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 CswCS-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 CswCS-bound Dos and (2) the ability of DosYY (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).

Nuclear import of activated D-ERK by DIM-7, an importin family member encoded by the gene moleskin

The initiation of gene expression in response to Drosophila receptor tyrosine kinase signaling requires the nuclear import of the MAP kinase, Rolled. However, the molecular details of Rolled translocation are largely unknown. In this regard, D-Importin-7 (DIM-7), the Drosophila homolog of vertebrate importin 7, and its gene moleskin have been identified. DIM-7 exhibits a dynamic nuclear localization pattern that overlaps the spatial and temporal profile of nuclear, activated Rolled. Co-immunoprecipitation experiments show that DIM-7 associates with phosphorylated Rolled in Drosophila S2 cells. Furthermore, moleskin mutations enhance hypomorphic and suppress hypermorphic rolled mutant phenotypes. Deletion or mutation of moleskin dramatically reduces the nuclear localization of activated Rolled. Directly linking DIM-7 to its nuclear import, this defect can be rescued by the expression of wild-type DIM-7. Mutations in the Drosophila Importin beta homolog Ketel also reduce the nuclear localization of activated Rolled. Together, these data indicate that DIM-7 and Ketel are components of the nuclear import machinery for activated Rolled (Lorenzen, 2001).

The activation of ERK represents the focal point of a conserved signaling module used by a diverse array of extracellular stimuli. The potency and duration of ERK activation and its accompanying translocation to the nucleus can profoundly affect the fate of a cell. This is apparent in PC12 cells where the decision to proliferate or differentiate depends upon the number and duration of receptors stimulated. Throughout development, cells respond to spatial and temporal signals and must interpret gradients to produce qualitative differences in gene expression. In Drosophila, the terminal system or Torso RTK signaling pathway illustrates one example whereby quantitative differences in D-ERK activity generate distinct cell fates. Distinct quantitative levels of D-ERK activity inside a cell may be achieved within the RTK pathway by modulating D-ERK phosphorylation. Moreover, it is apparent that mechanisms exist to control the localization of ERK activity by either regulating its retention in the cytoplasm and/or its nucleocytoplasmic shuttling. In this regard, nuclear translocation of dpERK is not always a compulsory consequence of RTK signaling. In Drosophila as photoreceptors are recruited into the developing retina, dpERK is held in the cytoplasm for up to several hours following EGFR and Sevenless RTK signaling. This raises the possibility that import is differentially controlled relative to D-ERK phosphorylation and/or dimerization. Interest in the active transport of dpERK also partly stems from the observation that dimer formation is a common property of mammalian MAP kinase family members (Lorenzen, 2001).

Presumably, D-ERK shares this property, since the residues involved in dimer formation have been conserved. Although monomeric dpERK can enter the nucleus passively, it has been shown that import of dimeric ERK is an active process. This study has clarified mechanistic issues in the nuclear relocalization of dpERK through the identification of DIM-7, the Drosophila homolog of importin 7 and member of the importin superfamily of nuclear transport proteins. DIM-7 exhibits several properties that establish its participation in RTK signaling. These include a physical interaction with CSW and dpERK, and the finding that DIM-7 is tyrosine phosphorylated in stimulated cells. Furthermore, alleles of msk, the gene that encodes DIM-7, dominantly interact with hypomorphic and hypermorphic alleles of D-ERK (Lorenzen, 2001).

The primary structure of DIM-7 originally suggested that it might play a role in nuclear transport. In addition to exhibiting significant sequence identity with its Xenopus and human homologs, DIM-7 also possesses a conserved Ran-binding domain. This latter property is a hallmark of nuclear transport proteins that display a RanGDP versus RanGTP regulated interaction with their cargo proteins. In addition to physically binding to phosphorylated D-ERK, DIM-7 and dpERK have overlapping nuclear localization patterns in developing tissues subject to RTK regulation. This made dpERK an attractive candidate cargo for DIM-7. To address this possibility during embryogenesis and in the absence of functional DIM-7, tracheal placodes and tracheal pits were assayed for defects in the accumulation of dpERK. Significantly, embryos in which the genomic interval encoding the msk gene is deleted or mutated exhibit a fivefold reduction in the number of dpERK-positive nuclei. Importantly and demonstrating that DIM-7 is essential for nuclear uptake of dpERK, expression of wild-type DIM-7 in a msk mutant background restores dpERK nuclear accumulation. These findings reinforce the idea that not only is dpERK actively transported through the nuclear pore complex but also that DIM-7 functions as either the transport receptor and/or adapter for dpERK (Lorenzen, 2001).

