Vav1 regulates phospholipase cgamma activation and calcium responses in mast cells

The hematopoietic cell-specific protein Vav1 is a substrate of tyrosine kinases activated following engagement of many receptors, including FcepsilonRI. Vav1-deficient mice contain normal numbers of mast cells but respond more weakly than their normal counterparts to a passive systemic anaphylaxis challenge. Vav1-deficient bone marrow-derived mast cells also exhibited reduced degranulation and cytokine production, although tyrosine phosphorylation of FcepsilonRI, Syk, and LAT (linker for activation of T cells) was normal. In contrast, tyrosine phosphorylation of phospholipase Cgamma1 (PLCgamma1) and PLCgamma2 and calcium mobilization were markedly inhibited. Reconstitution of deficient mast cells with Vav1 restored normal tyrosine phosphorylation of PLCgamma1 and PLCgamma2 and calcium responses. Thus, Vav1 is essential to FcepsilonRI-mediated activation of PLCgamma and calcium mobilization in mast cells. In addition to its known role as an activator of Rac1 GTPases, these findings demonstrate a novel function for Vav1 as a regulator of PLCgamma-activated calcium signals.     

Regulation of Ca2+ signaling in mast cells by tyrosine-phosphorylated and unphosphorylated non-T cell activation linker

Engagement of the FcepsilonRI in mast cells and basophils leads to a rapid tyrosine phosphorylation of the transmembrane adaptors LAT (linker for activation of T cells) and NTAL (non-T cell activation linker, also called LAB or LAT2). NTAL regulates activation of mast cells by a mechanism, which is incompletely understood. Here we report properties of rat basophilic leukemia cells with enhanced or reduced NTAL expression. Overexpression of NTAL led to changes in cell morphology, enhanced formation of actin filaments and inhibition of the FcepsilonRI-induced tyrosine phosphorylation of the FcepsilonRI subunits, Syk kinase and LAT and all downstream activation events, including calcium and secretory responses. In contrast, reduced expression of NTAL had little effect on early FcepsilonRI-induced signaling events but inhibited calcium mobilization and secretory response. Calcium response was also repressed in Ag-activated cells defective in Grb2, a major target of phosphorylated NTAL. Unexpectedly, in cells stimulated with thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) ATPase, the amount of cellular NTAL directly correlated with the uptake of extracellular calcium even though no enhanced tyrosine phosphorylation of NTAL was observed. The combined data indicate that NTAL regulates FcepsilonRI-mediated signaling at multiple steps and by different mechanisms. At early stages NTAL interferes with tyrosine phosphorylation of several substrates and formation of signaling assemblies, whereas at later stages it regulates the activity of store-operated calcium channels through a distinct mechanism independent of enhanced NTAL tyrosine phosphorylation.     

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.     

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.     

Silver activates calcium signals in rat basophilic leukemia-2H3 mast cells by a mechanism that differs from the Fc epsilon RI-activated response

We previously showed that silver stimulates degranulation and leukotriene (LT) C(4) production in rat basophilic leukemia mast cells and now show that silver induces these events by a mechanism that differs from the FcepsilonRI-mediated response. In common with FcepsilonRI cross-linking, silver induced tyrosine phosphorylation of extracellular signal-regulated kinases and furthermore, PD98059, a specific inhibitor of extracellular signal-regulated kinase kinase dose-dependently inhibited the silver-induced LTC(4) production. In contrast to FcepsilonRI cross-linking, silver had no effect on the production of IL-4 and TNF-alpha, indicating that different mechanisms are involved in the activation by these two stimuli. In line with this, silver had no or only marginal effect on the tyrosine phosphorylation of FcepsilonRIbeta, Lyn, Syk, and linker for activation of T cells, the early and crucial events in FcepsilonRI signaling. Silver induced calcium signals that were involved in the metal-induced degranulation, but not LTC(4) production. Unlike Ag, the silver-induced calcium signals were resistant to the depletion of thapsigargin-sensitive calcium stores and the inhibition of tyrosine kinases and phospholipase Cgamma. These findings indicate that silver activates mast cells by bypassing the early signaling events required for the induction of calcium influx. Our data strongly suggest the existence of an alternative pathway bypassing the early signaling events in mast cell activation and indicate that silver may be useful for analyses of such alternative mechanisms.     

