Membrane-targeting is critical for the phosphorylation of Vav2 by activated EGF receptor

Vav2 is a member of the Vav family that serves as guanine nucleotide exchange factors (GEFs) for the Rho family of Ras-related GTPases. Unlike Vav1, whose expression is restricted to cells of hematopoietic origin, Vav2 is broadly expressed. Recently, Vav2 has been identified as a substrate for the EGF receptor. Here, we show that in EGF-treated COS7 cells Vav2 is phosphorylated on tyrosine residues and associates with the EGF receptor. In addition, introducing point mutations into the SH2 domain of green fluorescens protein (GFP)-Vav2 fusion protein leads to the loss of Vav2 tyrosine phosphorylation in response to EGF. To investigate further the mechanism of Vav2 phosphorylation, N-terminal (NT) domain of Vav2 was transiently expressed in COS7 cells as GFP fusion protein. Whereas the NT domain of Vav2 is a preferred substrate for the activated EGF receptor in vitro, we could not detect tyrosine phosphorylation of the GFP-NT construct in EGF-treated cells. However, when the SH2 domain of Vav2 was fused to its NT domain, NT domain proved to be a substrate for the EGF receptor in vivo. These data suggest that membrane-targeting of Vav2 through its SH2 domain is an important event in the phosphorylation and activation of Vav2 in response to EGF.     

Association of Bcr-Abl with the proto-oncogene Vav is implicated in activation of the Rac-1 pathway.

Vav is a guanine nucleotide exchange factor for the Rho/Rac family predominantly expressed in hematopoietic cells and implicated in cell proliferation and cytoskeletal organization. The oncogenic tyrosine kinase Bcr-Abl has been shown to activate Rac-1, which is important for Bcr-Abl induced leukemogenesis. Previous studies by Matsuguchi et al. (Matsuguchi, T., Inhorn, R. C., Carlesso, N., Xu, G., Druker, B., and Griffin, J. D. (1995) EMBO J. 14, 257-265) describe enhanced phosphorylation of Vav in Bcr-Abl-expressing Mo7e cells yet fail to demonstrate association of the two proteins. Here, we report the identification of a direct complex between Vav and Bcr-Abl in yeast, in vitro and in vivo. Furthermore, we show tyrosine phosphorylation of Vav by Bcr-Abl. Mutational analysis revealed that the SH2 domain and the C-terminal SH3 domain as well as a tetraproline motif directly adjacent to the N-terminal SH3 domain of Vav are important for establishing this phosphotyrosine dependent interaction. Activation of Rac-1 by Bcr-Abl was abrogated by co-expression of the Vav C terminus encoding the SH3-SH2-SH3 domains as a dominant negative construct. Bcr-Abl transduced primary bone marrow from Vav knock-out mice showed reduced proliferation in a culture cell transformation assay compared with wild-type bone marrow. These results suggest, that Bcr-Abl utilizes Vav as a guanine nucleotide exchange factor to activate Rac-1 in a process that involves a folding mechanism of the Vav C terminus. Given the importance of Rac-1 activation for Bcr-Abl-mediated leukemogenesis, this mechanism may be crucial for the molecular pathogenesis of chronic myeloid leukemia and of importance for other signal transduction pathways leading to the activation of Rac-1.     

Tyrosine phosphorylation of p95Vav in myeloid cells is regulated by GM-CSF, IL-3 and steel factor and is constitutively increased by p210BCR/ABL.

