Lyn Kinase-Mediated Regulation of Allergic Inflammation

描述（由申请方提供）：过敏反应的症状部分是由过敏原诱导的预先形成和新合成的介质从嗜碱性粒细胞和携带IgE受体的肥大细胞中释放而产生的。通过IgE的高亲和力受体（FceRI）的信号转导的起始需要受体的β和γ链中的不同酪氨酸残基的磷酸化。Src家族激酶林恩负责受体磷酸化以及许多下游信号传导分子（例如Syk激酶）的活化。林恩的两种同种型（林恩A和林恩B）的一小部分与肥大细胞/嗜碱性细胞系中的非磷酸化IgE受体相关。多种方法表明，林恩的独特结构域与受体β链的C-末端相关。我们已经研究了转染的林恩独特结构域破坏FceRI信号传导和介体分泌的能力。与对照转染子中的分泌相比，在稳定表达林恩独特结构域的大鼠嗜碱性白血病（RBL）转染子中组胺和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降低、Syk活化延迟和Gab 2酪氨酸磷酸化降低。相反，与对照转染子相比，白三烯C4（LTC 4）分泌上调三至五倍，并伴随MAP激酶途径的活化增加。我们拟在Src家族激酶（林恩或Fyn）基因敲除小鼠的骨髓源性肥大细胞（BMMC）中表达林恩独特结构域，并监测信号传导和介质分泌。我们将研究磷酸酶PP 2A、SHIP-1和SHP-2在BMMC中LTC 4分泌中的作用。通过使用重组林恩的体外激酶测定，我们发现林恩B对底物肽的比活性是林恩A的三倍。我们将林恩A或林恩B重新引入林恩敲除的BMMC中以研究每种同种型对FceRI信号传导和分泌的具体贡献。我们还建议制备转基因林恩独特结构域小鼠，以研究引入的林恩独特结构域限制过敏性炎症、气道高反应性和BMMC介体分泌的能力。了解林恩独特结构域和FceRI β相互作用的分子细节可能允许开发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.