Gab proteins are scaffold proteins that are related to the insulin receptor substrates (IRS1/2/3), the fibroblast growth factor ( FGF) receptor substrate 2 (FRS2-α/β), the downstream of kinase (Dok), and the linker of T cell ( LAT). These proteins lack intrinsic enzymatic activity. Upon their recruitment to activated growth factor and cytokine receptors, they become tyrosine phosphorylated, providing binding sites for multiple proteins involved in signal transduction. By virtue of their ability to assemble multiprotein complexes, they act to modulate, amplify, and diversify the signals downstream from receptors (Pawson and Scott 1997; Schlessinger and Lemmon 2003).
Genetic and cell biology analyses have demonstrated that Gab proteins are crucial in RTK signaling and biology. dos and soc-1 were found to be essential for RTK signaling in the developing fly and worm. Gab1 null mice are embryonic lethal and show impaired migration of muscle precursor cells and muscle development, thinner placenta, impaired diaphragm formation, reduced liver size, defective heart development, skin problems, open eyelids, and liver regeneration defects (Schaeper et al. 2007). These phenotypes predominantly mimic Hgf null and Met null mice, but also include additional defects observed in Pdgf and Egf null mice, demonstrating the important role played by Gab1 in transduction of signals from these growth factor signals during development. In the adult, Gab1 regulates glucose uptake in the liver, bone homeostasis, and differentiation of myocytes and B cells.
Despite having similar overall structure and signaling capacity, Gab2 null mice are viable, have a normal life span, yet show severe defects in mast cell lineages (Gu and Neel 2003), whereas Gab3 null mice have no apparent phenotype. The marked differences in knockout phenotypes suggest that despite high similarity in their domain structure organization, Gab proteins play different roles in RTK signaling during development in mammals.
This entry will focus on the Gab1 protein, its role in signal transduction, and its implication in development and cancer initiation and progression.
Gab1 in Signal Transduction
In addition to the indirect recruitment via Grb2, Gab1 is also directly recruited to the Met receptor. This direct interaction is mediated by the Met Binding Motif (MBM) on Gab1 and requires the activated kinase domain of Met (Birchmeier et al. 2003; Peschard and Park 2007). The Gab1 MBM does not resemble classical SH2 or PTB domains and is not conserved in other members of the Gab family. The direct recruitment of Gab1 correlates with sustained phosphorylation of Gab1, Erk and Akt downstream from Met, and is required for the invasive morphogenic program induced by HGF (Peschard and Park 2007).
The N-terminal region of all Gab proteins contains a conserved, phosphoinositide-binding, pleckstrin homology (PH) domain that is important for both membrane targeting and protein function. The PH domain of Gab1 predominantly binds phosphoinositol-3,4,5-phosphate (PI3,4,5P3), which is required for its translocation from the cytoplasm to the cell cortex, lamellipodia, membrane ruffles, and cell–cell junctions in sheets of epithelial cells in response to receptor activation (Peschard and Park 2007). Gab1 mutants unable to bind PI3,4,5P3 or lacking the PH domain are poorly phosphorylated and defective in transducing signals and biological responses downstream from the EGF and Met RTKs (Peschard and Park 2007; Rodrigues et al. 2000). This is overcome by targeting Gab1 to the plasma membrane using a Src myristoylation sequence (Peschard and Park 2007), highlighting the importance of membrane localization for Gab1 signaling.
Shp2 and MAPK Activation (pY627, 659)
Gab1 binds to the phosphatase Shp2 in response to multiple growth factors and cytokines including EGF, insulin, FGF, PDGF, or HGF (Gu and Neel 2003; Liu and Rohrschneider 2002; Wohrle et al. 2009). Recruitment of Shp2 SH2 domains leads to the activation of Shp2 enzymatic activity and is required for full activation of the MAPK pathway by the EGFR (Birchmeier et al. 2003; Peschard and Park 2007). Knockin mice expressing a Gab1ΔShp2 mutant are embryonic lethal and display impaired migration of muscle progenitor cells to the limb buds and placental defects, a phenotype similar to Gab1, Met and Hgf null animals (Schaeper et al. 2007), highlighting the importance of the Gab1–Shp2 interaction during development. Different models for the function of the Gab–Shp2 complex in Ras signaling have been proposed; however, the exact mechanisms of this regulation still need to be defined. One model supported by data from D. melanogaster proposes that recruitment of p120Ras-GAP on tyrosines 307 and 317 of Gab1 promotes the return of Ras to its inactive, GDP-bound state. Shp2 recruitment to Gab1 promotes dephosphorylation of these p120Ras-GAP sites thus decreasing the negative regulation of Ras, resulting in sustained activation (Gu and Neel 2003; Wohrle et al. 2009).
