Signal Transduction in Mast Cells

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。本课题以培养的大鼠嗜碱性白血病细胞系RBL-2H3为模型，研究了这些细胞信号转导的分子机制。在以前的研究中，我们观察到蛋白质酪氨酸磷酸化是FceRI诱导脱颗粒的早期和关键信号。蛋白酪氨酸激酶Syk被发现是酪氨酸磷酸化，受体聚集后被激活。RBL-2H3肥大细胞的一个变体也被发现，它没有检测到Syk，并证明Syk对于受体诱导的炎症介质的释放是必不可少的。对这些细胞中信号通路的分析表明，FceRI诱导的细胞蛋白酪氨酸磷酸化大多位于Syk下游，需要具有酶活性的Syk。我们观察到，激活环酪氨酸对Syk介导的FceRI信号的传播是必不可少的，尽管它们对激酶活性并不是必需的。通过Syk突变体的表达，我们发现Syk连接区的一个酪氨酸可以负向调节其激酶活性，从而导致炎症介质的释放。该位点的突变导致细胞内Syk功能的显著增强，并促进了介质的释放。由于蛋白质分子的磷酸化水平是由于蛋白质酪氨酸激酶和磷酸酶活性之间的平衡，我们研究了蛋白质酪氨酸磷酸酶在肥大细胞信号转导中的作用。CD45和Src同源2结构域包含蛋白酪氨酸磷酸酶SHP-1和SHP-2，是肥大细胞中存在的蛋白酪氨酸磷酸酶。在这一年的实验中，我们观察到CD45对ZAP70是必不可少的，但对Syk来说不是，在肥大细胞中重建抗原受体启动的脱颗粒信号。此外，蛋白酪氨酸磷酸酶SHP-1被发现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

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