MOLECULAR-CLONING AND FUNCTIONAL-CHARACTERIZATION OF MURINE SPHINOGOSINE KINASE

Sphingosine-l-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, an d extracellularly, it is a ligand for the G protein-coupled receptor E dp-1. Based on peptide sequences obtained from purified rat kidney sph ingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammali an sphingosine kinases (murine SPHK1a and SPHK1b), Sequence analysis i ndicates 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 k inase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positiv ely charged motif, GGHGK, which may be part of the ATP binding site. F rom Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectab le levels in skeletal muscle and Liver. Human embryonic kidney cells a nd NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) i n sphingosine kinase activity. The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phos phatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosi ne or N,N-dimethylsphingosine. The latter two sphingolipids were compe titive inhibitors of sphingosine kinase in the transfected cells as wa s previously found with the purified rat kidney enzyme. Transfected ce lls also had a marked increase in mass levels of SPP with a concomitan t decrease in levels of sphingosine and, to a lesser extent, in cerami de 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.