The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xie, Z.-H.
Right arrow Articles by Siraganian, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xie, Z.-H.
Right arrow Articles by Siraganian, R. P.
The Journal of Immunology, 2002, 168: 4682-4691.
Copyright © 2002 by The American Association of Immunologists

The Adapter Molecule Gab2 Regulates Fc{epsilon}RI-Mediated Signal Transduction in Mast Cells

Zhi-Hui Xie*, Indu Ambudkar{dagger} and Reuben P. Siraganian1,*

* Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, and {dagger} Secretory Physiology Section, Gene Therapy and Therapeutics Branch, Department of Health and Human Services, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The recently cloned scaffolding molecule Gab2 can assemble multiple molecules involved in signaling pathways. Bone marrow-derived mast cells isolated from Gab2-/- mice have defective signaling probably due to the lack of the activation of phosphatidylinositol-3 kinase (PI3-kinase). In this study, we investigated the role of Gab2 using the rat basophilic leukemia 2H3 cell line mast cells. Fc{epsilon}RI aggregation induced the tyrosine phosphorylation of Gab2 and translocation of a significant fraction of it from the cytosol to the plasma membrane. As in other cells, Gab2 was found to associate with several signaling molecules including Src homology 2-containing protein tyrosine phosphatase 2, Grb2, Lyn, and phospholipase C{gamma} (PLC{gamma}). The association of Gab2 with Lyn and PLC{gamma} were enhanced after receptor aggregation. Overexpression of Gab2 in rat basophilic leukemia 2H3 cell line cells inhibited the Fc{epsilon}RI-induced tyrosine phosphorylation of the subunits of the receptor, and the phosphorylation and/or activation of Syk and mitogen-activated protein kinase. Downstream events such as calcium mobilization, degranulation, and induction of TNF-{alpha} and IL-6 gene transcripts were decreased in Gab2 overexpressing cells, although Akt phosphorylation as a measure of PI3-kinase activation was unaffected. These results suggest that in addition to the positive effects mediated by PI3-kinase that are apparent in Gab2-/- mast cells, Gab2 by interacting with Lyn and PLC{gamma} may have negative regulatory effects on Fc{epsilon}RI-induced mast cell signaling and functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Binding of ligands to cell surface receptors triggers critical biological processes including enzyme activation, tyrosine phosphorylation, recruitment of signaling molecules, and their translocation to different compartments of the cell. Beside enzymes, such as kinases and phosphatases, other adapter and/or docking molecules play a critical role in signal transduction and cell responses (1, 2). A common feature in intracellular signaling pathways is the assembly of multiprotein complexes involving extensive protein-protein interactions. Adapter molecules may provide docking sites for kinases and phosphatases to target their substrates, or to facilitate or block enzymatic reactions.

Gab2 is a newly identified 97-kDa molecule that is a member of the Dos/Gab adapter family which also includes Gab1, the insulin receptor substrates, and Drosophilia daughter of sevenless Dos (3, 4, 5). Gab2 contains an N-terminal Pleckstrin homology domain followed by a long region with multiple tyrosine-containing motifs and two proline-rich domains. The tyrosine-containing motifs, once phosphorylated, provide binding sites for Src homology (SH)2 2 domain-containing signaling molecules including p85 subunit of phosphatidylinositol-3 kinase (PI3-kinase), phospholipase C{gamma} (PLC{gamma}), and SH2-containing protein tyrosine phosphate 2 (SHP-2). The proline-rich regions of Gab2 are potential binding sites for SH3-domain-containing proteins such as Src family protein tyrosine kinases (4).

Gab2 has been shown to function in various signaling pathways activated by the binding of cytokine, Ag, and growth factor receptors (5, 6, 7). The importance of Gab2 has been demonstrated by its ability to assemble multiple proteins, which then couple receptors to downstream pathways. The IL-3R activates Akt via a Shc/Grb2/Gab2/PI3-kinase pathway to result in cell proliferation and survival (8). Gab2 is also involved in the regulation of Grb2/Ras/extracellular regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway (3, 7). Recent studies with bone marrow-derived mast cells (BMMC) from Gab2-deficient mice demonstrates an essential role for Gab2 in the regulation of mast cell functions through its coupling to PI3-kinase (9). However, because Gab2 is a scaffolding molecule that can couple to different signaling molecules including not only PI3-kinase, but also SHP-2, PLC{gamma}, Grb2, Shc, Src, and SH2-containing inositol phosphatase (3, 4, 10, 11), it could have complex roles in multiple signaling pathways.

Aggregation of the Fc{epsilon}RI on mast cells initiates a biochemical cascade that ultimately results in the release of inflammatory mediators and generation of cytokines (12, 13, 14, 15). Because Fc{epsilon}RI itself has no intrinsic tyrosine enzymatic activity, nonreceptor protein tyrosine kinases such as Lyn and Syk are essential in this signaling pathway (16, 17, 18, 19, 20, 21, 22, 23). Several adapter molecules such as LAT and Vav are also critical for Fc{epsilon}RI-mediated mast cell signal transduction (24, 25). The purpose of the present experiments was to study the role of another adaptor molecule, Gab2, in Fc{epsilon}RI signaling, using the rat basophilic leukemia 2H3 mast cell line (RBL-2H3). Fc{epsilon}RI aggregation induced the tyrosine phosphorylation of Gab2 and translocation of a significant fraction of Gab2 from the cytosol to the plasma membrane. Overexpression of Gab2 in RBL-2H3 cells inhibited the Fc{epsilon}RI-induced signal transduction. These results suggest that Gab2, in addition to the positive effects mediated by PI3-kinase that are apparent in the BMMC from Gab-/- mice, can regulate Fc{epsilon}RI-induced mast cell signaling by interacting with Lyn and PLC{gamma} negatively.


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

Aprotinin, Triton X-100, and protein A-conjugated agarose were obtained from Sigma Aldrich (St. Louis, MO). Polyvinylidene difluoride transfer membrane was purchased from Millipore (Bedford, MA) and the ECL reagent was from NEN (Boston, MA). The materials for electrophoresis were purchased from NOVEX (San Diego, CA). The mouse Gab2 cDNA in pEBB vector was kindly provided by Dr. B. G. Neel (Beth Israel Hospital, Boston, MA).

Antibodies

Rabbit polyclonal anti-Gab2 and anti-c-Jun N-terminal kinase (JNK) Abs, HRP-conjugated anti-phospho-Tyr (pTyr) mAb, 4G10, and mixed monoclonal anti-PLC{gamma}1 Abs were obtained from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal anti-phospho-ERK (phospho-Thr (pThr)202 and pTyr204), anti-phospho-p38 (pThr180 and pTry182), anti-phospho-Akt (phospho-Ser473), anti-Akt, anti-ERK, and anti-p38 Abs were purchased from New England Biolabs (Beverly, MA). Goat polyclonal anti-Gab2, rabbit polyclonal anti-PLC{gamma}2, and monoclonal anti-phospho-JNK (pThr183 and pTyr185) Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Stable transfection

A total of 20 µg of linearized Gab2 cDNA in pEBB vector or empty vector together with 2 µg of pSV2-neo plasmid were cotransfected into 5 x 106 RBL-2H3 cells by electroporation (960 µF, 310 V) as described previously (26). Clones were selected with 350 µg/ml of active G418 (Life Technologies, Rockville, MD). Cell lines were screened for the level of Gab2 expression by immunoblotting of total cell lysates with anti-Gab2 Ab, using blotting with anti-Fc{epsilon}RI{beta} Ab as an internal control. Cell lines were selected for additional experiments that expressed high levels of the Gab2 molecule.

Cell culture and activation

RBL-2H3 cells and transfectants were cultured as monolayers in Eagle’s MEM supplemented with 15% heat-inactivated FBS, penicillin, streptomycin, amphotericin, and glutamine (27). For activation, cells were cultured overnight with or without anti-trinitrophenyl-specific IgE. For histamine release assays, the cell monolayers were washed twice with MEM containing 0.1% BSA and 10 mM Tris (pH 7.4). The cells incubated with IgE were stimulated with Ag (DNP coupled to human serum albumin, 35:1 molar ratio), or with calcium ionophore A23187 in the same medium. After incubation for 45 min at 37°C, the medium was removed for histamine analysis (28). For RNA protection assays, cells were stimulated in culture medium for 1 or 2 h, and total RNA was isolated using the RNeasy kit (Qiagen, Santa Clarita, CA). In the Akt experiments, cells were cultured in MEM medium without FBS for 24 h before stimulation with Ag.

Immunoprecipitation and immunoblotting

After stimulation, the cell monolayers were rinsed once with ice-cold PBS containing Na3VO4 (1 mM) and protease inhibitors (2 mM aminoethyl-benzenesulfonyl-fluoride hydrochloride, 10 µg/ml leupeptin, 5 µg/ml pepstatin A, and 0.2 U/ml aprotinin). Cells were then solubilized in Triton lysis buffer (1% Triton X-100, 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM Na3VO4, and protease inhibitors). The supernatants after a 15 min 20,000 x g centrifugation were mixed with protein A-coupled agarose beads and then proteins were immunoprecipitated with Abs prebound to protein A-agarose beads. Rabbit anti-mouse IgG Ab was used to couple mouse mAb with protein A-agarose. After gentle rotation for 1 h at 4°C, the beads were washed four times with ice-cold Triton lysis buffer, and the precipitated proteins were eluted by boiling for 15 min with SDS-PAGE sample buffer containing 1% 2-ME. For the preparation of total cell lysates, monolayers were rinsed once with PBS as described above, and directly lysed by the addition of SDS-PAGE sample buffer containing 2-ME.

Immunoprecipitated proteins or whole-cell lysates were separated by SDS-PAGE under reducing conditions and electrotransferred to polyvinylidene difluoride membranes. The membrane was incubated with 4% BSA blocking buffer (10 mM Tris (pH 7.4), 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature and the tyrosine-phosphorylated proteins were detected by HRP-conjugated anti-pTyr Ab, 4G10. The membranes were then stripped and reprobed with primary Abs. In all these blots, the proteins were visualized by the ECL reagent (NEN).

RNA protection assay

Cytokine mRNA was measured by using a multiprobe rat cytokine RNA protection kit (RiboQuant kit; BD PharMingen, San Diego, CA) as recommended by the manufacturer. Briefly, by in vitro transcription, 32P-labeled RNA probes were synthesized using the set of cytokine cDNA templates. The synthesized probes were purified using SELECT-D (RF) spin chromatography column (5 Prime->3 Prime, Boulder, CO) and hybridized overnight at 56°C with 20 µg of RNA. After digestion with RNase, the protected RNA were purified, resolved by QuickPoint PAGE (Invitrogen, Carlsbad, CA), and visualized by autoradiography.

Intracellular free calcium concentration ([Ca2+]i) measurements

Fura 2 fluorescence in single cells was measured using an SLM Aminco 8000/DMX 100 spectrofluorometer (Jobin Yvon Horiba, Edison, NJ) attached to an inverted Nikon Diaphot microscope (Nikon, Melville, NY) with a Fluor x40 oil-immersion objective. Images were acquired using an enhanced charge-coupled device camera (CCD-72; Maryland Technologies, Michigan City, IN) and the Image-1 software (Universal Imaging, Downingtown, PA) at excitation wavelengths of 340 and 380 nm, with emission at 510 nm. Analog plots of the fluorescence ratio (340:380) in single cells are shown. Cells were grown overnight in culture medium on coverslips, and then washed twice with loading medium (medium 199; Biofluid, Rockville, MD) supplemented with 2 mM CaCl2 and 0.1% BSA, and loaded with 2 µM fura 2 for 45 min at 37°C. After loading, cells were washed four times with working medium (medium 199 containing 2 mM CaCl2, 10 mM Tris (pH 7.4), and 0.01% BSA). All other details are given in the text and figure legends.

Subcellular fractionation

For the preparation of cytosolic and membrane fractions, 5 x 106 cells were washed with ice-cold PBS containing 1 mM NaVO4, 0.5 mM PMSF, 5 µg/ml leupeptin, and resuspended on ice in hypotonic buffer (42 mM KCl, 10 mM HEPES (pH 7.4), 5 mM MgCl2, and protease inhibitors). Cell lysates were centrifuged (10 min at 200 x g), and the supernatants were centrifuged for 30 min at 100,000 x g. Supernatants of the second centrifugation were collected as the cytosolic fraction. The pellet was washed once with hypotonic buffer, then directly solubilized in sample buffer as the membrane fraction.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of Gab2 in RBL-2H3 mast cells

Gab2 is widely expressed in many tissues and cells including heart, lung, kidney, testis, spinal cord, blood leukocytes, T cells, some B cell lines, and recently shown to be present in mouse mast cells (4, 5, 9). Immunoblotting demonstrated the presence of Gab2 in the RBL-2H3 mast cell line, which is a useful model for defining Fc{epsilon}RI-mediated signaling pathways. To investigate the role of Gab2 in Fc{epsilon}RI-mediated signaling, we first examined the tyrosine phosphorylation status of this molecule in RBL-2H3 cells. There was some basal constitutive tyrosine phosphorylation of Gab2, which rapidly increased after Fc{epsilon}RI aggregation (Fig. 1GoA). This Fc{epsilon}RI-induced phosphorylation was clearly apparent at 3 min, was stronger at 10 min, and then gradually decreased to baseline by 45 min. These results, together with the data with the BMMC (9), indicate that Gab2 may play an important role in Fc{epsilon}RI-mediated signaling.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 1. Rapid tyrosine phosphorylation of Gab2 by Fc{epsilon}RI aggregation and the overexpression of Gab2 in RBL-2H3 mast cells. A, RBL-2H3 cells were cultured overnight with IgE and stimulated with 100 ng/ml of Ag for the indicated times. Lysates from 5 x 106 cells were immunoprecipitated with anti-Gab2 Ab and analyzed by immunoblotting with anti-pTyr and anti-Gab2 Abs. B, RBL-2H3 cells were transfected with Gab2 cDNA and stable clones were selected with 350 µg/ml G418. Total cell lysates (2 x 105 cell equivalents) from the RBL-2H3, Gab2-transfected, or empty vector-transfected cells were immunoblotted with anti-Gab2 and anti-Fc{epsilon}RI{beta} Abs, respectively. The anti-Fc{epsilon}RI{beta} blotting was used to control for equal loading. C, Tyrosine phosphorylation of Gab2 still occurs in overexpressing cells. Gab2-overexpressing cells and the control vector-transfected cells were either nonstimulated or stimulated for the indicated times with 100 ng/ml of Ag. Lysates from 5 x 106 cells were immunoprecipitated with anti-Gab2 Ab, and analyzed by immunoblotting with anti-pTyr and anti-Gab2 Abs. The exposure times for the immunoblots with the Gab2-transfected cells was shorter than that for the control cells.

 
To further investigate the role of Gab2 in mast cells, we transfected RBL-2H3 cells with an expression plasmid containing this molecule. The clones selected with G418 were screened by immunoblotting with anti-Gab2 Ab, and several stable transfected cell lines were isolated. In two of these lines (clone nos. 1 and 2) the expression level of Gab2 was >50-fold of that in the parental RBL-2H3 cells, while there were many other cloned lines in which expression level was only 2- to 7-fold increased (Fig. 1GoB and data not shown). We next examined whether the increased Gab2 protein in the transfected cells was still tyrosine phosphorylated after Fc{epsilon}RI aggregation (Fig. 1GoC). As in the control vector-transfected cells, receptor stimulation induced a rapid increase in phosphorylations that returned to baseline at 30 min. As there was much more Gab2 in these transfected cells, we would assume that there was actually an increase in the concentration of this molecule that was tyrosine-phosphorylated. Therefore, Gab2 is tyrosine phosphorylated in these transfected cells in the same time course as in the controls, and participates in the Fc{epsilon}RI signaling pathway.

Fc{epsilon}RI-stimulated histamine release and cytokine gene transcription are inhibited in Gab2-overexpressing cells

Fc{epsilon}RI-mediated activation of mast cells results in the release of histamine from cytoplasmic granules. Therefore, we examined the effect of Gab2 overexpression on Ag-stimulated histamine release. In transfected cells that expressed high levels of Gab2, the Ag-induced histamine release was significantly reduced to <50% of that in the RBL-2H3 parental cells (Fig. 2GoA). In contrast, there were no changes in the calcium ionophore-induced release (data not shown), suggesting that inhibition was at an early step after receptor activation, and that the cells were still capable of degranulation. There was no inhibition of Ag-induced histamine release in the control vector-transfected cells and in Gab2-transfected cell lines where the expression level of this protein was increased by only 2- to 7-fold (clone nos. 3 and 4 and data not shown). Further evidence that the inhibition of histamine release was related to the expression level of Gab2 were studies in a third cloned line that after transfection initially expressed high levels of Gab2 (43-fold increase) and had 45% inhibition of Ag-induced histamine release. When maintained in culture, there was a gradual decrease in the expression level of Gab2 (to 27-fold) and the histamine release reverted to that of the parental cells. These results indicate that overexpression of Gab2 negatively regulates Fc{epsilon}RI-mediated degranulation.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 2. Fc{epsilon}RI-induced histamine release and TNF-{alpha} and IL-6 mRNA expression are decreased by overexpression of Gab2. A, RBL-2H3 cells and the cells transfected with Gab2 or empty vector were cultured overnight with Ag-specific IgE, and then stimulated for 45 min with the indicated concentrations of the Ag. Histamine release was measured in the supernatants and calculated as the percentage of total histamine content. Data represents the average of three separate experiments and are expressed as the mean ± SEM. B, Gab2-overexpressing cells and the vector control cells were either nonstimulated or stimulated for the indicated times with Ag (10 ng/ml). Total RNA was prepared, and 20 µg was used for hybridization with 32P-labeled cytokine probes. After purification, protected RNA was resolved on denaturing polyacrylamide gels and visualized by autoradiography. C and D, The intensity of autoradiography signals was determined by densitometric analysis. The data for TNF-{alpha} (C) and IL-6 (D) are presented and expressed as the percentage at each time point compared with the maximum for vector-transfected control cells. Data represents the average of three separate experiments, and are expressed as the mean ± SEM.

 
Aggregation of Fc{epsilon}RI not only causes the release of pre-formed mediators such as histamine, but also stimulates the synthesis of various cytokines including TNF-{alpha}, IL-6, and IL-2 (29, 30, 31, 32). In RBL-2H3 cells, a multiprobe RNA protection assay demonstrates the Fc{epsilon}RI-induced increase in the mRNA for multiple cytokines, the most prominent of which are IL-4, IL-6, and TNF-{alpha} (33). Using this assay, there was significant suppression of the Fc{epsilon}RI aggregation-induced production of mRNA for TNF-{alpha} and IL-6 in the cells that overexpressed Gab2 (Fig. 2GoB). The inhibition of TNF-{alpha} and IL-6 mRNA production was more dramatic (73–94%, Fig. 2Go, C and D) than the inhibition of histamine release (50–66%, Fig. 2GoA). However, the changes in the mRNA for IL-4 were variable. These results, taken together with the inhibition of histamine release, suggested a negative regulatory role of Gab2 overexpression on Fc{epsilon}RI-mediated mast cell function.

Gab2 overexpression inhibits the Fc{epsilon}RI-induced increase in intracellular calcium

Aggregation of Fc{epsilon}RI results in the generation of inositol 1,4,5-triphosphate (IP3) that next results in the release of calcium from intracellular stores, and calcium influx through calcium release-activated calcium channels in the plasma membrane (34, 35). The inhibition of Fc{epsilon}RI-induced, but not ionophore-induced, histamine release by overexpression of Gab2 suggested that these effects of Gab2 were at an early step after Fc{epsilon}RI aggregation. Therefore, intracellular measurements in individual cells were used to monitor the effects of Gab2 expression on receptor-mediated [Ca2+]i (Fig. 3Go). Both the rapid and the sustained response to Ag stimulation were suppressed by ~50% in the Gab2-transfected cells. In the absence of extracellular calcium, there was again an inhibition of the calcium response. RBL-2H3 cells have a G protein-coupled seven-transmembrane receptor for thrombin that when activated, results in calcium influx. The thrombin-induced increase in intracellular calcium was similar in all the different cell lines. Furthermore, there were similar responses with thapsigargin (data not shown) and ionomycin in all the cell lines, indicating that the expression of Gab2 had no effects on intracellular calcium storage. These controls confirmed that the changes in calcium influx were limited to the Fc{epsilon}RI-activated pathway. The results indicate that the major inhibitory effects of Gab2 overexpression were upstream of the rise in intracellular calcium.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 3. Fc{epsilon}RI-induced calcium mobilization is inhibited by Gab2 overexpression. Fluorescence changes in fura 2-loaded cells were measured as described in Materials and Methods. Where indicated (-Ca2+ medium), cells were in calcium-free medium + 100 µM EGTA. Addition of agonists (100 ng/ml Ag or 1 U/ml thrombin) and 0.5 µM ionomycin is indicated by arrows. Each trace is an average of at least 25 cells and is representative of results obtained in 3–4 experiments. The bar graph shows average data of Ag-stimulated calcium mobilization. Peak and sustained increases in fluorescence in +Ca2+ medium and peak value in -Ca2+ medium are shown. The values marked by * are significantly different (p < 0.001, Student’s t test) from other values in the same set. These data were obtained from four experiments and represent fura 2 fluorescence in at least 80 cells in each case.

 
Overexpression of Gab2 inhibits cellular protein tyrosine phosphorylations

Cellular protein tyrosine phosphorylation is an early event after Fc{epsilon}RI stimulation, and is critical for the propagation of downstream signal transduction (16, 36, 37). The inhibition of the calcium release suggested that the overexpression of Gab2 had effects at an early stage of cell activation. As compared with the control vector-transfected cells, the Ag-induced total cellular protein tyrosine phosphorylation was decreased in Gab2-overexpressing cells (Fig. 4GoA). However, the general pattern was similar except for the phosphorylated band at about p97 kDa in the cells overexpressing Gab2, which was recognized by anti-Gab2 Ab (data not shown). In both the control and transfected cells, Gab2 was tyrosine phosphorylated under quiescent conditions and after receptor aggregation (Fig. 1GoC). The suppression of cellular protein tyrosine phosphorylation by Gab2 overexpression further suggests that Gab2 was regulating signal transduction at an early step after Fc{epsilon}RI activation.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 4. Overexpression of Gab2 inhibits cellular protein tyrosine phosphorylation. Gab2 overexpressing cells and the vector control cells were cultured with IgE overnight, and stimulated with 100 ng/ml of Ag for the indicated times. A, Total cell lysates (2 x 105 cell equivalents) were separated by SDS-PAGE (10% gels) and analyzed by immunoblotting with anti-pTyr Ab. B and C, Tyrosine phosphorylation of the subunits of Fc{epsilon}RI and Syk. Lysates from 5 x 106 cells were immunoprecipitated with anti-Fc{epsilon}RI{beta} Ab (B) or anti-Syk Ab (C), and analyzed by immunoblotting with anti-pTyr Ab (B and C), anti-Fc{epsilon}RI{beta} and {gamma} Abs (B), or anti-Syk (C) Ab. The two isoforms of Syk that are present in RBL-2H3 cells are apparent in the anti-Syk immunoblot.

 
Aggregation of Fc{epsilon}RI results in the rapid tyrosine phosphorylation of the {beta} and {gamma} receptor subunits (37). The phosphorylation of the tyrosines in the immunoreceptor tyrosine-based activation motif of these subunits then recruits signaling molecules such as Syk to transduce downstream activation events (18, 38, 39). The decreases in total cellular tyrosine phosphorylations suggest that Gab2 may interfere at a very early stage in the signaling cascade after receptor aggregation. Indeed, tyrosine phosphorylation of both the {beta}- and {gamma}-chains of Fc{epsilon}RI were decreased in Gab2-overexpressing cells compared with those of controls (Fig. 4GoB). Tyrosine phosphorylation of Syk was also clearly reduced in these cells (Fig. 4GoC). Phosphorylation of the activation loop tyrosines is a measure of Syk activation and critical for downstream propagation of signals (40, 41). In these cells, the phosphorylation of the tyrosines in the activation loop of Syk was also decreased as detected by an anti-phosphoactivation loop tyrosine-specific Ab (data not shown). The recruitment and activation of Syk by Fc{epsilon}RI is mainly by the phosphorylated immunoreceptor tyrosine-based activation motif of the {gamma} subunit (28, 42), suggesting that the decreased Syk activation is probably due to the changes in Fc{epsilon}RI{gamma} tyrosine phosphorylation. Therefore, overexpression of Gab2 by interfering with receptor phosphorylation, results in decreased Syk activation.

Gab2 associates with Lyn protein tyrosine kinase and translocates to the plasma membrane after Fc{epsilon}RI activation

The protein tyrosine kinase Lyn, a Src kinase family member, associates with Fc{epsilon}RI, and this association is increased after receptor aggregation (17, 21, 43). After Fc{epsilon}RI aggregation, Lyn is thought to phosphorylate the {beta} and {gamma} subunits of the receptor, initiating the intracellular signaling cascades (44). The SH3 domain of Src tyrosine kinase has also been shown to bind to Gab2 in vitro (4). Therefore, we investigated whether there was an interaction between Lyn and Gab2. Indeed, Gab2 was detected in Lyn immunoprecipitates from the lysates of Gab2-overexpressing cells, and this association increased after Fc{epsilon}RI aggregation, suggesting that Gab2 might be recruited to Lyn after receptor aggregation (Fig. 5GoA). However, this association was below the detection threshold in control cells that have much less Gab2. Lyn was constitutively tyrosine phosphorylated, and there were no changes in this phosphorylation after Fc{epsilon}RI stimulation in both the control and Gab2-transfected cells (data not shown). Therefore, Gab2, by interacting with Lyn, could interfere with the phosphorylation of downstream substrates such as the subunits of the receptor.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 5. Gab2 is coimmunoprecipitated with Lyn kinase and translocates to the plasma membrane after Ag stimulation. A, The Gab2-overexpressing and control cells were either nonstimulated or stimulated for the indicated times with 100 ng/ml of Ag. Lysates from 5 x 106 cells were immunoprecipitated with anti-Lyn Ab, and analyzed by immunoblotting with anti-Gab2 and anti-Lyn Abs. In the anti-Lyn immunoblot, the two slower migrating bands are the p56 and p53 isoforms of Lyn, and the faster migrating band is due to a cross-reaction of the secondary Ab with the H chain of the rabbit anti-Lyn Abs. B and C, Subcellular fractionation experiments were perfumed as described in Materials and Methods. Cytosol (C) and membrane (M) fractions from control cells (B) and Gab2-transfected cells (C) were separated by SDS-PAGE and analyzed by immunoblotting with anti-Gab2 Ab using Fc{epsilon}RI{beta} blotting as a marker of the membrane fraction.

 
The interaction of Gab2 with Lyn could be due to changes in their cellular localization. By cellular fractionation, Gab2 was essentially all in the cytosol in quiescent cells, whereas Fc{epsilon}RI{beta}, as expected, was only in the membrane fraction (Fig. 5Go, B and C). After receptor aggregation, there was rapid translocation of Gab2 from the cytosol to the plasma membrane, which was apparent 3 min after stimulation, the earliest time point examined. However, at all time points examined, ~70% of the Gab2 remained in the cytosol fraction after cell activation. A similar fraction of the total Gab2 was present in the plasma membrane of stimulated cells in both the control (Fig. 5GoB) and Gab2-transfected cells (Fig. 5GoC). Therefore, after Fc{epsilon}RI aggregation there was translocation of Gab2 to the plasma membrane, presumably due to its Pleckstrin homology domain, and interaction with Lyn. The interaction of Gab2 with Lyn kinase might play a role in Fc{epsilon}RI signaling.

Gab2 binds PLC{gamma}2 after Fc{epsilon}RI stimulation, and inhibits its tyrosine phosphorylation

The aggregation of Fc{epsilon}RI results in tyrosine phosphorylation and activation of PLC{gamma}, which generates IP3 that in turn mediates the increase in intracellular calcium. The dramatic decrease in the Fc{epsilon}RI-induced intracellular calcium response in the Gab2-transfected cells suggested that there could be changes in the activation of PLC{gamma}. The extent of the tyrosine phosphorylation of PLC{gamma}1 was similar in the control and the Gab2-overexpressing cells (Fig. 6GoA). However, the amount of PLC{gamma}1 protein precipitated in Gab2-overexpressing cells was much greater than that in vector control cells, suggesting that Gab2 may up-regulate the expression of PLC{gamma}1. Anti-PLC{gamma}1 Ab analysis of total cell lysates confirmed that the expression level of PLC{gamma}1 in Gab2-transfected cells was indeed higher than that in the controls (data not shown). Therefore, in the Gab2-transfected cells, there is a decrease in the fraction of the PLC{gamma}1 that is tyrosine phosphorylated after receptor activation. Similarly, Ag induced a rapid tyrosine phosphorylation of PLC{gamma}2 in control cells; however, this phosphorylation was dramatically decreased in Gab2-transfected cells (Fig. 6GoB). Therefore, there is decreased tyrosine phosphorylation of PLC{gamma} that could account for the decrease in the reduction in the signals that result in the intracellular calcium response.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 6. Gab2 recruits PLC{gamma}2 after Ag stimulation and suppressed tyrosine phosphorylation of PLC{gamma}. Gab2-overexpressing cells and the control cells were either nonstimulated or stimulated for the indicated times with 100 ng/ml of Ag. Lysates from 5 x 106 cells were immunoprecipitated with anti-PLC{gamma}1 (A) or PLC{gamma}2 (B) Abs, and analyzed by immunoblotting with anti-pTyr (A and B) and anti-PLC{gamma}1 (A) or anti-PLC{gamma}2 and anti-Gab2 (B) Abs.

 
There are two potential binding sites for PLC{gamma} on the Gab2 molecule, phosphorylation of tyrosines at these sites allows binding by the SH2 domain of PLC{gamma} (3). Indeed, Gab2 was coprecipitated with PLC{gamma}2, and this association was increased after activation (Fig. 6GoB), suggesting that PLC{gamma}2 is recruited to Gab2 after Ag stimulation. The association of Gab2 with PLC{gamma}2 was clearly evident in the Gab2-transfected cells, but was below the detection threshold in the control vector-transfected cells. There was also association of PLC{gamma}1, Grb2, and SHP-2 with Gab2 (data not shown). However, there was no significant change in the association of Gab2 with PLC{gamma}1 after receptor activation (data not shown). Therefore, these data suggest that interactions of PLC{gamma} with Gab2 may play a role in the inhibition of the receptor-induced increase in [Ca2+]i.

Overexpression of Gab2 inhibits MAPK pathways

Gab2, and its structurally related molecule Gab1, by interacting with Grb2/Sos/Shc plays a role in the activation of the ERK MAPK pathway (7, 45, 46), thereby regulating downstream transcription factors (3, 10). The suppression of Ag-induced TNF-{alpha} and IL-6 mRNA production in Gab2-overexpressing cells suggested that there could be changes in the MAPK pathways. Therefore, phospho-specific Abs were used to investigate the ERK, JNK, and p38 MAPK pathways. These Abs recognize phosphorylated Thr and Tyr residues that are critical for their activation (47, 48). The phosphorylation of ERK at 3 min after Fc{epsilon}RI activation was similar in the different cell lines; however, at 10 min it was decreased in the Gab2-transfected compared with the control cells (Fig. 7GoA). A very similar pattern was observed with p38 MAPK phosphorylation; strong signals that were similar to controls at 3 min in the Gab2-transfected cells, but a faster return to baseline (Fig. 7GoB). Therefore, there was a more rapid rate of dephosphorylation and inactivation of ERK and p38 in Gab2-overexpressing cells. However, the pattern was different with the JNK proteins; there was some inhibition of the Fc{epsilon}RI-induced phosphorylation in the Gab2-transfected cells, although the changes were not dramatic (Fig. 7GoC). Therefore, Fc{epsilon}RI stimulation resulted in a more transient activation of the ERK and p38 MAPK pathways in the Gab2-transfected cells.



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 7. The effects of Gab2 on MAPK phosphorylation. Gab2-overexpressing cells and the control cells were either nonstimulated or stimulated for the indicated times with 100 ng/ml of Ag. Total lysates from 2 x 105 cells were analyzed by immunoblotting with anti-phospho-ERK and anti-ERK Abs (A), anti-phospho-p38 and anti-p38 Abs (B), or anti-phospho-JNK and anti-JNK Abs (C).

 
Akt phosphorylation is not affected by overexpression of Gab2

Gab2 has multiple binding sites for the p85 subunit of PI3-kinase and this interaction is involved in the activation of the PI3-kinase in response to growth factors, cytokines, and Ag receptors (7). The activation of PI3-kinase results in the production of phosphatidylinositol 3,4,5-triphosphate, which plays an important role in the sustained influx of calcium and also recruits Akt to the plasma membrane where Akt is Thr/Ser-phosphorylated and activated (49, 50, 51). The decrease in the calcium response in the Gab2-transfected cells could be due to inhibition of receptor-mediated PI3-kinase stimulation. Therefore, we used an Ab specific for phosphorylated Ser473 that is critical for the activation of Akt as a measure of the simulation of the PI3-kinase pathway (50). There was significant Akt phosphorylation in cells not stimulated by receptor aggregation; however, this disappeared when cultures were maintained for 24 h under serum-free conditions (data not shown). Fc{epsilon}RI-induced phosphorylation of Akt was similar in the control and Gab2-transfected cells cultured for 24 h under serum-free conditions (Fig. 8Go). Similar results were observed in cells that had been cultured in regular media. Because there was neither suppression nor enhancement of Akt activation in Gab2 transfectants, it can be assumed that PI3-kinase activation is not affected in these cells. Therefore, these results suggest that the PI3-kinase/Akt pathway is not involved in the Gab2-mediated inhibition of mast cell histamine release and cytokine production.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 8. Akt phosphorylation is not affected by overexpression of Gab2. Gab2-overexpressing cells and the vector control cells were cultured for 24 h in serum-free medium, and either nonstimulated or stimulated for the indicated times with 100 ng/ml of Ag. Total lysates from 2 x 105 cells were analyzed by immunoblotting with anti-phospho-Akt and anti-Akt Abs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results indicate that Fc{epsilon}RI aggregation results in the tyrosine phosphorylation of Gab2 and translocation of a significant fraction of it from the cytosol to the plasma membrane. Gab2 associates with several signaling molecules including protein tyrosine phosphatase SHP-2, p85 subunit of PI3-kinase, Lyn, and PLC{gamma}. The coprecipitation of Gab2 with Lyn and PLC{gamma} were enhanced after receptor aggregation. The association of Gab2 with Lyn might be through proline-rich domains of Gab2 interacting with the SH3 domain of Lyn (4). Overexpression of Gab2 in RBL-2H3 cells inhibited the Fc{epsilon}RI-induced tyrosine phosphorylation of the receptor subunits, and the phosphorylation and activation of Syk and MAPK. Downstream events such as PLC{gamma} tyrosine phosphorylation, calcium mobilization, degranulation, and induction of TNF-{alpha} and IL-6 gene transcripts were similarly decreased in these cells. These results suggest that in addition to the positive effects mediated by PI3-kinase that are apparent in Gab2-/- mast cells, Gab2 by interacting with Lyn and PLC{gamma} may have negative regulatory effects on Fc{epsilon}RI-induced mast cell signaling and functions.

The results of the present experiments have to be explained in light of the recent description of defects in Fc{epsilon}RI-mediated signaling in BMMC from Gab2-/- mice (9). In these cells, there is a decrease in the receptor-induced activation of the PI3-kinase pathway with a decrease in the generation of phosphatidylinositol 3,4,5-triphosphate, the tyrosine phosphorylation of PLC{gamma}1, and the formation of IP3. There is also a decrease in the receptor-induced increase in [Ca2+]i, especially of the delayed response with parallel decreases in degranulation and cytokine generation. These data suggest that Gab2 recruits and activates the PI3-kinase, thus playing an important role in degranulation. Multiple signaling pathways are activated in cells in response to a particular stimulus. A scaffolding molecule such as Gab2 binds different signaling proteins, thereby coupling to several pathways, some of which could have positive or negative effects. The absence of Gab2 as a scaffolding molecule would affect the related signaling pathways such as PI3-kinase, PLC{gamma}, Lyn, SHP-2, and Grb2. The observed functional changes would represent the major positive role of the molecule. In BMMC from Gab2-/- mice, the Fc{epsilon}RI-induced PI3-kinase activation was decreased and mast cell functions were defective, suggesting that PI3-kinase is a major positive effector of Gab2 in Fc{epsilon}RI signaling. In contrast, overexpression of Gab2 in cells would amplify additional pathways dependent on other interactions, such as PLC{gamma} and Lyn. These inhibitory effects would not be apparent in the deficient cells if they act downstream of the positive effects of Gab2.

Although the expression level of Gab2 was quite high, the effects of Gab2 were specific for Fc{epsilon}RI signaling. The localization of Gab2 in transfected cells was similar to that in the controls both in the quiescent state and after Ag stimulation. This suggests that there are no changes in subcelluar distribution of Gab2 that could account for the inhibition of signal transduction in transfected cells. Similarly, thrombin, which binds to a seven-transmembrane G-protein-coupled receptor, induced calcium mobilization that was not inhibited in these cells suggesting a specific regulatory role of Gab2 in IgE receptor signaling. Calcium ionophore-induced calcium mobilization and histamine release were not affected in these cells, indicating a normal degranulation function of the cells and a specific action of Gab2 upstream of calcium mobilization in Fc{epsilon}RI signal transduction.

