Defining the Role of the SHP2 Protein Tyrosine Phosphatase in FcepsilonRI Signaling in Mast Cells

Abstract

Mast cells are granulocytes that are a key component of the innate and adaptive immune system, and contribute to allergic disorders. Mast cell activation following clustering of the high affinity IgE receptor (FcepsilonRI) by multivalent antigens requires reversible tyrosine phosphorylation of myriad signaling proteins. Activated mast cells rapidly release granule contents (eg. histamine and serine proteases) that cause vascular permeability, and in a more delayed manner they also synthesize and secrete eicosanoids and numerous cytokines (eg. IL-6 and TNFalpha) that recruit activated leukocytes. FcepsilonRI signaling is initiated by Lyn, a Src Family Kinase (SFK), that phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) found on the FcepsilonRI beta and gamma chains. This allows recruitment of Fyn SFK and Syk kinase that bind ITAMs and phosphorylate numerous downstream targets. Src Homology 2 domain-containing Phosphatase 2 (SHP2, encoded by ptpn11/shp2) is known to be recruited to several phosphorylated proteins following FcepsilonRI aggregation in mast cells, however attempts to define the role of SHP2 have been hampered by its essential role during embryonic development and hematopoiesis in mice. Recently, conditional SHP2 knock-out mice (shp2fl/fl) have been created allowing for shp2 inactivation in a tissue-specific manner by Cre recombinase. Here we describe the use of transgenic mice expressing a modified estrogen receptor-Cre Recombinase (TgCreER*) on a shp2fl/fl genetic background, that allows for maturation of bone marrow-derived mast cells (BMMCs) prior to shp2-inactivation using 4-hydroxytamoxifen (4OH-TM). SHP2-depleted BMMCs display reduced phosphorylation of the FcepsilonRI beta chain, but exhibit extended phosphorylation of Syk kinase. Additionally, SHP2-deficient cells display a defect in the activation of both Erk mitogen-activated protein kinase and Akt, which correlates with an observed defect in the production of TNFalpha and Leukotriene C4. Finally, we show that SHP2-deficient BMMCs display elevated FcepsilonRI-evoked phosphorylation of Csk-Binding Protein (Cbp or PAG) on residue Y317, which recruits C-terminal Src kinase (Csk) that phosphorylates SFKs on an inhibitory tyrosine. This hyperphosphorylation of Cbp correlates with elevated phosphorylation of the C-terminal inhibitory tyrosine on Fyn kinase. This study provides new insights into the role of SHP2 as a positive effector of FcepsilonRI signaling and cytokine production in mast cells.