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. The binding of Syk to the tyrosine phosphorylated ITAM of the beta and gamma subunits of FceRI results in a conformational change in Syk, with an increase in its enzymatic activity that leads to tyrosine phosphorylation of several proteins and the downstream propagation of signals. The conformational change of Syk exposes its carboxy-terminal region to binding by an antibody. Syk has three Tyr residues located four amino acids from its carboxy terminal. To characterize the role of these Tyr residues in mast cell signaling, mutant Syk with these three Tyr mutated to Phe was 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 decrease of FceRI-stimulated mast cell degranulation and signaling to NFAT and NF-kB activation with a parallel decrease in activation of the Erk and p38 MAP kinases. In vitro this mutated Syk had decreased kinase activity but when expressed in cells it was constitutively tyrosine phosphorylated with an increase in phosphorylation of the negative regulatory Y317, but decreased phosphorylation of the activation loop tyrosines. Mutation of each of the three Tyr residues separately showed that the last two and especially the third was the most important for Syk function. These results suggest that phosphorylation of these Tyr residues contribute to the activate state by keeping the molecule in an extended conformation. These Tyr residues may also contribute to signaling in the cell by providing docking sites after phosphorylation for other molecules. 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 mouse mast cells with small interference RNA (siRNA) to specifically decrease the expression of Fyn, Gab2 or Syk.. There was decreased activation of phosphoinositide-3-kinase (PI3K) pathway as indicated by a change in Akt phosphorylation after suppression of Gab2 but not Fyn demonstrating that Gab2 but not Fyn regulates this pathway. The decreased expression of Gab2 slightly enhanced degranulation whereas decreased Fyn levels did not have any effect. There were some minor changes in NFAT or NFkB activation in cells with decreased 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 TNFa. In contrast, decreased expression of Syk dramatically reduced FceRI-induced degranulation, activation of NFAT and NFkB. These results suggest 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. The mAb BD6 is a monoclonal antibody that inhibits the binding of IgE to FceRI, without directly reacting with this receptor. By expression cloning, mAb BD6 identified the alpha1,3-galactosyltransferase gene. Both galactose and melibiose decreased the binding of mAb BD6 in a dose dependent manner on RBL-2H3 cells and abolished its binding on alpha1,3-galactosyltransferase transfected PEAK cells. There was also partial competition between mAb BD6 and IB4, a galactose binding lectin. MAb BD6 recognized a low molecular weight lipid separated by thin layer chromatography. By sucrose gradient analysis mAb BD6 bound to the lipid rafts fractions. By repeated sorting and cloning, RBL-2H3 variants were isolated deficient in mAb BD6 binding;these cells lacked the GD1b ganglioside and had low expression of GM1. However, they still had normal FceRI induced degranulation. These results suggest that mAb BD6 inhibits IgE binding by reacting with a galactose containing glycolipid present in lipid rafts. 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 cells. Therefore, NFAT was used as readout for mast 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-postive 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 were used to screen for molecules that could inhibit or enhance the receptor-induced GFP response. In a screen of 300 plasmids, there were a number of positives;among these are genes that have effects on cellular housekeeping and several others that are similar to signaling molecules. 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. Following each target siRNA transfection, IgE-antigen induced mast cell degranulation was assayed 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, which defined three unambiguous hits, Pten, Mtmr4 and Ppp3r1 (calcineurin B). Furthermore, the mechanism of the inhibition of mast cell degranulation due to calcineurin B deficiency was investigated. Calcineurin B deficiency reduced the phosphorylation of MAP kinases and the phosphorylation of PKD/PKCmu and PKCdelta, which are involved in FceRI signaling. Therefore, this siRNA screen identified several new molecules that are critical for FceRI-induced degranulation. Blocking the function of these proteins may be potential targets for the treatment of asthma and allergic diseases. Compared to the studies using knock-out mice in which the targeted protein is absent, the results with siRNA transfection probably reproduce better what would occur with an inhibitor of a molecule in this signaling pathway and therefore is more useful for the design of pharmacological inhibitors. Immune receptor stimulated synthesis of cytokines depends on NFAT and NFkB transcription factors. To study FceRI induced activation of these pathways,mast cell lines that have NFAT or NFkB reporter systems were screened with a siRNA library that targets phosphatases. Among the 198 phosphatases, 31 enhanced or inhibited FceRI-initiated NFAT or NFkB activation. Among the positive hits, the siRNA for PPP2CA (catalytic subunit of Ser/Thr phosphatase 2A) slightly reduced FceRI-initiated NFAT activation, but dramatically enhanced NFkB activity. While the siRNA of PPP1CA (catalytic subunit of the Ser/Thr phosphatase 1A) slightly enhanced NFAT, but had no effect on NFkB activation. These results suggest that Ser/Thr phosphatases are involved in FceRI signaling with protein phosphatase 1 and 2 having divergent roles in mast cell functions contrary to what had been concluded from experiments with inhibitors that indiscriminately block both enzymes.

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National Institute of Dental & Craniofacial Research
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Barbu, Emilia Alina; Zhang, Juan; Siraganian, Reuben P (2010) The limited contribution of Fyn and Gab2 to the high affinity IgE receptor signaling in mast cells. J Biol Chem 285:15761-8
Grodzki, Ana Cristina; Berenstein, Elsa (2010) Introduction to the purification of antibodies. Methods Mol Biol 588:11-3
Grodzki, Ana Cristina; Berenstein, Elsa (2010) Antibody purification: ammonium sulfate fractionation or gel filtration. Methods Mol Biol 588:15-26
Grodzki, Ana Cristina; Berenstein, Elsa (2010) Antibody purification: affinity chromatography - protein A and protein G Sepharose. Methods Mol Biol 588:33-41
de Castro, Rodrigo O; Zhang, Juan; Jamur, Maria C et al. (2010) Tyrosines in the carboxyl terminus regulate Syk kinase activity and function. J Biol Chem 285:26674-84
Grodzki, Ana Cristina; Berenstein, Elsa (2010) Antibody purification: ion-exchange chromatography. Methods Mol Biol 588:27-32
Siraganian, Reuben P; de Castro, Rodrigo O; Barbu, Emilia A et al. (2010) Mast cell signaling: the role of protein tyrosine kinase Syk, its activation and screening methods for new pathway participants. FEBS Lett 584:4933-40
Zhang, Juan; Mendoza, Mary; Guiraldelli, Michel F et al. (2010) Small interfering RNA screen for phosphatases involved in IgE-mediated mast cell degranulation. J Immunol 184:7178-85
Grodzki, Ana Cristina G; Moon, Kyungduk D; Berenstein, Elsa H et al. (2009) FcepsilonRI-induced activation by low antigen concentrations results in nuclear signals in the absence of degranulation. Mol Immunol 46:2539-47
Sly, Laura M; Kalesnikoff, Janet; Lam, Vivian et al. (2008) IgE-induced mast cell survival requires the prolonged generation of reactive oxygen species. J Immunol 181:3850-60

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