Protein Interactions

The Torso pathway selectively masks the ability of DL to repress gene expression but has only a slight effect on activation. Intracellular kinases that are thought to function downstream of Torso, such as D-raf and the Rolled MAP kinase, mediate this selective block in repression. Constitutive activation of the Torso pathway causes severe embryonic defects, including disruptions in gastrulation and mesoderm differentiation, as a result of misregulation of dl target genes (Rusch, 1994).

The active state of receptor tyrosine kinases (RTKs) and the RTK signaling cascade pathways were followed in situ. This was achieved by monitoring, with a specific monoclonal antibody, the distribution of the active, dual phosphorylated form of MAP kinase (ERK). A dynamic pattern is observed during embryonic and larval phases of Drosophila development, which can be attributed, to a large extent, to the known RTKs. Torso-dependent, Egfr-dependent, Breathless-dependent, and Heartless dependent activation profiles have all been identified. This specific detection has enabled the determination of the time of receptor activation, the visualization of gradients and boundaries of activation, and has allowed the postulation of the distribution of active ligands. A novel pattern is observed in the visceral mesoderm at stage 11 that is not Heartless dependent, as patches of cells display activated ERK at normal intensity in heartless mutants. Since the antibody was raised against the phosphorylated form of a conserved ERK peptide containing the TEY motif, this approach is applicable to a wide spectrum of multicellular organisms (Gabay, 1997).

The regulatory region of Drosophila Proliferating cell nuclear antigen (PCNA) gene consists of a promoter region (-168 to +24 with respect to the transcription initiation site) and an upstream region containing three homeodomain protein binding sites (HDB) (-357 to -165). When male transgenic flies are crossed with female flies homozygous for a torso gain-of-function allele, repression of PCNA is observed in the central region of the embryo. Local changes of expression depend on the homeodomain protein binding region, suggesting that the promoter region is practically sufficient for expression of the PCNA gene and that the homeodomain protein binding region functions as a silencer when torso is activated ectopically (Yamaguchi, 1995).

Torso-like (TSL) has been considered a putative ligand for Torso. TSL is made by follicle cells in both the anterior and posterior of the egg, giving TSL the localized distribution required for a Torso ligand. In addition, mutations in tsl delete head and tail structures as do mutations in torso. Unrestricted expression of tsl induces terminal pattern elements and suppresses the formation of the abdomen in embryos. Torso-like does not have a sequence characteristic of a growth factor (Martin, 1994).

Trunk is a second candidate for the Torso ligand. Unlike TSL, it is uniformly distributed in the oocyte. Trunk sequence is related to Spätzle. TRK could be activated at the poles analogous to the activity of Spätzle, the Toll ligand in the ventral portion of the egg; that is, TSL may be part of an activation cascade resulting in the localized activation of TRK at the poles (Casanova, 1995).

Several genes are normally required for activating TOR and appear to define a system in which a gene product tethered to the extracellular vitelline membrane at each end of the egg provides a local source for an extracellular TOR ligand. This ligand would have to diffuse from the membrane to the cell surface of the embryo without losing its spatial localization. The failure to accumulate TOR protein at one or both poles leads to spatially inappropriate activity of a more centrally located receptor. The receptor not only transduces the spatial signal imparted by the TOR ligand, but also ensures its correct localization by sequestering the ligand (Casanova, 1993).

The ras pathway

The maternal Drosophila raf serine/threonine kinase acts downstream of Torso (Tor) for specification of cell fates at the embryonic termini. D-raf activity is also required in other signal transduction pathways and consistent with its pleiotropic role, accumulation of a 90-kD D-raf protein is found throughout embryonic development. Maternal D-raf proteins accumulate in 0-2-hr embryos derived from females with germ cells lacking D-raf activity. Accumulation of a 90-kD or truncated mutant D-raf protein is observed for some of these embryos, while others lack the maternal D-raf protein. To determine whether rescue of the Tor pathway is influenced by pools of nonfunctional maternal D raf, wild-type D-raf mRNA was injected into embryos that inherit maternal stores of inactive 90-kD of truncated D-raf protein. For embryos lacking the maternal D-raf protein, a high level of terminal rescue is obtained. In contrast, rescue is reduced or not observed for embryos that accumulate mutant maternal D-raf proteins. These findings suggest that mutant forms of D-raf may deplete the embryo of a positive activator and/or form inactive protein complexes that affect rescue of the Tor pathway (Radke, 1997).

Determination of anterior and posterior terminal structures of Drosophila embryos requires activation of two genes encoding putative protein kinases: torso and D-raf. Torso manifests intrinsic tyrosine kinase activity. It is transiently tyrosine phosphorylated (activated) at syncytial blastoderm stages. Torso proteins causing a gain-of-function phenotype are constitutively tyrosine phosphorylated, while Torso proteins causing a loss-of-function phenotype lack tyrosine kinase activity. The D-raf gene product, which is required for Torso function, is identified as a 90-kDa protein with intrinsic serine/threonine kinase activity. The phosphorylation state of D-RAF changes during development. D-RAF is hyperphosphorylated at 1 to 2 h after egg laying, and thereafter only the most highly phosphorylated form is detected. Embryos lacking Torso activity, however, show significant reductions in D-RAF protein expression rather than major alterations in the protein's phosphorylation state (Sprenger, 1993).

Tyrosines 630 and 918 are the major sites of TOR autophosphorylation. The efficiency of TOR signaling is decreased with the mutation of tyrosine 630, a site required for association with and tyrosine phosphorylation of the tyrosine phosphatase Corkscrew. In contrast, increased TOR signaling appears in mutations of Y918, a site capable of binding mammalian rasGAP and PLC-gamma1. When receptors contain mutations in both sites, TOR signaling is restored to wild-type levels (Cleghon, 1996).

The Torso receptor tyrosine kinase signal is mediated by the serine/threonine kinase D-raf and Corkscrew. Expression of an activated form of Ras1 during oogenesis results in embryos with tor gain-of-function phenotypes. The injection of activated mammalian p21v-ras (See Drosophila Ras) rescues the maternal-effect phenotypes of both tor and csw null mutations. The maternal-effect phenotype embryos lacking Son of sevenless (Sos) exhibit a terminal-class phenotype. The Drosophila p21ras, encoded by Ras1, is an intrinsic component of the tor signaling pathway, where it is both necessary and sufficient in specifying posterior terminal cell fates. p21ras/Ras1 operates upstream of the D-raf kinase in this signaling pathway (Lu, 1993).

Activation of Torso defines the spatial domains of expression of the transcription factors tailless and huckebein. Torso regulates tailless and huckebein through the elements of the ras pathway. Ras1 (p21ras) operates upstream of the D-Raf (Raf1) serine/threonine kinase in this signaling pathway. D-Raf can be activated by Torso in the complete absence of Ras1. This is supported by analysis of D-Raf activation in the absence of either the exchange factor Son of sevenless or the adaptor protein DRK (Grb2), as well as by the phenotype of a D-Raf mutation that abolishes the binding of Ras1 to D-Raf (Hou, 1995).

corkscrew (csw), is maternally required for normal determination of cell fates at the termini of the embryo. Double mutant and cellular analyses between csw, torso, D-raf, and tailless indicate that CSW acts downstream of Torso and in concert with D-raf to positively transduce the Torso signal via Tailless to downstream terminal targets. The csw gene encodes a putative nonreceptor protein tyrosine phosphatase covalently linked to two N-terminal SH2 domains, similar to the mammalian PTP1C protein (Perkins, 1992).

The role in patterning of quantitative variations of MAPK activity in signaling from the Drosophila Torso (Tor) receptor tyrosine kinase (RTK) has been examined. Activation of Tor at the embryonic termini leads to differential expression of the genes tailless and huckebein. Using a series of mutations in the signal transducers Corkscrew/SHP-2 and D-Raf, it has been demonstrated that quantitative variations in the magnitude of MAPK activity trigger both qualitatively and quantitatively distinct transcriptional responses. When terminal activity is progressively removed, there is a corresponding progressive malformation and eventual loss of terminal cuticular structures. The first terminal cuticular elements that are malformed or lost require the highest terminal activation (e.g., the anal tuft and posterior spiracles visualized by the presence of Filzkorper material). The next elements that are malformed or lost require intermediate levels of terminal signal (e.g., the abdominal 8 (A8) denticle belt and the posterior spiracles). Finally, the last elements that are malformed or lost require the lowest levels of terminal activity (e.g., posterior A7). While in the absence of D-raf activity, no activated MAPK (dp-ERK) is observed at the posterior pole. In csw null mutant embryos, where the tll and Hb expression domains are present though mispositioned, reduced levels of dp-ERK reactivity are observed. Collectively, these results reveal that a precise transcriptional response translates into a specific cell identity (Ghiglione, 1999).

Two chimeric receptors, Torextracellular-Egfrcytoplasmic and Torextracellular-Sevcytoplasmic, cannot fully functionally replace the wild-type Tor receptor, revealing that the precise activation of MAPK involves not only the number of activated RTK molecules but also the magnitude of the signal generated by the RTK cytoplasmic domain. For example, analysis of Torextracellular-Egfrcytoplasmic reveals that the posterior domain of Hunchback does not retract from the posterior pole, but rather remains as a terminal cap. Further, the anterior border of this posterior Hb domain is shifted posteriorly. Altogether, these results illustrate how a gradient of MAPK activity controls differential gene expression and thus, the establishment of various cell fates. The roles of quantitative mechanisms in defining RTK specificity are discussed. It is possible that in some instances, the generation of differing magnitudes of activity from the cytoplasmic domains of specific RTKs might be dependent on the specific affinities of the downstream signal transducers to the receptor. Csw binds through one of its SH2 domains to only one phosphotyrosine on Tor. Perhaps a higher or lower affinity of Csw to this site, or addition of another site that would also engage the second SH2 domain of Csw, would increase or decrease signal output. Presumably, in each individual cell there exists a mechanism built into the enhancer elements of the promoters of both tll and hkb that acts to read directly the magnitude of Tor signaling. In the tll promoter, a Tor-response element that mediates the repression of tll has been identified, indicating that the Tor signal activates tll by a mechanism of derepression. A putative candidate for this repressor activity is encoded by the transcription factor Grainyhead. Grainyhead binds to the Tor-response element and can be directly phosphorylated by MAPK in vitro: a decrease in Gh activity has been shown to cause tll expansion in early embryos. Further, the transcriptional corepressor Groucho is required for terminal patterning. Further characterization of how Gh and/or Gro activities are regulated by activated MAPK should clairify how differing levels of phosphorylation translate into derepression of terminal target genes (Ghiglione, 1999).

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)

Sprouty was identified in a genetic screen as an inhibitor of Drosophila EGF receptor signaling. The Egfr triggers cell recruitment in the eye, and sprouty minus eyes have excess photoreceptors, cone cells, and pigment cells. Tests provide evidence that Sprouty interacts specifically with the Egfr pathway. Halving the dose of sprouty (1) strongly enhances the rough eye caused by the misexpression of rhomboid, a specific activator of Egfr signaling; suppresses the rough eye caused by underrecruitment of photoreceptors in a hypomorphic allele of spitz, the TGF-like ligand of the Egfr; (3) suppresses the phenotypes of Egfr hypomorphic mutations both in the eye and the wing and (4) flies heterozygous for both sprouty and argos have mildly rough eyes, caused by a slight overrecruitment of all types of cell, although heterozygosity for either mutation alone causes no phenotype. Other genetic interactions between sprouty and the Egfr pathway are also detailed. All point to the same conclusion: Sprouty inhibits Egfr signaling (Casci, 1999).

Both full-length Sprouty and a truncated Sprouty containing residues 1-369 (i.e., without the cys-rich domain and C-terminal residues) were assayed for their ability to bind in vitro translated members of the Ras pathway. Strong interactions are detected between Sprouty and Drk, an SH2-SH3 containing adaptor protein homologous to mammalian Grb2, and between Sprouty and Gap1, a Ras GTPase-activating protein. No interactions were seen between Sprouty and several other proteins involved in the Ras pathway: Sos, Dos, Csw, Ras1, Raf, and Leo (14-3-3). The interactions with Drk and Gap1 did not require the presence of the C-terminal cysteine-rich domain, the region of Sprouty most conserved between flies and humans. Since the well-conserved cysteine-rich domain of Sprouty is not required for binding to Drk or Gap1, it might instead target the protein to the plasma membrane. To test this, two truncated forms of Sprouty were expressed in cultured cells. One form lacks the conserved cysteine-rich domain, whereas a second exclusively comprises the cysteine-rich domain. The form with the cysteine-rich domain is membrane associated and is indistinguishable from the wild-type protein. In sharp contrast, the form lacking the cysteine-rich domain is distributed uniformly throughout the cell, with no specific localization to membranes. Cell fractionation confirms these results. It is concluded that the 147-residue cysteine-rich domain in Sprouty, which corresponds to the most conserved region in the published human ESTs, is responsible for the specific localization of Sprouty to the plasma membrane (Casci, 1999).

Sprouty's function is, however, more widespread. It also interacts genetically with the receptor tyrosine kinases Torso and Sevenless, and it was first discovered through its effect on FGF receptor signaling. In contrast to an earlier proposal that Sprouty is extracellular, biochemical analysis suggests that Sprouty is an intracellular protein, associated with the inner surface of the plasma membrane. Sprouty binds to two intracellular components of the Ras pathway, Drk and Gap1. These indicate that Sprouty is a widespread inhibitor of Ras pathway signal transduction (Casci, 1999).

RNA localization and post-transcriptional regulation

Pattern formation in Drosophila depends initially on the translational activation of maternal messenger RNAs (mRNAs) whose protein products determine cell fate. Three mRNAs that dictate anterior, dorsoventral, and terminal specification--Bicoid, Toll, and Torso, respectively--shows increases in polyadenylate [poly(A)] tail length concomitant with translation. (Salles, 1994).

The Drosophila SHC adaptor protein is required for signaling by a subset of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) transduce signals via cytoplasmic adaptor proteins to downstream signaling components. Loss-of-function mutations in have been identified in the Drosophila homolog of the mammalian adaptor protein SHC. A point mutation in the phosphotyrosine binding (PTB) domain of the Drosophila SHC-adaptor protein (Shc) completely abolishes Shc function and provides in vivo evidence for the function of PTB domains. Unlike other adaptor proteins, Drosophila Shc is involved in signaling by only a subset of RTKs: shc mutants show defects in Torso and DER but not Sevenless signaling, as confirmed by epistasis experiments. By double-mutant analysis, the adaptors DOS, DRK, and Shc act in parallel to transduce the Torso signal. These results suggest that Shc confers specificity to receptor signaling (Luschnig, 2000).

During the development of multicellular organisms, receptor tyrosine kinases (RTKs) play an important role in transducing a variety of extracellular signals that trigger cellular responses, such as proliferation, differentiation, or cell survival. RTKs activate a cytoplasmic signaling cascade, the RAS-MAPK pathway, which has been highly conserved from invertebrates to mammals (Luschnig, 2000).

Three Drosophila RTKs have been studied extensively. Torso (TOR) is required for the specification of terminal cell fate at the embryonic poles. Sevenless (SEV) induces R7 photoreceptor cell fate in the developing eye. In contrast to TOR and SEV, each of which being required only for one specific process, the Drosophila EGF receptor (DER) has multiple functions during development. Most of the known components of the RAS-MAPK pathway (CSW, DOS, DRK, KSR, SOS, GAP1, RAS1, DRAF, DMEK, DMAPK) are required for signaling by all three receptors. It is an intriguing problem how different RTKs, which activate one apparently common cytoplasmic signaling cascade, can trigger specific biological responses (Luschnig, 2000).

Upon ligand binding, RTK monomers phosphorylate each other on specific tyrosine residues, thereby creating docking sites for cytoplasmic adaptor proteins with phosphotyrosine (pY) binding modules, such as SH2 (SRC homology 2) or phospho tyrosine binding (PTB). One such adaptor protein, DRK (GRB2 in mammals, SEM-5 in C. elegans) acts as a link between activated RTKs and the Son of Sevenless (SOS) protein, which in turn activates RAS by catalyzing GDP-GTP exchange. The identification of more factors involved in signaling between RTKs and RAS1 has led to the view of a complex signaling network rather than a simple linear cascade. Such factors include Daughter of Sevenless (DOS), which potentially acts as a docking site for several SH2 and SH3 domain proteins, and Corkscrew (CSW), a protein tyrosine phosphatase containing two SH2 domains (Luschnig, 2000).

The maternally derived TOR pathway in the Drosophila embryo is a particularly useful system to study RTK signaling because it marks the first time point in development when the RAS-MAPK pathway becomes essential. The TOR receptor is activated by a spatially restricted ligand at the embryonic poles and regulates, via the RAS-MAPK pathway, zygotic expression of the terminal gap genes, tailless (tll) and huckebein (hkb). Posterior expression of tll and hkb is exclusively regulated by TOR signaling and can serve as a quantitative measure for the strength of signal, while anterior expression depends on additional input by the morphogen Bicoid (Luschnig, 2000).

The loss-of-function phenotypes of signaling components acting between the TOR receptor and DRAF are weaker than those of mutations in TOR or DRAF themselves, and it was shown that DRAF can be activated in the absence of RAS1. This indicates that downstream of the receptor the signal splits into branches and that a RAS1-independent signal can activate DRAF. An analysis of tyrosine phosphorylation sites in the activated TOR receptor revealed that individual sites have distinct positive or negative regulatory effects on signaling, by acting as targets for specific molecules (Luschnig, 2000).

Another adaptor protein, SHC, was suggested by studies in mammalian cells to act in RTK signaling. SHC contains two pY-binding modules, an N-terminal PTB domain, and a C-terminal SH2 domain, separated by a region with similarity to α-1 collagen. Upon EGF stimulation, SHC binds to EGFR and becomes phosphorylated on a specific tyrosine, which creates a binding site for the SH2 domain of GRB2. Mammalian SHC can act as an adaptor to link RTKs via GRB2 to RAS activation (Luschnig, 2000 and refences therein).

A Drosophila SHC homolog, SHC, has been isolated and characterized biochemically. SHC associates with and becomes tyrosine phosphorylated by activated DER in vivo, and the PTB domain of SHC binds specifically to a NPXpY motif in DER. While the PTB and SH2 domains of SHC show a high degree of conservation between human and Drosophila, SHC lacks the high-affinity GRB2 site present in mammalian SHC, and no binding of SHC to DRK was observed in coimmunoprecipitation experiments. However, the presence of another conserved motif (YYND/S) in all known SHC proteins suggested that SHC could couple RTKs to other downstream targets besides GRB2 and the RAS pathway. Thus far, no functional evidence for the suggested roles of SHC proteins has been shown, since shc loss-of-function mutations have not been reported in any system (Luschnig, 2000).

This work presents functional evidence that a Drosophila SHC homolog is an essential component of distinct RTK signaling pathways in the fly. Mutations affecting either only the PTB domain or eliminating SHC protein cause the same strength of phenotypes in all assays applied. Based on genetic data, it cannot be distinguish whether both domains, PTB and SH2, bind to pY sites on the same activated RTK or on different proteins. DER contains one bona fide binding site for the SHC PTB domain (consensus NPXpY; NPEY1357 in DER), while in activated TOR two sites with a mismatch at the +2 position are phosphorylated on Tyr (NKGY644; NKEY698; Gayko, 1999). No motifs for binding by SH2 domains (consensus YIXI) are found in DER, TOR, or SEV (Luschnig, 2000).

The shc111-40 allele contains a single amino acid exchange (R152 > Y) in the PTB domain. Structural studies have revealed that the corresponding residue (Arg-175) of the human SHC PTB domain is crucial for binding of the PTB domain to pY-containing phosphopeptides: mutating the corresponding Arg-175 of the human Shc PTB domain (to Gln or Lys) completely abolished binding of SHC to the phosphopeptide, while exchanging amino acids at three other structurally relevant positions had less or no significant effect on binding. This work supports the structural data and provides functional evidence for the role of PTB domains in RTK signaling (Luschnig, 2000).

In the absence of SHC protein, TOR and DER signaling is only partially reduced. It has been shown for the TOR pathway that signaling downstream of the receptor does not occur in a simple linear cascade, based on the finding that components acting between TOR and DRAF show weaker loss-of-function phenotypes than TOR or DRAF (Luschnig, 2000).

Double-mutant analysis shows that the TOR signal splits into at least three parallel branches, represented by DOS, DRK, and SHC. Simultaneous removal of DOS and SHC function from the germline does not completely abolish the TOR signal, although it is strongly reduced. Double mutant phenotypes and epistasis experiments suggest that more TOR signal is transmitted via SHC than via DOS or DRK. However, genetic data do not exclude that direct interactions between any of these proteins may exist; for example, DOS contains putative binding sites for the SH2 domains of DRK and SHC (Luschnig, 2000).

