Lyn Kinase-Mediated Regulation of Allergic Inflammation

描述（由申请人提供）：过敏反应的症状部分是由含有IgE受体的嗜碱性细胞和肥大细胞释放预先形成的和新合成的介质引起的。通过IgE高亲和受体（FceRI）启动信号转导需要在受体的β链和γ链中磷酸化不同的酪氨酸残基。src家族激酶Lyn负责受体磷酸化以及许多下游信号分子（如Syk激酶）的激活。在肥大细胞/嗜碱性粒细胞细胞系中，Lyn的两个亚型（Lyn A和Lyn B）中的一小部分与未磷酸化的IgE受体相关。多种方法表明，Lyn的独特结构域与受体β链的c端有关。我们已经研究了转染的Lyn独特结构域破坏FceRI信号传导和介质分泌的能力。与对照组相比，稳定表达Lyn独特结构域的大鼠嗜碱性白血病（RBL）转染物中组胺和tnf - α的释放被部分抑制。这种抑制伴随着FceRI酪氨酸磷酸化的部分减少、Syk激活的延迟和Gab2酪氨酸磷酸化的减少。相反，白三烯C4 （LTC4）的分泌比对照转染物上调了3至5倍，并伴有MAP激酶途径的激活增加。我们建议在Src家族激酶（Lyn或Fyn）敲除小鼠的骨髓源性肥大细胞（BMMC）中表达Lyn独特结构域，并监测信号和介质的分泌。我们将研究磷酸酶PP2A、ship - 1和SHP-2在BMMC中LTC4分泌中的作用。通过对重组Lyn的体外激酶分析，我们发现Lyn B对底物肽的特异性活性是Lyn a的三倍。我们将重新引入Lyn a或Lyn B到Lyn敲除BMMC中，以研究每种亚型对FceRI信号传导和分泌的特异性贡献。我们还准备制备转基因Lyn独特结构域小鼠，以研究引入Lyn独特结构域限制过敏性炎症、气道高反应性和BMMC介质分泌的能力。了解Lyn独特结构域和FceRI β相互作用的分子细节可能有助于开发出限制过敏反应而不是减轻症状的药物。

DESCRIPTION (provided by applicant): The symptoms of an allergic reaction are generated in part by the allergen-induced release of preformed and newly synthesized mediators from basophils and mast cells bearing receptors for IgE. Initiation of signal transduction through the high affinity receptor for IgE (FceRI) requires phosphorylation of distinct tyrosine residues in the receptor's beta and gamma chains. The Src-family kinase, Lyn, is responsible for the receptor phosphorylation as well as the activation of numerous downstream signaling molecules, (e.g. Syk kinase). A small fraction of the two isoforms of Lyn (Lyn A and Lyn B) is associated with unphosphorylated IgE receptors in mast/basophil cell lines. A variety of approaches indicate that the unique domain of Lyn is associated with the C-terminus of the receptor's beta chain. We have investigated the ability of transfected Lyn unique domain to disrupt FceRI signaling and mediator secretion. The release of both histamine and TNF-alpha was partially inhibited in Rat Basophilic Leukemia (RBL) transfectants stably expressing Lyn unique domain, compared to secretion in control transfectants. This inhibition was accompanied by a partial decrease in FceRI tyrosine phosphorylation, delayed Syk activation, and reduced Gab2 tyrosine phosphorylation. Conversely, Leukotriene C4 (LTC4) secretion was upregulated three to five fold compared to control transfectants and accompanied by increased activation of the MAP kinase pathway. We propose to express Lyn unique domain in bone marrow-derived mast cells (BMMC) from Src family kinase (Lyn or Fyn) knockout mice and to monitor signaling and mediator secretion. We will investigate the role of the phosphatases PP2A, SHIP-l, and SHP-2 in LTC4 secretion in BMMC. By using in vitro kinase assays of recombinant Lyn, we have found that Lyn B has three times the specific activity towards a substrate peptide than Lyn A. We will reintroduce either Lyn A or Lyn B into Lyn knockout BMMC to investigate the specific contribution of each isoform to FceRI signaling and secretion. We also propose to prepare a transgenic Lyn unique domain mouse to investigate the ability of introduced Lyn unique domain to limit allergic inflammation, airway hyperreactivity, and BMMC mediator secretion. Understanding the molecular details of the interaction of the Lyn unique domain and FceRI beta may allow the development of drugs that will limit allergic reactions rather than mitigating symptoms.