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The Journal of Immunology, 2008, 180, 3689-3698
Copyright © 2008 by The American Association of Immunologists, Inc.
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Non-T Cell Activation Linker Promotes Mast Cell Survival by Dampening the Recruitment of SHIP1 by Linker for Activation of T Cells1

Karine Roget*,{dagger}, Marie Malissen{ddagger},§,¶, Odile Malbec*,{dagger}, Bernard Malissen{ddagger},§,¶ and Marc Daëron2,*,{dagger}

* Institut Pasteur, Département d’Immunologie, Unité d’Allergologie Moléculaire et Cellulaire, Paris, France; {dagger} Institut National de la Santé et de la Recherche Médicale (Inserm), Unité 760, Paris, France; {ddagger} Centre d’Immunologie de Marseille-Luminy, Université de la Méditerrannée, Marseille, France; § Inserm, Unité 631, Marseille, France; and Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6102, Marseille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The linker for activation of T cells (LAT) and the non-T cell activation linker (NTAL) are two transmembrane adapters which organize IgE receptor (Fc{epsilon}RI) signaling complexes in mast cells. LAT positively regulates, whereas NTAL negatively regulates mast cell activation. We previously found that the four distal tyrosines of LAT can generate negative signals. We show here that two of these tyrosines provide two binding sites for SHIP1, that LAT recruits SHIP1 in vivo, and that SHIP1 recruitment is enhanced in NTAL-deficient cells. We show that NTAL negatively regulates mast cell activation by decreasing the recruitment, by LAT, of molecules involved in Fc{epsilon}RI-dependent positive signaling. We show that NTAL also decreases the recruitment of SHIP1 by LAT, leading to an increased phosphorylation of the antiapoptotic molecule Akt, and positively regulates mast cell survival. We finally show that the positive effect of NTAL on Akt phosphorylation and mast cell survival requires LAT. Our data thus document the mechanisms by which LAT and NTAL can generate both positive and negative signals which differentially regulate mast cell activation and survival. They also provide molecular bases for the recruitment of SHIP1 in Fc{epsilon}RI signaling complexes. SHIP1 is a major negative regulator of mast cell activation and, hence, of allergic reactions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Allergies arise as consequences of the development of a local inflammatory response, initiated by the combined effects of cytokines, chemokines, lipid-derived mediators, proteolytic enzymes, and vasoactive mediators that are secreted and/or released by activated tissue mast cells. Mast cell activation is triggered by the aggregation of high-affinity IgE receptors (Fc{epsilon}RI) induced by the binding of a specific multivalent Ag to Fc{epsilon}RI-bound IgE Abs. Fc{epsilon}RI aggregation generates intracellular signaling complexes which build up around the intracytoplasmic domains of lipid raft-associated tyrosine-rich transmembrane adapters which function as scaffold proteins (1, 2). These include the linker for activation of T cells (LAT)3 (3) and the non-T cell activation linker (NTAL, also called LAB or LAT2) (4) which are both expressed in mast cells. Studies in mast cells derived from LAT–/– and from NTAL–/– mice demonstrated that LAT exerts a positive effect (5), whereas NTAL exerts a negative effect (6, 7, 8) on Fc{epsilon}RI signaling leading to cell activation. By contrast with the mechanisms responsible for the positive effects of LAT (9, 10), the mechanisms responsible for the negative effects of NTAL are poorly understood (6). Interestingly, both LAT and NTAL were shown to generate positive and negative signals. NTAL functions as a homolog of LAT in B cell lines where it positively regulates BCR signaling (11, 12). NTAL also positively regulates kit signaling in human mast cells (13). We found in a previous work conducted in mast cells from knockin mice, in which individual LAT tyrosines were mutated into phenylalanines (14, 15), that the four distal tyrosines of LAT could generate not only positive, but also negative, signals in mast cells. Indeed, whereas IgE-induced responses of bone marrow-derived mast cells (BMMC) from Y136F or of Y(175,195,235)F mice were markedly reduced, the responses of BMMC from Y(136,175,195,235)F mice were almost as intense as those of wild-type (WT) BMMC (16). Molecular mechanisms responsible for LAT-dependent negative signaling were not elucidated.

SHIP1 (17) is a major negative regulator of Fc{epsilon}RI signaling. Mast cells from SHIP1-deficient mice indeed respond more vigorously than WT mast cells to Fc{epsilon}RI engagement by low concentrations of IgE and/or Ag (18). Besides, inhibition of IgE-induced responses observed in excess of Ag is abrogated in SHIP1–/– mice (19). Also, SHIP1 was demonstrated to account for the negative properties of Fc{gamma}RIIB, an inhibitory IgG receptor previously shown to inhibit IgE-induced mast cell activation, when coaggregated with Fc{epsilon}RI by immune complexes (20, 21). SHIP1 negatively regulates Fc{epsilon}RI signaling by two mechanisms. Through its enzymatic activity, SHIP1 removes 5-phosphate groups in the inositol ring of 3-phosphorylated inositides and phosphatidylinositides (17). Its substrates include phosphatidylinositol (3, 4, 5)-trisphosphate (PI(3, 4, 5)P3) which mediates the membrane recruitment of molecules which contain a pleckstrin homology domain. SHIP1 can therefore prevent the PI(3, 4, 5)P3-dependent recruitment of critical signaling molecules (22, 23). SHIP1 can also negatively regulate the Ras pathway independently of its catalytic activity, by recruiting Dok-1 and RasGAP, when tyrosyl-phosphorylated upon membrane translocation (24, 25). As a consequence, SHIP1 is a potent inhibitor of mast cell activation when brought within signaling complexes.

The molecular bases of the recruitment of SHIP1 by Fc{gamma}RIIB is well-documented. SHIP1 contains one Src homology 2 (SH2) domain which was shown to bind in vitro to the phosphorylated ITIM of Fc{gamma}RIIB (26) and to mediate the in vivo recruitment of the phosphatase in signaling complexes underneath Fc{gamma}RIIB-Fc{epsilon}RI coaggregates. SHIP1 is constitutively associated, via C-terminal proline-rich sequences, to the C-terminal SH3 domain of cytosolic adapters, such as Grb2 and Grap, which were demonstrated to critically stabilize the recruitment of the phosphatase by murine Fc{gamma}RIIB (27). How SHIP1 is recruited by Fc{epsilon}RI is unclear. Although the SH2 domain of SHIP1 was reported to bind in vitro to a tyrosine residue located between the two YxxL in the ITAM of the FcRβ subunit (28), only minute amounts of SHIP1 coprecipitated with FcRβ in IgE-stimulated mast cells (29).

In this study, we show that the C-terminal end of LAT contains two tyrosine-based binding sites for the SH2 domain of SHIP1 and that, when tyrosyl-phosphorylated upon Fc{epsilon}RI engagement, LAT recruits SHIP1. Like that of other SH2 domain-containing molecules, the recruitment of SHIP1 by LAT was markedly enhanced in mast cells from NTAL–/– mice. As a consequence, not only LAT-dependent positive signals, but also LAT-dependent, SHIP1-mediated, negative signals were enhanced in these cells, resulting in a decreased phosphorylation of Akt and a reduced survival of IL-3-deprived mast cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

LAT–/– and NTAL–/– mice (6) were backcrossed for one and seven generations, respectively, on the C57BL/6 background. LAT–/–NTAL–/– mice were obtained by crossing LAT–/– mice with NTAL–/– mice. WT littermate mice were used as controls.

