Tyrosine phosphorylation of the linker for activator of T cells in mast cells by stimulation with the high affinity IgE receptor

Aggregation of the high affinity IgE receptors (FcepsilonRI) on basophils and mast cells, members of the immune receptor family, initiates a cascade of events that results in the release of inflammatory mediators. This pathway involves the activation of several protein-tyrosine kinases, including Lyn, Syk, Btk, and Fak that induce the tyrosine phosphorylation of various proteins. The linker for activation of T cells (LAT), was originally found as a ZAP-70 tyrosine kinase substrate that linked T cell receptors to cellular activation, and was expressed in T cells, NK cells and mast cells. Here we show that LAT expressed in the RBL-2H3 rat mast cell line is tyrosine-phosphorylated after aggregation of FcepsilonRI. The tyrosine phosphorylation of the LAT was dramatically enhanced after receptor aggregation. Furthermore, a tyrosine-phosphorylated 80-kDa protein associated with LAT transiently after receptor aggregation. GST fusion proteins containing parts of PLCgamma or PI3 kinase can bind LAT. These results suggest that LAT plays an important role not only in T cell, but also in mast cell activation, and that the association among these signaling molecules is critical for FcepsilonRI-mediated intracellular signal transduction in mast cells.     

A perspective: regulation of IgE receptor-mediated mast cell responses by a LAT-organized plasma membrane-localized signaling complex

BACKGROUND: To understand how the high-affinity IgE receptor (FcepsilonRI) communicates with downstream effectors, we focused on exploring the functional importance of the FcepsilonRI-mediated formation and localization of a signaling complex that contains the hematopoietic cell-specific scaffolding protein linker for activation of T cells (LAT) and the guanine nucleotide exchange factor Vav1. METHODS: Using the mast cell line RBL-2H3, we explored the localization of these proteins by confocal microscopy and cell fractionation. Additionally, the mechanism of function and the importance of LAT and Vav1 to mast cells was studied in genetically disrupted mice and in mast cells derived from their bone marrow. RESULTS: We found that LAT, Vav1 and the adapter molecule SLP-76 associated in detergent-resistant microdomains (lipid rafts) found in the plasma membrane upon FcepsilonRI stimulation. In the absence of LAT, mast cells showed a remarkable loss of the secretory response and reduced cytokine responses. Vav1 deficiency also affected secretion, although not to the extent of LAT deficiency, and inhibited IL-2 and IFN-gamma production. LAT- and Vav1-deficient mice showed reduced blood histamine levels after a systemic anaphylaxis challenge as compared to their normal counterparts. CONCLUSIONS: The results demonstrate that LAT is a central mediator in IgE receptor signaling by regulating multiple signaling pathways that affect mast cell degranulation and cytokine production. Vav1, a component of this LAT-containing signaling complex, regulates a specific subset of these responses.     

Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation

Fc epsilon RI activation of mast cells is thought to involve Lyn and Syk kinases proximal to the receptor and the signaling complex organized by the linker for activation of T cells (LAT). We report here that Fc epsilon RI also uses a Fyn kinase-dependent pathway that does not require Lyn kinase or the adapter LAT for its initiation, but is necessary for mast cell degranulation. Lyn-deficiency enhanced Fyn-dependent signals and degranulation, but inhibited the calcium response. Fyn-deficiency impaired degranulation, whereas Lyn-mediated signaling and calcium was normal. Thus, Fc epsilon RI-dependent mast cell degranulation involves cross-talk between Fyn and Lyn kinases.     

Topography of signaling molecules as detected by electron microscopy on plasma membrane sheets isolated from non-adherent mast cells

Immunolabeling of isolated plasma membrane (PM) sheets combined with high-resolution electron microscopy is a powerful technique for understanding the topography of PM-bound signaling molecules. However, this technique has been mostly confined to analysis of membrane sheets from adherent cells. Here we present a rapid, simple and versatile method for isolation of PM sheets from non-adherent cells, and show its use for examination of the topography of Fcepsilon receptor I (FcepsilonRI) and transmembrane adaptors, LAT (linker for activation of T cells) and NTAL (non-T cell activation linker), in murine bone marrow-derived mast cells (BMMC). The data were compared with those obtained from widely used but tumor-derived rat basophilic leukemia (RBL) cells. In non-activated cells, FcepsilonRI was distributed either individually or in small clusters of comparable size in both cell types. In multivalent antigen-activated BMMC as well as RBL cells, FcepsilonRI was internalized to a similar extent, but, strikingly, internalization in BMMC was not preceded by formation of large (~200 nm) aggregates of FcepsilonRI, described previously in activated RBL cells. On the other hand, downstream adaptor proteins, LAT and NTAL, were localized in independent domains in both BMMC and RBL cells before and after FcepsilonRI triggering. The combined data demonstrate unexpected properties of FcepsilonRI signaling assemblies in BMMC and emphasize the importance of studies of PM sheets isolated from non-tumor cells.     

