corkscrew

Targets of Activity

csw mutant embryos show altered expression of hunchback (hb), forkhead (fkh) and fushi tarazu (ftz). In csw mutant embryos, HB does not retract from the pole, but remains as a terminal cap. This altered pattern of hb expression is like that observed in huckebein mutant embryos where hb continues to be expressed at the pole due to lack of the Huckebein repressing activity. As in hkb mutants, csw mutant embryos exhibit a reduction in fkh posteriorly. In csw mutant embryos as in hkb mutants, the seventh ftz stripe, while present, is expanded posteriorly. Taken together, these data suggest that maternally provided csw positively affects the activity of tailless, as well as further downstream genes of the terminal system. Since the effect of an hyperactive torso allele can be negated by mutations in csw, it is concluded that csw is a member of the terminal class signal transduction pathway, and functions downstream of the receptor tyrosine kinase, Torso (Perkins, 1992).

tailless expression is severly reduced but not eliminated in csw mutants. Since D-raf acts downstream of torso, a test was made whether all terminal activity in csw null alleles could be deleted by reducing D-raf activity. Embryos null for csw, with a hypomorphic allele of raf show no tll activity. It is concluded that csw acts in concert with D-raf to regulate tll expression (Perkins, 1992).

Protein Interactions

Tyrosines 630 and 918 are the major sites of TOR autophosphorylation. Mutation of tyrosine 630, a site required for association with and tyrosine phosphorylation of the tyrosine phosphatase Corkscrew, decreases the efficiency of TOR signaling. In contrast, mutation of Y918, a site capable of binding mammalian rasGAP and PLC-gamma1, increases TOR signaling. When receptors contain mutations in both sites, TOR signaling is restored to wild-type levels. CSW becomes tyrosine phosphorylated as a direct result of TOR ligand induced activation. It is thought that Y630 functions as a binding site for the SH2 domain-containing CSW and that CSW is a substrate of TOR (Cleghon, 1996).

Four tyrosine residues (Y644, Y698, Y767, and Y772) are identifed that become phosphorylated after activation of the Torso (Tor) receptor tyrosine kinase. Phosphotyrosine sites P-Y630 and P-Y918 have also been identifed. Of the six P-Y sites identified, three (Y630, Y644, and Y698) are located in the kinase domain insert region; one (Y918) is located in the C-terminal tail region, and two (Y767 and Y772) are located in the activation loop of the kinase domain. To investigate the function of each P-Y residue in Tor signaling, transgenic Drosophila embryos expressing mutant Tor receptors containing either single or multiple tyrosine to phenylalanine substitutions were generated. Single P-Y mutations have either positive, negative, or no effect on the signaling activity of the receptor. Elimination of all P-Y sites within the kinase insert region results in the complete loss of receptor function, indicating that some combination of these sites is necessary for Tor signaling. Mutation of the C-terminal P-Y918 site reveals that this site is responsible for negative signaling or down-regulation of receptor activity. Mutation of the P-Y sites in the kinase domain activation loop demonstrates that these sites are essential for enzymatic activity. This analysis provides a detailed in vivo example of the extent of cooperativity between P-Y residues in transducing the signal received by a receptor tyrosine kinase and in vivo data demonstrating the function of P-Y residues in the activation loop of the kinase domain (Gayko,1999).