It was also shown that DRK and SHC have parallel activities, in agreement with the finding that the unique DRK/GRB2 binding site (YVNV) present in mammalian SHC proteins is missing in Drosophila SHC, and Drosophila SHC does not bind DRK in vitro. While Drosophila SHC lacks the DRK binding site, a central YYND motif is conserved between Drosophila and mammalian SHC proteins and is likely to serve as a docking site for an as yet unknown protein that triggers a RAS-independent signal. It has been shown that DRAF can be activated in the absence of RAS1, but the molecular nature of a RAS1-independent pathway had remained elusive. Based on genetic results and binding studies, Drosophila SHC is a good candidate to activate downstream targets via a DRK-/RAS1-independent pathway. The identification of signaling components acting independently of RAS will be of great interest for understanding the specificity of RTK signaling, and SHC should provide a useful entry point to study such a pathway (Luschnig, 2000).

It remains a puzzling question why a complex network of adaptor proteins has evolved, if all of these proteins were involved in a single common pathway activated by all RTKs. Interestingly, this study has found that SHC, unlike most other components, functions only in a subset of RTK pathways. DOS, DRK, KSR, and all components downstream of RAS1 are required for signaling by the RTKs TOR, DER, and SEV. Also, unlike all other signaling components, flies lacking SHC can survive to adults, which show a specific phenotype, suggesting that SHC plays a more specific role than other components (Luschnig, 2000).

In the Drosophila eye, DER can replace the function of SEV in R7 determination. This has led to the conclusion that the cytoplasmic signaling pathways triggered by DER and SEV were identical. However, these data are based on the analysis of constitutively activated forms of RTKs that might not faithfully reflect the situation during normal signaling. This study found that SEV signaling appears to be normal in flies lacking SHC protein, and the defects seen in shc mutant eyes appear to be due to impaired DER signaling. The expression of phenotypes caused by activated DER in the eye is very sensitive for the gene dosage of shc. In contrast, dosage-sensitive interactions of shc with activated forms of SEV were not seen, whereas the adaptors DOS and DRK were identified on the basis of their dosage sensitivity for signal transduction by activated SEV. Also, binding of SHC to SEV was not observed, and SEV does not contain consensus binding sites for PTB or SH2 domains of SHC, whereas SHC specifically binds via its PTB domain to DER. Based on these arguments, it is suggested that SHC is not involved in SEV signaling. Even in the absence of SHC protein, the effect of activated SEV was not abolished; however, this study found that the reduction of DER signaling by shc mutations does partially affect the efficiency of SEV-induced overrecruitment of photoreceptors in the anterior of the eye. Accordingly, removing one copy of the DER itself causes partial suppression of the activated SEV phenotype, indicating that induction of photoreceptor cell fate by the DER is necessary before SEV can trigger R7 development (Luschnig, 2000).

The anteriorly-posteriorly graded phenotype of shc mutant eyes can be explained by a common feature of DER signaling: the morphogenetic furrow moves from posterior to anterior across the eye disc and leaves in its wake cells that express Spitz (Spi), an activating DER ligand, and Argos (Aos), a presumptive inhibitory DER ligand. Both Spi and Aos are themselves target genes of DER signaling and are thought to act as diffusible ligands for DER. Hyperactivation of DER by overexpressing activating Spi, or by mutating inhibitory Aos, leads to photoreceptor overrecruitment and eye roughness, which is more pronounced in the posterior of the eye. This graded phenotype is presumably due to accumulation of diffusible Spi in the posterior of the eye disc. It is likely that the same effect accounts for the anterior to posterior graded eye phenotype of shc mutants: although DER signaling efficiency is reduced, Spi can accumulate to sufficient levels in the posterior to allow normal differentiation of photoreceptors. Toward the anterior margin, the level of Spi becomes limiting, and DER signaling activity is reduced below the threshold for photoreceptor differentiation, resulting in the loss of photoreceptors (Luschnig, 2000).

Interestingly, planar polarity defects were found in shc mutant eyes. Frizzled, Notch, and Jak-Stat signaling have been implicated in the establishment of planar polarity in the eye, but to date no role in this process has been assigned to any member of the RAS-MAPK signaling pathway (Luschnig, 2000).

How can different RTKs trigger specific responses by using an apparently common signaling pathway? Several models have been put forward to explain this paradox. In a 'quantitative' model, the kinetics (exact level and duration) of the MAPK signal is critical for the specificity of the response. A 'qualitative' model proposes the existence of cytoplasmic signaling components dedicated to a specific receptor or subset of receptors. Recently, the protein DOF has been shown to act specifically in Drosophila FGFR signaling pathways. Interestingly, DOF is exclusively expressed in cells where also FGFRs are expressed, namely mesoderm and the tracheal system. Tissue-specific expression of a signal transduction component might be one way to create specific responses (Luschnig, 2000).

In contrast, SHC is widely expressed throughout the embryo (Lai, 1995). Specificity appears to be mediated by selective binding of SHC to specific receptors. The curren data show that SHC is functionally dedicated to a subset of RTKs and suggest that the cytoplasmic events triggered by different RTKs (e.g., DER and SEV) can be different. This is a new aspect that could provide an additional explanation for the specificity of RTK signals. It is suggested that different receptors use specific combinations of cytoplasmic adaptor proteins, which have parallel activities, and potentially activate different downstream targets. This could result in quantitatively and/or qualitatively different signal properties. It will be of great interest for the understanding of RTK signaling to elucidate what downstream events are activated by SHC (Luschnig, 2000).

Endosomal trafficking of Torso: The role of Hrs

Signaling through tyrosine kinase receptors (TKRs) is thought to be modulated by receptor-mediated endocytosis and degradation of the receptor in the lysosome. However, factors that regulate endosomal sorting of TKRs are largely unknown. Here, one such factor is Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate). Electron microscopy studies of hrs mutant larvae reveal an impairment in endosome membrane invagination and formation of multivesicular bodies (MVBs). hrs mutant animals fail to degrade active epidermal growth factor (EGF) and Torso TKRs, leading to enhanced signaling and altered embryonic patterning. These data suggest that Hrs and MVB formation function to downregulate TKR signaling (Lloyd, 2002).

Membrane trafficking events are tightly regulated to ensure proper spatial and temporal delivery of membrane bound cargo. Fusion of intracellular vesicles with their target membrane requires the formation of a highly stable core complex. Regulation of the formation of this complex may modulate vesicle fusion. One proposed regulator of core complex assembly is Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate), which binds to the plasma membrane t-SNARE SNAP-25 and inhibits core complex formation in vitro. Addition of Hrs to a neuroendocrine cell assay inhibits neurotransmitter release, suggesting that Hrs may regulate Ca2+-triggered exocytosis. Interestingly, Hrs is predominantly localized to early endosomes, and Hrs mutant mice have enlarged endosomes. Furthermore, Hrs interacts with Eps15, a protein implicated in receptor-mediated endocytosis. Thus, Hrs has been proposed to play roles in both exo- and endocytosis (Lloyd, 2002 and references therein).

Hrs is homologous to yeast Vps27p (vacuolar protein sorting), which regulates protein trafficking from a prevacuolar compartment to the vacuole. Vps27p belongs to the Class E subset of VPS proteins, which are implicated in sorting proteins into the vacuole lumen. Hrs and Vps27p contain a FYVE domain that binds specifically to phosphatidyl-inositol-3-phosphate (PI3P), and this domain has been demonstrated to localize many proteins to the early endosome. Several FYVE domain-containing proteins have been implicated in endosomal trafficking, including early endosome autoantigen 1 (EEA1), which is essential for early endosome fusion, and Fab1p, which is required for sorting into MVBs. Thus, the FYVE domain may allow proteins to mediate membrane trafficking from or to the endosome through its interaction with PI3P (Lloyd, 2002 and references therein).

