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.
M'Rabet
L
L
Laboratory for Physiological Chemistry and Centre for Biomedical Genetics, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.
Coffer
P J
PJ
Wolthuis
R M
RM
Zwartkruis
F
F
Koenderman
L
L
Bos
J L
JL
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