FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.
Chemoattractants bind to seven transmembrane-spanning, G-protein-linked receptors on polymorphonuclear leukocytes (neutrophils) and induce a variety of functional responses, including activation of microtubule-associated protein (MAP) kinase. Although the pathways by which MAP kinases are activated in neutrophils are unknown, we hypothesized that activation of the Ras/Raf pathway leading to activation of MAP/ERK kinase (MEK) would be induced by the chemoattractant f-met-leu-phe. Human neutrophils exposed to 10 nM FMLP for 30 s exhibited an MAP kinase kinase activity coeluting with MEK-1. Immunoprecipitation of Raf-1 kinase after stimulation with FMLP revealed an activity that phosphorylated MEK, was detectable at 30 s, and peaked at 2-3 min. Immunoprecipitation of Ras from both intact neutrophils labeled with [32P]orthophosphate and electropermeabilized neutrophils incubated with [32P]GTP was used to determine that FMLP treatment was associated with activation of Ras. Activation of both Ras and Raf was inhibited by treatment of neutrophils with pertussis toxin, indicating predominant linkage to the Gi2 protein. Although phorbol esters activated Raf, activation induced by FMLP appeared independent of protein kinase C, further suggesting that Gi2 was linked to Ras and Raf independent of phospholipase C and protein kinase C. Dibutyryl cAMP, which inhibits many neutrophil functional responses, blocked the activation of Raf by FMLP, suggesting that interruption of the Raf/MAP kinase pathway influences neutrophil responses to chemoattractants. These data suggest that Gi2-mediated receptor regulation of the Ras/Raf/MAP kinase pathway is a primary response to chemoattractants.
Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
G S
GS
Worthen
N
N
Avdi
A M
AM
Buhl
N
N
Suzuki
G L
GL
Johnson
D000595:Amino Acid Sequence; D016227:Benzoquinones; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology; D011905:Genes, ras; D006801:Humans; D047029:Lactams, Macrocyclic; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D037342:Pertussis Toxin; D011493:Protein Kinase C / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D019908:Proto-Oncogene Proteins c-raf; D011519:Proto-Oncogenes; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D011518:Proto-Oncogene Proteins; D011809:Quinones; D010566:Virulence Factors, Bordetella; D017828:Rifabutin; D009240:N-Formylmethionine Leucyl-Phenylalanine; C020754:herbimycin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins