title: Activation of p58c-fgr and p53/56lyn in adherent human neutrophils: evidence for a role of divalent cations in regulating neutrophil adhesion and protein tyrosine kinase activities.
abstract: Tumor necrosis factor (TNF) stimulates generation of reactive oxygen intermediates, secretion of granule constituents, and rearrangement of the cytoskeleton in neutrophils (PMN); this response requires that PMN be adherent to plasma or extracellular matrix proteins, and is dependent on beta 2 integrins. Tyrosine phosphorylation of distinct proteins [Fuortes et al., J Cell Biol 120:777-784, 1993] and activation of the protein tyrosine kinase p58c-fgr [Berton et al., J Cell Biol 126:1111-1121, 1994] were recently recognized as signals involved in beta 2 integrin-dependent responses of TNF-treated PMN. As the integrin capability to bind their ligands is regulated by divalent cations we investigated whether modulation of PMN adhesion to fibrinogen by divalent cations also affected activation of protein tyrosine kinases. In the absence of divalent cations or in the presence of Ca2+ alone, PMN did not adhere to fibrinogen in response to TNF. However, Mg2+, either alone or together with Ca2+, promoted stimulated adhesion to fibrinogen. We also found that Mn2+ promoted PMN adhesion to fibrinogen without additional stimuli. Analysis of the activity of two src family tyrosine kinases, p58c-fgr and p53/56lyn, showed that their autophosphorylating kinase activity strictly correlated with adhesion. In fact, only in the presence of Mg2+, but not in the absence of divalent cations or in the presence of Ca2+ alone, TNF increased p58c-fgr and p53/56lyn kinase activities; and this was prevented by anti-CD18 antibodies. In addition, Mn2+ strongly promoted activation of p58c-fgr and p53/56lyn without additional stimuli. Analysis of tyrosine phosphorylated proteins with anti-phosphotyrosine immunoblots showed that divalent cations regulated adhesion and protein tyrosine phosphorylation in the same fashion. Detergent extraction of proteins showed that the Mg(2+)-dependent, TNF-stimulated adhesion redistributed p58c-fgr and p53/56lyn to a Triton-insoluble fraction. In addition, analysis of p58c-fgr activity allowed us to demonstrate that the fraction of p58c-fgr which became Triton-insoluble displayed a higher kinase activity. These findings establish that PMN adhesion signals for activation of two different src family tyrosine kinases. The evidence that Mn2+, a strong promoter of integrin function, induces adhesion and activation of tyrosine kinases without additional stimuli suggest the existence of a direct link between beta 2 integrins binding to fibrinogen and activation of tyrosine kinases in neutrophils.
authors.affiliation: Institute of General Pathology, University of Verona, Italy.
authors.forename: S R
authors.initials: SR
authors.lastname: Yan
authors.forename: L
authors.initials: L
authors.lastname: Fumagalli
authors.forename: G
authors.initials: G
authors.lastname: Berton
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D018821:CD18 Antigens / Q000276:immunology; D002413:Cations, Divalent / Q000378:metabolism*; D002448:Cell Adhesion; D018844:Cyclin-Dependent Kinases; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D017830:Octoxynol; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000502:physiology*
chemical_list: D000906:Antibodies; D018821:CD18 Antigens; D002413:Cations, Divalent; D014409:Tumor Necrosis Factor-alpha; D014443:Tyrosine; D017830:Octoxynol; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D017346:Protein Serine-Threonine Kinases; C447612:CDK11a protein, human; D018844:Cyclin-Dependent Kinases; D008274:Magnesium