daughter of sevenless


Soc-1, a Dos homolog in C. elegans

EGL-15 is a fibroblast growth factor receptor in the nematode C. elegans. Components that mediate EGL-15 signaling have been identified via mutations that confer a Clear (Clr) phenotype, indicative of hyperactivity of this pathway, or a suppressor-of-Clr (Soc) phenotype, indicative of reduced pathway activity. A gain-of-function allele of let-60 ras has been identified that confers a Clr phenotype and has implicated by their Soc phenotype both let-60 ras and components of a mitogen-activated protein kinase cascade in EGL-15 signaling . Epistasis analysis indicates that the gene soc-1 functions in EGL-15 signaling by acting either upstream of or independently of LET-60 RAS. soc-1 encodes a multisubstrate adaptor protein with an amino-terminal pleckstrin homology domain that is structurally similar to the DOS protein in Drosophila and mammalian GAB1. DOS is known to act with the cytoplasmic tyrosine phosphatase Corkscrew (CSW) in signaling pathways in Drosophila. Similarly, the C. elegans CSW ortholog PTP-2 is involved in EGL-15 signaling. Structure-function analysis of SOC-1 and phenotypic analysis of single and double mutants are consistent with a model in which SOC-1 and PTP-2 act together in a pathway downstream of EGL-15 and the Src homology domain 2 (SH2)/SH3-adaptor protein SEM-5/GRB2 contributes to SOC-1-independent activities of EGL-15 (Schutzman, 2001).

Genetic and structure-function analyses support the hypothesis that SOC-1 and PTP-2 function as a signaling cassette to transduce the EGL-15 signal. (1) It was found that SOC-1 Y408, a predicted SHP2/PTP-2 binding site, is critical for SOC-1 function. Experiments with Drosophila have demonstrated that a similar C-terminal CSW binding site is also critical for DOS function. (2) PTP-2 requires an intact SH2 domain(s) to mediate EGL-15 signaling. (3) A chimeric protein that fuses the C-terminal SH2 and phosphatase domains of PTP-2 to SOC-1(Y408F) can restore some of the lost signaling potential of SOC-1(Y408F). (4) Although both SOC-1 and PTP-2 function to mediate EGL-15 signaling, ptp-2; soc-1 double mutants do not reduce EGL-15 pathway activity to a greater extent than the single mutants. These data support a model in which SOC-1 and PTP-2 function together to mediate EGL-15 signaling. More specifically, in response to EGL-15 activation, SOC-1 is likely phosphorylated at Y408, enabling SOC-1 to recruit PTP-2 via a direct interaction between the C-terminal tail of SOC-1 and the SH2 domain(s) of PTP-2. Precisely how activation of EGL-15 triggers SOC-1 and SEM-5 to participate in EGL-15 signal transduction remains unresolved (Schutzman, 2001).

Two paradigms from other signaling systems provide a context for thinking about how a SOC-1/PTP-2 cassette might function in EGL-15 signaling. The first of these paradigms rests on the high degree of structural similarity between SOC-1 and the MSAs GAB1 and DOS. These proteins all have N-terminal PH domains followed by a series of tyrosyl and polyprolyl motifs in their C-terminal tails. These domains not only share a common structure but also appear to share common functions. The SOC-1 PH domain is most similar to the GAB1 PH domain, and the PH domains of GAB1 and DOS are functionally interchangeable, suggesting that a common function is carried out by these PH domains (Bausenwein, 2000). The C-terminal tails of SOC-1 and DOS perform a common function to recruit PTP-2/CSW. This function is likely to be conserved in mammals, since the association of GAB1 with SHP2 plays an important role during epithelial morphogenesis (Schutzman, 2001).

In addition to its association with SHP2, GAB1 has a well-established relationship with GRB2. Initially identified as the GRB2-associated binder 1, GAB1 associates with GRB2 by a phosphotyrosine-independent mechanism. A possible association between SOC-1 and SEM-5 was tested and it was found that neither the canonical SEM-5 SH2 binding sites nor the canonical SEM-5 SH3 binding sites in SOC-1 are essential for SOC-1 function. More recently, the association between GAB1 and GRB2 has been shown to be mediated in part by a noncanonical GRB2 SH3 domain binding site (termed GBS1) as well as a canonical GRB2 SH3 binding site (termed GBS2). This newly identified GBS1 motif is found in both DOS and SOC-1, further supporting the structural homology among these proteins. The GBS1 motif in SOC-1 presents an alternative mechanism by which SOC-1 may associate with SEM-5 in a manner similar to that described for GAB1 (Schutzman, 2001).

