SRC FAMILY PROTEIN TYROSINE KINASES IN HEMATOPOIESIS

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The Src and Syk family tyrosine kinases regulate a number of intracellular signaling pathways in leukocytes, including responses to immune complexes, cytokines and adhesion proteins. Macrophages from knockout mice lacking the Src-family kinases Hck, Fgr, and Lyn are defective in Fcgamma receptor-mediated phagocytosis due to an inability to form filamentous actin in phagocytic vesicles. Syk-deficient macrophages also have a complete block in FcgammaR dependent phagocytosis, however the defect in these cells occurs at a step subsequent to F-actin formation; syk+/- macrophages fail to complete closure of the phagocytic vesicle. Impaired F-actin formation in Src- family deficient neutrophils also leads impaired activation following crosslinking of beta2 or beta3 integrins, while macrophages from these animals manifest alterations in cytoskeletal structure, cell spreading and migration. Defects in myeloid cell function result in altered immune responses in knockout mice in vivo; hck-/-fgr-/- mutants suffer reduced tissue damage during endotoxemia and blunted development of inflammatory disease when crossed with motheaten mice. We hypothesize that impaired signaling leading to cellular F-actin formation in Src-family mutants is due to lack of activation of Rho-family GTPases, while Syk may be involved in PI-3 kinase dependent signaling events. We will test this hypothesis in the FcgammaR signaling pathway by examining GTPase activation in hck-/-fgr-/-lyn-/- macrophages versus syk-/- cells and attempting to rescue the functional defects in these pathways by retroviral gene transduction. Defects in FcgammaR signaling should lead to retarded development of immunoglobulin- mediated diseases in these mutant mice; we will test this using an autoAb hemolytic anemia model and an immune complex inflammation model. In contrast to the impairments in the above signaling responses, deficiency of the Src-family kinases, in particular the Lyn kinase, leads to enhanced responses to GM-CSF signaling. This unexpected observation may be due to the role of this kinase in signaling through inhibitory receptors. We will examine additional GM-CSF dependent functional responses, signaling and inhibitory receptor function in the mutant cells. Initial studies have also revealed that collagen-receptor (GP VI) mediated platelet activation is lost in fyn-/-lyn-/- mice. We will extend these studies by examining platelet function in other mutants and by crossing the Src- and Syk-deficient mice to the ALV-R animals to facilitate gene transduction studies. These projects will expand our understanding of the functions of Src-family and Syk kinases in hematopoietic cells and will validate whether these kinases are potential targets for therapeutics against inflammatory diseases.