Regulation of mast cell signaling through high-affinity IgE receptor by CD45 protein tyrosine phosphatase

The transmembrane tyrosine phosphatase CD45 regulates the activity of src family protein tyrosine kinases (PTK) and thereby influences the signaling via such receptors as T and B cell antigen receptors associated with these PTK. However, its implication in signaling through the mast cell receptor with high affinity for IgE (FcepsilonRI) is less clear, although Lyn, a member of the src family, plays an important role in FcepsilonRI-mediated signaling. To define a role for CD45 in FcepsilonRI signal transduction, we established CD45 high expressing rat basophilic leukemia cell lines (RBL-CD45H) and cell lines expressing trace amounts of CD45 (RBL-CD45L). We demonstrate that although all RBL-CD45L cell lines degranulate following IgE- and antigen-induced FcepsilonRI aggregation, the response is significantly reduced at a low dose of antigen. The cells show a delayed and slowed Ca(2+) mobilization even though at a higher dose where the cells degranulate to a similar extent as RBL-CD45H. This diminished Ca(2+) response is restored by reconstitution of RBL-CD45L with a chimeric molecule containing the cytoplasmic phosphatase domains of rat CD45. Furthermore, tyrosine phosphorylation of FcepsilonRI, association of FcepsilonRI with Lyn and PTK activity associated with FcepsilonRI, all of which are enhanced upon FcepsilonRI aggregation in RBL-CD45H, are impaired in RBL-CD45L. Finally, we show that FcepsilonRI is physically associated with CD45 in RBL-CD45H prior to receptor aggregation. Thus, we propose that, although not indispensable in mast cell degranulation, CD45 positively regulates the signaling through FcepsilonRI by promoting the activation of FcepsilonRI-associated Lyn.     

FcepsilonRI signaling observed from the inside of the mast cell membrane

Crosslinking the high affinity IgE receptor, FcrepsilonRI, on basophils and mast cells initiates cascades of biochemical events leading to degranulation, membrane ruffling and other physiological responses. Downstream of FcepsilonRI and its coupled tyrosine kinases, Lyn and Syk, scores of different proteins and lipids are implicated in these signaling cascades and new players are being identified continuously. Here, we use immunogold probes to label receptors and signaling proteins on the cytoplasmic face of membrane sheets prepared from RBL-2H3 mast cells and transmission electron microscopy to examine their distributions in relationship to each other and to features of the membrane. New topographical data are integrated with existing knowledge of the biochemistry of FcepsilonRI signaling and of cell shape during signaling to implicate at least two distinct membrane domains in FcepsilonRI signaling. "Primary signaling domains", also called osmiophilic patches, are recognized by their dark staining with osmium, adjacency to coated pits (previously mapped to planar membrane between lamellae) and by the characteristic presence of receptor, Syk and PLCgamma2, but not Lyn. "Secondary signaling domains" are characterized by the presence of large elliptical linker for activation of T cells (LAT) rafts and of PLCgamma1 (previously mapped to lamellae) but not receptor. The signaling proteins, Vav, Grb2, Cbl and Gab2, and the endocytic proteins, AP2 and clathrin, all map to the primary domains, while the p85 regulatory subunit of phosphatidylinositol 3 (PI 3)-kinase maps to both domains. Recognition that FcepsilonRI signaling is controlled not only by which chemical species are available for interaction, but also by where the interactions occur, may provide new opportunities for the modeling of signaling cascades and new targets for the development of drugs to treat allergies and asthma.     

Requirement of the tyrosines at residues 258 and 270 of MAIR-I in inhibitory effect on degranulation from basophilic leukemia RBL-2H3

Mast cells and basophils express the high affinity receptor for IgE (FcepsilonRI) and play a central role for IgE-associated immediate hypersensitivity reactions and allergic disorders. Cross-linking of FcepsilonRI-bound IgE with multivalent antigen initiates the activation of mast cells and basophils, resulting in the degranulation from these cells. We have recently identified a novel inhibitory receptor, myeloid-associated immunoglobulin-like receptor (MAIR)-I, which is expressed on mast cells as well as other myeloid cell lineages. Co-ligation of FcepsilonRI and MAIR-I inhibits IgE-mediated degranulation from mast cells. However, MAIR-I-mediated signaling pathways involved in the inhibition remain undetermined. Here, we demonstrate that the transfectant of rat basophil leukemia RBL-2H3 expressing wild-type MAIR-I is tyrosine phosphorylated and recruits SHP-1 and SHIP upon cross-linking of MAIR-I. By using RBL-2H3 transfectants expressing variable mutant MAIR-I at Y233, Y258, Y270 and/or Y299, we further demonstrate that both Y258 and Y270, but not Y233 and Y299, were phosphorylated and were essentially required for inhibition of IgE-mediated degranulation from the RBL-2H3 transfectant.     

