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.

The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid-and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, in cis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca 2؉ . However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcRI does not exhibit an increased association with lipid rafts, suggest that FcRI phosphorylation and early activation events can be initiated outside of lipid rafts.The high-affinity immunoglobulin E (IgE) receptor (FcεRI)-mediated activation of mast cells and basophils triggers a cascade of intracellular biochemical events that ultimately lead to the secretion of preformed pharmacological agents and the transcription of cytokine genes. This process is initiated by aggregation of the receptor by means of multivalent antigen (Ag)-IgE complexes, followed by tyrosine phosphorylation of the receptor subunits by Src family protein tyrosine kinases (14, 31).FcεRI has a tetrameric structure comprised of an IgE-binding ␣ subunit, a ␤ subunit, and a disulfide-bonded ␥ dimer (32). The ␤ and ␥ subunits possess immunoreceptor tyrosine-based activation motifs (ITAMs), which are rapidly phosphorylated by protein tyrosine kinase Lyn. Tyrosine-phosphorylated ITAMs of the the ␥ subunits serve as novel binding sites for Src homology 2 (SH2) domains of Syk kinase (6, 22, 28), leading to phosphorylation and activation of Syk. Thereafter, a number of other signaling and adaptor molecules become phosphorylated and recruited into the regions of activated FcεRI/Syk complexes. These include PLC␥1 (26), the proto-oncogene product Vav (41), PKC-␦ (18), and the linker for activation of T cells (LAT) (37, 52).Detailed molecular mechanisms of the initial engagement of the Lyn kinase and FcεRI are not completely understood, but two different models have been proposed. One model, based on protein-protein interactions, postulates that a small fraction of Lyn is constitutively associated with the ␤ subunit of the FcεRI prior...

Migration of mast cells is essential for their recruitment within target tissues where they play an important role in innate and adaptive immune responses. These processes rely on the ability of mast cells to recognize appropriate chemotactic stimuli and react to them by a chemotactic response. Another level of intercellular communication is attained by production of chemoattractants by activated mast cells, which results in accumulation of mast cells and other hematopoietic cells at the sites of inflammation. Mast cells express numerous surface receptors for various ligands with properties of potent chemoattractants. They include the stem cell factor (SCF) recognized by c-Kit, antigen, which binds to immunoglobulin E (IgE) anchored to the high affinity IgE receptor (FcεRI), highly cytokinergic (HC) IgE recognized by FcεRI, lipid mediator sphingosine-1-phosphate (S1P), which binds to G protein-coupled receptors (GPCRs). Other large groups of chemoattractants are eicosanoids [prostaglandin E2 and D2, leukotriene (LT) B4, LTD4, and LTC4, and others] and chemokines (CC, CXC, C, and CX3C), which also bind to various GPCRs. Further noteworthy chemoattractants are isoforms of transforming growth factor (TGF) β1–3, which are sensitively recognized by TGF-β serine/threonine type I and II β receptors, adenosine, C1q, C3a, and C5a components of the complement, 5-hydroxytryptamine, neuroendocrine peptide catestatin, tumor necrosis factor-α, and others. Here we discuss the major types of chemoattractants recognized by mast cells, their target receptors, as well as signaling pathways they utilize. We also briefly deal with methods used for studies of mast cell chemotaxis and with ways of how these studies profited from the results obtained in other cellular systems.

Thy-1 is a surface glycoprotein that is attached to the plasma membrane by a glycosyl-phosphatidylinositol anchor. Crosslinking of Thy-1 in rat mast cells and basophilic leukemia cefls (RBL-2H3) induces cell activation including histamine release and tyrosine phosphorylation of several proteins. Here we show that glycosyl-phosphatidylinositol-linked Thy-1 forms noncovalent complexes with srcrelated protein-tyrosine kinase p53/p56'Yn and other proteintyrosine kinases and/or their substrates. These complexes are resistant to solubilization by a nonionic detergent, sedimentable at 200,000 x g, and very large (>10 MDa) as determined by gel chromatography. Activation of RBL-2H3 cells by crosslinking of the high-affinity IgE receptors resulted in decreased recovery of the complexes. The combined data indicate the existence of large detergent-resistant domains in the surface membrane of mast cells that may play an important role in their activation.

The exposure of phosphatidylserine (PS) on the cell surface is a general marker of apoptotic cells. Non-apoptotic PS externalization is induced by several activation stimuli, including engagement of immunoreceptors. Immune cells can also be activated by aggregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs). However, it is unknown whether cell triggering through these proteins, lacking transmembrane and cytoplasmic domains, also leads to PS externalization. Here we show that engagement of GPI-APs in rodent mast cells induces a rapid and reversible externalization of PS by a non-apoptotic mechanism. PS externalization triggered by GPI-AP-specific monoclonal antibodies was dependent on the activity of H ؉ -ATP synthase and several other enzymes involved in mast cell signaling but was independent of cell degranulation, free cytoplasmic calcium up-regulation, and a decrease in lipid packing as determined by merocyanine 540 binding. Surprisingly, disruption of actin cytoskeleton by latrunculin B or plasma membrane integrity by methyl-␤-cyclodextrin had opposite effects on PS externalization triggered through GPI-AP or the high affinity IgE receptor. We further show that PS externalization mediated by GPI-APs was also observed in some other cells, and its extent varied with antibodies used. Interestingly, effects of different antibodies on PS externalization were additive, indicating that independent stimuli converge onto a signaling pathways leading to PS externalization. Our findings identify the cell surface PS exposure induced through GPI-AP as a distinct mechanism of cell signaling. Such a mechanism could contribute to "inside-out" signaling in response to pathogens and other external activators and/or to initiation of other functions associated with PS externalization.The plasma membrane exhibits a marked asymmetry in transbilayer distribution of phospholipids. Aminophospholipids, including phosphatidylserine (PS), 2 are usually restricted to the inner leaflet of the membrane. This phospholipid asymmetry is maintained by activity of energy-dependent flippases and floppases that mediate, respectively, inward-directed and outward-directed transfer of phospholipids (1-3). Furthermore, equilibration of phospholipids between the two plasma membrane leaflets seems to be regulated by lipid scramblase, which facilitates bi-directional migration of phospholipids across the bilayer (4, 5). In response to some stimuli, the phospholipid asymmetry is lost, and PS is translocated to the exoplasmic leaflet of plasma membrane (6). Externalized PS is observed in apoptotic, injured, infected, senescent, or necrotic cells and becomes a target for recognition by phagocytes (7-11). PS externalization is also detected at certain stages of cell development (12) and in the course of activation of immune cells by different stimuli, including engagement of immunoreceptors (13-16).It has been described that PS externalization is triggered by stimuli enhancing the concentration of free cytoplasmic calcium, which regulates ...

