Fer and Fps/Fes Participate in a Lyn-dependent Pathway from FcϵRI to Platelet-Endothelial Cell Adhesion Molecule 1 to Limit Mast Cell Activation

Abstract

Mast cells express the high affinity IgE receptor Fc⑀RI, which is composed of an IgE-binding ␣-chain, a tetramembrane spanning ␤ chain, and a dimeric ␥ chain (1). Signals are transduced via immunoreceptor tyrosine-based activation motifs (ITAMs) 3 that are present in both the ␤ and ␥ subunits and serve as docking sites for the recruitment of signaling molecules (2). Fc⑀RI signaling is initiated by binding of IgE, which is sufficient for induction of survival pathways as well as cytokine production (3, 4). Some highly cytokinergic IgEs can induce antigen-independent degranulation, survival, adhesion, and chemotaxis of mast cells (5). However, in most cases, aggregation of Fc⑀RI by multivalent antigens is required for a full mast cell response including degranulation, lipid mediator release, increased cell adhesion, and increased motility (6). The Src family protein-tyrosine kinase (PTK) Lyn is constitutively associated with Fc⑀RI (7), and upon antigen-mediated clustering of receptor chains, Lyn phosphorylates ITAMs on ␤-and ␥-chains. The ␤-chain ITAMs are thought to recruit additional Lyn and Fyn kinases, the p85 subunit of phosphatidylinositol 3-kinase, SH2-containing inositol 5Ј-phosphatase, and Shp2 phosphatase (8). Phosphorylated ITAMs on the ␥-chains recruit the dual SH2 domain-containing PTK Syk (9). Syk activity is essential for signal transduction downstream of Fc⑀RI, because Syk-deficient mast cells fail to degranulate, synthesize leukotrienes, and secrete cytokines following antigen challenge (10). Although Fc⑀RI-induced tyrosine phosphorylation is greatly reduced in Syk-deficient mast cells, phosphorylation of the receptor ITAMs and Lyn are maintained (10). Lyn-deficient mast cells, despite severely reduced tyrosine phosphorylation and delayed calcium flux, are able to degranulate and secrete cytokines (11). In fact, Lyn-deficient mast cells release more of the granule constituent ␤-hexosaminidase than do wild type mast cells. Further studies have shown that multiple responses to Fc⑀RI aggregation are delayed in Lyn-deficient mast cells, including tyrosine phosphorylation of receptor subunits, calcium flux, and phosphatidylinositol 3,4,5-trisphosphate production but persist far longer than in wild type mast cells (12). Other notable characteristics of Lyn-deficient mast cells are that Fyn kinase is hyperactivate, whereas SH2-containing inositol 5-phosphatase is completely inactive (12). Thus, in addition to initiating signaling downstream of Fc⑀RI, Lyn is also involved in signal termination at least partly through activation of SHZ-containing inositol 5-phosphatase, which hydrolyzes phosphatidylinositol 3,4,5-trisphosphate and thereby reduces the plasma membrane localization of pleckstrin homology domain-containing proteins.

Contributions of F-BAR and SH2 Domains of Fes Protein Tyrosine Kinase for Coupling to the FcεRI Pathway in Mast Cells

“…In mast cells, we have shown that Fer promotes activation of p38 mitogen-activated protein kinase and chemotaxis of mast cells (10). We also found that Fer and Fes PTKs contribute to FcεRI-evoked phosphorylation of platelet-endothelial cell adhesion molecule 1 (PECAM-1) (67).…”

“…An EcoRI restriction site encoding silent mutations was created adjacent to Y388/Y405 for screening, followed by verification by sequencing. GST-PECAM-1(CT), encoding the C-terminal tail of PECAM-1 was described previously (67).…”

“…Surprisingly, FcεRI-induced tyrosine phosphorylation of Fes and Fer does not require their kinase activities (55) and is almost entirely dependent on Lyn (67). Through the use of transgenic mouse models, evidence for both unique and redundant functions for Fes and Fer has been described in regulating hematopoiesis (55) and limiting the innate immune response (22,40,50,72).…”

SH2 Domain-Containing Phosphatase-2 Protein-Tyrosine Phosphatase Promotes FcεRI-Induced Activation of Fyn and Erk Pathways Leading to TNFα Release from Bone Marrow-Derived Mast Cells

“…3,10). FcRI signaling also leads to recruitment of SH2 domain-containing phosphatase-2 (SHP2) (PTPN11) phosphatase to FcRI ␤-subunit (11), Gab2 adaptor protein (12), PECAM-1 (13,14), and MAFA (15). SHP2 interaction with these ligands likely contributes to activation of SHP2 phosphatase activity, which was previously reported following FcRI aggregation in mast cells (16,17).…”

“…BMMC cultures were generated in a manner similar to what has been previously described (14,34). Femurs were isolated from shp2 fl/fl and shp2 fl/fl :Tg CreER* mice, flushed with BMMC growth medium (IMDM, 10% (v/v) FBS, 1% (v/v) antimicrobial-antimycotic solution (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 1% (v/v) nonessential amino acids (Invitrogen), 5% (v/v) conditioned medium from X63-IL-3 cells, 5% (v/v) conditioned medium from HEK293-SCF cells, and 50 M ␣-monothioylglycolate (Sigma-Aldrich)) and bone marrow cells were cultured for 4 wk to generate BMMC cultures.…”