Antibodies

The mouse IgE anti-DNP mAb 2682-I (30) was used as culture supernatant. Mouse anti-LAT and mouse anti-phospholipase C (PLC)-{gamma}1 Abs were obtained from Upstate Biotechnology; mouse anti-Grb2 Abs was obtained from BD Transduction Laboratories (BD Biosciences); rabbit anti-phospho-p65 (Ser536), rabbit anti-p65, rabbit anti-phospho-Akt (Ser473), and rabbit anti-Akt were obtained from Cell Signaling; rabbit anti-LAT, mouse anti-SHIP1, rabbit anti-phospho-PLC-{gamma}1 (Tyr783), HRP-conjugated goat anti-rabbit, and goat anti-mouse Ig Abs were obtained from Santa Cruz Biotechnology. The anti-phosphotyrosine mAb 4G10 and the anti-GST mAb ND2.1 were a gift of Dr. S. Latour (Institut National de la Santé et de la Recherche Médicale (Inserm) Unité 429, Hôpital Necker, Paris, France) and Dr. J. L. Teillaud (Inserm Unité 255, Centre de Recherches Biomédicales des Cordeliers, Paris, France).

Mast cells

BMMC were generated from bone marrow cells as described (31). Culture reagents were obtained from Invitrogen Life Technologies.

Cell stimulation

BMMC, sensitized for 1 h at 37°C with 1 µg/ml IgE anti-DNP, were washed and challenged at 37°C for the indicated times with 0.1–1 µg/ml DNP-BSA or treated with 100 mM pervanadate.

β-hexosaminidase release

IgE anti-DNP-sensitized mast cells were challenged for 10 min at 37°C with the indicated concentrations of DNP-BSA. β-hexosaminidase released in supernatants was measured as described (16).

Calcium mobilization

Intracellular-free Ca2+ concentration was determined using 5 mM fluo-3 AM (Molecular Probes/Invitrogen Life Technologies) and 0.2% pluronic F-127 (Sigma-Aldrich). Cells were stimulated with 1 µg/ml DNP-BSA, and the intracellular Ca2+ concentration ([Ca2+]i) was assessed using a FACSCalibur and the FCS Assistant 1.2.9 β software (BD Biosciences).

Cell survival

Cells were washed and cultured at 3 x 105 cells/ml in IL-3-free medium. At indicated days, cells were stained with annexin V-allophycocyanin (BD Biosciences). To determine viable cell counts, aliquots of stained cells were resuspended in a known volume, transferred into BD Trucount tubes (BD Biosciences) containing a known number of fluorescent beads, and analyzed by flow cytometry. A constant number of beads was acquired. Cell fluorescence was analyzed using a FACSCalibur (BD Biosciences).

Cell proliferation

Cells (1 x 106/ml) loaded with 1 µM CFSE (Invitrogen Life Technologies) were cultured with 4% supernatant from X63-IL-3 cells. Cell division was determined by monitoring cell fluorescence using a FACSCalibur (BD Biosciences).

Immunoprecipitation

Five x 107 cells were lysed in lysis buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 2 mM Na3VO4, 10 mM NaF, 10 mM sodium pyrophosphate, 0.4 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM PMSF (pH 7.4). Postnuclear lysates were immunoprecipitated with protein G-Sepharose beads (Amersham Biosciences) coupled to mouse anti-LAT Ab for 2 h at 4°C.

GST fusion proteins

GST-LAT. The cDNA encoding the intracellular domain of LAT was amplified by PCR using cDNA generated from WT BMMC RNA. Amplified cDNA was inserted in pGEX-6P-1 (Amersham Biosciences) and transfected into DH5-{alpha} Escherichia coli.

GST-SH2. GST-SHIP1 SH2 and GST-Grb2 SH2 fusion proteins were described previously (27). Fusion proteins were produced in DH5-{alpha} following isopropyl-1-thio-β-D-galactopyranoside induction.

In vitro phosphorylation

GST- and GST-LAT-coated beads were washed in kinase buffer containing 100 mM Tris, 125 mM MgCl2, 25 mM MnCl2, 2 mM EGTA, 2 mM DTT, and 0.25 mM Na3VO4 (pH 7.4). Beads were incubated in kinase buffer containing 100 µM ATP and 3 µl of Syk kinase (Biaffin) for 30 min at 30°C and washed in kinase buffer. One-third was subjected to SDS-PAGE and Western blotted with anti-phosphotyrosine Abs. Two-thirds were incubated with lysates from 1 x 107 cells for 1 h at 4°C. Eluates from beads were subjected to SDS-PAGE and Western blotted as described below.

Peptides and in vitro-binding assays

Phosphorylated or not phosphorylated biotinylated peptides corresponding to tyrosine motifs of LAT were purchased from NeoMPS. Their sequences were: Y67: RTPAVSYPLVTSF, Y136: DDYPNGYLVVLPD, Y175: VESCEDYVNVPES, Y195: LDGSREYVNVSPE, Y235: GEEAPDYENLQEL. Phosphorylated or not phosphorylated biotinylated peptides corresponding to the Fc{gamma}RIIB ITIM were purchased from Sigma-Genosys. Their sequence was: AENTITYSLLKHP. Peptides were coupled to streptavidin-coated agarose beads (Pierce). Peptide-coated beads were incubated overnight with lysates from 5 x 107 cells or with soluble GST-SHIP1 SH2 and GST-Grb2 SH2 fusion proteins. GST- and GST-LAT-coated beads were incubated for 1 h in lysate from 1 x 107 cells. Eluates from beads were Western blotted as described below.

Western blot analysis

Beads or cell lysates were boiled in sample buffer. Eluates were fractionated by SDS-PAGE, and transferred onto Immobilon-P membranes (Millipore). Membranes were saturated with either 5% BSA (Roche) or 5% skimmed milk (Régilait), diluted in Western buffer containing 150 mM NaCl, 10 mM Tris, 0.5% Tween 20 (Merck) (pH 7.4), and incubated with the indicated Abs followed by HRP-goat anti-rabbit or HRP-goat anti-mouse Abs. Labeled Abs were detected using an ECL kit (Amersham Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
LAT contains two binding sites for the SH2 domain of SHIP1

To investigate the possible interactions of SHIP1 with the four distal tyrosines of LAT which were previously observed to generate negative signals in mast cells from LAT knockin mice (16), agarose beads were coated with nonphosphorylated (Y) or phosphorylated (pY) peptides corresponding to 13-aa sequences of LAT straddling Y67, Y136, Y175, Y195, or Y235 (Fig. 1A). Y67 was included because it stands within a potential SHIP-binding consensus sequence (26). When peptide-coated beads were incubated with a BMMC lysate, SHIP1 was found to bind to pY235 and, in smaller amounts, with pY195 and pY136 peptides. As expected, PLC-{gamma}1 bound to pY136 and, in smaller amounts, to pY195 peptides, whereas Grb2 bound to pY195 and, in smaller amounts, to pY235 peptides. In some experiments, minute amounts of Grb2 also bound to the pY175 peptide (data not shown). None of the molecules examined detectably bound to pY67 or to nonphosphorylated peptides (Fig. 1B).


Figure 1
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  		FIGURE 1. SHIP1 binds to phosphorylated LAT tyrosines 136 and 235. A, Schematic structure of the cytoplasmic domain of murine LAT. The nine tyrosines are numbered, based on their position in the amino acid sequence. Peptides used for binding assays are shown. EC, Extracellular domain; TM, transmembrane domain; and IC, intracytoplasmic domain. B, In vitro binding of PLC-{gamma}1, SHIP1, and Grb2 to LAT peptides. Beads coated with phosphorylated or not-phosphorylated LAT peptides were incubated with BMMC lysate. Eluted material was fractionated by SDS-PAGE and Western blotted with anti-PLC-{gamma}1, anti-SHIP1, and anti-Grb2 Abs. C, In vitro binding of SHIP1 and Grb2 SH2 domains to LAT peptides. Beads coated with phosphorylated or not phosphorylated LAT peptides were incubated with GST-SHIP1 SH2 or GST-Grb2 SH2 fusion proteins. As controls, agarose beads coated with phosphorylated or not phosphorylated Fc{gamma}RIIB ITIM peptides were incubated with GST-SHIP1 SH2. Eluted material was fractionated by SDS-PAGE and Western blotted with anti-GST Abs. D, In vitro binding of SHIP1 and Grb2 to beads coated with a mixture of LAT Y195 and LAT Y235 peptides. Phosphorylated or not phosphorylated LAT Y195 and LAT Y235 peptides were mixed in variable proportions and mixtures containing equal amounts of peptides were used to coat agarose beads which were incubated with BMMC lysate. Eluted material was fractionated by SDS-PAGE and Western blotted with anti-SHIP1 and anti-Grb2 Abs.