In addition to a role in vesicle trafficking, Hrs has been proposed to play different roles in several signal transduction pathways. Hrs binds to Stam, a protein implicated in cytokine signaling, and Hrs and Stam both contain VHS (Vps27p, Hrs, Stam) domains present in several proteins implicated in membrane trafficking or signal transduction. Overexpression of Hrs inhibits IL-2-mediated cell growth, suggesting that Hrs may function with STAM to negatively regulate cytokine signaling. In contrast to this inhibitory role, Hrs has recently been proposed to play positive roles in both TGF-ß and Egfr signaling. Hrs binds to SMAD-2, and hrs mutant mouse embryos exhibit a reduced response to activin and TGF-ß. Furthermore, overexpression of Hrs in HeLa cells inhibits ligand-induced degradation of Egfr, suggesting that Hrs may normally promote Egfr signaling by inhibiting endosome to lysosome trafficking of the receptor (Lloyd, 2002 and references therein).

Thus, although numerous data suggest Hrs may play a role in vesicle trafficking and signal transduction, the precise function of Hrs in these processes is unclear. To further investigate the function of Hrs, effects of the loss of Hrs in Drosophila were investigated. The data suggest that Hrs regulates inward budding of endosome membrane and MVB formation. More importantly, hrs mutant animals are unable to degrade active Egfr and Torso TKRs leading to enhanced TKR signaling (Lloyd, 2002).

A single Hrs homolog was identified in the Drosophila genome, and sequence analysis of a 2.7 kb hrs cDNA predicts an open reading frame of 760 amino acids with several well-conserved domains. The hrs gene was mapped to cytological band 23A and is removed by deficiency Df(2L)N19 (Df). Alleles of several complementation groups mapping to Df(2L)N19 were previously isolated in an EMS mutagenesis screen for mutations in synaptotagmin. An 11.5 kb genomic DNA fragment containing the hrs gene or the hrs cDNA driven by the hsp70 promoter (hs-hrs) fully rescues the early pupal lethality of l(2)23AdD28/Df and l(2)23AdD28/l(2)23AdD28 animals but not other mutations in this region. These data demonstrate that the l(2)23AdD28 chromosome (hereafter referred to as hrs) contains a mutation in the hrs gene. Sequencing of DNA from mutant animals reveals a nonsense mutation at amino acid Q270 (Lloyd, 2002).

Polyclonal antibodies were generated to the full-length (anti-FL-Hrs) and amino-terminal half (aa 1-376, anti-N-Hrs) of the recombinant protein. Western analysis of fly extracts using the anti-FL-Hrs antibody detects a major band of 110 kDa in wild-type animals, whereas no protein is detected in hrs third-instar larvae (L3) or white prepupae (WPP). However, the anti-N-Hrs antibody recognizes a 30 kDa band in mutant animals in addition to the 110 kDa band in wild-type animals, suggesting that a 270 amino acid truncated protein is expressed in mutant animals. Furthermore, the presence of full-length Hrs protein in late stage 17 Df embryos suggests that maternally deposited Hrs protein is very stable and may compensate for the loss of zygotic Hrs in embryonic development. Indeed, embryos produced by mothers homozygous for hrs in their germline cells (maternal knockout or mKO) lack full-length Hrs protein and die early in embryogenesis. Thus, the effects of loss of Hrs function may be analyzed in zygotic mutant third-instar larvae/early pupae or in germline mutant embryos (Lloyd, 2002).

Analysis of the protein expression of Hrs suggests that Hrs is ubiquitously expressed. To determine the subcellular localization of Hrs, expression was examined in garland cells and muscle cells of third-instar larvae. Anti-Hrs labels vesicles enriched in perinuclear regions of muscle cells, whereas labeled vesicles are predominantly in the periphery of garland cells. These staining patterns are specific, since they are not seen in hrs mutant cells. Interestingly, overexpression of hrs leads to an enlargement or accumulation of Hrs-positive vesicles and a reduction in cell size (Lloyd, 2002).

Both Hrs antibodies also label type I synaptic boutons of the neuromuscular junction (NMJ). There is some colocalization of Hrs with the synaptic vesicle (SV) marker Synaptotagmin, but most staining appears to be outside SV-rich regions. However, electrophysiological analysis of wild-type and mutant neuromuscular junctions suggests that Hrs does not play an important role in regulating synaptic vesicle exocytosis. Furthermore, Hrs is not enriched in the synapse-rich neuropil of the larval brain, even when overexpressed in neurons using the elav-GAL4 driver (Lloyd, 2002).

To determine if Hrs functions in endocytosis, trafficking of internalized tracers was investigated in third-instar larval garland cells, large cells with a high rate of fluid-phase endocytosis. Wild-type garland cells show strong labeling of peripheral vesicles after a 5 min incubation with avidin-Cy3, indicating rapid internalization of dye into endosomes. Mutant cells are much larger than wild-type cells but show strong labeling of peripheral vesicles, suggesting that dye internalization is not significantly impaired. However, many labeled vesicles (endosomes) in mutant cells are much larger than those observed in wild-type cells. Furthermore, analysis of lysosomal markers suggest that while lysosomes are reduced in size in hrs mutant cells, smaller endosomes are capable of delivering avidin to low pH compartments at a rate similar to wild-type cells (Lloyd, 2002).

Next, hrs mutant endosomes were analyzed using transmission electron microscopy (TEM). Garland cells were incubated with HRP for 5 min, fixed, and sectioned for TEM. In wild-type garland cells, HRP labels the lumen and internal membrane of peripherally located endosomes. In hrs mutant larvae, mutant endosomes are dramatically enlarged, but do not show significant HRP labeling, despite their ability to internalize avidin dye. Rather, in mutant cells, HRP labels a vast tubulo-vesicular network at the periphery of mutant cells, which is not seen in wild-type cells. Finally, endosomes in wild-type garland cells undergo invagination of their limiting membrane to eventually collapse upon themselves. In hrs garland cells, there is a strong reduction in the relative number of invaginated endosomes (5-fold, p = 0.008) and collapsed endosomes (10-fold, p = 0.003). These data suggest that endosomes are enlarged in hrs mutant cells due to an inability of endosomes to invaginate their limiting membrane (Lloyd, 2002).

Inward budding of endosome membrane is believed to be the first step of multivesicular body (MVB) formation. To determine if MVB formation is impaired in hrs mutant animals, electron microscopy was performed at the NMJ. In wild-type synapses, large, classical MVBs are occasionally observed, and they are believed to be endosomal intermediates containing synaptic vesicle proteins destined to be delivered to somatic lysosomes. Much more frequently, though, 60-120 nm vesicles are observed that also appear to contain small internal vesicles. Remarkably, in hrs mutant synapses, there is a 5-fold reduction in the number of these small MVBs. These data suggest that Hrs regulates formation of MVBs at the synapse (Lloyd, 2002).