This analysis reveals that Tor autophosphorylates on tyrosine residues located in both noncatalytic (the kinase insert region and the C-terminal tail) and catalytic regions (the activation loop) of the molecule. In the kinase insert, three (Y630, Y644, and Y698) of the four tyrosines present are phosphorylated. Mutation of individual P-Y sites either has no effect on or reduces the level of Tor signaling, whereas the simultaneous mutation of all four tyrosines (Y630, Y644, Y656, and Y698) eliminates Tor signaling. These results demonstrate that the tyrosine residues within the kinase insert domain are essential for Tor signal transduction and that multiple P-Y sites act synergistically to propagate the Tor signal. The P-Y630 site serves as a binding site for Csw; however, it is not yet known what molecules bind to either P-Y644 or P-Y698. Of interest is what appears to be a redundancy between the Y644 and Y698 sites, because neither mutation of Y644 or Y698 alone is associated with a decrease in Tor activity. Finally, Y656, which is located within the insert region of Tor, does not appear to be phosphorylated and has no apparent activity in triggering downstream signaling events. Thus, it is proposed that Tor positive signaling is transduced by the synergistic activities of Y630 with Y644 and/or Y698 (Gayko,1999).

To date, the only members of the Tor pathway that contain SH2 domains are Csw and Drk. Csw, the Drosophila homolog of SHP-2, encodes a nonreceptor tyrosine phosphatase with two N-terminal SH2 motifs. Drk, the Drosophila homolog of Grb2, encodes an adapter protein containing one SH2 and two SH3 motifs and functions to recruit the exchange factor Sos to the activated RTK. Csw associates directly with P-Y630 and there are no direct binding sites for Drk on Tor. Csw has at least two distinct functions in Tor signaling: (1) it regulates positive signaling through Drk because P-Y666 on Csw is a Drk-binding site; (2) it blocks the activity of a negative regulator of Tor signaling that binds to the P-Y918 site. Mutation of Y918 leads to an increase in Tor activity, and P-Y918 is a binding site for a Drosophila Ras-GAP protein that contains two SH2 motifs. Csw dephosphorylates P-Y918 and thus prevents the negative regulator RasGAP from associating with Tor (Gayko, 1999 and references).

Three models by which P-Y644 and P-Y698 residues transduce the Tor signal are envisioned. In the first model, these P-Ys may bind an adapter molecule(s), which would recruit either Csw and/or Drk to Tor. This model predicts that activation of all downstream signaling events is mediated solely by Csw and Drk, a hypothesis that can be tested by examining the phenotype of embryos derived from germlines missing both Csw and Drk activities. Possible candidates for such an adapter include SHC and NCK/DOCK, although it is not known whether these proteins bind Csw/SHP-2 or Drk/Grb2. In a second model, Tor could transduce a signal through activation of Csw and Drk, as well as through Dos. Dos encodes a protein with an amino-terminal pleckstrin homology domain, a polyproline motif that may bind an SH3 domain, and 10 potential P-Y sites with consensus sequences for binding SH2 domains. Previous studies have implicated Dos as a component of the Tor signaling pathway because embryos derived from females that lack maternal Dos activity show a partial loss of function Tor phenotype, and other studies have demonstrated that Dos can bind Grb2 and Csw. In the third model, P-Y644 and P-Y698 could mediate activation of the Raf kinase in a pathway that does not require Ras1 activity. Previous work has suggested the existence of a Ras1-independent pathway of Raf activation. In this scenario, a novel, yet unidentified protein could bind to Y644 and Y698, through either SH2 domain or PTB domains, and could lead to Raf activation in a Ras1-independent manner (Gayko, 1999 and references).

A role for the phosphorylation of Y767 and Y772, two tyrosine residues located in the activation loop of the kinase domain has been demonstrated. Mutation of these P-Y sites completely eliminates Tor signaling. This phenotype is likely caused by the effect that these mutations have on Tor catalytic activity itself. Catalytic activity has been shown to be essential for Tor signaling. No tyrosine phosphorylation can be demonstrated of the Tor protein isolated from embryos expressing TorYY767+Y772FF, and tyrosine phosphorylation of TorYY767+Y772FF expressed in Sf9 cells is greatly reduced (at least 50-fold) when compared with the WT protein. Autophosphorylation of the corresponding tyrosine residues in the insulin receptor, the scatter factor/hepatocyte growth factor receptor, the nerve growth factor receptor, and the fibroblast growth factor receptor has been shown to be required for catalytic activity of these kinases. Based on crystal structure analysis of the fibroblast growth factor receptor and the insulin receptor, autophosphorylation of tyrosine residues in the activation loop results in a dramatic change in conformation, thus relieving an autoinhibitory mechanism and allowing unrestricted access to the binding sites for ATP and protein substrates. Data in trangenic animals is provided here supporting the role for the autophosphorylation of activation loop residues in RTK signaling (Gayko, 1999 and references).

