Increased expression of genes linked to FcepsilonRI Signaling and to cytokine and chemokine production in Lyn-deficient mast cells

Cross-linking the high-affinity IgE receptor, FcepsilonRI, on mast cells activates signaling pathways leading to the release of preformed inflammatory mediators and the production of cytokines and chemokines associated with allergic disorders. Bone marrow-derived mast cells (BMMCs) from Lyn-deficient (Lyn-/-) mice are hyperresponsive to FcepsilonRI cross-linking with multivalent Ag. Previous studies linked the hyperresponsive phenotype in part to increased Fyn kinase activity and reduced SHIP phosphatase activity in the Lyn-/- BMMCs in comparison with wild-type (WT) cells. In this study, we compared gene expression profiles between resting and Ag-activated WT and Lyn-/- BMMCs to identify other factors that may contribute to the hyperresponsiveness of the Lyn-/- cells. Among genes implicated in the positive regulation of FcepsilonRI signaling, mRNA for the tyrosine kinase, Fyn, and for several proteins contributing to calcium regulation are more up-regulated following Ag stimulation in Lyn-/- BMMCs than in WT BMMCs. Conversely, mRNA for the low-affinity IgG receptor (FcgammaRIIB), implicated in negative regulation of FcepsilonRI-mediated signaling, is more down-regulated in Ag-stimulated Lyn-/- BMMCs than in WT BMMCs. Genes coding for proinflammatory cytokines and chemokines (IL-4, IL-6, IL-13, CSF, CCL1, CCL3, CCL5, CCL7, CCL9, and MIP1beta) are all more highly expressed in Ag-stimulated Lyn-/- mast cells than in WT cells. These microarray data identify Lyn as a negative regulator in Ag-stimulated BMMCs of the expression of genes linked to FcepsilonRI signaling and also to the response pathways that lead to allergy and asthma.     

SHIP negatively regulates IgE + antigen-induced IL-6 production in mast cells by inhibiting NF-kappa B activity

We demonstrate in this study that IgE + Ag-induced proinflammatory cytokine production is substantially higher in Src homology-2-containing inositol 5'-phosphatase (SHIP)(-/-) than in SHIP(+/+) bone marrow-derived mast cells (BMMCs). Focusing on IL-6, we found that the repression of IL-6 mRNA and protein production in SHIP(+/+) BMMCs requires the enzymatic activity of SHIP, because SHIP(-/-) BMMCs expressing wild-type, but not phosphatase-deficient (D675G), SHIP revert the IgE + Ag-induced increase in IL-6 mRNA and protein down to levels seen in SHIP(+/+) BMMCs. Comparing the activation of various signaling pathways to determine which ones might be responsible for the elevated IL-6 production in SHIP(-/-) BMMCs, we found the phosphatidylinositol 3-kinase/protein kinase B (PKB), extracellular signal-related kinase (Erk), p38, c-Jun N-terminal kinase, and protein kinase C (PKC) pathways are all elevated in IgE + Ag-induced SHIP(-/-) cells. Moreover, inhibitor studies suggested that all these pathways play an essential role in IL-6 production. Looking downstream, we found that IgE + Ag-induced IL-6 production is dependent on the activity of NF-kappa B and that I kappa B phosphorylation/degradation and NF-kappa B translocation, DNA binding and transactivation are much higher in SHIP(-/-) BMMCs. Interestingly, using various pathway inhibitors, it appears that the phosphatidylinositol 3-kinase/PKB and PKC pathways elevate IL-6 mRNA synthesis, at least in part, by enhancing the phosphorylation of I kappa B and NF-kappa B DNA binding while the Erk and p38 pathways enhance IL-6 mRNA synthesis by increasing the transactivation potential of NF-kappa B. Taken together, our data are consistent with a model in which SHIP negatively regulates NF-kappa B activity and IL-6 synthesis by reducing IgE + Ag-induced phosphatidylinositol-3,4,5-trisphosphate levels and thus PKB, PKC, Erk, and p38 activation.     

