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The Src Family Kinases Hck and Fgr Regulate Neutrophil Responses to N-Formyl-Methionyl-Leucyl-Phenylalanine¹

Laura Fumagalli^*, Hong Zhang, Anna Baruzzi^*, Clifford A. Lowell and Giorgio Berton^2,*

^* Department of Pathology, Section of General Pathology, University of Verona, Verona, Italy; and Department of Laboratory Medicine, University of California, San Francisco, CA 94143

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-µm pore size Transwells was normal, deficiency, or inhibition, of Src kinases resulted in a failure of neutrophils to migrate through 1-µm 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 Ca²⁺ 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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from Ministero dell’Istruzione dell’Università e della Ricerca of Italy (COFIN, FIRB), Associazione Italiana Ricerca sul Cancro (Associazione Italiana per la Ricerca sul Cancro), and Fondazione CariVerona (Bando 2001/2005) (to G.B.), and by National Institutes of Health Grants AI065495 and AI065150 (to C.A.L.).

² Address correspondence and reprint requests to Dr. Giorgio Berton, Department of Pathology, Section of General Pathology, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy. E-mail address: giorgio.berton{at}univr.it

³ Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; fMLP, formyl-methionyl-leucyl-phenilalanine; PLC, phospholipase C; IP₃, inositol trisphosphate; PKC, protein kinase C; WT, wild type; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PAK, p21-activated protein kinase; GEF, guanine nucleotide exchange factor; KO, knockout; T, temperature; CB, cytochalasin B; PAO, phenylarsine oxide; SB, sample buffer; SAPK, stress-activated protein kinase; Csk, C-terminal Src kinase.

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