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The Journal of Immunology, 2006, 176: 2421-2429.
Copyright © 2006 by The American Association of Immunologists

Scaffolding Adapter Grb2-Associated Binder 2 Requires Syk to Transmit Signals from Fc{epsilon}RI1

Min Yu*, Cliff A. Lowell{dagger}, Benjamin G. Neel* and Haihua Gu2,*

* Cancer Biology Program, Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115; and {dagger} Department of Laboratory Medicine, University of California, San Francisco, CA 94143


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


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells are the major effector cells for the allergic response. Fc{epsilon}RI, the high-affinity receptor for IgE presents on mast cells and basophils, binds the Fc portion of IgE. Fc{epsilon}RI becomes activated when IgE prebound to Fc{epsilon}RI is cross-linked by the binding of multivalent Ag. Activated Fc{epsilon}RI triggers the release of performed granules (degranulation) and lipid-derived mediators that evoke the immediate hypersensitivity reaction. Fc{epsilon}RI cross-linking also results in activation of gene expression for various cytokines and chemokines that can further enhance the initial hypersensitivity response.

Fc{epsilon}RI consists of one ligand-binding {alpha}-chain and three associated subunits (one beta-chain and two {gamma}-chains). The beta- and {gamma}-chains contain ITAMs without any intrinsic enzymatic activity. It is generally accepted that upon Fc{epsilon}RI cross-linking, Fc{epsilon}RI-associated Src family protein tyrosine kinase (SFK)3 Lyn becomes activated and phosphorylates the beta- and {gamma}-chain ITAMs. Phosphorylated ITAMs in the beta-chain recruits and activates additional Lyn whereas phosphorylated ITAMs in the {gamma}-chain recruit and activate another key protein tyrosine kinase (PTK) Syk, which ultimately triggers various mast cell responses (1). A recent report showed that another SFK, Fyn, also is important for Fc{epsilon}RI-initiated signaling and biological responses (2). Subsequent to these initial signaling events, critical downstream signaling molecules, including PI3K, become activated, leading to various mast cell responses. Although much is known about initial PTK activation following Fc{epsilon}RI cross-linking, it is still not well-understood how this results in the activation of various mast cell responses.

Recent studies indicate that adapter proteins including linker for activation of T cells (LAT) (3), Src homology 2 domain-containing leukocyte protein of 76 kDa (4), Grb2-associated binder (Gab) 2 (5), and non-T cell activation linker/linker for activation of B cells (6, 7) play key roles in mediating Fc{epsilon}RI-evoked responses in mast cells. Gab2 belongs to the Gab2/daughter of sevenless family of scaffolding adapters that include mammalian Gab1, Gab2, and Gab3, Drosophila daughter of sevenless, and Caenorhabditis elegans Soc-1 (8, 9). Like its relatives, Gab2 contains an N-terminal pleckstrin homology (PH) domain, several proline-rich motifs, and multiple tyrosyl phosphorylation sites. The PH domain is a modular domain known to bind inositol phospholipids (10). The Gab1 PH domain preferentially binds phosphatidylinositol-3,4,5-trisphosphate (PIP3) (11, 12), and is essential for Gab1 function in branching morphogenesis (12). The Gab2 PH domain is critical for recruiting Gab2 to the phagocytic cup in macrophages (13), but dispensable for Gab2 function in IL-3/GM-CSF signaling (14). Although the lipid-binding specificity of Gab2 PH has not been determined, Gab2 PH domain recruitment to the phagocytic cup is sensitive to pretreatment with wortmannin, a specific inhibitor of PI3K, suggesting that Gab2 PH may also bind PI3K lipid products (13). Two of the proline-rich motifs in Gab2 are binding sites for the SH3 of Grb2 (15, 16). Grb2 binding to Gab2 plays a key role in recruiting Gab2 to its upstream receptors. In IL-3/GM-CSF signaling, the Gab2/Grb2 complex via Grb2 Src homology 2 (SH2) domain interacts with tyrosyl-phosphorylated Shc. Shc via its phosphotyrosine-binding (PTB) domain interacts with the tyrosyl-phosphorylated IL-3 beta common (betac). Therefore, Gab2 is recruited to the betac via the Shc/Grb2 complex (14). Gab2/Grb2 complex via the Grb2 SH2 domain is recruited to tyrosyl-phosphorylated BCR-Abelson tyrosine kinase and plays key role in mediating BCR-Abelson tyrosine kinase transformation (17).

Upon receptor activation, Gab2 becomes tyrosyl-phosphorylated and associated with SHP-2, a tyrosine phosphatase, required for optimal activation of Erk (18, 19) and immediate early gene transcription (20). Tyrosyl-phosphorylated Gab2 also binds p85, the regulatory subunit of PI3K, resulting in activation of the PI3K pathway (14, 21).

Previously, we found that Gab2 is required for Fc{epsilon}RI-triggered responses including degranulation and cytokine gene expression in bone marrow-derived mast cells (BMMC) (5). Gab2 is also required for mast cell development in specific tissues mostly likely via signaling through c-Kit (5, 22). One key roles of Gab2 in the Fc{epsilon}RI-initiated response is to activate the PI3K pathway (5), which is required for mast cell degranulation (23). Interestingly, Gab2 has also been found to be more important for Fc{epsilon}RI-evoked chemokine gene expression (24). A recent report also suggests that Gab2 regulates degranulation by activating small GTPase RhoA and microtubule polymerization (25). Consistent with the important role of Gab2 in Fc{epsilon}RI-signaling responses, Gab2 is found to be in the same specialized membrane domain (osmiophilic patches) with Fc{epsilon}RIbeta upon Fc{epsilon}RI cross-linking (26). However, it is still not clear how Gab2 is recruited to the activated Fc{epsilon}RI and becomes activated to participate in the Fc{epsilon}RI-initiated degranulation response. Furthermore, the PTK required for Gab2 tyrosyl phosphorylation remains controversial. One group reported that Gab2 tyrosyl phosphorylation is dramatically increased in Lyn–/– BMMC (2); another group claimed that Gab2 phosphorylation remains normal in Lyn–/– BMMC (27). Although Fyn is reportedly required for Gab2 phosphorylation (2), it is not clear whether Gab2 is a direct substrate of Fyn. In this study, we investigated how Gab2 becomes tyrosyl-phosphorylated and which PTKs are responsible for Gab2 tyrosyl phosphorylation.