 
To investigate whether the interactions of SHIP1 with pY peptides were direct or indirect, GST fusion proteins containing the SH2 domain of SHIP1 were incubated with beads coated with each of the three peptides which bound SHIP1 in cell lysates or, as a positive control, with a peptide corresponding to the Fc{gamma}RIIB ITIM (26). The SH2 domain of SHIP1 bound to pY136 and to pY235, but not to pY195 peptides. Under the same conditions, the SH2 domain of Grb2 primarily bound to pY195. Smaller amounts of Grb2-SH2 bound to pY235 and even smaller amounts to pY175 peptides (Fig. 1C).

SHIP1 was previously shown to associate constitutively, via its C-terminal proline-rich region, with the C-terminal SH3 domain of Grb2 (27). To investigate whether, due to this association, the two molecules could cooperatively bind to LAT tyrosines, Y195 and pY235, pY195 and Y235, or pY195 and pY235 peptides were mixed in variable proportions, and a constant amount of the three mixtures was used to coat beads. These were used to precipitate SHIP1 and Grb2 from a BMMC lysate (Fig. 1D). SHIP1 dose-dependently bound to beads coated with the pY235 peptide, but not to beads coated with Y195 (left panel), Y235, or pY195 (middle panel) peptides. Higher amounts of SHIP1 bound to beads coated with a mixture of pY235 and pY195 peptides (right panel). Grb2 also bound to pY235 (left panel) and even more to pY195 (middle panel), but not to Y195 (left panel) or to Y235 (middle panel) peptides. Comparable amounts of Grb2 bound to the pY195 peptide, whether mixed with Y235 (middle panel) or with pY235 (right panel) peptides.

The above results altogether demonstrate that SHIP1 can bind to phosphorylated Y136 and Y235, in the intracytoplasmic domain of LAT and that binding can occur via the SH2 domain of SHIP1. They also indicate that the binding of SHIP1 to phosphorylated LAT tyrosines can be enhanced when Grb2 binds simultaneously via its SH2 domain to adjacent phosphorylated tyrosines. As PLC-{gamma}1 and Grb2 also bind to pY136 and pY235, respectively, they can possibly compete with SHIP1 for the two SHIP1-binding sites in intact LAT.

LAT recruits SHIP1 upon Fc{epsilon}RI engagement and SHIP1 recruitment by LAT is enhanced by the deletion of NTAL

To investigate whether SHIP1 could bind to phosphorylated LAT, a GST fusion protein containing the intracytoplasmic domain of LAT (GST-LAT) (i.e., containing all potential tyrosyl-phosphorylation sites) was constructed, phosphorylated or not in vitro with the protein tyrosine kinase Syk which was previously shown to phosphorylate LAT in vivo (32), and incubated with a BMMC lysate. A GST protein, treated similarly, was used as a negative control. As expected, PLC-{gamma}1 and Grb2, but also SHIP1, bound to phosphorylated GST-LAT (Fig. 2A). None of the three molecules bound to nonphosphorylated GST-LAT.


Figure 2
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  		FIGURE 2. Tyrosyl-phosphorylated LAT recruits SHIP1. A, Pull-down assay of PLC-{gamma}1, SHIP1, and Grb2 with in vitro-phosphorylated GST-LAT fusion proteins. GST or GST-LAT bound onto glutathione-agarose beads were subjected to an in vitro kinase assay using the Syk kinase and incubated with BMMC lysate. Eluted material was fractionated by SDS-PAGE and Western blotted with anti-phosphotyrosine (pY), anti-PLC-{gamma}1, anti-SHIP1, and anti-Grb2 Abs. B, Coprecipitation of PLC-{gamma}1, SHIP1, and Grb2 with LAT. WT (left panel) and NTAL–/– (right panel) BMMC, previously sensitized with mouse mAb IgE anti-DNP, were untreated, challenged with DNP-BSA, or treated with pervanadate. Cells were lysed, and LAT was immunoprecipitated. Immunoprecipitates were fractionated by SDS-PAGE and Western blotted with anti-phosphotyrosine (pY), anti-LAT, anti-PLC-{gamma}1, anti-SHIP1, and anti-Grb2 Abs.

 
To investigate whether the same three molecules could be recruited by LAT, when phosphorylated upon Fc{epsilon}RI engagement, their coprecipitation with LAT was searched for in BMMC sensitized with IgE anti-DNP and challenged with DNP-BSA. The same cells that were either not challenged or treated with pervanadate were used as negative and positive controls, respectively. As expected, LAT was mildly phosphorylated following Ag stimulation, and much more heavily following pervanadate treatment. PLC-{gamma}1 and, to a lower extent, SHIP1 and Grb2 coprecipitated with LAT in Ag-stimulated cells but, surprisingly, not in pervanadate-treated cells (Fig. 2B).

The deletion of NTAL was previously reported to enhance Fc{epsilon}RI-dependent LAT phosphorylation and, as a consequence, the recruitment of SH2 domain-containing molecules (6, 7). BMMC from NTAL–/– mice were therefore compared with BMMC from WT mice in the same experiment. As expected, LAT phosphorylation was enhanced, and higher amounts of PLC-{gamma}1 and Grb2 coprecipitated with phosphorylated LAT in Ag-stimulated NTAL-deficient cells. The Ag-induced coprecipitation of SHIP1 with LAT was also markedly enhanced in the same cells (Fig. 2B). Noticeably, the low molecular mass isoform of SHIP1 predominantly coprecipitated with LAT.

These results altogether indicate that SHIP1 cannot only bind in vitro to phosphorylated LAT, but also be recruited in vivo by LAT, when phosphorylated upon Fc{epsilon}RI engagement. Even though competition may occur between SHIP1, PLC-{gamma}1, and Grb2, for phosphorylated LAT-binding sites, it did not prevent the three molecules from binding in vitro to a phosphorylated fusion protein containing the whole intracytoplasmic domain of LAT and from being recruited in vivo by phosphorylated LAT. Like that of PLC-{gamma}1 and Grb2, the recruitment of SHIP1 by LAT was markedly enhanced in NTAL-deficient mast cells.

NTAL deletion enhances not only LAT-dependent positive signals which control cell activation, but also LAT-dependent negative signals which control cell survival

NTAL was described as an inhibitory molecule for Fc{epsilon}RI signaling (6). NTAL-dependent inhibitory signals, however, remain unknown. As previously reported, Ag-induced PLC-{gamma}1 phosphorylation, which was markedly reduced in LAT–/– BMMC, was enhanced in NTAL–/– BMMC (Fig. 3A). When phosphorylated and activated, PLC-{gamma}1 cleaves PI(4, 5)P2 into inositol Tris-phosphate and diacylglycerol. Inositol Tris-phosphate initiates the Ca2+ response while diacylglycerol initiates a protein kinase C-mediated pathway leading to NF-{kappa}B phosphorylation and activation (33, 34). Both the Ag-induced Ca2+ response (Fig. 3C) and NF-{kappa}B phosphorylation (Fig. 3B), which were reduced in LAT–/– BMMC, were enhanced in NTAL–/– BMMC. As a consequence and, as previously observed, Ag-induced release of β-hexosaminidase was decreased in LAT–/– BMMC, but enhanced in NTAL–/– BMMC (Fig. 3D). The enhancing effect of NTAL deletion on IgE-dependent mast cell biological responses may therefore primarily result from an enhancement of LAT-dependent positive signals.