Regulation of signaling in B cells through the phosphorylation of Syk on linker region tyrosines. A mechanism for negative signaling by the Lyn tyrosine kinase

The B cell antigen receptor (BCR) is coupled to the mobilization of Ca(2+) by the protein-tyrosine kinase, Syk. Syk, recruited to the clustered BCR, becomes phosphorylated on three tyrosines (Tyr-317, Tyr-342, and Tyr-346) located within the linker region that separates the C-terminal catalytic domain from the N-terminal tandem Src homology 2 domains. Phosphorylation within the linker region can be either activating or inhibitory to Ca(2+) mobilization depending on the sites that are modified. Syk that is not phosphorylated on linker region tyrosines couples the BCR to Ca(2+) mobilization through a phosphoinositide 3-kinase-dependent pathway. The phosphorylation of Tyr-342 and -346 enhances the phosphorylation and activation of phospholipase C-gamma and the early phase of Ca(2+) mobilization via a phosphoinositide 3-kinase-independent pathway. The phosphorylation of Tyr-317 strongly dampens the Ca(2+) signal. In cells that lack the Src family kinase, Lyn, the phosphorylation of the inhibitory Tyr-317 is suppressed leading to elevated production of inositol 1,4,5-trisphosphate and an amplified Ca(2+) signal. This provides a novel mechanism by which Lyn functions as an inhibitor of BCR-stimulated signaling. Thus, Syk and Lyn combine to determine the pathway through which the BCR is coupled to Ca(2+) mobilization as well as the magnitude and duration of the Ca(2+) flux.     

Point mutation of a tyrosine in the linker region of Syk results in a gain of function

The protein tyrosine kinase Syk plays an essential role in Fc epsilon RI-mediated histamine release in mast cells by regulating the phosphorylation of other proteins. We investigated the functional role of a putative Syk phosphorylation site, Tyr317. This tyrosine in the linker region of Syk is a possible site for binding by the negative regulator Cbl. Syk with Tyr317 mutated to Phe (Y317F) was expressed in a Syk-negative variant of the RBL-2H3 mast cells. Compared with cells expressing wild-type Syk, expression of the Y317F mutant resulted in an increase in the Fc epsilon RI-mediated tyrosine phosphorylation of phospholipase C-gamma and a dramatic enhancement of histamine release. The in vivo Fc epsilon RI-induced tyrosine phosphorylation of wild-type Syk and that of the Y317F mutant were similar. Although the Fc epsilon RI-induced tyrosine phosphorylation of total cellular proteins was enhanced in the cells expressing the Y317F Syk, the phosphorylation of some other molecules, including the receptor subunits, Vav and mitogen-activated protein kinase, was not increased. The Fc epsilon RI-induced phosphorylation of Cbl was downstream of Syk kinase activity and was unchanged by expression of the Y317F mutation. These data indicate that Tyr317 in the linker region of Syk functions to negatively regulate the signals leading to degranulation.     

Recombinant anti-CD4 antibody 13B8.2 blocks membrane-proximal events by excluding the Zap70 molecule and downstream targets SLP-76, PLC gamma 1, and Vav-1 from the CD4-segregated Brij 98 detergent-resistant raft domains

The biological effects of rIgG(1) 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-kappaB nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG(1) 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG(1) 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37 degrees C, together with recruitment of TCR, CD3zeta, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase Ctheta, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase Cgamma1, and p95(vav). Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr(292) and Tyr(319) phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10-30 s following rIgG(1) 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-kappaB nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG(1) 13B8.2 acts to induce its biological effects.     

