Gab1 was originaly identified as a Grb2 binding protein by Wang and his colleagues. At the same time Drosophila Dos (daughter of sevenless)was identified as a substrate of Corkscrew (CWS), which is a substrate of the protein tyrosine phosphatase or downstream molecule of CWS in Drosophila and found to be genetically essential molecule for photoreceptor development (see a review Hibi and Hirano , Leukemia and Lymphoma 37, 299-307, 2000). We also independently identified Gab1 as a 110KDa molecule which is tyrosine phophorylated uupon the stimulation of gp130 (Mol. Cell. Biol. 18, 4109-4117,1998). Gab1 has structural similarities with DOS. Both Gab1 and DOS have a pleckstrin homology domain and tyrosine residues, potential binding sites for various SH2 domain-containing adapter molecules when they are phosphorylated. We found that Gab1 was tyrosine phosphorylated in response to various cytokines, such as interleukin-6 (IL-6), IL-3, alpha interferon (IFN-alpha), and IFN-gamma. Upon the stimulation of IL-6 or IL-3, Gab1 was found to form a complex with phosphatidylinositol (PI)-3 kinase and SHP-2, a homolog of Corkscrew. Mutational analysis of gp130, the common subunit of IL-6 family cytokine receptors, revealed that neither tyrosine residues of gp130 nor its carboxy terminus was required for tyrosine phosphorylation of Gab1. Expression of Gab1 enhanced gp130-dependent mitogen-activated protein (MAP) kinase ERK2 activation. A mutation of tyrosine 759, the SHP-2 binding site of gp130, abrogated the interactions of Gab1 with SHP-2 and PI-3 kinase as well as ERK2 activation. Furthermore, ERK2 activation was inhibited by a dominant negative p85 PI-3 kinase, wortmannin, or a dominant negative Ras. These observations suggest that Gab1 acts as an adapter molecule in transmitting signals to ERK MAP kinase for the cytokine receptor gp130 and that SHP-2, PI-3 kinase, and Ras are involved in Gab1-mediated ERK activation. We further identified another adapter molecule (100 kD) interacting with SHP-2 and PI-3 kinase in response to various stimuli (Blood, 93, 1806-1816, 1999). The molecule displays striking homology to Gab1 at the amino acid level; thus, we named it Gab2. Gab1 is phosphorylated on tyrosine upon stimulation through the thrombopoietin receptor (TPOR), stem cell factor receptor (SCFR), and T-cell and B-cell antigen receptors (TCR and BCR, respectively), in addition to IL-3R and gp130. Tyrosine phosphorylation of Gab2 was induced by stimulation through gp130, IL-2R, IL-3R, TPOR, SCFR, and TCR. Gab1 and Gab2 were shown to be substrates for SHP-2 in vitro. Overexpression of Gab2 enhanced the gp130 or Src-related kinases-mediated ERK2 activation as that of Gab1 did. These data indicate that Gab-family molecules act as adapters for transmitting various signals. In addition to Gab1 and Gab2, Gab3 was also identified recently. To reveal the functions of Gab1 in vivo, we generated mice lacking Gab1 by gene targeting (Mol. Cell Biol. 20, 3695-3704, 2000). Gab1-deficient embryos died in utero and displayed developmental defects in the heart, placenta, and skin, which were similar to phenotypes observed in mice lacking signals of the hepatocyte growth factor/scatter factor, platelet-derived growth factor, and epidermal growth factor pathways. Consistent with these observations, extracellular signal-regulated kinase mitogen-activated protein (ERK MAP) kinases were activated at much lower levels in cells from Gab1-deficient embryos in response to these growth factors or to stimulation of the cytokine receptor gp130. These results indicate that Gab1 is a common player in a broad range of growth factor and cytokine signaling pathways linking ERK MAP kinase activation. We further made Gab2 deficient mice to clarify the in vivo roles of Gab2 (Blood 99, 1866-1869, 2002). We found that Gab2 is essential for in vivo development of mast cells and required for stemm cell factor (c-kit ligand). Furtheremore, we disected the pathwyas of degranulation of mast cells: microtuble-dependent calcium-independent granule translocation step and calcium-dependent granule-plasma membrane fusion and exocytosis step. We showed that calcium-independent pathway is regulated by the Syk-Gab2-Rho pathway (J Cell Bilo., 2005, in press).
