Grb2-associated binder 2, Gab2, belongs to the Gab/DOS family of scaffolding adaptors that include mammalian Gab1, Gab3, Drosophila Daughter of Sevenless (DOS), and Caenorhabditis elegans Suppressor of Clear 1 (SOC1) (Gu and Neel 2003; Wohrle et al. 2009). Gab1, the first identified member of this family of adaptors, was discovered in search of protein ligands for Grb2 SH3 domain (Holgado-Madruga et al. 1996). DOS was identified as a potential substrate for Corkscrew (Csw), the Drosophila ortholog of the SH2 domain containing protein tyrosine phosphatase 2 (Shp2) (Herbst et al. 1996). Gab2, the third member identified in this family, was initially cloned as a binding protein and substrate of Shp2 (Gu et al. 1998). Gab2 gene is located on human chromosome 11q14.1. Two other groups later cloned Gab2 by searching DNA database for protein with sequence homology to Gab1 (Nishida et al. 1999; Zhao et al. 1999). SOC1 was uncovered in a screen for suppressors of hyperactive signaling from Egl-15, an FGF receptor ortholog (Schutzman et al. 2001). Gab3 was identified by sequence similarity to Gab1 and Gab2 (Wolf et al. 2002). Gab1 and Gab2 are expressed ubiquitously. However, Gab2 is expressed at relatively higher level in myeloid cells and low in lymphoid tissues and fibroblasts (Gu et al. 1998). Gab3 also has a widespread expression pattern, although it is highly expressed in lymphoid tissue (Wolf et al. 2002). GenBank database also contains a human cDNA clone (Acession Number AB076978), termed Gab4. However, it is unclear how this “Gab4” cDNA sequence information was obtained. Although mRNAs for parts of the “Gab4” cDNA are found as ESTs in testis specifically, no report has shown that the full-length “Gab4” protein is expressed 10 years after the sequence was deposited into the GenBank. Thus, it is unclear whether this “Gab4” clone represents a bona fide Gab protein.
Gab2 Domain Structures
Gab2 in Cell Signaling
Gab2 functions to mediate the activation of critical cell signaling pathways by multiple cell surface receptors for growth factors, cytokines, antigens, and immunoglobulins (Fig. 2). The current model is that upon receptor activation, Gab2 is recruited to the activated receptor, becomes tyrosyl phosphorylated, binds SH2 domain-containing signaling molecules, and activates key downstream signaling pathways. Gab2-activated signaling events can be turned off at least by serine and threonine phosphorylation in Gab2 (Gu and Neel 2003; Wohrle et al. 2009).
Gab2 Recruitment to the Receptor
Gab2 is recruited to the activated receptors through other signaling intermediates because Gab2 does not contain modular domain that can directly interact with the receptor. The main route of Gab2-receptor interaction is mediated through Grb2. Grb2 has one SH2 domain flanked by two SH3 domains. The two RXXK motifs in Gab2 (Harkiolaki et al. 2009) lead to constitutive interaction of Gab2 with the Grb2 C-SH3 domain (Lock et al. 2000). The Grb2 SH2 domain mediates the targeting of the Grb2-Gab2 complex to the activated receptor (i.e., Flt3-ITD) or upstream activator (i.e., BCR-ABL) that contains Grb2-SH2 domain binding sites (YXNX) (Masson et al. 2009; Sattler et al. 2002). However, in cell signaling initiated by receptors for IL3/IL5/GM-CSF/IL2 and FcɛRI that lacks direct Grb2 binding to their cytoplasmic tails, another adapter protein Shc, brings the Grb2-Gab2 complex to these receptors that contain binding sites for the Shc PTB or SH2 domain (Gu et al. 2000; Yu et al. 2006a). Shc also has three Grb2-SH2 binding sites which, when phosphorylated, recruit the Grb2-Gab2 complex to the receptor (Gu and Neel 2003). A Gab2 mutant with the two RXXK mutated cannot be tyrosyl phosphorylated efficiently and is impaired in its ability to mediate receptor signaling (Brummer et al. 2006; Yu et al. 2006a).
The PH domain provides another route for Gab2 recruitment. PI3,4,5P3 produced by the receptor-activated PI3K recruits Gab2 via its PH domain to the subcellular compartment where the activated receptor is located. The importance of Grb2 C-SH3 binding sites and PH domain in mediating Gab2 recruitment and functions depends on the receptor involved. The Grb2 C-SH3 binding sites play a dominant role in Gab2 function in EGFR and Flt3 signaling (Brummer et al. 2006; Masson et al. 2009) whereas in the FcγR-induced signaling response, the PH domain exerts a major role (Yu et al. 2006a). However, both Grb2 C-SH3 binding sites and PH domain contribute to Gab2 action in FcɛRI-induced signaling (Yu et al. 2006a).
Protein Tyrosine Kinases That Phosphorylate Gab2
Upon recruited to the activated receptors or receptor signaling complexes, Gab2 undergoes tyrosyl phosphorylation in a receptor-dependent manner. Although it is likely that receptor tyrosine kinase may directly phosphorylate on certain tyrosine residues in Gab2 (Lee and States 2000), this scenario has not been carefully examined. JAK2, the receptor-associated protein tyrosine kinase, plays an essential role in cytokine receptor signaling. However, JAK2 is not required for the G-CSF-induced Gab2 tyrosyl phosphorylation (Zhu et al. 2004). Instead, most of the studies show that the cytoplasmic tyrosine kinases are responsible for Gab2 tyrosine phosphorylation. In T cell antigen receptor (TCR) signaling, ZAP-70 forms a complex with Gab2 and phosphorylates it (Yamasaki et al. 2001). Syk is required for Gab2 tyrosyl phosphorylation and is associated with Gab2 upon FcɛRI activation (Yu et al. 2006a). Members of Src Family protein tyrosine Kinase (SFK) such as Src and Lyn are also responsible for Gab2 tyrosyl phosphorylation upon activation of CSF1R (Lee and States 2000) and G-CSFR (Zhu et al. 2004).
Signaling Pathways Activated by Gab2
Tyrosyl-phosphorylated Gab2 can activate multiple downstream signaling pathways by recruiting SH2 domain-containing signaling proteins. The major Gab2-induced signaling pathways include Shp2 and PI3K (Fig. 2). Recent studies also reveal additional signaling pathways activated by Gab2.
Role in Shp2 pathway. Gab2 contains two tyrosine motifs VDYXXV/L (where X = any amino acid) (Fig. 1), when phosphorylated, which that bind Shp2. Shp2 has low basal activity due to allosteric inhibition of its PTP domain by the N-terminal SH2 domain. Upon binding to tyrosyl-phosphorylated Gab2, the basal inhibition is relieved, resulting in strong activation of Shp2 (Neel et al. 2003). Vertebrates express another SH2 domain containing PTP, Shp1. The SH2 domains of Shp1 and Shp2 recognize similar phosphorylated tyrosine containing motifs. However, the interaction between the tyrosyl-phosphorylated Gab2 and Shp1 has not been observed. It is not well understood why Gab2 binds preferentially to Shp2. Nevertheless, this deferential binding ability helps explain the distinct biological functions of Shp1 and Shp2 (Neel et al. 2003).
Gab2, acting via Shp2, is required for full activation of Erk downstream of many receptors. Expression of a Gab2 mutant with Y->F in the VDYXXV/L motifs impairs Erk activation in response to CSF-1 (Liu et al. 2001), IL2 (Arnaud et al. 2004b), and EGF stimulation (Brummer et al. 2006) as well as under three-dimensional culture condition (Bentires-Alj et al. 2006). In addition, SCF-induced full activation of Ras and Erk requires Gab2 interaction with Shp2 (Yu et al. 2006b), indicating that Gab2/Shp2 contribute to the efficient Erk activation at the level of Ras. Gab2/Shp2 complexes may serve as “amplifiers” of initial Ras/Erk pathway activation (Gu and Neel 2003).