Finally, this study has implicated KET in the nuclear import of dpERK. As vertebrate importin 7 and importin b form an abundant heterodimeric complex, it was asked whether the Drosophila homolog of importin b, KET participates in the dpERK transport mechanism. Supporting a model whereby DIM-7 and KET function together in the nuclear transport of dpERK, it was found that nuclear localization of dpERK is impaired in homozygous ket mutant embryos. Although there are other possibilities, two models are envisioned by which DIM-7 could function in the nuclear import cycle of dpERK. In one, DIM-7 and KET could function together in the same import cycle, where DIM-7 and KET serve as the import adapter and import receptor, respectively. Alternatively, DIM-7 and KET could function independently of each other in separate import cycles that could be, at least partially, redundant. However, it is unlikely that DIM-7 and KET serve totally redundant functions for two reasons. First, each individual locus when deleted (msk) or mutated (msk and ket) reduces substantially the number of dpERK-positive nuclei, and, second, both msk and ket mutations, alone, are lethal (Lorenzen, 2001).

In the literature there appears to be an increasing number of transport receptors with complex cargo specificities. If importin 7 is the functional vertebrate homolog of DIM-7, then this transport factor can import at least three different proteins, dpERK, histone H1 and rpL23a. An additional point concerning specificity of DIM-7 regards its nearest homolog, Nmd5p, in Saccharomyces cerevisiae. Nmd5p is essential for the nuclear import of HOG1, a p38 MAP kinase family member that is activated in response to osmotic stress. Interestingly, the movement of HOG1 into the nucleus does not require the importin b homolog, RSL1. This work raises the possibility that DIM-7 might also mediate the nuclear transport of one or more other Drosophila MAPkinase family members, D-p38a (Mpk2), D-p38b (Mpk34C) and D-JNK (JUN kinase). The combinatorial use of different transport factors may provide a means for specific recognition of the various MAP kinase family members. For example, DIM-7 alone may bind and import D-p38; however, recognition of dpERK may require the simultaneous pairing of DIM-7 and KET. Determining the mechanism(s) employed to establish recognition between a cargo and its transport receptor will require a precise molecular dissection of the interactions between several transport receptor/cargo pairs (Lorenzen, 2001).

CSW was first demonstrated to have a positive function during embryogenesis in the Torso RTK pathway. In this pathway CSW serves two functions. First, the adapter protein DRK does not bind Torso; instead, CSW functions as an adapter linking Torso to RAS. Second, CSW is able to dephosphorylate the Torso autophosphorylation site that binds RasGAP. The work presented in this paper suggests a third function for CSW as an adapter to facilitate the physical interaction of DIM-7 with its import cargo dpERK. When two, or three, of these CSW functions are used within one signaling module, interpreting the epistatic relationships of CSW with various signaling components could become problematic. For example, previous epistasis experiments have suggested that CSW carries out its function either upstream or downstream of RAS1 and/or D-RAF. Now it appears these differences could simply reflect the differential use of the various signaling capabilities of CSW within a given RTK pathway (Lorenzen, 2001).

Whether or not the association of DIM-7 with CSW constitutes part of a regulatory process at the level of the receptor that governs D-ERK redistribution has yet to be determined. However, it appears that nuclear import is a fundamental mechanism used by cells to modulate incoming signals throughout development. It is expected, then, that the development of reagents to modulate the nuclear entry of specific molecules may have profound effects for controling both disease and oncogenic states (Lorenzen, 2001).

Genetic interaction between integrins and moleskin, a gene encoding a Drosophila homolog of Importin-7

The Drosophila PS1 and PS2 integrins are required to maintain the connection between the dorsal and ventral wing epithelia. αPS 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 αPS2 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 αPS2 subunits with mutations that inhibit ligand binding, and gain-of-function, hyperactivated mutant αPS2 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-α, 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 αLß2 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 RasV12 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 RasV12 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 (RasV12) 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 CswG547E, consistent with the model, it did not suppress the milder phenotype of dominant-negative CswC583S. 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 CswC583S is proposed to result from its ability to partially mimic the effect of dephosphorylating a substrate by binding to it tightly. Spry binds CswC583S and could be such a substrate. If so, this could explain the lack of suppression of CswC583S 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 CswC583S (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).