Corkscrew (Csw) is required for signaling by receptor tyrosine kinases, including Sevenless (Sev), which directs Drosophila R7 photoreceptor cell development. To investigate the role of the different domains of Csw, domain-specific csw mutations were constructed and their effects on Csw function were assayed. Csw SH2 domain function is essential, but either of the Csw SH2 domains can fulfill this requirement. Csw and activated Sev are associated in vivo in a manner that does not require either Csw SH2 domain function or tyrosine phosphorylation of Sev. Thus, Sev is unlikely to be a binding partner for the Csw SH2 domains. Evaluation cannot presently be made as to whether the observed phosphotyrosine-independent association of Csw and Sev actually occurs in the developing R7 cell or is important for Sev signaling. In contrast, the interaction between Csw and Daughter of sevenless, a Csw substrate, is dependent on SH2 domain function. These results suggest that the role of the Csw SH2 domains during Sev signaling is to bind Daughter of Sevenless rather than activated Sev. Although Csw protein-tyrosine phosphatase activity is required for full Csw function, a catalytically inactive Csw is capable of providing partial function. In addition, deletion of either the Csw protein-tyrosine phosphatase insert or the entire Csw carboxyl terminus, which includes a conserved Drk/Grb2 SH2 domain binding sequence, does not abolish Csw function (Allard, 1998).

Expression of a catalytically inactive 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).

The pleckstrin homology (PH) domain-containing protein Daughter of Sevenless (Dos) is an essential component of the Sevenless receptor tyrosine kinase (SEV) signaling cascade, which specifies R7 photoreceptor development in the Drosophila eye. Previous results have suggested that Dos becomes tyrosine phosphorylated during Sev signaling and collaborates with the protein tyrosine phosphatase Csw. This possibility was investigated by identifying tyrosine residues 801 and 854 of Dos as the phosphorylated binding sites for the Csw SH2 domains. These sites become phosphorylated in response to Sev activation and phosphorylation of both sites is required to allow Csw to bind Dos. Mutant Dos proteins in which either Y801 or Y854 of Dos has been changed to phenylalanine are unable to function during signaling by Sev and other receptor tyrosine kinases. In contrast, a mutant Dos protein in which all tyrosine phosphorylation sites except Y801 and Y854 have been removed is able effectively to provide Dos function during Sev signaling and to rescue the lethality associated with dos loss-of-function mutations. These results indicate that a primary role for Dos during signaling by Sev and other receptor tyrosine kinases is to become phosphorylated at Y801 and Y854 and then recruit Csw (Herbst, 1999).

Structural and enzymatic studies of SHP-2, the mammalian homolog of Csw, have shown that binding of the N-terminal SH2 domain to a phosphorylated tyrosine-containing peptide leads to a marked increase in SHP-2 catalytic activity. Thus, an expected consequence of the binding of Csw to Dos is a substantial increase in Csw catalytic activity. Given the requirement for the Csw catalytic activity during Sev signaling, a key question is the identity of the key Csw target(s) whose dephosphorylation is required for R7 photoreceptor development. Dos itself might be a crucial target of Csw. In particular, Dos might contain sites of tyrosine phosphorylation that serve as docking sites for proteins inhibitory to Sev signaling and these sites might be dephosphorylated by Csw. This idea is based both on studies showing that Dos is a potential Csw substrate and on genetic studies indicating that reduced Csw or Dos function decreases the strength of Sev signaling. Given this model, an expectation is that mapping the sites of tyrosine phosphorylation in catalytically inactive Csw^CS-bound Dos might identify both the Csw SH2 domain-binding sites and additional Csw target sites. Instead, this analysis reveals phosphorylation of only the two Csw SH2 domain-binding sites, Y801 and Y854. Together, the (1) failure to find additional sites of Dos tyrosine phosphorylation in Csw^CS-bound Dos and (2) the ability of Dos^YY (in which all tyrosines outside of the Dos PH domain except Y801 and Y854 are either removed by deletion or changed to phenylalanine) to provide Dos function during Sev signaling, suggest that the primary role of Dos may be to activate the catalytic activity of Csw towards other, as yet unidentified, proteins. In this model, the role of Csw's dephosphorylation of Dos would be to down-regulate signaling by eliminating its own binding sites (Herbst, 1999).

