Differential fMet-Leu-Phe- and platelet-activating factor-induced signaling toward Ral activation in primary human neutrophils.
We have measured the activation of the small GTPase Ral in human neutrophils after stimulation with fMet-Leu-Phe (fMLP), platelet activating factor (PAF), and granulocyte macrophage-colony stimulating factor and compared it with the activation of two other small GTPases, Ras and Rap1. We found that fMLP and PAF, but not granulocyte macrophage-colony stimulating factor, induce Ral activation. All three stimuli induce the activation of both Ras and Rap1. Utilizing specific inhibitors we demonstrate that fMLP-induced Ral activation is mediated by pertussis toxin-sensitive G-proteins and partially by Src-like kinases, whereas fMLP-induced Ras activation is independent of Src-like kinases. PAF-induced Ral activation is mediated by pertussis toxin-insensitive proteins, Src-like kinases and phosphatidylinositol 3-kinase. Phosphatidylinositol 3-kinase is not involved in PAF-induced Ras activation. The calcium ionophore ionomycin activates Ral, but calcium depletion partially inhibits fMLP- and PAF-induced Ral activation, whereas Ras activation was not affected. In addition, 12-O-tetradecanoylphorbol-13-acetate-induced activation of Ral is completely abolished by inhibitors of protein kinase C, whereas 12-O-tetradecanoylphorbol-13-acetate-induced Ras activation is largely insensitive. We conclude that in neutrophils Ral activation is mediated by multiple pathways, and that fMLP and PAF induce Ral activation differently.
Laboratory for Physiological Chemistry and Centre for Biomedical Genetics, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.
L
L
M'Rabet
P J
PJ
Coffer
R M
RM
Wolthuis
F
F
Zwartkruis
L
L
Koenderman
J L
JL
Bos
D002118:Calcium / Q000097:blood; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000097:blood; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000097:blood; D019204:GTP-Binding Proteins / Q000097:blood*; D020690:GTPase-Activating Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000494:pharmacology*; D011493:Protein Kinase C / Q000097:blood; D011506:Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020725:ral GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D010972:Platelet Activating Factor; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020725:ral GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium