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RI Ε 2 Requires Syk to Transmit Signals from Fc Scaffolding Adapter Grb2-Associated Binder

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RI Ε 2 Requires Syk to Transmit Signals from Fc Scaffolding Adapter Grb2-Associated Binder Scaffolding Adapter Grb2-Associated Binder 2 Requires Syk to Transmit Signals from Fc ε RI This information is current as Min Yu, Cliff A. Lowell, Benjamin G. Neel and Haihua Gu of October 2, 2021. J Immunol 2006; 176:2421-2429; ; doi: 10.4049/jimmunol.176.4.2421 http://www.jimmunol.org/content/176/4/2421 Downloaded from References This article cites 49 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/176/4/2421.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Scaffolding Adapter Grb2-Associated Binder 2 Requires Syk to Transmit Signals from Fc⑀RI1 Min Yu,* Cliff A. Lowell,† Benjamin G. Neel,* and Haihua Gu2* Scaffolding adapter Grb2-associated binder 2 (Gab2) is a key component of Fc⑀RI signaling in mast cells, required for the activation of PI3K. To understand how Gab2 is activated in Fc⑀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. Further- more, the Grb2-SH3 binding sites and PH domain binding to PI3K lipid products are required for Gab2 function in Fc⑀RI-evoked Downloaded from degranulation and Akt activation. Our data strongly suggest a model for Gab2 action in Fc⑀RI signaling. The Grb2 SH3 binding sites play a critical role in bringing Gab2 to Fc⑀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⑀RI-evoked degranulation of mast cells. The Journal of Immunology, 2006, 176: 2421–2429. http://www.jimmunol.org/ ast cells are the major effector cells for the allergic whereas phosphorylated ITAMs in the ␥-chain recruit and activate response. Fc⑀RI, the high-affinity receptor for IgE pre- another key protein tyrosine kinase (PTK) Syk, which ultimately M sents on mast cells and basophils, binds the Fc portion triggers various mast cell responses (1). A recent report showed of IgE. Fc⑀RI becomes activated when IgE prebound to Fc⑀RI is that another SFK, Fyn, also is important for Fc⑀RI-initiated sig- cross-linked by the binding of multivalent Ag. Activated Fc⑀RI naling and biological responses (2). Subsequent to these initial triggers the release of performed granules (degranulation) and lip- signaling events, critical downstream signaling molecules, includ- id-derived mediators that evoke the immediate hypersensitivity re- ing PI3K, become activated, leading to various mast cell re- action. Fc⑀RI cross-linking also results in activation of gene ex- sponses. Although much is known about initial PTK activation by guest on October 2, 2021 pression for various cytokines and chemokines that can further following Fc⑀RI cross-linking, it is still not well-understood how enhance the initial hypersensitivity response. this results in the activation of various mast cell responses. Fc⑀RI consists of one ligand-binding ␣-chain and three associ- Recent studies indicate that adapter proteins including linker for ated subunits (one ␤-chain and two ␥-chains). The ␤- and ␥-chains activation of T cells (LAT) (3), Src homology 2 domain-containing contain ITAMs without any intrinsic enzymatic activity. It is gen- leukocyte protein of 76 kDa (4), Grb2-associated binder (Gab) 2 erally accepted that upon Fc⑀RI cross-linking, Fc⑀RI-associated (5), and non-T cell activation linker/linker for activation of B cells Src family protein tyrosine kinase (SFK)3 Lyn becomes activated (6, 7) play key roles in mediating Fc⑀RI-evoked responses in mast and phosphorylates the ␤- and ␥-chain ITAMs. Phosphorylated cells. Gab2 belongs to the Gab2/daughter of sevenless family of ITAMs in the ␤-chain recruits and activates additional Lyn scaffolding adapters that include mammalian Gab1, Gab2, and Gab3, Drosophila daughter of sevenless, and Caenorhabditis el- egans Soc-1 (8, 9). Like its relatives, Gab2 contains an N-terminal *Cancer Biology Program, Division of Hematology/Oncology, Department