCH activity and expression induced by TNF-alpha. Surprisingly, we found that, although C6 cells can secrete SPP, which is enhanced by TNF-alpha, treatment of C6 cells with exogenous SPP or dihydro-SPP had no affect on BH(4) biosynthesis. However, both SPP and dihydro-SPP markedly stimulated ERK 1/2 in C6 cells, which express cell surface SPP receptors. Interestingly, although this ERK activation was blocked by PD98059, which also reduced cellular proliferation induced by enforced expression of sphingosine kinase, PD98059 had no effect on GTPCH activity. Collectively, these results suggest that only intracellularly generated SPP plays a role in regulation of GTPCH and BH(4) levels.     

Sphingosine kinase-mediated Ca2+ signalling by G-protein-coupled receptors.

Formation of inositol 1,4,5-trisphosphate (IP3) by phospholipase C (PLC) with subsequent release of Ca2+ from intracellular stores, is one of the major Ca2+ signalling pathways triggered by G-protein-coupled receptors (GPCRs). However, in a large number of cellular systems, Ca2+ mobilization by GPCRs apparently occurs independently of the PLC-IP3 pathway, mediated by an as yet unknown mechanism. The present study investigated whether sphingosine kinase activation, leading to production of sphingosine-1-phosphate (SPP), is involved in GPCR-mediated Ca2+ signalling as proposed for platelet-derived growth factor and FcepsilonRI antigen receptors. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by m2 and m3 muscarinic acetylcholine receptors (mAChRs) expressed in HEK-293 cells without affecting mAChR-induced PLC stimulation. Activation of mAChRs rapidly and transiently stimulated production of SPP in HEK-293 cells. Finally, intracellular injection of SPP induced a rapid and transient Ca2+ mobilization in HEK-293 cells which was not antagonized by heparin. We conclude that mAChRs utilize the sphingosine kinase-SPP pathway in addition to PLC-IP3 to mediate Ca2+ mobilization. As Ca2+ signalling by various, but not all, GPCRs in different cell types was likewise attenuated by the sphingosine kinase inhibitors, we suggest a general role for sphingosine kinase, besides PLC, in mediation of GPCR-induced Ca2+ signalling.     

Sphingosine inhibition of tyrosine aminotransferase and tryptophan oxygenase induction by dexamethasone in primary culture of rat hepatocytes

The effect of sphingosine, a known selective inhibitor of protein kinase C, on the induction of tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) by dexamethasone was studied in the primary culture of rat hepatocytes to determine the possible involvement of protein kinase C in the expression of glucocorticoid action. Sphingosine inhibits the induction of TAT by dexamethasone in a concentration- and time-dependent manner in primary culture of rat hepatocytes. It does not inhibit the induction of TAT by Bt2cAMP. Sphingosine inhibits also the induction of TO by dexamethasone in a manner similar to TAT inhibition. It has no effect on the activity of lactate dehydrogenase, a cytosolic marker enzyme and on the protein content of the cultured hepatocytes. These findings indicate that endogenous modulator of protein kinase C, such as sphingosine, may influence the expression of glucocorticoid action in rat hepatocytes.     

Simultaneous quantitative analysis of sphingoid base 1-phosphates in biological samples by o-phthalaldehyde precolumn derivatization after dephosphorylation with alkaline phosphatase

This paper describes a simultaneous analytical method for the measurement of sphingoid base 1-phosphates and sphingoid bases from a variety of biological samples. This method consists of two steps of sample pretreatment: the enzymatic dephosphorylation of sphingoid base 1-phosphates by alkaline phosphatase (APase) and the subsequent analysis of o-phthalaldehyde (OPA) derivatives of the liberated sphingoid bases by HPLC. By introducing C17-sphingosine 1-phosphate and C17-sphingosine as internal standards, not only phytosphingosine 1-phosphate, sphingosine 1-phosphate, and sphinganine 1-phosphate but also phytosphingosine, sphingosine, and sphinganine present in a sample could be quantified in 12 min on a C18 reversed-phase column with a simple mobile phase of acetonitrile:deionized distilled water (90:10, v/v). With this HPLC method, we could reproducibly analyze the levels of sphingoid base 1-phosphates over a broad range of concentrations from 0.5 to 100.0 pmol from various biological samples including serum, cultured cells, and rat tissue homogenates. The conversion of sphingoid base 1-phosphates into sphingoid bases increased the stability of the OPA adducts. Thus, this indirect measurement of sphingoid base 1-phosphates increased the sensitivity and reproducibility of the method. This HPLC method was also used to measure the changes in the levels of sphingoid base 1-phosphates in cultured cells after treatment with 1,25-(OH)2D3, a sphingosine kinase activator, or with fumonisin B1, a sphinganine N-acyltransferase inhibitor.     