Regulation of microtubule formation in activated mast cells by complexes of gamma-tubulin with Fyn and Syk kinases

Aggregation of the high-affinity IgE receptors (FcepsilonRIs) on the surface of granulated mast cells initiates a chain of signaling events culminating in the release of allergy mediators. Although microtubules are involved in mast cell degranulation, the molecular mechanism that controls microtubule rearrangement after FcepsilonRI triggering is poorly understood. In this study, we show that the activation of bone marrow-derived mast cells (BMMCs) induced by FcepsilonRI aggregation or treatment with pervanadate leads to a rapid polymerization of microtubules. This polymerization was not dependent on the presence of Lyn kinase as determined by experiments with BMMCs isolated from Lyn-negative mice. One of the key regulators of microtubule polymerization is gamma-tubulin. Immunoprecipitation experiments revealed that gamma-tubulin from activated cells formed complexes with Fyn and Syk protein tyrosine kinases and several tyrosine phosphorylated proteins from both wild-type and Lyn(-/-) BMMCs. Pretreatment of the cells with Src-family or Syk-family selective tyrosine kinase inhibitors, PP2 or piceatannol, respectively, inhibited the formation of microtubules and reduced the amount of tyrosine phosphorylated proteins in gamma-tubulin complexes, suggesting that Src and Syk family kinases are involved in the initial stages of microtubule formation. This notion was corroborated by pull-down experiments in which gamma-tubulin complex bounds to the recombinant Src homology 2 and Src homology 3 domains of Fyn kinase. We propose that Fyn and Syk kinases are involved in the regulation of binding properties of gamma-tubulin and/or its associated proteins, and thus modulate the microtubule nucleation in activated mast cells.     

Scaffolding adapter Grb2-associated binder 2 requires Syk to transmit signals from FcepsilonRI

Scaffolding adapter Grb2-associated binder 2 (Gab2) is a key component of FcepsilonRI signaling in mast cells, required for the activation of PI3K. To understand how Gab2 is activated in FcepsilonRI signaling, we asked which protein tyrosine kinase is required for Gab2 phosphorylation. We found that Gab2 tyrosyl phosphorylation requires Lyn and Syk. In agreement with published results, we found that Fyn also contributes to Gab2 tyrosyl phosphorylation. However, Syk activation is defective in Fyn(-/-) mast cells, suggesting that Syk is the proximal kinase responsible for Gab2 tyrosyl phosphorylation. Then, we asked which domains in Gab2 are required for Gab2 tyrosyl phosphorylation. We found that the Grb2-Src homology 3 (SH3) binding sites are required for, whereas the pleckstrin homology (PH) domain contributes to, Gab2 tyrosyl phosphorylation. Using a protein/lipid overlay assay, we determined that the Gab2 PH domain preferentially binds the PI3K lipid products, PI3, 4,5P3 and PI3, 4P2. Furthermore, the Grb2-SH3 binding sites and PH domain binding to PI3K lipid products are required for Gab2 function in FcepsilonRI-evoked degranulation and Akt activation. Our data strongly suggest a model for Gab2 action in FcepsilonRI signaling. The Grb2 SH3 binding sites play a critical role in bringing Gab2 to FcepsilonRI, whereupon Gab2 becomes tyrosyl-phosphorylated in a Syk-dependent fashion. Phosphorylated Gab2 results in recruitment and activation of PI3K, whose lipid products bind the PH domain of Gab2 and acts in positive feedback loop for sustained PI3K recruitment and phosphatidylinositol-3,4,5-trisphosphate production, required for FcepsilonRI-evoked degranulation of mast cells.     

Protein kinase C-delta is a negative regulator of antigen-induced mast cell degranulation

Regulation of mast cell degranulation is dependent on the subtle interplay of cellular signaling proteins. The Src homology 2 (SH2) domain-containing inositol-5'-phosphatase (SHIP), which acts as the gatekeeper of degranulation, binds via both its SH2 domain and its phosphorylated NPXY motifs to the adapter protein Shc via the latter's phosphorylated tyrosines and phosphotyrosine-binding domain, respectively. This theoretically leaves Shc's SH2 domain available to bind proteins, which might be part of the SHIP/Shc complex. In a search for such proteins, protein kinase C-delta (PKC-delta) was found to coprecipitate in mast cells with Shc and to interact with Shc's SH2 domain following antigen or pervanadate stimulation. Phosphorylation of PKC-delta's Y(332), most likely by Lyn, was found to be responsible for PKC-delta's binding to Shc's SH2 domain. Using PKC-delta(-/-) bone marrow-derived mast cells (BMMCs), we found that the antigen-induced tyrosine phosphorylation of Shc was similar to that in wild-type (WT) BMMCs while that of SHIP was significantly increased. Moreover, increased translocation of PKC-delta to the membrane, as well as phosphorylation at T505, was observed in SHIP(-/-) BMMCs, demonstrating that while PKC-delta regulates SHIP phosphorylation, SHIP regulates PKC-delta localization and activation. Interestingly, stimulation of PKC-delta(-/-) BMMCs with suboptimal doses of antigen yielded a more sustained calcium mobilization and a significantly higher level of degranulation than that of WT cells. Altogether, our data suggest that PKC-delta is a negative regulator of antigen-induced mast cell degranulation.     

The role of SHIP in growth factor induced signalling

The recently cloned, hemopoietic-specific, src homology 2 (SH2)-containing inositol phosphatase, SHIP, is rapidly gaining prominence as a potential regulator of all phosphatidylinositol (PI)-3 kinase mediated events since it has been shown both in vitro and in vivo to hydrolyze the 5′ phosphate from phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P₃). Thus SHIP, and its more widely expressed counterpart, SHIP2, could play a central role in determining PI-3,4,5-P₃ and PI-3,4-P₂ levels in many cell types. To explore the in vivo function of SHIP further we recently generated a SHIP knock out mouse and in this review we discuss experiments carried out with bone marrow derived mast cells (BMMCs) from these animals.     

Protein-tyrosine phosphatase alpha regulates stem cell factor-dependent c-Kit activation and migration of mast cells

The role of protein-tyrosine phosphatase alpha (PTPalpha) in mast cell function was investigated in tissues and cells from PTPalpha-deficient mice. Bone marrow-derived mast cells (BMMCs) lacking PTPalpha exhibit defective stem cell factor (SCF)-dependent polarization and migration. Investigation of the molecular basis for this reveals that SCF/c-Kit-stimulated activation of the Fyn tyrosine kinase is impaired in PTPalpha(-/-) BMMCs, with a consequent inhibition of site-specific c-Kit phosphorylation at tyrosines 567/569 and 719. Although c-Kit-mediated activation of phosphatidylinositol 3-kinase and Akt is unaffected, profound defects occur in the activation of downstream signaling proteins, including mitogen-activated protein kinases and Rho GTPases. Phosphorylation and interaction of Fyn effectors Gab2 and Shp2, which are linked to Rac/JNK activation in mast cells, are impaired in PTPalpha(-/-) BMMCs. Thus, PTPalpha is required for SCF-induced c-Kit and Fyn activation, and in this way regulates a Fyn-based c-Kit signaling axis (Fyn/Gab2/Shp2/Vav/PAK/Rac/JNK) that mediates mast cell migration. These defective signaling events may underlie the altered tissue-resident mast cell populations found in PTPalpha(-/-) mice.     