Gab1 is a member of the Gab/DOS (Daughter of Sevenless) family of adapter molecules, which contain a pleckstrin homology (PH) domain and potential binding sites for SH2 and SH3 domains. Gab1 is tyrosine phosphorylated upon stimulation of various cytokines, growth factors, and antigen receptors in cell lines and interacts with signaling molecules, such as SHP-2 and phosphatidylinositol 3-kinase, although its biological roles have not yet been established. To reveal the functions of Gab1 in vivo, we generated mice lacking Gab1 by gene targeting. Gab1-deficient embryos died in utero and displayed developmental defects in the heart, placenta, and skin, which were similar to phenotypes observed in mice lacking signals of the hepatocyte growth factor/scatter factor, platelet-derived growth factor, and epidermal growth factor pathways. Consistent with these observations, extracellular signal-regulated kinase mitogen-activated protein (ERK MAP) kinases were activated at much lower levels in cells from Gab1-deficient embryos in response to these growth factors or to stimulation of the cytokine receptor gp130. These results indicate that Gab1 is a common player in a broad range of growth factor and cytokine signaling pathways linking ERK MAP kinase activation. (Mol. Cell Biol. 20, 3695-3704, 2000).
Mast cells are thought to participate in a variety of immune responses, such as parasite resistance and the allergic reaction. Mast cell development depends on stem cell factor (Kit ligand) and its receptor, c-Kit. Gab2 is an adaptor molecule containing a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab2 is phosphorylated on tyrosine after stimulation with cytokines and growth factors, including KitL. Gab2-deficient mice were created to define the physiological requirement for Gab2 in KitL/c-Kit signaling and mast cell development. In Gab2-deficient mice, the number of mast cells was reduced markedly in the stomach and less severely in the skin. Bone marrow-derived mast cells (BMMCs) from the Gab2-deficient mice grew poorly in response to KitL. KitL-induced ERK MAP kinase and Akt activation were impaired in Gab2-deficient BMMCs. These data indicate that Gab2 is required for mast cell development and KitL/c-Kit signaling. (Blood 99, 1866-1869, 2002 )
Molecular mechanism of mast cell degranulation
Mast cells and basophils are granulated cells that play a pivotal role in allergy and inflammation. Their granules contain inflammatory mediators such as histamine, proteases, lipid mediators, and cytokines. The activation of mast cells induces exocytosis and fusion of cytoplasmic granules with the plasma membrane, followed by the release of inflammatory mediators within minutes of stimulation. One potent stimulus is the aggregation of high affinity receptors (FceRI) by the Ag-IgE complexes. FceRI-stimulation initiates a signaling cascade that includes activation of tyrosine kinases, such as Syk, Lyn, Fyn, and BTK, and phosphorylation of numerous adapter proteins. These adapters include the linker for the activation of T-cells (LAT), SH2 domain-containing leukocyte protein of 76 kDa (SLP-76), Grb2-associated binder2 (Gab2), MIST/Clnk, 3BP2, and adhesion-and degranulation-promoting adapter protein.
As described above, Gab2 is a member of the Gab family of adapter proteins and displays sequence similarity with Drosophila DOS, a substrate for Corkscrew, a homologue of SHP2, a mammalian protein tyrosine phosphatase (see a review Leukemia and Lymphoma. 37:299-307,2000; Cancer Science. 94:1029-1033,2003). The Gab family adapter proteins are involved in signal transduction through a variety of cytokine and growth factor receptors including c-kit (Blood. 93:1809-1816,1999; Blood. 99:1866-1869,2002). These FceRI-associated adapter proteins regulate the generation of downstream second messengers, such as phosphatidylinositol-3,4,5-trisphosphate (PIP3) and the induction of calcium influx. The deletion of adapter proteins LAT, SLP-76 or Gab2 results in decreased FceRI-mediated mast cell activation. Collectively, these reports indicate that adapter proteins play important roles in mast cell degranulation.
However, It is still unclear how FceRI-mediated signal transduction ultimately regulates the reorganization of the cytoskeleton and how these events lead to degranulation.
We dissected degranulation process of mast cells. First, FceRI stimulation triggers microtubule polymerization and granule translocation to the plasma membrane in a calcium-independent manner. Second, the granules fuse with the plasma membrane in a well-characterized calcium-dependent manner.
Mast cells are so full of granules that degranulation was thought to occur through granule-to-plasma membrane fusion and granule-to-granule fusion, without need for granule translocation. However, our data suggested that microtubules were involved, including evidence that inhibition of microtubule polymerization blocked degranulation.To examine the role of microtubules in degranulation, we stimulated mast cells and observed by confocal microscopy. Tubulin staining increased in response to FceRI stimulation and CD63-GFP (as a granule marker) translocated to the plasma membrane prior to exocytosis. The deprivation of calcium from the culture medium prevented granule fusion to the membrane but had no effect on microtubule polymerization or granule translocation, suggesting that there are two distinct steps required for degranulation. Finally, we showed that the Fyn/Gab2/RhoA, but not Lyn/SLP-76 signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway. J Cell Biol. 170:115-126, 2005 (PubMed). (JCB) (In This Issue)
From these finding, we are currently performing several projects as follows:
Project
1, Identification of molecules involved in FceRI-induced microtubule formation and granule translocation
2, Molecular mechanism of Gab2 which can relay FceRI to microtubule formation
3, Elucidation of the roles of RhoA and downstream signaling molecules in mast cell
4, Application of ENU-induced mutagenesis for allergy diseases