The Drosophila PS1 and PS2 integrins are required to maintain the connection between the dorsal and ventral wing epithelia. aPS subunits are inappropriately expressed during early pupariation via the Blistermaker chromosome (containing a PS2 gene driven by the wing pouch enhancer trap, 684). Inappropriate expression of aPS2 results in the separation of epithelia, causing a wing blister. Two lines of evidence indicate that this apparent loss-of-function phenotype is not a dominant negative effect, but is due to inappropriate expression of functional integrins: wing blisters are not generated efficiently by misexpression of loss-of-function aPS2 subunits with mutations that inhibit ligand binding, and gain-of-function, hyperactivated mutant aPS2 proteins cause blistering at expression levels well below those required by wild-type proteins. A genetic screen was carried out for dominant suppressors of Blistermaker induced wing blisters. Suppression was induced by null alleles of a gene named moleskin, which encodes the protein DIM-7. DIM-7, a Drosophila homolog of vertebrate importin-7, has been shown to bind the SHP-2 tyrosine phosphatase homolog Corkscrew and to be important in the nuclear translocation of activated D-ERK (Rolled). Consistent with this latter finding, homozygous mutant clones of moleskin fail to grow in the wing. Genetic tests suggest that the moleskin suppression of wing blisters is not directly related to inhibition of D-ERK nuclear import (Baker, 2002).

The ß-importin family of proteins is principally linked with nuclear import of protein cargos. However, recently other functions have been associated with members of the importin superfamily. For example, importin-ß, in some cases with importin-a, functions in vertebrates to sequester microtubule polymerization factors early in mitosis. Mitotic microtubule formation can be triggered by the release of the polymerizaion regulators by RanGTP, just as RanGTP binding to importin-ß leads to release of cargos inside the nucleus. DIM-7 protein can be detected immunologically at the cell cortex, both in early Drosophila embryos and in S2 cells in culture. It thus seems reasonable to consider a more direct connection between the peripheral DIM-7 and integrin regulation. Additionally, it appears that a mutation in corkscrew, the Drosophila SHP-2 homolog, can also suppress Blistermaker and that Corkscrew protein binds directly to DIM-7. Although Corkscrew has been implicated primarily in signaling events downstream of receptor tyrosine kinases, vertebrate SHP-2 has been implicated in signaling via a host of growth factor receptors, cytokines, hormones, and antigens. Most relevant to this study, SHP-2, often in association with the membrane glycoproteins PECAM-1 or SHPS-1, has been shown to be involved in many integrin-dependent signaling events and also to be important in regulating integrin-mediated cell adhesion, spreading, or migration. While SHP-2 is a cytoplasmic tyrosine phosphatase, some experiments suggest that it can serve as a scaffolding protein at or near the plasma membrane. For example, a Corkscrew protein mutated in the phosphatase domain retains significant wild-type activity in situ, and this activity is increased if the protein is targeted to the plasma membrane (Baker, 2002).

