Dos protein is located in the membranes of cells within and immediately posterior to the morphogenetic furrow. Further behind the furrow, Dos protein is only membrane associated in a few cells of each ommatidial cluster. Membrane-localized Dos protein is restricted to a small region just below the apical surface of the cells, which probably corresponds to the adherens junctions (Raabe, 1996).
Expression of a catalytically inactive Corkscrew (CSW) was used to trap CSW in a complex with a 115 kDa tyrosine-phosphorylated substrate. This substrate was purified and identified as the product of the daughter of sevenless (dos) gene. Mutations of dos were identified in a screen for dominant mutations that enhance the phenotype caused by overexpression of inactive CSW during photoreceptor development. Analysis of dos mutations indicates that DOS is a positive component of the SEV signaling pathway and suggests that DOS dephosphorylation by CSW may be a key event during signaling by SEV (Herbst, 1996).
One mechanism used by receptor tyrosine kinases to relay a signal to different downstream effector molecules is to use adaptor proteins that provide docking sites for a variety of proteins. The daughter of sevenless (dos) gene was isolated in a genetic screen for components acting downstream of the Sevenless (Sev) receptor tyrosine kinase. Dos contains an N-terminally located PH domain and several tyrosine residues within consensus binding sites for a number of SH2 domain containing proteins. The structural features of Dos and experiments demonstrating tyrosine phosphorylation of Dos upon Sev activation have suggested that Dos belongs to the family of multisite adaptor proteins that includes the Insulin Receptor Substrate (IRS) proteins, Gab1, and Gab2. The structural requirements for Dos function in receptor tyrosine kinase mediated signaling processes has been studied by expressing mutated dos transgenes in the fly. Mutant Dos proteins lacking the putative binding sites for the SH2 domains of Shc, PhospholipaseC-gamma (PLC-gamma) and the regulatory subunit of Phosphoinositide 3-kinase (PI3-K) can substitute the loss of endogenous Dos function during development. In contrast, tyrosine 801, corresponding to a predicted Corkscrew (Csw) tyrosine phosphatase SH2 domain binding site, is essential for Dos function. Furthermore, assays were performed to see whether the Pleckstrin homology (PH) domain is required for Dos function and localization. Evidence is provided that deletion or mutation of the PH domain interferes with the function but not with localization of the Dos protein. The Dos PH domain can be replaced by the Gab1 PH domain but not by a heterologous membrane anchor, suggesting a specific function of the PH domain in regulating signal transduction (Bausenwein, 2000).
The Drosophila nonreceptor protein tyrosine phosphatase, Corkscrew, functions positively in multiple receptor tyrosine kinase pathways, including signaling by the epidermal growth factor receptor. Detailed phenotypic analyses of csw mutations have revealed that Csw activity is required in many of the same developmental processes that require Egfr function. However, it is still unclear where in the signaling hierarchy Csw functions relative to other proteins whose activities are also required downstream of the receptor. To address this issue, genetic interaction experiments were performed to place csw gene activity relative to the Egfr, spitz, rhomboid, daughter of sevenless, kinase-suppressor of ras, ras1, D-raf, pointed, and moleskin. The Egfr-dependent formation of VA2 muscle precursor cells was followed as a sensitive assay for these genetic interaction studies: (1) it was established that Csw has a positive function during mesoderm development; (2) it was found that tissue-specific expression of a gain-of-function csw construct rescues loss-of-function mutations in other positive signaling genes upstream of rolled/MAPK in the Egfr pathway; (3) it was possible to infer levels of Egfr signaling in various mutant backgrounds during myogenesis (Hamlet, 2001).
This work extends previous studies of Csw during Torso and Sevenless RTK signaling to include an in-depth analysis of the role of Csw in the Egfr signaling pathway. It has been reported that a direct physical interaction between DOS and Csw is important for signaling in the Sevenless (Sev) pathway (Herbst, 1999; Bausenwein,, 2000). Interestingly, the genetic data are consistent with a close interaction between DOS and Csw on the basis of their similar phenotypes. For example, VA2 precursor cells form in 25% of hemisegments in mutant embryos expressing the putative dominant-negative DOS mutation, DOSR31 (Bausenwein, 2000), which is in the same range statistically as the percentage of VA2 precursor formation in cswVA199/Y embryos. Likewise, expression of UAS-cswsrc90 with twi-Gal4 in these DOS mutant embryos increases VA2 development to 41%, a statistically significant rescue that places Csw function downstream of DOS. Further, this rescue is in the same range as Cswsrc90-induced rescue of cswVA199 mutant embryos. In summary, these data are consistent with a close association between Csw and DOS and suggest that Csw function is required downstream of DOS function (Hamlet, 2001).