Figure 3
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  		FIGURE 3. LAT positively regulates, whereas NTAL negatively regulates, signals leading to cell activation. A, PLC{gamma}-1 activation. WT, NTAL–/–, and LAT–/– BMMC, previously sensitized with mouse mAb IgE anti-DNP, were untreated or challenged with DNP-BSA for the indicated time and lysed. Equal amounts of proteins were fractionated by SDS-PAGE and Western blotted with anti-pPLC-{gamma}1 and anti-PLC-{gamma}1 Abs. B, NF-{kappa}B activation. WT, NTAL–/–, and LAT–/– BMMC, previously sensitized with mouse mAb IgE anti-DNP, were untreated or challenged with DNP-BSA for the indicated time and lysed. Equal amounts of proteins were fractionated by SDS-PAGE and Western blotted with anti-pP65-NF-{kappa}B and anti-P65-NF-{kappa}B. C, Ca2+ mobilization. WT, NTAL–/–, and LAT–/– BMMC, previously sensitized with mouse mAb IgE anti-DNP, were loaded with Fluo-3 and stimulated with DNP-BSA at the indicated time (arrow), and intracellular Ca2+ concentration was measured. D, β-hexosaminidase release. WT, NTAL–/–, and LAT–/– BMMC, previously sensitized with mouse mAb IgE anti-DNP, were challenged with the indicated concentrations of DNP-BSA. β-hexosaminidase was measured in supernatant by an enzymatic assay.

 
If, however, LAT can recruit SHIP1 and if the deletion of NTAL enhances the recruitment of SHIP1 by LAT, we anticipated that it should decrease PI(3, 4, 5)P3-dependent signals. The concomitant recruitment of Akt and of phosphoinositide-dependent protein kinase1 (PDK1) by PI(3, 4, 5)P3 enables PDK1 to phosphorylate Akt (35). Indeed, Ag-induced Akt phosphorylation was decreased in NTAL–/– BMMC (Fig. 4A) but increased in LAT–/– BMMC (Fig. 4B). To investigate the respective roles of LAT and NTAL on the regulation of Akt phosphorylation, we compared LAT–/–NTAL–/– double-deficient BMMC with LAT–/– and with NTAL–/– single-deficient BMMC. The deletion of LAT in NTAL-deficient cells increased Ag-induced Akt phosphorylation (Fig. 4C). The deletion of NTAL in LAT-deficient cells, however, had no detectable effect on Ag-induced Akt phosphorylation (Fig. 4D). It follows that LAT can negatively regulate Akt phosphorylation in the absence of NTAL, whereas NTAL requires LAT for enhancing Akt phosphorylation.


Figure 4
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  		FIGURE 4. NTAL positively regulates, whereas LAT negatively regulates, Akt activation. BMMC, previously sensitized with mouse mAb IgE anti-DNP, were untreated or challenged with DNP-BSA for the indicated time and lysed. Equal amounts of proteins were fractionated by SDS-PAGE and Western blotted with anti-pAkt and anti-Akt. A, Effect of NTAL deletion in the presence of LAT. B, Effect of LAT deletion in the presence of NTAL. C, Effect of LAT deletion in the absence of NTAL. D, Effect of NTAL deletion in the absence of LAT.

 
When phosphorylated, Akt acts as an antiapoptotic molecule (36, 37). We therefore investigated the survival rates of BMMC when deprived from growth factors. We found that the viability of LAT–/– BMMC decreased similarly with time as that of WT BMMC when cultured without IL-3 (Fig. 5A). The viability of NTAL–/– BMMC, however, decreased more rapidly than that of WT BMMC, when cultured under the same conditions (Fig. 5B). Noticeably, the viability of LAT–/–NTAL–/– double-deficient BMMC was similar as that of WT BMMC (Fig. 5C). NTAL therefore enhances the survival of IL-3-deprived mast cells, and this effect requires the presence of LAT. When cultured in IL-3-containing medium, the growth (Fig. 6, A and C) and proliferation (Fig. 6, B and D) of LAT–/– or NTAL–/– BMMC were similar as those of WT BMMC.


Figure 5
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  		FIGURE 5. NTAL positively regulates mast cell survival. A, Effect of LAT deletion on cell survival. B, Effect of NTAL deletion on cell survival. C, Effect of LAT and NTAL deletion on cell survival. BMMC were cultured for various periods of time in the absence of IL-3, stained with annexin V-allophycocyanin, and mixed with fluorescent beads. Daily FACS analyses were performed to evaluate the percentages of cells remaining in culture and the percentages of viable and apoptotic cells among remaining cells. Left panels, Dot plots at days 0 and 3. Gate 3 shows fluorescent beads used for cell counts. Middle panels, Percentage of annexin V-negative cells remaining in culture as a function of time (number of cells in gate 1: number of cells at the initiation of culture). Right panels, Percentage of annexin V-positive cells among cells remaining in culture (number of cells in gate 1: number of cells in gate 1 + gate 2).

 

Figure 6
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  		FIGURE 6. LAT and NTAL do not affect IL-3-dependent mast cell growth and proliferation. A and C, Mast cell growth. BMMC were generated by culturing bone marrow cells (1 femur equivalent) from WT, LAT–/– (A), and NTAL–/– (C) mice in IL-3-containing medium. The figure represents the number of cells that could have been obtained if all cells had been kept in culture for the indicated times. Two independent sets of cultures (1 and 2) are shown. B and D, Mast cell proliferation. WT, LAT–/– (B), and NTAL–/– (D) BMMC were loaded with CFSE at day 0 and cultured in the presence of IL-3. The relative decrease of CFSE staining was monitored by flow cytometry. Curves shown in the right panels represent the mean fluorescence intensity of histograms shown in the left panels.

 
These results altogether indicate that the deletion of NTAL amplifies not only positive but also negative LAT-dependent, SHIP1-mediated signals. LAT-dependent positive signals affect primarily mast cell activation whereas LAT-dependent negative signals affect primarily mast cell survival in the absence of IL-3. LAT-dependent negative signals are counterbalanced by the positive effects of NTAL in WT BMMC, and NTAL-dependent positive signals require LAT to increase cell survival.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We show here that LAT contains two binding sites for the SH2 domain of SHIP1 and that, when tyrosyl-phosphorylated upon Fc{epsilon}RI engagement, it recruits SHIP1. LAT may thus bring SHIP1 into Fc{epsilon}RI signaling complexes. This phosphatase plays a prominent role in the negative regulation of IgE-induced mast cell activation. Our findings provide a molecular basis for the negative signaling properties of the four distal tyrosines of LAT that we previously unraveled in mast cells from LAT knockin mice. We also show that NTAL negatively regulates mast cell activation, but positively regulates mast cell survival, and that both effects depend on LAT. NTAL negatively regulates mast cell activation by decreasing the recruitment of molecules involved in the generation of positive signals by LAT. It positively regulates mast cell survival by decreasing the recruitment of SHIP1 by LAT, leading to an increased activation of the antiapoptotic molecule Akt.

Compelling evidence that SHIP1 negatively regulates Fc{epsilon}RI signaling was obtained when studying SHIP1–/– mice, in which IgE-induced anaphylactic reactions were more severe, and the mast cells of which degranulated more vigorously and secreted more cytokines than mast cells from WT mice, when sensitized with IgE Abs and challenged with Ag (38). Consistent with the previously recognized ability of SHIP1 to hydrolyze PI(3, 4, 5)P3 when brought into signaling complexes (22) and to recruit Dok-1/rasGAP when phosphorylated upon Fc{epsilon}RI aggregation (25), IgE-induced Ca2+ responses and MAPK activation were enhanced in these cells. On the basis of these data, SHIP1 was described as "the gatekeeper of mast cell degranulation" (18). How SHIP1 is recruited into Fc{epsilon}RI signaling complexes is, however, unclear.