It is likely therefore that cell surface receptors mediate a localized Corkscrew/SHP-2 activation of cortical DIM-7. This active DIM-7, in combination with associated factors such as D-ERK, could then function more directly in integrin regulation. A more direct connection between DIM-7 and integrin function is also consistent with the fact that moleskin mutations were especially common among the suppressors isolated in the screen. A key question for future work, therefore, will be defining the subcellular location at which DIM-7 functions with respect to integrin-related phenotypes (Baker, 2002).

Recently, evidence has begun to appear that integrin engagement with the ECM can regulate nuclear import of regulatory molecules. For example, there is an association between aLß2 and the c-Jun coactivator JAB1; this connection is suggested to regulate the nuclear localization of JAB1. More directly relevant to these results, ERK nuclear translocation in fibroblasts is dependent on an integrin-mediated event, also involving the actin cytoskeleton. Also, primary mouse embryo fibroblasts with a ß1 integrin cytoplasmic mutant show reduced nuclear translocation of phosphorylated ERK. Regardless of the importance of nuclear transport in Blistermaker suppression, the genetic data indicate a functional connection between integrins and a specific importin-ß that can transport activated ERK and suggest another potential molecular mechanism whereby integrin and growth factor signals can be integrated by the cell (Baker, 2002).

Downstream-of-FGFR is a fibroblast growth factor-specific scaffolding protein and recruits Corkscrew upon receptor activation

Fibroblast growth factor (FGF) receptor (FGFR) signaling controls the migration of glial, mesodermal, and tracheal cells in Drosophila melanogaster. Little is known about the molecular events linking receptor activation to cytoskeletal rearrangements during cell migration. A functional characterization has been performed of Downstream-of-FGFR (Dof), a putative adapter protein that acts specifically in FGFR signal transduction in Drosophila. By combining reverse genetic, cell culture, and biochemical approaches, it was demonstrated that Dof is a specific substrate for the two Drosophila FGFRs. After defining a minimal Dof rescue protein, two regions were identified that are important for Dof function in mesodermal and tracheal cell migration. The N-terminal 484 amino acids are strictly required for the interaction of Dof with the FGFRs. Upon receptor activation, tyrosine residue 515 becomes phosphorylated and recruits the phosphatase Corkscrew (Csw). Csw recruitment represents an essential step in FGF-induced cell migration and in the activation of the Ras/MAPK pathway. However, the results also indicate that the activation of Ras is not sufficient to activate the migration machinery in tracheal and mesodermal cells. Additional proteins binding either to the FGFRs, to Dof, or to Csw appear to be crucial for a chemotactic response (Petit, 2004).

Genetic epistasis experiments have shown that Dof functions downstream of the activated FGFRs and upstream or in parallel to Ras. However, the biochemical function of Dof in the interpretation of the chemotactic response to FGFR signaling has not been addressed so far. Using in vivo rescue assays, a minimal Dof protein containing the first 600 amino acids of Dof was identified that allows rescue of both mesodermal and tracheal cell migration. Although the rescue in the tracheal system is not as efficient as the rescue observed with the wild-type construct, all six branches can migrate out, demonstrating that the first 600 amino acids of Dof retain the capacity to read out the local activation state of the FGFRs and to relay the signal to the migration machinery, albeit with somewhat reduced efficiency. Deletion from the C terminus of this dof minigene, as well as internal deletions, results in loss of rescue capacity, thus identifying regions of functional importance (Petit, 2004).