The present experiments strongly suggest that overexpression of Gab2 interferes at a very early stage in Fc{epsilon}RI signal transduction. The reduction in the tyrosine phosphorylation of subunits of Fc{epsilon}RI and Syk kinase would implicate the step at the Lyn-mediated phosphorylation of the receptor subunits. Because Lyn is localized to the plasma membrane, it is possible that binding of Gab2 with Lyn may directly interfere with its capacity to phosphorylate the receptor subunits. It is also possible that Gab2 may indirectly interfere with receptor subunit phosphorylation by recruiting multiple other proteins such as SHP-2, PI3-kinase, PLC{gamma}, or Shc and keep these away from Fc{epsilon}RI-organized signaling complexes (42, 52). Although Gab2 and SHP-2 were associated in RBL-2H3 cells, we could not detect a significant change in membrane translocation of SHP-2 and overexpression of SHP-2 in RBL-2H3 cells did not inhibit histamine release (data not shown). Therefore, the decrease in the tyrosine phosphorylation of subunits of Fc{epsilon}RI and PLC{gamma}2 do not appear to be due to Gab2-mediated membrane recruitment of SHP-2. Nevertheless, whatever the mechanism, the decrease in receptor phosphorylation would then be a major contributor to the inhibition of the subsequent signaling cascade.

The tyrosine phosphorylation and activation of PLC{gamma}1 and PLC{gamma}2 for the generation of IP3 are downstream of Syk (26). The Fc{epsilon}RI-induced tyrosine phosphorylation of PLC{gamma}2 was dramatically decreased by over-expression of Gab2. In contrast, although the extent of the total tyrosine phosphorylation of PLC{gamma}1 was similar in control and Gab2-transfected cells, there was a decrease in the fraction of the total PLC{gamma}1 that was phosphorylated. These results suggest that the interaction of Gab2 with PLC{gamma} inhibited the phosphorylation, and therefore, the activation of PLC{gamma}. The binding of Gab2 to PLC{gamma} might prevent its tyrosine phosphorylation by mechanisms such as blocking tyrosine residues, sequestering the molecule away from kinases, or by promoting dephosphorylation by phosphatases. The difference in the extent of the decrease in the phosphorylation of PLC{gamma}1 and PLC{gamma}2 may be due to variation in their interaction with Gab2 or their subcellular locations. By electron microscopy, PLC{gamma}1 and PLC{gamma}2 isoforms are differentially distributed in the RBL-2H3 cells with the PLC{gamma}2 inherently associated with the membrane, whereas PLC{gamma}1 is recruited to membrane ruffles only after receptor aggregation (53, 54). Studies with inhibitors suggest that the Fc{epsilon}RI-induced tyrosine phosphorylation and activation of PLC{gamma}1, but not of PLC{gamma}2, depends on PI3-kinase (54). Therefore, the minimal changes in the phosphorylation of PLC{gamma}1 compared with PLC{gamma}2 would further indicate that in the Gab2-expressing cells the PI3-kinase pathway is still active.

The overexpression of Gab2 also had dramatic effects on the Fc{epsilon}RI-induced rise in intracellular calcium. There was ~50% inhibition in both the initial and sustained increase in intracellular calcium, which would indicate a decrease in release from intracellular stores as well as in influx from the extracellular medium. The PI3-kinase-generated lipid products play an important role in the rise in intracellular calcium by recruiting PLC{gamma} and Tec protein tyrosine kinases to the membrane (55, 56, 57). The Tec protein kinase Btk then regulates the protein tyrosine phosphorylation and activation of PLC{gamma} (57). Similarly, PI3-kinase inhibitors block the rise in intracellular calcium (53). The normal Fc{epsilon}RI-induced activation of Akt in the Gab2-transfected cells suggests that PI3-kinase was not inhibited. Therefore, the decreased calcium response to Ag in these cells is probably due to the reduced activation of PLC{gamma}, and would contribute to the subsequent inhibition of mast cell functions.

Similar to its role in signaling from cytokine and growth factor receptors, Gab2 is important for Fc{epsilon}RI-induced activation of the PI3-kinase pathway (9). Therefore, it was surprising that PI3-kinase pathway appeared to be unchanged in Gab2-overexpressing cells, as demonstrated by the phosphorylation of Akt. Because tyrosine phosphorylation of the {beta} and {gamma} subunits of the receptor (as well as Syk) was inhibited by Gab2 overexpression, it would be assumed that receptor-induced PI3-kinase/Akt pathway should also be suppressed. However, the normal Akt phosphorylation might reflect two opposing effects on the PI3-kinase pathway: 1) reduced activation on the one hand, due to the decrease in Syk activity; and 2) increased activation due to overexpression of Gab2. PI3-kinase could also be both an upstream regulator and downstream effector of Gab2, as has been shown with the closely related molecule of Gab1 (58). Therefore, our data supports a positive role of Gab2 in regulating PI3-kinase activation.

In summary, these experiments demonstrate that Fc{epsilon}RI induced the tyrosine phosphorylation of Gab2 and its translocation to the membrane. Gab2 interacted with several signaling molecules the binding to two of which (PLC{gamma}2 and Lyn) was increased after Ag stimulation. These interactions appear to play a negative regulatory role in Fc{epsilon}RI signaling. Overexpression of Gab2 dramatically suppressed Fc{epsilon}RI-induced signal transduction, except for the activation of the PI3-kinase pathway as evidenced by Akt phosphorylation. These results demonstrate complex positive and negative regulatory effects of Gab2 in Fc{epsilon}RI signaling in mast cells.


   Acknowledgments
 
We thank Drs. Juan Zhang and Daniel Vial for reviewing the manuscript and for helpful suggestions. We also thank Greta Bader for histamine analysis.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Reuben P. Siraganian, Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, Department of Health and Human Services, National Institute of Dental and Craniofacial Research, National Institute of Health, Building 10, Room 1N106, Bethesda, MD 20892. E-mail address: RS53X{at}nih.gov Back

2 Abbreviations used in this paper: SH, Src homology; BMMC, bone marrow-derived mast cells; JNK, c-Jun N-terminal kinase; ERK, extracellular regulated kinase; MAPK, mitogen-activated protein kinase; pTyr, phosphotyrosine; pThr, phosphothreonine; RBL-2H3, rat basophilic leukemia 2H3 cell line; SHP-2, SH2-containing protein tyrosine phosphotase 2; PLC{gamma}, phospholipase C{gamma}; PI3-kinase, phosphatidylinositol-3 kinase; [Ca2+]i, intracellular free calcium concentration; IP3, inositol 1,4,5-trisphosphate. Back

