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
Showing the most recent 10 out of 27 publications