The genetic interaction data between csw and DOS are consistent with a model whereby a direct interaction between Csw and DOS is essential for Drosophila EGFR signaling. When the predominant Tyr residues are mutated in DOS, only DOS protein lacking the phosphorylated Tyr (pTyr) site(s) in consensus to bind the Csw SH2 domain significantly abrogated Sev signaling (Herbst, 1999; Bausenwein, 2000). Conversely, a DOS protein containing only the pTyr sites that bind to the Csw SH2 domains is sufficient to provide wild-type DOS function (Herbst, 1999). A vertebrate DOS homolog, Gab1, and SHP-2 associate upon activation of the vertebrate EGFR, the result of which is an increase in MAPK signaling. The readout from the putative DOS dominant-negative mutant embryos is in the same range as that of dominant-negative csw mutant embryos. The identical genetic interaction of csw and DOS with cswsrc90 places their function in a category separate from that of the other signaling genes analyzed and suggests that they both function at the same level in the EGFR pathway. Interestingly, DOS mutant embryos phenocopy the putative dominant-negative csw mutant embryos but not the protein null csw mutant embryos. These results suggest that the dominant-negative csw mutant phenotype reflects loss of DOS function. Since the cswVA199 mutation generates a truncated Csw protein where only the SH2 domains are expressed, perhaps the SH2 domains still bind to and sequester DOS function away from the signaling pathway (Hamlet, 2001).
A variety of genetic interaction experiments between gain- and loss-of-function mutations and/or constructs in genes involved in Egfr signaling has resulted in three principal findings. (1) Consistent with findings in the developing retina, Cswsrc90 functions like a bona fide gain-of-function protein in several Egfr-initiated developmental processes during oogenesis, embryogenesis, and metamorphosis. (2) Csw plays a positive role in Egfr signaling during myogenesis. (3) Tracking the formation of VA2 precursor cells serves as a sensitive assay to infer levels of Egfr signaling in various mutant genetic backgrounds (Hamlet, 2001).
Genetic interaction data between csw and Dos are consistent with a model whereby a direct interaction between Csw and Dos is essential for Drosophila Egfr signaling. A Dos protein containing only the pTyr sites that bind to the Csw SH2 domains is sufficient to provide wild-type Dos function. A vertebrate Dos homolog, Gab1, and SHP-2 associate upon activation of the vertebrate Egfr, results in an increase in MAPK signaling (Hamlet, 2001 and references therein).
The readout from the putative Dos dominant-negative mutant embryos is in the same range as that of dominant-negative csw mutant embryos. The identical genetic interaction of csw and Dos with cswsrc90 places their function in a category separate from that of the other signaling genes analyzed and suggests that they both function at the same level in the Egfr pathway (Hamlet, 2001).
Interestingly, Dos mutant embryos phenocopy the putative dominant-negative csw mutant embryos but not the protein null csw mutant embryos. These results suggest that the dominant-negative csw mutant phenotype reflects loss of Dos function. Since the cswVA199 mutation generates a truncated Csw protein where only the SH2 domains are expressed, perhaps the SH2 domains still bind to and sequester Dos function away from the signaling pathway (Hamlet, 2001).
Bausenwein, B. S., Schmidt, M., Mielke, B. and Raabe, T. (2000). In vivo functional analysis of the Daughter of Sevenless protein in receptor tyrosine kinase signaling. Mech. Dev. 90: 205-215. 10640704
Bisotto, S. and Fixman, E. D. (2001). Src-family tyrosine kinases, phosphoinositide 3-kinase and Gab1 regulate extracellular signal-regulated kinase 1 activation induced by the type A endothelin-1 G-protein-coupled receptor. Biochem. J. 360(Pt 1): 77-85. 11695994
Cunnick, J. M., et al. (2001). Phosphotyrosines 627 and 659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) conferring binding and activation of SHP2. J. Biol. Chem. 276(26): 24380-7. 11323411
Cunnick, J. M., et al. (2002). Regulation of the mitogen-activated protein kinase signaling pathway by SHP2. J. Biol. Chem. 277(11): 9498-504. 11779868
Fan, S., et al. (2001). The multisubstrate adapter Gab1 regulates hepatocyte growth factor (scatter factor)-c-Met signaling for cell survival and DNA repair. Mol. Cell. Biol. 21(15): 4968-84. 11438654
Feller, S. M., et al. (2002). SH3 domain-mediated binding of the Drk protein to Dos is an important step in signaling of Drosophila receptor tyrosine kinases. Mech. Dev. 116: 129-139. 12128212
Fixman, M., et al. (1997). Efficient cellular transformation by the Met oncoprotein requires a functional Grb2 binding site and correlates with phosphorylation of the Grb2-associated proteins, Cbl and Gab1. J. Biol. Chem. 272: 20167-20172. 9242692
Gu, H., et al. (2001). Essential role for Gab2 in the allergic response. Nature 412(6843): 186-90. 11449275
Hamlet, M. R. J. and Perkins, L. A. (2001). Analysis of Corkscrew signaling in the Drosophila Epidermal growth factor receptor pathway during myogenesis. Genetics 159: 1073-1087. 11729154
Herbst, R., et al. (1996). Daughter of sevenless is a substrate of the phosphotyrosine phosphatase Corkscrew and functions during sevenless signaling. Cell 85: 899-909
Herbst, R., et al. (1999). Recruitment of the protein tyrosine phosphatase CSW by DOS is an essential step during signaling by the Sevenless receptor tyrosine kinase. EMBO J. 18: 6950-6961.