Although structurally distinct from SOC-1, the MSA protein FRS2 offers a second paradigm to consider as a functional analog for SOC-1. FRS2 plays a key role in FGFR signaling by linking the FGFR to GRB2 and the RAS/MAPK cascade. In addition to recruiting GRB2 directly, FRS2 can associate with SHP2, offering an indirect means to recruit GRB2. This recruitment could be mediated via known GRB2 binding sites within the C terminus of SHP2. SOC-1 does not act similarly for two reasons: (1) since its phosphotyrosine motifs predicted to bind SEM-5 are not essential for SOC-1 function, SOC-1 does not appear to recruit SEM-5 directly; (2) although the data suggest a direct interaction between PTP-2 and SOC-1 that is important for SOC-1 function, SOC-1 is unlikely to recruit SEM-5 via PTP-2, since PTP-2 does not have canonical GRB2 binding sites within its C-terminal tail. Thus, if SOC-1 recruits SEM-5 to activate RAS signaling, it must accomplish this by a different mechanism (Schutzman, 2001).

If SOC-1 does not play a direct role in linking EGL-15 signaling to the activation of the RAS/MAPK cascade, how might this link be made? Unlike mammalian FGFRs, EGL-15 may be able to associate directly with GRB2 through phosphotyrosyl motifs within its intracellular region. EGL-15 contains two potential phosphotyrosine motifs predicted to bind the SH2 domain of SEM-5, although these do not appear to be essential for EGL-15 function. Alternatively, a C. elegans predicted gene, F54D12.6, that has similarity to FRS2 may perform an FRS2-like function to recruit GRB2/SOS/RAS during EGL-15 signaling. The role of this gene in EGL-15 signaling is currently being investigated (Schutzman, 2001).

It is not yet clear which of these two paradigms SOC-1 follows most closely. Similar to FRS2, SOC-1 appears to be a relatively specific adaptor for EGL-15 signaling. This is in contrast to GAB1 and DOS, which have been implicated in a number of RTK pathways and may represent more promiscuous adaptor proteins. Mechanistically, however, SOC-1 appears to function most like DOS in that one of its critical functions is to recruit PTP-2/CSW during signal transduction. Although DOS has not been implicated in FGFR signaling to date, a role for GAB1 was recently identified in linking FGFR activation with phosphatidylinositol 3-kinase signaling. The identification of SOC-1 as an important component of EGL-15 FGFR signaling implicates GAB1 or DOS in FGFR signaling in their respective systems as well (Schutzman, 2001).

These data establish that the Clr/Soc EGL-15 pathway utilizes a RAS/MAPK signaling cascade and also define a role for the SOC-1/PTP-2 cassette in EGL-15 FGFR signaling. Several lines of evidence suggest that SOC-1/PTP-2 may act in a parallel signaling branch that has a modulatory effect on RAS signaling downstream of EGL-15; (1) a strong synergism has been observed between sem-5 mutations and null alleles of soc-1; (2) a supporting parallel pathway might explain why two different let-60 gain-of-function mutations confer only a late-onset Clr phenotype rather than the full Clr phenotype observed in clr-1 mutants; (3) a parallel-pathway model can help explain why so many of these soc genes, including let-60, ptp-2, and soc-1, require strong mutations to confer a Soc phenotype; (4) this model is supported by data for Drosophila that suggest a complex interdependence of the RAS and DOS/CSW pathways. Unraveling the precise molecular mechanisms by which SOC-1/PTP-2 contributes to signaling will be crucial to a more complete understanding of RTK signal transduction (Schutzman, 2001).

Gab family proteins - Interactions with Grb2 and Shp2

The adapter Grb2 is an important mediator of normal cell proliferation and oncogenic signal transduction events. It consists of a central SH2 domain flanked by two SH3 domains. While the binding specificities of the Grb2 SH2 and N-terminal SH3 domain [Grb2 SH3(N)] have been studied in detail, binding properties of the Grb2 SH3(C) domain remained poorly defined. Gab1, a receptor tyrosine kinase substrate which associates with Grb2 and the c-Met receptor binds Grb2 via a region which lacks a Grb2 SH3(N)-typical motif (P-x-x-P-x-R). Precipitation experiments with the domains of Grb2 show that Gab1 can bind stably to the Grb2 SH3(C) domain. Gab1 mutants were generated by PCR to test in vivo residues thought to be crucial for Grb2 SH3(C) binding. The Grb2 SH3(C) binding region of Gab1 has significant homology to a region of the adapter protein SLP-76. Peptides corresponding to epitopes SLP-76, Gab1, Sos and other proteins with related sequences, as well as mutant peptides were synthesized and analysed by tryptophan-fluorescence spectrometry and by in vitro competition experiments. These experiments define a 13 amino acid sequence with the unusual consensus motif P-x-x-x-R-x-x-K-P as required for a stable binding to the SH3(C) domain of Grb2. Additional analyses point to a distinct binding specificity of the Grb2-homologous adapter protein Mona (Gads), indicating that the proteins of the Grb2 adapter family may have partially overlapping, yet distinct protein binding properties (Lewitzky, 2001).

