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.     

Conformational changes induced in the protein tyrosine kinase p72syk by tyrosine phosphorylation or by binding of phosphorylated immunoreceptor tyrosine-based activation motif peptides.

A critical event in signaling in immune cells is the interaction of Syk or ZAP-70 protein tyrosine kinases with multisubunit receptors that contain an approximately 18-amino-acid domain called the immunoreceptor tyrosine-based activation motif (ITAM). Tyrosine-phosphorylated Syk from activated cells was in a conformation different from that in nonstimulated cells as demonstrated by changes in immunoreactivity. The addition of tyrosine-diphosphorylated ITAM peptides resulted in a similar conformational change in Syk from nonactivated cells. The peptides based on FcepsilonRIgamma were more active than those based on Fcepsilon RIbeta. In vitro autophosphorylation of Syk was dramatically enhanced by the addition of the diphosphorylated ITAM peptides. The conformational change and the enhanced autophosphorylation required the presence of both phosphorylated tyrosines on the same molecule. These conformational changes in Syk by tyrosine phosphorylation or binding to diphosphorylated ITAM could be critical for Syk activation and downstream propagation of intracellular signals.     

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.     

Switching between low and high affinity for the Syk tandem SH2 domain by irradiation of azobenzene containing ITAM peptidomimetics

Spleen tyrosine kinase (Syk) plays an essential role in IgE receptor signaling (FcεRI), which leads to mast cell degranulation. Divalent binding of the tandem SH2 domain (tSH2) of Syk to the intracellular ITAM motif of FcεRI activates the kinase domain of Syk, and thereby initiates cell degranulation. The inter SH2 domain distance in Syk tSH2 might be important for Syk kinase activation. In this study, photoswitchable ITAM peptidomimetics containing an azobenzene moiety were synthesized. Irradiation of these constructs changes the distance between the two SH2 binding epitopes and therefore, they may be used as photoswitches. The affinity of the cis‐ and trans‐isomer for tSH2 was assayed with SPR. The ITAM peptidomimetic with the smallest linker displayed the largest difference in affinity between the two isomers (at least 100‐fold), and the affinity of the cis‐isomer was comparable to monovalent binding. The ITAM mimics with larger photoswitchable linkers displayed modest differences. These results indicate that Syk tSH2 is able to adapt the inter SH2 domain distance to ligands larger than native ITAM, but not to smaller ones. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Differential dephosphorylation of the FcRgamma immunoreceptor tyrosine-based activation motif tyrosines with dissimilar potential for activating Syk

The cell surface-expressed gamma chain of the high affinity receptor for IgE (FcepsilonRI) can be phosphorylated on two tyrosine residues of the immunoreceptor tyrosine-based activation motif (ITAM), leading to recruitment and activation of spleen tyrosine kinase (Syk), a kinase that is essential for mast cell signaling and allergic responses. However, it is not known whether preferential phosphorylation or dephosphorylation of the two individual FcRgamma tyrosines (the N-terminal Tyr47 and C-terminal Tyr58) could regulate Syk activation. Herein we report that phosphorylation of only Tyr58 was able to elicit Syk phosphorylation and a weak rise in intracellular calcium, suggesting that Tyr58 phosphorylation may be distinctively important for Syk activation. In vitro and in vivo studies revealed that both Tyr47 and Tyr58 could be similarly phosphorylated. However, mass spectrometric analysis of the phosphorylated FcepsilonRgamma from bone marrow-derived mast cells showed that phosphorylation at Tyr47 was at least 2-fold greater than at Tyr58. This suggested that, once phosphorylated, Tyr58 is preferentially dephosphorylated. In vitro studies demonstrated more efficient dephosphorylation of Tyr58 (by the receptor-associated phosphatases SHP-1 and SHP-2) than of Tyr47. Analysis of Syk binding to wild type and mutant phosphorylated FcepsilonRI revealed that mutation at Tyr58 almost completely ablated Syk binding, whereas mutation at Tyr47 moderately reduced Syk binding. The findings argue for a novel regulatory mechanism, where dephosphorylation of phospho-Tyr58 is likely to promote the down-regulation of Syk activation and suppression of mast cell responses.     

Phosphorylation on Syk Y342 is important for both ITAM and hemITAM signaling in platelets