Regulation of the Tyrosine Phosphorylation of Phospholipid Scramblase 1 in Mast Cells That Are Stimulated through the High-Affinity IgE Receptor

Engagement of high-affinity immunoglobulin E receptors (FcεRI) activates two signaling pathways in mast cells. The Lyn pathway leads to recruitment of Syk and to calcium mobilization whereas the Fyn pathway leads to phosphatidylinositol 3-kinase recruitment. Mapping the connections between both pathways remains an important task to be completed. We previously reported that Phospholipid Scramblase 1 (PLSCR1) is phosphorylated on tyrosine after cross-linking FcεRI on RBL-2H3 rat mast cells, amplifies mast cell degranulation, and is associated with both Lyn and Syk tyrosine kinases. Here, analysis of the pathway leading to PLSCR1 tyrosine phosphorylation reveals that it depends on the FcRγ chain. FcεRI aggregation in Fyn-deficient mouse bone marrow-derived mast cells (BMMC) induced a more robust increase in FcεRI-dependent tyrosine phosphorylation of PLSCR1 compared to wild-type cells, whereas PLSCR1 phosphorylation was abolished in Lyn-deficient BMMC. Lyn association with PLSCR1 was not altered in Fyn-deficient BMMC. PLSCR1 phosphorylation was also dependent on the kinase Syk and significantly, but partially, dependent on detectable calcium mobilization. Thus, the Lyn/Syk/calcium axis promotes PLSCR1 phosphorylation in multiple ways. Conversely, the Fyn-dependent pathway negatively regulates it. This study reveals a complex regulation for PLSCR1 tyrosine phosphorylation in FcεRI-activated mast cells and that PLSCR1 sits at a crossroads between Lyn and Fyn pathways.     

PTPalpha activates Lyn and Fyn and suppresses Hck to negatively regulate FcepsilonRI-dependent mast cell activation and allergic responses

Mast cell activation via FcεRI involves activation of the Src family kinases (SFKs) Lyn, Fyn, and Hck that positively or, in the case of Lyn, negatively regulate cellular responses. Little is known of upstream activators of these SFKs in FcεRI-dependent signaling. We investigated the role of receptor protein tyrosine phosphatase (PTP)α, a well-known activator of SFKs in diverse signaling systems, FcεRI-mediated mast cell activation, and IgE-dependent allergic responses in mice. PTPα(-/-) bone marrow-derived mast cells hyperdegranulate and exhibit increased cytokine and cysteinyl leukotriene secretion, and PTPα(-/-) mice display enhanced IgE-dependent anaphylaxis. At or proximal to FcεRI, PTPα(-/-) cells have reduced IgE-dependent activation of Lyn and Fyn, as well as reduced FcεRI and SHIP phosphorylation. In contrast, Hck and Syk activation is enhanced. Syk hyperactivation correlated with its increased phosphorylation at positive regulatory sites and defective phosphorylation at a negative regulatory site. Distal to FcεRI, we observed increased activation of PI3K and MAPK pathways. These findings demonstrate that PTPα activates the FcεRI-coupled kinases Lyn and Fyn and suppresses Hck activity. Furthermore, the findings indicate that hyperactivation of PTPα(-/-) mast cells and enhanced IgE-dependent allergic responses of PTPα(-/-) mice are due to the ablated function of PTPα as a critical regulator of Lyn negative signaling.     

Detergent-resistant membrane domains are required for mast cell activation but dispensable for tyrosine phosphorylation upon aggregation of the high affinity receptor for IgE

Aggregation of the high affinity receptor for IgE (FceRI) on mast cells results in the rapid phosphorylation of tyrosines on the beta and gamma chains of the receptor by the Src family kinase Lyn, which initiates the signaling cascades leading to secretion of inflammatory mediators. The detergent-resistant membranes (DRMs) have been implicated in FcepsilonRI signaling because aggregated receptors emigrate to DRMs that are enriched in certain signaling components. We evaluated the role of DRMs in FcepsilonRI signaling by disruption of DRMs using a cholesterol-binding agent, methyl-beta-cyclodextrin (MBCD). While treatment of rat basophilic leukemia cells with MBCD inhibits degranulation and Ca(2+) mobilization upon aggregation of FcepsilonRI, MBCD hardly affects the aggregation-induced tyrosine phosphorylation of FcepsilonRI as well as other signaling molecules such as phospholipase C-gamma1 (PLC-gamma1). MBCD delocalizes phosphatidylinositol 4,5-bisphosphate from DRMs, which may prevent MBCD-treated cells from producing inositol 1,4,5-trisphosphate by means of activated PLC-gamma1. These data suggest an indispensable role for DRMs in the Ca(2+) response rather than tyrosine phosphorylation, and support a model of receptor phosphorylation in which aggregated FcepsilonRI is tyrosine phosphorylated outside DRMs by constitutively associated Src family kinase Lyn via a transphosphorylation mechanism.     

