CHARACTERIZATION OF SPHINGOSINE KINASE (SK) ACTIVITY IN SACCHAROMYCES-CEREVISIAE AND ISOLATION OF SK-DEFICIENT MUTANTS

Sphingosine kinase (SK) catalyses the phosphorylation of sphingosine t o generate sphingosine 1phosphate, which is a second messenger involve d in the proliferative responses of mammalian cells. Although the yeas t Saccharomyces cerevisiae has similar phosphorylated sphingoid bases which appear to be involved in growth regulation and the response to s tress, SK activity had not been previously demonstrated in yeast. In t his study, an in vitro system was set up to characterize yeast SK acti vity. Activity was detected in the cytosol at neutral pH and 37 degree s C. Yeast SK phosphorylated the sphingoid bases sphingosine, dihydros phingosine and phytosphingosine. (D,L)-threo-dihydrosphingosine, an in hibitor of mammalian SK, did not inhibit the yeast enzyme. Unique prop erties of yeast SK were an optimal temperature of 43 degrees C, and in vivo activation during nutrient deprivation. Spontaneous mutants with diminished SK activity were isolated utilizing a screen for resistanc e 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 prote in in yeast.