Gab2/Shp2 complexes also have additional signaling roles besides activation of the Ras/Erk pathway. Expression of Gab2 mutants that cannot bind Shp2 fails to activate IL3-induced immediate early gene (i.e., fos) transcription without affecting Erk activation (Gu et al. 1998). The Gab2/Shp2 complex appears to activate the Rac-JNK pathway in response to SCF in mast cells (Yu et al. 2006b). Gab2 via Shp2 promotes migration and invasion of mammary epithelial cells by recruiting p190RhoGAP and inhibiting RhoA activation (Abreu et al. 2011).
Role in PI3K pathway. Gab2 has three potential binding sites (YXXM) for the SH2 domain of p85. By binding p85, Gab2 mediates the activation of the PI3K pathway for receptors that lack the p85 binding sites. For example, Gab2 is implicated in PI3K activation from the receptor for IL-3/GM-CSF (Gu et al. 2000), EGFR (Kong et al. 2000), FcγR (Gu et al. 2003), and FcɛRI (Gu et al. 2001). Gab2 also recruits PI3K in response to stimulation of receptor systems, such as the TCR (Nishida et al. 1999; Pratt et al. 2000) and BCR (Nishida et al. 1999), in which co-receptors recruit PI3K. In these systems, Gab2/p85 probably serves to amplify receptor-evoked PI3K activity. Gab2-induced activation of PI3K can have different signaling effects depending on the cell type. Recruitment of PI3K to Gab2 is critical for FcɛRI-induced degranulation of mast cell (Gu et al. 2001). In contrast, Gab2/PI3K complexes inhibit TCR-induced IL2 production (Pratt et al. 2000; Yamasaki et al. 2001).
Role in other signaling pathways. Gab2 has been implicated in the activation of the Stat pathway. Gab2 has been shown in complexes with Stat5 (Nyga et al. 2005). However, it is still unclear how Stat5 interacts with Gab2 and whether Gab2 is required for Stat5 activation. It appears that the constitutively activated mutant of Stat5 via interaction with Gab2 activates the PI3K-Akt and Erk pathways in BaF3 cells (Nyga et al. 2005). A recent study also reveals that Stat3 interacts with the phosphorylated tyrosine residue in 195YLHQ motif of Gab2. Gab2 is required for Stat3 activation, which is important for Gab2-dependent transformation of primary hematopoietic cells by Stk/Ron in response to Friend virus infection (Ni et al. 2007). Gab2 association with PLCγ2 has been observed in osteoclasts upon RANK activation (Mao et al. 2006). It appears that PLCγ2 acts as an adaptor mediating Gab2 tyrosyl phosphorylation (Mao et al. 2006). Gab2 contributes to efficient activation of NF-κB induced by RANKL (Wada et al. 2005). However, it is unknown whether PLCγ2 association with Gab2 is required for RANK signaling. Although Gab2 via Shp2 can inhibit RhoA activation in mammary epithelial cells, a report shows that Gab2 activates RhoA, which is important for microtubule-dependent granule transport to plasma membrane in mast cells. It is unclear how Gab2 activates RhoA in mast cells, which is likely independent of Shp2 (Nishida et al. 2005). Yeast two-hybrid screens have identified CrkL (Crouin et al. 2001) that can bind Gab2. The functional significance of this interaction remains unclear.
Turning Off Gab2 Signaling Activity
Gab2 activates cell signaling pathways through phosphotyrosine interaction with SH2 domains. One way to terminate Gab2-initiating signaling is to dephosphorylate tyrosine residues in Gab2 by protein tyrosine phosphatases. Although Gab2 was identified as potential substrate for Shp2 (Gu et al. 2000), the functional consequence of Gab2 dephosphorylation by Shp2 is still unclear. Recent studies reveal that serine and threonine phosphorylation of Gab2 function as negative feedback mechanism to inhibit Gab2 signaling activities. Phosphorylation of three amino acid residues contributes to negative regulation of Gab2 tyrosyl phosphorylation and Gab2-involved responses. Ser159, phosphorylated by Akt, is involved in reducing Gab2 tyrosyl phosphorylation by ErbB2 (Lynch and Daly 2002). Gab2 Ser210 and Thr391, phosphorylated partially by Akt or an Akt-dependent kinase, recruit 14-3-3 protein, which reduces tyrosyl phosphorylation of Gab2 and Gab2-initiated signaling responses (Brummer et al. 2008). In addition, Ser623 phosphorylation by Erk results in reduced Gab2 association with Shp-2 and Erk activation induced by IL2 (Arnaud et al. 2004a). However, the mechanism by which Ser/Thr phosphorylation inhibits Gab2 signaling is still not well understood.
