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Clustering of the high affinity IgE receptor (Fc(epsilon)RI) in mast cells leads to degranulation and production of numerous cytokines and lipid mediators that promote allergic inflammation. Initiation of FFc(epsilon)RI signaling involves rapid tyrosine phosphorylation of Fc(epsilon)RI and membrane-localized adaptor proteins that recruit additional SH2 domain-containing proteins that dynamically regulate downstream signaling. SH2 domain-containing phosphatase-2 (SHP2) is a protein-tyrosine phosphatase implicated in Fc(epsilon)RI signaling, but whose function is not well defined. In this study, using a mouse model allowing temporal shp2 inactivation in bone marrow-derived mast cells (BMMCs), we provide insights into SHP2 functions in the Fc(epsilon)RI pathway. Although no overt defects in Fc(epsilon)RI-induced tyrosine phosphorylation were observed in SHP2 knock-out (KO) BMMCs, several proteins including Lyn and Syk kinases displayed extended phosphorylation kinetics compared with wild-type BMMCs. SHP2 was dispensable for Fc(epsilon)RI-induced degranulation of BMMCs, but was required for maximal activation of Erk and Jnk mitogen-activated protein kinases. SHP2 KO BMMCs displayed several phenotypes associated with reduced Fyn activity, including elevated phosphorylation of the inhibitory pY531 site in Fyn, impaired signaling to Grb2-associated binder 2, Akt/PKB, and IkappaB kinase, and decreased TNF-alpha release compared with control cells. This is likely due to elevated Lyn activity in SHP2 KO BMMCs, and the ability of Lyn to antagonize Fyn activity. Overall, our study identifies SHP2 as a positive effector of Fc(epsilon)RI-induced activation of Fyn/Grb2-associated binder 2/Akt and Ras/Erk pathways leading to TNF-alpha release from mast cells.

This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcepsilonRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcepsilonRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcepsilonRI beta chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcepsilonRI signaling and potential regulation the actin reorganization in mast cells.