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。本研究以大鼠嗜碱性粒细胞白血病细胞系RBL-2 H3为模型，探讨了这些细胞中信号转导的分子机制。在先前的研究中，我们观察到蛋白酪氨酸磷酸化是FceRI诱导的脱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和激活后，受体聚集。还鉴定了RBL-2 H3肥大细胞的变体，其没有可检测的Syk，并证明Syk对于受体诱导的炎症介质释放是必需的。对这些细胞中的信号传导途径的分析表明，大多数FceRI诱导的细胞蛋白酪氨酸磷酸化是Syk的下游，并且需要酶活性SykSyk阴性肥大细胞用于检查Syk调节的机制。我们观察到活化环酪氨酸对于Syk介导的FceRI信号传导的传播是必需的，尽管它们对于激酶活性不是必需的。通过表达Syk的突变体，我们发现Syk的接头区中的酪氨酸起负调节其激酶活性的作用，从而调节导致炎症介质释放的信号。该位点的突变导致细胞中Syk功能的显著增加，并增强了介质的释放。由于分子的蛋白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，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CD 45和Src同源2结构域的蛋白酪氨酸磷酸酶SHP-1和SHP-2是存在于肥大细胞中的蛋白酪氨酸磷酸酶。在今年的实验中，我们观察到CD 45对于ZAP 70是必需的，而不是Syk，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调节肥大细胞细胞因子的产生，但不调节其他介质如组胺的释放。- 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