Signal Transduction in Mast Cells

肥大细胞中的信号转导

• 批准号: 6227917
• 负责人: Reuben P. Siraganian
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6227917
• 关键词: CD antigens; basophils; biological signal transduction; cell line; cytokine; enzyme activity; immunomodulators; mast cell; protein tyrosine kinase; protein tyrosine phosphatase; site directed mutagenesis

The activation of mast cells and basophils by immunoglobulin cell surface receptors results in the release of an array of inflammatory mediators. This project investigates the molecular mechanisms involved in signal transduction in these cells using as a model the rat basophilic leukemia RBL-2H3 cultured cell line. In previous studies we observed that protein tyrosine phosphorylation is an early and critical signal for FceRI induced degranulation. The protein tyrosine kinase Syk was found to be tyrosine phosphorylated and activated after receptor aggregation. A variant of the RBL-2H3 mast cells that has no detectable Syk was also identified and demonstrated that Syk is essential for the receptor-induced release of inflammatory mediators. Analysis of signaling pathways in these cells indicate that most of the FceRI- induced cellular protein tyrosine phosphorylation is downstream of Syk and requires enzymatically active Syk.The Syk negative mast cells were used to examine the mechanism of Syk regulation. We observed that the activation loop tyrosines were essential for Syk mediated propagation of FceRI signaling although they were not essential for the kinase activity. By expression of a mutant of Syk we found that a tyrosine in the linker region of Syk functions to negatively regulate its kinase activity and thereby the signals leading to the release of inflammatory mediators. Mutation of this site resulted in a dramatic gain of the function of Syk in cells with enhancement of the release of mediators. As the level of protein phosphorylation of molecules is due to the balance between protein tyrosine kinase and phosphatase activities, we investigated the role of protein tyrosine phosphatases in signal transduction in mast cells. CD45 and Src homology 2 domain containing protein tyrosine phosphatases SHP-1 and SHP-2 are among the protein tyrosine phosphatase present in mast cells. In experiments during this year we observed that that CD45 is essential for ZAP70, but not for Syk, to reconstitute antigen receptor-initiated degranulation signals in mast cells. Furthermore the protein tyrosine phosphatase SHP-1 was found to regulate mast cell cytokine production but not the release of other mediators such as histamine. - Mast Cells, Basophils, Signal transduction, FceRI, protein tyrosine kinases/phosphatases, Syk

免疫球蛋白细胞表面受体激活肥大细胞和嗜碱性粒细胞，导致一系列炎症介质的释放。该项目以大鼠嗜碱性白血病 RBL-2H3 培养细胞系为模型，研究这些细胞中信号转导所涉及的分子机制。在之前的研究中，我们观察到蛋白质酪氨酸磷酸化是 FceRI 诱导脱粒的早期且关键的信号。发现蛋白酪氨酸激酶Syk在受体聚集后被酪氨酸磷酸化并激活。还鉴定出了一种未检测到 Syk 的 RBL-2H3 肥大细胞变体，并证明 Syk 对于受体诱导的炎症介质释放至关重要。对这些细胞中信号通路的分析表明，大多数 FceRI 诱导的细胞蛋白酪氨酸磷酸化位于 Syk 下游，并且需要具有酶活性的 Syk。使用 Syk 阴性肥大细胞来检查 Syk 调节机制。我们观察到激活环酪氨酸对于 Syk 介导的 FceRI 信号传播至关重要，尽管它们对于激酶活性不是必需的。通过 Syk 突变体的表达，我们发现 Syk 连接区中的酪氨酸可负向调节其激酶活性，从而导致炎症介质释放的信号。该位点的突变导致细胞中 Syk 功能的显着增强，并增强介质的释放。由于分子的蛋白质磷酸化水平是由于蛋白酪氨酸激酶和磷酸酶活性之间的平衡，因此我们研究了蛋白酪氨酸磷酸酶在肥大细胞信号转导中的作用。含有 CD45 和 Src 同源 2 结构域的蛋白质酪氨酸磷酸酶 SHP-1 和 SHP-2 属于存在于肥大细胞中的蛋白质酪氨酸磷酸酶。在今年的实验中，我们观察到 CD45 对于 ZAP70 而言是必需的，但对于 Syk 而言并非如此，以重建肥大细胞中抗原受体启动的脱粒信号。此外，发现蛋白酪氨酸磷酸酶 SHP-1 可以调节肥大细胞细胞因子的产生，但不能调节组胺等其他介质的释放。 - 肥大细胞、嗜碱性粒细胞、信号转导、FceRI、蛋白酪氨酸激酶/磷酸酶、Syk