Mast Cell Signaling Research Articles

Distinct Roles for the Linker Region Tyrosines of Syk in FcϵRI Signaling in Primary Mast Cells

Stimulation of FcepsilonRI, the high affinity IgE receptor of mast cells results in the rapid binding of the Syk tyrosine kinase to cytoplasmic domains of FcepsilonRI and to its subsequent activation. Syk plays an essential role in signal transduction from FcepsilonRI as shown by Syk-deficient mast cells, which are defective in receptor-induced degranulation, cytokine synthesis, and intracellular pathways. However the mechanism by which Syk activates these pathways remains unclear. Activation of Syk is associated with its phosphorylation on several tyrosine residues, including the linker tyrosines Tyr317, Tyr342, and Tyr346. These residues have been proposed to play important roles in the transduction of signals by binding to other signaling proteins. To test these hypotheses in primary murine mast cells, we used retroviral infection of Syk-deficient mast cells to generate cells expressing Syk proteins bearing mutations in the linker tyrosines. We show that Tyr342 and Tyr346 contribute positively to the function of Syk and have both overlapping as well as distinct functions. Mutations in either Tyr342 or Tyr346 alone had no effect on FcepsilonRI-induced degranulation or calcium flux, whereas mutation of both residues caused a significant reduction in both pathways. In contrast, phosphorylation of PLCgamma1, PLCgamma2, and Vav1 was strongly decreased by a mutation in Tyr342 alone, whereas phosphorylation of ERK and Akt was more dependent on Tyr346. Finally we show that Tyr317 functions as a negative regulatory site and that its mutation can partially compensate for the loss of both Tyr342 and Tyr346.

Reconstitution of Regulated Phosphorylation of FcϵRI by a Lipid Raft-excluded Protein-tyrosine Phosphatase

To examine the exquisite regulation of IgE-FcepsilonRI tyrosine phosphorylation by Lyn kinase that is stimulated by antigen-mediated cross-linking, we utilized co-expression of FcepsilonRI and Lyn in Chinese hamster ovary cells, which results in high basal levels of Lyn kinase activity and spontaneous phosphorylation of FcepsilonRI. We found that co-expression of a lipid raft-excluded transmembrane tyrosine phosphatase, PTPalpha, suppresses Lyn kinase activity and markedly reduces the level of spontaneous phosphorylation of FcepsilonRI, while facilitating its antigen-stimulated phosphorylation. Other tyrosine phosphatases, including SHP-1, CD45, and a lipid raft-preferring chimeric version of PTPalpha fail to reconstitute antigen-dependent FcepsilonRI phosphorylation. We concluded that both substrate specificity and submembrane location are critical to phosphatase-mediated regulation of Lyn kinase activity that supports activation of FcepsilonRI.

Genetically engineered negative signaling molecules in the immunomodulation of allergic diseases

This review summarizes current knowledge regarding the control of human mast cell and basophil signaling and recent developments using a new therapeutic platform consisting of a human bifunctional gamma and epsilon heavy chain (Fc gamma-Fc epsilon) protein to inhibit allergic reactivity. Crosslinking of Fc gamma RIIb to Fc epsilon RI on human mast cells and basophils by a genetically engineered Fc gamma-Fc epsilon protein (GE2) leads to the inhibition of mediator release upon Fc epsilon RI challenge. GE2 protein was shown to inhibit cord blood-derived mast cell and peripheral blood basophil mediator release in vitro in a dose-dependent fashion, including inhibition of human IgE reactivity to cat. IgE-driven mediator release from lung tissue was also inhibited by GE2. The mechanism of inhibition in mast cells included alterations in IgE-mediated Ca mobilization, spleen tyrosine kinase phosphorylation and the formation of downstream of kinase-growth factor receptor-bound protein 2-SH2 domain-containing inositol 5-phosphatase (dok-grb2-SHIP) complexes. Proallergic effects of Langerhan's like dendritic cells and B-cell IgE switching were also inhibited by GE2. In vivo, GE2 was shown to block passive cutaneous anaphylaxis driven by human IgE in mice expressing the human Fc epsilon RI and inhibit skin test reactivity to dust mite antigen in a dose-dependent manner in rhesus monkeys. The balance between positive and negative signaling controls mast cell and basophil reactivity, which is critical in the expression of human allergic diseases. This approach using a human Fc gamma-Fc epsilon fusion protein to co-aggregate Fc epsilon RI with the Fc gamma RII holds promise as a new therapeutic platform for the immunomodulation of allergic diseases and potentially other mast cell/basophil-dependent disease states.