A novel conserved phosphotyrosine motif in the Drosophila fibroblast growth factor signaling adaptor Dof with a redundant role in signal transmission

The fibroblast growth factor receptor (FGFR) signals through adaptors constitutively associated with the receptor. In Drosophila melanogaster, the FGFR-specific adaptor protein Downstream-of-FGFR (Dof) becomes phosphorylated upon receptor activation at several tyrosine residues, one of which recruits Corkscrew (Csw), the Drosophila homolog of SHP2, which provides a molecular link to mitogen-activated protein kinase (MAPK) activation. However, the Csw pathway is not the only link from Dof to MAPK. This study identified a novel phosphotyrosine motif present in four copies in Dof and also found in other insect and vertebrate signaling molecules. These motifs are phosphorylated and contribute to FGF signal transduction. They constitute one of three sets of phosphotyrosines that act redundantly in signal transmission: (1) a Csw binding site, (2) four consensus Grb2 recognition sites, and (3) four novel tyrosine motifs. Src64B binds to Dof and Src kinases contribute to FGFR-dependent MAPK activation. Phosphorylation of the novel tyrosine motifs is required for the interaction of Dof with Src64B. Thus, Src64B recruitment to Dof through the novel phosphosites can provide a new link to MAPK activation and other cellular responses. This may give a molecular explanation for the involvement of Src kinases in FGF-dependent developmental events (Csiszar, 2010).

Mutational analysis of Dof, which was used as an indirect approach to map tyrosines that are phosphorylated in the presence of an activated FGFR, showed that consensus tyrosine motifs for PI3K and Csw binding at amino acid positions 486 and 515 were substrates of phosphorylation. In addition, three tyrosine residues at positions 592, 613, and 629 were identified as phosphorylation targets, but these do not conform to known conserved tyrosine motifs. Finally, the last 200 amino acids of Dof also contain several phosphorylation target sites. Mutational analyses of this type do not prove that the same residues are phosphorylated in the wild-type situation, but the tyrosine at position 515 is required for the binding of Csw upon FGFR activation, and this study has shown that mutation of most of the identified sites resulted in impaired activity of the molecule in vivo, supporting the notion that these tyrosines are physiologically relevant phosphorylation targets in Dof (Csiszar, 2010).

The three tyrosine-containing motifs at positions 592, 613, and 629 do not resemble known conserved tyrosine motifs, but their positions and their sequences are conserved in Anopheles Dof. In Drosophila the sites are very close together, so that they could act as tandem interaction surfaces, but the fact that they are separated by longer insertions in Anopheles makes this unlikely. The motifs at 613 and 629 resemble each other, and the same sequence motif is present two more times in the C termini of both Drosophila and Anopheles Dof. Protein database searches with the consensus sequence motif Y-X3-P-X3-P, generated from these eight related sites, showed that this motif is present in several signaling molecules, in many cases as known phosphorylation target sites (e.g., in Shc and the mammalian FGFR-1). Mammalian Shc contains two consensus Grb2 binding sites. One, conserved in vertebrate Shc proteins, has been shown to bind Grb2 and activate the Ras-MAPK pathway. The other, part of a double-phosphorylation site of two adjacent tyrosines and conserved in all members of the Shc family from insects to vertebrates, does not interact with Grb2, and its function in mitogenic activation and apoptosis protection does not depend on Ras. This site is part of the novel tyrosine motif. In vitro kinase assays showed that this pair of adjacent tyrosines can be phosphorylation targets for EGFR and Src as well, but no proteins have been identified as binding partners. The highly conserved sequence patch around these two adjacent phosphorylated tyrosines in Shc goes beyond the Grb2 consensus site and outlines exactly the novel Y-X3-P-X3-P motif, suggesting that the whole motif is important for an evolutionarily conserved biological function (Csiszar, 2010).

In the mammalian FGFR-1, the novel tyrosine motif surrounds the one conserved autophosphorylation site at position Y583 and thus is located in the loop separating the small and large lobes of the kinase domain (28). This is the most variable intracellular sequence part within the FGFR family, and no other mammalian FGFRs share this motif. In spite of the fact that most autophosphorylation sites in the FGFR are conserved, many of these tyrosines are dispensable for signal propagation and only a few proteins that associate with these sites have been identified to date. Phosphorylated Y583 has no known binding partners, and no signaling function has been linked to it so far (Csiszar, 2010).