Cross-linking of FcepsilonRI causes Ca2+ mobilization via a sphingosine kinase pathway in a clathrin-dependent manner

Clathrin-coated pits are now recognized to be involved in cell signaling in addition to receptor down-regulation. Here we tried to identify signaling pathways that might be dependent on clathrin. Our initial data with pharmacological inhibitors of formation of clathrin-coated pits or lipid-rafts indicated that Ca(2+) response evoked by cross-linking of the high affinity receptors for IgE (FcepsilonRI) was dependent on clathrin. To confirm this finding, we created clathrin-knockdown cells by transfecting the mast cell line RBL-2H3 with a shRNA-clathrin heavy chain construct. In these cells, the FcepsilonRI-mediated Ca(2+) response was almost completely abolished, which was accompanied by the inhibition of sphingosine 1-phosphate (S1P) production with no changes in inositol 1,4,5-trisphosphate (IP(3)) production. This suggests that the Ca(2+) signaling pathway via a sphingosine kinase (SK) is dependent on clathrin. Furthermore, antigen-induced tyrosine phosphorylation of p85 and p110 subunits of PI3K was almost completely inhibited in clathrin-knockdown cells. In contrast, antigen-induced tyrosine phosphorylation of phospholipase Cgamma was not affected by clathrin-knockdown and tyrosine phosphorylation of Syk and degranulation were partially inhibited in clathrin-knockdown cells. The present study identifies the SK/Ca(2+) pathway to be dependent on clathrin.     

Bivalent ligands with rigid double-stranded DNA spacers reveal structural constraints on signaling by Fc epsilon RI

Degranulation of mast cells and basophils during the allergic response is initiated by Ag-induced cross-linking of cell surface IgE-Fc epsilon RI receptor complexes. To investigate how separation distances between cross-linked receptors affect the competency of signal transduction, we synthesized and characterized bivalent dinitrophenyl (DNP)-modified dsDNA oligomers with rigid spacing lengths of approximately 40-100 A. All of these bivalent ligands effectively bind and cross-link anti-DNP IgE with similar affinities in the nanomolar range. The 13-mer (dsDNA length of 44 A), 15-mer (51 A), and flexible 30-mer ligands stimulate similar amounts of cellular degranulation, about one-third of that with multivalent Ag, whereas the 20-mer (68 A) ligand is less effective and the rigid 30-mer (102 A) ligand is ineffective. Surprisingly, all stimulate tyrosine phosphorylation of Fc epsilon RI beta, Syk, and linker for activation of T cells to similar extents as multivalent Ag at optimal ligand concentrations. The magnitudes of Ca(2+) responses stimulated by these bivalent DNP-dsDNA ligands are small, implicating activation of Ca(2+) mobilization by stimulated tyrosine phosphorylation as a limiting process. The results indicate that structural constraints on cross-linked IgE-Fc epsilon RI complexes imposed by these rigid DNP-dsDNA ligands prevent robust activation of signaling immediately downstream of early tyrosine phosphorylation events. To account for these results, we propose that activation of a key downstream target is limited by the spacing between cross-linked, phosphorylated receptors and their associated components.     

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.     

The Fc receptor gamma-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen.

Activation of mouse platelets by collagen is associated with tyrosine phosphorylation of multiple proteins including the Fc receptor gamma-chain, the tyrosine kinase Syk and phospholipase Cgamma2, suggesting that collagen signals in a manner similar to that of immune receptors. This hypothesis has been tested using platelets from mice lacking the Fc receptor gamma-chain or Syk. Tyrosine phosphorylation of Syk and phospholipase Cgamma2 by collagen stimulation is absent in mice lacking the Fc receptor gamma-chain. Tyrosine phosphorylation of phospholipase Cgamma2 by collagen stimulation is also absent in mice platelets which lack Syk, although phosphorylation of the Fc receptor gamma-chain is maintained. In contrast, tyrosine phosphorylation of platelet proteins by the G protein-coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor gamma-chain or Syk. The absence of Fc receptor gamma-chain or Syk is accompanied by a loss of secretion and aggregation responses in collagen- but not thrombin-stimulated platelets. These observations provide the first direct evidence of an essential role for the immunoreceptor tyrosine-based activation motif (ITAM) in signalling by a non-immune receptor stimulus.     