Targeted disruption of SHIP leads to Steel factor-induced degranulation of mast cells.

To investigate the role of the src homology 2 (SH2)-containing inositol 5' phosphatase (SHIP) in growth factor-mediated signalling, we compared Steel factor (SF)-induced events in bone marrow-derived mast cells (BMMCs) from SHIP-/- and SHIP+/+ littermates. We found SF alone stimulated massive degranulation from SHIP-/- but none from SHIP+/+ BMMCs. This SF-induced degranulation, which was not due to higher c-kit levels in SHIP-/- cells, correlated with higher intracellular calcium than that in SHIP+/+ cells and was dependent on the influx of extracellular calcium. Both this influx and subsequent degranulation were completely inhibited by PI-3-kinase inhibitors, indicating that SF-induced activation of PI-3-kinase was upstream of extracellular calcium entry. A comparison of phosphatidylinositol-3,4,5-trisphosphate (PIP3) levels following SF stimulation of SHIP+/+ and SHIP-/- BMMCs suggested that SHIP restricted this entry by hydrolyzing PIP3. Although PI-3-kinase inhibitors blocked the release of intracellular calcium, implicating PIP3, and PLCgamma-2 was slightly more tyrosine phosphorylated in SHIP-/- cells, the increase in inositol-1,4,5-trisphosphate (IP3) and intracellular calcium levels were identical in SHIP-/- and SHIP+/+ BMMCs. These results suggest that SHIP prevents SF from triggering degranulation of normal BMMCs, and does so by hydrolyzing PIP3, which in turn limits extracellular calcium entry at a step after the release of intracellular calcium.     

SHIP1 and Lyn Kinase Negatively Regulate Integrin alpha IIb beta 3 signaling in platelets

Integrin alpha(IIb)beta(3) plays a critical role in platelet function, promoting a broad range of functional responses including platelet adhesion, spreading, aggregation, clot retraction, and platelet procoagulant function. Signaling events operating downstream of this receptor (outside-in signaling) are important for these responses; however the mechanisms negatively regulating integrin alpha(IIb)beta(3) signaling remain ill-defined. We demonstrate here a major role for the Src homology 2 domain-containing inositol 5-phosphatase (SHIP1) and Src family kinase, Lyn, in this process. Our studies on murine SHIP1 knockout platelets have defined a major role for this enzyme in regulating integrin alpha(IIb)beta(3)-dependent phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) accumulation, necessary for a cytosolic calcium response and platelet spreading. SHIP1 phosphorylation and PtdIns(3,4,5)P(3) metabolism is partially regulated through Lyn kinase, resulting in an enhanced calcium flux and spreading response in Lyn-deficient mouse platelets. Analysis of platelet adhesion dynamics under physiological blood flow conditions revealed an important role for SHIP1 in regulating platelet adhesion on fibrinogen. Specifically, SHIP1-dependent PtdIns(3,4,5)P(3) metabolism down-regulates the stability of integrin alpha(IIb)beta(3)-fibrinogen adhesive bonds, leading to a decrease in the proportion of platelets forming shear-resistant adhesion contacts. These studies define a major role for SHIP1 and Lyn as negative regulators of integrin alpha(IIb)beta(3) adhesive and signaling function.     

Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation of FcepsilonRI and their association with detergent-resistant membranes.

