ACTIVATION OF P58(C-FGR) AND P53 56(LYN) IN ADHERENT HUMAN NEUTROPHILS - EVIDENCE FOR A ROLE OF DIVALENT-CATIONS IN REGULATING NEUTROPHIL ADHESION AND PROTEIN-TYROSINE KINASE-ACTIVITIES/

Tumor necrosis factor (TNF) stimulates generation of reactive oxygen i ntermediates, 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 depend ent on beta 2 integrins. Tyrosine phosphorylation of distinct proteins [Fuortes et al., J Cell Biol 120: 777-784, 1993] and activation of th e protein tyrosine kinase p58(c-fgr) [Berton et al., J Cell Biol 126:1 111-1121, 1994] were recently recognized as signals involved in beta 2 integrin-dependent responses of TNF-treated PMN. As the integrin capa bility to bind their ligands is regulated by divalent cations we inves tigated whether modulation of PMN adhesion to fibrinogen by divalent c ations also affected activation of protein tyrosine kinases. lit the a bsence of divalent cations or in the presence of Ca2+ alone, PMN did n ot adhere to fibrinogen in response to TNF. However, Mg2+, either alon e or together with Ca2+, promoted stimulated adhesion to fibrinogen. W e also found that Mn2+ promoted PMN adhesion to fibrinogen without add itional stimuli Analysis of the activity of two src family tyrosine ki nases, p58(c-fgr) and p53/56(lyn), showed that their autophosphorylati ng kinase activity strictly correlated with adhesion. In fact, only in the presence of Mg2+, but not in the absence of divalent cations or i n the presence of Ca2+ alone, TNF increased p58(c-fgr) and p53/56(lyn) kinase activities; and this was prevented by anti-CD18 antibodies. In addition, Mn2+ strongly promoted activation of p58(c-fgr) and p53/56( lyn) without additional stimuli. Analysis of tyrosine phosphorylated p roteins with anti-phosphotyrosine immunoblots showed that divalent cat ions regulated adhesion and protein tyrosine phosphorylation in the sa me fashion. Detergent extraction of proteins showed that the Mg2+-depe ndent, TNF-stimulated adhesion redistributed p58(c-fgr) and p53/56(lyn ) to a Triton-insoluble fraction. In addition, analysis of p58(c-fgr) activity allowed us to demonstrate that the fraction of p58(c-fgr) whi ch became Triton-insoluble displayed a higher kinase activity. These f indings establish that PMN adhesion signals for activation of two diff erent src family tyrosine kinases. The evidence that Mn2+, a strong pr omoter of integrin function, induces adhesion and activation of tyrosi ne kinases without additional stimuli suggest the existence of a direc t link between beta 2 integrins binding to fibrinogen and activation o f tyrosine kinases in neutrophils. (C) 1995 Wiley-Liss, Inc.