To maintain various T cell responses and immune equilibrium, activation signals triggered by T cell antigen receptor (TCR) must be regulated by inhibitory signals. Gab2, an adaptor protein of the insulin receptor substrate-1 family, has been shown to be involved in the downstream signaling from cytokine receptors. The functional role of Gab2 in TCR-mediated signal transduction has been examined. Gab2 is phosphorylated by ZAP-70 and co-precipitates with phosphoproteins, such as ZAP-70, LAT, and CD3zeta, upon TCR stimulation. Overexpression of Gab2 in Jurkat cells or antigen-specific T cell hybridomas results in the inhibition of NF-AT activation, interleukin-2 production, and tyrosine phosphorylation. The structure-function relationship of Gab2 was analyzed by mutants of Gab2. The Gab2 mutants lacking SHP-2-binding sites mostly abrogate the inhibitory activity of Gab2, but Gab2's inhibitory function is restored by fusing to active SHP-2 as a chimeric protein. A mutant with defective phosphatidylinositol 3-kinase binding capacity also impairs the inhibitory activity, and the pleckstrin homology domain-deletion mutant reveals a crucial function of the pleckstrin homology domain for localization to the plasma membrane. These results suggest that Gab2 is a substrate of ZAP-70 and functions as a switch molecule toward inhibition of TCR signal transduction by mediating the recruitment of inhibitory molecules to the TCR signaling complex (Yamasaki, 2001).

SHP-2, a nontransmembrane-type protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains, is thought to participate in growth factor signal transduction pathways via SH2 domain interactions. To determine the role of each region of SHP-2 in platelet-derived growth factor signaling assayed by Elk-1 activation, six deletion mutants of SHP-2 were generated. The large SH2 domain deletion SHP-2 mutant composed of amino acids 198-593 [SHP-2-(198-593)], but not the smaller SHP-2-(399-593), shows significantly higher SHP-2 phosphatase activity in vitro. In contrast, the SHP-2-(198-593) mutant inhibits wild type SHP-2 phosphatase activity, whereas SHP-2-(399-593) mutant increases activity. To understand these functional changes, focus was placed on the docking protein Gab1 that assembles signaling complexes. Pull-down experiments with Gab1 suggest that the C-terminal region of SHP-2 as well as the SH2 domains (N-terminal region) associate with Gab1, but the SHP-2-(198-593) mutant does not associate with Gab1. SHP-2-(1-202) or SHP-2-(198-593) inhibits platelet-derived growth factor-induced Elk-1 activation, but SHP-2-(399-593) increases Elk-1 activation. Co-expression of SHP-2-(1-202) with SHP-2-(399-593) inhibits SHP-2-(399-593)/Gab1 interaction, and the SHP-2-(399-593) mutant induces SHP-2 phosphatase and Elk-1 activation, supporting the autoinhibitory effect of SH2 domains on the C-terminal region of SHP-2. These data suggest that both SHP-2/Gab1 interaction in the C-terminal region of SHP-2 and increased SHP-2 phosphatase activity are important for Elk-1 activation. Furthermore, a novel sequence for SHP-2/Gab1 interactions was identified in the C-terminal region of SHP-2 (Huang, 2002).

A major Grb2-associated binder-1 (Gab1) binding partner in epidermal growth factor (EGF)-stimulated cells is protein-tyrosine phosphatase (PTPase) SHP2, which contains tandem SH2 domains. The SHP2 PTPase activity is required for activation of the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinase by EGF. To investigate the mechanism by which Gab1 and SHP2 mediate ERK activation, the Gab1-SHP2 interaction was characterized. Both Tyr-627 and Tyr-659 of Gab1 are required for SHP2 binding to Gab1 and for ERK2 activation by EGF. Far Western blot analysis suggested that the tandem SH2 domains of SHP2 bind to Gab1 in a specific orientation, in which the N-SH2 domain binds to phosphotyrosine Tyr(P)-627 and the C-SH2 domain binds to Tyr(P)-659. When assayed with peptide substrates, SHP2 PTPase is activated by a bisphosphopeptide containing both Tyr(P)-627 and Tyr(P)-659, but not by monophosphopeptides containing Tyr(P)-627 or Tyr(P)-659 or a mixture of these monophosphopeptides. These results suggest that Tyr(P)-627 and Tyr(P)-659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) that binds and activates SHP2. Remarkably, while a constitutively active SHP2 (SHP2DeltaN) can not rescue the defect of a SHP2-binding defective Gab1 (Gab1FF) in ERK2 activation, expression of a Gab1FF-SHP2DeltaN chimera results in constitutive activation of ERK2 in transfected cells. Thus, physical association of activated SHP2 with Gab1 is necessary and sufficient to mediate the ERK mitogen-activated protein kinase activation. Phosphopeptides derived from Gab1 are dephosphorylated by active SHP2 in vitro. Consistently, substrate-trapping experiments with a SHP2 catalytic inactive mutant suggests that Gab1 is a SHP2 PTPase substrate in the cells. Therefore, Gab1 not only is a SHP2 activator but also is a target of its PTPase (Cunnock, 2001).

