REGULATION OF SPHINGOSINE-ACTIVATED PROTEIN-KINASES - SELECTIVITY OF ACTIVATION BY SPHINGOID BASES AND INHIBITION BY NONESTERIFIED FATTY-ACIDS

Sphingosine has been shown to activate protein kinases in Jurkat T cel l cytosol [Pushkareva, Khan, Alessenko, Sahyoun and Hannun (1992) J. B iol. Chem. 267, 15246-15251]. In this study, two sphingosine-activated protein kinases were distinguished by their substrate specificity, th eir dose-response to sphingosine and the specificity of their activati on by sphingosine and dihydrosphingosine stereoisomers. A p32-sphingos ine-activated protein kinase responded to low concentrations of D-eryt hro-spingosine with an initial activation observed at 2.5 muM and a pe ak activity at 10-20 muM. This kinase showed a modest specificity for D-erythro-sphingosine over other sphingosine stereoisomers, and a pref erence for sphingosines over dihydrosphingosines. Phosphorylation of a p18 substrate required higher concentrations of sphingosine (20-100 m uM) and showed a significant preference for the erythro isomers of sph ingosine and dihydrosphingosine over the threo isomers. The ability of other lipids to modulate sphingosine activation of these kinases was also examined. Oleic acid, but not oleic alcohol or the methyl ester, induced the phosphorylation of a distinct set of substrates (probably through the activation of protein kinase C), and inhibited sphingosine -induced phosphorylation with an IC50 of approximately 20 muM. Oleic a nhydride failed to induce changes in basal protein phosphorylation but inhibited sphingosine-activated protein kinases, thus distinguishing the effects of fatty acids on protein kinase C from the inhibition of sphingosine-induced phosphorylation. These studies define two distinct sphingosine-activated protein kinases and reveal an important interac tion between two classes of putative lipid second messengers.