Vav is a recently described proto-oncogene expressed only in hematopoietic cells which contains an SH2 and two SH3 domains and shares homology with the Dbl GDP-GTP exchange factor and BCR. p95Vav is phosphorylated on tyrosine residues in response to stimulation of the T cell antigen receptor, cross-linking of IgE or IgM receptors and stimulation of immature hematopoietic cells by Steel factor. Monoclonal antibodies to human Vav were generated and used to examine the events which regulate tyrosine phosphorylation of p95Vav in myeloid cells. In the factor-dependent MO7e cell line, p95Vav was rapidly phosphorylated on tyrosine residues in a dose- and time-dependent manner by GM-CSF, IL-3 and Steel factor. Introduction of the BCR/ABL oncogene into this cell line resulted in factor-independent proliferation and constitutive phosphorylation of p95Vav. Tyrosine phosphorylation of p95Vav was also substantially increased by treatment of cytokine-deprived cells with the tyrosine phosphatase inhibitor sodium vanadate. Since many of the cytokines known to induce tyrosine phosphorylation of p95Vav are also known to activate JAK family tyrosine kinases, we looked for an interaction of p95Vav with JAK kinases. p95Vav co-precipitated with JAK2 in MO7e cells stimulated with GM-CSF, but not in unstimulated cells. Also, JAK2 was found to be constitutively associated with p95Vav in vivo when expressed at high levels in insect cells using baculovirus vectors. A fusion protein consisting of glutathione-S-transferase and the SH2 domain of p95Vav (GST-Vav-SH2) precipitated JAK2, suggesting that this interaction is mediated by the SH2 domain of p95Vav.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two closely spaced tyrosines regulate NFAT signaling in B cells via Syk association with Vav

Activated Syk, an essential tyrosine kinase in B cell signaling, interacts with Vav guanine nucleotide exchange factors and regulates Vav activity through tyrosine phosphorylation. The Vav SH2 domain binds Syk linker B by an unusual recognition of two closely spaced Syk tyrosines: Y342 and Y346. The binding affinity is highest when both Y342 and Y346 are phosphorylated. An investigation in B cells of the dependence of Vav phosphorylation and NFAT activation on phosphorylation of Y342 and Y346 finds that cellular response levels match the relative binding affinities of the Vav1 SH2 domain for singly and doubly phosphorylated linker B peptides. This key result suggests that the uncommon recognition determinant of these two closely spaced tyrosines is a limiting factor in signaling. Interestingly, differences in affinities for binding singly and doubly phosphorylated peptides are reflected in the on rate, not the off rate. Such a control mechanism would be highly effective for regulating binding among competing Syk binding partners. The nuclear magnetic resonance (NMR) structure of Vav1 SH2 in complex with a doubly phosphorylated linker B peptide reveals diverse conformations associated with the unusual SH2 recognition of two phosphotyrosines. NMR relaxation indicates compensatory changes in loop fluctuations upon binding, with implications for nonphosphotyrosine interactions of Vav1 SH2.     

The BPS domain of Grb10 inhibits the catalytic activity of the insulin and IGF1 receptors

Grb7, Grb10 and Grb14 comprise a family of adaptor proteins that interact with numerous receptor tyrosine kinases upon receptor activation. Between the pleckstrin homology (PH) domain and the Src homology 2 (SH2) domain of these proteins is a region of approximately 50 residues known as the BPS (between PH and SH2) domain. Here we show, using purified recombinant proteins, that the BPS domain of Grb10 directly inhibits substrate phosphorylation by the activated tyrosine kinase domains of the insulin receptor and the insulin-like growth factor 1 (IGF1) receptor. Although inhibition by the BPS domain is dependent on tyrosine phosphorylation of the kinase activation loop, peptide competition experiments indicate that the BPS domain does not bind directly to phosphotyrosine. These studies provide a molecular mechanism by which Grb10 functions as a negative regulator of insulin- and/or IGF1-mediated signaling.     

Structural basis for recruitment of the adaptor protein APS to the activated insulin receptor

The adaptor protein APS is a substrate of the insulin receptor and couples receptor activation with phosphorylation of Cbl to facilitate glucose uptake. The interaction with the activated insulin receptor is mediated by the Src homology 2 (SH2) domain of APS. Here, we present the crystal structure of the APS SH2 domain in complex with the phosphorylated tyrosine kinase domain of the insulin receptor. The structure reveals a novel dimeric configuration of the APS SH2 domain, wherein the C-terminal half of each protomer is structurally divergent from conventional, monomeric SH2 domains. The APS SH2 dimer engages two kinase molecules, with pTyr-1158 of the kinase activation loop bound in the canonical phosphotyrosine binding pocket of the SH2 domain and a second phosphotyrosine, pTyr-1162, coordinated by two lysine residues in beta strand D. This structure provides a molecular visualization of one of the initial downstream recruitment events following insulin activation of its dimeric receptor.     

