The Syk tyrosine kinase family plays an essential role in immunoreceptor tyrosine-based activation motif (ITAM) signaling. The binding of Syk to tyrosine phosphorylated ITAM subunits of immunoreceptors, such as high affinity IgE receptor (FceRI) on mast cells, results in a conformational change, with an increase in its enzymatic activity. This conformational change exposes the COOH terminal tail region of Syk which has three conserved Tyr residues (Tyr-623, Tyr-624, Tyr-625 of rat Syk). To understand the role of these residues in signaling, wild-type and mutant Syk with these three Tyr mutated to Phe was expressed in Syk-deficient mast cells. There was decreased FceRI-induced degranulation, NFAT and NF-kB activation with the mutated Syk together with reduced phosphorylation of MAP kinases p38 and p42/44 ERK. In non-stimulated cells, the mutated Syk was more tyrosine phosphorylated predominantly as a result of autophosphorylation. In vitro, there was reduced binding of mutated Syk to phosphorylated ITAM due to this increased phosphorylation. This mutated Syk from non-stimulated cells had significantly reduced kinase activity while its autophosphorylation capacity was not affected. However, the kinase activity and the autophosphorylation capacity of this mutated Syk were dramatically decreased when the protein was dephosphorylated before the in vitro kinase reaction. Furthermore, mutation of these tyrosines in the COOH-terminal region of Syk transforms it to an enzyme, similar to its homolog Zap-70, that depends on other tyrosine kinases for optimal activation. In testing Syk mutated singly at each one of the tyrosines, Y624 but especially Y625 had the major role in these reactions. Therefore, these results indicate that these tyrosines in the tail region play a critical role in regulating the kinase activity and function of Syk. Several studies with mast cells from knockout mice have suggested that the tyrosine kinase Fyn and its downstream substrate Gab2 may play a role in FceRI-mediated mast cell activation. To examine the relative role of these two molecules and compare them to that of Syk, we transiently transfected mast cells with small interference RNA (siRNA) targeted to Fyn, Gab2 or Syk to specifically decrease their expression. The siRNA suppression of Gab2 but not Fyn reduced activation of phosphoinositide-3-kinase (PI3K) as indicated by the change in phosphorylation of Akt demonstrating that Gab2 but not Fyn regulates this pathway. The decreased expression of Gab2 and Fyn had minor effects on degranulation. There were also some minor changes in NFAT or NF-kB activation in cells with reduced expression of Fyn or Gab2. Decreased Gab2 but not Fyn reduced the FceRI-induced activation of the Erk, Jnk and p38 MAP kinases and the release of TNF-a. In contrast, decreased expression of Syk dramatically reduced FceRI-induced degranulation, activation of NFAT and NF-kB. Therefore these experiments indicate that Syk is the major regulator of FceRI-mediated reactions while Fyn has minor if any effects and Gab2 regulates primarily late events including MAP kinase activation and release of cytokines. FceRI stimulation results in an increase in intracellular calcium that activates the serine phosphatase calcineurin, which then dephosphorylates the nuclear factor of activated T cells (NFAT). The dephosphorylated NFAT rapidly translocates into the nucleus and induces the transcription of various cytokine genes in T, B and mast cells. Therefore, NFAT was used as readout for immune cell activation. A plasmid containing three tandem NFAT binding sites fused to the cDNA of enhanced green fluorescent protein (GFP) was transfected into the RBL-2H3 cells and a cell line was selected that became strongly GFP+ only after FceRI stimulation. Transient transfection of a plasmid containing the cDNA for the NH2-half of Syk that lacks the enzymatic domain (Syk-SH2) inhibits this GFP response. Transient transfection of these cells with plasmids from an RBL-2H3 cDNA library was used to screen for molecules that could regulate the receptor-induced GFP response. In a screen of 300 plasmids, there were a number of confirmed positives that were also tested for their capacity to regulate degranulation;three were found to consistently decrease release. Experiments will determine the effect of transfection with these plasmids on the expression of the specific proteins and will explore how and where they interact with signaling in cells. The pathways leading from FceRI aggregation to cellular responses depend on protein phosphorylations regulated by both kinases and phosphatases. To gain an understanding on the functions played by phosphatases in IgE-mediated mast cell activation, a siRNA library that targets all mouse phosphatase genes was screened in a mouse mast cell line, MMC-1. Following each target siRNA transfection, IgE-antigen induced mast cell degranulation was assayed continuously for three days as a functional readout of targeted protein knock-down. Out of 198 phosphatases, 10 enhanced and 7 inhibited FceRI-induced mast cell mediator release. For 7 of the strongest hits, four different siRNAs per target were tested, and at least 2 out of the 4 single siRNA per target had similar effects as the pool suggesting that these were true hits. Bone marrow derived mast cells (BMMC) from normal mice further validated these results for six definite positive targets. The mechanism of the reduced mast cell degranulation due to calcineurin B deficiency was investigated. Calcineurin B deficiency reduced the phosphorylation of MAP kinases and the phosphorylation of PKD/PKCu and PKC-delta, which are involved in FceRI signaling. The screen therefore, has identified several new molecules that are critical for FceRI-induced degranulation. Regulating the function of these proteins may be potential targets for the treatment of allergic inflammation. The synthesis and secretion of cytokines after immune receptor activation depends on NFAT and NF-kB transcription factors. To understand regulation of FceRI induced activation of these pathways, stable mouse mast cell lines that have NFAT or NF-kB reporter systems were developed, and screened with a siRNA library that targets all known and predicted mouse phosphatases. IgE-antigen-induced NFAT and NF-kB activations were examined for three days following each siRNA pool transfection. There was no correlation in the effect of these siRNA on the NFAT, NF-kB or degranulation responses, suggesting that distinct phosphatases regulate the FceRI-mediated early and late responses. Among the 198 phosphatases, 16 consistently enhanced or inhibited the FceRI-initiated NFAT or NF-kB activation. The positive hits were validated by testing in BMMC for their effects on FceRI-induced degranulation and the release of the 3 cytokines, TNF-a, IL-13 and MIP-1b. There were 12 hits that enhanced the NFAT or NF-kB response;all of these enhanced the release of at least one cytokine in these primary BMMC cells. Therefore, these genes are positive regulator of the FceRI-induced late cellular responses. The largest increase was after the knockdown of Inpp5d (SHIP-1), a known negative regulator of the FceRI pathway. Among four hits that inhibited the activation of NFAT or NF-kB, three inhibited the receptor induced cytokine generation and two of these also the degranulation response in BMMC. This strongly suggests that these two molecules control FceRI-induced immediate and late cellular events. These screens using degranulation, NFAT or NF-kB end-points have identified molecules not previously associate with the FceRI pathway. These experiments also demonstrate the utility of these systems in screening for molecules that play a role in complex receptor-induced signaling.
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