Gab1-SHP2 association is required for Erk mitogen-activated protein kinase activation by several growth factors. Gab1-SHP2 interaction activates SHP2. However, an activated SHP2 still needs to associate with Gab1 to mediate Erk activation. It was unclear whether SHP2 is required to dephosphorylate a negative phosphorylation site on Gab1 or whether SHP2 needs the Gab1 pleckstrin homology (PH) domain to target it to the plasma membrane. Expression of a fusion protein consisting of the Gab1 PH domain and an active SHP2 (Gab1PH-SHP2DeltaN) induces constitutive Mek1 and Erk2 activation. Linking the active SHP2DeltaN to the PDK1 PH domain or the FRS2beta myristoylation sequence also induces Mek1 activation. Mek1 activation by Gab1PH-SHP2DeltaN is inhibited by an Src inhibitor and by Csk. Significantly, Gab1PH-SHP2DeltaN induces Src activation. Gab1PH-SHP2DeltaN expression activates Ras, and the Gab1PH-SHP2DeltaN-induced Mek1 activation is blocked by RasN17. These findings suggest that Gab1PH-SHP2DeltaN activates a signaling step upstream of Src and Ras. The SHP2 tyrosine phosphatase activity is essential for the function of the fusion protein. Together, these data show that the Gab1 sequence, besides the PH domain and SHP2 binding sites, is dispensable for Erk activation, suggesting that the primary role of Gab1 association with an activated SHP2 is to target it to the membrane (Cunnick, 2002).

Gab family proteins - Functions downstream of receptors

The multisubstrate docking protein, growth-factor-receptor-bound protein 2-associated binder 1 (Gab1), which is phosphorylated on tyrosine residues following activation of receptor tyrosine kinases and cytokine receptors, regulates cell proliferation, survival and epithelial morphogenesis. Gab1 is also tyrosine phosphorylated following activation of G-protein-coupled receptors (GPCRs) where its function is poorly understood. To elucidate the role of Gab1 in GPCR signaling, the mechanism by which the type A endothelin-1 (ET-1) GPCR induces tyrosine phosphorylation of Gab1 was investigated. Tyrosine phosphorylation of Gab1 induced by endothelin-1 is inhibited by PP1, a pharmacological inhibitor of Src-family tyrosine kinases. ET-1-induced Gab1 tyrosine phosphorylation is also inhibited by LY294002, which inhibits phosphoinositide 3-kinase (PI 3-kinase) enzymes. Inhibition of Src-family tyrosine kinases or PI 3-kinase also inhibits ET-1-induced activation of the mitogen activated protein kinase family member, extracellular signal-regulated kinase (ERK) 1. Thus it was determined whether Gab1 regulates ET-1-induced ERK1 activation. Overexpression of wild-type Gab1 potentiates ET-1-induced activation of ERK1. Structure-function analyses of Gab1 indicates that mutant forms of Gab1 that do not bind the Src homology (SH) 2 domains of the p85 adapter subunit of PI 3-kinase or the SH2-domain-containing protein tyrosine phosphatase 2 (SHP-2) are impaired in their ability to potentiate ET-1-induced ERK1 activation. Taken together, these data indicate that PI 3-kinase and Src-family tyrosine kinases regulate ET-1-induced Gab1 tyrosine phosphorylation, which, in turn, induces ERK1 activation via PI 3-kinase- and SHP-2-dependent pathways (Bisotto, 2001).

Fms is the receptor for macrophage colony-stimulating factor (M-CSF) and contains intrinsic tyrosine kinase activity. Expression of exogenous Fms in a murine myeloid progenitor cell line, FDC-P1 (FD-Fms), results in M-CSF-dependent growth and macrophage differentiation. A 100-kDa protein is tyrosine phosphorylated upon M-CSF stimulation of FD-Fms cells. This 100-kDa protein is the recently cloned scaffolding protein Gab2. Gab2 associates with several molecules involved in M-CSF signaling, including Grb2, SHP2, the p85 subunit of phosphatidylinositol 3'-kinase, SHIP, and SHC. Tyrosine phosphorylation of Gab2 in response to M-CSF requires the kinase activity of Fms, but not that of Src. Overexpression of Gab2 in FD-Fms cells enhances both mitogen-activated protein kinase (MAPK) activity and macrophage differentiation, but reduces proliferation, in response to M-CSF. In contrast, a mutant of Gab2 that is unable to bind SHP2 does not potentiate MAPK activity. Furthermore, overexpression of this mutant in FD-Fms cells inhibits macrophage differentiation and results in a concomitant increase in growth potential in response to M-CSF. These data indicate that Gab2 is involved in the activation of the MAPK pathway and that the interaction between Gab2 and SHP2 is essential for the differentiation signal triggered by M-CSF (Liu, 2001).