p85 and PI3K Signaling (pY 447, 472, 589)
Gab1 plays a crucial role in the activation and amplification of PI3K signaling downstream of RTKs and cytokine receptors. The majority of RTK-dependent PI3K activity is associated with Gab1 and depends on its ability to recruit the p85 regulatory subunit of PI3K through three YXXM motifs that include tyrosines 447, 472, and 589. Gab1-dependent PI3K activation is critical for cell survival and proliferation through the Akt pathway in many biological contexts. This is particularly important for RTKs that are unable to directly recruit p85, including EGFR, VEGFR-2, and FGFR. The ability of Gab1 to activate the PI3K pathway is also required for actin remodeling and formation of membrane ruffles, cell migration, and tubulogenesis downstream from several RTKs. This possibly occurs through generation of PI3,4,5P3-rich membranes following localized Gab1-dependent PI3K activation and subsequent activation of guanine exchange factors (GEFs) for the Rac GTPase. A Gab1-p85 interaction is required for cell scatter and epithelial morphogenesis induced by HGF (Peschard and Park 2007), to induce Rac activation and lamellipodia formation downstream of the Ret RTK (Maeda et al. 2004), and for the formation of HGF-induced circular dorsal ruffles (Abella et al. 2010). During development, Gab1/p85 interaction is also required for keratinocyte migration as well as EGF-induced eyelid closure (Schaeper et al. 2007).
Crk (pY242, 259, 307, 317, 373, 406)
The interaction between Gab1 and Crk relies on six YxxP motifs in Gab1 and the SH2 domain of Crk. The Gab1–Crk interaction controls cytoskeleton rearrangement, cell motility, adhesion, morphology and polarity downstream of RTKs, antigen and cytokine receptors. These biological outcomes possibly depend on the ability of Crk to regulate the activation of the small GTPases Rac and Rap1 via the recruitment of the Rac GEF Dock180 and the Rap1 GEF C3G to their SH3 domains (Peschard and Park 2007).
Gab1 overexpression also enhances HGF- and EGF-induced JNK activation. Downstream of Met, this effect is abrogated by co-expression of a dominant negative Crk mutant, suggesting that Met activates JNK through a Gab1–Crk complex. In addition, Gab1–Crk signaling to JNK is correlated with anchorage independent growth, and MMP-1 secretion, supporting a role for Gab1–Crk signaling in cell transformation and invasion (Lamorte et al. 2000).
PLCγ (pY307, 373, 406)
Structure-function analysis identified that the SH2 domain of PLCγ was recruited to three of the Crk binding sites (pY307, 373, 406). These studies have proposed a requirement for the Gab1–PLCγ interaction for branching morphogenesis in response to HGF/Met signaling (Birchmeier et al. 2003) and for endothelial cell migration (Laramee et al. 2007) downstream from the VEGFR-2.
Nck1 SH2 domain directly interacts with tyrosine 406 on Gab1 downstream from Met, EGF, and PDGF RTKs activation. This interaction is required for induction of dorsal ruffles downstream of EGF, PDGF, and Met RTKs, and for Met-dependent Rac activation and for epithelial morphogenesis in MDCK cells and provides a mechanism through which Gab1 binding to RTKs can signal to the actin cytoskeleton (Abella et al. 2010). Although these biological outcomes were originally attributed to Crk and/or PLCγ, which also bind Gab1 on tyrosine 406, it is now important to assess the relative contribution of each signaling molecule to these biological processes.
Pak4 (ASM, aa. 116–234)
In addition to proteins containing SH2 domains, the P21-activated serine/threonine kinase 4 (Pak4) is recruited to Gab1 downstream from the Met RTK. This interaction has been mapped to amino acids 116 to 234 of Gab1 (Fig. 1) and requires phosphorylation. If this association is direct, however, remains to be addressed. Pak4 association with Gab1 is required for HGF-induced epithelial cell dispersion, migration, and invasive growth, likely through the ability of Pak4 to phosphorylate cofilin and promote remodeling of the actin cytoskeleton (Paliouras et al. 2009).
Feedback Loops Regulating Gab1 Signaling
Cross Talk Between PI3K and Shp2
The most important arms of Gab1 signaling network are the Shp2/MAPK arm and the PI3K/Akt arm. Activation of these pathways can influence each other through negative and positive feedback loops. As described earlier, Gab1 is a substrate for the Shp2 phosphatase. Recruitment of Shp2 to Gab1 leads to the dephosphorylation of the p85 binding sites on Gab1, which attenuates PI3K signaling.