Huang, Q., et al. (2002). The novel role of the c-terminal region of SHP-2. Involvement of Gab1 AND SHP-2 Phosphatase activity in Elk-1 activation. J. Biol. Chem. 277(32): 29330-41. 12011040
Ingham, R. J., et al. (2001). The Gab1 docking protein links the B cell antigen receptor to the phosphatidylinositol 3-kinase/Akt signaling pathway and to the SHP2 tyrosine phosphatase. J. Biol. Chem. 276(15): 12257-65. 11278704
Kameda, H., et al. (2001). Expression of Gab1 lacking the pleckstrin homology domain is associated with neoplastic progression. Mol. Cell. Biol. 21(20): 6895-905. 11564873
Lewitzky, M., et al. (2001). The C-terminal SH3 domain of the adapter protein Grb2 binds with high affinity to sequences in Gab1 and SLP-76 which lack the SH3-typical P-x-x-P core motif. Oncogene 20(9): 1052-62. 11314042
Liu, Y., et al. (2001). Scaffolding protein Gab2 mediates differentiation signaling downstream of Fms receptor tyrosine kinase. Mol. Cell. Biol. 21(9): 3047-56. 11287610
Lock, L. S., et al. (2000). Identification of an atypical Grb2 carboxyl-terminal SH3 domain binding site in Gab docking proteins reveals Grb2-dependent and -independent recruitment of Gab1 to receptor tyrosine kinases. J. Biol. Chem. 275: 31536-31545. 10913131
Lock, L. S., et al. (2002). Distinct recruitment and function of Gab1 and Gab2 in Met receptor-mediated epithelial morphogenesis. Mol. Biol. Cell 13(6): 2132-46. 12058075
Nguyen, L., et al. (1997). Association of the multisubstrate docking protein Gab1 with the hepatocyte growth factor requires a functional Grb2 binding site involving tyrosine 1356. J. Biol. Chem. 272: 20811-20819. 9252406
Ong, S. H., et al. (2001). ShcA and Grb2 mediate polyoma middle T antigen-induced endothelial transformation and Gab1 tyrosine phosphorylation. EMBO J. 20(22): 6327-36. 11707404
Raabe, T., et al. (1995). Biochemical and genetic analysis of the Drk SH2/SH3 adaptor protein of Drosophila. EMBO J. 14: 2509-2518. 7781603
Raabe, T., et al. (1996). Dos, a novel pleckstrin homology domain-containing protein required for signal transduction between sevenless and Ras1 in Drosophila. Cell 85: 911-920. 8681385
Schaeper, U., et al. (2000). Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses. J. Cell Biol. 149: 1419-1432. 10871282
Schutzman, J. L., et al. (2001). The Caenorhabditis elegans EGL-15 signaling pathway implicates a DOS-like multisubstrate adaptor protein in fibroblast growth factor signal transduction. Mol. Cell. Biol. 21(23): 8104-16. 11689700
Shinohara, M., et al. (2001). Roles of cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1. J. Biol. Chem. 276(22): 18941-6. 11262408
Takahashi-Tezuka, M., et al. (1998). Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase. Mol. Cell. Biol. 18(7): 4109-17.
Wolf, I., et al. (2002). Gab3, a new DOS/Gab family member, facilitates macrophage differentiation. Mol. Cell. Biol. 22(1): 231-44. 11739737
Yamasaki, S., et al. (2001). Docking protein Gab2 is phosphorylated by ZAP-70 and negatively regulates T cell receptor signaling by recruitment of inhibitory molecules. J. Biol. Chem. 276(48): 45175-83. 11572860
Yu, C. F., et al. (2001). ERK regulates the hepatocyte growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase. J. Biol. Chem. 276(35): 32552-8. 11445578
Yu, C. F., Liu, Z. X. and Cantley, L. G. (2002). ERK negatively regulates the epidermal growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase. J. Biol. Chem. 277(22): 19382-8. 11896055
Zhang, S. Q., et al. (2002). Receptor-specific regulation of phosphatidylinositol 3'-kinase activation by the protein tyrosine phosphatase Shp2. Mol. Cell. Biol. 22(12): 4062-72. 12024020
Zhao, C., et al. (1999). Gab2, a new pleckstrin homology domain-containing adapter protein, acts to uncouple signaling from ERK kinase to Elk-1. J. Biol. Chem. 274: 19649-19654.
date revised: 20 August 2002
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