Distinct regulation of C3a-induced MCP-1/CCL2 and RANTES/CCL5 production in human mast cells by extracellular signal regulated kinase and PI3 kinase

Complement component C3a causes a robust degranulation in human mast cells. Whether C3a also stimulates chemokine production in human mast cells and what signaling pathway it activates is not known. In the present study, we demonstrate that CD34 +cell-derived primary mast cells and a human mast cell line LAD 2 express surface C3a receptors at similar levels. Furthermore, C3a caused ∼50% internalization of cell surface C3a receptors in both cell types. We therefore used LAD 2 cells as a model to study C3a-induced biological responses and signaling in human mast cells. We found that C3a stimulated substantial degranulation and induced chemokine monocyte chemoattractant protein 1 (MCP-1/CCL2) and regulated upon activation, normal T-cell expressed and secreted (RANTES/CCL5) production in LAD 2 mast cells. C3a caused a rapid and sustained extracellular-signal-regulated kinase (ERK) phosphorylation and Akt phosphorylation in LAD 2 mast cells. Furthermore, U0126 and LY294002, which respectively inhibit MEK-induced ERK phosphorylation and PI3 kinase-mediated Akt phosphorylation had distinct effects on C3a-induced responses. Thus, U0126, which blocked C3a-induced RANTES/CCL5 production by 50.6 ± 2.3%, inhibited MCP-1/CCL2 generation by 85.2 ± 0.6%. In contrast, LY294002 had no effect on C3a-induced RANTES/CCL5 production but blocked MCP-1/CCL2 generation by 83.7 ± 1.5%. These data demonstrate that C3a activates divergent signaling pathways to induce chemokine production in human mast cells.

Negative regulation of mast cell signaling and function by the adaptor LAB/NTAL.

Engagement of the Fcɛ receptor I (FcɛRI) on mast cells and basophils initiates signaling pathways leading to degranulation. Early activation events include tyrosine phosphorylation of two transmembrane adaptor proteins, linker for activation of T cells (LAT) and non–T cell activation linker (NTAL; also called LAB; a product of Wbscr5 gene). Previous studies showed that the secretory response was partially inhibited in bone marrow–derived mast cells (BMMCs) from LAT-deficient mice. To clarify the role of NTAL in mast cell degranulation, we compared FcɛRI-mediated signaling events in BMMCs from NTAL-deficient and wild-type mice. Although NTAL is structurally similar to LAT, antigen-mediated degranulation responses were unexpectedly increased in NTAL-deficient mast cells. The earliest event affected was enhanced tyrosine phosphorylation of LAT in antigen-activated cells. This was accompanied by enhanced tyrosine phosphorylation and enzymatic activity of phospholipase C γ1 and phospholipase C γ2, resulting in elevated levels of inositol 1,4,5-trisphosphate and free intracellular Ca2+. NTAL-deficient BMMCs also exhibited an enhanced activity of phosphatidylinositol 3-OH kinase and Src homology 2 domain–containing protein tyrosine phosphatase-2. Although both LAT and NTAL are considered to be localized in membrane rafts, immunogold electron microscopy on isolated membrane sheets demonstrated their independent clustering. The combined data show that NTAL is functionally and topographically different from LAT.