All of the constructs were examined in a Drosophila S2 cell culture assay, in which either the FGFR or the Torso signaling system was activated. Both full-length Dof and Dof600 are phosphorylated on tyrosine residues upon FGF signaling, but Torso cannot use Dof as a substrate. These results are consistent with in vivo data showing that Dof is exclusively needed for FGF-mediated signal transduction and that Torso is able to activate the MAPK cascade in the absence of Dof in dof mutant embryos. Using coimmunoprecipitation experiments, it was shown that Dof forms a complex with both FGFRs and that the first 484 amino acids, although not phosphorylated upon FGF signaling, are required and sufficient for the association with the FGFRs, demonstrating that phosphorylation of Dof is not necessary for complex formation. Cell culture analysis is in line with studies showing that the N-terminal part of Dof directly interacts with the kinase domains of Btl and Htl in yeast two hybrid assays. In addition, it was observed that both the juxtamembrane and the C terminus of Btl can be deleted without affecting considerably the quality of the rescue capacity of the receptor. Thus, it appears that Dof directly docks onto the kinase domain of the FGF receptor, in contrast to the vertebrate FGFR adapter SNT/FRS2, which interacts with a sequence motif in the juxtamembrane region of the receptor (Petit, 2004).

Since Dof becomes phosphorylated upon FGFR signaling in S2 cells, it was asked whether it was possible to identify functionally important phosphorylation sites, the proteins recognizing these sites in the phosphorylated state, and confirm the results in vivo by making use of the rescue assay and genetic analysis. Two potential phosphorylation target sites were identified by sequence analysis in the essential region comprising amino acids 485 to 600. While mutation of each individual site results in reduced phosphorylation of Dof600 in S2 cells upon FGFR signaling, mutation of only one of these sites, tyrosine 515, abolished the migration rescue capacity in vivo. Since the functionally required tyrosine residue was part of a putative consensus binding site for the SH2 domain of the nonreceptor tyrosine phosphatase Csw/SHP-2, the interaction of Csw with Dof was tested using coimmunoprecipitation experiments; Csw is indeed recruited to the activated signaling complex via Dof. It was found in rescue assays that both the region 485 to 600 as well as the region from 600 to the C terminus (construct dofDelta485-600) are able to confer function to the signaling-deficient N terminus (residues 1 to 484). It is known that the C-terminal sequences also recruit the Csw phosphatase in the absence of tyrosine 515, but it is not known know whether they do so directly or indirectly. Further deletion analyses and biochemical studies will be required to address this question (Petit, 2004).

Genetic evidence supporting an interaction between Dof and Csw was provided some time ago by the finding that mutations in csw produce a phenotype identical to bnl, btl, and dof; i.e., tracheal and mesodermal cells fail to migrate. The sum of these results clearly assign a crucial role for both Dof and Csw downstream of the FGFRs in the migratory response, indicating that the ligand-dependent phosphorylation of Dof leads to the recruitment of Csw to the signaling complex, ultimately triggering cell locomotion. SHP-2, the vertebrate homologue of Csw, has been shown to be required at the initial steps of gastrulation, as mesodermal cells migrate away from the primitive streak in response to chemotactic signals initiated by fibroblast growth factors. In addition, SHP-2 has also been found to be crucial for tubulogenesis and for the sustained stimulation of the ERK/MAPK pathway upon induction of another chemotactic factor, the hepatocyte growth factor/scatter factor, thus placing SHP-2/Csw as a key player in branching morphogenesis induced by diverse chemotactic factors. Therefore, it appears that both in invertebrates and vertebrates, SHP-2/Csw plays a major role in RTK signaling in the control of cell migration. The similarity of the Drosophila FGF signal transduction pathway to the vertebrate FGF pathway make the fly system accessible to address future issues not resolved in vertebrates, such as the targets of SHP-2/Csw involved in Ras activation and/or cell migration (Petit, 2004).

Using the dpERK antibody as a readout for the activation of the Ras/MAPK pathway in vivo, it was found that abolishing the interaction between the Dof600 minimal protein and Csw abolishes the activation of the MAPK cascade upon FGFR signaling. The strong correlation found between migration and MAPK activation when analyzing all mutant dof constructs in this assay might indicate that local activation of the Ras/MAPK pathway in tracheal tip cells is sufficient to trigger the migratory response upon Btl signaling. However, two lines of evidence suggest that this might not be the case (Petit, 2004).