The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN-and/or a FYNdependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved. Mast cells are widely distributed in the body, where they play important roles in innate as well as adaptive immune responses (1). To fulfill their role in adaptive immune responses, the cells express the high-affinity IgE receptor FcεRI on their plasma membranes. Aggregation of this tetrameric immunoreceptor, ␣␤␥2, induces cell signaling events leading to the release of preformed inflammatory mediators and the de novo synthesis and release of leukotrienes, cytokines, and chemokines. The first welldefined biochemical step after FcεRI triggering is tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motifs in the cytoplasmic tail of the FcεRI ␤ and ␥ subunits by the SRC family protein tyrosine kinase (PTK) LYN (2, 3). The phosphorylated ␤ and ␥ subunits then serve as binding and activation sites for LYN kinase and spleen tyrosine kinase (SYK), respectively. These two enzymes, together with FYN and other kinases, then phosphorylate various adaptor proteins and enzymes with a variety of functions in signal transduction pathways. The exact molecular events preceding LYN-mediated tyrosine phosphorylation of the FcεRI ␤ subunit are not clear, and several models have been proposed, including the transphosphorylation model (4), the lipid raft model (5), and the PTK-protein tyrosine phosphatase (PTP) interplay model (6).Our previous study with murine bone marrow-derived mast cells (BMMCs) showed that FcεRI triggering induced transient hyperphosphorylation of LYN kinase on its C-terminal regulatory tyrosine (Tyr 487), leading to the formation of a closed inactive conformation where the SRC homology 2 (...

The molecular mechanisms controlling microtubule formation in cells with non-centrosomal microtubular arrays are not yet fully understood. The key component of microtubule nucleation is gamma-tubulin. Although previous results suggested that tyrosine kinases might serve as regulators of gamma-tubulin function, their exact roles remain enigmatic. In the present study, we show that a pool of gamma-tubulin associates with detergent-resistant membranes in differentiating P19 embryonal carcinoma cells, which exhibit elevated expression of the Src family kinase Fyn (protein tyrosine kinase p59(Fyn)). Microtubule-assembly assays demonstrated that membrane-associated gamma-tubulin complexes are capable of initiating the formation of microtubules. Pretreatment of the cells with Src family kinase inhibitors or wortmannin blocked the nucleation activity of the gamma-tubulin complexes. Immunoprecipitation experiments revealed that membrane-associated gamma-tubulin forms complexes with Fyn and PI3K (phosphoinositide 3-kinase). Furthermore, in vitro kinase assays showed that p85alpha (regulatory p85alpha subunit of PI3K) serves as a Fyn substrate. Direct interaction of gamma-tubulin with the C-terminal Src homology 2 domain of p85alpha was determined by pull-down experiments and immunoprecipitation experiments with cells expressing truncated forms of p85alpha. The combined results suggest that Fyn and PI3K might take part in the modulation of membrane-associated gamma-tubulin activities.

Previous studies using cytochalasins and latrunculin B, inhibitors of actin polymerization, showed that filamentous (F)-actin had a negative regulatory role in Fc 4 receptor I (Fc 4 RI) signaling. How F-actin is involved in regulating the activation of mast cells is unknown. In this study we investigated the role of F-actin in mast cell activation induced by aggregation of the glycosylphosphatidylinositol (GPI)-anchored proteins Thy-1 and TEC-21, and compared it to activation via Fc 4 RI. Pretreatment of rat basophilic leukemia cells with latrunculin B inhibited the Thy-1-induced actin polymerization and elevated the Thy-1-mediated secretory and calcium responses. Inhibition of actin polymerization followed by Thy-1 aggregation resulted in an increased tyrosine phosphorylation of Syk, phospholipase C + (PLC + ), Gab2 and linker for activation of T cells (LAT) adapters, and some other signaling molecules. Enzymatic activities of phosphatidylinositol 3-kinase, PLC + , and phosphatase SHP-2 were also upregulated, but tyrosine phosphorylation of ezrin was inhibited. Similar changes were observed in Fc 4 RI-activated cells. Significant changes in intracellular distribution, tyrosine phosphorylation, and/or enzymatic activities of signaling molecules occurred in latrunculinpretreated cells before cell triggering. The combined data suggest that actin polymerization is critical for setting the thresholds for mast cell signaling via aggregation of both Fc 4 RI and GPI-anchored proteins.