Products of sphingolipid catabolism block activation of the p21-activated protein kinases in neutrophils.
Neutrophils stimulated with the chemoattractant FMLP are known to exhibit a rapid and transient activation of two p21-activated protein kinases (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-adenine dinucleotide phosphate-oxidase complex (p47-phox) fixed within a gel. In this study, we demonstrate that N-acetylsphingosine (C2-ceramide) and a variety of sphingoid bases (e.g., D-erythrosphingosine) block activation of the 63- and 69-kDa Paks in neutrophils. The concentrations of these lipids that were effective in blocking Pak activation were similar to those that inhibit a variety of neutrophil responses. Activation of the 63- and 69-kDa Paks was also markedly reduced in neutrophils treated with sphingomyelinase before stimulation. Moreover, we report that addition of C2-ceramide or D-erythrosphingosine to neutrophils after stimulation with FMLP markedly enhances the rate of Pak inactivation. These effects were not mimicked by arachidonate, which is a potent disorganizing agent of neutrophil membranes. These data support and extend the proposal that sphingoid bases may establish a set point in neutrophils for positive stimuli.
Arthritis Unit, Massachusetts General Hospital, Boston 02114, USA.
J P
JP
Lian
R
R
Huang
D
D
Robinson
J A
JA
Badwey
D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D018375:Neutrophil Activation / Q000187:drug effects*; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D054462:p21-Activated Kinases
D004791:Enzyme Inhibitors; C064769:N-acetylsphingosine; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D013110:Sphingosine