N,N-DIMETHYLSPHINGOSINE IS A POTENT COMPETITIVE INHIBITOR OF SPHINGOSINE KINASE BUT NOT OF PROTEIN-KINASE-C - MODULATION OF CELLULAR-LEVELSOF SPHINGOSINE 1-PHOSPHATE AND CERAMIDE

Sphingosine 1-phosphate (SPP), a lipid second messenger formed by the action of sphingosine kinase, has been implicated in regulating divers e biological processes, including growth, survival, and differentiatio n. N,N-Dimethylspingosine (DMS) inhibits sphingosine kinase and has be en used to investigate the biological roles of SPP; however, little is known of the mechanism of inhibition of sphingosine kinase by DMS. In addition, DMS has been shown to inhibit protein kinase C in vitro. He re we report that DMS is a competitive inhibitor of sphingosine kinase from U937 monoblastic leukemia cells, Swiss 3T3 fibroblasts, and PC12 pheochromocytoma cells. DMS decreases basal levels of SPP and prevent s increases in SPP in response to physiological stimuli known to activ ate sphingosine kinase. DMS also effectively increases cellular levels of ceramide in a variety of cell types, and resetting of the ceramide /SPP rheostat may account for the pro-apoptotic effects of DMS. Moreov er, DMS, at concentrations which effectively inhibit sphingosine kinas e, has no effect on protein kinase C activity or its membrane transloc ation. Thus, DMS acts as a specific competitive inhibitor of sphingosi ne kinase in diverse cell types and is a useful tool to elucidate the role of SPP as an intracellular second messenger.