Linker for Activation of T Cells Integrates Positive and Negative Signaling in Mast Cells

The transmembrane adapter linker for activation of T cells (LAT) is thought to couple immunoreceptors to intracellular signaling pathways. In mice, its intracytoplasmic domain contains nine tyrosines which, when phosphorylated upon receptor aggregation, recruit Src-homology 2 domain-containing cytosolic enzymes and adapters. The four distal tyrosines are critical for both TCR and FcepsilonRI signaling. Unexpectedly, knock-in mice expressing LAT with a point mutation of the first or of the last three of these tyrosines exhibited an abnormal T cell development characterized by a massive expansion of TH2-like alphabeta or gammadelta T cells, respectively. This phenotype suggests that, besides positive signals, LAT might support negative signals that normally regulate terminal T cell differentiation and proliferation. We investigated here whether LAT might similarly regulate mast cell activation, by generating not only positive but also negative signals, following FcR engagement. To this end, we examined IgE- and/or IgG-induced secretory and intracellular responses of mast cells derived from knock-in mice expressing LAT with combinations of tyrosine mutations (Y136F, Y(175, 195, 235)F, or Y(136, 175, 195, 235)F). A systematic comparison of pairs of mutants enabled us to dissect the respective roles played by the five proximal and the four distal tyrosines. We found that LAT tyrosines differentially contribute to exocytosis and cytokine secretion and differentially regulate biological responses of mucosal- and serosal-type mast cells. We also found that, indeed, both positive and negative signals may emanate from distinct tyrosines in LAT, whose integration modulates mast cell secretory responses.

Adenosine up‐regulation of the mucin gene, MU2, in asthma

Mucus hypersecretion is a hallmark of asthma that contributes to airway obstruction. While the etiology is not well understood, hypersecretion has been linked to the presence of cytokines such as IL-4, IL-5, IL-9, and IL-13 in the inflamed airway. The presence of adenosine has also been noted in asthmatic airways, and adenosine-mediated signaling in mast cells has been implicated in the severe bronchoconstriction and inflammation prevalent in these patients (1, 2). Here we examine the possibility that adenosine also contributes to mucus hypersecretion by airway epithelial cells. Results in cultured airway epithelial cells showed that MUC2 mucin expression increased in response to adenosine. This appeared to be mediated by a pathway initiated at the adenosine A1 receptor that transduced signals through a Ca2+-activated Cl- channel and EGFR. That this signaling cascade is relevant to asthmatic hypersecretion was indicated by results showing that mucin induction by asthmatic tracheal aspirates was reduced by A1, CLCA1, and EGFR inhibitors. These results suggest that adenosine cooperates with inflammatory cytokines to stimulate mucin production in the asthmatic airway and supports the use of A1, CLCA1, and EGFR inhibitors in the treatment of asthma.

Enteric neuroimmunophysiology and pathophysiology1,2

Minute-to-minute behavior of the bowel, whether it is normal or disordered, is determined by integrative functions of the enteric nervous system (ENS). Information input processed by the ENS is derived from local sensory receptors, the central nervous system, and immune/inflammatory cells including mast cells. Enteric mast cells use the power of the immune system for detection of antigenic threats and for long-term memory of the identity of the specific antigens. Specific antibodies attach to the mast cells and enable the mast cell to detect sensitizing antigens when they reappear in the gut lumen. Should the sensitizing antigen reappear, mast cells detect it and signal its presence to the ENS. The ENS interprets the mast cell signal as a threat and calls up from its program library secretory and propulsive motor behavior that is organized to eliminate the threat rapidly and effectively. Operation of the alarm program protects the individual, but at the expense of symptoms that include cramping abdominal pain, fecal urgency, and diarrhea. Enteric mast cells use immunologic memory functions to detect foreign antigens as they appear and reappear throughout the life of the individual. Mast cells use paracrine signaling for the transfer of chemical information to the neural networks of the ENS. Integrative circuits in the ENS receive and interpret the chemical signals from the mast cells. Signals from the mast cells are interpreted by the ENS as a labeled code for the presence of a threat in the intestinal lumen.

Molecular regulation of interleukin-13 and monocyte chemoattractant protein-1 expression in human mast cells by interleukin-1beta.