Hepatocyte growth factor (scatter factor) (HGF/SF) is a pleiotrophic mediator of epithelial cell motility, morphogenesis, angiogenesis, and tumorigenesis. HGF/SF protects cells against DNA damage by a pathway from its receptor c-Met to phosphatidylinositol 3-kinase (PI3K) to c-Akt, resulting in enhanced DNA repair and decreased apoptosis. Protection against the DNA-damaging agent adriamycin (ADR; topoisomerase IIalpha inhibitor) requires the Grb2-binding site of c-Met, and overexpression of the Grb2-associated binder Gab1 (a multisubstrate adapter required for epithelial morphogenesis) inhibits the ability of HGF/SF to protect MDCK epithelial cells against ADR. In contrast to Gab1 and its homolog Gab2, overexpression of c-Cb1, another multisubstrate adapter that associates with c-Met, does not affect protection. Gab1 blocks the ability of HGF/SF to cause the sustained activation of c-Akt and c-Akt signaling (FKHR phosphorylation). The Gab1 inhibition of sustained c-Akt activation and of cell protection does not require the Gab1 pleckstrin homology or SHP2 phosphatase-binding domain but does require the PI3K-binding domain. HGF/SF protection of parental MDCK cells is blocked by wortmannin, expression of PTEN, and dominant negative mutants of p85 (regulatory subunit of PI3K), Akt, and Pak1; the protection of cells overexpressing Gab1 is restored by wild-type or activated mutants of p85, Akt, and Pak1. These findings suggest that the adapter Gab1 may redirect c-Met signaling through PI3K away from a c-Akt/Pak1 cell survival pathway (Fan, 2001).

B cell antigen receptor (BCR) signaling causes tyrosine phosphorylation of the Gab1 docking protein. This allows phosphatidylinositol 3-kinase (PI3K) and the SHP2 tyrosine phosphatase to bind to Gab1. The hypothesis that Gab1 acts as an amplifier of PI3K- and SHP2-dependent signaling in B lymphocytes has been tested. By overexpressing Gab1 in the WEHI-231 B cell line, it was found that Gab1 can potentiate BCR-induced phosphorylation of Akt, a PI3K-dependent response. Gab1 expression also increases BCR-induced tyrosine phosphorylation of SHP2 as well as the binding of Grb2 to SHP2. The pleckstrin homology (PH) domain of Gab1 is required for BCR-induced phosphorylation of Gab1 and for Gab1 participation in BCR signaling. Moreover, using confocal microscopy, it has been shown that BCR ligation can induce the translocation of Gab1 from the cytosol to the plasma membrane and that this requires the Gab1 PH domain as well as PI3K activity. These findings are consistent with a model in which the binding of the Gab1 PH domain to PI3K-derived lipids brings Gab1 to the plasma membrane, where it can be tyrosine-phosphorylated and then act as an amplifier of BCR signaling (Ingham, 2001).

The Gab family of docking proteins (Gab1 and Gab2) are phosphorylated in response to various cytokines and growth factors. Gab1 acts to diversify the signal downstream from the Met receptor tyrosine kinase through the recruitment of multiple signaling proteins, and is essential for epithelial morphogenesis. To determine whether Gab1 and Gab2 are functionally redundant, the role of Gab2 in epithelial cells was examined. Both Gab1 and Gab2 are expressed in epithelial cells and localize to cell-cell junctions. However, whereas overexpression of Gab1 promotes a morphogenic response, the overexpression of Gab2 fails to induce this response. Gab2 recruitment to the Met receptor is dependent on the Grb2 adapter protein. In contrast, Gab1 recruitment to Met is both Grb2 dependent and Grb2 independent. The latter requires a novel amino acid sequence present in the Met-binding domain of Gab1 but not Gab2. Mutation of these residues in Gab1 impairs both association with the Met receptor and the ability of Gab1 to promote a morphogenic response, whereas their insertion into Gab2 increases Gab2 association with Met, but does not confer on Gab2 the ability to promote epithelial morphogenesis. It is proposed that the Grb2-independent recruitment of Gab proteins to Met is necessary but not sufficient to promote epithelial morphogenesis (Lock, 2002).

Receptor tyrosine kinases (RTKs) play distinct roles in multiple biological systems. Many RTKs transmit similar signals, raising questions about how specificity is achieved. One potential mechanism for RTK specificity is control of the magnitude and kinetics of activation of downstream pathways. The protein tyrosine phosphatase Shp2 regulates the strength and duration of phosphatidylinositol 3'-kinase (PI3K) activation in the epidermal growth factor (EGF) receptor signaling pathway. Shp2 mutant fibroblasts exhibit increased association of the p85 subunit of PI3K with the scaffolding adapter Gab1, compared to that for wild-type (WT) fibroblasts or Shp2 mutant cells reconstituted with WT Shp2. Far-Western analysis suggests increased phosphorylation of p85 binding sites on Gab1. Gab1-associated PI3K activity is increased and PI3K-dependent downstream signals are enhanced in Shp2 mutant cells following EGF stimulation. Analogous results are obtained in fibroblasts inducibly expressing dominant-negative Shp2. These results suggest that, in addition to its role as a positive component of the Ras-Erk pathway, Shp2 negatively regulates EGF-dependent PI3K activation by dephosphorylating Gab1 p85 binding sites, thereby terminating a previously proposed Gab1-PI3K positive feedback loop. Activation of PI3K-dependent pathways following stimulation by other growth factors is unaffected or decreased in Shp2 mutant cells. Thus, Shp2 regulates the kinetics and magnitude of RTK signaling in a receptor-specific manner (Zhang, 2002).