LAT is required for tyrosine phosphorylation of phospholipase cgamma2 and platelet activation by the collagen receptor GPVI

In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cgamma2 (PLCgamma2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcgammaRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCgamma2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCgamma2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin alpha(IIb)beta(3) in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCgamma2, leading to downstream responses such as alpha-granule secretion and activation of integrin alpha(IIb)beta(3). The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCgamma2. We propose a model in which LAT and SLP-76 are required for PLCgamma2 phosphorylation but are regulated through independent pathways downstream of Syk.     

Surface plasmon resonance biosensor detects the downstream events of active PKCbeta in antigen-stimulated mast cells

Surface plasmon resonance (SPR) biosensors detect large changes of angle of resonance (AR) when RBL-2H3 mast cells are cultured on a sensor chip and stimulated with antigen. However, the detail of molecular events that are responsible for such large changes of AR remained unknown. In this study, we investigated the relationship between intracellular signaling events induced by antigen and the change of AR, by genetic manipulation of intracellular signaling molecules; spleen tyrosine kinase (Syk), src-like adaptor protein (SLAP), linker for activation of T cells (LAT), growth-factor-receptor-bound protein 2 (Grb2), Grb2-related adaptor protein (Gads), and isotypes of protein kinase C (PKC). RBL-2H3 mast cells overexpressing dominant-negative Syk or SLAP, which both interfere with active Syk, exhibited only minimal increase of AR in response to antigen stimulation. Likewise, the interference of the activation of LAT and Gads, by expressing dominant-negative LAT and Gads, respectively, resulted in nearly complete suppression of the antigen-induced increase of AR. The cells overexpressing PKCs, apart from PKCbeta, showed a reduced extent of increase of AR in response to antigen stimulation. Moreover, the introduction of the small interfering RNA targeted against PKCbeta suppressed the antigen-induced increase of AR. These results indicate that the activation of Syk, LAT, Gads, and subsequent PKCbeta is indispensable for the antigen-induced increase of AR of mast cells detected by SPR biosensors.     

Interaction of linker for activation of T cells with multiple adapter proteins in platelets activated by the glycoprotein VI-selective ligand, convulxin

The snake venom toxin convulxin activates platelets through the collagen receptor glycoprotein VI (GPVI)/Fc receptor gamma-chain (FcR gamma-chain) complex leading to tyrosine phosphorylation and activation of the tyrosine Syk and phospholipase Cgamma2 (PLCgamma2). In the present study, we demonstrate that convulxin is a considerably more powerful agonist than collagen or the GPVI-selective collagen-related peptide (CRP). Confirmation that the response to convulxin is mediated solely via Syk was provided by studies on Syk-deficient platelets. The increase in phosphorylation of the FcR gamma-chain is associated with marked increases in tyrosine phosphorylation of downstream proteins including Syk, linker for activation of T cells (LAT), SLP-76, and PLCgamma2. The transmembrane adapter LAT coprecipitates with SLP-76 and PLCgamma2, as well as with a number of other adapter proteins, some of which have not been previously described in platelets, including Cbl, Grb2, Gads, and SKAP-HOM. Gads is constitutively associated with SLP-76 and is probably the protein bridging its association with LAT. There was no detectable association between Grb2 and SLP-76 in control or stimulated cells, suggesting that the interaction of LAT with Grb2 is present in a separate complex to that of LAT-Gads-SLP-76. These results show that the trimeric convulxin stimulates a much greater phosphorylation of the FcR gamma-chain and subsequent downstream responses relative to CRP and collagen, presumably because of its ability to cause a greater degree of cross-linking of GPVI. The adapter LAT appears to play a critical role in recruiting a number of other adapter proteins to the surface membrane in response to activation of GPVI, presumably at sites of glycolipid-enriched microdomains, enabling an organized signaling cascade that leads to platelet activation.     