Mechanical stretch-induced RhoA activation is mediated by the RhoGEF Vav2 in mesangial cells

Increased intraglomerular pressure is an important hemodynamic determinant of glomerulosclerosis, and can be modelled in vitro by exposing mesangial cells (MC) to cyclic mechanical stretch. We have previously shown that the GTPase RhoA mediates stretch-induced fibronectin production. Here we investigate the role of the RhoGEF Vav2 in the activation of RhoA by stretch. Primary rat MC were exposed to 1 Hz cyclic stretch, previously shown to induce maximal RhoA activation at 1 min. Total Vav2 tyrosine phosphorylation and specific phosphorylation on Y172, required for activation, were increased by 1 min of stretch. Overexpression of dominant-negative Vav2 Y172/159F in COS-1 cells or downregulation of Vav2 by siRNA in MC prevented stretch-induced RhoA activation. Vav2 is known to be activated in response to growth factors, and we have previously shown the epidermal growth factor receptor (EGFR) to be transactivated by stretch in MC. Both Vav2 Y172 phosphorylation and RhoA activation were blocked by the EGFR inhibitor AG1478 and prevented in MC overexpressing kinase inactive EGFR. Stretch led to physical association between the EGFR and Vav2, and this was dependent on EGFR activation. EGFR Y992 phosphorylation, required for growth factor-induced Vav2 phosphorylation, was also induced by stretch. Activation of both Src and PI3K were necessary upstream mediators of stretch-induced Vav2 Y172 phosphorylation and RhoA activation. In summary, stretch-induced RhoA activation is dependent on transactivation of the EGFR and activation of the RhoGEF Vav2. Src and PI3K are both required upstream of Vav2 and RhoA activation.     

An active form of Vav1 induces migration of mammary epithelial cells by stimulating secretion of an epidermal growth factor receptor ligand

Background

Vav proteins are guanine nucleotide exchange factors (GEF) for Rho family GTPases and are activated following engagement of membrane receptors. Overexpression of Vav proteins enhances lamellipodium and ruffle formation, migration, and cell spreading, and augments activation of many downstream signaling proteins like Rac, ERK and Akt. Vav proteins are composed of multiple structural domains that mediate their GEF function and binding interactions with many cellular proteins. In this report we examine the mechanisms responsible for stimulation of cell migration by an activated variant of Vav1 and identify the domains of Vav1 required for this activity.

Results

We found that expression of an active form of Vav1, Vav1Y3F, in MCF-10A mammary epithelial cells increases cell migration in the absence or presence of EGF. Vav1Y3F was also able to drive Rac1 activation and PAK and ERK phosphorylation in MCF-10A cells in the absence of EGF stimulation. Mutations in the Dbl homology, pleckstrin homology, or cysteine-rich domains of Vav1Y3F abolished Rac1 or ERK activation in the absence of EGF and blocked the migration-promoting activity of Vav1Y3F. In contrast, mutations in the SH2 and C-SH3 domains did not affect Rac activation by Vav1Y3F, but reduced the ability of Vav1Y3F to induce EGF-independent migration and constitutive ERK phosphorylation. EGF-independent migration of MCF-10A cells expressing Vav1Y3F was abolished by treatment of cells with an antibody that prevents ligand binding to the EGF receptor. In addition, conditioned media collected from Vav1Y3F expressing cells stimulated migration of parental MCF-10A cells. Lastly, treatment of cells with the EGF receptor inhibitory antibody blocked the Vav1Y3F-induced, EGF-independent stimulation of ERK phosphorylation, but had no effect on Rac1 activation or PAK phosphorylation.

Conclusion

Our results indicate that increased migration of active Vav1 expressing cells is dependent on Vav1 GEF activity and secretion of an EGF receptor ligand. In addition, activation of ERK downstream of Vav1 is dependent on autocrine EGF receptor stimulation while active Vav1 can stimulate Rac1 and PAK activation independent of ligand binding to the EGF receptor. Thus, stimulation of migration by activated Vav1 involves both EGF receptor-dependent and independent activities induced through the Rho GEF domain of Vav1.     