These findings lead to the speculation that the mammalian FGF-R1 has the ability to recruit a molecule directly that in the case of the Drosophila FGFR is recruited indirectly via Dof and, in the other mammalian FGF receptors, perhaps via other interactors, such as Shc (Csiszar, 2010).

The rescue experiments used to assay the functional relevance of the tyrosine mutations that influenced the phosphorylation levels of Dof in vitro yielded two important findings: first, three independent functional units in Dof were identified that contribute to signal propagation, and second, these units act redundantly, in that any one of them is sufficient to provide significant biological activity. However, it cannot be ruled out that the overexpression system used might have masked potential minor qualitative differences and therefore exaggerated the redundancy. Similarly, two of the phosphorylated tyrosines showed no functional relevance in this or previous studies. One is the tyrosine of a PI3K consensus site at position 486 for which there was no requirement in any of the assays, employing either full-length Dof or mutant forms retaining only the first 600 amino acids. The other identified phosphotyrosine site without an identified function is located at position 592. Though it is conserved in Anopheles Dof, including several surrounding amino acids, the mutation of this tyrosine alone or in combination with other tyrosines did not affect the biological function of Dof. Again, perhaps subtle effects of the loss of these sites might have been missed. Nevertheless, the presence of several copies of docking sites for downstream signaling molecules and the availability of alternative routes to activate the same signaling cascade may provide Dof with options for fine-tuning of signaling strength and duration (Csiszar, 2010).

Csw has previously been shown to interact with Dof. The phosphotyrosine of the Csw consensus site was required for efficient interaction and for MAPK activation in the context of a Dof construct that lacked any of the other phosphorylation sites. This study shows that the Csw site is indeed important only if other parts of Dof with MAPK activating capacity are deleted or mutated. The same is true for the phosphorylation sites in the C-terminal domain, which this study shows to be the consensus Grb2 binding sites (Csiszar, 2010).

The four novel phosphotyrosine motifs contributed to the activation of the MAPK cascade, although they were essential only if other parts of the molecule with MAPK activation capability were deleted or mutated. Phosphorylation of these tyrosines was essential for the efficient interaction of Dof with the protein kinase Src64B, and Src activity was in turn required for Dof-dependent MAPK activation in S2 cells. Src kinases can activate mitogenic signaling in many different ways. Recently, Drosophila Src64B has been shown to be essential in the regulation of Raf activity by phosphorylating a regulatory tyrosine residue in Raf, which is also conserved in mammalian B-Raf. Thus, it is reasonable to postulate that upon FGFR-dependent phosphorylation the novel tyrosine motif in Dof is utilized to recruit Src64B, which can then contribute to MAPK activation via Raf activation (Csiszar, 2010).

In addition to Src64B, a tyrosine phosphorylated protein of 29 kDa (p29) was found that coprecipitated with Dof802 and required the phosphorylated tyrosines of the novel motif for this interaction. This raises the possibility that other proteins might use these sites as docking surfaces as well, although no evidence was found that this protein directly binds these phosphosites (Csiszar, 2010).

Since the residues surrounding the tyrosines in the novel motifs are conserved, it was expected that they would be found to be important for function. However, while mutating the tyrosines had measurable effects on the function of Dof in vivo, replacing the prolines had only moderate or no effects in the same assays. Similarly, Src64B binding to Dof802 in S2 cells was strongly reduced when the tyrosines of the novel motifs were mutated (in the background of mutated Csw and PI3K sites) but not when the prolines were mutated (Csiszar, 2010).

The finding that the impact of tyrosine mutations in the novel motifs on Dof function was greater than that of the proline mutations agrees with the known general characteristics of the interaction of phosphotyrosine motifs with phosphotyrosine binding domains. The driving force of these interactions is the phosphorylated tyrosine itself, with additional lower-affinity interactions of surrounding residues contributing to specificity for the different phosphotyrosine binding domains, as has also been found for dissociation constants when measuring interactions of SH2 domains in phosphopeptide library interaction screens. Indeed, it has been proposed that the modest selectivity of SH2 domains to phosphotyrosine containing linear peptides (5- to 20-fold) is not sufficient to explain selectivity of signaling pathways in living cells. Recent work has identified additional components of these type of interactions and shows that the selectivity of phospholipase Cγ binding and signaling via activated FGFR-1 are determined by interactions between a secondary binding site on an SH2 domain and a region in the FGFR-1 kinase domain in a phosphorylation-independent manner. These data suggest that the mutation of two amino acids in a tyrosine motif might have only mild consequences compared to the loss of the phosphotyrosine site in the context of whole protein-protein interactions, based on the complexity of different binding interfaces and their different affinities of this interaction (Csiszar, 2010).