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

The Gab2R32C mutant cDNA was generated by PCR using the pBluescript (Stratagene) HA-Gab2 as template and the following primers: 1) 5'-A CCA GCA TTT CTT CCA CGC GT A GCG-3' is the primer encoding the R->C 32 mutation; 2) 5'-ATC GGA TCC CCG AAT ATG AGC GGC-3' encoding the 5' end of Gab2 cDNA; and 3) 5'-GCT CTA GAA CTA GTG GAT CCA-3': encoding the 3' end of Gab2 and the hemagglutinin (HA) epitope Tag. The PCR product was ligated into pBluescript and verified by DNA sequencing. The HA-Gab2 wild-type (WT) and R32C fragments released by restriction enzyme digestion from pBluescript and the HA-Gab2{Delta}Grb2 fragment released from a pEBB plasmid (17) were cloned into the retroviral vector pMXs-puro (a gift from T. Kitomura, Tokyo University, Tokyo, Japan) or MSCV-IRES-GFP (13). To generate the pGex-4T-3 Gab2PH WT and Gab2 PH R32C constructs, Gab2 PH WT (1–120 aa; WT) and PH R32C cDNAs were amplified by PCR with the following primers from pBluescript HA-Gab2WT and Gab2R32C, respectively: 5'-ATC GGA TCC CCG AAT ATG AGC GGC-3' and 5'-TCA CTC GAG TTA GAA GCC GCA GAT CTG GCA G-3'. The PCR fragments were cloned into BamHI- and XhoI-digested pGex-4T-3. The GST-Shc-SH2 and GST-Shc-PTB domain constructs were provided by Dr. K. S. Ravichandran (University of Virginia Medical School, Charlottesville, VA). The GST-Grp1 PH plasmid is a gift from Dr. S Field (Beth Israel Deaconess Medical Center, Boston, MA).

Mice and cell culture

Gab2–/– mice (129sv/J x C57BL/6J mixed background) were generated as described previously (5). Lyn–/– mice (129sv/J x C57BL/6J mixed background) were provided by Dr. T. Yamamoto (University of Tokyo, Tokyo, Japan). Fyn–/– mice (C57BL/6J x 129S mixed background) were purchased from The Jackson Laboratory. All the mice work was followed to a protocol approved by the Harvard Medical Area Standing Committee on Animals. BMMC were cultured as described previously (5). Briefly, bone marrow cells from 2- to 4-mo-old mice were incubated in IMDM with 10% heat-inactivated FBS (HyClone), 2 mM L-glutamine, 0.1 mM nonessential amino acids (NEAA), 1 mM sodium pyruvate, 1000 U/ml penicillin, 1000 µg/ml streptomycin, 50 µM 2-ME, 4 ng/ml recombinant murine IL-3 (BioSource International). For Fyn–/– mice or Lyn–/– mice and WT littermates, 4 ng/ml IL-3 and 20 ng/ml stem cell factor (SCF) were used to obtain BMMC from littermate WT+/+ and Fyn–/– or Lyn–/– mice as described (2). After 5 wk, these cultures consist of ~95% mast cells reflected by surface expression of Fc{epsilon}RI and c-Kit. Five- to 8-wk-old BMMC cultures were used for biochemical and degranulation assays. Syk–/– BMMC were generated from bone marrow chimeric mice (C57BL/6J) transplanted with Syk–/– fetal liver cells as described (28). Three months after transplant, chimeric mice were killed and the bone marrows were used to obtain Syk–/– BMMC as described above. Three-month-old WT C57BL/6J mice were used to generate Syk+/+ BMMC. The MC/9 murine mast cell line, purchased from the American Type Culture Collection, was passaged in RPMI 1640 supplemented with 10% FBS, 5% T-Stim supplement (BD Biosciences), 0.1 mM NEAA, 2 mM L-glutamine, 0.1 mM NEAA, 1 mM sodium pyruvate, 1000 U/ml penicillin, 1000 µg/ml streptomycin, and 50 µM 2-ME. The anti-DNP IgE hybridoma cell line (106/ml), a gift from Dr. F.-T. Liu (University of California–Davis, Sacramento, CA) (29), was grown in PFHM-II protein-free hybridoma medium (Invitrogen Life Technologies) for 4–5 days. The resultant supernatant that typically contains 10–20 µg/ml anti-DNP-IgE was used to sensitize mast cells at a 1/10–20 dilution overnight. For cell stimulation, sensitized BMMC were washed, starved in IMDM + 1% BSA for 3–4 h at 37°C, and resuspended in modified Tyrode’s buffer (135 mM NaCl, 5 mM KCl, 1 mM MgCl, 1.8 mM CaCl2, 10 mM HEPES (pH 7.4), 5.6 mM glucose, and 0.1% BSA) before stimulation with indicated concentrations of DNP (Sigma-Aldrich).