Mast cells play pivotal roles in immunoglobulin (Ig) E-mediated airway inflammation, expressing interleukin (IL)-13 and monocyte chemoattractant protein-1 (MCP-1), which in turn regulate IgE synthesis and/or inflammatory cell recruitment. The molecular effects of IL-1beta on cytokine expression by human mast cells (HMC) have not been studied well. In this report, we provide evidence that human umbilical cord blood-derived mast cells (CBDMC) and HMC-1 cells express the type 1 receptor for IL-1. We also demonstrate that IL-1beta and tumor necrosis factor-alpha are able to induce, individually or additively, dose-dependent expression of IL-13 and MCP-1 in these cells. The induction of IL-13 and MCP-1 gene expression by IL-1beta was accompanied by the activation of IL-1 receptor-associated kinase and translocation of the transcription factor, nuclear factor (NF) kappaB into the nucleus. Accordingly, Bay-11 7082, an inhibitor of NF-kappaB activation, inhibited IL-1beta-induced IL-13 and MCP-1 expression. IL-1beta also induced IL-13 promoter activity while enhancing the stability of IL-13 messenger RNA transcripts. Dexamethasone, a glucocorticoid, inhibited IL-1beta-induced nuclear translocation of NF-kappaB and also the secretion of IL-13 from mast cells. Our data suggest that IL-1beta can serve as a pivotal costimulus of inflammatory cytokine synthesis in human mast cells, and this may be partly mediated by IL-1 receptor-binding and subsequent signaling via nuclear translocation of NF-kappaB. Because IL-1beta is a ubiquitously expressed cytokine, these findings have important implications for non-IgE-mediated signaling in airway mast cells as well as for innate immunity and airway inflammatory responses, such as observed in extrinsic and intrinsic asthma.

Tyk2 Is Essential for IFN-α-Induced Gene Expression in Mast Cells

Mast cells are recognized not only as the major effector cells of type I hypersensitivity reactions but also as an important player of innate immune response against bacterial infection. Type I IFNs are also involved in the response against bacterial infection. However, the role of type I IFNs and their associated Janus kinase Tyk2 in mast cell functions remains to be determined. In this study, we addressed this issue using Tyk2-deficient (Tyk2^–/–) bone marrow-derived mast cells (BMMCs). When BMMCs from wild-type (WT) mice were stimulated with IFN-α, they expressed mRNA for IFN-γ-inducible protein 10 (IP-10) and monocyte chemoattractant protein-5 (MCP-5). Interestingly, IFN-α-induced expression of IP-10 and MCP-5 was severely decreased in Tyk2^–/– BMMCs. In addition, IFN-α-induced Stat1 phosphorylation was decreased in Tyk2^–/– BMMCs. On the other hand, IFN-α-induced Stat1 phosphorylation and IP-10 and MCP-5 expression were normal in Tyk2^–/– fibroblasts. These results indicate that IFN-α induces the expression of TNF-α and the chemokines IP-10 and MCP-5 in mast cells and thatTyk2 plays a nonredundant role in IFN-α signaling in mast cells.

Modulation of the Fcepsilon receptor I signaling by tyrosine kinase inhibitors: search for therapeutic targets of inflammatory and allergy diseases.

Mast cells and basophils are major effector cells in the immunoglobulin E (IgE)-dependent allergic reactions as well as in the innate immunity. They are distributed throughout the body and, upon allergen exposure, are stimulated via the high affinity IgE receptor (FcepsilonRI) to release several pro-inflammatory mediators such as leukotrienes, immunoregulatory cytokines and histamine. FcepsilonRI-mediated signaling is initiated by tyrosine phosphorylation of FcepsilonRI subunits by Src family kinase Lyn, which is followed by an activation of Syk/Zap family kinase Syk. The activated kinases then in turn phosphorylate and activate other enzymes [phospholipase Cgamma (PLCgamma) isoforms, phosphatidylinositol-3 kinase (PI3K) isoforms, protein kinase C (PKC) isoforms, Bruton's tyrosine kinase (Btk) and others], adaptors [linker for activation of T cells (LAT), Cbl, Grb2 and others] and GTP exchange factors/GTPases (Vav, Ras, Rho, and others), and subsequently induce the mobilization of stored and extracellular Ca(2+). These and other biochemical events lead within seconds and minutes to the secretory response and later to the production of chemokines. This review is focused on the use of tyrosine kinase inhibitors specific for Src family kinases (PP1/PP2, SU6656 and CT5269), Syk kinase (piceatannol, ER-27319 and BAY 61-3606) and Btk (terreic acid and LFM-A13) for a modulation of FcepsilonRI-mediated signaling in mast cells. Potential use of the inhibitors in the treatment of inflammatory and allergy diseases as well as future directions in the development of highly specific tyrosine kinases inhibitors of new generations and their use in an intended modulation of mast cell signaling are discussed.