Cell adhesion and Gab proteins

Gab-1 is a multiple docking protein that is tyrosine phosphorylated by receptor tyrosine kinases such as c-Met, hepatocyte growth factor/scatter factor receptor, and epidermal growth factor receptor. Cell-cell adhesion also induces marked tyrosine phosphorylation of Gab-1. Disruption of cell-cell adhesion results in its dephosphorylation. An anti-E-cadherin antibody decreases cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas the expression of E-cadherin specifically induces tyrosine phosphorylation of Gab-1. A relatively selective inhibitor of Src family kinases reduces cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increases it. Disruption of cell-cell adhesion, which reduced tyrosine phosphorylation of Gab-1, also reduced the activation of mitogen-activated protein kinase and Akt in response to cell-cell adhesion. These results indicate that E-cadherin-mediated cell-cell adhesion induces tyrosine phosphorylation by a Src family kinase of Gab-1, thereby regulating the activation of Ras/MAP kinase and phosphatidylinositol 3-kinase/Akt cascades (Shinohara, 2001).

Signaling downstream of Gab proteins

Gab1 has structural similarities to Drosophila Dos; Dos is a substrate of the protein tyrosine phosphatase Corkscrew. 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. Gab1 is 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 is found to form a complex with phosphatidylinositol PI3 kinase and SHP-2, a homolog of Corkscrew. Mutational analysis of gp130, the common subunit of IL-6 family cytokine receptors, reveals that neither tyrosine residues of gp130 nor its carboxy terminus is required for tyrosine phosphorylation of Gab1. Expression of Gab1 enhances gp130-dependent mitogen-activated protein (MAP) kinase ERK2 activation. A mutation of tyrosine 759, the SHP-2 binding site of gp130, abrogates the interactions of Gab1 with SHP-2 and PI-3 kinase as well as ERK2 activation. Furthermore, ERK2 activation is 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 (Takahashi-Tezuka, 1998).

A novel human adapter molecule is described containing a pleckstrin homology (PH) domain at the N terminus that is closely related to human Grb2-associated binder 1 (Gab1) and Drosophila Daughter of sevenless. This protein has been designated as Gab2. Northern blot analysis indicates that Gab2 is widely expressed and has an overlapping but distinctive expression pattern as compared with Gab1, with high levels of Gab2 mRNA detected in the heart, brain, placenta, spleen, ovary, peripheral blood leukocytes, and spinal cord. Upon tyrosine phosphorylation, Gab2 physically interacts with Shp2 tyrosine phosphatase and Grb2 adapter protein. Strikingly, Gab2 has an inhibitory effect on the activation of Elk-1-dependent transcription triggered by a dominant active Ras mutant (RasV12) or under growth factor stimulation, whereas Gab1 acts to potentiate slightly the Elk-1 activity in the same system. In contrast to the reciprocal effects of Gab1 and Gab2 in mediating Elk-1 induction, these two molecules have a similar function in extracellular signal-regulated kinase activation induced by either oncogenic Ras or growth factor stimulation. Taken together, these results argue that Gab1 and Gab2, two closely related PH-containing adapter proteins, might have distinct roles in coupling cytoplasmic-nuclear signal transduction. This is the first evidence that an intracellular molecule with a PH domain operates as a negative effector in signal relay to the regulation of gene expression (Zhao, 1999).

Although the physiological function of Gab1 has not been fully understood, it apparently acts to promote cell growth and transformation. Overexpression of Gab1 leads to enhanced cell division and anchorage-independent cell growth in soft agar. Tyrosine phosphorylation of Gab1 and its association with Grb2 might also play an important role in cellular transformation by Tpr-Met, an oncoprotein consisting of the catalytic domain of hepatocyte growth factor receptor tyrosine kinase fused with sequences encoded by the tpr gene. A mutant Tpr-Met protein (Tyr489 to Phe) that fails to bind to Grb2 shows significantly impaired transforming activity. Although it is unclear how Gab1 functions in cytoplasmic signaling, the molecular architecture of this protein strongly suggests that Gab1 is tyrosine phosphorylated at numerous sites and therefore is engaged in the formation of a complex that consists of many proteins, including phospholipase Cgamma, Shp2, and phosphatidylinositol 3-kinase. In this regard, Gab1 and Gab2 might work in cells in a similar manner as the family of insulin receptor substrates, IRS1-4, by coupling to multiple signaling pathways. However, the difference is the possession of a PTB domain following the PH domain in the IRS proteins: this is missing from Gab1 and Gab2. Without a PTB domain, Gab1/2 might interact with growth factor receptors indirectly via other SH2-containing molecules, such as Grb2 (Zhao, 1999).

Gab1 and Gab2 share structural similarity with the Drosophila Daughter of sevenless protein. Not only is Daughter of sevenless physically associated with Corkscrew, but Daughter of sevenless is the major phosphoprotein trapped by a catalytically inactive mutant of Corkscrew that is overexpressed in cells. Both Gab1 and Gab2 have been detected in association with Shp2, the mammalian homolog of Drosophila Corkscrew. Now the critical issue is to determine whether they are the physiological substrates of Shp2 and how this dephosphorylation event could contribute to the Ras signaling pathway. It is possible that these two molecules may serve as substrates of the ubiquitously expressed Shp2 phosphatase in different cell types. Further, these proteins may also be potential substrates of another Shp2-related phosphatase, Shp1, which is predominantly expressed in hematopoietic cells (Zhao, 1999).