Abs, reagents, immunoprecipitation, and Western blots

Anti-Gab2 Abs were generated as described (20). Anti-HA mAb (12CA5) was described previously (14). Anti-murine Fc{epsilon}RI beta-chain mAb was provided by Dr. J. Rivera (National Institutes of Health, Bethesda, MD). Rabbit anti-Syk Abs were provided by Dr. J. Cambier (National Jewish Medical and Research Center, Denver, CO) or purchased from Cell Signaling Technology. Anti-Grb2, Akt1/2, Lyn, SHP-2 rabbit Abs, and anti-GST mAb were purchased from Santa Cruz Biotechnology. Anti-phospho-Akt (Ser473)- and -Syk Abs were purchased from Cell Signaling Technology. Monoclonal anti-phosphotyrosine Ab (4G10) was obtained from Upstate Biotechnology. Anti-Shc rabbit Abs were purchased from BD Transduction Laboratories. The Syk inhibitor piceatannol was obtained from Calbiochem. Cells were lysed in 1% Nonidet-P40 lysis buffer as described previously (5). Total cell lysates or immunoprecipitates were resolved by SDS-PAGE, immunoblotted with indicated primary Abs, followed by HRP-conjugated anti-rabbit or anti-mouse IgG (Amersham Biosciences), and developed by ECL (Amersham Biosciences). Bands in Western blots were quantified by densitometry analysis using NIH Image 1.63F software. The intensities of phospho-Akt, phospho-Gab2, and phospho-Syk were normalized to the corresponding total Akt, Gab2, and Syk signal, respectively.

Flow cytometry analysis (FACS)

BMMCs were prebound with anti-DNP IgE, washed with PBS containing 3% FBS, and stained with FITC-anti-mouse IgE rat mAb R35-72 (IgG1) or FITC-rat monoclonal IgG1 as control. Stained cells were analyzed using a FACScan (BD Biosciences). BMMC were stained with rat mAb FITC-anti-CD117 or FITC-isotype control Ab to measure surface expression of c-Kit. All these FITC-conjugated Abs were purchased from BD Pharmingen.

Mast cell degranulation assays

BMMC (5 x 105) were sensitized with anti-DNP IgE supernatants overnight, washed, resuspended in modified Tyrode’s buffer, stimulated with 10 ng/ml DNP for 7 min, and centrifuged (4°C; 300 x g, 5 min). Supernatants were assayed for beta-hexosaminidase activity as described (30). Degranulation is quantified as the percent of beta-hexosaminidase released into the medium as a fraction of the total cellular beta-hexosaminidase.

Retroviral infection of BMMC and MC9

PMXs-puro and MSCV-IRES-GFP retroviral plasmids were transfected into the ecotrophic packaging cell line PlateE (31) (provided by T. Kitamura, Tokyo University, Tokyo, Japan) using Fugene reagent (Roche). Virus-containing culture supernatants were collected 2 days later. Bone marrow cells cultured in IL-3-containing IMDM medium for 12 days were spin-infected (2500 rpm, 90 min) with pMXs-puro virus supernatants in the presence of 4 µg/ml polybrene, and then incubated at 37°C for 18–24 h. Infected cells were selected in the presence of 0.8 µg/ml puromycin for 10–14 days, and then cultured in the absence of puromycin for 4–5 wk. For infection of MC9 with MSCV-IRES-GFP viruses, MC9 cells were resuspended in fresh medium and incubated with an equal volume of virus supernatant in the presence of 4 µg/ml polybrene at 37°C overnight. One week later, GFP-positive infected cells were purified by FACS.

Protein/lipid overlay assay

Bacterial-expressing GST fusion proteins were purified as described (32). GST-fusion proteins purified by glutathione agarose beads (Sigma-Aldrich) were washed, eluted with an equal volume of 20 mM glutathione (Sigma-Aldrich), resolved by SDS-PAGE, and quantified by staining with Coomassie brilliant blue.

Phosphatidylinositol (PI) lipids were purchased from Echelon Biosciences, reconstituted in chloroform:methanol:water (1:2:0.8), and spotted onto HyBond C extra membranes (Amersham Biosciences) at 100, 50, 25, and 12.5 pmol/spot, respectively. Membranes were air-dried, blocked in TBST (150 mM NaCl, 10 mM Tris-Cl (pH 8.0), 0.05% Tween 20) + 3% fat-free BSA (Sigma-Aldrich)) for 1 h, incubated with 1 µg/ml purified GST-fusion proteins in TBST + 3% fat-free BSA overnight, and then blotted with anti-GST mAb.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Lyn is required for Gab2 phosphorylation

We first asked which PTKs are required for Gab2 tyrosyl phosphorylation. It is generally believed that Lyn is the initial tyrosine kinase activated upon Fc{epsilon}RI cross-linking (1). However, whether Lyn is required for Gab2 tyrosyl phosphorylation remains controversial. Flow cytometry analysis showed that Lyn+/+ and Lyn–/– BMMC have similar surface expression of IgE receptor and c-Kit (Fig. 1A). However, while Gab2 was robustly tyrosyl-phosphorylated in Lyn+/+ BMMC, it was hardly tyrosyl-phosphorylated upon Fc{epsilon}RI cross-linking in Lyn–/– BMMC (Fig. 1B). We also found, in agreement with published reports (33), that Syk activation is severely inhibited in Lyn–/– BMMC (data not shown). These data suggest that Lyn and/or Syk are required for Gab2 tyrosyl phosphorylation. Interestingly, Fc{epsilon}RI cross-linking still induced similar mobility shift of Gab2 in Lyn+/+ and Lyn–/– BMMC (Fig. 1B). In response to various stimuli, Gab2 also shows a decreased in mobility shift in SDS-PAGE, which is mainly due to serine and threonine phosphorylation (14, 34, 35). This result suggests that loss of Lyn does not affect serine and threonine phosphorylation (Gab2).


Figure 1
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  		FIGURE 1. Fc{epsilon}RI-evoked tyrosyl phosphorylation of Gab2 is impaired in Lyn–/– BMMC. A, Lyn+/+ and Lyn–/– BMMC express similar surface IgE receptor and c-Kit. BMMC from Lyn+/+ and Lyn–/– mice were analyzed for cell surface IgE binding (top) and c-Kit (bottom) by FACS as described in Materials and Methods. B, Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation is impaired in Lyn–/– BMMC. Lyn+/+ and Lyn–/– BMMC were sensitized by anti-DNP-IgE, starved, and stimulated with 10 ng/ml DNP for the indicated times. Lysates were immunoprecipitated with anti-Gab2 serum, immunoblotted with anti-phosphorylation tyrosine Ab (pTyr), and reprobed with anti-Gab2 Abs. Data shown here represent one of the three independent experiments with similar results.

 
Syk is required for Gab2 phosphorylation

Because Lyn is required for Syk activation in response to Fc{epsilon}RI activation, we asked whether Gab2 tyrosyl phosphorylation also depends on Syk. We first examined Gab2 tyrosyl phosphorylation in WT BMMC pretreated with piceatannol, a pharmacological inhibitor of Syk. Piceatannol pretreatment completely eliminated Fc{epsilon}RI-evoked Syk and Gab2 tyrosyl phosphorylation, and Gab2 mobility shift, respectively (Fig. 2A). To further support a role of Syk in Gab2 tyrosyl phosphorylation, we assessed Gab2 tyrosyl phosphorylation in Syk+/+ and Syk–/– BMMC. FACS analysis indicated that Syk+/+ and Syk–/– BMMC express similar levels of cell surface IgE receptor and c-Kit (Fig. 2B), suggesting that loss of Syk has no effect on mast cell differentiation (data not shown). However, in agreement with the effect of piceatannol, Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation and mobility shift was completely blocked in Syk–/– BMMC (Fig. 2C). We also found that Syk becomes coimmunoprecipitated with Gab2 in WT BMMC upon Fc{epsilon}RI cross-linking (Fig. 2D). Gab2 association with Syk has also been reported when BaF3 cells were stimulated through integrin cross-linking (36). In contrast, SCF-stimulated Gab2 tyrosyl phosphorylation occurred normally in Syk–/– BMMC compared with Syk+/+ BMMC (data not shown). Collectively, these data suggest that Syk, rather a SFK, is the proximal PTK required specifically for Fc{epsilon}RI-evoked Gab2 tyrosyl and serine/threonine phosphorylation.


Figure 2
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  		FIGURE 2. Syk is required for Fc{epsilon}RI-evoked tyrosyl phosphorylation of Gab2. A, Syk inhibitor blocks Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation. DNP-IgE-sensitized WT BMMC were pretreated with DMSO (–) or 100 µg/ml Syk inhibitor piceatannol (+) for 1 h, and stimulated with 10 ng/ml DNP for 5 min. Lysates were immunoprecipitated with anti-Gab2 or Syk serum, immunoblotted with anti-pTyr, and reprobed with anti-Gab2 and Syk Abs, respectively. B, Syk+/+ and Syk–/– BMMC express similar IgE receptor and c-Kit. Syk+/+ and Syk–/– BMMC were analyzed for cell surface IgE binding (top) and c-Kit (bottom) by FACS as described in Materials and Methods. C, Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation is completely inhibited in Syk–/– BMMC. BMMC were sensitized by anti-DNP-IgE, starved, and stimulated with 10 ng/ml DNP for the indicated times. Gab2 tyrosyl phosphorylation was analyzed as in Fig. 1B. D, Syk becomes associated with Gab2 upon Fc{epsilon}RI cross-linking. WT BMMC were sensitized with anti-DNP-IgE, starved, and stimulated with 10 ng/ml DNP for 1 min. Lysates were immunoprecipitated with anti-Gab2 serum, immunoblotted with anti-pTyr, and reprobed with anti-Gab2 or Syk Abs. Data shown here represent one of the three independent experiments with similar results.

 
Decreased Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation correlates with decreased Syk activation in Fyn–/– BMMC

A recent report suggested that Fyn is the kinase that responsible for Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation and activation of PI3K (2). Therefore, we examined Fc{epsilon}RI evoked Gab2 tyrosyl phosphorylation and Akt activation in Fyn+/+ and Fyn–/– BMMC. In agreement with the previous report, loss of Fyn did not affect surface expression of IgE receptor and c-Kit in BMMC (Fig. 3A). In addition, Fc{epsilon}RI-evoked degranulation (data not shown) and Akt phosphorylation (Fig. 3B) were decreased in Fyn–/– BMMC compared with Fyn+/+ BMMC as reported (2). Gab2 tyrosyl phosphorylation was also reduced in Fyn–/– BMMC (Fig. 3C), but not completely inhibited as we found in Syk–/– BMMC. Importantly, Fc{epsilon}RI-evoked total Syk tyrosyl phosphorylation (Fig. 3D) was decreased in Fyn–/– BMMC compared with Fyn+/+ BMMC. These data suggest that the Fc{epsilon}RI-evoked Syk activation is impaired in Fyn–/– BMMC. Collectively, these data indicate that Fyn is required for the optimal activation of Syk in response to Fc{epsilon}RI engagement. Since we found that Syk is absolutely required for Gab2 tyrosyl phosphorylation (Fig. 2, A and B), our data strongly support a model that Fyn, via Syk, indirectly contributes to the Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation.