Several proteins were described to associate with SHIP1. The adapters Dok-1 and Shc bind, via their phospho tyrosine-binding domain (39), while the adapter CrkL (40) and the protein tyrosine phosphatase SHP-2 bind, via their SH2 domains (41), to tyrosyl-phosphorylated SHIP1. These molecular interactions are unlikely to contribute to the recruitment of SHIP1 into signaling complexes. SHIP1 indeed becomes phosphorylated after it has been recruited and brought close to Lyn (42). Grb2 constitutively associates with C-terminal proline-rich sequences of SHIP1 via its C-terminal SH3 domain (27). As a consequence, Grb2 may bring SHIP1 into signaling complexes, when this adapter is recruited by LAT or by another tyrosyl-phosphorylated molecule. SHIP1 was indeed recently described as forming a multimolecular complex with Dok-1, Dok-2, and Grb2 that associated with LAT upon TCR engagement in human T cells (43). SHIP1 was also reported to interact with phosphorylated FcRβ when examined by a yeast triple hybrid assay (44) and to bind in vitro to phosphopeptides corresponding to the FcR{gamma} and FcRβ ITAMs via its SH2 domain (45). The FcRβ ITAM contains a third tyrosine residue, in the NVYSPI sequence that separates the YEEL and YSEL sequences of the canonical ITAM motif. Pull-down experiments showed that both Lyn and SHIP1 could bind in vitro to this tyrosine, and mutational analysis showed that it is involved in the negative regulation of ERK, p38, and NF-{kappa}B activation and of the secretion of IL-6, IL-13, and TNF-{alpha} induced by IgE (28). SHIP1 was less phosphorylated following Fc{epsilon}RI engagement in cells bearing a mutation of this tyrosine. On the basis of these data, FcRβ was proposed to bring molecules with negative properties, including SHIP1, in signaling complexes, and a faint coprecipitation of SHIP1with FcRβ was reported in one article (29). We show here that LAT can recruit SHIP1 in mast cells.

When tyrosyl-phosphorylated upon receptor engagement, LAT provides multiple docking sites for SH2 domain-containing molecules which contribute to the Fc{epsilon}RI signaling complex. These include enzymes such as PLC-{gamma}, protein tyrosine kinases of the Tec family, and the p85 subunit of PI3K, exchange factors of the Vav family and cytosolic adapters, among which are Gads, Grap, and Grb2 (10, 32, 46). Works based on mutational analysis identified the four distal tyrosines of LAT (Y136, Y175, Y195, and Y235) as being critical for the recruitment of these molecules in T cells (10, 47) and mast cells (48). We identified two binding sites for the SH2 domain of SHIP1 among these four tyrosines. These are within sequences containing Y136 and Y235. As described previously (10), the four distal tyrosines of LAT also provided two binding sites for PLC-{gamma}1 and two binding sites for Grb2 and, not surprisingly, binding required that these sites be tyrosyl-phosphorylated. Noticeably however, each molecule bound preferentially to one of these two binding sites: SHIP1 bound to pY235 more than to pY136, PLC-{gamma}1 bound to pY136 more than to pY195, and Grb2 bound to pY195 more than to pY235. GST fusion proteins containing the SH2 domains of SHIP1 and Grb2 bound to the same phosphorylated peptides as SHIP1 and Grb2 contained in cell lysates, and with the same hierarchy.

The three binding sites having an affinity for the SH2 domains of more than one molecule (PLC-{gamma}1 and SHIP1 bind to pY136, Grb2 and PLC-{gamma}1 bind to pY195, SHIP1 and Grb2 bind to pY235), SHIP1 shares its two binding sites with molecules which are likely to compete with the phosphatase for being recruited by LAT (Fig. 7A). Cooperative interactions, however, occur, which facilitate the in vivo recruitment of signaling molecules by LAT. Thus, when interacting with pY(175,195,235)-bound Gads, SLP-76 stabilizes the binding of PLC-{gamma}1 to pY136 (10, 47, 49). We found that, when binding to pY195, Grb2 enhanced the binding of SHIP1 to pY235. This finding is reminiscent of our previous demonstration that the recruitment of SHIP1 by Fc{gamma}RIIB in B cells requires the cooperative binding of SHIP1 to the phosphorylated ITIM and of Grb2 to another phosphorylated tyrosine-based motif, in the C-terminal end of mouse Fc{gamma}RIIB (27). This cooperation, which can be explained by the affinity of the C-terminal SH3 domain of Grb2 for proline-rich sequences of SHIP1, is likely to compensate for competition. Indeed, all three molecules could be pulled down by a GST fusion protein containing the whole intracytoplasmic domain of LAT that was previously in vitro phosphorylated by Syk. More importantly, SHIP1, Grb2, and PLC-{gamma}1 coprecipitated with LAT in BMMC, following Fc{epsilon}RI engagement by IgE and Ag. LAT was weakly phosphorylated, and small but detectable amounts of all three molecules coprecipitated with LAT under these conditions. Surprisingly, SHIP1, Grb2, and PLC-{gamma}1 failed to detectably coprecipitate with LAT in cells treated with pervanadate, although LAT was massively phosphorylated. This suggests that, although necessary, LAT phosphorylation may not be sufficient for SH2 domain-containing molecules to be recruited. Receptors and signaling molecules which reside in distinct subcellular compartments need to be brought close to each other for being able to interact. We showed previously that, when aggregated, Fc{epsilon}RI becomes associated with the F-actin skeleton where a fraction of SHIP1 is constitutively associated with the actin-binding molecule Filamin-1 (50). Other conditions, which determine the intensity of LAT phosphorylation, markedly affected the recruitment of SH2 domain-containing molecules. Among these is the presence/absence of NTAL.


Figure 7
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  		FIGURE 7. The recruitment of SHIP1 by LAT and its consequences on signaling pathways in WT, NTAL–/–, and LAT–/– mast cells. A, Binding sites for SHIP1, PLC-{gamma}, and Grb2 in the distal end of LAT. Main (large characters) and secondary (small characters) binding sites are shown. B, Effects of the recruitment of SHIP1 by LAT on signaling pathways in mast cells. The Lyn/Syk/LAT and the Fyn/Gab2/PI3K pathways are triggered upon Fc{epsilon}RI engagement and lead to mast cell activation and survival. The deletion of NTAL enhances LAT-dependent positive and negative signals (in bold). Due to increased LAT phosphorylation, downstream positive signals (PLC-{gamma}1 and NF-{kappa}B activation, Ca2+ mobilization) and mast cell activation are increased (in bold). Due to increased PI(3,4,5)P3 degradation by the increased amount of SHIP1 that is recruited by hyperphosphorylated LAT (in bold), Akt phosphorylation and mast cell survival are decreased (in gray). The deletion of LAT abrogates the dampening effects of NTAL on LAT-dependent positive and negative signals. Due to the absence of recruitment of SHIP1 by LAT, PI(3,4,5)P3-dependent positive signals are enhanced in LAT-deficient cells. These partially compensate the lack of LAT-dependent positive signals leading to cell activation (in gray) and, as a consequence, activation is reduced, but not abolished. Akt phosphorylation is increased (in bold). It, however, does not fully compensate decreased survival signals (e.g., NF-{kappa}B-dependent signals) and, as a consequence, cell survival of LAT-deficient cells is similar as that of WT cells.

 
The deletion of NTAL indeed enhanced IgE-induced LAT phosphorylation. Although this effect has been known for some time (6), its mechanisms remains unknown. Competition between the two adapters for the kinase Syk is one possibility. If, as suggested, LAT and NTAL reside in distinct lipid microdomains, these should merge following receptor engagement (6). The dephosphorylation of LAT by SH2 domain-containing tyrosine phosphatases, possibly recruited by NTAL, is another possibility. However, neither SHP-1 nor SHP-2 was reported to coprecipitate with NTAL. Whatever the mechanism by which NTAL affects LAT phosphorylation, the recruitment of SH2 domain-containing molecules by LAT is enhanced by the deletion of NTAL (6, 7). IgE-induced activation of LAT-dependent intracellular pathways and, as a consequence, secretory responses were indeed markedly enhanced in BMMC from NTAL-deficient mice. PLC-{gamma} phosphorylation and Ca2+ responses, which were decreased in LAT–/– cells, were increased in NTAL–/– cells, as well as β-hexosaminidase release. These effects may account for the negative regulation of Fc{epsilon}RI signaling that was assigned to NTAL in mast cells: rather (or more) than having a negative effect per se, NTAL can simply dampen LAT-dependent positive signals. Supporting this conclusion, the enhancing effect of NTAL deletion on IgE-induced responses was apparently not seen in LAT-deficient BMMC (6).