of Med- pleckstrin homology (PH) domain, several proline-rich motifs, and icine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115; and †Department of Laboratory Medicine, University of California, San multiple tyrosyl phosphorylation sites. The PH domain is a mod- Francisco, CA 94143 ular domain known to bind inositol phospholipids (10). The Gab1 Received for publication September 29, 2005. Accepted for publication November PH domain preferentially binds phosphatidylinositol-3,4,5- 28, 2005. trisphosphate (PIP3) (11, 12), and is essential for Gab1 function in The costs of publication of this article were defrayed in part by the payment of page branching morphogenesis (12). The Gab2 PH domain is critical for charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. recruiting Gab2 to the phagocytic cup in macrophages (13), but 1 This work was supported by National Institutes of Health Grants R01-AI51612 (to dispensable for Gab2 function in IL-3/GM-CSF signaling (14). H.G) and R01-DK50693 (to B.G.N). H.G. is a recipient of the Junior Faculty Scholar Although the lipid-binding specificity of Gab2 PH has not been Award from the American Association of Hematology. determined, Gab2 PH domain recruitment to the phagocytic cup is 2 Address correspondence and reprint requests to Dr. Haihua Gu, Harvard Medical sensitive to pretreatment with wortmannin, a specific inhibitor of School, New Research Building, NRB 1030N, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: [email protected] PI3K, suggesting that Gab2 PH may also bind PI3K lipid products (13). Two of the proline-rich motifs in Gab2 are binding sites for 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 the SH3 of Grb2 (15, 16). Grb2 binding to Gab2 plays a key role binder; PH, pleckstrin homology; PIP3, phosphatidylinositol-3,4,5-trisphosphate; SH, in recruiting Gab2 to its upstream receptors. In IL-3/GM-CSF sig- Src homology; PTB, phosphotyrosine binding; ␤c, ␤ common; BMMC, bone mar- row-derived mast cell; HA, hemagglutinin; WT, wild type; NEAA, nonessential naling, the Gab2/Grb2 complex via Grb2 Src homology 2 (SH2) amino acid; SCF, stem cell factor; PI, phosphatidylinositol. domain interacts with tyrosyl-phosphorylated Shc. Shc via its Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 2422 Gab2 TRANSMIT SIGNALS FROM Fc⑀RI IN MAST CELLS phosphotyrosine-binding (PTB) domain interacts with the tyrosyl- purchased from The Jackson Laboratory. All the mice work was followed phosphorylated IL-3 ␤ common (␤c). Therefore, Gab2 is recruited to a protocol approved by the Harvard Medical Area Standing Committee to the ␤c via the Shc/Grb2 complex (14). Gab2/Grb2 complex via 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 the Grb2 SH2 domain is recruited to tyrosyl-phosphorylated BCR- 10% heat-inactivated FBS (HyClone), 2 mM L-glutamine, 0.1 mM nones- Abelson tyrosine kinase and plays key role in mediating BCR- sential amino acids (NEAA), 1 mM sodium pyruvate, 1000 U/ml penicillin, Abelson tyrosine kinase transformation (17). 1000 ␮g/ml streptomycin, 50 ␮M 2-ME, 4 ng/ml recombinant murine IL-3 Ϫ/Ϫ Ϫ/Ϫ Upon receptor activation, Gab2 becomes tyrosyl-phosphory- (BioSource International). For Fyn mice or Lyn mice and WT lit- termates, 4 ng/ml IL-3 and 20 ng/ml stem cell factor (SCF) were used to lated and associated with SHP-2, a tyrosine phosphatase, required ϩ ϩ Ϫ Ϫ Ϫ Ϫ obtain BMMC from littermate WT / and Fyn / or Lyn / mice as for optimal activation of Erk (18, 19) and immediate early gene described (2). After 5 wk, these cultures consist of ϳ95% mast cells re- transcription (20). Tyrosyl-phosphorylated Gab2 also binds p85, flected by surface expression of Fc⑀RI and c-Kit. Five- to 8-wk-old BMMC Ϫ Ϫ the regulatory subunit of PI3K, resulting in activation of the PI3K cultures were used for biochemical and degranulation assays. Syk / BMMC were generated from bone marrow chimeric mice (C57BL/6J) pathway (14, 21).
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