SIGNALING BY ADHESION IN HUMAN NEUTROPHILS - ACTIVATION OF THE P72(SYK) TYROSINE KINASE AND FORMATION OF PROTEIN COMPLEXES CONTAINING P72(SYK) AND SRC FAMILY KINASES IN NEUTROPHILS SPREADING OVER FIBRINOGEN

Src family kinases are implicated in signaling by integrins in polymor phonuclear neutrophils (PMN). To identify proteins present in complexe s with Src family kinases, we subjected p58(c-fgr) or p53/56(lyn) immu noprecipitates from Triton X-100 lysates of PMN incubated on fibrinoge n-coated surfaces to in vitro kinase assays. Assays on p58(c-fgr) or p 53/56(lyn) immunoprecipitates from Triton lysates of PMN induced to sp read over fibrinogen in response to TNF-alpha showed that several prot eins form complexes with Src family kinases and can be phosphorylated in vitro. Immunoblot analysis showed that the p72(syk) tyrosine kinase is one of these proteins. Formation of protein complexes containing S rc family kinases and p72(syk) required PMN spreading because p72(syk) was not detected in p58(c-fgr) or p53/56(lyn) immunoprecipitates from PMN, which were stimulated with TNF-alpha, in suspension. In addition , induction of PMN spreading with Mn2+ in the absence of TNF-alpha pro moted the formation of protein complexes containing Src family kinases and p72(syk). We also found that p72(syk)-autophosphorylating kinase activity was enhanced, and a fraction of p72(syk) was translocated to a Triton-insoluble cytoskeletal fraction in PMN induced to spread over fibrinogen by TNF-alpha or Mn2+. In vitro kinase assays on CD18 (beta (2) integrin subunit) immunoprecipitates from Triton lysates of spread PMN showed that several proteins formed complexes with CD18 and could be phosphorylated in vitro. Immunoblot analysis of CD18 immunoprecipi tates allowed us to identify p72(syk) as one of these proteins. These findings show that PMN spreading is accompanied by activation of p72(s yk) and formation of multimolecular complexes in which Src family kina ses and p72(syk) colocalize.