The spleen tyrosine kinase (Syk) in human disease, implications for design of tyrosine kinase inhibitor based therapy.

The spleen tyrosine kinase Syk is an enigmatic protein tyrosine kinase functional in a number of diverse cellular processes. It is best known as a non receptor protein tyrosine kinase involved in signal transduction in cells of hematopoietic origin and plays a crucial role in signaling in most of these cells. It is involved in B and T-cell function, platelet aggregation, mast cell signaling, neutrophils and macrophages. Recently it has been found in tissues outside of the hematopoietic lineage. Perhaps the most interesting non-traditional role of Syk is that of a potential tumor suppressor in breast cancer. Absence of Syk protein in primary breast tumors is correlated with poor outcomes. Syk deficient cells have increased motility which is restored to normalcy by replacement with wild-type Syk. Syk also associates with the actin and tubulin cytoskeleton and is an alpha-tubulin kinase. The central role that Syk has in a number of cellular processes makes it an ideal starting point for broad therapeutic targeting.

Differential requirement for adapter proteins Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa and adhesion- and degranulation-promoting adapter protein in FcepsilonRI signaling and mast cell function.

The adapter molecule Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is essential for FcepsilonRI-mediated signaling, degranulation and IL-6 production in mast cells. To test the structural requirements of SLP-76 in mast cell signaling and function, we have studied the functional responses of murine bone marrow-derived mast cells (BMMCs) expressing mutant forms of SLP-76. We found that the N-terminal tyrosines as well as the central proline-rich region of SLP-76 are required for participation of SLP-76 in FcepsilonRI-mediated signaling and function. The C-terminal SH2 domain of SLP-76 also contributes to optimal function of SLP-76 in mast cells. Another adapter molecule, adhesion- and degranulation-promoting adapter protein (ADAP), is known to bind the SH2 domain of SLP-76, and cell line studies have implicated ADAP in mast cell adhesion and FcepsilonRI-induced degranulation. Surprisingly, we found that mast cells lacking ADAP expression demonstrate no defects in FcepsilonRI-induced adhesion, granule release, or IL-6 production, and that ADAP-deficient mice produce a normal passive systemic anaphylactic response. Thus, failure to bind ADAP does not underlie the functional defects exhibited by SLP-76 SH2 domain mutant-expressing mast cells.

The effects of the calcineurin inhibitors cyclosporin and ascomycin on an in-vitro assay for autoimmune urticaria

Rationale Since the calcineurin inhibitor, cyclosporin, has been used successfully in the treatment of chronic nonallergic urticaria, we examined the effect of cyclosporin (CSA) and ascomycin (ASM) on the release of histamine from basophils when stimulated with sera from patients with chronic autoimmune urticaria. Methods Healthy volunteer leukocytes were isolated and incubated in varying concentrations of CSA and ASM (0.1–10000nM) for 30 minutes prior to stimulation with serum from patients with urticaria known to have functional IgG antibodies directed against the alpha subunit of the IgE receptor. Results Pre-incubating cells with CSA and ASM produced dose-dependent inhibition of histamine release from sera of urticaria patients. Inhibition was not prevented by C5a receptor blocking antibodies. Dose-dependent inhibition was seen to purified IgG from serum but not from complement C5a. Conclusions Calcineurin inhibitors such as CSA and ASM may work by interfering with intracellular signalling in basophils and mast cells following cross-linking of the IgE receptor but not by stimulation of the C5a receptor.

