SRC FAMILY PROTEIN TYROSINE KINASES IN HEMATOPOIESIS

Src和Syk家族酪氨酸激酶调节白细胞的许多细胞内信号通路，包括对免疫复合物、细胞因子和粘附蛋白的反应。来自缺乏src家族激酶Hck、Fgr和Lyn的敲除小鼠的巨噬细胞由于无法在吞噬囊泡中形成丝状肌动蛋白，在fcγ受体介导的吞噬中存在缺陷。syk缺陷型巨噬细胞也完全阻断fgammar依赖性吞噬，然而这些细胞的缺陷发生在f -肌动蛋白形成之后的一个步骤；Syk +/-巨噬细胞不能完全闭合吞噬囊泡。Src家族缺陷中性粒细胞f -肌动蛋白形成受损也导致β 2或β 3整合素交联后活化受损，而这些动物的巨噬细胞表现出细胞骨架结构、细胞扩散和迁移的改变。骨髓细胞功能缺陷导致基因敲除小鼠体内免疫反应改变当与母鼠杂交时，Hck -/-fgr-/-突变体在内毒素血症中遭受的组织损伤减少，炎症性疾病的发展减弱。我们假设src家族突变体中导致细胞f -肌动蛋白形成的信号通路受损是由于rho家族gtpase缺乏激活，而Syk可能参与PI-3激酶依赖的信号通路事件。我们将通过检测hck-/-fgr-/-lyn-/-巨噬细胞与syk-/-细胞中GTPase的激活情况，在FcgammaR信号通路中验证这一假设，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。在这些突变小鼠中，fgammar信号缺陷可能导致免疫球蛋白介导疾病的发展迟缓；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进行测试。与上述信号反应的损伤相反，src家族激酶，特别是Lyn激酶的缺乏，会导致对GM-CSF信号的反应增强。这一意想不到的观察结果可能是由于该激酶通过抑制受体在信号传导中的作用。我们将在突变细胞中检查其他GM-CSF依赖的功能反应，信号传导和抑制受体功能。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-/-lyn-/-小鼠中丢失。我们将通过检测其他突变体的血小板功能，并将Src和syk缺陷小鼠与ALV-R动物杂交，以促进基因转导研究，从而扩展这些研究。这些项目将扩大我们对src家族和Syk激酶在造血细胞中的功能的理解，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.