Mechanism of epidermal growth factor regulation of Vav2, a guanine nucleotide exchange factor for Rac

Vav2 is a member of the Vav family that serves as a guanine nucleotide exchange factor for the Rho family of Ras-related GTPases. Unlike Vav1, whose expression is restricted to cells of hematopoietic origin, Vav2 is broadly expressed. Recently, Vav2 has been identified as a substrate for the epidermal growth factor (EGF) receptor; however, the mechanism by which Vav2 is activated in EGF-treated cells is unclear. By the means of an in vitro protein kinase assay, we show here that purified and activated EGF receptor phosphorylates Vav2 exclusively on its N-terminal domain. Furthermore, EGF receptor phosphorylates Vav2 on all three possible phosphorylation sites, Tyr-142, Tyr-159, and Tyr-172. In intact cells we also show that Vav2 associates with the activated EGF receptor in an Src homology 2 domain-dependent manner, with Vav2 Src homology 2 domain binding preferentially to autophosphorylation sites Tyr-992 and Tyr-1148 of the EGF receptor. Treatment of cells with EGF results in stimulation of exchange activity of Vav2 as measured on Rac; however, the intensity of the exchange activity does not show any correlation with the level of Vav2 tyrosine phosphorylation. Introducing a point mutation into the Vav2 pleckstrin homology domain or treatment of cells with the phosphatidylinositol 3-kinase inhibitor LY294002 prior to EGF stimulation inhibits Vav2 exchange activity. Although phosphorylation mutants of Vav2 can readily induce actin rearrangement in COS7 cells, pleckstrin homology domain mutant does not stimulate membrane ruffling. These results suggest that EGF regulates Vav2 activity basically through phosphatidylinositol 3-kinase activation, whereas tyrosine phosphorylation of Vav2 may rather be necessary for mediating protein-protein interactions.     

GEF at work: Vav in protruding filopodia.

The Dbl family proto-oncogene vav is a nucleotide exchange factor for Rho family GTPases and is involved in triggering cytoskeletal changes contributing to the alterations of cell shape and motility, as well as in the induction of gene expression. In vitro and in vivo Vav is regulated by multiple tyrosine phosphorylation and binding to phosphatidylinositol phosphates. Although recruitment of Vav to the plasma membrane appears important for the activation of Vav function, there is little information on the precise subcellular localization of Vav in living cells. Employing live video fluorescence and immunoelectron microscopy, we show that GFP-tagged full-length Vav, and several mutants in which the N-terminal regulatory calponin homology (CH) domain has been deleted, specifically localize to the tips of filopodia. This localization was congruent with a high content of tyrosine phosphorylation in these regions. Consistent with earlier observations, mutants lacking the C-terminal SH domain region were unable to translocate to the filopodia tips. The enrichment in filopodial tips persisted despite their lateral movement but was dependent on forward growth. Upon retraction, the signal was rapidly lost, indicating that Vav undergoes a specific and transient translocation in response to actin-based, protrusive events in filopodia.     

Vav2 activates Rac1, Cdc42, and RhoA downstream from growth factor receptors but not beta1 integrins

The Rho family of GTPases plays a major role in the organization of the actin cytoskeleton. These G proteins are activated by guanine nucleotide exchange factors that stimulate the exchange of bound GDP for GTP. In their GTP-bound state, these G proteins interact with downstream effectors. Vav2 is an exchange factor for Rho family GTPases. It is a ubiquitously expressed homologue of Vav1, and like Vav1, it has previously been shown to be activated by tyrosine phosphorylation. Because Vav1 becomes tyrosine phosphorylated and activated following integrin engagement in hematopoietic cells, we investigated the tyrosine phosphorylation of Vav2 in response to integrin-mediated adhesion in fibroblasts and epithelial cells. However, no tyrosine phosphorylation of Vav2 was detected in response to integrin engagement. In contrast, treating cells with either epidermal growth factor or platelet-derived growth factor stimulated tyrosine phosphorylation of Vav2. We have examined the effects of overexpressing either wild-type or amino-terminally truncated (constitutively active) forms of Vav2 as fusion proteins with green fluorescent protein. Overexpression of either wild-type or constitutively active Vav2 resulted in prominent membrane ruffles and enhanced stress fibers. These cells revealed elevated rates of cell migration that were inhibited by expression of dominant negative forms of Rac1 and Cdc42. Using a binding assay to measure the activity of Rac1, Cdc42, and RhoA, we found that overexpression of Vav2 resulted in increased activity of each of these G proteins. Expression of a carboxy-terminal fragment of Vav2 decreased the elevation of Rac1 activity induced by epidermal growth factor, consistent with Vav2 mediating activation of Rac1 downstream from growth factor receptors.     