Tyrosine phosphorylation of the high affinity immunoglobulin (Ig)E receptor (FcepsilonRI) by the Src family kinase Lyn is the first known biochemical step that occurs during activation of mast cells and basophils after cross-linking of FcepsilonRI by antigen. The hypothesis that specialized regions in the plasma membrane, enriched in sphingolipids and cholesterol, facilitate the coupling of Lyn and FcepsilonRI was tested by investigating functional and structural effects of cholesterol depletion on Lyn/FcepsilonRI interactions. We find that cholesterol depletion with methyl-beta-cyclodextrin substantially reduces stimulated tyrosine phosphorylation of FcepsilonRI and other proteins while enhancing more downstream events that lead to stimulated exocytosis. In parallel to its inhibition of tyrosine phosphorylation, cholesterol depletion disrupts the interactions of aggregated FcepsilonRI and Lyn on intact cells and also disrupts those interactions with detergent-resistant membranes that are isolated by sucrose gradient ultracentrifugation of lysed cells. Importantly, cholesterol repletion restores receptor phosphorylation together with the structural interactions. These results provide strong evidence that membrane structure, maintained by cholesterol, plays a critical role in the initiation of FcepsilonRI signaling.     

Regulation of insulin signaling and glucose transporter 4 (GLUT4) exocytosis by phosphatidylinositol 3,4,5-trisphosphate (PIP3) phosphatase, skeletal muscle, and kidney enriched inositol polyphosphate phosphatase (SKIP)

The glucose transporter 4 (GLUT4) is responsible for glucose uptake in the skeletal muscle. Insulin-induced translocation of GLUT4 to the plasma membrane requires phosphatidylinositol 3-kinase activation-mediated generation of phosphatidylinositol 3,4,5-trisphosphate PIP(3) and subsequent activation of Akt. Previous studies suggested that skeletal muscle and kidney enriched inositol polyphosphate phosphatase (SKIP) has negative effects on the regulation of insulin signaling in the skeletal muscle cells. Here, we compared its effects on insulin signaling by selective inhibition of SKIP, SHIP2, and phosphatase and tensin homologue on chromosome 10 (PTEN) by short interfering RNA in the C2C12 myoblast cells. Suppression of SKIP significantly increased the insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate levels and Akt phosphorylation. Furthermore, silencing of SKIP, but not of PTEN, increased the insulin-dependent recruitment of GLUT4 vesicles to the plasma membrane. Taken together, these results imply that SKIP negatively regulates insulin signaling and glucose uptake by inhibiting GLUT4 docking and/or fusion to the plasma membrane.     

The docking properties of SHIP2 influence both JIP1 tyrosine phosphorylation and JNK activity

SHIP2 (SH2-containing inositol polyphosphate 5-phosphatase 2) is an ubiquitously expressed phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase which contains various motifs susceptible to mediate protein-protein interaction. In cell models, evidence has been provided that SHIP2 plays a role in insulin and growth factor signaling, cytoskeletal organization, cell adhesion and migration. Herein we describe the c-Jun NH2-terminal kinase (JNK)-interacting protein 1 (JIP1) as a new protein partner of SHIP2. The interaction between SHIP2 and JIP1 was confirmed in both overexpression systems and native cells. Without modifying the association of JIP1 with the MAPKs in the scaffold complex and with no apparent change of Akt phosphorylation, SHIP2 positively modulated the MLK3/JIP1-mediated JNK1 activation. Moreover, SHIP2 positively regulated the tyrosine phosphorylation of JIP1. This up-regulation was prevented by inhibitors of the Src family and Abl kinases, PP2 and Glivec. The effects of SHIP2 on JNK activity and JIP1 tyrosine phosphorylation were independent of the SHIP2 phosphoinositide 5-phosphatase activity, as similar results were obtained when using a SHIP2 catalytic inactive mutant instead of wild-type SHIP2. Together, these data suggest that by its docking properties, SHIP2 can modulate JIP1-mediated JNK pathway signaling.     

Phosphatidylinositol (3,4,5)P3 is essential but not sufficient for protein kinase B (PKB) activation; phosphatidylinositol (3,4)P2 is required for PKB phosphorylation at Ser-473: studies using cells from SH2-containing inositol-5-phosphatase knockout mice.