Gab2 mainly plays positive roles in regulating cell proliferation, survival, differentiation, and migration in a cell type-specific manner. While Gab2 activation of PI3K contributes to proliferation of hematopoietic cells (Gu et al. 2000), and hepatocytes (Kong et al. 2000), Gab2 via Shp-2 is required for the proliferation of mammary epithelial cells in three-dimensional culture (Bentires-Alj et al. 2006) and mast cells (Yu et al. 2006b). Likewise, while Gab2 activation of PI3K plays a critical role in differentiation and survival of neuronal cells (Mao and Lee 2005), Gab2 via association with Shp-2 and activation of Erk promotes differentiation of macrophages (Liu et al. 2001) and myeloid cells (Dorsey et al. 2002). In addition, Gab2 expression also contributes to cell migration in different cell types (Horst et al. 2009; Meng et al. 2005). Despite its major role as a positive signal transducer in regulating cell responses, some studies also suggest that Gab2 plays a negative role in cell signaling. Gab2 mediates the suppression of TCR-evoked IL2 gene expression in T lymphocytes (Pratt et al. 2000; Yamasaki et al. 2001).
Studies using mice with targeted disruption of the Gab2 gene (Gab2–/–) help elucidate the functions of Gab2 in vivo. Gab2 is not required for the normal development of mice since Gab2–/– mice are viable and healthy (Gu et al. 2001; Nishida et al. 2002). However, detailed analyses of the Gab2–/– mice reveal that Gab2 expression is required for the growth and functions of a variety of hematopoietic cells. Gab2–/– mast cells are defective in FcɛRI-evoked degranulation and cytokine gene expression (Gu et al. 2001), which contributes to the impaired allergy response of the Gab2–/– mice (Gu et al. 2001). In addition, mast cell development is comprised in Gab2–/– mice due to impaired c-Kit-evoked signaling of Gab2–/– mast cells (Nishida et al. 2002; Yu et al. 2006b). Although differentiation of the Gab2–/– macrophage seems to be normal, FcγR-induced phagocytosis is diminished in Gab2–/– macrophages (Gu et al. 2003). Eosinophil number in the peripheral blood from Gab2–/– mice is also significantly reduced (Gu and Neel 2003). Gab2–/– mice also display mild osteopetrosis and decreased bone resorption due to defective RANK-evoked signaling in Gab2–/– osteoclasts and osteoclast differentiation from Gab2–/– progenitor cells (Wada et al. 2005). Hematopoietic stem cells from Gab2–/– mice show reduced survival and self-renewal capability (Zhang et al. 2007). Further studies of mutant mice that lack the expression of both Gab1 and Gab2 in cardiomyocytes uncover a role of Gab2 in contributing to the normal cardiac function in adult mice through mediating neuregulin1/ErbB signaling (Nakaoka et al. 2007).
Gab2 in Cancer and Alzheimer’s Disease
Gab2 has been implicated in different types of cancers including solid tumors and leukemia. Gab2 protein overexpression has been reported in breast cancer cells and breast tumors (Bentires-Alj et al. 2006; Daly et al. 2002) and melanoma (Horst et al. 2009). In breast tumors, Gab2 overexpression is associated with early stage of breast cancer (Fleuren et al. 2010). Gab2 gene amplification is at least one mechanism contributing to Gab2 protein overexpression in breast cancer (Bentires-Alj et al. 2006) and melanoma (Horst et al. 2009). Gab2 overexpression is associated with metastatic melanoma (Horst et al. 2009). Studies by manipulating the expression of Gab2 in cell lines and mice indicate that Gab2 expression alone or in combination with other oncogenes promote breast tumor cell growth (Bentires-Alj et al. 2006; Brummer et al. 2006), migration, invasion, and metastasis in vitro and in mice (Bentires-Alj et al. 2006; Ke et al. 2007). Gab2-regulated Shp2-Erk pathway contributes to the proliferation, invasion, and metastasis of breast cancer cells (Bentires-Alj et al. 2006; Ke et al. 2007). In contrast, Gab2-regulated PI3K pathway promotes migration, invasion, and metastasis of melanoma cells (Horst et al. 2009). Although Gab2 protein overexpression or gene amplification has been reported in gastric carcinomas (Lee et al. 2007), acute myeloid leukemia (Zatkova et al. 2006), and ovarian cancer (Brown et al. 2008), the functional roles of Gab2 in these cancers remain unexplored.