SLAM-associated protein as a potential negative regulator in Trk signaling

Neurotrophin signaling plays important roles in regulating the survival, differentiation, and maintenance of neurons in the nervous system. Binding of neurotrophins to their cognate receptors Trks induces transactivation and phosphorylation of the receptor at several tyrosine residues. These phosphorylated tyrosine residues then serve as crucial docking sites for adaptor proteins containing a Src homology 2 or phosphotyrosine binding domain, which upon association with the receptor initiates multiple signaling events to mediate the action of neurotrophins. Here we report the identification of a Src homology 2 domain-containing molecule, SLAM-associated protein (SAP), as an interacting protein of TrkB in a yeast two-hybrid screen. SAP was initially identified as an adaptor molecule in SLAM family receptor signaling for regulating interferon-gamma secretion. In the current study, we found that SAP interacted with TrkA, TrkB, and TrkC receptors in vitro and in vivo. Binding of SAP required Trk receptor activation and phosphorylation at the tyrosine 674 residue, which is located in the activation loop of the kinase domain. Overexpression of SAP with Trk attenuated tyrosine phosphorylation of the receptors and reduced the binding of SH2B and Shc to TrkB. Moreover, overexpression of SAP in PC12 cells suppressed the nerve growth factor-dependent activation of extracellular signal-regulated kinases 1/2 and phospholipase Cgamma, in addition to inhibiting neurite outgrowth. In summary, our findings demonstrated that SAP may serve as a negative regulator of Trk receptor activation and downstream signaling.     

The Gammaherpesvirus m2 protein manipulates the Fyn/Vav pathway through a multidocking mechanism of assembly

To establish latent infections in B-cells, gammaherpesviruses express proteins in the infected B-cells of the host that spuriously activate signalling pathways located downstream of the B-cell receptor. One such protein is M2, a murine gammaherpesvirus 68-encoded molecule that activates the Vav1/Rac1 pathway via the formation of trimolecular complexes with Scr family members. Previous reports have shown that the formation of this heteromolecular complex involves interactions between a proline rich region of M2 and the Vav1 and Fyn SH3 domains. Here, we show that the optimal association of these proteins requires a second structural motif encompassing two tyrosine residues (Tyr120 and 129). These residues are inducibly phosphorylated by Fyn in non-hematopoietic cells and constitutively phosphorylated in B-cells. We also demonstrate that the phosphorylation of Tyr120 creates specific docking sites for the SH2 domains of both Vav1 and Fyn, a condition sine qua non for the optimal association of these two signalling proteins in vivo. Interestingly, signaling experiments indicate that the expression of M2 in B-cells promotes the tyrosine phosphorylation of Vav1 and additional signaling proteins, a biological process that requires the integrity of both the M2 phosphotyrosine and proline rich region motifs. By infecting mice with viruses mutated in the m2 locus, we show that the integrity of each of these two M2 docking motifs is essential for the early steps of murine gammaherpesvirus-68 latency. Taken together, these results indicate that the M2 phosphotyrosine motif and the previously described M2 proline rich region work in a concerted manner to manipulate the signaling machinery of the host B-cell.     

CrkL is recruited through its SH2 domain to the erythropoietin receptor and plays a role in Lyn-mediated receptor signaling.