The effect of a novel inhibitor of mast cell activation on mediators, symptoms and nasal patency in allergic rhinitis

Rationale Examine the efficacy and safety of a single dose of R926112, an inhibitor of the tyrosine kinase Syk critical for mast cell signaling, on symptoms, mediator release, and nasal volumes in allergic rhinitis. Methods Twenty out-of-season volunteers with grass or ragweed pollen induced allergic rhinitis were enrolled in this double blind randomized crossover trial. Subjects underwent a screening nasal allergen challenge to establish the extract dose eliciting a Bousquet score of 5. One dose of R926112 or vehicle was given intranasally followed by nasal allergen challenge 15 minutes later. Symptom scores were obtained at baseline, after treatment and after challenge. Histamine and PGD 2 content of nasal secretions was determined at baseline, 5 and 10 minutes, and tryptase content at baseline and 10 minutes after allergen challenge. Bilateral nasal volumes (cm 3) and minimal cross sectional area (MCA) were assessed by acoustic rhinometry before and twenty minutes after challenge. Results Adverse event frequency was similar between treatments. R926112 resulted in a significant decrease in PGD 2 at 5 minutes vs. vehicle (93.4 ± 23 pg/ml vs. 171.6 ± 23 pg/ml p=0.03) that correlated with rhinorrhea (p=0.05). PGD 2 and tryptase at 10 minutes tended to decrease with R926112 administration (46.8 ± 9.2 pg/ml vs. 68.6 ± 9.2 p=0.1, and 9.5 ± 2.7 ng/ml vs. 16.6 ± 2.9, p=0.09). Treatment improved rhinorrhea but symptom scores were not significantly different between groups. There were no differences in histamine concentrations between the treatment arms or acoustic rhinometry determined volume or cross-sectional area. Conclusions R926112 appears to be safe and reduces mediator release in subjects with allergic rhinitis.

The Src kinase Lyn is a negative regulator of mast cell proliferation.

Previous investigators have reported that deletion of the protein tyrosine kinase Lyn alters mast cell (MC) signaling responses but does not affect or reduces the cytokine-mediated proliferation of mouse bone marrow-derived MC (BMMC) precursors and of mature MC. We observed that Lyn-deficient mice have more peritoneal MC than wild-type (WT) mice. Studies to explore this unexpected result showed that Lyn(-/-) BM cells expand faster than WT cells in response to interleukin (IL)-3 and stem-cell factor over the 4-5 weeks required to produce a >95% pure population of granular, receptor with high affinity for immunoglobulin E-positive BMMC. Furthermore, differentiated Lyn(-/-) BMMC continue to proliferate more rapidly than WT BMMC and undergo less apoptosis in response to cytokine withdrawal. Additionally, Lyn(-/-) BMMC support greater IL-3-mediated phosphorylation of the prosurvival kinase, Akt, and the proliferative kinase, extracellular-regulated kinase 1/2. These results identify Lyn as a negative regulator of murine MC survival and proliferation.

IgE alone-induced actin assembly modifies calcium signaling and degranulation in RBL-2H3 mast cells.

In the mast cell signaling pathways, the binding of immunoglobulin E (IgE) to FcepsilonRI, its high-affinity receptor, is generally thought to be a passive step. In this study, we examined the effect of IgE alone, that is, without antigen stimulation, on the degranulation in mast cells. Monomeric IgE (500-5,000 ng/ml) alone increased cytosolic Ca2+ level ([Ca2+]i) and induced degranulation in rat basophilic leukemia (RBL)-2H3 mast cells. Monomeric IgE (5,000 ng/ml) alone also increased [Ca2+]i and induced degranulation in bone marrow-derived mast cells. Interestingly, monomeric IgE (5-50 ng/ml) alone, in concentrations too low to induce degranulation, increased filamentous actin content in RBL-2H3 mast cells. We next examined whether actin dynamics affect the IgE alone-induced RBL-2H3 mast cell activation pathways. Cytochalasin D inhibited the ability of IgE alone (50 ng/ml) to induce de novo actin assembly. In cytochalasin D-treated cells, IgE (50 ng/ml) alone increased [Ca2+]i and induced degranulation. We have summarized the current findings into two points. First, IgE alone increases [Ca2+]i and induces degranulation in mast cells. Second, IgE, at concentrations too low to increase either [Ca2+]i or degranulation, significantly induces actin assembly, which serves as a negative feedback control in the mast cell Ca2+ signaling and degranulation.

