sphingosine-kinase and phytosphingosine

Sphingosine kinase (SPHK) phosphorylates sphingosine to form a bioactive lipid mediator, sphingosine 1-phosphate (S1P). S1P mediates such diverse biological processes as regulation of cell differentiation, motility, and apoptosis both extracellularly, via S1P (Edg) family receptors, and intracellularly, through unidentified targets. In cells S1P is short-lived, so the synthetic process catalyzed by sphingosine kinase may be important in maintaining the cellular levels of the compound. Thus far, two sphingosine kinases have been reported, with SPHK1 exhibiting the higher activity. However, several studies suggest the existence of unidentified sphingosine kinases. Therefore, to further elucidate the role of SPHK1 in the formation of S1P, we investigated its contribution to the total sphingosine kinase activity in mouse tissues. We found that SPHK1 is a major sphingosine kinase in many tissues, especially in brain, heart, and colon. However, some tissues such as spleen, small intestine, and lung contained sphingosine kinase activity that was not attributable to SPHK1 or SPHK2, as determined by immunodepletion assays. Furthermore, the presence of other sphingosine kinases with different properties, i.e., higher activity toward phytosphingosine and a different subcellular distribution, is suggested.

Topics: Animals; Apoptosis; Blood Platelets; Catalysis; Cell Differentiation; Cell Movement; CHO Cells; Cricetinae; Immunoblotting; Male; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Precipitin Tests; Sphingosine; Subcellular Fractions; Tissue Distribution

Topics: Blood Platelets; Cell Line; Chromatography, High Pressure Liquid; Humans; Indicators and Reagents; o-Phthalaldehyde; Phosphotransferases (Alcohol Group Acceptor); Spectrometry, Fluorescence; Sphingosine

Sphingosine kinase (SK) catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, which is a second messenger involved in the proliferative responses of mammalian cells. Although the yeast Saccharomyces cerevisiae has similar phosphorylated sphingoid bases which appear to be involved in growth regulation and the response to stress, SK activity had not been previously demonstrated in yeast. In this study, an in vitro system was set up to characterize yeast SK activity. Activity was detected in the cytosol at neutral pH and 37 degreesC. Yeast SK phosphorylated the sphingoid bases sphingosine, dihydrosphingosine and phytosphingosine. (d,l)-threo-dihydrosphingosine, an inhibitor of mammalian SK, did not inhibit the yeast enzyme. Unique properties of yeast SK were an optimal temperature of 43 degreesC, and in vivo activation during nutrient deprivation. Spontaneous mutants with diminished SK activity were isolated utilizing a screen for resistance to sphingosine in a sphingosine-phosphate-lyase deletion background. Abnormal growth and heat sensitivity were observed in these mutants. These findings suggest that SK may function as a stress-response protein in yeast.

Topics: Cell Division; Cell Survival; Enzyme Activation; Enzyme Inhibitors; Heat-Shock Proteins; Hydrogen-Ion Concentration; Kinetics; Lysophospholipids; Mutation; Phenotype; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Saccharomyces cerevisiae; Sphingosine; Spores; Temperature