Immune cells express receptors bearing an immune tyrosine activation motif (ITAM) containing two YXXL motifs or hemITAMs containing only one YXXL motif. Phosphorylation of the ITAM/hemITAM is mediated by Src family kinases allowing for the binding and activation of spleen tyrosine kinase (Syk). It is believed that Syk must be phosphorylated on tyrosine residues for activation, and Tyr342, а conserved tyrosine in the interdomain B region, has been shown to be critical for regulating Syk in FcεR1-activated mast cells. Syk is a key mediator of signaling pathways downstream of several platelet pathways including the ITAM bearing glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor and the hemITAM containing C-type lectin-like receptor-2 (CLEC-2). Since platelet activation is a crucial step in both hemostasis and thrombosis, we evaluated the importance of Syk Y342 in these processes by producing an Syk Y342F knock-in mouse. When using a CLEC-2 antibody as an agonist, reduced aggregation and secretion were observed in Syk Y342F mouse platelets when compared with control mouse platelets. Platelet reactivity was also reduced in response to the GPVI agonist collagen-related peptide. Signaling initiated by either GPVI or CLEC-2 was also greatly inhibited, including Syk Y519/520 phosphorylation. Hemostasis, as measured by tail bleeding time, was not altered in Syk Y342F mice, but thrombus formation in response to FeCl₃ injury was prolonged in Syk Y342F mice. These data demonstrate that phosphorylation of Y342 on Syk following stimulation of either GPVI or CLEC-2 receptors is important for the ability of Syk to transduce a signal.     

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.     

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.     

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.     

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.     

Hantavirus pulmonary syndrome-associated hantaviruses contain conserved and functional ITAM signaling elements

Hantaviruses infect human endothelial and immune cells, causing two human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). We have identified key signaling elements termed immunoreceptor tyrosine-based activation motifs (ITAMs) within the G1 cytoplasmic tail of all HPS-causing hantaviruses. ITAMs direct receptor signaling within immune and endothelial cells and the presence of ITAMs in all HPS-causing hantaviruses provides a means for altering normal cellular responses which maintain vascular integrity. The NY-1 G1 ITAM was shown to coprecipitate a complex of phosphoproteins from cells, and the G1 ITAM is a substrate for the Src family kinase Fyn. The hantavirus ITAM coprecipitated Lyn, Syk, and ZAP-70 kinases from T or B cells, while mutagenesis of the ITAM abolished these interactions. In addition, G1 ITAM tyrosines directed intracellular interactions with Syk by mammalian two-hybrid analysis. These findings demonstrate that G1 ITAMs bind key cellular kinases that regulate immune and endothelial cell functions. There is currently no means for establishing the role of the G1 ITAM in hantavirus pathogenesis. However, the conservation of G1 ITAMs in all HPS-causing hantaviruses and the role of these signaling elements in immune and endothelial cells suggest that functional G1 ITAMs are likely to dysregulate normal immune and endothelial cell responses and contribute to hantavirus pathogenesis.     

Molecular dissection of the FcRbeta signaling amplifier

Human high affinity IgE receptors are expressed as two different isoforms: the tetrameric isoform, alphabetagamma(2), or the trimeric isoform, alphagamma(2). The alpha chain is the IgE binding subunit, whereas the FcRbeta and FcRgamma chains are the signaling modules. Both FcRbeta and FcRgamma contain immunoreceptor tyrosine-based activation motifs (ITAM), but the beta ITAM differs from canonical ITAMs in two ways; the spacing between the two canonical tyrosines harbors a third tyrosine, and it is one amino acid shorter than in canonical ITAMs, making it unfit to bind the tandem SH2 of Syk. We have shown that FcRbeta functions as an amplifier of the FcRgamma signaling function. However, the molecular mechanism of this amplification remains unclear. Here we show that mutation of the three tyrosines (Tyr-219, Tyr-225, and Tyr-229) in the beta ITAM essentially converts alphabetagamma(2)into an alphagamma(2) complex in terms of Lyn recruitment, FcRgamma phosphorylation, Syk activation, and calcium mobilization. Tyr-219 is the most critical residue in this regard. In addition, a detailed analysis of the dynamics of calcium mobilization suggests a possible inhibitory role for Tyr-225, which becomes apparent when Tyr-219 is mutated. Thus, the signaling amplification function of FcRbeta is mainly encoded in Tyr-219 and in its capacity to recruit Lyn. In turn, this Tyr-219-mediated Lyn recruitment enhances gamma chain phosphorylation, Syk activation, and calcium mobilization. The two other tyrosines appear to have a modulating function that remains to be fully assessed.     

Phosphorylation of Tyr342 in the linker region of Syk is critical for Fc epsilon RI signaling in mast cells

The linker region of Syk and ZAP70 tyrosine kinases plays an important role in regulating their function. There are three conserved tyrosines in this linker region; Tyr317 of Syk and its equivalent residue in ZAP70 were previously shown to negatively regulate the function of Syk and ZAP70. Here we studied the roles of the other two tyrosines, Tyr342 and Tyr346 of Syk, in Fc epsilon RI-mediated signaling. Antigen stimulation resulted in Tyr342 phosphorylation in mast cells. Syk with Y342F mutation failed to reconstitute Fc epsilon RI-initiated histamine release. In the Syk Y342F-expressing cells there was dramatically impaired receptor-induced phosphorylation of multiple signaling molecules, including LAT, SLP-76, phospholipase C-gamma2, but not Vav. Compared to wild-type Syk, Y342F Syk had decreased binding to phosphorylated immunoreceptor tyrosine-based activation motifs and reduced kinase activity. Surprisingly, mutation of Tyr346 had much less effect on Fc epsilon RI-dependent mast cell degranulation. An anti-Syk-phospho-346 tyrosine antibody indicated that antigen stimulation induced only a very minor increase in the phosphorylation of this tyrosine. Therefore, Tyr342, but not Tyr346, is critical for regulating Syk in mast cells and the function of these tyrosines in immune receptor signaling appears to be different from what has been previously reported for the equivalent residues of ZAP70.     

