The Syk tyrosine kinase family plays an essential role in immunoreceptor tyrosine-based activation motif signaling (ITAM). The binding of Syk to tyrosine phosphorylated ITAM subunits of immunoreceptors, such as 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), two of which (Tyr-624 and Tyr-625) are auto-phosphorylated after FceRI stimulation. Previously we observed that the Tyr-624 and Tyr-625 are important in regulating the activity of Syk by maintaining the enzyme in an auto-inhibitory state. Once phosphorylated these sites contribute to the increase in the enzymatic activity but also could provide docking sites for other molecules thereby having a role in signal transduction. To investigate this possibility, biotinylated synthetic peptides representing the last 10 amino acids of the tail of Syk with these two tyrosines either phosphorylated or non-phosphorylated were used to search for molecules that bind to these sites. The bound proteins were separated by 2-D gel electrophoresis and then analyzed by mass spectrometry with the identity of the proteins further confirmed by immunoblotting. SHIP-1, SLP-76, NCK-1, GADS and GRB-2 were identified as proteins that bound to the phosphorylated peptide. By far-westerns, using the peptides as probes, there was direct binding of the phosphorylated peptide to NCK-1 and NCK-2. By immunoprecipitation there were two molecular complexes after FceRI activation;one with Syk, SLP-76, NCK and GRB-2 and the other involving Syk, SHIP-1, NCK and GRB-2. These results indicate that the phosphorylation of Tyr-624 and Tyr-625 of Syk creates sites for the binding of the adaptor protein NCK. This leads to the formation of signaling complexes important for Syk-mediated FceRI activation in mast cells. The pathways leading from FceRI aggregation to cellular responses depend on protein phosphorylations regulated by both kinases and phosphatases. To gain an understanding of the functions played by phosphatases in IgE-mediated mast cell activation, small interference RNA (siRNA) library that targets all mouse phosphatase genes was used in screens to detect molecules that regulate FceRI-induced signaling. Previously we found 17 positive hits that either enhanced or inhibited FceRI-induced mast cell degranulation. Receptor stimulation also results in the synthesis and secretion of cytokines which depends on NFAT and NF-κB transcription factors. To understand regulation of FceRI induced activation of these pathways, stable mouse mast cell lines that have NFAT or NF-κB reporter systems were developed, and screened with the same siRNA phosphatase library. IgE-antigen-induced NFAT and NF-κB 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-κB or degranulation responses, suggesting that distinct phosphatases regulate the FceRI-mediated early and late responses. Among the 198 phosphatases, 12 consistently enhanced or inhibited the FceRI-initiated NFAT or NF-κB activation. The positive hits were then validated by testing in bone marrow derived primary mast cells. Four of the siRNA had similar effects;knockdown of three (Rwdd2, Dusp7 and Ptpn23) inhibited while that of Inpp5d (SHIP-1), a known negative regulator of FceRI signaling, enhanced degranulation and cytokine responses. With the other hits the mast cell responses were variable;knockdown of several of these had minimal or no effects on degranulation but enhanced or inhibited the cytokine response. There were also differences in the cytokine responses;some of the positive hits enhanced release of one cytokine but inhibited that of another. These results demonstrate the utility of these systems in screening for molecules that are involved in the receptor activated pathway leading to the synthesis of cytokines. The results also indicate the effectiveness of NFAT or NF-κB screens for identifying molecules that were not recognized by the degranulation screen. These different screening techniques have recognized molecules not previously associated with the FceRI pathway. Mast cell activation results in the release of stored and newly synthesized inflammatory mediators. We found that Zeb2, a zinc finger transcription factor, regulates these mast cell responses. Transient transfection with small interference RNA (siRNA) was used to specifically knock down Zeb2 expression. The siRNA-induced decrease in Zeb2 expression correlated with decreased FceRI-mediated degranulation;with a parallel reduction in receptor induced activation of NFAT and NF-kB transcription factors, but an enhanced response to the LPS-mediated activation of NF-kB. The antigen-mediated release of cytokines TNFa, IL-13 and CCL-4 was reduced;however this was not as dramatic as the decrease in degranulation. This suggests that low Zeb2 expression differentially regulates signaling pathways in mast cells. The Zeb2 siRNA treated mast cells had altered cell cycle progression, as well as decreased expression of several molecules including the beta subunit of FceRI, Gab2, phospholipase-Cg1 and phospholipase-Cg2, all of which are required for signaling from FceRI. The results indicate that the transcription factor Zeb2 controls the expression of molecules thereby regulating signaling in mast cells.

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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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