PRODUCTS OF SPHINGOLIPID CATABOLISM BLOCK ACTIVATION OF THE P21-ACTIVATED PROTEIN-KINASES IN NEUTROPHILS

Neutrophils stimulated with the chemoatttractant FMLP are known to exh ibit a rapid and transient activation of two p21-activated protein kin ases (Paks) with molecular masses of approximately 63 and 69 kDa, Paks can be detected by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297 to 331 of the 47-kDa subunit of the nicotinamide-ade nine dinucleotide phosphate-oxidase complex (p47-phox) fixed within a gel. In this study, we demonstrate that N-acetylsphingosine (C-2-ceram ide) and a variety of sphingoid bases (e.g., D-erythrosphingosine) blo ck activation of the 63- and 69-kDa Paks in neutrophils. The concentra tions of these lipids that were effective in blocking Pak activation w ere similar to those that inhibit a variety of neutrophil responses. A ctivation of the 63- and 69-kDa Paks was also markedly reduced in neut rophils treated with sphingomyelinase before stimulation. Moreover, we report that addition of C-2-ceramide or D-erythrosphingosine to neutr ophils after stimulation with FMLP markedly enhances the rate of Pak i nactivation. These effects were not mimicked by arachidonate, which is a potent disorganizing agent of neutrophil membranes, These data supp ort and extend the proposal that sphingoid bases may establish a set p oint int neutrophils for positive stimuli.