title: The Src family kinases Hck and Fgr regulate neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine.
abstract: The chemotactic peptide formyl-methionyl-leucyl-phenilalanine (fMLP) triggers intracellular protein tyrosine phosphorylation leading to neutrophil activation. Deficiency of the Src family kinases Hck and Fgr have previously been found to regulate fMLP-induced degranulation. In this study, we further investigate fMLP signaling in hck-/-fgr-/- neutrophils and find that they fail to activate a respiratory burst and display reduced F-actin polymerization in response to fMLP. Additionally, albeit migration of both hck-/-fgr-/-mouse neutrophils and human neutrophils incubated with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) through 3-microm pore size Transwells was normal, deficiency, or inhibition, of Src kinases resulted in a failure of neutrophils to migrate through 1-microm pore size Transwells. Among MAPKs, phosphorylation of ERK1/2 was not different, phosphorylation of p38 was only partially affected, and phosphorylation of JNK was markedly decreased in fMLP-stimulated hck-/-fgr-/- neutrophils and in human neutrophils incubated with PP2. An increase in intracellular Ca(2+) concentration and phosphorylation of Akt/PKB occurred normally in fMLP-stimulated hck-/-fgr-/- neutrophils, indicating that activation of both phosphoinositide-specific phospholipase C and PI3K is independent of Hck and Fgr. In contrast, phosphorylation of the Rho/Rac guanine nucleotide exchange factor Vav1 and the Rac target p21-activated kinases were markedly reduced in both hck-/-fgr-/- neutrophils and human neutrophils incubated with a PP2. Consistent with these findings, PP2 inhibited Rac2 activation in human neutrophils. We suggest that Hck and Fgr act within a signaling pathway triggered by fMLP receptors that involves Vav1 and p21-activated kinases, leading to respiratory burst and F-actin polymerization.
authors.affiliation: Department of Pathology, Section of General Pathology, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Fumagalli
authors.forename: Hong
authors.initials: H
authors.lastname: Zhang
authors.forename: Anna
authors.initials: A
authors.lastname: Baruzzi
authors.forename: Clifford A
authors.initials: CA
authors.lastname: Lowell
authors.forename: Giorgio
authors.initials: G
authors.lastname: Berton
mesh_terms: D000818:Animals; D015550:Cell Degranulation / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D018375:Neutrophil Activation / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D009504:Neutrophils / Q000276:immunology*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000276:immunology*; D051057:Proto-Oncogene Proteins c-akt / Q000276:immunology; D051572:Proto-Oncogene Proteins c-hck / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000276:immunology*; D051567:Proto-Oncogene Proteins c-vav / Q000276:immunology; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000276:immunology*
chemical_list: C412373:AG 1879; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; D011743:Pyrimidines; C496619:Vav1 protein, mouse; D009240:N-Formylmethionine Leucyl-Phenylalanine; C496632:Hck protein, mouse; D051572:Proto-Oncogene Proteins c-hck; C053102:proto-oncogene proteins c-fgr; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt