A photoswitchable ITAM peptidomimetic: synthesis and real time surface plasmon resonance (SPR) analysis of the effects of cis-trans isomerization on binding

The Syk protein plays an important role in immune receptor signaling. The Syk tandem SH2 domain (tSH2)-ITAM interaction is important for recruiting Syk to the receptor complex and for Syk kinase activation. A peptidomimetic ligand for tSH2 was synthesized in which a photoswitchable azobenzene moiety was incorporated. Such a photoswitchable moiety may regulate the distance between the two phosphotyrosine containing ITAM sequences, which bind to tSH2. Different affinities of the cis and trans isomer of the ligand were found by surface plasmon resonance (SPR). By in situ irradiation during SPR measurements the effect of the cis-trans isomerization on binding could be monitored in real time.     

Comparison of the anti-allergic activity of Syk inhibitors with optimized Syk siRNAs in FcεRI-activated RBL-2H3 basophilic cells

Spleen tyrosine kinase (Syk) binds ITAM-bearing receptors in a wide variety of cell types. One such example is the activation of mast cells, basophils and eosinophils via the stimulation of the FcεRI receptor by IgE/allergen complexes. The possible role of Syk in inflammatory signaling cascades has led to the development of pharmacological agents designed to block the Syk catalytic domain as potential novel therapeutics. Whilst the enzymatic activity of Syk lends towards the design of small-molecule inhibitors, other attention has focused on the possibility of targeting Syk expression using anti-sense oligonucleotides as an alternate means of anti-inflammatory therapy. In this study, we compared the ability of multiple optimized Syk siRNA sequences and small-molecule Syk inhibitors to block FcεRI-mediated signal transduction, degranulation and TNFα secretion in the basophilic cell line RBL-2H3. We also characterized the specificity of each siRNA sequence with regards to off-target induction of the interferon-inducible gene IFIT1. We identified a single siRNA sequence, which displayed a favorable profile of efficient Syk knockdown, blockage of FcεRI-mediated signal transduction, degranulation and TNFα secretion and a lack of IFIT1 induction. The effect of this siRNA was comparable to that of the Syk kinase domain inhibitors BAY61-3606 and R406. The identification of an active and specific Syk siRNA could be a basis for the development of therapeutic Syk siRNAs against inflammatory diseases.     

Activation of the high-affinity immunoglobulin E receptor Fc epsilon RI in RBL-2H3 cells is inhibited by Syk SH2 domains.

Antigen-mediated aggregation of the high-affinity receptor for immunoglobulin E, Fc epsilon RI, results in the activation of multiple signaling pathways, leading to the release of mediators of the allergic response. One of the earliest responses to receptor stimulation is the tyrosine phosphorylation of the beta and gamma subunits of Fc epsilon RI and the association of the tyrosine kinase Syk with the phosphorylated receptor. This association is mediated by the SH2 domains of Syk and is believed to be critical for activating signaling pathways resulting in mediator release. To examine the importance of the interaction of Syk with Fc epsilon RI in signaling events following receptor activation, we introduced a protein containing the SH2 domains of Syk into streptolysin O-permeabilized RBL-2H3 cells. The Syk SH2 domains completely inhibited degranulation and leukotriene production following receptor aggregation, and they blocked the increase in protein tyrosine phosphorylation observed after receptor activation. Inhibition was specific for Fc epsilon RI-mediated signaling, since degranulation of cells activated by alternative stimuli was not blocked by the Syk SH2 domains. A protein containing a point mutation in the carboxy-terminal SH2 domain which abolishes phosphotyrosine binding was not inhibitory. In addition, inhibition of degranulation was reversed by pretreatment of the SH2 domains with a tyrosine phosphorylated peptide corresponding to the tyrosine-based activation motif found in the gamma subunit of Fc epsilon RI, the nonphosphorylated peptide had no effect. The association of Syk with the tyrosine-phosphorylated gamma subunit of the activated receptor was blocked by the Syk SH2 domains, and deregulation in cells activated by clustering of Syk directly without Fc epsilon RI aggregation was not affected by the Syk SH2 domains. These results demonstrate that the association of Syk with the activated Fc epsilon RI is critical for both early and late events following receptor activation and confirm the key role Syk plays in signaling through the high-affinity IgE receptor.     

Crystal structure and NMR studies of the apo SH2 domains of ZAP-70: two bikes rather than a tandem

The protein kinase ZAP-70 is involved in T-cell activation, and interacts with tyrosine-phosphorylated peptide sequences known as immunoreceptor tyrosine activation motifs (ITAMs), which are present in three of the subunits of the T-cell receptor. We have studied the tandem SH2 (tSH2) domains of ZAP-70, by both X-ray and NMR. Here, we present the crystal structure of the apoprotein, i.e., the tSH2 domain in the absence of ITAM. Comparison with the previously reported complex structure reveals that binding to the ITAM peptide induces surprisingly large movements between the two SH2 domains and within the actual binding sites. The conformation of the ITAM-free protein is partly governed by a hydrophobic cluster between the linker region and the C-terminal SH2 domain. Our data suggest that the two SH2 domains are able to undergo large interdomain movements. The proposed relative flexibility of the SH2 domains is further supported by the finding that no NMR signals could be detected for the two helices connecting the SH2 domains; these are likely to be broadened beyond detection due to conformational exchange. It is likely that this conformational reorientation induced by ITAM binding is the main signaling event activating the kinase domain in ZAP-70. Another NMR observation was that the N-terminal SH2 domain could bind tetrapeptides derived from the ITAM sequence, apparently without the need to interact with the C-terminal domain. In contrast, the C-terminal domain has little affinity for tetrapeptides. The opposite situation is true for binding to plain phosphotyrosine, where the C-terminal domain has a higher affinity. Distinct features in the crystal structure, showing the interdependence of both domains, explain these binding data.     

Interaction of p72syk with the gamma and beta subunits of the high-affinity receptor for immunoglobulin E, Fc epsilon RI.

Activation of protein tyrosine kinases is one of the initial events following aggregation of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72syk (Syk), which contains two Src homology 2 (SH2) domains, is activated and associates with phosphorylated Fc epsilon RI subunits after receptor aggregation. In this report, we used Syk SH2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the tandem Syk SH2 domains selectively associate with tyrosine-phosphorylated forms of the gamma and beta subunits of Fc epsilon RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc epsilon RI subunits than did the amino-proximal domain. When in tandem, the Syk SH2 domains showed enhanced binding to phosphorylated gamma and beta subunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the gamma and beta subunits, characterized by two YXXL/I sequences in tandem, represent potential high-affinity binding sites for the dual SH2 domains of Syk. Peptide competition studies indicated that Syk exhibits a higher affinity for the phosphorylated tyrosine activation motif of the gamma subunit than for that of the beta subunit. In addition, we show that Syk is the major protein in RBL-2H3 cells that is affinity isolated with phosphorylated peptides corresponding to the phosphorylated gamma subunit motif. These data suggest that Syk associates with the gamma subunit of the high-affinity receptor for immunoglobulin E through an interaction between the tandem SH2 domains of SH2 domains of Syk and the phosphorylated tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Fc epsilon RI tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Dc epsilon tyrosine activation motifs in RBL-2H3 cells.     

FcεRI-induced mast cell cytokine production critically involves an aspartic acid residue (D234) in the C-terminal intracellular domain of the FcεRIβ chain

The high affinity IgE Fc receptor (FcεRI) β chain is well implicated as a signal amplifier through the immunoreceptor tyrosine-based activation motif (ITAM) in its C-terminal intracellular region. Our previous study, however, demonstrated that mutation in all of the three tyrosine residues within the FcεRIβ ITAM did not impair FcεRI-induced cytokine production, suggesting a possible functional region other than the ITAM. To investigate the ITAM-independent mechanism by which FcεRIβ regulates FcεRI-induced cytokine production, mouse mast cells expressing various FcεRIβ mutants were generated. We observed that truncation of the FcεRIβ C-terminus downstream of the ITAM resulted in a considerable decrease in FcεRI-induced IL-6 production but not degranulation. Furthermore, mutagenesis of a single C-terminal aspartic acid (D234) to alanine (β-D234A) also significantly impaired IL-6 production. In addition, the similarity between the circular dichroism (CD) spectra of the wild type and β-D234A suggests that the secondary structure of the FcεRIβ C-terminus was not affected by the D234A mutation. Consistently, we did not observe any effect of this mutation on FcεRI-induced tyrosine phosphorylation of FcεRIβ. These observations strongly suggest a novel signaling pathway mediated by the cytoplasmic tail downstream of the FcεRIβ ITAM.     

Analysis of Proteins Binding to the ITAM Motif of the β-Subunit of the High-Affinity Receptor for IgE (FcεRI)

Aggregation of the multichain (α β γ₂) high-affinity IgE receptor (Fcε RI) initiates a signaling cascade that results in the release of allergic mediators. The cytoplasmic tails of the Fcε RI-β and -γ subunits contain immunoreceptor tyrosine-based activation motifs (ITAMs). Phosphorylation of the γ ITAM mediates activation of Syk kinase and is sufficient for triggering the responses induced by Fcε RI crosslinking. Phosphorylation of the β ITAM is insufficient to mediate cell activation. The rat β ITAM contains three tyrosines (Tyr²¹⁸, Tyr²²⁴, and Tyr²²⁸) with an intermediate noncanonical tyrosine. Synthetic peptides based on the ITAM of the Fcε RI-β subunit were used to investigate the role of each phosphotyrosine in the binding of signaling proteins to this motif. Among the proteins that bind to phosphorylated β ITAM are Syk, Grb2, Shc, SHIP, and SHP-1, and binding does not depend on previous cell activation. Nonphosphorylated peptides do not bind these proteins. Syk binding to β -peptides is dependent on the number and position of phosphotyrosines in the ITAM. Phosphorylation of Tyr²¹⁸ seems to be most important for Syk binding. Recruitment of Syk and other signaling proteins to the β -subunit might be important for its amplifier role.     

Once phosphorylated, tyrosines in carboxyl terminus of protein-tyrosine kinase Syk interact with signaling proteins, including TULA-2, a negative regulator of mast cell degranulation

Activation of the high affinity IgE-binding receptor (FcεRI) results in the tyrosine phosphorylation of two conserved tyrosines located close to the COOH terminus of the protein-tyrosine kinase Syk. Synthetic peptides representing the last 10 amino acids of the tail of Syk with these two tyrosines either nonphosphorylated or phosphorylated were used to precipitate proteins from mast cell lysates. Proteins specifically precipitated by the phosphorylated peptide were identified by mass spectrometry. These included the adaptor proteins SLP-76, Nck-1, Grb2, and Grb2-related adaptor downstream of Shc (GADS) and the protein phosphatases SHIP-1 and TULA-2 (also known as UBASH3B or STS-1). The presence of these in the precipitates was further confirmed by immunoblotting. Using the peptides as probes in far Western blots showed direct binding of the phosphorylated peptide to Nck-1 and SHIP-1. Immunoprecipitations suggested that there were complexes of these proteins associated with Syk especially after receptor activation; in these complexes are Nck, SHIP-1, SLP-76, Grb2, and TULA-2 (UBASH3B or STS-1). The decreased expression of TULA-2 by treatment of mast cells with siRNA increased the FcεRI-induced tyrosine phosphorylation of the activation loop tyrosines of Syk and the phosphorylation of phospholipase C-γ2. There was parallel enhancement of the receptor-induced degranulation and activation of nuclear factor for T cells or nuclear factor κB, indicating that TULA-2, like SHIP-1, functions as a negative regulator of FcεRI signaling in mast cells. Therefore, once phosphorylated, the terminal tyrosines of Syk bind complexes of proteins that are positive and negative regulators of signaling in mast cells.     

Differences in the dynamics of the tandem‐SH2 modules of the Syk and ZAP‐70 tyrosine kinases

The catalytic activity of Syk‐family tyrosine kinases is regulated by a tandem Src homology 2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation that stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to phosphorylated immunoreceptor tyrosine‐based activation motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP‐70, that its conformation more closely resembles that of the peptide‐bound state, rather than the autoinhibited state. Hydrogen–deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP‐70 differ, with most of these differences occurring in the C‐terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP‐70 have been tuned differently, such that the autoinhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP‐70, consistent with tighter control of downstream signaling pathways in T cells.     

Molecular mechanism of selective recruitment of Syk kinases by the membrane antigen-receptor complex

ZAP-70 and Syk are essential tyrosine kinases in intracellular immunological signaling. Both contain an inhibitory SH2 domain tandem, which assembles onto the catalytic domain. Upon binding to doubly phosphorylated ITAM motifs on activated antigen receptors, the arrangement of the SH2 domains changes. From available structures, this event is not obviously conducive to dissociation of the autoinhibited complex, yet it ultimately translates into kinase activation through a mechanism not yet understood. We present a comprehensive theoretical study of this molecular mechanism, using atomic resolution simulations and free-energy calculations, totaling >10 μs of simulation time. Through these, we dissect the microscopic mechanism coupling stepwise ITAM engagement and SH2 tandem structural change and reveal key differences between ZAP-70 and Syk. Importantly, we show that a subtle conformational bias in the inter-SH2 connector causes ITAM to bind preferentially to kinase-dissociated tandems. We thus propose that phosphorylated antigen receptors selectively recruit kinases that are uninhibited and that the resulting population shift in the membrane vicinity sustains signal transduction.     

Structural and Biophysical Characterization of the Syk Activation Switch

Syk is an essential non-receptor tyrosine kinase in intracellular immunological signaling, and the control of Syk kinase function is considered as a valuable target for pharmacological intervention in autoimmune or inflammation diseases. Upon immune receptor stimulation, the kinase activity of Syk is regulated by binding of phosphorylated immune receptor tyrosine-based activating motifs (pITAMs) to the N-terminal tandem Src homology 2 (tSH2) domain and by autophosphorylation with consequences for the molecular structure of the Syk protein. Here, we present the first crystal structures of full-length Syk (fl-Syk) as wild type and as Y348F,Y352F mutant forms in complex with AMP-PNP revealing an autoinhibited conformation. The comparison with the crystal structure of the truncated Syk kinase domain in complex with AMP-PNP taken together with ligand binding studies by surface plasmon resonance (SPR) suggests conformational differences in the ATP sites of autoinhibited and activated Syk forms. This hypothesis was corroborated by studying the thermodynamic and kinetic interaction of three published Syk inhibitors with isothermal titration calorimetry and SPR, respectively. We further demonstrate the modulation of inhibitor binding affinities in the presence of pITAM and discuss the observed differences of thermodynamic and kinetic signatures. The functional relevance of pITAM binding to fl-Syk was confirmed by a strong stimulation of in vitro autophosphorylation. A structural feedback mechanism on the kinase domain upon pITAM binding to the tSH2 domain is discussed in analogy of the related family kinase ZAP-70 (Zeta-chain-associated protein kinase 70). Surprisingly, we observed distinct conformations of the tSH2 domain and the activation switch including Tyr348 and Tyr352 in the interdomain linker of Syk in comparison to ZAP-70.     

The Src family kinase Fgr is critical for activation of mast cells and IgE-mediated anaphylaxis in mice

Mast cells are critical for various allergic disorders. Mast cells express Src family kinases, which relay positive and negative regulatory signals by Ag. Lyn, for example, initiates activating signaling events, but it also induces inhibitory signals. Fyn and Hck are reported to be positive regulators, but little is known about the roles of other Src kinases, including Fgr, in mast cells. In this study, we define the role of Fgr. Endogenous Fgr associates with FcεRI and promotes phosphorylation of Syk, Syk substrates, which include linkers for activation of T cells, SLP76, and Gab2, and downstream targets such as Akt and the MAPKs in Ag-stimulated mast cells. As a consequence, Fgr positively regulates degranulation, production of eicosanoids, and cytokines. Fgr and Fyn appeared to act in concert, as phosphorylation of Syk and degranulation are enhanced by overexpression of Fgr and further augmented by overexpression of Fyn but are suppressed by overexpression of Lyn. Moreover, knockdown of Fgr by small interfering RNAs (siRNAs) further suppressed degranulation in Fyn-deficient bone marrow-derived mast cells. Overexpression of Fyn or Fgr restored phosphorylation of Syk and partially restored degranulation in Fyn-deficient cells. Additionally, knockdown of Fgr by siRNAs inhibited association of Syk with FcεRIγ as well as the tyrosine phosphorylation of FcεRIγ. Of note, the injection of Fgr siRNAs diminished the protein level of Fgr in mice and simultaneously inhibited IgE-mediated anaphylaxis. In conclusion, Fgr positively regulates mast cell through activation of Syk. These findings help clarify the interplay among Src family kinases and identify Fgr as a potential therapeutic target for allergic diseases.     

Anti-Human IgE Monoclonal Antibodies Recognizing Epitopes Related to the Binding Sites of High and Low Affinity IgE Receptors

Anti-human IgE monoclonal antibodies (mAbs) were produced and eight clones recognizing epitopes on native IgE were selected. Epitopes were mapped by a competitive inhibition enzyme-linked immunosorbent assay, Western blotting and a multi-pin peptide technology. Four sites (one each in the Cε1, Cε2, Cε2/Cε3 junction and Cε3) were recognized by the mAbs. The relationship between the four epitopes and the binding sites of high and low affinity IgE receptors (FcεRI and FcεRII, respectively) was studied using a monovalent Fab fragment of each mAb as a binding inhibitor. The IgE-FcεRII binding was clearly inhibited by the mAb recognizing the Cε2/Cε3 junction, suggesting that FcεRII binds to a rather limited area around the Cε2/Cε3 junction. The IgE-FcεRI binding, on the other hand, was scarcely inhibited by any single mAb. However, the binding was inhibited when the epitope in Cε2 was blocked simultaneously with that at the Cε2/Cε3 junction or with that in Cε3, indicating that these three distinct epitopes are related to the FcεRI binding sites. When these three epitopes were shown in the stereograph of human IgE, the FcεRI binding area was spread largely on the groove side between Cε2 and Cε3 domains. These results suggest that FcεRI acquires the high affinity through multiple bindings.     

Investigation of early events in Fc epsilon RI-mediated signaling using a detailed mathematical model

Aggregation of Fc epsilon RI on mast cells and basophils leads to autophosphorylation and increased activity of the cytosolic protein tyrosine kinase Syk. We investigated the roles of the Src kinase Lyn, the immunoreceptor tyrosine-based activation motifs (ITAMs) on the beta and gamma subunits of Fc epsilon RI, and Syk itself in the activation of Syk. Our approach was to build a detailed mathematical model of reactions involving Fc epsilon RI, Lyn, Syk, and a bivalent ligand that aggregates Fc(epsilon)RI. We applied the model to experiments in which covalently cross-linked IgE dimers stimulate rat basophilic leukemia cells. The model makes it possible to test the consistency of mechanistic assumptions with data that alone provide limited mechanistic insight. For example, the model helps sort out mechanisms that jointly control dephosphorylation of receptor subunits. In addition, interpreted in the context of the model, experimentally observed differences between the beta- and gamma-chains with respect to levels of phosphorylation and rates of dephosphorylation indicate that most cellular Syk, but only a small fraction of Lyn, is available to interact with receptors. We also show that although the beta ITAM acts to amplify signaling in experimental systems where its role has been investigated, there are conditions under which the beta ITAM will act as an inhibitor.     

DAP12 couples c-Fms activation to the osteoclast cytoskeleton by recruitment of Syk

We examined the mechanism by which M-CSF regulates the cytoskeleton and function of the osteoclast, the exclusive bone resorptive cell. We show that binding of M-CSF to its receptor c-Fms generates a signaling complex comprising phosphorylated DAP12, an adaptor containing an immunoreceptor tyrosine-based activation motif (ITAM) and the nonreceptor tyrosine kinase Syk. c-Fms tyrosine 559, the exclusive binding site of c-Src, is necessary for regulation of DAP12/Syk signaling. Deletion of either of these molecules yields osteoclasts that fail to reorganize their cytoskeleton. Retroviral transduction of null precursors with wild-type or mutant DAP12 or Syk reveals that the SH2 domain of Syk and the ITAM tyrosine residues and transmembrane domain of DAP12 mediate M-CSF signaling. Our data provide genetic and biochemical evidence that uncovers an epistatic signaling pathway linking the receptor tyrosine kinase c-Fms to the immune adaptor DAP12 and the cytoskeleton.     

Fyn kinase controls FcεRI receptor-operated calcium entry necessary for full degranulation in mast cells

IgE-antigen-dependent crosslinking of the high affinity IgE receptor (FcεRI) on mast cells leads to degranulation, leukotriene synthesis and cytokine production. Calcium (Ca²⁺) mobilization is a sine qua non requisite for degranulation, allowing the rapid secretion of stored pro-inflammatory mediators responsible for allergy symptoms. Fyn is a Src-family kinase that positively controls FcεRI-induced mast cell degranulation. However, our understanding of the mechanism connecting Fyn activation to secretion of pre-synthesized mediators is very limited. We analyzed FcεRI-dependent Ca²⁺ mobilization in bone marrow-derived mast cells (BMMCs) differentiated from WT and Fyn −/− knock out mice. Fyn −/− BMMCs showed a marked defect in extracellular Ca²⁺ influx after FcεRI crosslinking but not after thapsigargin addition. High concentrations of Gadolinium (Gd³⁺) partially blocked FcεRI-induced Ca²⁺ influx in WT cells but, in contrast, completely inhibited Ca²⁺ mobilization in Fyn −/− cells. Low concentrations of an inhibitor of the canonical transient receptor potential (TRPC) Ca²⁺ channels (2-aminoethoxyphenyl-borane, 2-APB) blocked FcεRI-induced maximal Ca²⁺ rise in WT but not in Fyn −/− cells. Ca²⁺ entry through Fyn-controlled, 2-APB sensitive channels was found to be important for full degranulation and IL-2 mRNA accumulation in WT cells. Immunoprecipitation assays showed that Fyn kinase interacts with TRPC 3/6/7 channels after IgE-antigen stimulation, but its association is not related to protein tyrosine phosphorylation. Results indicate Fyn kinase mediates the receptor-dependent activation of TRPC channels that contribute to degranulation in FcεRI-stimulated mast cells.     

Gab2, via PI-3K, regulates ARF1 in FcεRI-mediated granule translocation and mast cell degranulation

Mast cells are major players in allergic responses. IgE-dependent activation through FcεR leads to degranulation and cytokine production, both of which require Gab2. To clarify how the signals diverge at Gab2, we established Gab2 knock-in mice that express Gab2 mutated at either the PI3K or SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) binding sites. Examination of these mutants showed that both binding sites were required for the degranulation and anaphylaxis response but not for cytokine production or contact hypersensitivity. Furthermore, the PI3K, but not the SHP2, binding site was important for granule translocation during degranulation. We also identified a small GTPase, ADP-ribosylation factor (ARF)1, as the downstream target of PI3K that regulates granule translocation. FcεRI stimulation induced ARF1 activation, and this response was dependent on Fyn and the PI3K binding site of Gab2. ARF1 activity was required for FcεRI-mediated granule translocation. These data indicated that Fyn/Gab2/PI3K/ARF1-mediated signaling is specifically involved in granule translocation and the anaphylaxis response.     

Oxysterol represses high-affinity IgE receptor-stimulated mast cell activation in Liver X receptor-dependent and -independent manners

Oxysterols activating liver X receptors (LXRs) repress expression of pro-inflammatory genes and have anti-inflammatory effects. Here, we show for the first time that bone marrow-derived murine mast cells (BMMCs) predominantly express LXRβ. 25-hydroxycholesterol, a representative LXR activating oxysterol, suppressed IL-6 production and degranulation response in BMMCs following engagement of high-affinity IgE receptor (FcεRI). Interestingly, 25-hydroxycholesterol reduced cell-surface FcεRI expression by inhibiting assembly of FcεRIα and FcεRIβ. We demonstrate that LXR activation was involved in the suppression of IL-6 production in BMMCs, but that reduced FcεRI expression and degranulation response was mediated in an LXR-independent manner.     

Tyrosines in the Carboxyl Terminus Regulate Syk Kinase Activity and Function

The Syk tyrosine kinase family plays an essential role in immunoreceptor tyrosine-based activation motif (ITAM) signaling. The binding of Syk to tyrosine-phosphorylated ITAM subunits of immunoreceptors, such as FcϵRI on mast cells, results in a conformational change, with an increase of enzymatic activity of Syk. This conformational change exposes the COOH-terminal tail of Syk, which has three conserved Tyr residues (Tyr-623, Tyr-624, and Tyr-625 of rat Syk). To understand the role of these residues in signaling, wild-type and mutant Syk with these three Tyr mutated to Phe was expressed in Syk-deficient mast cells. There was decreased FcϵRI-induced degranulation, nuclear factor for T cell activation and NFκB activation with the mutated Syk together with reduced phosphorylation of MAP kinases p38 and p42/44 ERK. In non-stimulated cells, the mutated Syk was more tyrosine phosphorylated predominantly as a result of autophosphorylation. In vitro, there was reduced binding of mutated Syk to phosphorylated ITAM due to this increased phosphorylation. This mutated Syk from non-stimulated cells had significantly reduced kinase activity toward an exogenous substrate, whereas its autophosphorylation capacity was not affected. However, the kinase activity and the autophosphorylation capacity of this mutated Syk were dramatically decreased when the protein was dephosphorylated before the in vitro kinase reaction. Furthermore, mutation of these tyrosines in the COOH-terminal region of Syk transforms it to an enzyme, similar to its homolog ZAP-70, which depends on other tyrosine kinases for optimal activation. In testing Syk mutated singly at each one of the tyrosines, Tyr-624 but especially Tyr-625 had the major role in these reactions. Therefore, these results indicate that these tyrosines in the tail region play a critical role in regulating the kinase activity and function of Syk.     

Structural requirements of SLP-76 in signaling via the high-affinity immunoglobulin E receptor (Fc epsilon RI) in mast cells

The adapter SLP-76 plays an essential role in Fc epsilon RI signaling, since SLP-76(-/-) bone marrow-derived mast cells (BMMC) fail to degranulate and release interleukin-6 (IL-6) following Fc epsilon RI ligation. To define the role of SLP-76 domains and motifs in Fc epsilon RI signaling, SLP-76(-/-) BMMC were retrovirally transduced with SLP-76 and SLP-76 mutants. The SLP-76 N-terminal and Gads binding domains, but not the SH2 domain, were critical for Fc epsilon RI-mediated degranulation and IL-6 secretion, whereas all three domains are essential for T-cell proliferation following T-cell receptor (TCR) ligation. Unexpectedly, the three tyrosine residues in SLP-76 critical for TCR signaling, Y112, Y128, and Y145, were not essential for IL-6 secretion, but were required for degranulation and mitogen-activated protein kinase activation. Furthermore, a Y112/128F SLP-76 mutant, but not a Y145F mutant, strongly reconstituted mast cell degranulation, suggesting a critical role for Y145 in Fc epsilon RI-mediated exocytosis. These results point to important differences in the function of SLP-76 between T cells and mast cells.