Since SH2 domains preferentially interact with residues C-terminal to the tyrosine, and these are the conserved residues in the novel motif, the motif is expected to interact with SH2-type domains. Why has the motif described in this study not been found in the extensive searches for SH2 target motifs? The answer may lie in the fact that no motifs with important conserved amino acids at positions +4 and +8 are known at all, and this may be primarily because of the way they have been screened for. The degenerate phosphotyrosine peptide libraries that have been used to determine SH2 domain specificities screened only positions +1 to +3, and the furthest that other studies have gone was up to position +5 (Csiszar, 2010).

It is not known if the SH2 domain of Src64B is involved in the interaction with the novel phosphomotif of Dof, but if so, it is not clear whether this motif could be accommodated by the same interaction surface as the one that binds to the consensus Src SH2 domain recognition sequence pYEEI, which has been defined by phosphopeptide library screening and structural studies on peptide-bound Src SH2 domains (Csiszar, 2010).

Finally, little is known about the interaction of Src family kinases with other vertebrate signaling molecules bearing the novel tyrosine motif. For example, the motif in Shc is a phosphorylation target of Src, but no interaction studies have been performed, and there are conflicting reports about direct interaction between FGFR-1 and Src. The results of this study suggest that Src might be a good candidate for interacting with mammalian FGFR-1 and other vertebrate signaling molecules via the novel motif. It should be worth probing the general validity of Src binding to this novel phosphotyrosine motif in the future (Csiszar, 2010).

Negative regulation of glial engulfment activity by Draper terminates glial responses to axon injury

Neuronal injury elicits potent cellular responses from glia, but molecular pathways modulating glial activation, phagocytic function and termination of reactive responses remain poorly defined. Here we show that positive or negative regulation of glial responses to axon injury is molecularly encoded by unique isoforms of the Drosophila melanogaster engulfment receptor Draper. Draper-I promotes engulfment of axonal debris through an immunoreceptor tyrosine-based activation motif (ITAM). In contrast, Draper-II, an alternative splice variant, potently inhibits glial engulfment function. Draper-II suppresses Draper-I signaling through a previously undescribed immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain and the tyrosine phosphatase Corkscrew (Csw). Intriguingly, loss of Draper-II-Csw signaling prolongs expression of glial engulfment genes after axotomy and reduces the ability of glia to respond to secondary axotomy. This work highlights a novel role for Draper-II in inhibiting glial responses to neurodegeneration, and indicates that a balance of opposing Draper-I and Draper-II signaling events is essential to maintain glial sensitivity to brain injury (Logan, 2012).

This study has shown how a single receptor, Draper, can positively or negatively influence glial responses to axonal injury. The work reveals a crucial role for ITAM and ITIM-like signaling events in regulating the activation, termination and maintenance of engulfment signal transduction cascades during glial responses to axonal injury. In addition, direct evidence is provided that negative regulation of glial responses to neurodegeneration is essential for glia to reset their responses after an initial injury, and thereby remain competent to respond efficiently to subsequent brain trauma (Logan, 2012).

The unique intracellular domains of Draper-I and Draper-II determine their effects on glial responses to axonal injury. Whereas Draper-I promotes engulfment of axonal debris, Draper-II completely inhibits glial clearance of degenerating axons, and the inhibitory activity mapped to a Draper-II-specific intracellular motif that contains an ITIM-like domain. It is noted that this insertion also produces two ITAMs in Draper-II, raising the possibility that one or both of these may function as an inhibitory ITAM (ITAMi). Recent work has shown that some ITAMs function in a dual manner, recruiting activating or inhibitory effectors in response to changes in receptor configuration. However, a model is favored in which Draper-II acts exclusively as an inhibitory ITIM-like receptor, as the ITIM-like Draper-II domain is not a functional activator in any context examined in vivo (Logan, 2012).