Noticeably, the inducible coprecipitation of SHIP1 with LAT observed upon Fc{epsilon}RI engagement was also markedly enhanced in BMMC from NTAL–/– mice, suggesting that LAT-dependent negative signals might also be increased in the absence of NTAL. If the recruitment of SHIP1 by LAT may, at least in part, account for these negative signals, one anticipates that PI(3, 4, 5)P3-dependent signaling events be enhanced in LAT-deficient cells. Akt phosphorylation, which requires that Akt and PDK1 be corecruited to the membrane by PI(3, 4, 5)P3 via their pleckstrin homology domain, was indeed increased in LAT–/– cells and decreased in NTAL–/– cells. Akt phosphorylation therefore appears as the first signaling event described as being negatively regulated by LAT. Noticeably, the deletion of NTAL did not affect Akt phosphorylation in the absence of LAT, indicating that the positive effect of NTAL on Akt phosphorylation requires LAT. By contrast, the deletion of LAT still increased Akt phosphorylation in the absence of NTAL, indicating that the negative effects of LAT on Akt phosphorylation do not require NTAL. Although NTAL deletion enhanced the recruitment of SHIP1 by LAT, SHIP1 phosphorylation was not detectably enhanced in whole lysates from NTAL–/– mast cells (data not shown). One possible explanation is that LAT recruits only a small proportion of the intracellular pool of SHIP1. Alternatively, SHIP1 may not be phosphorylated when recruited by LAT. The tyrosine-rich region is located in the C-terminal end which is spliced out in the low molecular mass isoforms of SHIP1. By contrast with Fc{gamma}RIIB, which we previously found to predominantly recruit the high molecular mass isoform of SHIP1 (50), LAT seems to predominantly recruit the low molecular mass isoform of SHIP1. If so, SHIP1 could hydrolyze PI(3, 4, 5)P3 and decrease Akt phosphorylation when recruited by LAT, but not inhibit ERK phosphorylation. SHIP1 indeed needs to be phosphorylated to recruit Dok-1/RasGAP. Supporting this possibility, the phosphorylation of ERK was marginally reduced in LAT–/– BMMC, and not detectably enhanced in NTAL–/– BMMC (data not shown).

If it also contributes to mast cell activation and cytokine production (51), Akt plays a major role in mast cell survival (52). Because we found that Akt phosphorylation was up-regulated by NTAL and down-regulated by LAT, we examined the survival of mast cells when they were deprived of growth factor. We observed that the numbers of live NTAL–/– BMMC decreased more rapidly than the numbers of live WT BMMC, when cultured in the absence of IL-3. At every time point, the percentage of annexin V-positive cells was higher in NTAL–/– cells than in WT cells. The same was observed when cells were sensitized with IgE Abs or not and when IgE-sensitized cells were challenged with Ag or not (data not shown). This suggests that LAT-dependent, SHIP1-mediated negative regulation of mast cell survival operates constitutively, in the absence of Fc{epsilon}RI engagement. Consistent with this hypothesis, minute amounts of SHIP1 could be seen in LAT precipitates from nonstimulated cells. Surprisingly, however, the deletion of LAT did not affect the survival of IL-3-deprived BMMC. Although cell survival depends on antiapoptotic factors other than Akt, this observation suggests that, like NTAL-dependent negative regulation of mast cell activation, NTAL-dependent positive regulation of mast cell survival requires the presence of LAT. Supporting this interpretation, we found that the defect in survival observed in NTAL-deficient cells was restored in LAT–/– NTAL–/– double deficient cells. Finally, the growth rates of LAT–/–, NTAL–/–, and WT BMMC in IL-3-containing medium were similar and IL-3-induced proliferation of LAT–/–, NTAL–/–, and WT BMMC were identical as assessed by CFSE staining. This suggests that LAT-dependent, SHIP1-mediated negative signals are not involved in SHIP1-dependent negative regulation of IL-3-induced mast cell proliferation observed in SHIP1–/– BMMC (38).

Taken together, our results support the conclusion that NTAL negatively regulates cell activation but positively regulates cell survival and that both effects of NTAL depend on LAT (Fig. 7B). The deletion of NTAL enhances both LAT-dependent positive and negative signals. As a consequence, mast cell secretory responses are enhanced because LAT-dependent positive signals that affect cell activation are dominant over LAT-dependent negative signals. NTAL deletion, however, also enhances LAT-dependent negative signals which are mediated by SHIP1 and affect primarily cell survival. Finally, our results provide a molecular mechanism to the LAT-dependent negative effects previously observed in mast cells from LAT knockin mice (16). These can indeed be explained by the presence of two SHIP1-binding sites in LAT (Fig. 7A). Mast cell activation results from the integration of LAT-dependent positive and negative (SHIP1-mediated) signals and of LAT-independent positive signals (mast cell responses are indeed reduced, but not abrogated in LAT–/– mast cells). LAT-independent positive signals result in part from the PI(3, 4, 5)P3-dependent recruitment of molecules with a pleckstrin homology domain such as PLC-{gamma}1 and Btk (Fig. 7B). By removing the main PLC-{gamma}-binding site and the secondary SHIP1-binding site, the LAT Y136F mutation abrogated most of PLC-{gamma}-dependent positive signals and a part of SHIP1-dependent negative signals. It left intact LAT-independent positive signals, Grb2-dependent positive signals and most SHIP1-dependent negative signals. Remaining SHIP1-dependent negative signals could not affect LAT-dependent positive signals because these were virtually abrogated, but they could affect LAT-independent positive signals. By removing all Grb2-binding sites and the main SHIP1-binding site, the LAT Y(175,195,235)F mutations abrogated Grb2-dependent positive signals and most SHIP1-dependent negative signals. They left intact LAT-independent positive signals, PLC-{gamma}-dependent positive signals, and a part of the SHIP1-dependent negative signals. The remaining SHIP1-dependent negative signals could affect PLC-{gamma}-dependent and LAT-independent positive signals. By removing all PLC-{gamma}-, Grb2-, and SHIP1-binding sites, the LAT Y(136,175,195,235)F mutations abrogated all LAT-dependent positive and negative signals. They left intact LAT-independent positive signals which were not any more negatively regulated by SHIP1. As a consequence, the secretory responses of LAT Y(136,175,195,235)F BMMC were more intense than those of LAT Y136F BMMC and than those of LAT Y(175,195,235)F BMMC (16).


   Acknowledgments
 
We are grateful to Drs P. Dubreuil (Institut de Cancérologie et d’Immunologie, Marseille, France) for IL-3-secreting cells; S. Latour (Inserm Unité 429, Hôpital Necker, Paris, France) for 4.G10 Abs; J.-L. Teillaud (Inserm Unité 255, Centre de Recherches Biomédicales des Cordeliers, Paris, France) for ND2.1 Abs; and S. A. Susin and C. Artus (Institut Pasteur, Paris, France) for discussion. LAT- and NTAL-deficient mice were generated at the Plate-forme Réseau Inter Organisme-Marseille Nice Génopole (RIO-MNG).