Figure 3
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  		FIGURE 3. Decreased Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation is correlated to reduced Syk phosphorylation in Fyn–/– BMMC. A, Fyn+/+ and Fyn–/– BMMC express similar IgE receptor and c-Kit. BMMC derived from Fyn+/+ and Fyn–/– mice were analyzed for cell surface IgE binding (top) and c-Kit (bottom) by FACS as described in Materials and Methods. B, Fc{epsilon}RI-evoked Akt activation is reduced in Fyn–/– BMMC. Anti-DNP IgE-sensitized BMMC were starved and stimulated with 10 ng/ml for the indicated time. Total cell lysates were immunoblotted with anti-phospho-Akt (Ser473) and reprobed with anti-Akt Abs. C, Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation is reduced in Fyn–/– BMMC. BMMC were sensitized by anti-DNP-IgE, starved, and stimulated with 10 ng/ml DNP for the indicated times. Gab2 tyrosyl phosphorylation was analyzed as in Fig. 1B (top). Gab2 tyrosyl phosphorylation quantified by densitometry was shown (bottom). D, Fc{epsilon}RI-evoked-Syk tyrosyl phosphorylation is reduced in Fyn–/– BMMC. BMMC were sensitized by anti-DNP-IgE, starved, and stimulated with 10 ng/ml DNP for the indicated times. Total Syk tyrosyl phosphorylation was analyzed as in Fig. 2C (top). Syk tyrosyl phosphorylation quantified by densitometry was shown (bottom). Data shown here represent one of the three independent experiments with similar results.  

 
Grb2-SH3 domain binding sites are required for Gab2 tyrosyl phosphorylation

Next, we asked which domain(s) in Gab2 is required for Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation. To address the role of Grb2 SH3 binding sites, we expressed HA-tagged Gab2 WT or a mutant of Gab2 (Gab2{Delta}Grb2), in which the Grb2-SH3 domain binding sites are mutated, in the MC/9 mast cell line. Upon Fc{epsilon}RI cross-linking, HA-tagged Gab2 WT or Gab2{Delta}Grb2 were immunoprecipitated by anti-HA Ab followed by anti-phosphorylation tyrosine immunoblotting (Fig. 4A). HA-Gab2 WT became robustly tyrosyl-phosphorylated and associated with Shc and Grb2. By contrast, HA-Gab2{Delta}Grb2 showed only weak tyrosyl phosphorylation (~75% decrease). HA-Gab2{Delta}Grb2 also failed to associate with Shc and Grb2. Thus, the Grb2-SH3 domain binding sites are required for Gab2 association with Shc and the majority of the Gab2 tyrosyl phosphorylation in response to Fc{epsilon}RI cross-linking.


Figure 4
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  		FIGURE 4. Grb2-SH3 binding sites are required for Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation and association with Shc. A, MC9 cells were infected MSCV-HA Gab2WT and MSCV-HA-Gab2 {Delta}Grb2. Infected cells were sensitized with anti-DNP-IgE, starved, and stimulated with DNP for 5 and 10 min. Lysates were immunoprecipitated with anti-HA Ab, immunoblotted with anti-pTyr, and reprobed with anti-HA, Shc, and Grb2 Abs. Gab2 tyrosyl phosphorylation quantified by densitometry was shown (middle). B, Shc SH2 domain can bind to Fc{epsilon}RI beta upon Fc{epsilon}RI cross-linking. BMMC were sensitized, starved, and stimulated with DNP. Lysates were incubated with GST alone, and GST-ShcPTB or GST-ShcSH2 fusion protein, immunoblotted with anti-pTyr, and reprobed with anti-Fc{epsilon}RI beta Ab. Data shown here represent one of the three independent experiments with similar results.

 
Shc was reported to bind a phosphorylated ITAM peptide derived from the Fc{epsilon}RI beta-chain (37). However, it is not clear whether the Shc SH2 domain or the PTB domain interacts with the tyrosyl-phosphorylated Fc{epsilon}RI beta-chain. To address this question, we incubated purified GST-fusion proteins containing either the Shc SH2 domain or PTB with lysates from unstimulated or Fc{epsilon}RI-activated mast cells. Only the GST-Shc SH2 domain was able to immunoprecipitate the Fc{epsilon}RI beta-chain, as validated by anti-phosphotyrosine and anti-Fc{epsilon}RI beta immunoblotting (Fig. 4B). These data suggest that Shc, via its SH2 domain, is recruited to Fc{epsilon}RI beta-chain. Shc then becomes tyrosyl-phosphorylated and recruits the Grb/Gab2 complex.

Gab2 PH domain also contributes to Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation

Our previously published data suggest that the Gab2 PH domain may bind PI3K lipid products, and is critical for recruiting Gab2 to the phagocytic cup in macrophages (13). However, the phospholipid-binding specificity of Gab2 PH has not been determined. To address this question, we performed protein/lipid overlay assays by incubating the GST-Gab2 PH domain fusion protein with membranes spotted with different amounts of PI lipids (Fig. 5A). The GST-Gab2 PH domain preferentially interacted PIP3 and P3,4P2, and to a lesser extent PI5P and PI4,5P2. As a control for the lipids spotted on the membrane, the GST-Grp1PH domain interacted only with PIP3, consistent with published reports about this PH domain’s specificity (38). To verify the PI lipid-binding specificity of Gab2 PH, we generated a mutant (R32C) Gab2 PH domain in which R32 is mutant to C. R32, a residue found conserved in other PH domains, is required for PI lipid binding (39). Importantly, the GST-Gab2 PH R32C fusion protein failed to bind PIP3 and PI3,4P2 in the lipid overlay assay (Fig. 5A). These data indicate that the Gab2 PH domain binds preferentially to PI3,4,5P3 and PI3,4P2, the lipid products of class IA PI3K, similar to the Gab1 PH domain as reported (11, 12).


