SRC FAMILY PROTEIN TYROSINE KINASES IN HEMATOPOIESIS

Src和Syk家族酪氨酸激酶调节白细胞中的许多细胞内信号传导途径，包括对免疫复合物、细胞因子和粘附蛋白的应答。来自缺乏Src家族激酶Hck、Fgr和林恩的敲除小鼠的巨噬细胞由于不能在吞噬囊泡中形成丝状肌动蛋白而在Fc γ受体介导的吞噬作用中有缺陷。Syk缺陷型巨噬细胞在FcgammaR依赖性吞噬作用中也具有完全阻断，然而这些细胞中的缺陷发生在F-肌动蛋白形成之后的步骤; syk+/-巨噬细胞不能完全关闭吞噬囊泡。Src家族缺陷型中性粒细胞中F-肌动蛋白形成受损也导致β 2或β 3整联蛋白交联后活化受损，而这些动物的巨噬细胞表现出细胞骨架结构、细胞铺展和迁移的改变。骨髓细胞功能缺陷导致基因敲除小鼠体内免疫反应改变; hck-/-fgr-/-突变体在内毒素血症时组织损伤减少，与蛀虫小鼠杂交时炎症性疾病发展减弱。我们假设，受损的信号传导导致细胞F-肌动蛋白形成Src家族突变体是由于缺乏激活的Rho家族GTP酶，而Syk可能参与PI-3激酶依赖的信号传导事件。我们将通过检查hck-/-fgr-/-林恩-/-巨噬细胞与syk-/-细胞中的GTPase激活来验证FcgammaR信号通路中的这一假设，并试图通过逆转录病毒基因转导来挽救这些通路中的功能缺陷。Fc γ R信号传导的缺陷应导致这些突变小鼠中免疫球蛋白介导的疾病发展迟缓;我们将使用自身抗体溶血性贫血模型和免疫复合物炎症模型对此进行测试。与上述信号传导应答中的损伤相反，Src家族激酶，特别是林恩激酶的缺乏导致对GM-CSF信号传导的应答增强。这种意想不到的观察结果可能是由于这种激酶通过抑制性受体在信号传导中的作用。我们将研究额外的GM-CSF依赖的功能反应，信号传导和抑制性受体功能的突变细胞。初步研究还表明，胶原蛋白受体（GP VI）介导的血小板活化在fyn-/-林恩-/-小鼠中丢失。我们将通过检查其他突变体中的血小板功能以及将Src和Syk缺陷小鼠与ALV-R动物杂交以促进基因转导研究来扩展这些研究。这些项目将扩大我们对Src家族和Syk激酶在造血细胞中功能的理解，并将验证这些激酶是否是治疗炎症性疾病的潜在靶点。

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.