One proposed function of multivesicular bodies is to partition transmembrane proteins and lipids destined for delivery to the lysosome from those destined to be recycled back to the surface of the cell. While most cell surface proteins are recycled from endosomes, activated TKRs such as the Egfr are trafficked inside MVBs for degradation in the lysosome. Because Hrs is phosphorylated in response to TKR activation, the possibility that Hrs regulates TKR degradation and signaling in Drosophila was investigated (Lloyd, 2002).

The first TKR pathway analyzed was the Torso pathway, which functions very early in development to pattern the embryonic termini. The Torso receptor is present throughout the surface of the early embryo, but it only binds ligand at the poles. Ligand binding of the Torso receptor triggers the ras/MAPK cascade leading to the phosphorylation of ERK/MAPK. A diphospho-ERK (dpMAPK)-specific antibody has been used to investigate TKR signaling in the early embryo. In wild-type embryos, terminal dpMAPK staining initiates approximately 2 hr after egg laying (AEL), peaks about 30 min later, and then rapidly terminates during the initiation of gastrulation. In cellular blastoderm hrs maternal knockout (mKO) embryos, dpMAPK staining is enhanced and spatially broadened when compared to wild-type embryos. Furthermore, dpMAPK expression remains elevated at the anterior and posterior termini during early gastrulation when compared to wild-type. Hence, Torso signaling is both spatially broadened and temporally prolonged (Lloyd, 2002).

MAPK activation induces the expression of the terminal gap genes huckebein (hkb) and tailless (tll), and the pattern of expression of these genes can be used as a quantitative readout for Torso receptor activation. In mutant embryos, both anterior and posterior domains of hkb are significantly expanded by 23% and 50%, respectively. Similarly, the anterior stripe of tll is moved posteriorly by 50%, and the posterior stripe is expanded by 25%. Thus, these data confirm that Torso signaling is enhanced and broadened both spatially and temporally in mutant embryos and demonstrate that Hrs negatively regulates signaling of the Torso TKR pathway (Lloyd, 2002).

Next, whether an increase in Torso signaling was due to the failure of receptor degradation was investigated. Total levels of the Torso receptor in early (0-4 hr) embryos are similar between wild-type and hrs mKO embryos. However, a 56 kDa band corresponding to the cytoplasmic domain of Torso (500 aa) is prevalent in mutant animals but barely detectable in wild-type embryos. These data suggest that a failure to degrade the active cytoplasmic portion of the receptor may underlie the increased Torso signaling observed in early embryos. To further examine degradation of the Torso receptor, the kinetics of receptor protein expression were examined in wild-type and mutant embryos. In wild-type embryos, Torso protein expression is maximal just prior to receptor activation (1-2 hr AEL) and is rapidly downregulated such that it is barely detectable by 5 hr AEL. However, in hrs mKO embryos, Torso protein levels fail to decline until 9-10 hr AEL, at which time zygotic expression of hrs is abundant. These data demonstrate that hrs is essential for Torso TKR degradation (Lloyd, 2002).

During early gastrulation, hrs mKO embryos display numerous morphological defects. Some mKO embryos fail to complete posterior terminal cellularization, and their yolk appears to be extruded posteriorly during gastrulation. During germ band elongation, most hrs mKO embryos undergo highly abnormal development and exhibit aberrant folding or twisting. The vast majority of mutant embryos arrest morphological development prior to germband retraction, and few secrete cuticle. These phenotypes suggest that hrs may be required to regulate multiple early embryonic signaling pathways (Lloyd, 2002).

Recent evidence suggests that ubiquitination of endosomal TKRs may be a signal for trafficking to the lysosome rather than recycling to the surface. However, factors that bind ubiquitinated TKRs and sort them into MVBs are unknown. Recently, a 20 amino acid ubiquitin-interacting motif (UIM) conserved in family members of the proteosome subunit 5A (S5A) has been found in a large number of proteins, including several proteins implicated in endocytic trafficking. The UIM present in Hrs is highly conserved among all species examined, so it was determined whether or not Hrs interacts with ubiquitin, using GST pull-down assays. GST-ubiquitin but not GST readily pulls down the full-length Hrs protein from pupal extract. This interaction is direct, since GST-ubiquitin also binds purified recombinant N-Hrs (aa 1-376) protein containing the UIM. These data demonstrate that Hrs binds ubiquitin and suggest that Hrs may regulate endosomal sorting of TKRs via a direct interaction of Hrs with ubiquitinated receptors (Lloyd, 2002).

Extracellular signals are communicated to cells with remarkable temporal and spatial resolution. The rapid kinetics of signal amplification and termination are critical to the precision of signal transduction. One mechanism thought to mediate signal downregulation is the internalization and degradation of cell surface receptors. Although internalization of receptors may inhibit ligand binding, many receptors are still active, or in some cases, more active, after internalization. Once inside the early endosome, TKRs may either be recycled back to the surface of the cell or sorted into the multivesicular body (MVB) for degradation in the lysosome (Lloyd, 2002).

It has long been proposed that lysosomal delivery of cell surface receptors is a negative feedback mechanism for downregulation of receptor signaling. However, there is little in vivo evidence for this model, and it remains possible that deactivation of the receptor or downstream components may compensate for a failure to downregulate active receptor. The data suggest that trafficking of TKRs into the MVB plays an important role in signal attenuation. Interestingly, several of the morphological phenotypes observed in hrs mKO embryos are also seen in mutations affecting the torso pathway. For example, posterior cellularization defects are also observed in fs(1)polehole and l(1)polehole/D-raf embryos, and twisted gastrulation phenotypes are also observed in torso embryos (Lloyd, 2002).

Recently, overexpression of Hrs in HeLa cells has been shown to inhibit ligand-mediated degradation of Egfr, suggesting that Hrs may function to prolong Egfr signaling. In contrast, the data in this study suggest the opposite function for Hrs, namely that it functions to attenuate TKR signaling by promoting degradation of the tyrosine-phosphorylated, or active, receptor. Interestingly, although active Egfr is upregulated in hrs mutants, total levels of the receptor are decreased, suggesting that Hrs is specifically required for degradation of active receptors. This reduction in total receptor is likely due to a well-characterized negative feedback mechanism whereby Egfr hyperactivation inhibits receptor transcription (Lloyd, 2002).

In summary, the following model is proposed for Hrs function. (1) Endocytosis of activated tyrosine kinase receptors (2) leads to the phosphorylation of Hrs on the early endosome membrane. Phosphorylation may enhance the activity of Hrs, which then (3) leads to localized invagination of endosomal membrane. Ubiquitinated receptors may be sorted into the invagination directly via an interaction with the UIM of Hrs or indirectly through an interaction with Hrs binding proteins SNX1, clathrin, or Eps15. Finally, (4) the membrane is pinched off to form a MVB, and (5) the internalized vesicles are trafficked to the lysosome for degradation. This process of MVB formation leads to a reversal of membrane topology such that the cytoplasmic portion of TKRs is now inside the MVB and unable to signal to downstream components. In this model, receptor-mediated activation of Hrs and MVB formation serves a critical role in attenuating tyrosine kinase receptor signaling (Lloyd, 2002).