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Institut Pasteur, Institut National de la Santé et de la Recherche Médicale and the Fondation pour la Recherche Médicale (Program Défis de la Recherche en Allergologie). K.R. was financially supported by the Ministère de l’Education Nationale, de la Recherche et de la Technologie and by a Pasteur-Weizmann fellowship. Back

2 Address correspondence and reprint requests to Dr. Marc Daëron, Unité d’Allergologie Moléculaire et Cellulaire, Département d’Immunologie, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, France. E-mail address: daeron{at}pasteur.fr Back

3 Abbreviations used in this paper: LAT, linker for activation of T cells; NTAL, non-T cell activation linker; BMMC, bone marrow-derived mast cell; WT, wild type; PI(3,4,5)P3, phosphatidylinositol (3, 4, 5)-trisphosphate; SH2, Src homology 2; PLC, phospholipase C; PDK, phosphoinositide-dependent protein kinase. Back

Received for publication August 15, 2007. Accepted for publication January 5, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Leo, A., J. Wienands, G. Baier, V. Horejsi, B. Schraven. 2002. Adapters in lymphocyte signaling. J. Clin. Invest. 109: 301-309. [Medline]
  2. Samelson, L. E.. 2002. Signal transduction mediated by the T cell antigen receptor: the role of adapter proteins. Annu. Rev. Immunol. 20: 371-394. [Medline]
  3. Finco, T. S., T. Kadlecek, W. Zhang, L. E. Samelson, A. Weiss. 1998. LAT is required for TCR-mediated activation of PLC{gamma}1 and the Ras pathway. Immunity 9: 617-626. [Medline]
  4. Brdicka, T., M. Imrich, P. Angelisova, N. Brdickova, O. Horvath, J. Spicka, I. Hilgert, P. Luskova, P. Draber, P. Novak, et al 2002. Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling. J. Exp. Med. 196: 1617-1626. [Abstract/Free Full Text]
  5. Saitoh, S., R. Arudchandran, T. S. Manetz, W. Zhang, C. L. Sommers, P. E. Love, J. Rivera, L. E. Samelson. 2000. LAT is essential for Fc{epsilon}RI-mediated mast cell activation. Immunity 12: 525-535. [Medline]
  6. Volna, P., P. Lebduska, L. Draberova, S. Simova, P. Heneberg, M. Boubelik, V. Bugajev, B. Malissen, B. S. Wilson, V. Horejsi, et al 2004. Negative regulation of mast cell signaling and function by the adptor LAB/NTAL. J. Exp. Med. 200: 1001-1013. [Abstract/Free Full Text]
  7. Zhu, M., Y. Liu, S. Koonpaew, O. Granillo, W. Zhang. 2004. Positive and negative regulation of Fc{epsilon}RI-mediated signaling by the adaptor protein LAB/NTAL. J. Exp. Med. 200: 991-1000. [Abstract/Free Full Text]
  8. Rivera, J.. 2005. NTAL/LAB and LAT: a balancing act in mast-cell activation and function. Trends Immunol. 26: 119-122. [Medline]
  9. Zhang, W., B. J. Irvin, R. P. Trible, R. T. Abraham, L. E. Samelson. 1999. Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient cell line. Int. Immunol. 11: 943-950. [Abstract/Free Full Text]
  10. Zhang, W., R. P. Trible, M. Zhu, S. K. Liu, C. J. McGlade, L. E. Samelson. 2000. Association of Grb2: Gads and phopsholipase C-{gamma}1 with phosphorylated LAT tyrosine residues: effect of LAT tyrosine mutations on T cell antigen receptor signaling. J. Biol. Chem. 275: 23355-23361. [Abstract/Free Full Text]
  11. Stork, B., M. Engelke, J. Frey, V. Horejsi, A. Hamm-Baarke, B. Schraven, T. Kurosaki, J. Wienands. 2004. Grb2 and the non-T cell activation linker NTAL constitute a Ca2+-regulating signal circuit in B lymphocytes. Immunity 21: 681-691. [Medline]
  12. Janssen, E., M. Zhu, B. Craven, W. Zhang. 2004. Linker for activation of B cells: a functional equivalent of a mutant linker for activation of T cells deficient in phospholipase C-{gamma}1 binding. J. Immunol. 172: 6810-6819. [Abstract/Free Full Text]
  13. Tkaczyk, C., V. Horejsi, S. Iwaki, P. Draber, L. E. Samelson, A. B. Satterthwaite, D. H. Nahm, D. D. Metcalfe, A. M. Gilfillan. 2004. NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and Fc{epsilon}RI aggregation. Blood 104: 207-214. [Abstract/Free Full Text]
  14. Aguado, E., S. Richelme, S. Nunez-Cruz, A. Miazek, A. M. Mura, M. Richelme, X. J. Guo, D. Sainty, H. T. He, B. Malissen, M. Malissen. 2002. Induction of T helper type 2 immunity by a point mutation in the LAT adaptor. Science 296: 2036-2040. [Abstract/Free Full Text]
  15. Nuez-Cruz, S., E. Aguado, S. Richelme, B. Chetaille, A.-M. Mura, M. Richelme, L. Pouyet, E. Jouvin-Marche, L. Xerri, B. Malissen, M. Malissen. 2003. LAT regulates {gamma}{delta} T cell homeostasis and differentiation. Nat. Immunol. 4: 999-1008. [Medline]
  16. Malbec, O., M. Malissen, I. Isnardi, R. Lesourne, A.-M. Mura, W. H. Fridman, B. Malissen, M. Daëron. 2004. Linker for activation of T cells integrates positive and negative signaling in mast cells. J. Immunol. 173: 5086-5094. [Abstract/Free Full Text]
  17. Damen, J. E., L. Liu, P. Rosten, R. K. Humphries, A. B. Jefferson, P. W. Majerus, G. Krystal. 1996. The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase. Proc. Natl. Acad. Sci. USA 93: 1689-1693. [Abstract/Free Full Text]
  18. Huber, M., C. D. Helgason, J. E. Damen, L. Liu, R. K. Humphries, G. Krystal. 1998. The src homology 2-containing inositol phosphatase (SHIP) is the gatekeeper of mast cell degranulation. Proc. Natl. Acad. Sci. USA 95: 11330-11335. [Abstract/Free Full Text]
  19. Gimborn, K., E. Lessmann, S. Kuppig, G. Krystal, M. Huber. 2005. SHIP down-regulates Fc{epsilon}R1-induced degranulation at supraoptimal IgE or antigen levels. J. Immunol. 174: 507-516. [Abstract/Free Full Text]
  20. Daëron, M., O. Malbec, S. Latour, M. Arock, W. H. Fridman. 1995. Regulation of high-affinity IgE receptor-mediated mast cell activation by murine low-affinity IgG receptors. J. Clin. Invest. 95: 577-585. [Medline]
  21. Fong, D. C., O. Malbec, M. Arock, J. C. Cambier, W. H. Fridman, M. Daëron. 1996. Selective in vivo recruitment of the phosphatidylinositol phosphatase SHIP by phosphorylated Fc{gamma}RIIB during negative regulation of IgE-dependent mouse mast cell activation. Immunol. Lett. 54: 83-91. [Medline]
  22. Scharenberg, A. M., O. El-Hillal, D. A. Fruman, L. O. Beitz, Z. Li, S. Lin, I. Gout, L. C. Cantley, D. J. Rawlings, J.-P. Kinet. 1998. Phosphatidylinositol-3,4,5-triphosphate (PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals. EMBO J. 17: 1961-1972. [Medline]
  23. Scharenberg, A. M., J. P. Kinet. 1998. PtdIns-3,4,5-P3: a regulatory nexus between tyrosine kinases and sustained calcium signals. Cell 94: 5-8. [Medline]
  24. Tamir, I., J. C. Stolpa, C. D. Helgason, K. Nakamura, P. Bruhns, M. Daëron, J. C. Cambier. 2000. The RasGAP-binding protein p62dok is a mediator of inhibitory Fc{gamma}RIIB signals in B cells. Immunity 12: 347-358. [Medline]
  25. Ott, V. L., I. Tamir, M. Niki, P. P. Pandolfi, J. C. Cambier. 2002. Downstream of kinase, p62dok, is a mediator of Fc{gamma}IIB inhibition of Fc{epsilon}RI signaling. J. Immunol. 168: 4430-4439. [Abstract/Free Full Text]