Using bone marrow derived mast cells from SH2-containing inositol-5-phosphatase (SHIP) +/+ and minus sign/minus sign mice, we found that the loss of SHIP leads to a dramatic increase in Steel Factor (SF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)), a substantial reduction in PI(3,4)P(2), and no change in PI(4,5)P(2) levels. We also found that SF-induced activation of protein kinase B (PKB) is increased and prolonged in SHIP -/- cells, due in large part to more PKB associating with the plasma membrane in these cells. Pretreatment of SHIP -/- cells with 25 microm LY294002 resulted in complete inhibition of SF-induced PI(3,4)P(2), while still yielding PI(3,4,5)P(3) levels similar to those achieved in SHIP+/+ cells. This offered a unique opportunity to study the regulation of PKB by PI(3,4,5)P(3), in the absence of PI(3,4)P(2). Under these conditions, PKB activity was markedly reduced compared with that in SF-stimulated SHIP+/+ cells, even though more PKB localized to the plasma membrane. Although phosphoinositide-dependent kinase 1 mediated phosphorylation of PKB at Thr-308 was unaffected by LY294002, phosphorylation at Ser-473 was dramatically reduced. Moreover, intracellular delivery of PI(3,4)P(2) to LY294002-pretreated, SF-stimulated SHIP -/- cells increased phosphorylation of PKB at Ser-473 and increased PKB activity. These results are consistent with a model in which SHIP serves as a regulator of both activity and subcellular localization of PKB.     

Cutting edge: genetic variation influences Fc epsilonRI-induced mast cell activation and allergic responses

Mast cell responses are influenced by a diverse array of environmental factors, but little is known about the effect of genetic background. In this study, we report that 129/Sv mice had high levels of circulating IgE, increased expression of the high-affinity receptor for IgE (Fc epsilonRI), and greater sensitivity to anaphylaxis when compared with C57BL/6 mice. Bone marrow-derived mast cells (BMMCs) from 129/Sv mice showed more robust degranulation upon the engagement of Fc epsilonRI. Deficiency of the Src family kinase Lyn enhanced degranulation in 129/Sv BMMCs but inhibited this response in C57BL/6 cells. C57BL/6 lyn(-/-) BMMCs had reduced expression of the Src family kinase Fyn, and increasing its expression markedly enhanced degranulation. In human mast cells the silencing of Lyn or Fyn expression resulted in hyperdegranulation or hypodegranulation, respectively. The findings demonstrate a genetic influence on the extent of a mast cell's response and identify Fyn kinase as a contributory determinant.     

Positive and negative regulation of mast cell activation by Lyn via the FcepsilonRI

Aggregation of the high affinity receptor for IgE (FcepsilonRI) induces activation of mast cells. In this study we show that upon low intensity stimulation of FcepsilonRI with monomeric IgE, IgE plus anti-IgE, or IgE plus low Ag, Lyn (a Src family kinase) positively regulates degranulation, cytokine production, and survival, whereas Lyn works as a negative regulator of high intensity stimulation with IgE plus high Ag. Low intensity stimulation suppressed Lyn kinase activity and its association with FcepsilonRI beta subunit, whereas high intensity stimulation enhanced Lyn activity and its association with FcepsilonRI beta. The latter induced much higher levels of FcepsilonRI beta phosphorylation and Syk activity than the former. Downstream positive signaling molecules, such as Akt and p38, were positively and negatively regulated by Lyn upon low and high intensity stimulations, respectively. In contrast, the negative regulators, SHIP and Src homology 2 domain-containing protein tyrosine phosphatase-1, interacted with FcepsilonRI beta, and their phosphorylation was controlled by Lyn. Therefore, we conclude that Lyn-mediated positive vs negative regulation depends on the intensity of the stimuli. Studies of mutant FcepsilonRI beta showed that FcepsilonRI beta subunit-ITAM (ITAM motif) regulates degranulation and cytokine production positively and negatively depending on the intensity of FcepsilonRI stimulation. Furthermore, Lyn-mediated negative regulation was shown to be exerted via the FcepsilonRI beta-ITAM.