Two distinct but convergent groups of cells trigger Torso receptor tyrosine kinase activation by independently expressing torso-like

Cell fate determination is often the outcome of specific interactions between adjacent cells. However, cells frequently change positions during development, and thus signaling molecules might be synthesized far from their final site of action. This study analyzed the regulation of the torso-like gene, which is required to trigger Torso receptor tyrosine kinase activation in the Drosophila embryo. Whereas torso is present in the oocyte, torso-like is expressed in the egg chamber, at the posterior follicle cells and in two separated groups of anterior cells, the border cells and the centripetal cells. JAK/STAT signaling regulates torso-like expression in the posterior follicle cells and border cells but not in the centripetal cells, where torso-like is regulated by a different enhancer. The border and centripetal cells, which are originally apart, converge at the anterior end of the oocyte, and both groups contribute to trigger Torso activation. These results illustrate how independently acquired expression of a signaling molecule can constitute a mechanism by which distinct groups of cells act together in the activation of a signaling pathway (Furriols, 2007; full text of article).

Although tsl is expressed in three different groups of follicle cells, these cells are not completely unrelated. Thus, for example, both the BCs and the CCs are derived from a common pool of anterior follicle cells and express and require some of the same genes for their development. Likewise, many similarities have also been recognized between the BCs and the PFCs. This raises the possibility that a common mechanism could single out these cells for tsl expression. Alternatively, each of these groups of follicle cells could be independently targeted to express tsl. As a first attempt to address how these distinct groups of follicle cells acquire the ability to express a common signaling factor, an analysis of the tsl promoter was undertaked (Furriols, 2007).

As a first indication of what constitutes the tsl regulatory region, the P-element insertion carrying the lacZ gene upstream of the 5'-UTR exon in the tsl0617 mutant, thereafter tsl0617-lacZ, was know to reproduced all of the features of tsl expression in the follicle cells, as judged by comparison with the tsl in situ hybridization pattern. By transformation of lacZ reporter constructs using different regions upstream of the coding sequences of tsl, it was found that a single fragment of ~1,500 bp upstream of the 5'-UTR exon (see Distinct Enhancers Regulate tsl Expression in Specific Groups of Follicle Cells) reproduces the tsl wild-type pattern. Further dissection allowed splitomg the tsl promoter into two nonoverlapping regions responsible for a different subset of the tsl expression pattern. In particular, it was found that a 604-bp sequence drives expression only in the CCs, hereafter referred to as the CC enhancer, whereas an adjacent 954-bp sequence drives expression in both the BCs and the PFCs. Comparison between the different constructs suggested that the enhancer for BCs and PFCs could be further refined to a region of 298 bp (fragment K). This assumption was confirmed by establishing that two copies of fragment K are sufficient to drive expression in BCs and PFCs, hereafter referred to as the BC/PFC enhancer. Thus, in summary, two different regions of the tsl promoter are responsible for distinct subsets of tsl expression. It is remarkable that a single promoter fragment (fragment K) drives tsl expression in two independent group of follicle cells (the BCs and PFCs), whereas separate enhancers (fragments K and F) are responsible for tsl expression in the BCs and CCs, which are derived from a common pool of anterior follicle cells (Furriols, 2007).

This study found that tsl expression is controlled by different cis-regulatory regions and different transactivating factors independently in different cell populations: a single promoter fragment responds to JAK/STAT signaling and activates tsl expression in both the BCs and PFCs, whereas another enhancer drives tsl expression in the CCs. Moreover, putative STAT binding sites (consensus TTCNNNGAA) were found in the identified BC/PFC enhancer. Mutations in those sites greatly reduce tsl-lacZ expression in the BCs and PFCs, pointing to a direct regulation by the JAK/STAT pathway. The fact that some reporter expression can occasionally be detected in those cells even when these sites are mutated could be attributed to regulation by other factors, which could also contribute to tsl expression in BCs and PFCs. In this regard, microarray analysis has shown that activity of the slbo transcription factor, which has been shown to function as a simple transcriptional activator and whose expression is also dependent on the JAK/STAT pathway, induces a 2-fold increase of tsl expression. In summary, these results show that the JAK/STAT pathway acts as a primary regulator of tsl expression in the BCs and PFCs (Furriols, 2007).

The JAK/STAT pathway is triggered in the Drosophila egg chamber by localized expression of its ligand, Upd, in two polar cells at each end of the chamber. Signaling from this pathway is responsible for the patterning of the follicle cells at both ends of the egg chamber, and the results show now that it is also responsible for tsl expression in the BCs and the PFCs. Thus, these results indicate that a common mechanism is responsible for initially patterning the egg chamber terminal epithelium and later triggering the mechanism that specifies the embryonic terminal regions (Furriols, 2007).

At the anterior end of the egg chamber, three populations of follicle cells can be distinguished: the BCs, the CCs, and the stretched cells in between. Among those, BCs and CCs, but not stretched cells, express tsl. Although the role of the JAK/STAT pathway in patterning the follicle cells at both ends of the egg chamber is well established, there are conflicting data about whether a gradient of its ligand, Upd, could indeed be responsible for patterning all of the anterior follicle cells. If that was the case, it might be expected that the JAK/STAT pathway could play a role in tsl expression in both the BCs and the CCs. In this scenario, absence of tsl expression in the stretched cells could be due to specific mechanisms of tsl gene repression in those cells. Conversely, the current results show that the JAK/STAT pathway does not have a specific role in the activation of tsl in the CCs. These results do not necessarily argue against a gradient of Upd. It could be argued, for example, that lower levels of JAK/STAT signaling in the CCs might not be sufficient to trigger activation of the BC/PFC enhancer. Alternatively, it could also be the case that a specific repressor element in this enhancer might inhibit its expression in the CCs. However, irrespective of a role of the Upd gradient in patterning the follicle cells, the results show that tsl expression in the CCs is independent of JAK/STAT. This result indicates that there are JAK/STAT-independent differences within the anterior epithelial cells of the egg chamber, as has been hypothesized (Furriols, 2007).

The results show that the two groups of anterior cells, the BCs and the CCs, contribute to trigger anterior Tor activation. Moreover, they indicate that this is accomplished by independent regulation of tsl in each of these cell populations. At first glance, either the BCs or the CCs appear to be sufficient to trigger Tor activation. Thus, GAL4-driven expression of tsl in either the BCs or the CCs is able to promote normal development of the terminal anterior structures in embryos derived from otherwise tsl mutant females. Additionally, RNAi-mediated inactivation of tsl in either the BCs or the CCs is not able to generate an anterior tsl phenotype, whereas inactivation in both the BCs and CCs produces embryos with anterior tsl mutant phenotypes. Thus, tsl expression in the BCs and CCs might be redundant. However, there are some caveats to those experiments that should be considered. First, GAL4-driven expression might generate higher tsl levels than the normal in the BCs or CCs. Second, in these experiments, RNAi-mediated inactivation does not completely impair tsl function; this is clearly observed because ~40% of the embryos develop anterior terminal structures even when UAStsldsRNA is expressed in both the BCs and the CCs using the C306 and 55B drivers (Furriols, 2007).

