Sphingosine Kinase: Assay and Product Analysis

The phosphorylation of long chain sphingoid bases on the primary alcohol group occurs in cells by the action of sphingosine kinase (1 –3 ). Sphingosine kinase is present in the cytosolic fraction of most cells (4 –7 ) and in the membrane fraction of certain tissues and organisms (8 ,9 ). The reaction product, sphingosine-1-phosphate (SPP), was considered for more than 20 yr to be merely an intermediate in the catabolism of long-chain sphingoid bases to palmitaldehyde and phosphoethanolamine (3 ,10 ). Studies in our lab stimulated new interest in the potential roles of SPP as a second messenger. Initially, we found that exogenous SPP initiated cell division of quiescent Swiss 3T3 fibroblasts (11 ) and induced inositol trisphosphate-independent release of calcium from intracellular stores (11 ,12 ). SPP also has been shown to affect several signal transduction pathways including phospholipase D activation (13 ), stimulation of the Raf/MEK/ERK signaling pathway (14 ,15 ), and inhibition of ceramide-induced activation of stress-activated protein kinase (SAPK/JNK) that leads to apoptotic responses (15 ). Additional effects of SPP include stimulation of tyrosine phosphorylation of focal adhesion kinase (FAK) and the cytoskeleton-associated protein paxillin (16 ). These tyrosine phosphorylations are mediated through the activation of the small G protein rho, which also mediates stress fiber formation induced by SPP (16 ).