Signal Transduction In Mast Cells

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The activation of the high affinity IgE receptor (FceRI) results in rapid protein tyrosine phosphorylation and activation of the cytoplasmic protein tyrosine kinase Syk which is essential for this receptor-mediated signaling in mast cells. Because of the importance of Syk in signaling, there is much interest in understanding its regulation. The tandem Src homology 2 (SH2) domains in the N-terminal half of Syk are involved in its association with subunits of FceRI after receptor aggregation. The binding of Syk to the tyrosine phosphorylated ITAM of subunits of FceRI by its two SH2 domains results in a conformational change in Syk, with an increase in its enzymatic activity. This leads to the downstream propagation of signals such as the tyrosine phosphorylation of phospholipase C, LAT and the influx of calcium. Previously we found that the conformational change exposes the carboxy terminal region of Syk so that it becomes accessible to binding with an antibody to this region of the molecule. Syk has three Tyr residues located 4 amino acids from the COOH terminal; at least one of these Tyr residues appears to get phosphorylated. The present studies aim to characterize the roles of the three Tyr residues of Syk in mast cell signaling. Therefore, mutant Syk with these three Tyr mutated to Phe were expressed in a Syk-deficient variant of the RBL-2H3 mast cells. Compared with wild-type Syk, mutation of the three Tyr residues resulted in a dramatic reduction of IgE-stimulated mast cell degranulation and nuclear signaling through NFAT and NF-kB activation. The planned experiments will define the effect of these mutations on the signaling events in mast cells such as the increase in intracellular calcium, phosphorylation of adaptor molecules and the kinase activity of Syk. Several studies have suggested that the tyrosine kinase Fyn and its downstream substrate Gab2 may play a role in mast cell activation. Adherence of RBL-2H3 cells to fibronectin resulted in the tyrosine phosphorylation of Gab2, FAK and paxillin, but not Syk. In adherent RBL-2H3 cells, Gab2, FAK and paxillin were already phosphorylated, and antigen-stimulation further enhanced the tyrosine phosphorylation of these molecules. Syk was essential for antigen-, but not integrin-, induced tyrosine phosphorylation of Gab2, FAK and paxillin while FAK was not critical for integrin-induced Gab2 tyrosine phosphorylation. Transient transfection with small interference RNA (siRNA) targeting Fyn or Gab2 decreased their expression as confirmed by western blot analysis. There was decreased activation of phosphoinositide-3-kinase (PI3K) as indicated by the change in phosphorylation of Akt after siRNA suppression of Gab2 but not Fyn suggesting that Gab2 but not Fyn may regulate PI3K. However there was no change in antigen-induced degranulation, and some minor changes in NFAT or NF-kB activation. Decreased Gab2 expression had minimal effects on fibronectin-induced mast cell adhesion, but it did slightly reduce fibronectin-initiated paxillin and Akt phosphorylation. Therefore, even though both the integrin- and FceRI-stimulation resulted in the tyrosine phosphorylation of Gab2, this molecule does not play a critical role in the integrin and FceRI-mediated signaling pathways in mast cells. These results also suggest that Fyn itself does not regulate Gab2 and that these two molecules have minor effects on receptor induced activation of transcription factors and limited contribution to the FceRI signaling network. FceRI-induced mast cell activation was analyzed at a single cell basis using a rat basophilic leukemia (RBL-2H3) cell line transfected with a reporter plasmid containing three tandem NFAT binding sites fused to enhanced green fluorescent protein (GFP). Surprisingly, with this sensitive detection system, there is activation of IgE sensitized cells at concentrations of antigen as low as 10pg/ml, which was 10-fold lower than was detected by degranulation. There were also differences in signaling pathways leading to degranulation compared to NFAT-mediated gene activation. Both signaling to NFAT activation and degranulation required Syk and calcineurin. However inhibitors of the phosphatidylinositol 3-kinase pathway blocked degranulation but did not inhibit NFAT activation. Therefore, FceRI activation can result in nuclear signals in the absence of the release of mediators. These results suggest differences between the pathways that result in nuclear gene expression from those that induce degranulation and the release of inflammatory mediators. With the sequencing of the genome of several species and the development of tools to perturb gene function in cells it has become possible to study the role of different genes in signaling pathways. Without direct functional testing, molecular biological tools are inefficient in these studies. Therefore we are currently using functional genomics to find the role of novel molecules in the intracellular signal transduction pathways. These experiments use phenotype-oriented identification that allows a direct link between the molecule and the signaling pathway. The pathways leading from FceRI stimulation to cellular responses depend on protein phosphorylations regulated by both kinases and phosphatases. To gain a comprehensive view on the functions played by protein phosphatases on FceRI signaling, a siRNA library was screened, which contains 198 pools of 4 siRNA duplexes per gene targeting all the mouse enzymes with known or predicted phosphatase activity. IgE-antigen-induced mast cell degranulation was tested repeatedly for 3 days following each siRNA transfection to increase the possibility that the functional assay was performed at the time at the lowest expression of the targeted protein. Out of 198 targets, 26 enhanced or inhibited FceRI-induced degranulation at greater than 2 S.D. Retesting of all the positive hits showed similar results. To rule out the possibility of off target effects, each of the four different siRNAs included in the original siRNA pools were tested in the case of the 7 strongest hits. The results showed that among the 7 hits, 3 were probably due to off target effects of single siRNA species. Where antibodies were available, Western blotting confirmed the siRNAs results. The screen therefore, has identified several new molecules involved in FceRI signaling. The results also indicate that this is an efficient system for screening for molecules that are important in immune-receptor signaling pathways leading to granular secretion.