Besides Gab2 overexpression, endogenous level of Gab2 plays critical roles in leukemogenesis induced by BCR-ABL and juvenile myelomonocytic leukemia (JMML)-associated Shp2 mutants. BCR-ABL is the causative oncogene for chronic myelogenous leukemia (CML). Gab2 via Grb2 interacts with BCR-ABL through Y177 (a Grb2 SH2 domain binding site) and mediates BCR-ABL-evoked PI3K-Akt and Erk activation, and oncogenic transformation of myeloid progenitor cells (Sattler et al. 2002). Importantly, Gab2 is required for BCR-ABL-induced CML-like disease in mice (Gu and Neel 2003). Further supporting a role for Gab2 in CML, a recent study shows that Gab2 protein expression is significantly enhanced in bone marrow samples of CML patients with accelerated phase and blast crisis diseases (Aumann et al. 2011). About 35% of JMML has somatic Shp2 mutations. Transplantation of wild type, not Gab2–/–, bone marrow progenitor cells transduced with retroviruses expressing the JMML-associated Shp2 mutants induces JMML-like disease in mice, indicating that Gab2 is required for leukemogenesis induced by these Shp2 mutants (Mohi et al. 2005). Lastly, only Gab2, not Gab1, mediates oncogenic transformation of fibroblasts by the V-Sea oncogene (Ischenko et al. 2003) and induction of erytholeukemia by the Stk receptor tyrosine kinase in response to Friend virus infection (Teal et al. 2006).
A recent study using genome-wide association analysis reveals that several Gab2 polymorphic alleles can increase the risk of Alzheimer’s disease (AD) onset in patients carrying the APOE epsilon4 allele (Reiman et al. 2007). Subsequent studies by more than ten different groups indicate that the association of Gab2 polymorphic alleles with increase AD onset depends on patient cohorts, with about half of those studies supporting Gab2 polymorphic allele as a risk factor for AD in APOE epsilon4 carriers whereas roughly the other half of the studies fail to demonstrate significant association of Gab2 polymorphic allele with increased risk of AD. It is not understood how the polymorphic alleles of Gab2 contribute to Alzheimer disease onset. These polymorphic alleles are located in the intron regions of Gab2 gene. They are more likely to change the expression of Gab2, not the protein structure of Gab2. Knockdown of Gab2 expression by siRNA increases GSK-3 and Tau phosphorylation in neuronal cell culture, suggesting that polymorphic alleles of Gab2 may involve in Alzheimer disease onset by modulating Tau phosphorylation (Reiman et al. 2007). Future study by changing the expression level of Gab2 in the brain of mouse model of AD should help clarify the role of Gab2 in AD.
Since the discovery of Gab2 13 years ago, results from molecular, biochemical, cellular, and genetic studies have revealed Gab2 as a critical regulator of multiple important cell signaling pathways including PI3K-Akt and Shp2-Ras-Erk. One important question needs to be addressed is how Gab2 via Shp2 regulates the activation of the Ras-Erk pathway. In addition, it will also be important to understand the detailed mechanism of the negative regulation on Gab2 signaling by serine/threonine phosphorylation. Gab2 overexpression or expression is functionally implicated in various diseases including cancer, allergy, and Alzheimer’s. Because Gab2 belongs to the so-called undruggable target, it will be a challenge to find agents or small molecule inhibitors that can decrease Gab2 expression or blocking Gab2 activation of downstream signaling pathways. However, with the new technological advances in the drug discovery field, drugging Gab2 should provide potential new therapeutics for treating human diseases with minimal side effects since mice lacking Gab2 expression are generally healthy.