Figure 5
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  		FIGURE 5. Gab2 PH domain binding to PI3K lipid products contributes to Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation. A, Gab2 PH domain preferentially binds to PIP3 and PI3,4P2 in a protein/lipid overlay assay in vitro. Membranes were spotted with PI lipids at 2-fold less in mass sequentially, incubated with GST-Gab2 PH, GST-Gab2-PH R32C, and GST-Grp1PH domain fusion proteins, and probed with anti-GST Abs. B, Gab2 R32C mutant showed transient Fc{epsilon}RI-evoked tyrosyl phosphorylation. BMMC were infected with vector alone, Gab2WT, and Gab2R32C pMXs-puro retrovirus. Infected cells were starved, sensitized with IgE, and stimulated with anti-DNP for the indicated times. Gab2 immunoprecipitates or total cell lysates from infected BMMC were immunoblotted with anti-pTyr, and probed with anti-Gab2 Abs. Gab2 tyrosyl phosphorylation quantified by densitometry was shown (bottom). Data shown here represent one of the three independent experiments with similar results.

 
Next, we assessed the role of the Gab2 PH domain in Fc{epsilon}RI-triggered tyrosyl phosphorylation. We expressed HA-tagged Gab2WT or Gab2 R32C in BMMC using retroviral transduction. Although HA-Gab2 R32C tyrosyl phosphorylation occurred normally initially (1 min), its phosphorylation was decreased by ~40% at later time points compared with Gab2 WT (Fig. 5B). This result indicates that Gab2 binding to PIP3 is critical for the sustained tyrosyl phosphorylation of Gab2 in Fc{epsilon}RI signaling.

Gab2 Grb2 SH3 binding sites and PH domain-binding PI lipids are required for Fc{epsilon}RI-evoked mast cell responses

To assess the role of the Grb2-SH3 domain binding sites and PH domain binding to PIP3 in Fc{epsilon}RI-evoked mast cell responses, we re-expressed Gab2WT, Gab2{Delta}Grb2, and Gab2R32C in Gab2–/– BMMC using retroviral transduction, and measured Fc{epsilon}RI-evoked mast cell degranulation by analyzing the release of beta-hexosaminidase. Re-expression of Gab2 WT increased degranulation by ~2.5-fold compared with vector control (Fig. 6A). This difference in degranulation shows comparable difference in degranulation between Gab2+/+ and Gab2–/– BMMC reported earlier (5). Re-expression of either Gab2{Delta}Grb2 or Gab2R32C only slightly increased degranulation (~50%) compared with vector control (Fig. 6A). Furthermore, re-expression of Gab2WT also increased Fc{epsilon}RI-evoked Akt phosphorylation dramatically (Fig. 6, B and C). However, compared with Gab2WT expressing cells, phosphorylation of Akt was decreased by ~60% in Gab2{Delta}Grb2 cells and Gab2R32C cells (at 10–15 min). As Akt is a downstream effector of PI3K, the results suggest that both Gab2 mutants of Gab2 cannot fully activate PI3K. Thus, both Grb2 SH3 binding sites and the Gab2 PH domain binding to PI3K lipid products are critical for Fc{epsilon}RI-evoked responses such as activation of PI3K and degranulation.


Figure 6
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  		FIGURE 6. Grb2-SH3 domain binding sites and the Gab2 PH domain are required for Fc{epsilon}RI-evoked degranulation and Akt activation in BMMC. A, Expression of Gab2{Delta}Grb2 or Gab2 R32C cannot rescue the defective Fc{epsilon}RI-evoked degranulation in Gab2–/– BMMC. Gab2–/– BMMC were infected with vector, Gab2WT, Gab2{Delta}Grb2, and Gab2R32C pMXs-puro-viruses as described in Materials and Methods. BMMC were sensitized with anti-DNP-IgE, and stimulated with DNP for 10 min. Degranulation was measured as percentage of beta-hexosaminidase release (top). Cell lysates were immunoblotted with anti-Gab2 Abs (bottom), indicating similar expression of Gab2WT, Gab2{Delta}Grb2, and Gab2R32C. B, Expression of Gab2{Delta}Grb2 cannot rescue the defective Akt activation compared with Gab2WT in Gab2–/– BMMC. BMMC were sensitized with anti-DNP-IgE, starved, and stimulated with DNP for the indicated times. Lysates were immunoblotted with Abs against phospho-Akt and Gab2, and reprobed with Abs against Akt. Akt phosphorylation was quantified by densitometry was shown (bottom). C, Expression of Gab2 R32C cannot rescue the defective Akt activation compared with Gab2 WT in Gab2–/– BMMC. BMMC were sensitized with anti-DNP-IgE, starved, and stimulated with DNP for the indicated times. Lysates were immunoblotted with Abs against phospho-Akt and Gab2, and reprobed with Abs against Akt. Akt phosphorylation quantified by densitometry was shown (bottom). Data shown here represent one of the three independent experiments with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Gab2 is one key tyrosyl-phosphorylated scaffolding adapter protein that plays a critical role in Fc{epsilon}RI-evoked mast cell responses including degranulation, cytokine, and chemokine gene transcription (5, 24, 25). In our present study, we have shown that Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation requires Syk by using the pharmacologic inhibitor of Syk and Syk-deficient mast cells. Although Lyn and Fyn are also critical for Gab2 tyrosyl phosphorylation, they act likely through activation of Syk. By expressing the mutant of the Gab2 protein in mast cells, we found that the Grb2-SH3 domain-binding sties and the PH domain are required for Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation, Akt activation, and degranulation. We showed that the Grb2-SH3 binding sites are required for Gab2 interaction with Shc while the Gab2 PH domain can preferentially bind the PIP3 and PI3,4P2.

Gab2 recruitment to Fc{epsilon}RI

A previous study using immunogold labeling suggested that Gab2 becomes associated with a pool of the membrane domain containing Fc{epsilon}RI beta and p85, the regulatory submit of PI3K, upon Fc{epsilon}RI cross-linking (26). Our study provides a biochemical explanation for how Gab2 is recruited to the Fc{epsilon}RI beta complex. Upon Fc{epsilon}RI cross-linking, the ITAM in the cytoplasmic tail of the beta-chain becomes tyrosyl-phosphorylated. Shc is recruited to the Fc{epsilon}RI beta via its SH2 domain and becomes tyrosyl-phosphorylated. Phosphorylated Shc provides a docking site for the Grb2 SH2 domain and recruits Gab2 that is in a constitutive complex with Grb2 via the Grb2 SH3 domain (16, 20). Subsequently, Gab2 becomes tyrosyl-phosphorylated in an Syk-dependent manner and recruits SH2 domain-containing signaling molecules including PI3K. The Gab2-activated PI3K lipid products bind to the Gab2 PH domain around the Fc{epsilon}RI beta, further potentiating recruitment. In this positive feedback loop, Gab2 contributes to the sustained activation of PI3K, required for Fc{epsilon}RI-evoked mast cell responses (Fig. 7). This model is similar to the role of Shc in recruiting Gab2 to IL-3/GM-CSF betac although Shc binds betac via its PTB domain (14). Likewise, the function of Gab2 PH binding to PIP3 is analogous to the proposed model for Gab1 action in epidermal growth factor receptor signaling (11).


Figure 7
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  		FIGURE 7. Model of Gab2 action in Fc{epsilon}RI signaling. Upon cross-linking of IgE prebound Fc{epsilon}RI by Ag, Fc{epsilon}RI-associated SFK (Lyn and Fyn) are activated. Then, the ITAM in beta-chain becomes phosphorylated and recruits Shc via the Shc SH2 domain. Shc becomes phosphorylated and interacts with Grb2 via the Grb2 SH2 domain, and subsequently recruits Gab2, which is in a constitutive complex with Grb2 via the Grb2 SH3 domain. Gab2 becomes tyrosyl-phosphorylated in a Syk-dependent manner and becomes associated with PI3K that produces PIP3. The Gab2 PH domain binds to PIP3, resulting in a sustained Gab2-signaling complex around Fc{epsilon}RI and Fc{epsilon}RI-evoked mast cell responses.

 
Previously, phosphorylated Fc{epsilon}RI beta was reported to interact with adapter protein Shc (37). Shc is also reported to become tyrosyl-phosphorylated and associated with Grb2 upon Fc{epsilon}RI cross-linking (40). However, it is not clear how Shc interacts with Fc{epsilon}RI beta and what role Shc plays in Fc{epsilon}RI signaling. We showed here that Shc can interact with the phosphorylated Fc{epsilon}RI beta specifically via its SH2 domain, not the PTB domain (Fig. 4B). This result is consistent with the predication by Scansite (41) that the cytoplasmic tail of Fc{epsilon}RI beta only contains tyrosine-containing motifs (Tyr217, Tyr223, and Tyr227) that may bind the Shc SH2 domain. Consistent with Shc recruiting Gab2 to Fc{epsilon}RI beta (Fig. 7), we found that Gab2 increases its association with Shc upon Fc{epsilon}RI cross-linking. A mutant of Gab2 ({Delta}Grb2), that cannot bind Grb2, lost its association with Shc and tyrosyl phosphorylation (Fig. 4A). Therefore, the Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation requires Gab2 association with the Shc/Grb2 complex.

A previous study strongly suggested that the Gab2 PH domain binds PI3K lipid products and is critical for Gab2 function in Fc{gamma}RI-mediated phagocytosis (13). Here, we showed that the Gab2 PH domain can preferentially bind PIP3, and PI3,4P2, the lipid products of PI3K, and the mutant Gab2 PH R32C domain lost its binding to these lipids (Fig. 5A). Gab1 and Gab2 PH domains share ~70% of the sequence identity. Especially, the regions in PH domain involved in direct PI lipid binding are conserved (20). Using the same protein/lipid overlay assay, we found that the Gab1 PH domain also binds preferentially PIP3 and PI3,4P2 (data not shown), in agreement with previously published work using a lipid vesicle-binding assay (11), validating the method of protein/lipid overlay assay in determining PI lipid-binding specificity for the Gab2 PH domain. We also found consistently, although to a lesser extent, that the GST-Gab2 PH domain binds to PI5P (Fig. 5A), a PI lipid with essentially unknown function (42). Likewise, the Gab1 PH domain binds to PI5P in the similar preference in this in vitro-binding assay (data not shown). Future study is needed to look into how PI5P binds to the Gab PH domain and whether this regulates Gab protein function.

Kinases phosphorylate Gab2

Our data strongly suggest that Syk is the major tyrosine kinase responsible for Gab2 tyrosyl phosphorylation upon Fc{epsilon}RI engagement whereas Lyn and Fyn contribute to Gab2 tyrosyl phosphorylation by acting upstream of Syk. A previous study suggested that Fc{epsilon}RI-triggered Gab2 tyrosyl phosphorylation requires Fyn while Syk is dispensable. Furthermore, Gab2 became associated with Fyn upon Fc{epsilon}RI engagement (2). However, under the condition that we immunoprecipitated almost all of Gab2 or Fyn from mast cell lysates using Abs against Gab2 or Fyn, we were unable to detect Gab2 association with Fyn (data not shown). Importantly, we found that Fc{epsilon}RI-triggered Syk tyrosyl phosphorylation was reduced accordingly in Fyn–/– BMMC compared with Fyn+/+ BMMC (Fig. 3D), suggesting that Fyn is required for optimal activation of Syk.