The most striking finding in this report is the reciprocal effects of Gab1 and Gab2 in mediating Ras-responsive Elk-1 activation in cells. Gab2 does not seem to block the ERK kinase activation by either receptor tyrosine kinase or dominant active Ras, but rather it acts to uncouple signals from activated ERK to Elk-1, a member of the ternary complex factors family. Expression of murine p97/Gab2 in Ba/F3 cells also leads to a suppression of IL-3-induced Elk-1 activity, whereas ERK1 kinase activation is not affected. This inhibitory effect of mouse Gab2 on IL-3-stimulated Elk-1 activity is exacerbated by the introduction of a C-terminal truncation mutation that abolishes its binding to Shp2 (Zhao, 1999 and references).

Dos/Gab family scaffolding adapters (Dos, Gab1, Gab2) bind several signal relay molecules, including the protein-tyrosine phosphatase Shp-2 and phosphatidylinositol-3-OH kinase (PI(3)K); they are also implicated in growth factor, cytokine and antigen receptor signal transduction. Mice lacking Gab1 die during embryogenesis and show defective responses to several stimuli. Gab2-/- mice are viable and generally healthy; however, there is a defective response (for example, degranulation and cytokine gene expression) of Gab2-/- mast cells to stimulation of the high affinity immunoglobulin-epsilon (IgE) receptor Fc(epsilon)RI. Accordingly, allergic reactions such as passive cutaneous and systemic anaphylaxis are markedly impaired in Gab2-/- mice. Biochemical analyses reveal that signaling pathways dependent on PI(3)K, a critical component of Fc(epsilon)RI signaling, are defective in Gab2-/- mast cells. These data identify Gab2 as the principal activator of PI(3)K in response to Fc(epsilon)RI activation, thereby providing genetic evidence that Dos/Gab family scaffolds regulate the PI(3)K pathway in vivo. Gab2 and/or its associated signaling molecules may be new targets for developing drugs to treat allergy (Gu, 2001).

Based on the observations that active ERK associates with and phosphorylates Gab1 in response to HGF, and the prediction that the ERK phosphorylation site is adjacent to one of the phosphatidylinositol 3-kinase (PI3K) SH2 binding motifs, the possibility that ERK phosphorylation can regulate the Gab1/PI3K association was examined. The HGF-mediated association of Gab1 with either full-length GST-p85 or its isolated N- or C-terminal SH2 domains was inhibited by approximately 50% in the setting of ERK inhibition, a result confirmed by co-immunoprecipitation of the native proteins. A 14-amino acid peptide encoding (472)YVPMTP(477) (one of the major p85 binding sites in Gab1 and the predicted ERK phosphorylation site) was synthesized with either phosphotyrosine alone (pY), or phosphotyrosine + phosphothreonine (pYT). In both pull-down assays and competition assays, pYT demonstrates a higher affinity for p85 than does pY alone. Finally, examination of the phosphorylation state of Akt after HGF stimulation revealed that ERK inhibition results in a decrease in Akt activation at both 5 and 10 min. These results suggest that activated ERK can phosphorylate Gab1 in response to HGF stimulation and thereby potentiate the Gab1/PI3K association and subsequent PI3K activation (Yu, 2001).

The ability of epidermal growth factor (EGF)-stimulated ERK activation to regulate Grb2-associated binder-1 (Gab1)/phosphatidylinositol 3-kinase (PI3K) interactions has been examined. Inhibiting ERK activation with the MEK inhibitor U0126 increases the EGF-stimulated association of Gab1 with either full-length glutathione S-transferase-p85 or the p85 C-terminal Src homology 2 (SH2) domain, a result reproduced by co-immunoprecipitation of the native proteins from intact cells. This increased association of Gab1 and the PI3K correlates with an increase in PI3K activity and greater phosphorylation of Akt. This result is in direct contrast to what has been reported following HGF stimulation where MEK inhibition decreases the HGF-stimulated association of Gab1 and p85. In support of this divergent effect of ERK on Gab1/PI3K association following HGF and EGF stimulation, U0126 decreases the HGF-stimulated association of p85 and the Gab1 c-Met binding domain but does not alter the EGF-stimulated association of p85 and the c-Met binding domain. An examination of the mechanism of this effect revealed that the treatment of cells with EGF + U0126 increases the tyrosine phosphorylation of Gab1 as well as its association with another SH2-containing protein, SHP2. Furthermore, overexpression of a catalytically inactive form of SHP2 or pretreatment with pervanadate markedly increases EGF-stimulated Gab1 tyrosine phosphorylation. These experiments demonstrate that EGF and HGF-mediated ERK activation result in divergent effects on Gab1/PI3K signaling. HGF-stimulated ERK activation increases the Gab1/PI3K association, whereas EGF-stimulated ERK activation results in a decrease in the tyrosine phosphorylation of Gab1 and a decreased association with the PI3K. SHP2 is shown to associate with and dephosphorylate Gab1, suggesting that EGF-stimulated ERK might act through the regulation of SHP2 (Yu, 2002).