In one case, it has been observed that under conditions in which all tracheal cells sustain high levels of Ras/MAPK activity (upon Ras^V12 overexpression), tracheal cells migrate normally in wild-type embryos. In sharp contrast, ectopic expression of the Bnl ligand leads to a complete disruption of directed migration. Therefore, high levels of Ras/MAPK activity do not appear to produce the same migratory response as ligand-activated FGFR signaling. Indeed, and again in contrast to ectopic Bnl, overexpression of Ras^V12 in wild-type embryos does not produce significant filopodial activity in DT tracheal cells, confirming that the activation of Ras is not sufficient to produce cytoskeletal rearrangements by itself (Petit, 2004).

In the other case, it was also observed that while the Dof600 protein lacking the ankyrin repeats did allow FGFR-dependent activation of the Ras/MAPK pathway and downstream nuclear response genes, this protein failed to induce migration. Thus, even local Ras activation under the control of the endogenous ligand Bnl, Btl, and Dof600DeltaAR is unable to activate the migratory machinery. Interestingly, it has also been reported that Ras activation is insufficient to guide RTK-mediated border cell migration during Drosophila oogenesis (Petit, 2004).

Is Ras activation then required at all for cells to produce a cytoskeletal response and migrate directionally? Unfortunately, genetic analysis cannot be used to directly address this question in the embryo since maternal and zygotic loss of Ras activity results in embryos that do not develop far enough to analyze the tracheal system. However, when activated Ras (Ras^V12) is expressed in the tracheal system or in the mesoderm of dof mutant embryos, a certain rescue of migration can be obtained. This suggests that Ras signaling is essential but not sufficient for efficient FGFR-dependent cell migration; additional proteins binding to the receptor, to Dof or to Csw appear to be crucial for a chemotactic response. To analyze the role of Ras experimentally and in detail, mitotic clones lacking Ras activity should be analyzed with regard to their migration properties. Recent reports concerning the role of FGF signaling in the migration of mesodermal and tracheal cells during late larval development might provide the basis for such analyses (Petit, 2004).

Sprouty proteins are in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases

Drosophila Corkscrew protein and its vertebrate ortholog SHP-2 (now known as Ptpn11) positively modulate receptor tyrosine kinase (RTK) signaling during development, but how these tyrosine phosphatases promote tyrosine kinase signaling is not well understood. Sprouty proteins are tyrosine-phosphorylated RTK feedback inhibitors, but their regulation and mechanism of action are also poorly understood. This study shows that Corkscrew/SHP-2 proteins control Sprouty phosphorylation and function. Genetic experiments demonstrate that Corkscrew/SHP-2 and Sprouty proteins have opposite effects on RTK-mediated developmental events in Drosophila and an RTK signaling process in cultured mammalian cells, and the genes display dose-sensitive genetic interactions. In cultured cells, inactivation of SHP-2 increases phosphorylation on the critical tyrosine of Sprouty 1. SHP-2 associates in a complex with Sprouty 1 in cultured cells and in vitro, and a purified SHP-2 protein dephosphorylates the critical tyrosine of Sprouty 1. Substrate-trapping forms of Corkscrew bind Sprouty in cultured Drosophila cells and the developing eye. These results identify Sprouty proteins as in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases and show how Corkscrew/SHP-2 proteins can promote RTK signaling by inactivating a feedback inhibitor. It is proposed that this double-negative feedback circuit shapes the output profile of RTK signaling events (Jarvis, 2006).