Stat6-protease but not Stat5-protease is inhibited by an elastase inhibitor ONO-5046

A short isoform of Stat6 (65-kDa Stat6), a product of proteolytic processing by an undefined protease (Stat6-protease) in the nucleus, downregulates Stat6-mediated signaling in mast cells. Similarly, Stat5-mediated signaling is downregulated by Stat5-protease in myeloid progenitors. These proteases share a number of characteristics, including their nuclear localization and susceptibility to protease inhibitors. Here, we further investigated these Stat proteases. Interestingly, the activity of Stat6-protease but not of Stat5-protease was inhibited by ONO-5046, an elastase inhibitor that inhibits the activity of neutrophil elastase (NE) and NE-related protease proteinase 3 (PR3). Although both NE and PR3 were able to cleave Stat6 in vitro, the cleavage sites of Stat6 by NE or PR3 differed from that by Stat6-protease in mast cells. In addition, both NE and PR3 could also cleave Stat5, but they differed from Stat5-protease in myeloid progenitors. These results suggest that Stat6-protease may belong to the elastase family but differs from NE or PR3.

Role of phosphoinositide 3-kinase signaling in mast cells: new insights from knockout mouse studies.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases essential for diverse physiological reactions. In recent years a series of gene-targeted mice lacking different types of PI3Ks and related molecules have been generated which enable us to understand the role of PI3K pathways, particularly class I members, in vivo. Analyses of such gene-targeted mice have led to major discoveries in the physiological roles of PI3K signaling in mast cell biology. In particular the role of PI3Ks has been extensively studied in signaling through the high-affinity IgE receptor (FcepsilonRI), since mast cells are the main effector cells in type I allergic reaction associated with IgE-dependent mechanisms. Furthermore, the knockout mice have provided significant information concerning the role of PI3K signals in mast cell differentiation. This review presents several new insights into mast cell biology, which have been elucidated by the analyses of these knockout mice.

Adaptor Protein 3BP2 Is a Potential Ligand of Src Homology 2 and 3 Domains of Lyn Protein-tyrosine Kinase

Adaptor protein 3BP2, a c-Abl-Src homology 3 (SH3) domain-binding protein, is known to play a regulatory role in T-cell receptor-mediated transcriptional activation of nuclear factor of activated T cell and activator protein 1 by interacting with Syk/ZAP-70 protein-tyrosine kinase. We have previously demonstrated that aggregation of high affinity IgE receptor (FcepsilonRI) induces tyrosine phosphorylation of 3BP2, and overexpression of the 3BP2-SH2 domain suppresses antigen-induced degranulation in rat basophilic leukemia RBL-2H3 mast cell line. In this report, we attempt to analyze the biological relevance of 3BP2 tyrosine phosphorylation. By using the transient expression system in COS-7 cells, we have demonstrated that 3BP2 was predominantly phosphorylated on Tyr174, Tyr183, and Tyr446 when it was coexpressed with Syk. An in vitro binding study revealed that phosphorylation of Tyr446 by Syk was likely to create a binding site for the Lyn-SH2 domain in RBL-2H3 cells. In addition, proline-rich region of 3BP2 bound to the Lyn-SH3 domain. Conformational microscopic analysis showed that Lyn and 3BP2 are constitutively colocalized in RBL-2H3 cells. Overexpression of 3BP2 in RBL-2H3 cells resulted in an enhancement of Lyn autophosphorylation. These results suggest that the adaptor protein 3BP2 is a potential regulator of Lyn protein-tyrosine kinase as a ligand of its SH3/SH2 domains in FcepsilonRI-mediated signaling in mast cells.