Distinct roles for the linker region tyrosines of Syk in FcepsilonRI 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.     

Differential and multiple binding of signal transducing molecules to the ITAMs of the TCR-ζ chain

A biotin-streptavidin-based technique was developed for high affinity, unidirectional, and specific immobilization of synthetic peptides to a solid phase. Biotinylated 23-mer carboxamide peptides corresponding to the three immunoreceptor tyrosine-based activation motifs (ITAMs) of the T cell antigen receptor associated ζ-chain (TCR-ζ) in their bis-, mono-, or unphosphorylated forms were used to study the binding of cellular proteins from human Jurkat T cells to these signal transduction motifs. The protein tyrosine kinase ZAP-70 bound specifically to all bisphosphorylated peptides but not to the mono- or unphosphorylated peptides. In contrast, Shc, phosphatidylinositol 3-kinase (P13K), Grb2, and Ras-GTPase activating protein (GAP) bound with different affinities to the bis- or monophosphorylated peptides, while the Src family protein tyrosine kinase (PTK) Fyn did not bind specifically to any of the tested peptides. The different preferences of the studied signaling molecules for distinct ITAMs, and in particular the binding of some of them preferentially to monophosphorylated peptides, suggests that the TCR-ζ may bind multiple signaling molecules with each ITAM binding a unique set of such molecules. In addition, partial phosphorylation of the ITAMs may result in recruitment of different proteins compared to double phosphorylation. This may be crucial for coupling of the TCR to various effector functions under different conditions of receptor triggering. © 1996 Wiley-Liss, Inc.     

Structure and function of Syk protein-tyrosine kinase

Non-receptor type of protein-tyrosine kinase Syk contains 2 Src homology 2 (SH2) domains in tandem and multiple autophosphorylation sites. Syk is activated upon binding of tandem SH2 domains to immunoreceptor tyrosine-based activating motif (ITAM) and plays an essential role in lymphocyte development and activation of immune cells. Syk is critical for tyrosine phosphorylation of multiple proteins which regulate important pathways leading from the receptor, such as Ca(2+) mobilization and mitogen-activated protein kinase (MAPK) cascades. Recent findings reveal that expression of Syk appears to be involved in a wide variety of cellular functions and pathogenesis of malignant cancer. These observations have demonstrated that Syk is a key molecule that controls multiple physiological functions in cells.     

A peptide library approach identifies a specific inhibitor for the ZAP-70 protein tyrosine kinase

We utilized a novel peptide library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase involved in T cell activation. By screening more than 6 billion peptides oriented by a common Tyr residue for their ability to bind to ZAP-70, we determined a consensus optimal peptide. A Phe-for-Tyr substituted version of the peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates (K(I) of 2 microM). The related protein Tyr kinases, Lck and Syk, were not significantly inhibited by the peptide. When introduced into intact T cells, the peptide blocked signaling downstream of ZAP-70, including ZAP-70-dependent gene induction, without affecting upstream Tyr phosphorylation. Thus, screening Tyr-oriented peptide libraries can identify selective peptide inhibitors of protein Tyr kinases.     

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.     

The LMP2A ITAM is essential for providing B cells with development and survival signals in vivo

In Epstein-Barr virus-transformed B cells, known as lymphoblastoid cell lines (LCLs), LMP2A binds the tyrosine kinases Syk and Lyn, blocking B-cell receptor (BCR) signaling and viral lytic replication. SH2 domains in Syk mediate binding to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) in LMP2A. Mutation of the LMP2A ITAM in LCLs eliminates Syk binding and allows for full BCR signaling, thereby delineating the significance of the LMP2A-Syk interaction. In transgenic mice, LMP2A causes a developmental alteration characterized by a block in surface immunoglobulin rearrangement resulting in BCR-negative B cells. Normally B cells lacking cognate BCR are rapidly apoptosed; however, LMP2A transgenic B cells develop and survive without a BCR. When bred into the recombinase activating gene 1 null (RAG(-/-)) background, all LMP2A transgenic lines produce BCR-negative B cells that develop and survive in the periphery. These data indicate that LMP2A imparts developmental and survival signals to B cells in vivo. In this study, LMP2A ITAM mutant transgenic mice were generated to investigate whether the LMP2A ITAM is essential for the survival phenotype in vivo. LMP2A ITAM mutant B cells develop normally, although transgene expression is comparable to that in previously described nonmutated LMP2A transgenic B cells. Additionally, LMP2A ITAM mutant mice are unable to promote B-cell development or survival when bred into the RAG(-/-) background or when grown in methylcellulose containing interleukin-7. These data demonstrate that the LMP2A ITAM is required for LMP2A-mediated developmental and survival signals in vivo.