Ceramide/sphingosine/sphingosine 1-phosphate metabolism on the cell surface and in the extracellular space

Sphingolipid metabolites, ceramide, sphingosine, and sphingosine 1-phosphate, have emerged as a new class of lipid biomodulators of various cell functions. These metabolites are known to function not only as intracellular second messengers, but also in the extracellular space. Sphingosine 1-phosphate especially has numerous functions as an important extracellular mediator that binds to cell surface S1P receptors. Recent studies have also shown that sphingolipid-metabolizing enzymes function not only in intracellular organelles but also in the extracellular spaces, including the outer leaflet of the plasma membrane. This review focuses on the metabolic enzymes (acid and alkaline sphingomyelinases, neutral ceramidase, and sphingosine kinase) that are involved in the production of the sphingolipid metabolites in these extracellular spaces, and on the metabolic pathway itself.     

Characterization of the Drosophila sphingosine kinases and requirement for Sk2 in normal reproductive function

Sphingosine kinase is a highly conserved enzyme that catalyzes the synthesis of sphingosine 1-phosphate and reduces cellular levels of sphingosine and ceramide. Although ceramide is pro-apoptotic and sphingosine is generally growth-inhibitory, sphingosine 1-phosphate signaling promotes cell proliferation, survival, and migration. Sphingosine kinase is thus in a strategic position to regulate important cell fate decisions which may contribute to normal animal development. To facilitate studies examining the potential role of sphingosine kinase and long chain base metabolism in Drosophila development, we characterized two putative Drosophila sphingosine kinase genes, Sk1 and Sk2. Both genes functionally and biochemically complement a yeast sphingosine kinase mutant, express predominantly cytosolic activities, and are capable of phosphorylating a range of endogenous and non-endogenous sphingoid base substrates. The two genes demonstrate overlapping but distinct temporal and spatial expression patterns in the Drosophila embryo, and timing of expression is consistent with observed changes in long chain base levels throughout development. A null Sk2 transposon insertion mutant demonstrated elevated long chain base levels, impaired flight performance, and diminished ovulation. This is the first reported mutation of a sphingosine kinase in an animal model; the associated phenotypes indicate that Sk1 and Sk2 are not redundant in biological function and that sphingosine kinase is essential for diverse physiological functions in this organism.     

Do mammals make all their own inositol hexakisphosphate?

A highly specific and sensitive mass assay for inositol hexakisphosphate (InsP6) was characterized. This centres around phosphorylating InsP6 with [32P]ATP using a recombinant InsP6 kinase from Giardia lambia, followed by HPLC of the 32P-labelled products with an internal [3H]InsP7 standard. This assay was used to quantify InsP6 levels in a variety of biological samples.Concentrations of InsP6 in rat tissues varied from 10-20 microM (assuming 64% of wet weight of tissue is cytosol water), whereas using the same assumption axenic Dictyostelium discoideum cells contained 352 +/- 11 microM InsP6. HeLa cells were seeded at low density and grown to confluence, at which point they contained InsP6 levels per mg of protein similar to rat tissues. This amounted to 1.952 +/- 0.117 nmol InsP6 per culture dish, despite the cells being grown in serum shown to contain no detectable(less than 20 pmol per dish) InsP6. These results demonstrate that mammalian cells synthesize all their own InsP6. Human blood was analysed, and although the white cell fraction contained InsP6 at a concentration comparable with other tissues, in serum and platelet-free plasma no InsP6 was detected (<1 nM InsP6). Human urine was also examined, and also contained no detectable (<5 nM) InsP6. These results suggest that dietary studies purporting to measure InsP6 at micromolar concentrations in human plasma or urine may not have been quantifying this inositol phosphate. Therefore claims that administrating InsP6 in the diet or applying it topically can produce health benefits by increasing extracellular InsP6 levels may require reassessment.