The erythropoietin (Epo) receptor transduces its signals by activating physically associated tyrosine kinases, mainly Jak2 and Lyn, and thereby inducing tyrosine phosphorylation of various substrates including the Epo receptor (EpoR) itself. We previously demonstrated that, in Epo-stimulated cells, an adapter protein, CrkL, becomes tyrosine-phosphorylated, physically associates with Shc, SHP-2, and Cbl, and plays a role in activation of the Ras/Erk signaling pathway. Here, we demonstrate that Epo induces binding of CrkL to the tyrosine-phosphorylated EpoR and SHIP1 in 32D/EpoR-Wt cells overexpressing CrkL. In vitro binding studies showed that the CrkL SH2 domain directly mediates the EpoR binding, which was specifically inhibited by a synthetic phosphopeptide corresponding to the amino acid sequences at Tyr(460) in the cytoplasmic domain of EpoR. The CrkL SH2 domain was also required for tyrosine phosphorylation of CrkL in Epo-stimulated cells. Overexpression of Lyn induced constitutive phosphorylation of CrkL and activation of Erk, whereas that of a Lyn mutant lacking the tyrosine kinase domain attenuated the Epo-induced phosphorylation of CrkL and activation of Erk. Furthermore, Lyn, but not Jak2, phosphorylated CrkL on tyrosine in in vitro kinase assays. Together, the present study suggests that, upon Epo stimulation, CrkL is recruited to the EpoR through interaction between the CrkL SH2 domain and phosphorylated Tyr(460) in the EpoR cytoplasmic domain and undergoes tyrosine phosphorylation by receptor-associated Lyn to activate the downstream signaling pathway leading to the activation of Erk and Elk-1.     

Vav1 acidic region tyrosine 174 is required for the formation of T cell receptor-induced microclusters and is essential in T cell development and activation

Vav proteins are multidomain signaling molecules critical for mediating signals downstream of several surface receptors, including the antigen receptors of T and B lymphocytes. The catalytic guanine nucleotide exchange factor (GEF) activity of the Vav Dbl homology (DH) domain is thought to be controlled by an intramolecular autoinhibitory mechanism involving an N-terminal extension and phosphorylation of tyrosine residues in the acidic region (AC). Here, we report that the sequences surrounding the Vav1 AC: Tyr(142), Tyr(160), and Tyr(174) are evolutionarily conserved, conform to consensus SH2 domain binding motifs, and bind several proteins implicated in TCR signaling, including Lck, PI3K p85alpha, and PLCgamma1, through direct interactions with their SH2 domains. In addition, the AC tyrosines regulate tyrosine phosphorylation of Vav1. We also show that Tyr(174) is required for the maintenance of TCR-signaling microclusters and for normal T cell development and activation. In this regard, our data demonstrate that while Vav1 Tyr(174) is essential for maintaining the inhibitory constraint of the DH domain in both developing and mature T cells, constitutively activated Vav GEF disrupts TCR-signaling microclusters and leads to defective T cell development and proliferation.     

Vav1 and vav3 have critical but redundant roles in mediating platelet activation by collagen

Vav family proteins are guanine nucleotide exchange factors for the Rho/Rac family of small GTP-binding proteins. In addition, they have domains that mediate protein-protein interactions, including one Src homology 2 (SH2) and two Src homology 3 (SH3) domains. Vav1, Vav2, and Vav3 play a crucial role in the regulation of phospholipase C gamma (PLC gamma) isoforms by immuno-tyrosine-based activation motif (ITAM)-coupled receptors, including the T- and B-cell antigen receptors. We have reported in platelets, however, that Vav1 and Vav2 are not required for activation of PLC gamma 2 in response to stimulation of the ITAM-coupled collagen receptor glycoprotein VI (GPVI). Here we report that Vav3 is tyrosinephosphorylated upon activation of GPVI but that Vav3-deficient platelets also exhibit a normal response upon activation of the ITAM receptor. In sharp contrast, platelets deficient in both Vav1 and Vav3 show a marked inhibition of aggregation and spreading upon activation of GPVI, which is associated with a reduction in tyrosine phosphorylation of PLC gamma 2. The phenotype of Vav1/2/3 triple-deficient platelets is similar to that of Vav1/3 double-deficient cells. These results demonstrate that Vav3 and Vav1 play crucial but redundant roles in the activation of PLC gamma 2 by GPVI. This is the first time that absolute redundancy between two protein isoforms has been observed with respect to the regulation of PLC gamma 2 in platelets.     

Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr¹⁷⁴, Tyr¹⁸³ and Tyr⁴⁴⁶ in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr¹⁸³ and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr¹⁷⁴, Tyr¹⁸³ and Tyr⁴²⁶ of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr⁴²⁶ is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr⁴²⁶ was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr⁴²⁶ following BCR stimulation.     

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.