Received for publication September 13, 2001. Accepted for publication February 15, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tomlinson, M. G., J. Lin, A. Weiss. 2000. Lymphocytes with a complex: adapter proteins in antigen receptor signaling. Immunol. Today 21:584.[Medline]
  2. Van Leeuwen, J. E. M., L. E. Samelson. 1999. T cell antigen-receptor signal transduction. Curr. Opin. Immunol. 11:242.[Medline]
  3. Gu, H. H., J. C. Pratt, S. J. Burakoff, B. G. Neel. 1998. Cloning of p97/Gab2, the major SHP2-binding protein in hematopoietic cells, reveals a novel pathway for cytokine-induced gene activation. Mol. Cell 2:729.[Medline]
  4. Zhao, C. M., D. H. Yu, R. Shen, G.S. Feng. 1999. Gab2, a new pleckstrin homology domain-containing adapter protein, acts to uncouple signaling from ERK kinase to Elk-1. J. Biol. Chem. 274:19649.[Abstract/Free Full Text]
  5. Nishida, K., Y. Yoshida, M. Itoh, T. Fukada, T. Ohtani, T. Shirogane, T. Atsumi, M. Takahashi-Tezuka, K. Ishihara, M. Hibi, T. Hirano. 1999. Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T- and B-cell antigen receptors. Blood 93:1809.[Abstract/Free Full Text]
  6. Gadina, M., C. Sudarshan, R. Visconti, Y. J. Zhou, H. H. Gu, B. G. Neel, J. J. O’Shea. 2000. The docking molecule Gab2 is induced by lymphocyte activation and is involved in signaling by interleukin-2 and interleukin-15 but not other common {gamma} chain-using cytokines. J. Biol. Chem. 275:26959.[Abstract/Free Full Text]
  7. Hibi, M., T. Hirano. 2000. Gab-family adapter molecules in signal transduction of cytokine and growth factor receptors, and T and B cell antigen receptors. Leuk. Lymphoma 37:299.[Medline]
  8. Gu, H. H., H. Maeda, J. J. Moon, J. D. Lord, M. Yoakim, B. H. Nelson, B. G. Neel. 2000. New role for Shc in activation of the phosphatidylinositol 3- kinase/Akt pathway. Mol. Cell. Biol. 20:7109.[Abstract/Free Full Text]
  9. Gu, H. H., K. Saito, L. D. Klaman, J. Q. Shen, T. Fleming, Y. P. Wang, J. C. Pratt, G. S. Lin, B. Lim, J. P. Kinet, B. G. Neel. 2001. Essential role for Gab2 in the allergic response. Nature 412:186.[Medline]
  10. Liu, Y., B. Jenkins, J. L. Shin, L. R. Rohrschneider. 2001. Scaffolding protein Gab2 mediates differentiation signaling downstream of Fms receptor tyrosine kinase. Mol. Cell. Biol. 21:3047.[Abstract/Free Full Text]
  11. Crouin, C., M. L. Arnaud, F. Gesbert, J. Camonis, J. Bertoglio. 2001. A yeast two-hybrid study of human p97/Gab2 interactions with its SH2 domain-containing binding partners. FEBS Lett. 495:148.[Medline]
  12. Stevens, R. L., K. F. Austen. 1989. Recent advances in the cellular and molecular biology of mast cells. Immunol. Today 10:381.[Medline]
  13. Gordon, J. R., P. R. Burd, S. J. Galli. 1990. Mast cells as a source of multifunctional cytokines. Immunol. Today 11:458.[Medline]
  14. Beaven, M. A., H. Metzger. 1993. Signal transduction by Fc receptors: the Fc{epsilon}RI case. Immunol. Today 14:222.[Medline]
  15. Siraganian, R. P.. 1993. Mechanism of IgE-mediated hypersensitivity. E. J. Middleton, and C. E. Reed, and E. F. Ellis, and N. F. J. Adkinson, and J. W. Yunginger, and W. W. Busse, eds. Allergy: Principles and Practice 105. Mosby, St. Louis.
  16. Benhamou, M., J. S. Gutkind, K. C. Robbins, R. P. Siraganian. 1990. Tyrosine phosphorylation coupled to IgE receptor-mediated signal transduction and histamine release. Proc. Natl. Acad. Sci. USA 87:5327.[Abstract/Free Full Text]
  17. Eiseman, E., J. B. Bolen. 1992. Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases. Nature 355:78.[Medline]
  18. Benhamou, M., N. J. P. Ryba, H. Kihara, H. Nishikata, R. P. Siraganian. 1993. Protein-tyrosine kinase p72Syk in high affinity IgE receptor signaling: identification as a component of pp72 and association with the receptor {gamma} chain. J. Biol. Chem. 268:23318.[Abstract/Free Full Text]
  19. Hutchcroft, J. E., R. L. Geahlen, G. G. Deanin, J. M. Oliver. 1992. Fc{epsilon}RI-mediated tyrosine phosphorylation and activation of the 72-kDa protein-tyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells. Proc. Natl. Acad. Sci. USA 89:9107.[Abstract/Free Full Text]
  20. Fukamachi, H., Y. Kawakami, M. Takei, T. Ishizaka, K. Ishizaka, T. Kawakami. 1992. Association of protein-tyrosine kinase with phospholipase C{gamma}1 in bone marrow-derived mouse mast cells. Proc. Natl. Acad. Sci. USA 89:9524.[Abstract/Free Full Text]
  21. Yamashita, T., S.-Y. Mao, H. Metzger. 1994. Aggregation of the high-affinity IgE receptor and enhanced activity of p53/56Lyn protein-tyrosine kinase. Proc. Natl. Acad. Sci. USA 91:11251.[Abstract/Free Full Text]
  22. Shiue, L., J. Green, O. M. Green, J. L. Karas, J. P. Morgenstern, M. K. Ram, M. K. Taylor, M. J. Zoller, L. D. Zydowsky, J. B. Bolen, J. S. Brugge. 1995. Interaction of p72Syk with the {gamma} and {beta} subunits of the high-affinity receptor for immunoglobulin E, Fc{epsilon}RI. Mol. Cell. Biol. 15:272.[Abstract]
  23. Jouvin, M. H., R. P. Numerof, J. P. Kinet. 1995. Signal transduction through the conserved motifs of the high affinity IgE receptor Fc{epsilon}RI. Semin. Immunol. 7:29.[Medline]
  24. Saitoh, S., R. Arudchandran, T. S. Manetz, W. G. 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.[Medline]
  25. Manetz, T. S., C. Gonzalez-Espinosa, R. Arudchandran, S. Xirasagar, V. Tybulewicz, J. Rivera. 2001. Vav1 regulates phospholipase C{gamma} activation and calcium responses in mast cells. Mol. Cell. Biol. 21:3763.[Abstract/Free Full Text]
  26. Zhang, J., E. H. Berenstein, R. L. Evans, R. P. Siraganian. 1996. Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor mediated degranulation in a Syk negative variant of rat basophilic leukemia RBL-2H3 cells. J. Exp. Med. 184:71.[Abstract/Free Full Text]
  27. Barsumian, E. L., C. Isersky, M. G. Petrino, R. P. Siraganian. 1981. IgE-induced histamine release from rat basophilic leukemia cell lines: isolation of releasing and nonreleasing clones. Eur. J. Immunol. 11:317.[Medline]
  28. Siraganian, R. P.. 1975. Refinements in the automatic fluorometric histamine analysis system. J. Immunol. Methods 7:283.[Medline]
  29. Teramoto, H., P. Salem, K. C. Robbins, X. R. Bustelo, J. S. Gutkind. 1997. Tyrosine phosphorylation of the vav proto-oncogene product links Fc{epsilon}RI to the Rac1-JNK pathway. J. Biol. Chem. 272:10751.[Abstract/Free Full Text]
  30. Hata, D., Y. Kawakami, N. Inagaki, C. S. Lantz, T. Kitamura, W. N. Khan, M. Maeda-Yamamoto, T. Miura, W. Han, S. E. Hartman, et al 1998. Involvement of Bruton’s tyrosine kinase in Fc{epsilon}RI-dependent mast cell degranulation and cytokine production. J. Exp. Med. 187:1235.[Abstract/Free Full Text]