  26. Bruhns, P., F. Vély, O. Malbec, W. H. Fridman, E. Vivier, M. Daëron. 2000. Molecular basis of the recruitment of the SH2 domain-containing inositol 5-phosphatases SHIP1 and SHIP2 by Fc{gamma}RIIB. J. Biol. Chem. 275: 37357-37364. [Abstract/Free Full Text]
  27. Isnardi, I., R. Lesourne, P. Bruhns, W. H. Fridman, J. C. Cambier, M. Daëron. 2004. Two distinct tyrosine-based motifs enable the inhibitory receptor Fc{gamma}RIIB to cooperatively recruit the inositol phosphatases SHIP1/2 and the adapters Grb2/Grap. J. Biol. Chem. 279: 51931-51938. [Abstract/Free Full Text]
  28. Furumoto, Y., S. Nunomura, T. Terada, J. Rivera, C. Ra. 2004. The Fc{epsilon}RIβ immunoreceptor tyrosine-based activation motif exerts inhibitory control on MAPK and I{kappa}B kinase phosphorylation and mast cell cytokine production. J. Biol. Chem. 279: 49177-49187. [Abstract/Free Full Text]
  29. Xiao, W., H. Nishimoto, H. Hong, J. Kitaura, S. Nunomura, M. Maeda-Yamamoto, Y. Kawakami, C. A. Lowell, C. Ra, T. Kawakami. 2005. Positive and negative regulation of mast cell activation by Lyn via the Fc{epsilon}RI. J. Immunol. 175: 6885-6892. [Abstract/Free Full Text]
  30. Liu, F. T., J. W. Bohn, E. L. Ferry, H. Yamamoto, C. A. Molinaro. 1980. Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation and characterization. J. Immunol. 124: 2728-2737. [Medline]
  31. Malbec, O., W. H. Fridman, M. Daëron. 1999. Negative regulation of c-kit-mediated cell proliferation by Fc{gamma}RIIB. J. Immunol. 162: 4424-4429. [Abstract/Free Full Text]
  32. Zhang, W., J. Sloan-Lancaster, J. Kitchen, R. P. Trible, L. E. Samelson. 1998. LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92: 83-92. [Medline]
  33. Shirakawa, F., S. B. Mizel. 1989. In vitro activation and nuclear translocation of NF-{kappa}B catalyzed by cyclic AMP-dependent protein kinase and protein kinase C. Mol. Cell. Biol. 9: 2424-2430. [Abstract/Free Full Text]
  34. Kim, M. J., E. Kim, S. H. Ryu, P. G. Suh. 2000. The mechanism of phospholipase C-{gamma}1 regulation. Exp. Mol. Med. 32: 101-109. [Medline]
  35. Alessi, D. R., S. R. James, C. P. Downes, A. B. Holmes, P. R. Gaffney, C. B. Reese, P. Cohen. 1997. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B{alpha}. Curr. Biol. 7: 261-269. [Medline]
  36. Datta, S. R., H. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh, M. E. Greenberg. 1997. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91: 231-241. [Medline]
  37. Liu, Q., T. Sasaki, I. Kozieradzki, A. Wakeham, A. Itie, D. J. Dumont, J. M. Penninger. 1999. SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation and myeloid cell survival. Genes Dev. 13: 786-791. [Abstract/Free Full Text]
  38. Helgason, C. D., J. E. Damen, P. Rosten, R. Grewal, P. Sorensen, S. M. Chappel, A. Borowski, F. Jirik, G. Krystal, R. K. Humphries. 1998. Targeted disruption of SHIP leads to hemopoietic perturbations, lung pathology, and shortened life span. Genes Dev. 12: 1610-1620. [Abstract/Free Full Text]
  39. Lioubin, M. N., P. A. Algate, S. Tsai, K. Carlberg, R. Aebersold, L. R. Rohrschneider. 1996. p150 SHIP, a signal transduction molecule with inositol polyphosphate-5-phosphatase activity. Genes Dev. 10: 1084-1095. [Abstract/Free Full Text]
  40. Sattler, M., S. Verma, Y. B. Pride, R. Salgia, L. R. Rohrschneider, J. D. Griffin. 2001. SHIP1, an SH2 domain containing polyinositol-5-phosphatase, regulates migration through two critical tyrosine residues and forms a novel signaling complex with DOK1 and CRKL. J. Biol. Chem. 276: 2451-2458. [Abstract/Free Full Text]
  41. Liu, L., J. E. Damen, M. D. Ware, G. Krystal. 1997. Interleukin-3 induces the association of the inositol 5-phopshatase SHIP with SHP2. J. Biol. Chem. 272: 10998-11001. [Abstract/Free Full Text]
  42. Bolland, S., R. N. Pearse, T. Kurosaki, J. V. Ravetch. 1998. SHIP modulates immune receptor responses by regulating membrane association of Btk. Immunity 8: 509-516. [Medline]
  43. Dong, S., B. Corre, E. Foulon, E. Dufour, A. Veillette, O. Acuto, F. Michel. 2006. T cell receptor for antigen induces linker for activation of T cell-dependent activation of a negative signaling complex involving Dok-2, SHIP-1, and Grb-2. J. Exp. Med. 203: 2509-2518. [Abstract/Free Full Text]
  44. Osborne, M. A., G. Zenner, M. Lubinus, X. Zhang, Z. Songyang, L. C. Cantley, P. Majerus, P. Burn, J. P. Kochan. 1996. The inositol 5'-phosphatase SHIP binds to immunoreceptor signaling motifs and responds to high affinity IgE receptor aggregation. J. Biol. Chem. 271: 29271-29278. [Abstract/Free Full Text]
  45. Kimura, T., H. Sakamoto, E. Appella, R. P. Siraganian. 1997. The negative signaling molecule SH2 domain-containing inositol-polyphosphate 5-phosphatase (SHIP) binds to the tyrosine-phosphorylated β subunit of the high affinity IgE receptor. J. Biol. Chem. 272: 13991-13996. [Abstract/Free Full Text]
  46. Weber, J. R., S. Orstavik, K. M. Torgersen, N. C. Danbolt, S. F. Berg, J. C. Ryan, K. Tasken, J. B. Imboden, J. T. Vaage. 1998. Molecular cloning of the cDNA encoding pp36, a tyrosine-phosphorylated adaptor protein selectively expressed by T cells and natural killer cells. J. Exp. Med. 187: 1157-1161. [Abstract/Free Full Text]
  47. Zhu, M., E. Janssen, W. Zhang. 2003. Minimal requirements of tyrosine residues of linker for activation of T cells in TCR signaling and thymocyte development. J. Immunol. 170: 325-333. [Abstract/Free Full Text]
  48. Saitoh, S.-I., S. Odom, G. Gomez, C. L. Sommers, H. A. Young, J. Rivera, L. E. Samelson. 2003. The four distal tyrosines are required for LAT-dependent signaling in FceRI-mediated mast cell activation. J. Exp. Med. 198: 831-843. [Abstract/Free Full Text]
  49. Yablonski, D., T. Kadlecek, A. Weiss. 2001. Identification of a phospholipase C-{gamma}1 (PLC-{gamma}1) SH3 domain-binding site in SLP-76 required for T-cell receptor-mediated activation of PLC-{gamma}1 and NFAT. Mol. Cell. Biol. 21: 4208-4218. [Abstract/Free Full Text]
  50. Lesourne, R., W. H. Fridman, M. Daëron. 2005. Dynamic interactions of Fc{gamma}RIIB with filamn-bound SHIP1 amplify filamentous actin-dependent negative regulation of Fc{epsilon}RI signaling. J. Immunol. 174: 1365-1373. [Abstract/Free Full Text]
  51. Kitaura, J., K. Asai, M. Maeda-Yamamoto, Y. Kawakami, U. Kikkawa, T. Kawakami. 2000. Akt-dependent cytokine production in mast cells. J. Exp. Med. 192: 729-740. [Abstract/Free Full Text]
  52. Alfredsson, J., H. Puthalakath, H. Martin, A. Strasser, G. Nilsson. 2005. Proapoptotic Bcl-2 family member Bim is involved in the control of mast cell survival and is induced together with Bcl-xL upon IgE-receptor activation. Cell Death Differ. 12: 136-144. [Medline]




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