Given these results, it is proposed that an absolute level of tsl expression may be crucial to trigger Tor signaling. Therefore, it might not be so important whether tsl is supplied by the BCs or the CCs, provided it reaches an absolute amount. This would explain why overexpression of tsl in either the BCs or the CCs can rescue the anterior tsl mutant phenotype. It would also explain the additive effects of lowering tsl activity from the BCs and the CCs to generate an anterior tsl phenotype. Besides, it has to be considered that too much Tsl could also be damaging. In this regard, it has to be noted that tsl overexpression driven by the slboGAL4 driver produces head involution defects in many embryos. Taking this into account, expression of tsl from both the BCs and CCs could be a means to reach a minimum amount of Tsl product, but also not to exceed a certain limit (Furriols, 2007).

To understand how such a mechanism could have been established, the differences in ovary organization among insects should be considered. Although all insect ovaries consist of morphologically and physiologically discrete entities (the ovarioles), there are differences on how the oocyte is positioned in reference to the follicle cells. In more ancient insects, the oocyte is surrounded by a monolayer of somatic follicle cells. Conversely, in more evolved insects, a group of nurse cells are clustered at the anterior end of the oocyte and it is only later that the anterior side of the oocyte is separated from the nurse cells and contacts the follicle cells. In Tribolium, an insect with a more primitive ovary in which tsl expression has been examined, tsl is precisely expressed in the follicle cells overlying both edges of the oocyte. Therefore, Drosophila tsl expression in the BCs and PFCs may represent an adaptation, or the remnant, of a more ancient pattern of tsl expression. The difference in Drosophila is that the anterior tsl-expressing follicle cells, initially separated from the oocyte, have acquired the capacity to migrate through the nurse cells to reach the anterior end of the oocyte. Thus, two insects with different type of ovaries share a common pattern of tsl expression in two groups of follicle cells at both ends of the oocyte, although the mechanism to position these cells next to the oocyte differ in both insects. Conversely, tsl expression in the CCs of Drosophila appears to be a more recent acquisition. The CCs are a new particularly evolved set of follicular cells that migrate to separate the oocyte from the adjacent nurse cells. In this context, concomitant tsl expression in the CCs in Drosophila may have been independently attained by the acquisition of a new distinct enhancer in the tsl promoter (Furriols, 2007).

Therefore, the complex pattern of tsl expression could provide a means to ensure the full triggering and robustness of Tor receptor tyrosine kinase activation and illustrates a mechanism by which the full response of a receptor cell can be accomplished by the independent acquisition of signaling capacity in distinct cell populations and their combined action (Furriols, 2007).

Conserved and divergent elements in Torso RTK activation in Drosophila development

The repeated use of signalling pathways is a common phenomenon but little is known about how they become co-opted in different contexts. This study examined this issue by analysing the activation of Drosophila Torso receptor in embryogenesis and in pupariation. While its putative ligand differs in each case, Torso-like, but not other proteins required for Torso activation in embryogenesis, is also required for Torso activation in pupariation. In addition, it was demonstrated that distinct enhancers control torso-like expression in both scenarios. It is concluded that repeated Torso activation is linked to a duplication and differential expression of a ligand-encoding gene, the acquisition of distinct enhancers in the torso-like promoter and the recruitment of proteins independently required for embryogenesis. A combination of these mechanisms is likely to allow the repeated activation of a single receptor in different contexts (Grillo, 2012).

This study provided evidences that Tsl participates in Tor activation both in the embryo and in the prothoracic gland. In the embryo, the TrkC108 cleaved form activates Tor in the absence of tsl function, thereby suggesting that the latter is directly or indirectly involved in the processing of the Trk protein. Given the similarity between Trk and Ptth, the effect of Tsl in dpERK accumulation in the prothoracic gland and the effect of TrkC-108 and Tsl in advancing and delaying pupariation respectively, it is proposed that Tsl is similarly involved in Ptth processing in the prothoracic gland. It should be noted that in the prothoracic gland Tsl and Tor proteins are produced in the same cells while during embryogenesis Tor accumulates in the embryo upon synthesis while Tsl is synthesized and secreted from cells surrounding the oocyte. However, tor and tsl expression in distinct cell types is not an absolute requirement for Tor activation in embryogenesis, it has been shown that tsl expression in the germline is also functional in Tor activation. Thus, Tsl is detected in the cytoplasm of the cells where it is synthesised both in the ovary and in the prothoracic gland, although the presence of a signal peptide in the protein suggests that it is secreted in both cases. Indeed, secreted Tsl is detected, upon specific processing, in the vitelline membrane, a particular type of extracellular matrix in the early embryo; yet, it has not been possible to detect Tsl at the extracellular matrix of the prothoracic gland cells (Grillo, 2012).

As Tsl lacks any feature indicating that it has protease activity, it has been suggested that this protein participates in the activation or nucleation of such an enzymatic complex. In this scenario, similar proteins that could equally be activated/nucleated by Tsl could carry out the processing of Trk and Ptth. In this case, Tsl would be the common module in both events of Tor activation. Alternatively, the same players could be involved in both Trk and Ptth processing, in which case, the common module for Tor activation should be enlarged to also encompass the same processing complex. Final clarification of these two possibilities awaits the identification of the Trk (and Ptth) processing mechanism, which still remains elusive (Grillo, 2012).

Conversely, fs(1)N, fs(1)ph and clos are required for Tor activation only in the early embryo indicating that Tsl does not need the function of these gene products to exert its function outside the embryo. Indeed, a relevant function of these three proteins is in vitelline membrane morphogenesis. Therefore, it is likely that these proteins are recruited to anchor Tsl at the vitelline membrane and thus they participate in Tor activation exclusively in embryonic patterning. Of note, several observations have led to the proposal that anchorage of Tsl in the vitelline membrane serves to store it in a restricted domain until Tor activation in the early embryo (Grillo, 2012).

As for Tor, Toll signalling is a transduction pathway that was initially thought to act only in early embryonic patterning but does in fact participate in other signalling events. However, in the case of Toll signalling, a single putative ligand, Spätzle (spz), acts both during embryonic patterning and in immunity, while for Tor signalling different putative ligands are responsible for its activation in embryonic patterning and in the control of pupariation. Spz triggers Toll activation in many scenarios because the spz promoter drives its expression in several groups of cells, possibly by distinct enhancers. In contrast, in the case of the Tor pathway a likely duplication might have generated two genes each with a distinct expression pattern and encoding the corresponding ligand for one of the two Tor activation events. The observation that Coleoptera but not Hymenopthera possess both trk and ptth orthologues suggest the putative duplication to have occurred at the origin of holometabolous insects. However and regarding tsl as the key element in ligand activation, multiple usage of the Tor pathway appears to have evolved by recruiting independent enhancers responsible for the distinct expression of the same gene (Grillo, 2012).

In summary, Tor activation in oogenesis and in the prothoracic gland is linked to the following: a duplication and subsequent differential expression of trk and ptth; the acquisition of independent specific oogenesis and prothoracic gland enhancers in the tsl promoter; and the recruitment of proteins independently required for organ morphogenesis, in particular for eggshell assembly. The Drosophila EGFR resembles the case of Tor as another example of the repetitive use of the same receptor by different ligands in different contexts: Gurken in oogenesis and Spitz, Vein, and Keren during other stages of development. Thus, it is proposed a combination of gene duplication, enhancer diversification and cofactor recruitment to be common mechanisms that allow the co-option of a single receptor-signalling pathway in distinct developmental and physiological functions (Grillo, 2012).

torso: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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