Topography of plasma membrane microdomains and its consequences for mast cell signaling

Thy-1 (CD90) is a glycoprotein bound to the plasma membrane by a GPI anchor. Aggregation of Thy-1 in mast cells and basophils induces activation events independent of the expression of Fcepsilon receptor I (FcepsilonRI). Although we and others have previously suggested that plasma membrane microdomains called lipid rafts are implicated in both Thy-1 and FcepsilonRI signaling, properties of these microdomains are still poorly understood. In this study we used rat basophilic leukemia cells and their transfectants expressing both endogenous Thy-1.1 and exogenous Thy-1.2 genes and analyzed topography of the Thy-1 isoforms and Thy-1-induced signaling events. Light microscopy showed that both Thy-1 isoforms were in the plasma membrane distributed randomly and independently. Electron microscopy on isolated membrane sheets and fluorescence resonance energy transfer analysis indicated cross-talk between Thy-1 isoforms and between Thy-1 and FcepsilonRI. This cross-talk was dependent on actin filaments. Thy-1 aggregates colocalized with two transmembrane adaptor proteins, non-T cell activation linker (NTAL) and linker for activation of T cells (LAT), which had been shown to inhabit different membrane microdomains. Thy-1 aggregation led to tyrosine phosphorylation of these two adaptors. The combined data indicate that aggregated GPI-anchored proteins can attract different membrane proteins in different clusters and thus can trigger different signaling pathways.     

LAT is essential for Fc(epsilon)RI-mediated mast cell activation

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement of T cells. LAT is also expressed in platelets, NK, and mast cells. Although LAT-deficient mice contain normal numbers of mast cells, we found that LAT-deficient mice were resistant to IgE-mediated passive systemic anaphylaxis. LAT-deficient bone marrow-derived mast cells (BMMC) showed normal growth and development. Whereas tyrosine phosphorylation of Fc(epsilon)RI, Syk, and Vav was intact in LAT-deficient BMMCs following Fc(epsilon)RI engagement, tyrosine phosphorylation of SLP-76, PLC-gamma1, and PLC-gamma2 and calcium mobilization were dramatically reduced. LAT-deficient BMMCs also exhibited profound defects in activation of MAPK, degranulation, and cytokine production after Fc(epsilon)RI cross-linking. These results show that LAT plays a critical role in Fc(epsilon)RI-mediated signaling in mast cells.     

Signaling assemblies formed in mast cells activated via Fcepsilon receptor I dimers

Although aggregation of the Fcepsilon receptor I (FcepsilonRI) is necessary for Ag-mediated mast cell triggering, the relationship between the extent of the FcepsilonRI aggregation and subsequent biochemical and topographical events is incompletely understood. In this study, we analyzed the activation events induced by FcepsilonRI dimers, elicited by binding of anti-FcepsilonRI mAb to rat basophilic leukemia cells. We found that, in contrast to extensively aggregated FcepsilonRI, receptor dimers (1) induced a less extensive association of FcepsilonRI with detergent-resistant membranes, (2) delayed the tyrosine phosphorylation and membrane recruitment of several signaling molecules, (3) triggered a slower but more sustained increase in concentration of free cytoplasmic calcium, (4) induced degranulation which was not inhibited at higher concentrations of the cross-linking mAb, and (5) failed to produce clusters of FcepsilonRI, Syk kinase and Grb2 adapter in osmiophilic membranes, as detected by immunogold electron microscopy on membrane sheets. Despite striking differences in the topography of FcepsilonRI dimers and multimers, biochemical differences were less pronounced. The combined data suggest that FcepsilonRI-activated mast cells propagate signals from small signaling domains formed around dimerized/oligomerized FcepsilonRI; formation of large FcepsilonRI aggregates in osmiophilic membranes seems to promote both strong receptor triggering and rapid termination of the signaling responses.     

Association of 3BP2 with SHP-1 regulates SHP-1-mediated production of TNF-α in RBL-2H3 cells

Adaptor protein 3BP2, a c-Abl Src homology 3 (SH3) domain-binding protein, is tyrosine phosphorylated and positively regulates mast cell signal transduction after the aggregation of the high affinity IgE receptor (FcεRI). Overexpression of the Src homology 2 (SH2) domain of 3BP2 results in the dramatic suppression of antigen-induced degranulation in rat basophilic leukemia RBL-2H3 cells. Previously, a linker for activation of T cells (LAT) was identified as one of the 3BP2 SH2 domain-binding protein. In this report, to further understand the functions of 3BP2 in FcεRI-mediated activation of mast cell, we explored the protein that associates with the SH2 domain of 3BP2 and found that SH2 domain-containing phosphatase-1 (SHP-1) inducibly interacts with the SH2 domain of 3BP2 after the aggregation of FcεRI. The phosphorylation of Tyr(564) in the carboxy (C)-terminal tail region of SHP-1 is required for the direct interaction of SHP-1 to the SH2 domain of 3BP2. The expression of the mutant form of SHP-1 which was unable to interact with 3BP2 resulted in the significant reduction in SHP-1-mediated tumor necrosis factor-α (TNF-α) production without any effects on the degranulation in antigen-stimulated RBL-2H3 cells. These findings suggest that 3BP2 directly interacts with Tyr(564) -phosphorylated form of SHP-1 and positively regulates the function of SHP-1 in FcεRI-mediated signaling in mast cells.     

The protein tyrosine kinase syk activity is reduced by clustering the mast cell function-associated antigen

The mast cell function-associated antigen (MAFA) is a glycoprotein first identified on the membrane of rat mucosal-type mast cells (RBL-2H3 line). MAFA clustering causes a dose-dependent inhibition of these cells' secretory response to the type I Fcepsilon receptor (FcepsilonRI) stimulus. The inhibition has earlier been shown to take place upstream to the production step of inositol phosphates in the FcepsilonRI coupling cascade. To resolve further the mechanism of action of MAFA, we have investigated the events prior to the activation of phospholipase C. Activities of the non-receptor protein tyrosine kinases Lyn and Syk in untreated cells were compared with those where the FcepsilonRI, MAFA or both were clustered. Syk tyrosine phosphorylation and activation, as well as LAT (linker for activation of T cells) tyrosine phosphorylation, both induced by FcepsilonRI clustering, were found to be reduced upon MAFA clustering. In contrast, the activity of the Src homology domain 2 (SH2)-containing protein tyrosine phosphatase (SHP-2) increased. MAFA clustering also enhanced the co-isolation of SHP-2 and Syk with tyrosine-phosphorylated MAFA in both untreated and FcepsilonRI-stimulated cells. SHP-2 caused a decline in the FcepsilonRI-induced tyrosine phosphorylation of Syk, at least under in vitro conditions. Taken together, these results suggest that one possible mechanism by which MAFA affects the FcepsilonRI stimulation cascade is suppression of Syk activity, i.e. MAFA clustering leads SHP-2 to act on Syk, thereby reducing its tyrosine phosphorylation and its activity.     

Functionality of the IgA Fc receptor (FcalphaR, CD89) is down-regulated by extensive engagement of FcepsilonRI

Besides mast cells and basophils, the high-affinity IgE Fc receptor (FcepsilonRI) is exclusively expressed on certain FcalphaR (IgA Fc receptor)-expressing immune cells such as neutrophils in allergic patients. Transfected rat basophilic leukemia cell line (RBL-2H3) co-expressing FcepsilonRI and FcalphaR was analyzed for effects of simultaneous receptor engagement by their specific antibodies on degranulation and signaling. Whereas supraoptimal FcepsilonRI engagement decreased degranulation, which is known as a bell-shaped dose-response curve, such inhibitory effect was not observed with FcalphaR engagement. However, simultaneous engagement of FcepsilonRI and FcalphaR showed that supraoptimal FcepsilonRI engagement down-regulates FcalphaR-mediated degranulation. This inhibition was associated with extensive phosphorylation of inositol polyphosphate 5'-phosphatase SHIP1 and FcepsilonRIbeta, and reversed by adding actin-depolymerizing drug, latrunculin B. The results suggest an endogenous mechanism by which FcalphaR functionality is down-regulated in an 'allergic environment' where FcepsilonRI is co-expressed and extensively cross-linked on FcalphaR-expressing effector cells.     

SWAP-70-deficient mast cells are impaired in development and IgE-mediated degranulation

Cross-linking of the high-affinity IgE receptor (FcepsilonRI) on mast cell activates signaling pathways that trigger degranulation and the release of multiple pro-inflammatory mediators. Mature,immature and precursor mast cells are degranulation competent. We show here that the signaling protein SWAP-70 has a function in mast cell biology. While not found in many cell types, we find that apart from B cells, mast cells also express SWAP-70. In activated B cells, SWAP-70 shuttles between cytoplasm and nucleus, but in mast cells it is confined to the cytoplasm. SWAP-70(ko/ko) (double knockout) mice have reduced numbers of mature mast cells, and these are degranulation competent. However, although immature mast cells from SWAP-70(ko/ko) mice respond normally to SCF and IL-3 and have functional granules, their FcepsilonRI-mediated degranulation is inhibited. Thus, in mast cells SWAP-70 plays a role both in establishing the initial competence to degranulate and to develop into mature mast cells.     

Regulation of lymphocyte development and activation by the LAT family of adapter proteins

Summary:  Transmembrane adapter proteins (TRAPs) are critical components of signaling pathways in lymphocytes, linking antigen receptor engagement to downstream cellular processes. While these proteins lack intrinsic enzymatic activity, their phosphorylation following receptor ligation allows them to function as scaffolds for the assembly of multi-molecular signaling complexes. Many TRAPs have recently been discovered, and numerous studies demonstrate their roles in the positive and negative regulation of lymphocyte maturation, activation, and differentiation. One such example is the linker for activation of T cells (LAT) family of adapter proteins. While LAT has been shown to play an indispensable role in T-cell and mast cell function, the other family members, linker for activation of B cells (LAB) and linker for activation of X cells (LAX), are necessary to fine-tune immune responses. In addition to its well-established role in the positive regulation of lymphocyte activation, LAT exerts an inhibitory effect on T-cell receptor-mediated signaling. Furthermore, LAT, along with LAB and LAX, plays a crucial role in establishing and maintaining tolerance. Here, we review recent data concerning the regulation of lymphocyte development and activation by the LAT family of proteins.     

Amplification mechanisms for the enhancement of antigen-mediated mast cell activation

Activation of mast cells in the allergic inflammatory response occurs via the high affinity receptor for IgE (FcepsilonRI) following receptor aggregation induced by antigen-mediated cross-linking of IgE-occupied FcepsilonRI. Recent observations suggest this response is profoundly influenced by other factors that reduce the threshold for, and increase the extent of, mast cell activation. For example, under experimental conditions, cell surface receptors such as KIT and specific G protein-coupled receptors synergistically enhance FcepsilonRI-mediated mast cell degranulation and cytokine production. Activating mutations in critical signaling molecules may also contribute to such responses. In this review, we describe our research exploring the mechanisms regulating these synergistic interactions and, furthermore, discuss the relevance of our observations in the context of clinical considerations.     

Negative regulation of T cell activation and autoimmunity by the transmembrane adaptor protein LAB

LAB (linker for activation of B cells), also known as NTAL (non-T cell activation linker), is a LAT (linker for activation of T cells)-like adaptor protein that is expressed in B, NK, and mast cells. Its role in lymphocytes has not been clearly demonstrated. Here, we showed that aged LAB-deficient (Lat2(-/-)) mice developed an autoimmune syndrome. Lat2(-/-) T cells were hyperactivated and produced more cytokines than Lat2(+/+) T cells. Even though LAB was absent in naive T cells, LAB could be detected in activated Lat2(+/+) T cells. LAT-mediated signaling events were enhanced in Lat2(-/-) T cells; however, they were suppressed in T cells that overexpressed LAB. Mice with the Lat2 gene conditionally deleted from T cells also developed the autoimmune syndrome like Lat2(-/-) mice. Together, these data demonstrated an important role of LAB in limiting autoimmune response and exposed a mechanism regulating T cell activation.     

A tale of two TRAPs: LAT and LAB in the regulation of lymphocyte development, activation, and autoimmunity

Transmembrane adaptor proteins (TRAPs) link antigen receptor engagement to downstream cellular processes. Although these proteins typically lack intrinsic enzymatic activity, they are phosphorylated on multiple tyrosine residues following lymphocyte activation, allowing them to function as scaffolds for the assembly of multi-molecular signaling complexes. Among the many TRAPs that have been discovered in recent years, the LAT (linker for activation of T cells) family of adaptor proteins plays an important role in the positive and negative regulation of lymphocyte maturation, activation, and differentiation. Of the two members in this family, LAT is an indispensable component controlling T cell and mast cell activation and function; LAB (linker for activation of B cells), also called NTAL, is necessary to fine-tune lymphocyte activation and may be a key regulator of innate immune responses. Here, we review recent advances on the function of LAT and LAB in the regulation of development and activation of immune cells.