There are two unique Draper extracellular domains that are likely to be involved in recognition of engulfment targets. These are fully interchangeable in the engulfment assay, indicating that neither extracellular domain contains inherent inhibitory activity. It is possible that both domains recognize the same molecule, perhaps a ligand presented by degenerating axons. Alternatively, each extracellular domain may recognize a unique ligand. Identifying specific factors that associate with the extracellular region of each Draper isoform after axotomy will provide key insight into these post-injury neuron-glia communication events (Logan, 2012).

Following axotomy, engulfment molecules (Draper and Ced-6) are robustly upregulated in responding glia and return to baseline levels once axonal debris has been cleared. Notably, it was found that Csw signaling is essential to restore Draper and Ced-6 to basal levels as glia terminate responses to axotomy and that glia lacking Draper-II-Csw signaling fail to respond to secondary injuries in the brain. These data highlight previously unknown in vivo requirements for Draper-I and Draper-II-Csw signaling in coordinating the activation, termination and maintenance of glial cellular responses to axonal injury (Logan, 2012).

It is proposed that Draper-I, acting via Src42A and Shark, promotes the expression of engulfment genes after axotomy and phagocytosis of degenerating axons; such upregulation of engulfment genes is probably essential for rapid clearance of degenerating axons. It is also proposed that Draper-II and Csw then negatively regulate Draper-I signaling to terminate reactive glial responses and allow glia to return to a resting state. The data showing differential regulation of Draper-I and Draper-II transcripts, with Draper-I preceding Draper-II by several hours, supports the idea that Draper-II-Csw signaling may be delayed relative to Draper-I-ITAM signaling, thereby allowing for potent activation and temporal regulation of responses. Curiously, csw transcript levels seem to decrease ~4 h after injury, suggesting that although Draper-II and Csw are both influenced by post-injury signaling, they are differentially regulated (Logan, 2012).

It will be important in the future to identify additional factors functioning within the Draper-II-Csw inhibitory signaling pathway. Following engagement of mammalian ITIM receptors, activated SHP-1 or SHP-2 can target the Src-family kinases that phosphorylate the crucial ITAM tyrosine residues required for ITAM receptor signaling. Thus, Src42A, the kinase that phosphorylates the Draper-I ITAM, may also be an in vivo Csw target. SHP phosphatases can also influence gene transcription within immune cells, although it is largely unclear how this occurs at the molecular level (Logan, 2012).

The discovery that unique Draper isoforms execute opposing functions during the engulfment of degenerating axons raises interesting questions regarding the putative function of Draper homologs in other species. The related mammalian receptors Jedi-1 and MEGF10 are expressed in glia and play a conserved role in glial phagocytic activity (Cahoy, 2008; Singh, 2010; Wu, 2009). Jedi and MEGF10 drive engulfment in peripheral glia that clear large numbers of apoptotic neurons in the developing dorsal root ganglia. It is unknown whether splice variants of Jedi or MEGF10 exist. Notably, the role of Draper-II may be specific to adults; Draper-II transcript was not detected at embryonic or larval stages, which may indicate that the exuberant phagocytic activity of glia that occurs during nervous system development does not require negative regulation (Logan, 2012).

It is predicted that inhibitory constraints on reactive gliosis will be common to many organisms, and that these constraints will be particularly important in the context of neuropathological conditions. It is well known that reactive microglia can actively destroy neurons through phagocytic activity and reactive glia release a number of factors to promote neuronal death or inhibit neuronal function. Maintaining glial cells in an activated state might exacerbate these effects. The roles of MEGF10 and/or Jedi in glial response to adult neural injury have not yet been examined, but there is evidence that ITIM-mediated pathways may provide neuroprotection. For example, in the SHP-1 knockout mouse motheaten there is striking evidence of enhanced microglial activation and neurodegeneration after acute neuronal damage (Logan, 2012).

Promoting glial termination of reactive responses may be particularly important in neurodegenerative disease, which amounts to a continuous series of neural injuries. Reactive gliosis is certainly a hallmark of nearly all neurodegenerative diseases and there is growing evidence that glia help to promote disease pathology in mouse models. The Draper-II-Csw signaling pathway is remarkably specific in its negative regulation of glial responses to axonal injury, and it provides an exciting molecular entry point to understanding how glial cells terminate cellular and molecular responses to neural trauma (Logan, 2012).

corkscrew continued: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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