Gab proteins and differentiation

Using the FDC-P1 cell line expressing the exogenous macrophage colony-stimulating factor (M-CSF) receptor, Fms, the role of a new mammalian DOS/Gab-related signaling protein, called Gab3, has been analyzed in macrophage cell development of the mouse. Gab3 contains an amino-terminal pleckstrin homology domain, multiple potential sites for tyrosine phosphorylation and SH2 domain binding, and two major polyproline motifs potentially interacting with SH3 domains. Among the growing family of Gab proteins, Gab3 exhibits a unique and overlapping pattern of expression in tissues of the mouse compared with Gab1 and Gab2. Gab3 is more restricted to the hematopoietic tissues such as spleen and thymus but is detectable at progressively lower levels within heart, kidney, uterus, and brain. Like Gab2, Gab3 is tyrosine phosphorylated after M-CSF receptor stimulation and associates transiently with the SH2 domain-containing proteins p85 and SHP2. Overexpression of exogenous Gab3 in FD-Fms cells dramatically accelerates macrophage differentiation upon M-CSF stimulation. Unlike Gab2, which shows a constant mRNA expression level after M-CSF stimulation, Gab3 expression is initially absent or low in abundance in FD cells expressing the wild-type Fms, but Gab3 mRNA levels are increased upon M-CSF stimulation. Moreover, M-CSF stimulation of FD-FmsY807F cells (which grow but do not differentiate) fails to increase Gab3 expression. These results suggest that Gab3 is important for macrophage differentiation and that differentiation requires the early phosphorylation of Gab2 followed by induction and subsequent phosphorylation of Gab3 (Wolf, 2002).

Gab proteins and transformation

An in vitro transformation system of carcinogen-treated Syrian hamster embryo (SHE) cell cultures represents multistep genetic and nongenetic changes that develop during the neoplastic progression of normal cells to tumor cells in vivo. During this neoplastic progression, SHE cells demonstrate an altered response to epidermal growth factor (EGF). The role of the adapter protein Gab1 (Grb2-associated binder-1) in the neoplastic progression of SHE cells has been examined. Two asbestos-transformed SHE cell clones in different neoplastic stages were used: a 10W+8 clone, which is immortal and retains the ability to suppress the tumorigenicity of tumor cells in cell-cell hybrid experiments, and a 10W-1 clone, which has lost this tumor suppressor ability. 10W+8 cells express full-length 100-kDa Gab1 and associated 5.2-kb mRNA. Upon repeated cell passaging, 10W-1 cells show increasing expression of a novel 87-kDa form of Gab1 as well as 4.6-kb mRNA with diminishing expression of the original 100-kDa Gab1. cDNA encoding the 87-kDa Gab1 predicts a form of Gab1 lacking the amino-terminal 103 amino acids [Gab1(Delta1-103)], which corresponds to loss of most of the pleckstrin homology (PH) domain. Gab1(Delta1-103) retains the ability to be phosphorylated in an EGF-dependent manner and to associate with the EGF receptor and SHP-2 upon EGF stimulation. The endogenous expression of Gab1(Delta1-103) in 10W-1 cells appears closely related to EGF-dependent colony formation in soft agar. Moreover, transfection and expression of Gab1(Delta1-103), but not Gab1, in 10W+8 cells enhances their EGF-dependent colony formation in soft agar. These results demonstrate that Gab1 is a target of carcinogen-induced transformation of SHE cells and that the expression of a Gab1 variant lacking most of the PH domain plays a specific role in the neoplastic progression of SHE cells (Kameda, 2001).

Middle T antigen (PymT) is the principal transforming component of polyomavirus, and rapidly induces hemangiomas in neonatal mice. PymT, a membrane-associated scaffold, recruits and activates Src family tyrosine kinases, and, once tyrosine phosphorylated, binds proteins with PTB and SH2 domains such as ShcA, phosphatidylinositol 3-kinase (PI3K) and phospholipase Cgamma-1 (PLCgamma-1). To explore the pathways required for endothelial transformation in vivo, PymT mutant forms were introduced into mice. PymT variants unable to bind PI3K and PLCgamma-1 directly induce hemangiomas similarly to wild type, but a mutant unable to bind ShcA is transformation compromised. Requirement for a ShcA PTB domain-binding site is suppressed by replacing this motif in PymT with YXN sequences, which bind the Grb2 SH2 domain upon phosphorylation. Surprisingly, PymT recruitment of ShcA and Grb2 correlate with PI3K activation. PymT mimics activated receptor tyrosine kinases by forming a ShcA-Grb2-Gab1 complex, thus inducing Gab1 tyrosine phosphorylation, which itself is associated with PI3K. Therefore, PymT activation of ShcA-Grb2 signaling is critical for endothelial transformation, and PymT can stimulate Grb2 signaling to both the MAP kinase and PI3K pathways (Ong, 2001).

daughter of sevenless: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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