Molecular Cloning and Functional Characterization of Murine Sphingosine Kinase

Abstract

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.The sphingolipid metabolite, sphingosine-1-phosphate (SPP), 1 is emerging as a prototype of a new class of lipid second messengers, which has both intracellular and extracellular actions (1-4). Ample evidence indicates that SPP can serve as an intracellular second messenger; SPP modulates intracellular pathways important for diverse biological processes including cell growth, survival, motility, and cytoskeletal changes (reviewed in Ref. 5). Moreover, because SPP antagonizes apoptosis mediated by ceramide, a stress-induced sphingolipid metabolite (3, 6), we have proposed that the relative intracellular levels of these two sphingolipid metabolites is an important factor that determines whether cells will survive or die (3). In support of this idea, it has recently been shown that unfertilized mouse oocytes exposed to the anticancer drug doxorubicin undergo ceramide-mediated apoptosis that is inhibited by SPP (7). In addition, it seems that this ceramide/SPP rheostat is an evolutionarily conserved stress...

CXCL4‐induced monocyte survival, cytokine expression, and oxygen radical formation is regulated by sphingosine kinase 1

“…Activation of SphK1 was determined by an SphK1-specific in vitro-phosphorylation assay using sphingosine as exogenous substrate, essentially as described [43].…”

Sphingolipids and membrane biology as determined from genetic models

“…Thus, the evolution of S1P into a signaling ligand at the cell surface is of recent origin and might contribute to the sophistication and complexity of these phyla. S1P is generated by the phosphorylation of sphingosine by sphingosine kinase (SphK) [147,148]. Because recent reviews have dealt extensively with the S1P family receptors we will make only brief mention of them here, focusing on the findings of in vivo studies with an emphasis on membrane biology [149,150].…”

Cloning and characterization of rat sphingosine kinase 1 with an N-terminal extension

“…Cytosolic SK activity was measured by incubating various concentration of D-erythrosphingosine in the reaction mixture containing 0.125% Triton X-100, 10 mM MgCl 2 , and [γ-32 P]ATP (5 μCi, 1 mM) at 30 °C for 20 minutes as previously described [9].…”

“…SKs are highly conserved throughout various organisms from plants to mammals [8]. Two isoforms, SK1 and SK2, have been cloned and characterized so far in mammals [9][10][11][12][13][14]. Although these two enzymes have a high degree of sequence similarity, they are different in kinetic properties and tissue distribution and originate from different genes [2,15].…”

“…SK1 was initially isolated from rat kidney as a 49 kDa protein [13]. Subsequently, two SK1s, SPHK1a and SPHK1b, were cloned from mice [9]. Although these two isoforms are differ only in a few N-terminal amino acid residues, they show different characteristics including stability, intracellular distribution, and tissue distribution [20].…”