Because our result shows that Syk is absolutely required for Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation (Fig. 2C), there are several possibilities regarding how Fyn and Syk control Gab2 tyrosyl phosphorylation. A simple model is that Fyn regulates the activation of Syk, which phosphorylates Gab2 directly (Fig. 7). Alternatively, Syk phosphorylates Gab2 at several sites including the site that recruits Fyn. Upon interacting with Gab2, Fyn may phosphorylate other tyrosine residues in Gab2. Lastly, Syk may contribute to Gab2 tyrosyl phosphorylation by controlling the phosphorylation of the tyrosine residue in Fc{epsilon}RI beta that recruits the Shc/Grb2/Gab2 complex as discussed above (Fig. 7). Consistent with this idea, a previous study revealed that the Fc{epsilon}RI-evoked Shc/Grb2 complex depends on Syk (40). At present, we could not determine whether Syk also indirectly contributes to tyrosyl phosphorylation of Gab2 through activation of other protein kinases, such as Tec family kinase member Btk and Itk (1), present in mast cells. Syk is required for activation of Btk in mast cells (43). The use of Btk and/or Itk-deficient mast cells are required to address the potential role of Tec family kinase Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation in the future.

Our results also indicate that Lyn is required for Gab2 phosphorylation. Because Lyn acts upstream of Syk in Fc{epsilon}RI signaling (1), it is logical to conclude that Lyn controls Gab2 phosphorylation mainly through activation of Syk. We cannot exclude the possibility that Lyn may phosphorylate Gab2 directly. Consistent with this idea, Gab2 has been suggested to mediate G-CSF-invoked differentiation in an Lyn-dependent manner (44). However, our result does not agree with published reports from Rivera’s group (2) which showed that Lyn negatively regulates Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation through the activation of C-terminal Src kinase, an inhibitory PTK for SFK, which inhibits Fc{epsilon}RI-evoked Fyn activation in mast cells (45). There are several possibilities that may account for this discrepancy. The conditions for generating BMMC are different. We typically use only IL-3-containing medium to generate BMMC from bone marrow. However, Fc{epsilon}RI-evoked Gab2 tyrosyl phosphorylation was still severely impaired in Lyn–/– BMMC derived from the same medium condition (IL-3 + SCF) as described (2). The Lyn–/– mice used in our study were generated differently from the Lyn–/– mice used in the Parravicini et al. (2) study. In the former case, the promoter of region of the Lyn gene was disrupted (46). In the latter case, exon 3-7 of the Lyn gene was eliminated (47). However, both Lyn–/– mice are null for Lyn protein. The possibility that we favor is the difference of genetic background of these two Lyn–/– mice. The Lyn–/– mice used in our study was in the mixed background of sv129/J and C57BL6 while the mice used in the Parravicini et al. (2) study was in the pure C57BL6 background, which could contribute to the different responses of the mast cells derived. Notably, the Fc{epsilon}RI-evoked degranulation is impaired in Lyn–/– BMMC in our study (data not shown) while the degranulation was enhanced in Lyn–/– BMMC used in the Parravicini et al. (2) study. One possible implication from this discrepancy is the genetic background that may contribute to different expression and/or activation of different SFKs that can activate parallel pathways critical for Fc{epsilon}RI-evoked responses in mast cells (48).

Besides tyrosyl phosphorylation, our data also indicates that Syk is required, while Lyn or Fyn is dispensable, for Fc{epsilon}RI-evoked serine/threonine phosphorylation. Published reports showed that activation of the PI3K (14, 34) and/or Erk (35) pathways contributes to serine phosphorylation in Gab2. Serine phosphorylation on Gab2 has been suggested as a negative feedback mechanism to turn off the Gab2-initiated positive signaling by tyrosyl phosphorylation in response to growth factor and cytokine stimulation (34, 35). It will be important to study in the future how serine/threonine phosphorylation affects Gab2 functions in Fc{epsilon}RI-evoked responses in mast cells.

The Lyn/Syk/LAT pathway and the alternative pathway involving Fyn/Gab2/PI3K have been suggested to play critical roles in Fc{epsilon}RI-evoked degranulation of mast cells (2). Our result indicates that Syk, at least a pool of Syk, is critical for the activation of the Gab2/PI3K in the Fc{epsilon}RI-evoked degranulation response (Fig. 7). Furthermore, our data provide a further biochemical basis for developing potential drugs that specifically inhibit Syk activation (49) to treat allergy.


   Acknowledgments
 
We thank Naoko Imanaka, Yongping Wang, and Wenkai Yang for excellent technical assistance.


   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 National Institutes of Health Grants R01-AI51612 (to H.G) and R01-DK50693 (to B.G.N). H.G. is a recipient of the Junior Faculty Scholar Award from the American Association of Hematology. Back

2 Address correspondence and reprint requests to Dr. Haihua Gu, Harvard Medical School, New Research Building, NRB 1030N, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: hgu{at}bidmc.harvard.edu Back

3 Abbreviations used in this paper: SFK, Src family protein tyrosine kinase; PTK, protein tyrosine kinase; LAT, linker for activation of T cells; Gab, Grb2-associated binder; PH, pleckstrin homology; PIP3, phosphatidylinositol-3,4,5-trisphosphate; SH, Src homology; PTB, phosphotyrosine binding; betac, beta common; BMMC, bone marrow-derived mast cell; HA, hemagglutinin; WT, wild type; NEAA, nonessential amino acid; SCF, stem cell factor; PI, phosphatidylinositol. Back

Received for publication September 29, 2005. Accepted for publication November 28, 2005.


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 Abstract
 Introduction
 Materials and Methods
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