Src-dependent Syk activation controls CD69-mediated signaling and function on human NK cells

CD69 C-type lectin receptor represents a functional triggering molecule on activated NK cells, capable of directing their natural killing function. The receptor-proximal signaling pathways activated by CD69 cross-linking and involved in CD69-mediated cytotoxic activity are still poorly understood. Here we show that CD69 engagement leads to the rapid and selective activation of the tyrosine kinase Syk, but not of the closely related member of the same family, ZAP70, in IL-2-activated human NK cells. Our results indicate the requirement for Src family kinases in the CD69-triggered activation of Syk and suggest a role for Lck in this event. We also demonstrate that Syk and Src family tyrosine kinases control the CD69-triggered tyrosine phosphorylation and activation of phospholipase Cgamma2 and the Rho family-specific exchange factor Vav1 and are responsible for CD69-triggered cytotoxicity of activated NK cells. The same CD69-activated signaling pathways are also observed in an RBL transfectant clone, constitutively expressing the receptor. These data demonstrate for the first time that the CD69 receptor functionally couples to the activation of Src family tyrosine kinases, which, by inducing Syk activation, initiate downstream signaling pathways and regulate CD69-triggered functions on human NK cells.     

Phospholipid scramblase 1 modulates a selected set of IgE receptor-mediated mast cell responses through LAT-dependent pathway

Engagement of the IgE receptor (FcepsilonRI) on mast cells leads to the release of preformed and newly formed mediators as well as of cytokines. The signaling pathways responsible for these responses involve tyrosine phosphorylation of multiple proteins. We previously reported the phosphorylation on tyrosine of phospholipid scramblase 1 (PLSCR1) after FcepsilonRI aggregation. Here, PLSCR1 expression was knocked down in the RBL-2H3 mast cell line using short hairpin RNA. Knocking down PLSCR1 expression resulted in significantly impaired degranulation responses after FcepsilonRI aggregation and release of vascular endothelial growth factor, whereas release of MCP-1 was minimally affected. The release of neither leukotriene C4 nor prostaglandin D2 was altered by knocking down of PLSCR1. Analysis of FcepsilonRI-dependent signaling pathways revealed that whereas tyrosine phosphorylation of ERK and Akt was unaffected, tyrosine phosphorylation of LAT was significantly reduced in PLSCR1 knocked down cells. Tyrosine phosphorylation of phospholipase Cgamma1 and consequently the mobilization of calcium were also significantly reduced in these cells. In nonactivated mast cells, PLSCR1 was found in part in lipid rafts where it was further recruited after cell activation and was constitutively associated with Lyn and Syk but not with LAT or Fyn. Altogether, these data identify PLSCR1 as a novel amplifier of FcepsilonRI signaling that acts selectively on the Lyn-initiated LAT/phospholipase Cgamma1/calcium axis, resulting in potentiation of a selected set of mast cell responses.     

Phosphorylation of Syk activation loop tyrosines is essential for Syk function. An in vivo study using a specific anti-Syk activation loop phosphotyrosine antibody

Syk is an important protein-tyrosine kinase in immunoreceptor signaling. FcepsilonRI aggregation in mast cells induces tyrosine phosphorylation and increased enzymatic activity of Syk. The two adjacent tyrosines in the Syk activation loop are thought to be important for the propagation of FcepsilonRI signaling. To evaluate the phosphorylation of these tyrosines in vivo and further understand the relationship of Syk tyrosine phosphorylation with its function, an antibody was developed specific for phosphorylated tyrosines in the activation loop of Syk. FcepsilonRI aggregation on mast cells induced the phosphorylation of both tyrosine residues of the activation loop. The kinase activity of Syk played the major role in phosphorylating its activation loop tyrosines both in vivo and in vitro. In FcepsilonRI-stimulated mast cells, the total Syk tyrosine phosphorylation paralleled the phosphorylation of its activation loop tyrosines and downstream propagation of signals for histamine release. In contrast, the cell surface binding of anti-ganglioside monoclonal antibody AA4 induced only strong general tyrosine phosphorylation of Syk and minimal histamine release and weak phosphorylation of activation loop tyrosines. These results demonstrate that phosphorylation of the activation loop tyrosines is important for mediating receptor signaling and is a better marker of Syk function than is total Syk tyrosine phosphorylation.