Four lines of evidence support the conclusion that Csw/SHP-2 inactivate Spry proteins by direct binding and dephosphorylation. First, genetic experiments in developing Drosophila eye and trachea and HEK293 cells demonstrated that Csw/SHP-2 and Spry act in the same RTK signaling events but in opposite directions. Indeed, manipulating their activity in opposite directions caused similar Drosophila phenotypes and similar effects on MAPK activation in HEK293 cells, and reducing spry dose suppressed the csw loss-of-function phenotype in the eye and enhanced the gain-of-function phenotype, supporting the idea that they regulate the same step in signaling. Second, molecular epistasis experiments in HEK293 cells demonstrated that SHP-2 functions upstream of, and negatively regulates, phosphorylation of the critical tyrosine residue (Y53) of Spry1. Third, biochemical studies of extracts of HEK293 cells, Drosophila S2 cells, and eye discs demonstrated that Csw/SHP-2 proteins associate in complexes with Spry proteins. Interaction was enhanced in S2 cells and eye discs when a substrate-trapping Csw was used. Interaction involves more than just binding of Csw/SHP-2 to the crucial tyrosine, because complex formation was observed with SHP-2 mutants lacking the phosphatase domain and with a Spry mutant lacking the tyrosine. Finally, purified SHP-2 selectively dephosphorylated Spry1 in vitro. These data support the conclusion that Spry proteins are direct targets of Csw/SHP-2 in all three systems examined (Jarvis, 2006).

One genetic result did not readily fit with the model that Csw functions by inactivating Spry by dephosphorylation. Whereas reduction of spry dose suppressed the eye phenotype of a hypomorphic csw allele and dominant-negative Csw^G547E, consistent with the model, it did not suppress the milder phenotype of dominant-negative Csw^C583S. This catalytically inactive, substrate trapping form of Csw has unusual properties: it behaves in a dominant-negative fashion, interfering with wild-type Csw function, but also retains some wild-type Csw function because it partially rescues other dominant-negative and hypomorphic csw alleles. This residual activity of Csw^C583S is proposed to result from its ability to partially mimic the effect of dephosphorylating a substrate by binding to it tightly. Spry binds Csw^C583S and could be such a substrate. If so, this could explain the lack of suppression of Csw^C583S phenotype by reduction in spry dose: decreasing spry levels would not reduce spry function under conditions in which it is already trapped in an inactive or partially inactive form by Csw^C583S (Jarvis, 2006).

Csw/SHP-2 binding and dephosphorylation of Spry creates an interesting regulatory circuit downstream of RTKs. Both components of the circuit are induced and activated following receptor activation, Csw/SHP-2 by SH2 domain interactions with phosphotyrosines, and Spry proteins by transcriptional induction of their genes and tyrosine phosphorylation of the proteins. One induced component (Spry) is a signaling inhibitor, the other (Csw/SHP-2) is a signaling promoter that acts by inactivating the inhibitor (Jarvis, 2006).

Why does a signaling pathway induce both a feedback inhibitor and a protein that inactivates it? One possibility is that this double-negative circuit provides a mechanism for rapidly resetting the signaling system: the inhibitor terminates signaling and the deactivator restores the inhibitor to its original (inactive) state, readying the cell for another round of signaling. This may be important when cells experience successive waves of signaling, such as the waves of EGFR and Sevenless signaling in eye development (Jarvis, 2006).

Another possibility is that the double-negative circuit allows precise control of the signal output profile. In the absence of feedback, the response to a signal is simple and sustained, increasing monotonically until reaching saturation. If a basic negative-feedback system is operative, the magnitude and duration of the response are limited, generating a parabolic response profile. However, if the pathway contains both a feedback inhibitor (Spry) and an inducible component (Csw/SHP-2) that deactivates it, this creates more complex output profiles, such as the irregularly shaped curve observed for MAPK activation following FGFR activation in HEK293 cells. By altering activity of individual feedback components, other complex profiles can be generated. If cells can distinguish different profiles, as some cells distinguish different calcium oscillations, this could lead to different outcomes. The shape of the RTK response profile could be as important to outcome as the magnitude and duration of the response. In a similar way, differential induction of individual components of a double-negative feedback circuit can transform simple signaling gradients into complex spatial patterns of signal output (Jarvis, 2006).

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.