  31. Kawakami, Y., S. E. Hartman, P. M. Holland, J. A. Cooper, T. Kawakami. 1998. Multiple signaling pathways for the activation of JNK in mast cells: involvement of Bruton’s tyrosine kinase, protein kinase C, and JNK kinases, SEK1 and MKK7. J. Immunol. 161:1795.[Abstract/Free Full Text]
  32. Chang, E. Y., Z. Szallasi, P. Acs, V. Raizada, P. C. Wolfe, C. Fewtrell, P. M. Blumberg, J. Rivera. 1997. Functional effects of overexpression of protein kinase C-{alpha}, -{beta}, -{delta}, -{epsilon}, and -{eta} in the mast cell line RBL-2H3. J. Immunol. 159:2624.[Abstract]
  33. Xie, Z. H., J. Zhang, R. P. Siraganian. 2000. Positive regulation of c-Jun N-terminal kinase and TNF-{alpha} production but not histamine release by SHP-1 in RBL-2H3 mast cells. J. Immunol. 164:1521.[Abstract/Free Full Text]
  34. Zhang, L., M. A. McCloskey. 1995. Immunoglobulin E receptor-activated calcium conductance in rat mast cell. J. Physiol. 483:59.[Medline]
  35. McCloskey, M. A., L. Zhang. 2000. Potentiation of Fc{epsilon}RI-activated Ca2+ current (I-CRAC) by cholera toxin: possible mediation by ADP ribosylation factor. J. Cell Biol. 148:137.[Abstract/Free Full Text]
  36. Benhamou, M., R. P. Siraganian. 1992. Protein-tyrosine phosphorylation: an essential component of Fc{epsilon}RI signaling. Immunol. Today 13:195.[Medline]
  37. Paolini, R., M. H. Jouvin, J. P. Kinet. 1991. Phosphorylation and dephosphorylation of the high-affinity receptor for immunoglobulin E immediately after receptor engagement and disengagement. Nature 353:855.[Medline]
  38. Kurimoto, Y., A. L. De Weck, C. A. Dahinden. 1991. The effect of interleukin 3 upon IgE-dependent and IgE-independent basophil degranulation and leukotriene generation. Eur. J. Immunol. 21:361.[Medline]
  39. Kimura, T., H. Sakamoto, E. Appella, R. P. Siraganian. 1996. Conformational changes induced in the protein tyrosine kinase p72Syk by tyrosine phosphorylation or by binding of phosphorylated immunoreceptor tyrosine-based activation motif peptides. Mol. Cell. Biol. 16:1471.[Abstract]
  40. Zhang, J., T. Kimura, R. P. Siraganian. 1998. Mutations in the activation loop tyrosines of protein tyrosine kinase Syk abrogate intracellular signaling but not kinase activity. J. Immunol. 161:4366.[Abstract/Free Full Text]
  41. Zhang, J., M. L. Billingsley, R. L. Kincaid, R. P. Siraganian. 2000. Phosphorylation of Syk activation loop tyrosines is essential for Syk function: an in vivo study using a specific anti-Syk activation loop phosphotyrosine Ab. J. Biol. Chem. 275:35442.[Abstract/Free Full Text]
  42. Kimura, T., H. Kihara, S. Bhattacharyya, H. Sakamoto, E. Appella, R. P. Siraganian. 1996. Downstream signaling molecules bind to different phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) peptides of the high affinity IgE receptor. J. Biol. Chem. 271:27962.[Abstract/Free Full Text]
  43. Pribluda, V. S., C. Pribluda, H. Metzger. 1994. Transphosphorylation as the mechanism by which the high-affinity receptor for IgE is phosphorylated upon aggregation. Proc. Natl. Acad. Sci. USA 91:11246.[Abstract/Free Full Text]
  44. Scharenberg, A. M., S. Lin, B. Cuenod, H. Yamamura, J. P. Kinet. 1995. Reconstitution of interactions between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor clustering. EMBO J. 14:3385.[Medline]
  45. Itoh, M., Y. Yoshida, K. Nishida, M. Narimatsu, M. Hibi, T. Hirano. 2000. Role of Gab1 in heart, placenta, and skin development and growth factor- and cytokine-induced extracellular signal-regulated kinase mitogen-activated protein kinase activation. Mol. Cell. Biol. 20:3695.[Abstract/Free Full Text]
  46. Harada, S., G. L. Esch, M. Holgado-Madruga, A. J. Wong. 2001. Grb-2-associated binder-1 is involved in insulin-induced egr-1 gene expression through its phosphatidylinositol 3'-kinase binding site. DNA Cell Biol. 20:223.[Medline]
  47. Derijard, B., M. Hibi, I. H. Wu, T. Barrett, B. Su, T. L. DENG, M. Karin, R. J. Davis. 1994. JNK1: a protein-kinase stimulated by UV-light and HA-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76:1025.[Medline]
  48. Han, J., J. D. Lee, L. Bibbs, R. J. Ulevitch. 1994. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cell. Science 265:808.[Abstract/Free Full Text]
  49. Franke, T. F., S. I. Yang, T. O. Chan, K. Datta, A. Kazlauskas, D. K. Morrison, D. R. Kaplan, P. N. Tsichlis. 1995. The protein-kinase encoded by the Akt protooncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81:727.[Medline]
  50. Franke, T. F., D. R. Kaplan, L. C. Cantley. 1997. PI3K: downstream AKTion blocks apoptosis. Cell 88:435.[Medline]
  51. Burgering, B. M. T., P. J. Coffer. 1995. Protein-kinase B (c-Akt) in phosphatidylinositol 3-OH kinase signal transduction. Nature 376:599.[Medline]
  52. Kimura, T., H. Sakamoto, E. Appella, R. P. Siraganian. 1997. The negative signaling molecule inositol polyphosphate 5-phosphatase, SHIP, binds to tyrosine phosphorylated {beta} subunit of the high affinity IgE receptor. J. Biol. Chem. :272.
  53. Barker, S. A., D. Lujan, B. S. Wilson. 1999. Multiple roles for PI3-kinase in the regulation of PLC{gamma} activity and Ca2+ mobilization in antigen-stimulated mast cells. J. Leukocyte Biol. 65:321.[Abstract]
  54. Barker, S. A., K. K. Caldwell, J. R. Pfeiffer, B. S. Wilson. 1998. Wortmannin-sensitive phosphorylation, translocation, and activation of PLC{gamma}1, but not PLC{gamma}2, in antigen-stimulated RBL-2H3 mast cells. Mol. Biol. Cell 9:483.[Abstract/Free Full Text]
  55. Falasca, M., S. K. Logan, V. P. Lehto, G. Baccante, M. A. Lemmon, J. Schlessinger. 1998. Activation of phospholipase C{gamma} by PI3-kinase-induced PH domain-mediated membrane targeting. EMBO J. 17:414.[Medline]
  56. Bae, Y. S., L. G. Cantley, C. S. Chen, S. R. Kim, K. S. Kwon, S. G. Rhee. 1998. Activation of phospholipase C{gamma} by phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 273:4465.[Abstract/Free Full Text]
  57. Scharenberg, A. M., O. El-Hillal, D. A. Fruman, L. O. Beitz, Z. M. Li, S. Q. Lin, I. Gout, L. C. Cantley, D. J. Rawlings, J. P. Kinet. 1998. Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P-3) Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals. EMBO J. 17:1961.[Medline]
  58. Rodrigues, G. A., M. Falasca, Z. T. Zhang, S. H. Ong, J. Schlessinger. 2000. A novel positive feedback loop mediated by the docking protein Gab1 and phosphatidylinositol 3-kinase in epidermal growth factor receptor signaling. Mol. Cell. Biol. 20:1448.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page