Activation of PI3K potentiates activation of the MAPK pathway by increasing Shp2 recruitment to Gab1. p85 binding to Gab1 leads to an increase in local PI3,4,5P3 formation, resulting in increased Gab1 membrane targeting and tyrosine phosphorylation. Downstream of EGFR, VEGFR, and EPO-R, a Gab1Δp85 mutant has a reduced ability to activate the MAPK pathway due to a decreased ability to recruit Shp2 (Liu and Rohrschneider 2002; Nishida and Hirano 2003; Wohrle et al. 2009). These data indicate the existence of Gab1-dependent cross talk between the PI3K and MAPK signaling pathways.
Growth factor and cytokine stimulation induces a decrease in the mobility of Gab1 on an SDS PAGE that is attributed to increased serine and threonine phosphorylation. The biological role of this Ser/Thr phosphorylation depends on the cell type and receptor activated. Insulin and EGF stimulation induces Erk-dependent phosphorylation of Gab1 on Ser454, Ser581, Ser597, and Thr476, which are located in the vicinity of the p85 binding sites. This Ser/Thr phosphorylation correlates with reduced Tyr phosphorylation and attenuated PI3K signaling. In contrast, Met activation induces Erk-dependent phosphorylation of Thr476 and is associated with an increase in PI3K signaling (Wohrle et al. 2009). In melanocytes, however, HGF induces a PKCßII-dependent Ser/Thr phosphorylation of Gab1 associated with attenuated PI3K activation, as well as a decrease in cell migration and invasion (Oka et al. 2008). Furthermore, MAPK-dependent phosphorylation of Ser551 positively regulates the function of the PH domain of Gab1 in response to IL-6, and therefore enhances its recruitment to the plasma membrane (Wohrle et al. 2009). Taken together, these data suggest that Gab1 Tyr and Ser/Thr phosphorylation can influence each other by creating feedback loops allowing fine-tuned responses by modulating protein–protein interactions and subcellular localization.
Gab1 in Cancer
Even though Gab1 is essential in embryonic development, it has not been strongly linked to specific disease. Disease-linked phenotypes due to germline mutations in GAB1 may not be visible because Gab1 is essential during development. Somatic mutations of GAB1 have been described in cancers; however, their significance for tumorigenesis remains to be addressed. Nevertheless, in vitro and in vivo data have identified a role for Gab1 protein in cellular transformation and cancer progression in various systems due its critical role in RTK signaling. Expression of a mutant EGFR unable to efficiently phosphorylate Gab1 delays the onset of ErbB2-driven mammary tumor in mouse model (Gillgrass et al. 2003). In addition, expression of a Gab1 mutant uncoupled from Shp2, and Shp2 knock down impairs invasion and migration of Met overexpressing colorectal cancer cells in matrigel and prevents tumor formation when these cells are subcutaneously injected into nude mice (Seiden-Long et al. 2008). Furthermore, recent studies demonstrated that Gab1 plays a role in resistance to EGFR tyrosine kinase inhibitors induced by HGF and Met in NSCLC patients and cell lines by promoting cell survival (Turke et al. 2010).
In contrast to Gab1, Gab2 is often over-expressed in breast, ovarian, gastric, and skin cancer. In breast epithelial cell lines, over-expression of Gab2 cooperates with ErbB2 over-expression to promote cell survival and proliferation (Gu and Neel 2003; Wohrle et al. 2009). The differences between the roles of Gab1 and Gab2 in tumor initiation and progression may reflect that despite having similar signaling capacity, these two proteins have nonredundant functions as supported by the different phenotypes observed in the respective knockout mice. Alternatively, it may reflect the genetic instability of the GAB2 locus located on chromosome 11q14.1 that is often amplified in human cancers.
The Gab1 scaffold protein is a critical node in signaling networks initiated by RTKs, cytokine and immune receptors. Recruitment of signaling molecules to Gab1 leads to two different outcomes. First, activation of PI3K/Akt, Shp2/MAPK, and JNK pathways engages a transcriptional program regulating cell survival, proliferation, morphogenesis, and invasion. Second, the recent discoveries that Pak4, Nck, and Crk are recruited to Gab1 downstream of RTKs indicate that Gab1 is a crucial scaffold for the regulation of the actin cytoskeleton and cell motility.
It is becoming evident that the role of Gab1 in signal transduction is more complex and dynamic than the classic definition of adaptor molecules. Recent data demonstrated that Gab1 does not only amplify and diversify receptor signals, but spatially organizes these signals in microdomains such as dorsal ruffles (Abella et al. 2010).
Gab1 associates with numerous receptors, binds many different downstream signaling molecules, and shows dynamic relocalization upon stimulation. Gab1 therefore likely engages in complexes of different composition and localization over time. In the future, it will be important to dissect the spatial and temporal regulation of these signaling complexes.