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.Sphingosine 1-phosphate (S1P) 1 is a bioactive sphingolipid metabolite that provides directional cues to migrating cells (1) and exerts proliferative and anti-apoptotic effects on many cell types (2). S1P signals are transduced through both extracellular (receptor-mediated) and intracellular mechanisms. Intracellularly, S1P signaling affects cytoskeletal organization, calcium homeostasis, DNA synthesis, and the apoptotic machinery. S1P is generated by the phosphorylation of sphingosine, a reaction catalyzed by sphingosine kinase (SK). It is eliminated through dephosphorylation catalyzed by either S1P phosphatase (S1PP) or type 2 phosphatidate phosphohydrolases or through its irreversible degradation to a long chain aldehyde and ethanolamine phosphate, catalyzed by sphingosine 1-phosphate lyase (SPL) (Fig. 1).SK is a member of a growing class of lipid kinases, including diacylglycerol kinases and phosphatidylinositol 3-kinases, that participate in cell signaling. Many studies have implicated SK activation and S1P generation in mediating angiogenesis, tumorigenicity, metastasis, cell proliferation, motility, lymphocyte trafficking, endocytosis, and survival (reviewed in Refs. 3-10). SK activation has been reported in response to many stimuli and regulates diverse functions such as heat stress response in yeast (11) and stomatal closure in Arabidopsis (12). Thus, SK activation appears to be a widely employed mechanism for propagating mitogenic and survival signals in eukaryotes. Known do...

BackgroundS1P3 is a lipid-activated G protein-couple receptor (GPCR) that has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. Currently, there are no available high-affinity, subtype-selective drug compounds that can block activation of S1P3. We have developed a monoclonal antibody (7H9) that specifically recognizes S1P3 and acts as a functional antagonist.Methodology/Principal FindingsSpecific binding of 7H9 was demonstrated by immunocytochemistry using cells that over-express individual members of the S1P receptor family. We show, in vitro, that 7H9 can inhibit the activation of S1P3-mediated cellular processes, including arrestin translocation, receptor internalization, adenylate cyclase inhibiton, and calcium mobilization. We also demonstrate that 7H9 blocks activation of S1P3 in vivo, 1) by preventing lethality due to systemic inflammation, and 2) by altering the progression of breast tumor xenografts.Conclusions/SignificanceWe have developed the first-reported monoclonal antibody that selectively recognizes a lipid-activated GPCR and blocks functional activity. In addition to serving as a lead drug compound for the treatment of sepsis and breast cancer, it also provides proof of concept for the generation of novel GPCR-specific therapeutic antibodies.